tetrodotoxin and Disease-Models--Animal

Voltage-gated sodium ion channel subtype 1.7 (Na

Topics: Analgesics; Animals; Disease Models, Animal; Humans; NAV1.7 Voltage-Gated Sodium Channel; Pain; Protein Isoforms; Signal Transduction; Sodium Channel Blockers; Sulfonamides

The sequelae of traumatic brain injury, including posttraumatic epilepsy, represent a major societal problem. Significant resources are required to develop a better understanding of the underlying pathophysiologic mechanisms as targets for potential prophylactic therapies. Posttraumatic epilepsy undoubtedly involves numerous pathogenic factors that develop more or less in parallel. We have highlighted two potential "prime movers": disinhibition and development of new functional excitatory connectivity, which occur in a number of animal models and some forms of epilepsy in humans. Previous experiments have shown that tetrodotoxin (TTX) applied to injured cortex during a critical period early after lesion placement can prevent epileptogenesis in the partial cortical ("undercut") model of posttraumatic epilepsy. Here we show that such treatment markedly attenuates histologic indices of axonal and terminal sprouting and presumably associated aberrant excitatory connectivity. A second finding in the undercut model is a decrease in spontaneous inhibitory events. Current experiments show that this is accompanied by regressive alterations in fast-spiking gamma-aminobutyric acid (GABA)ergic interneurons, including shrinkage of dendrites, marked decreases in axonal length, structural changes in inhibitory boutons, and loss of inhibitory synapses on pyramidal cells. Other data support the hypothesis that these anatomic abnormalities may result from loss of trophic support normally provided to interneurons by brain-derived neurotrophic factor (BDNF). Approaches that prevent these two pathophysiologic mechanisms may offer avenues for prophylaxis for posttraumatic epilepsy. However, major issues such as the role of these processes in functional recovery from injury and the timing of the critical period(s) for application of potential therapies in humans need to be resolved.

Topics: Animals; Anticonvulsants; Brain Injuries; Cerebral Cortex; Disease Models, Animal; Epilepsy, Post-Traumatic; gamma-Aminobutyric Acid; Humans; Interneurons; Nerve Net; Nerve Regeneration; Neural Inhibition; Neuronal Plasticity; Pyramidal Cells; Tetrodotoxin

Cervical spinal cord injury results in significant functional impairment. It is important to understand the neuroplasticity in response to inactivity of respiratory muscles in order to prevent any associated effects that limit functional recovery. Recent studies have examined the mechanisms involved in inactivity-induced neuroplasticity of diaphragm motor units. Both spinal hemisection at C2 (C2HS) and tetrodotoxin (TTX)-induced phrenic nerve blockade result in diaphragm paralysis and inactivity of axon terminals. However, phrenic motoneurons are inactive with C2HS but remain active after TTX. Diaphragm muscle fibers ipsilateral to C2HS display minimal changes post-injury. Neuromuscular transmission is enhanced following C2HS but impaired following TTX. Synaptic vesicle pool size at diaphragm neuromuscular junctions increases after C2HS, but decreases after TTX. Thus, inactivity-induced neuromuscular plasticity reflects specific adaptations that depend on inactivity at the motoneuron rather than at axon terminals or muscle fibers. These results indicate that neuromuscular transmission and functional properties of diaphragm fibers can be maintained after spinal cord injury, providing a substrate for functional recovery and/or specific therapeutic approaches such as phrenic pacing.

Topics: Adaptation, Physiological; Anesthetics, Local; Animals; Cervical Vertebrae; Diaphragm; Disease Models, Animal; Humans; Neuromuscular Junction; Neuronal Plasticity; Phrenic Nerve; Respiratory Muscles; Respiratory Paralysis; Spinal Cord Injuries; Synaptic Transmission; Tetrodotoxin

Topics: Animals; Brain; Disease Models, Animal; Elapid Venoms; Glutamates; Glutamic Acid; Humans; Hydroxydopamines; Kainic Acid; Nervous System Diseases; Neurons; Neurotoxins; Organ Specificity; Rats; Receptors, Neurotransmitter; Tetrodotoxin

Infantile spasms are associated with a wide variety of clinical conditions, including perinatal brain injuries. We have created a model in which prolonged infusion of tetrodotoxin (TTX) into the neocortex, beginning in infancy, produces a localized lesion and reproduces the behavioral spasms, electroencephalogram (EEG) abnormalities, and drug responsiveness seen clinically. Here, we undertook experiments to explore the possibility that the growth factor IGF-1 plays a role in generating epileptic spasms.. We combined long-term video EEG recordings with quantitative immunohistochemical and biochemical analyses to unravel IGF-1's role in spasm generation. Immunohistochemistry was undertaken in surgically resected tissue from infantile spasms patients. We used viral injections in neonatal conditional IGF-1R knock-out mice to show that an IGF-1-derived tripeptide (1-3)IGF-1, acts through the IGF-1 receptor to abolish spasms.. Immunohistochemical methods revealed widespread loss of IGF-1 from cortical neurons, but an increase in IGF-1 in the reactive astrocytes in the TTX-induced lesion. Very similar changes were observed in the neocortex from patients with spasms. In animals, we observed reduced signaling through the IGF-1 growth pathways in areas remote from the lesion. To show the reduction in IGF-1 expression plays a role in spasm generation, epileptic rats were treated with (1-3)IGF-1. We provide 3 lines of evidence that (1-3)IGF-1 activates the IGF-1 signaling pathway by acting through the receptor for IGF-1. Treatment with (1-3)IGF-1 abolished spasms and hypsarrhythmia-like activity in the majority of animals.. Results implicate IGF-1 in the pathogenesis of infantile spasms and IGF-1 analogues as potential novel therapies for this neurodevelopmental disorder. ANN NEUROL 2022;92:45-60.

Topics: Animals; Disease Models, Animal; Electroencephalography; Humans; Infant; Insulin-Like Growth Factor I; Mice; Rats; Spasm; Spasms, Infantile; Tetrodotoxin

Epileptic spasms are a hallmark of severe seizure disorders. The neurophysiological mechanisms and the neuronal circuit(s) that generate these seizures are unresolved and are the focus of studies reported here.. In the tetrodotoxin model, we used 16-channel microarrays and microwires to record electrophysiological activity in neocortex and thalamus during spasms. Chemogenetic activation was used to examine the role of neocortical pyramidal cells in generating spasms. Comparisons were made to recordings from infantile spasm patients.. Current source density and simultaneous multiunit activity analyses indicate that the ictal events of spasms are initiated in infragranular cortical layers. A dramatic pause of neuronal activity was recorded immediately prior to the onset of spasms. This preictal pause is shown to share many features with the down states of slow wave sleep. In addition, the ensuing interictal up states of slow wave rhythms are more intense in epileptic than control animals and occasionally appear sufficient to initiate spasms. Chemogenetic activation of neocortical pyramidal cells supported these observations, as it increased slow oscillations and spasm numbers and clustering. Recordings also revealed a ramp-up in the number of neocortical slow oscillations preceding spasms, which was also observed in infantile spasm patients.. Our findings provide evidence that epileptic spasms can arise from the neocortex and reveal a previously unappreciated interplay between brain state physiology and spasm generation. The identification of neocortical up states as a mechanism capable of initiating epileptic spasms will likely provide new targets for interventional therapies. ANN NEUROL 2021;89:226-241.

Topics: Animals; Brain Waves; Disease Models, Animal; Electrocorticography; Female; Humans; Infant; Male; Neocortex; Pyramidal Cells; Rats; Rats, Wistar; Seizures; Sodium Channel Blockers; Spasm; Spasms, Infantile; Tetrodotoxin; Thalamus

Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).

Topics: Animals; Astrocytes; Calcium Signaling; Disease Models, Animal; Disease Progression; Disease Susceptibility; Epilepsy; Gliosis; Inositol 1,4,5-Trisphosphate Receptors; Interleukin-1beta; Mice; Microglia; Muscarinic Agonists; Organic Chemicals; Pilocarpine; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; Sodium Channel Blockers; Status Epilepticus; Tetrodotoxin; Time Factors; Tumor Necrosis Factor-alpha

The primary studies have shown that scorpion analgesic peptide N58A has a significant effect on voltage-gated sodium channels (VGSCs) and plays an important role in neuropathic pain. The purpose of this study was to investigate the analgesic effect of N58A on trigeminal neuralgia (TN) and its possible mechanism. The results showed that N58A could significantly increase the threshold of mechanical pain and thermal pain and inhibit the spontaneous asymmetric scratching behavior of rats. Western blotting results showed that N58A could significantly reduce the protein phosphorylation level of ERK1/2, P38, JNK, and ERK5/CREB pathways and the expression of Nav1.8 and Nav1.9 proteins in a dose-dependent manner. The changes in current and kinetic characteristics of Nav1.8 and Nav1.9 channels in TG neurons were detected by the whole-cell patch clamp technique. The results showed that N58A significantly decreased the current density of Nav1.8 and Nav1.9 in model rats, and shifted the activation curve to hyperpolarization and the inactivation curve to depolarization. In conclusion, the analgesic effect of N58A on the chronic constriction injury of the infraorbital (IoN-CCI) model rats may be closely related to the regulation of the MAPK pathway and Nav1.8 and Nav1.9 sodium channels.

Topics: Analgesics; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Female; MAP Kinase Signaling System; NAV1.8 Voltage-Gated Sodium Channel; NAV1.9 Voltage-Gated Sodium Channel; Pain; Patch-Clamp Techniques; Peptides; Rats; Rats, Sprague-Dawley; Scorpion Venoms; Scorpions; Tetrodotoxin; Trigeminal Neuralgia

Severe arrhythmias-such as ventricular arrhythmias-can be fatal, but treatment options are limited. The effects of a combined formulation of tetrodotoxin (TTX) and lidocaine (LID) on severe arrhythmias were studied. Patch clamp recording data showed that the combination of LID and TTX had a stronger inhibitory effect on voltage-gated sodium channel 1.5 (Nav1.5) than that of either TTX or LID alone. LID + TTX formulations were prepared with optimal stability containing 1 μg of TTX, 5 mg of LID, 6 mg of mannitol, and 4 mg of dextran-40 and then freeze dried. This formulation significantly delayed the onset and shortened the duration of arrhythmia induced by aconitine in rats. Arrhythmia-originated death was avoided by the combined formulation, with a decrease in the mortality rate from 64% to 0%. The data also suggests that the anti-arrhythmic effect of the combination was greater than that of either TTX or LID alone. This paper offers new approaches to develop effective medications against arrhythmias.

Topics: Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Disease Models, Animal; Drug Combinations; Drug Stability; Excipients; Female; Freeze Drying; Lidocaine; Male; NAV1.5 Voltage-Gated Sodium Channel; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Voltage-Gated Sodium Channel Blockers

Neuropathic pain is a significant public health challenge, yet the underlying mechanisms remain poorly understood. Painful small fiber neuropathy (SFN) may be caused by gain-of-function mutations in Nav1.8, a sodium channel subtype predominantly expressed in peripheral nociceptive neurons. However, it is not clear how Nav1.8 disease mutations induce sensory neuron hyperexcitability. Here we studied two mutations in Nav1.8 associated with hypersensitive sensory neurons: G1662S reported in painful SFN; and T790A, which underlies increased pain behaviors in the

Topics: Action Potentials; Animals; Disease Models, Animal; Gain of Function Mutation; Humans; Ion Channel Gating; Ion Transport; Male; Mice; Mice, Neurologic Mutants; Mice, Transgenic; Mutation, Missense; NAV1.8 Voltage-Gated Sodium Channel; Neuralgia; Nociception; Patch-Clamp Techniques; Peripheral Nervous System Diseases; Point Mutation; Rats; Rats, Sprague-Dawley; Recombinant Proteins; RNA Interference; Sensory Receptor Cells; Sodium; Tetrodotoxin

The rostral ventromedial medulla (RVM) is highly involved in pain signal transmissions. Previous studies have shown that thalidomide is anti-nociceptive. Thus, we evaluated the neurobiological mechanisms of thalidomide in the RVM in the regulation of postoperative pain. We used a rat model of postoperative pain to investigate the effects of intra-RVM thalidomide treatments on postoperative pain, and evaluate the role of cannabinoid receptors in the effects of intra-RVM thalidomide treatments on GABAergic neurotransmission in the RVM neurons. We found intra-RVM thalidomide treatments reduced incisional surgery induced mechanical allodynia. This phenomenon was associated with attenuation of the frequency and amplitude of miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous IPSCs (sIPSCs) in RVM neurons. Furthermore, applications of WIN 55,212-3 mesylate, a non-selective cannabinoid receptor antagonist reversed the effects of repeated thalidomide treatment on the frequency but not the amplitude of mIPSCs and sIPSCs. Finally, we found that repeated thalidomide treatment robustly enhanced CB2 receptor expression, but slightly reduced CB1 receptor expression, in the RVM. These results suggested that the antinociceptive effects of thalidomide in the RVM likely involve the attenuation of GABA release, which are critically regulated by cannabinoid receptors.

Topics: Analgesics; Animals; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; Hyperalgesia; Inhibitory Postsynaptic Potentials; Male; Medulla Oblongata; Neurons; Pain Measurement; Pain Threshold; Pain, Postoperative; Quinoxalines; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin; Thalidomide; Valine

The physiological role of cardiac late Na

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Cardiac Pacing, Artificial; Disease Models, Animal; Female; Heart Rate; Heart Ventricles; In Vitro Techniques; Isolated Heart Preparation; Kinetics; Myocytes, Cardiac; Piperidines; Pyridines; Rabbits; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Triazoles

Homeostatic synaptic plasticity (HSP) serves as a gain control mechanism at central nervous system (CNS) synapses, including those between the dentate gyrus (DG) and CA3. Improper circuit control of DG-CA3 synapses is hypothesized to underlie epileptogenesis. Here, we sought to (1) identify compounds that preferentially modulate DG-CA3 synapses in primary neuronal culture and (2) determine if these compounds would delay or prevent epileptogenesis in vivo.. We previously developed and validated an in vitro assay to visualize the behavior of DG-CA3 synapses and predict functional changes. We used this "synapse-on-chip" assay (quantification of synapse size, number, and type using immunocytochemical markers) to dissect the mechanisms of HSP at DG-CA3 synapses. Using chemogenetic constructs and pharmacological agents we determined the signaling cascades necessary for gain control at DG-CA3 synapses. Finally, we tested the implicated cascades (using kappa opioid receptor (OR) agonists and antagonists) in two models of epileptogenesis: electrical amygdala kindling in the mouse and chemical (pentylenetetrazole) kindling in the rat.. In vitro, synapses between DG mossy fibers (MFs) and CA3 neurons are the primary homeostatic responders during sustained periods of activity change. Kappa OR signaling is both necessary and sufficient for the homeostatic elaboration of DG-CA3 synapses, induced by presynaptic DG activity levels. Blocking kappa OR signaling in vivo attenuates the development of seizures in both mouse and rat models of epilepsy.. This study elucidates mechanisms by which synapses between DG granule cells and CA3 pyramidal neurons undergo activity-dependent homeostatic compensation, via OR signaling in vitro. Modulation of kappa OR signaling in vivo alters seizure progression, suggesting that breakdown of homeostatic closed-loop control at DG-CA3 synapses contributes to seizures, and that targeting endogenous homeostatic mechanisms at DG-CA3 synapses may prove useful in combating epileptogenesis.

Topics: Animals; Cells, Cultured; Central Nervous System Stimulants; Convulsants; Disease Models, Animal; Disks Large Homolog 4 Protein; Dose-Response Relationship, Drug; Embryo, Mammalian; Epilepsy; Green Fluorescent Proteins; Hippocampus; Kindling, Neurologic; Male; Mice; Narcotic Antagonists; Narcotics; Neurons; Pentylenetetrazole; Picrotoxin; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Opioid, kappa; Repressor Proteins; Synapses; Synaptophysin; Tetrodotoxin; Transfection; Tumor Suppressor Proteins

Functional deactivation of the prefrontal cortex (PFC) is a critical step in the neuropathic pain phenotype. We performed optogenetic circuit dissection to study the properties of ventral hippocampal (vHipp) and thalamic (MDTh) inputs to L5 pyramidal cells in acute mPFC slices and to test whether alterations in these inputs contribute to mPFC deactivation in neuropathic pain. We found that: (1) both the vHipp and MDTh inputs elicit monosynaptic excitatory and polysynaptic inhibitory currents. (2) The strength of the excitatory MDTh input is uniform, while the vHipp input becomes progressively stronger along the dorsal-ventral axis. (3) Synaptic current kinetics suggests that the MDTh inputs contact distal, while the vHipp inputs contact proximal dendritic sections. (4) The longer delay of inhibitory currents in response to vHipp compared to MDTh inputs suggests that they are activated by feedback and feed-forward circuitries, respectively. (5) One week after a peripheral neuropathic injury, both glutamatergic inputs are modified: MDTh responses are smaller, without evidence of presynaptic changes, while the probability of release at vHipp-mPFC synapses becomes lower, without significant change in current amplitude. Thus, dysregulation of both these inputs likely contributes to the mPFC deactivation in neuropathic pain and may impair PFC-dependent cognitive tasks.

Topics: Action Potentials; Animals; Animals, Newborn; Channelrhodopsins; Disease Models, Animal; Excitatory Amino Acid Antagonists; Functional Laterality; Glutamic Acid; Hippocampus; Male; Nerve Net; Neural Inhibition; Neural Pathways; Neuralgia; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin; Thalamus; Valine

Recently, we have reported that animals with telomerase reverse transcriptase (TERT) overexpression exhibit reduced social interaction, decreased preference for novel social interaction and poor nest-building behaviors symptoms that mirror those observed in human autism spectrum disorders (ASD). Overexpression of TERT also alters the excitatory/inhibitory (E/I) ratio in the medial prefrontal cortex. However, the effects of TERT overexpression on hippocampal-dependent learning and synaptic efficacy have not been investigated. In the present study, we employed electrophysiological approaches in combination with behavioral analysis to examine hippocampal function of TERT transgenic (TERT-tg) mice and FVB controls. We found that TERT overexpression results in enhanced hippocampal excitation with no changes in inhibition and significantly impairs long-term synaptic plasticity. Interestingly, the expression levels of phosphorylated CREB and phosphory-lated CaMKIIα were significantly decreased while the expression level of CaMKIIα was slightly increased in the hippocampus of TERT-overexpressing mice. Our observations highlight the importance of TERT in normal synaptic function and behavior and provide additional information on a novel animal model of ASD associated with TERT overexpression.

Topics: Animals; Autism Spectrum Disorder; CA1 Region, Hippocampal; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cyclic AMP Response Element-Binding Protein; Disease Models, Animal; Excitatory Postsynaptic Potentials; Gene Expression; Hippocampus; Inhibitory Postsynaptic Potentials; Male; Maze Learning; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neuronal Plasticity; Neurotoxins; Patch-Clamp Techniques; Pyramidal Cells; Recombinant Proteins; Synaptic Transmission; Telomerase; Tetrodotoxin

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Carbazoles; Cells, Cultured; Cerebral Cortex; Cuprizone; Disease Models, Animal; Egtazic Acid; Encephalomyelitis, Autoimmune, Experimental; Excitatory Amino Acid Antagonists; Female; Freund's Adjuvant; Hyperkinesis; Membrane Potentials; Mice; Mice, Transgenic; Microglia; Myelin Proteolipid Protein; Peptide Fragments; Proto-Oncogene Proteins c-fos; Quinoxalines; Recovery of Function; Sodium Channel Blockers; Tetrodotoxin

Approximately 30% of patients with epilepsy are refractory to existing antiseizure drugs (ASDs). Given that the properties of the central nervous systems of these patients are likely to be altered due to their epilepsy, tissues from rodents that have undergone epileptogenesis might provide a therapeutically relevant disease substrate for identifying compounds capable of attenuating pharmacoresistant seizures. To facilitate the development of such a model, this study describes the effects of classical glutamate receptor antagonists and 20 ASDs on recurrent epileptiform discharges (REDs) in brain slices derived from the kainate-induced status epilepticus model of temporal lobe epilepsy (KA-rats).. Horizontal brain slices containing the medial entorhinal cortex (mEC) were prepared from KA-rats, and REDs were recorded from the superficial layers. 6-cyano-7-nitroquinoxaline-2,3-dione, (2R)-amino-5-phosphonovaleric acid, tetrodotoxin, or ASDs were bath applied for 20 minutes. Concentration-dependent effects and half maximal effective concentration values were determined for RED duration, frequency, and amplitude.. ASDs targeting sodium and potassium channels (carbamazepine, eslicarbazepine, ezogabine, lamotrigine, lacosamide, phenytoin, and rufinamide) attenuated REDs at concentrations near their average therapeutic plasma concentrations. γ-aminobutyric acid (GABA)ergic synaptic transmission-modulating ASDs (clobazam, midazolam, phenobarbital, stiripentol, tiagabine, and vigabatrin) attenuated REDs only at higher concentrations and, in some cases, prolonged RED durations. ASDs with other/mixed mechanisms of action (bumetanide, ethosuximide, felbamate, gabapentin, levetiracetam, topiramate, and valproate) and glutamate receptor antagonists weakly or incompletely inhibited RED frequency, increased RED duration, or had no significant effects.. Taken together, these data suggest that epileptiform activity recorded from the superficial layers of the mEC in slices obtained from KA-rats is differentially sensitive to existing ASDs. The different sensitivities of REDs to these ASDs may reflect persistent molecular, cellular, and/or network-level changes resulting from disease. These data are expected to serve as a foundation upon which future therapeutics may be differentiated and assessed for potentially translatable efficacy in patients with refractory epilepsy.

Topics: Animals; Anticonvulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Kainic Acid; Male; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin

Topics: Animals; Astrocytes; Connexin 30; Disease Models, Animal; Epilepsy, Temporal Lobe; Excitatory Postsynaptic Potentials; Female; Kainic Acid; Luminescent Proteins; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Receptors, Purinergic P2Y1; Signal Transduction; Sodium Channel Blockers; Synapses; Tetrodotoxin; Tumor Necrosis Factor-alpha

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G

Topics: Animals; Anti-Anxiety Agents; Anxiety; Brain Mapping; Cannabinoid Receptor Antagonists; Clozapine; Dark Adaptation; Disease Models, Animal; Estrenes; Excitatory Postsynaptic Potentials; Exploratory Behavior; Green Fluorescent Proteins; GTP-Binding Protein alpha Subunits, Gq-G11; In Vitro Techniques; Male; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Phosphodiesterase Inhibitors; Piperazines; Pyrrolidinones; Receptors, Drug; Rimonabant; RNA, Messenger; Septal Nuclei; Serotonin Receptor Agonists; Signal Transduction; Sodium Channel Blockers; Tetrodotoxin; Vesicular Inhibitory Amino Acid Transport Proteins

Catamenial epilepsy is a common central nervous system disease in female, which is influenced by the 17-β-estradiol (estrogen) level during the menstrual cycle. Low level (<0.05ng/ml) of estrogen normally accompanies with the perimenstrual classification of catamenial epilepsy, however, without clear mechanism. In previous studies, estrogen has been demonstrated to possess widely regulatory effects on potassium channels. Here, the effect of 17-β-estradiol on modulating inwardly rectifying K. In this research, null-estrogen cultures and spaying animals were used to mimicked the low level estrogen condition in menstrual period. Patch clamp recordings, western blotting and pharmacological experiments were performed to detect the effects of estrogen receptors and the underlying mechanisms.. Compared to those neurons in normal medium (with 0.1ng/ml estrogen), null-estrogen cultures or neurons treated by estrogen receptor blocker (ICI 182,780) both had significant suppressed Kir currents. The expression level of G protein-gated inwardly rectifying K. Taken together, 17-β-estradiol, by the activation of estrogen receptors, is essential for the maintenance of Kir currents, and thus has an inhibitory effect on the epileptiform bursting activities in cultured hippocampal neurons, whereas GIRK1 is the major intermedial mediator. This research provides a new mechanism for the pathogenesis of catamenial epilepsy, particularly in the menstrual period and the early section of follicular phase.

Topics: Animals; Bee Venoms; Cells, Cultured; Disease Models, Animal; Embryo, Mammalian; Epilepsy; Estradiol; Estrogen Receptor Antagonists; Female; Fulvestrant; G Protein-Coupled Inwardly-Rectifying Potassium Channels; Gene Expression Regulation; Hippocampus; Membrane Potentials; Neurons; Ovariectomy; Potassium Channel Blockers; Potassium Channels, Inwardly Rectifying; Pregnancy; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin

It has been postulated that glia play a critical role in modifying neuronal activity, mediating neurovascular coupling, and in seizure initiation. We investigated the role of glia in ictogenesis and neurovascular coupling through wide-field multicell and 2-photon single cell imaging of calcium and intrinsic signal imaging of cerebral blood volume in an in vivo rat model of focal neocortical seizures. Ictal events triggered a slowly propagating glial calcium wave that was markedly delayed after both neuronal and hemodynamic onset. Glial calcium waves exhibited a stereotypical spread that terminated prior to seizure offset and propagated to an area ~60% greater than the propagation area of neural and vascular signals. Complete blockage of glial activity with fluoroacetate resulted in no change in either neuronal or hemodynamic activity. These ictal glial waves were blocked by carbenoxolone, a gap junction blocker. Our in vivo data reveal that ictal events trigger a slowly propagating, stereotypical glial calcium wave, mediated by gap junctions, that is spatially and temporally independent of neuronal and hemodynamic activities. We introduce a novel ictally triggered propagating glial calcium wave calling into question the criticality of glial calcium wave in both ictal onset and neurovascular coupling.

Topics: 4-Aminopyridine; Animals; Brain Mapping; Calcium; Calcium Signaling; Carbenoxolone; Diagnostic Imaging; Disease Models, Animal; Epilepsy; Evoked Potentials, Somatosensory; Male; Neuroglia; Neurons; Neurovascular Coupling; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Somatosensory Cortex; Tetrodotoxin

Cardiac arrhythmias associated with intracellular calcium inhomeostasis are refractory to antiarrhythmic therapy. We hypothesized that late sodium current (I

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Arrhythmias, Cardiac; Calcium; Calcium Channel Agonists; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Disease Models, Animal; Male; Muscle Contraction; Oxazepines; Phosphorylation; Rabbits; Sodium Channels; Tetrodotoxin

Striatal neurons forming the indirect pathway (iSPNs) are particularly vulnerable in Huntington's disease (HD). In this study we set out to investigate morphological and physiological alterations of iSPNs in two mouse models of HD with relatively slow disease progression (long CAG repeat R6/2 and zQ175-KI). Both were crossed with a transgenic mouse line expressing eGFP in iSPNs. Using the open-field and rotarod tests, we first defined two time points in relation to the occurrence of motor deficits in each model. Then, we investigated electrophysiological and morphological properties of iSPNs at both ages. Both HD models exhibited increased iSPN excitability already before the onset of motor deficits, associated with a reduced number of primary dendrites and decreased function of Kir- and voltage-gated potassium channels. Alterations that specifically occurred at symptomatic ages included increased calcium release by back-propagating action potentials in proximal dendrites, due to enhanced engagement of intracellular calcium stores. Moreover, motorically impaired mice of both HD models showed a reduction in iSPN spine density and progressive formation of huntingtin (Htt) aggregates in the striatum. Our study therefore reports iSPN-specific alterations relative to the development of a motor phenotype in two different mouse models of HD. While some alterations occur early and are partly non-progressive, others potentially provide a pathophysiological marker of an overt disease state.

Topics: Action Potentials; Animals; Cadmium Chloride; Cesium; Chlorides; Corpus Striatum; Dendrites; Disease Models, Animal; Exploratory Behavior; Huntingtin Protein; Huntington Disease; Mice; Mice, Inbred C57BL; Mice, Transgenic; Movement Disorders; Neurons; Potassium; Psychomotor Performance; Sodium Channel Blockers; Tetrodotoxin; Trinucleotide Repeat Expansion

Visceral pain is very common and represents a major unmet clinical need for which current pharmacological treatments are often insufficient. Tetrodotoxin (TTX) is a potent neurotoxin that exerts analgesic actions in both humans and rodents under different somatic pain conditions, but its effect has been unexplored in visceral pain. Therefore, we tested the effects of systemic TTX in viscero-specific mouse models of chemical stimulation of the colon (intracolonic instillation of capsaicin and mustard oil) and intraperitoneal cyclophosphamide-induced cystitis. The subcutaneous administration of TTX dose-dependently inhibited the number of pain-related behaviors in all evaluated pain models and reversed the referred mechanical hyperalgesia (examined by stimulation of the abdomen with von Frey filaments) induced by capsaicin and cyclophosphamide, but not that induced by mustard oil. Morphine inhibited both pain responses and the referred mechanical hyperalgesia in all tests. Conditional nociceptor‑specific Na

Topics: Analgesics; Animals; Capsaicin; Colon; Cystitis; Disease Models, Animal; Female; Hyperalgesia; Male; Mice; Mice, Knockout; Morphine; Mustard Plant; Nociceptors; Pain Measurement; Plant Oils; Sodium Channels; Tetrodotoxin; Visceral Pain

Schizophrenia is a complex and devastating neuropsychiatric disease thought to result from impaired connectivity between several integrative regions, stemming from developmental failures. In particular, the left prefrontal cortex of schizophrenia patients seems to be targeted by such early developmental disturbances. Data obtained over the last three decades support the hypothesis of a dopaminergic dysfunction in schizophrenia. Striatal dopaminergic dysregulation in schizophrenia may result from a dysconnection between the prefrontal cortex and the striatum (dorsal and ventral) involving glutamatergic N-methyl-d-aspartate (NMDA) receptors. In the context of animal modeling of the pathophysiology of schizophrenia, the present study was designed to investigate the effects of MK 801 (dizocilpine) on locomotor activity and dopaminergic responses in the left core part of the nucleus accumbens (ventral striatum) in adult rats following neonatal tetrodotoxin inactivation of the left prefrontal cortex (infralimbic/prelimbic region) at postnatal day 8. Dopaminergic variations were recorded in the nucleus accumbens by means of in vivo voltammetry in freely moving adult animals. Following MK 801 administration, and in comparison to control (PBS) animals, animals microinjected with tetrodotoxin display locomotor hyperactivity and increased extracellular dopamine levels in the core part of the nucleus accumbens. These findings suggest neonatal functional inactivation of the prefrontal cortex may lead to a dysregulation of dopamine release in the core part of the nucleus accumbens involving NMDA receptors. The results obtained may provide new insight into the involvement of NMDA receptors in the pathophysiology of schizophrenia and suggest that future studies should look carefully at the core of the nucleus accumbens.

Topics: Animals; Animals, Newborn; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Dopamine; Dopamine Agents; Locomotion; Male; Neostriatum; Nucleus Accumbens; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Schizophrenia; Tetrodotoxin

Oral Bulleyaconitine A (BLA) is effective for treating neuropathic pain in human patients, but the underlying mechanism is poorly understood. Here, we tested whether BLA blocked voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons. Compelling evidence shows that voltage-gated sodium channels are upregulated in uninjured DRG neurons but downregulated in injured ones following peripheral nerve injury. We found that BLA preferably inhibited Na currents in uninjured DRG neurons in neuropathic rats. Compared to sham rats, IC50 values for resting and inactivated Na currents were 113 and 74 times lower in injured and uninjured neurons of L4-6 DRGs in spared nerve injury (SNI) rats (4.55 and 0.56 nM) and were 688 and 518 times lower in the uninjured L4 and L6 DRG neurons of L5 spinal nerve ligation (L5-SNL) rats. The use-dependent blockage of BLA on Na currents was more potent in neuropathic rats compared to sham rats. Bulleyaconitine A facilitated the inactivation of Na channels in each group. IC50 values for resting and inactivated tetrodotoxin-sensitive (TTX-S) channels were 1855 and 1843 times lower than those for TTX-resistant channels in the uninjured neurons of L5 spinal nerve ligation rats. The upregulation of protein kinase C was associated with the preferable effect of BLA on TTX-S Na channels in the uninjured DRG neurons. Local application of BLA onto L4-6 DRGs at 0.1 to 10 nM dose-dependently alleviated the mechanical allodynia and thermal hyperalgesia in L5 spinal nerve ligation model. Thus, preferable blockage of TTX-S Na channels in uninjured DRG neurons may contribute to BLA's antineuropathic pain effect.

Topics: Aconitine; Animals; Cadmium Chloride; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Male; Neuralgia; Patch-Clamp Techniques; Protein Kinase C; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Tetrodotoxin; Time Factors; Voltage-Gated Sodium Channels

Extracellular matrix protein-integrin interaction on neurons plays an important role in the development of neuroplasticity in the brain. However, the role of fibronectin-integrin signaling in epilepsy is elusive. Here, we examined the functional role of fibronectin-integrin signaling by utilizing a combination approach involving atomic force microscopy (AFM), immunocytochemistry, and pharmacology in epileptic mouse dentate gyrus granule cells (DGGCs). There was marked increase in the fibronectin receptor α5β1-integrin staining intensity in DGGCs in epileptic mice. In the AFM study, the unbinding force and binding probability between the fibronectin-coated AFM probe and the membrane integrins were significantly reduced; while the cell stiffness was strikingly increased in epileptic DGGCs. Pretreatment with α5β1-integrin monoclonal antibody partially reversed this membrane dysfunction. In patch-clamp recordings, fibronectin significantly inhibited GABA current, while RGD, which is known to disrupt fibronectin-integrin-dependent cell adhesive events, strikingly enhanced GABA tonic currents in DGGCs in hippocampal slices. The α5β1-integrin antibody significantly reduced 4-aminopyridine-induced epileptiform discharges in brain slices. In systemic behavioral studies, susceptibility to hippocampus kindling epileptogenesis was significantly attenuated in mice treated with RGD or β1-integrin antibody. These pilot studies provide new insights on the functional role of integrin receptor signaling in epileptogenesis and may help identify novel targets for the prevention and treatment of epilepsy.

Topics: 4-Aminopyridine; Action Potentials; Animals; Disease Models, Animal; Epilepsy; Excitatory Amino Acid Antagonists; Fibronectins; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Integrin alpha6beta1; Kynurenic Acid; Male; Mice; Mice, Inbred C57BL; Microscopy, Atomic Force; Neuronal Plasticity; Neurons; Potassium Channel Blockers; Signal Transduction; Sodium Channel Blockers; Tetrodotoxin

During cortical development, plasticity reflects the dynamic equilibrium between increasing and decreasing functional connectivity subserved by synaptic sprouting and pruning. After adult cortical deafferentation, plasticity seems to be dominated by increased functional connectivity, leading to the classical expansive reorganization from the intact to the deafferented cortex. In contrast, here we show a striking "decrease" in the fast cortical responses to high-intensity forepaw stimulation 1-3 months after complete thoracic spinal cord transection, as evident in both local field potentials and intracellular in vivo recordings. Importantly, this decrease in fast cortical responses co-exists with an "increase" in cortical activation over slower post-stimulus timescales, as measured by an increased forepaw-to-hindpaw propagation of stimulus-triggered cortical up-states, as well as by the enhanced slow sustained depolarization evoked by high-frequency forepaw stimuli in the deafferented hindpaw cortex. This coincidence of diminished fast cortical responses and enhanced slow cortical activation offers a dual perspective of adult cortical plasticity after spinal cord injury.

Topics: Action Potentials; Afferent Pathways; Analysis of Variance; Anesthetics, Local; Animals; Biophysics; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Hindlimb; Male; Neurons; Rats; Rats, Wistar; Somatosensory Cortex; Spinal Cord Injuries; Tetrodotoxin; Time Factors

The intergeniculate leaflet (IGL) of the thalamus is a retinorecipient structure implicated in orchestrating circadian rhythmicity. The IGL network is highly GABAergic and consists mainly of neuropeptide Y-synthesising and enkephalinergic neurons. A high density of GFAP-immunoreactive astrocytes has been observed in the IGL, with a probable function in guarding neuronal inhibition. Interestingly, putatively enkephalinergic IGL neurons generate action potentials with an infra-slow oscillatory (ISO) pattern in vivo in urethane anesthetised Wistar rats, under light-on conditions only. Absence epilepsy (AE) is a disease characterised by spike-wave discharges present in the encephalogram, directly caused by hypersynchronous thalamo-cortical oscillations. Many pathologies connected with the arousal system, such as abnormalities in sleep architecture and an insufficient brain sleep-promoting system accompany the epileptic phenotype. We hypothesise that disturbances in the function of biological clock structures, controlling this rhythmic physiological process, may be responsible for the observed pathomechanism. To test this hypothesis, we performed an in vitro patch-clamp study on WAG/Rij rats, a well-validated genetic model of AE, in order to assess dampened GABAergic synaptic transmission in the IGL expressed as a lower IPSC amplitude and reduced sIPSC frequency. Moreover, our in vivo extracellular recordings showed higher firing rate of ISO IGL neurons with an abnormal reaction to a change in constant illumination (maintenance of rhythmic neuronal activity in darkness) in the AE model. Additional immunohistochemical experiments indicated astrogliosis in the area of the IGL, which may partially underlie the observed changes in inhibition. Altogether, the data presented here show for the first time the disinhibition of IGL neurons in a model of AE, thereby proposing the possible involvement of circadian-related brain structures in the epileptic phenotype.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Animals; Animals, Newborn; Disease Models, Animal; Epilepsy, Absence; Excitatory Amino Acid Antagonists; GABA Agents; Geniculate Bodies; Inhibitory Postsynaptic Potentials; Male; Nerve Net; Neural Inhibition; Neuropeptide Y; Rats; Rats, Mutant Strains; Rats, Wistar; Sodium Channel Blockers; Tetrodotoxin; Valine

Abdominal pain is one of the major symptoms in bowel obstruction (BO); its cellular mechanisms remain incompletely understood. We tested the hypothesis that mechanical stress in obstruction upregulates expression of nociception mediator nerve growth factor (NGF) in gut smooth muscle cells (SMCs), and NGF sensitizes primary sensory nerve to contribute to pain in BO. Partial colon obstruction was induced with a silicon band implanted in the distal bowel of Sprague-Dawley rats. Colon-projecting sensory neurons in the dorsal root ganglia (T13 to L2) were identified for patch-clamp and gene expression studies. Referred visceral sensitivity was assessed by measuring withdrawal response to stimulation by von Frey filaments in the lower abdomen. Membrane excitability of colon-projecting dorsal root ganglia neurons was significantly enhanced, and the withdrawal response to von Frey filament stimulation markedly increased in BO rats. The expression of NGF mRNA and protein was increased in a time-dependent manner (day 1-day 7) in colonic SMC but not in mucosa/submucosa of the obstructed colon. Mechanical stretch in vitro caused robust NGF mRNA and protein expression in colonic SMC. Treatment with anti-NGF antibody attenuated colon neuron hyperexcitability and referred hypersensitivity in BO rats. Obstruction led to significant increases of tetrodotoxin-resistant Na currents and mRNA expression of Nav1.8 but not Nav1.6 and Nav1.7 in colon neurons; these changes were abolished by anti-NGF treatment. In conclusion, mechanical stress-induced upregulation of NGF in colon SMC underlies the visceral hypersensitivity in BO through increased gene expression and activity of tetrodotoxin-resistant Na channels in sensory neurons.

Topics: Animals; Antibodies; Bowen's Disease; Cells, Cultured; Colon; Disease Models, Animal; Ganglia, Spinal; Male; Membrane Potentials; Myocytes, Smooth Muscle; NAV1.6 Voltage-Gated Sodium Channel; NAV1.7 Voltage-Gated Sodium Channel; Nerve Growth Factor; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Tetrodotoxin; Up-Regulation

The gastrointestinal tract transports the food bolus by peristalsis. Gut motility starts at an early age in the developing embryo, well before it is required for nutrition of the organism. We present a comprehensive kinematic study of the emergence and physiological development of gut motility in all regions of the lower digestive tract of the chicken embryo from embryonic days E5 through E9. We characterized motility emergence time, propagation patterns, speed, frequency and amplitude of peristalsis waves. We found that the emergence of an uninterrupted circular ring of smooth muscle correlated with the appearance of propagative contractile waves, at E6 in the hindgut and midgut, and at E9 in the caecal appendix. We show that peristalsis at these stages is critically dependent on calcium and is not mediated by neurons as gut motility is insensitive to tetrodotoxin and takes place in the hindgut in the absence of neurons. We further demonstrate that motility also matures in ex-vivo organ culture. We compare our results to existing literature on zebrafish, mouse and human motility development, and discuss their chronological relationship with other major developmental events occurring in the chicken embryonic gut at these stages. Our work sets a baseline for further investigations of motility development in this important animal model.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Movement; Chick Embryo; Cobalt; Disease Models, Animal; Hirschsprung Disease; Intestines; Muscle, Smooth; Myenteric Plexus; Neural Crest; Organ Culture Techniques; Peristalsis; Tetrodotoxin; Time-Lapse Imaging

Chronic visceral pain is a defining feature of irritable bowel syndrome (IBS). IBS patients often show alterations in innate and adaptive immune function which may contribute to symptoms. Immune mediators are known to modulate the activity of viscero-sensory afferent nerves, but the focus has been on the innate immune system. Interleukin-2 (IL-2) is primarily associated with adaptive immune responses but its effects on colo-rectal afferent function in health or disease are unknown.. Myeloperoxidase (MPO) activity determined the extent of inflammation in health, acute trinitrobenzene-sulfonic acid (TNBS) colitis, and in our post-TNBS colitis model of chronic visceral hypersensitivity (CVH). The functional effects of IL-2 on high-threshold colo-rectal afferents and the expression of IL-2R and NaV 1.7 mRNA in colo-rectal dorsal root ganglia (DRG) neurons were compared between healthy and CVH mice.. MPO activity was increased during acute colitis, but subsided to levels comparable to health in CVH mice. IL-2 caused direct excitation of colo-rectal afferents that was blocked by tetrodotoxin. IL-2 did not affect afferent mechanosensitivity in health or CVH. However, an increased proportion of afferents responded directly to IL-2 in CVH mice compared with controls (73% vs 33%; p < 0.05), and the abundance of IL-2R and NaV 1.7 mRNA was increased 3.5- and 2-fold (p < 0.001 for both) in colo-rectal DRG neurons.. IL-2, an immune mediator from the adaptive arm of the immune response, affects colo-rectal afferent function, indicating these effects are not restricted to innate immune mediators. Colo-rectal afferent sensitivity to IL-2 is increased long after healing from inflammation.

Topics: Adaptive Immunity; Afferent Pathways; Animals; Colitis; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Interleukin-2; Irritable Bowel Syndrome; Mice; NAV1.7 Voltage-Gated Sodium Channel; Neurons, Afferent; Peroxidase; Real-Time Polymerase Chain Reaction; Receptors, Interleukin-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium Channel Blockers; Tetrodotoxin; Trinitrobenzenesulfonic Acid; Visceral Pain

Dry eye disease (DED) affects >10% of the population worldwide, and it provokes an unpleasant sensation of ocular dryness, whose underlying neural mechanisms remain unknown. Removal of the main lachrymal gland in guinea pigs caused long-term reduction of basal tearing accompanied by changes in the architecture and density of subbasal corneal nerves and epithelial terminals. After 4 weeks, ongoing impulse activity and responses to cooling of corneal cold thermoreceptor endings were enhanced. Menthol (200 μM) first excited and then inactivated this augmented spontaneous and cold-evoked activity. Comparatively, corneal polymodal nociceptors of tear-deficient eyes remained silent and exhibited only a mild sensitization to acidic stimulation, whereas mechanonociceptors were not affected. Dryness-induced changes in peripheral cold thermoreceptor responsiveness developed in parallel with a progressive excitability enhancement of corneal cold trigeminal ganglion neurons, primarily due to an increase of sodium currents and a decrease of potassium currents. In corneal polymodal nociceptor neurons, sodium currents were enhanced whereas potassium currents remain unaltered. In healthy humans, exposure of the eye surface to menthol vapors or to cold air currents evoked unpleasant sensations accompanied by increased blinking frequency that we attributed to cold thermoreceptor stimulation. Notably, stimulation with menthol reduced the ongoing background discomfort of patients with DED, conceivably due to use-dependent inactivation of cold thermoreceptors. Together, these data indicate that cold thermoreceptors contribute importantly to the detection and signaling of ocular surface wetness, and develop under chronic eye dryness conditions an injury-evoked neuropathic firing that seems to underlie the unpleasant sensations experienced by patients with DED.

Topics: Action Potentials; Adult; Animals; Blinking; Cold Temperature; Cornea; Disease Models, Animal; Dry Eye Syndromes; Female; Humans; Male; Middle Aged; Neurons, Afferent; Nociceptors; Potassium Channel Blockers; Sensation; Sensory Receptor Cells; Sodium Channel Blockers; Swine; Tears; Tetraethylammonium; Tetrodotoxin; Thermoreceptors; Trigeminal Ganglion; Young Adult

Clinical studies have shown that post-traumatic stress disorder (PTSD) remission, induced by selective serotonin reuptake inhibitor (SSRI) treatment, is associated with increased prefrontal activation during post-treatment symptom provocation. Other studies have shown that continuation SSRI treatment after remitting from PTSD reduces the rate of relapse. The aim of the present preclinical study was to investigate the relationship between post-treatment prefrontal changes and PTSD relapse prevention. Avoidance conditioning (with a 1.5-mA foot-shock), avoidance extinction and a trauma priming exposure (with a 0.3-mA foot-shock) were used in mice to induce, suppress and reactivate PTSD-like symptoms (including avoidance, fear sensitization, enhanced contextual fear, and anxiety-like behavior), respectively. Paroxetine, injected at 8 mg/kg/day (7 days), was used as SSRI treatment. PTSD-like symptoms were present for at least 30 days and resistant to paroxetine treatment. However, after extinction training (suppressing all PTSD-like symptoms), paroxetine treatment prevented symptom reactivation. Paroxetine treatment also induced infralimbic neuronal activation. However, infralimbic functional tetrodotoxin inactivation abolished the preventive effect of paroxetine treatment on symptom reactivation. The data reveal a potential ability of treatments inducing infralimbic activation to provide prophylactic protection against PTSD relapse.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Antidepressive Agents, Second-Generation; Avoidance Learning; Cerebral Cortex; Conditioning, Classical; Cues; Disease Models, Animal; Electroshock; Freezing Reaction, Cataleptic; Male; Maze Learning; Mice; Oncogene Proteins v-fos; Paroxetine; Recurrence; Stress Disorders, Post-Traumatic; Tetrodotoxin; Time Factors

Each year, approximately 3.8 million people suffer mild to moderate traumatic brain injuries (mTBI) that result in an array of neuropsychological symptoms and disorders. Despite these alarming statistics, the neurological bases of these persistent, debilitating neuropsychological symptoms are currently poorly understood. In this study we examined the effects of mTBI on the amygdala, a brain structure known to be critically involved in the processing of emotional stimuli. Seven days after lateral fluid percussion injury (LFPI), mice underwent a series of physiological and behavioral experiments to assess amygdala function. Brain-injured mice exhibited a decreased threat response in a cued fear conditioning paradigm, congruent with a decrease in amygdala excitability determined with basolateral amygdala (BLA) field excitatory post-synaptic potentials together with voltage-sensitive dye imaging (VSD). Furthermore, beyond exposing a general decrease in the excitability of the primary input of the amygdala, the lateral amygdala (LA), VSD also revealed a decrease in the relative strength or activation of internuclear amygdala circuit projections after LFPI. Thus, not only does activation of the LA require increased stimulation, but the proportion of this activation that is propagated to the primary output of the amygdala, the central amygdala, is also diminished following LFPI. Intracellular recordings revealed no changes in the intrinsic properties of BLA pyramidal neurons after LFPI. This data suggests that mild to moderate TBI has prominent effects on amygdala function and provides a potential neurological substrate for many of the neuropsychological symptoms suffered by TBI patients.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Brain Injuries; Brain Mapping; Conditioning, Psychological; Cues; Disease Models, Animal; Electric Stimulation; Escape Reaction; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Fear; Male; Mice; Mice, Inbred C57BL; Sodium Channel Blockers; Tetrodotoxin; Voltage-Sensitive Dye Imaging

Neuronal activity sculpts brain development by inducing the transcription of genes such as brain-derived neurotrophic factor (Bdnf) that modulate the function of synapses. Sensory experience is transduced into changes in gene transcription via the activation of calcium signaling pathways downstream of both L-type voltage-gated calcium channels (L-VGCCs) and NMDA-type glutamate receptors (NMDARs). These signaling pathways converge on the regulation of transcription factors including calcium-response factor (CaRF). Although CaRF is dispensable for the transcriptional induction of Bdnf following the activation of L-VGCCs, here we show that the loss of CaRF leads to enhanced NMDAR-dependent transcription of Bdnf as well as Arc. We identify the NMDAR subunit-encoding gene Grin3a as a regulatory target of CaRF, and we show that expression of both Carf and Grin3a is depressed by the elevation of intracellular calcium, linking the function of this transcriptional regulatory pathway to neuronal activity. We find that light-dependent activation of Bdnf and Arc transcription is enhanced in the visual cortex of young CaRF knockout mice, suggesting a role for CaRF-dependent dampening of NMDAR-dependent transcription in the developing brain. Finally, we demonstrate that enhanced Bdnf expression in CaRF-lacking neurons increases inhibitory synapse formation. Taken together, these data reveal a novel role for CaRF as an upstream regulator of NMDAR-dependent gene transcription and synapse formation in the developing brain. NMDARs promote brain development by inducing the transcription of genes, including brain-derived neurotrophic factor (BDNF). We show that the transcription factor calcium-response factor (CaRF) limits NMDAR-dependent BDNF induction by regulating expression of the NMDAR subunit GluN3A. Loss of CaRF leads to enhanced BDNF-dependent GABAergic synapse formation indicating the importance of this process for brain development. Our observation that both CaRF and GluN3A are down-regulated by intracellular calcium suggests that this may be a mechanism for experience-dependent modulation of synapse formation.

Topics: Animals; Animals, Newborn; Brain; Brain-Derived Neurotrophic Factor; Calcium Channel Blockers; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Female; Gene Expression Regulation, Developmental; Male; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Tetrodotoxin; Transcription Factors; Valine; Visual Cortex

Cannabinoids have anti-inflammatory effects and can produce bronchodilation in the airways. We have investigated the effects of cannabinoids on tracheal hyperreactivity and airway inflammation in dinitrofluorobenzene (DNFB)-induced experimental non-atopic asthma in mice. 5-hydroxytryptamine (5-HT)-induced contraction response was enhanced while carbachol- and electrical field stimulation-induced contractions, and isoprenaline-induced relaxation responses were remained unchanged in DNFB group. The increased 5-HT-induced contractions were inhibited by incubation with either atropine or tetrodotoxin. DNFB application resulted in increased macrophage number in the bronchoalveolar lavage fluid (BALF). In vivo ACEA (CB1 agonist) treatment prevented the increase in 5-HT contractions, while JWH133 (CB2 agonist) had no effect. However, neither ACEA nor JWH133 prevented the increase in macrophage number in BALF. In vitro ACEA incubation also inhibited the increase in 5-HT contraction in DNFB group. These results show that cannabinoid CB1 receptor agonist can prevent tracheal hyperreactivity to 5-HT in DNFB-induced non-atopic asthma in mice.

Topics: Acetylcholine; Animals; Anti-Asthmatic Agents; Asthma; Atropine; Cannabinoid Receptor Agonists; Cannabinoids; Carbachol; Dinitrofluorobenzene; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Female; Macrophages; Mice; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Serotonin; Tetrodotoxin; Trachea

Arginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome.. These studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

Topics: Action Potentials; Ammonia; Animals; Animals, Newborn; Arginase; Disease Models, Animal; Genetic Therapy; Hyperargininemia; In Vitro Techniques; Mice; Mice, Transgenic; Motor Cortex; Nerve Net; Neurons; Picrotoxin; Recovery of Function; Sodium Channel Blockers; Synapses; Tetrodotoxin

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) can cause Parkinson's disease (PD), and the most common disease-associated mutation, G2019S, increases kinase activity. Because LRRK2 expression levels rise during synaptogenesis and are highest in dorsal striatal spiny projection neurons (SPNs), we tested the hypothesis that the LRRK2-G2019S mutation would alter development of excitatory synaptic networks in dorsal striatum. To circumvent experimental confounds associated with LRRK2 overexpression, we used mice expressing LRRK2-G2019S or D2017A (kinase-dead) knockin mutations. In whole-cell recordings, G2019S SPNs exhibited a fourfold increase in sEPSC frequency compared with wild-type SPNs in postnatal day 21 mice. Such heightened neural activity was increased similarly in direct- and indirect-pathway SPNs, and action potential-dependent activity was particularly elevated. Excitatory synaptic activity in D2017A SPNs was similar to wild type, indicating a selective effect of G2019S. Acute exposure to LRRK2 kinase inhibitors normalized activity, supporting that excessive neural activity in G2019S SPNs is mediated directly and is kinase dependent. Although dendritic arborization and densities of excitatory presynaptic terminals and postsynaptic dendritic spines in G2019S SPNs were similar to wild type, G2019S SPNs displayed larger spines that were matched functionally by a shift toward larger postsynaptic response amplitudes. Acutely isolating striatum from overlying neocortex normalized sEPSC frequency in G2019S mutants, supporting that abnormal corticostriatal activity is involved. These findings indicate that the G2019S mutation imparts a gain-of-abnormal function to SPN activity and morphology during a stage of development when activity can permanently modify circuit structure and function.. Mutations in the kinase domain of leucine-rich repeat kinase 2 (LRRK2) follow Parkinson's disease (PD) heritability. How such mutations affect brain function is poorly understood. LRRK2 expression levels rise after birth at a time when synapses are forming and are highest in dorsal striatum, suggesting that LRRK2 regulates development of striatal circuits. During a period of postnatal development when activity plays a large role in permanently shaping neural circuits, our data show how the most common PD-causing LRRK2 mutation dramatically alters excitatory synaptic activity and the shape of postsynaptic structures in striatum. These findings provide new insight into early functional and structural aberrations in striatal connectivity that may predispose striatal circuitry to both motor and nonmotor dysfunction later in life.

Topics: Animals; Animals, Newborn; Corpus Striatum; Dendrites; Disease Models, Animal; Excitatory Postsynaptic Potentials; Female; Gene Expression Regulation, Developmental; In Vitro Techniques; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neurons; Parkinson Disease; Patch-Clamp Techniques; Receptors, Dopamine D1; Sodium Channel Blockers; Tetrodotoxin

After spinal cord injury, central neuropathic pain develops in the majority of spinal cord injury patients. Spinal hemisection in rats, which has been developed as an animal model of spinal cord injury in humans, results in hyperexcitation of spinal dorsal horn neurons soon after the hemisection and thereafter. The hyperexcitation is likely caused by permanent elimination of the descending pain systems. We examined the change in synaptic transmission of substantia gelatinosa neurons following acute spinal hemisection by using an in vivo whole-cell patch-clamp technique.. An increased spontaneous action potential firings of substantia gelatinosa neurons was detected in hemisected rats compared with that in control animals. The frequencies and amplitudes of spontaneous excitatory postsynaptic currents and of evoked excitatory postsynaptic currentss in response to non-noxious and noxious stimuli were not different between hemisected and control animals. On the contrary, the amplitude and frequency of spontaneous inhibitory postsynaptic currents of substantia gelatinosa neurons in hemisected animals were significantly smaller and lower, respectively, than those in control animals (P < 0.01). Large amplitude and high-frequency spontaneous inhibitory postsynaptic currents, which could not be elicited by mechanical stimuli, were seen in 44% of substantia gelatinosa neurons in control animals but only in 17% of substantia gelatinosa neurons in hemisected animals. In control animals, such large amplitude spontaneous inhibitory postsynaptic currents were suppressed by spinal application of tetrodotoxin (1 µM). Cervical application of lidocaine (2%, 10 µl) also inhibited such large amplitude of inhibitory postsynaptic currents. The proportion of multi-receptive substantia gelatinosa neurons, which exhibit action potential firing in response to non-noxious and noxious stimuli, was much larger in hemisected animals than in control animals.. These suggest that substantia gelatinosa neurons receive tonic inhibition by spinal inhibitory interneurons which generate persistent action potentials. Spinal hemisection results in hyperexcitation of substantia gelatinosa neurons at least in part by eliminating the tonic descending control of spinal inhibitory interneurons from supraspinal levels.

Topics: Anesthetics, Intravenous; Animals; Bicuculline; Disease Models, Animal; Electric Stimulation; Functional Laterality; Hyperalgesia; Male; Neurons; Neurotransmitter Agents; Patch-Clamp Techniques; Physical Stimulation; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Strychnine; Substantia Gelatinosa; Synaptic Transmission; Tetrodotoxin; Urethane

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes.

Topics: Acetamides; Action Potentials; Animals; Cells, Cultured; Computer Simulation; Disease Models, Animal; Electron Transport Complex IV; Ganglia, Spinal; Lacosamide; Male; Models, Neurological; Nociceptors; p38 Mitogen-Activated Protein Kinases; Pain; Patch-Clamp Techniques; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Tumor Necrosis Factor-alpha

KCNQ5/Kv7.5, a low-threshold noninactivating voltage-gated potassium channel, is preferentially targeted to excitatory endings of auditory neurons in the adult rat brainstem. Endbulds of Held from auditory nerve axons on the bushy cells of the ventral cochlear nucleus (VCN) and calyces of Held around the principal neurons in the medial nucleus of the trapezoid body (MNTB) are rich in KCNQ5 immunoreactivity. We have previously shown that this synaptic distribution occurs at about the time of hearing onset. The current study tests whether this localization in excitatory endings depends on the peripheral activity carried by the auditory nerve. Auditory nerve activity was abolished by cochlear removal or intracochlear injection of tetrodotoxin (TTX). Presence of KCNQ5 was analyzed by immunocytochemistry, Western blotting, and quantitative reverse transcription polymerase chain reaction. After cochlear removal, KCNQ5 immunoreactivity was virtually undetectable at its usual location in endbulbs and calyces of Held in the anteroventral CN and in the MNTB, respectively, although it was found in cell bodies in the VCN. The results were comparable after intracochlear TTX injection, which drastically reduced KCNQ5 immunostaining in MNTB calyces and increased immunolabeling in VCN cell bodies. Endbulbs of Held in the VCN also showed diminished KCNQ5 labeling after intracochlear TTX injection. These results show that peripheral activity from auditory nerve afferents is necessary to maintain the subcellular distribution of KCNQ5 in synaptic endings of the auditory brainstem. This may contribute to adaptations in the excitability and neurotransmitter release properties of these presynaptic endings under altered input conditions.

Topics: Anesthetics, Local; Animals; Auditory Diseases, Central; Brain Stem; Calbindin 2; Cochlear Diseases; Disease Models, Animal; Evoked Potentials, Auditory, Brain Stem; Female; Fluoresceins; Gene Expression Regulation; KCNQ Potassium Channels; Male; Nerve Degeneration; Neurons; Rats; Rats, Wistar; RNA, Messenger; Tetrodotoxin; Time Factors

New therapeutic approaches to improve cardiac contractility without severe risk would improve the management of acute heart failure. Increasing systolic sodium influx can increase cardiac contractility, but most sodium channel activators have proarrhythmic effects that limit their clinical use. Here, we report the cardiac effects of a novel positive inotropic peptide isolated from the toxin of the Black Judean scorpion that activates neuronal tetrodotoxin-sensitive sodium channels.. All venoms and peptides were isolated from Black Judean Scorpions (Buthotus Hottentotta) caught in the Judean Desert. The full scorpion venom increased left ventricular function in sedated mice in vivo, prolonged ventricular repolarization, and provoked ventricular arrhythmias. An inotropic peptide (BjIP) isolated from the full venom by chromatography increased cardiac contractility but did neither provoke ventricular arrhythmias nor prolong cardiac repolarization. BjIP increased intracellular calcium in ventricular cardiomyocytes and prolonged inactivation of the cardiac sodium current. Low concentrations of tetrodotoxin (200 nmol/L) abolished the effect of BjIP on calcium transients and sodium current. BjIP did not alter the function of Nav1.5, but selectively activated the brain-type sodium channels Nav1.6 or Nav1.3 in cellular electrophysiological recordings obtained from rodent thalamic slices. Nav1.3 (SCN3A) mRNA was detected in human and mouse heart tissue.. Our pilot experiments suggest that selective activation of tetrodotoxin-sensitive neuronal sodium channels can safely increase cardiac contractility. As such, the peptide described here may become a lead compound for a new class of positive inotropic agents.

Topics: Animals; Disease Models, Animal; Heart; Heart Failure; Heart Ventricles; Mice; Myocardial Contraction; Myocytes, Cardiac; Pilot Projects; Sodium; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents (Kv) and outward delayed-rectifier potassium currents (IK), but not outward transient potassium currents (IA), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced IK increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of IK via activation of the PI3-K signaling pathway.

Topics: 4-Aminopyridine; Animals; Cerebral Infarction; Disease Models, Animal; Enzyme Inhibitors; Hippocampus; In Vitro Techniques; Infarction, Middle Cerebral Artery; Kv1.2 Potassium Channel; Male; Membrane Potentials; Neurons; Phosphatidylinositol 3-Kinases; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Signal Transduction; Sodium Channel Blockers; Tetrodotoxin; Vascular Endothelial Growth Factor A

Rufinamide is a structurally novel, antiepileptic drug approved for the treatment of Lennox-Gastaut syndrome. Its mechanism of action involves inhibition of voltage-gated Na+ channels (VGSCs) with possible membrane-stabilizing effects. VGSCs play a significant role in the pathogenesis of neuropathic pain. Therefore, we investigated the effects of rufinamide on tetrodotoxin-resistant sodium current (TTX-R I(Na)) in acutely dissociated rat dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats by using whole-cell voltage-clamp configuration. In addition, the functional and behavioural nociceptive parameters were evaluated to assess its potential in diabetic neuropathy. Diabetic rats demonstrated the mechanical allodynia and thermal hyperalgesia with reduced nerve perfusion and conduction velocity as compared to control. Rufinamide treatments (3 and 10 mg/kg) significantly improved these functional and nociceptive deficits. Diabetic rat DRG neurons exhibited increased TTX-R I(Na) density as compared to control. The voltage-dependent activation and steady-state inactivation curves for TTX-R I(Na) in DRG neurons from diabetic rats were shifted negatively as compared to control. Rufinamide treatments significantly blocked the TTX-R Na+ channel activity as evident from significant reduction in I(Na) density and hyperpolarizing shift in activation and inactivation curves as compared to diabetic control. This suggests that rufinamide acts on TTX-R Na+ channels, reduces channel activity and attenuates nerve functional and behavioral parameters in diabetic rats. Altogether, these results indicate therapeutic potential of rufinamide in the treatment of diabetic neuropathy.

Topics: Animals; Biophysical Phenomena; Blood Glucose; Diabetes Mellitus, Experimental; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Ganglia, Spinal; Mental Disorders; Neural Conduction; Neurons; Patch-Clamp Techniques; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Triazoles

Corneal injury can produce photophobia, an aversive sensitivity to light. Using topical application of lidocaine, a local anesthetic, and tetrodotoxin (TTX), a selective voltage-sensitive sodium channel blocker, we assessed whether enhanced aversiveness to light induced by corneal injury in rats was caused by enhanced activity in corneal afferents. Eye closure induced by 30 seconds of exposure to bright light (460-485 nm) was increased 24 hours after corneal injury induced by de-epithelialization. Although the topical application of lidocaine did not affect the baseline eye closure response to bright light in control rats, it eliminated the enhancement of the response to the light stimulus after corneal injury (photophobia). Similarly, topical application of TTX had no effect on the eye closure response to bright light in rats with intact corneas, but it markedly attenuated photophobia in rats with corneal injury. Given the well-established corneal toxicity of local anesthetics, we suggest TTX as a therapeutic option to treat photophobia and possibly other symptoms that occur in clinical diseases that involve corneal nociceptor sensitization. Perspective: We show that lidocaine and TTX attenuate photophobia induced by corneal injury. Although corneal toxicity limits use of local anesthetics, TTX may be a safer therapeutic option to reduce the symptom of photophobia associated with corneal injury.

Topics: Administration, Topical; Analysis of Variance; Anesthetics, Local; Animals; Corneal Injuries; Disease Models, Animal; Dose-Response Relationship, Drug; Lidocaine; Male; Photic Stimulation; Photophobia; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

Diabetes mellitus is a common metabolic disease in human beings with characteristic symptoms of hyperglycemia, chronic inflammation and insulin resistance. One of the most common complications of early-onset diabetes mellitus is peripheral diabetic neuropathy, which is manifested either by loss of nociception or by allodynia and hyperalgesia. Dietary fatty acids, especially polyunsaturated fatty acids, have been shown the potential of anti-inflammation and modulating neuron excitability. The present study investigated the effects of docosahexaenoic acid (DHA) on the excitability of dorsal root ganglion (DRG) neurons in streptozotocin (STZ)-induced diabetes rats. The effects of DHA on the allodynia and hyperalgesia of diabetic rats were also evaluated. Dietary DHA supplementation effectively attenuated both allodynia and hyperalgesia induced by STZ injection. DHA supplementation decreased the excitability of DRG neurons by decreasing the sodium currents and increasing potassium currents, which may contribute to the effect of alleviating allodynia and hyperalgesia in diabetic rats. The results suggested that DHA might be useful as an adjuvant therapy for the prevention and treatment of painful diabetic neuropathy.

Topics: Action Potentials; Animals; Diabetes Mellitus, Experimental; Disease Models, Animal; Docosahexaenoic Acids; Ganglia, Spinal; Hyperalgesia; Ion Channels; Male; Neurons; Pain Measurement; Pain Threshold; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Streptozocin; Tetrodotoxin; Time Factors

SHANK3 (also known as PROSAP2) is a postsynaptic scaffolding protein at excitatory synapses in which mutations and deletions have been implicated in patients with idiopathic autism, Phelan-McDermid (aka 22q13 microdeletion) syndrome, and other neuropsychiatric disorders. In this study, we have created a novel mouse model of human autism caused by the insertion of a single guanine nucleotide into exon 21 (Shank3(G)). The resulting frameshift causes a premature STOP codon and loss of major higher molecular weight Shank3 isoforms at the synapse. Shank3(G/G) mice exhibit deficits in hippocampus-dependent spatial learning, impaired motor coordination, altered response to novelty, and sensory processing deficits. At the cellular level, Shank3(G/G) mice also exhibit impaired hippocampal excitatory transmission and plasticity as well as changes in baseline NMDA receptor-mediated synaptic responses. This work identifies clear alterations in synaptic function and behavior in a novel, genetically accurate mouse model of autism mimicking an autism-associated insertion mutation. Furthermore, these findings lay the foundation for future studies aimed to validate and study region-selective and temporally selective genetic reversal studies in the Shank3(G/G) mouse that was engineered with such future experiments in mind.

Topics: Animals; Disease Models, Animal; Excitatory Postsynaptic Potentials; Exons; Exploratory Behavior; Female; Grooming; Hippocampus; Locomotion; Male; Maze Learning; Mental Disorders; Mice; Mice, Transgenic; Microfilament Proteins; Mutagenesis, Insertional; Mutation; N-Methylaspartate; Nerve Tissue Proteins; Nesting Behavior; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

A afferent fibers have been reported to participate in the development of the central sensitization induced by inflammation and injuries. Current evidence suggests that myofascial trigger points (MTrPs) induce central sensitization in the related spinal dorsal horn, and clinical studies indicate that A fibers are associated with pain behavior. Because most of these clinical studies applied behavioral indexes, objective evidence is needed. Additionally, MTrP-related neurons in dorsal root ganglia and the spinal ventral horn have been reported to be smaller than normal, and these neurons were considered to be related to A fibers. To confirm the role of A fibers in MTrP-related central changes in the spinal dorsal horn, we studied central sensitization as well as the size of neurons associated with myofascial trigger spots (MTrSs, equivalent to MTrPs in humans) in the biceps femoris muscle of rats and provided some objective morphological evidence. Cholera toxin B subunit-conjugated horseradish peroxidase was applied to label the MTrS-related neurons, and tetrodotoxin was used to block A fibers specifically. The results showed that in the spinal dorsal horn associated with MTrS, the expression of glutamate receptor (mGluR1α/mGluR5/NMDAR1) increased, while the mean size of MTrS-related neurons was smaller than normal. After blocking A fibers, these changes reversed to some extent. Therefore, we concluded that A fibers participated in the development and maintenance of the central sensitization induced by MTrPs and were related to the mean size of neurons associated with MTrPs in the spinal dorsal horn.

Topics: Afferent Pathways; Animals; Basement Membrane; Cholera Toxin; Disease Models, Animal; Electromyography; Horseradish Peroxidase; Male; Muscle, Skeletal; Myofascial Pain Syndromes; Nerve Fibers, Myelinated; Neurons, Afferent; Rats; Rats, Wistar; Receptors, Glutamate; Spinal Cord Dorsal Horn; Statistics, Nonparametric; Tetrodotoxin

Patients that suffer mild traumatic brain injuries (mTBI) often develop cognitive impairments, including memory and learning deficits. The hippocampus shows a high susceptibility to mTBI-induced damage due to its anatomical localization and has been implicated in cognitive and neurological impairments after mTBI. However, it remains unknown whether mTBI cognitive impairments are a result of morphological and pathophysiological alterations occurring in the CA1 hippocampal region. We investigated whether mTBI induces morphological and pathophysiological alterations in the CA1 using the controlled cortical impact (CCI) model. Seven days after CCI, animals subjected to mTBI showed cognitive impairment in the passive avoidance test and deficits to long-term potentiation (LTP) of synaptic transmission. Deficiencies in inducing or maintaining LTP were likely due to an observed reduction in the activation of NMDA but not AMPA receptors. Significant reductions in the frequency and amplitude of spontaneous and miniature GABAA-receptor mediated inhibitory postsynaptic currents (IPSCs) were also observed 7 days after CCI. Design-based stereology revealed that although the total number of neurons was unaltered, the number of GABAergic interneurons is significantly reduced in the CA1 region 7 days after CCI. Additionally, the surface expression of α1, ß2/3, and γ2 subunits of the GABAA receptor were reduced, contributing to a reduced mIPSC frequency and amplitude, respectively. Together, these results suggest that mTBI causes a significant reduction in GABAergic inhibitory transmission and deficits to NMDA receptor mediated currents in the CA1, which may contribute to changes in hippocampal excitability and subsequent cognitive impairments after mTBI.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Avoidance Learning; Brain Injuries; CA1 Region, Hippocampal; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; GABAergic Neurons; Glucose Transporter Type 1; Glutamate Decarboxylase; Inhibitory Postsynaptic Potentials; Interneurons; Male; Memory Disorders; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, GABA-A; Sodium Channel Blockers; Tetrodotoxin; Time Factors

Glucose uptake in neurons depends on their cellular/physiological activity and the extracellular concentration of glucose around the cell. High concentration of extra-cellular glucose, as under hyperglycemic conditions or pathological condition in diabetes, may persist for extended periods of time in neurons and trigger cellular damage by altering voltage-gated sodium channels (VGSCs), the exact mechanism of which remains unclear. Therefore, we hypothesized that high glucose may directly affect kinetics of the VGSCs in the dorsal root ganglion (DRG) neurons. DRG neurons were exposed to normal glucose (NG: 5.5 mM) and high glucose (HG: 30 mM) for 24 h. In another set of experiments, diabetic DRG neurons were also isolated from streptozotocin-induced diabetic rats. Effects of sodium channel blockers on nociceptive parameters and tetrodotoxin-resistant (TTX-R) Na(+) channel kinetics were elucidated by whole-cell patch-clamp in HG exposure and diabetes-induced rat DRG neurons. HG exposure and diabetes-induced DRG neurons demonstrated significant increase in TTX-R Na(+) current (INa) densities in comparison to the control. Both HG-exposed and diabetic DRG neurons demonstrated similar biophysical characteristics of INa. Lidocaine and tetracaine significantly decreased TTX-R INa density in a concentration- and voltage-dependent manner. Steady-state fast inactivation of INa was shifted in the hyperpolarizing direction whereas voltage-dependent activation was shifted in the rightward direction. Diabetic rats treated with lidocaine and tetracaine (3 mg/kg, i.p.) significantly improved thermal hyperalgesia, mechanical allodynia and motor nerve conduction velocity with a significant inhibition of TTX-R INa density as compared to the diabetic control. These results suggest that HG exposure increases the TTX-R Na(+) channel activity sensitive to Na(+) channel blockers, lidocaine and tetracaine.

Topics: Animals; Antibiotics, Antineoplastic; Cells, Cultured; Diabetes Mellitus, Experimental; Disease Models, Animal; Drug Interactions; Ganglia, Spinal; Glucose; Lidocaine; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Streptozocin; Sweetening Agents; Tetrodotoxin; Voltage-Gated Sodium Channels

The citrus flavonoid hesperidin exerts neuroprotective effects and could cross the blood-brain barrier. Given the involvement of glutamate neurotoxicity in the pathogenesis of neurodegenerative disorders, this study was conducted to evaluate the potential role of hesperidin in glutamate release and glutamate neurotoxicity in the hippocampus of rats. In rat hippocampal nerve terminals (synaptosomes), hesperidin inhibited the release of glutamate and elevation of cytosolic free Ca(2+) concentration evoked by 4-aminopyridine (4-AP), but did not alter 4-AP-mediated depolarization. The inhibitory effect of hesperidin on evoked glutamate release was prevented by chelating the extracellular Ca(2+) ions and blocking the activity of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels or protein kinase C. In hippocampal slice preparations, whole-cell patch clamp experiments showed that hesperidin reduced the frequency of spontaneous excitatory postsynaptic currents without affecting their amplitude, indicating the involvement of a presynaptic mechanism. In addition, intraperitoneal (i.p.) injection of kainic acid (KA, 15 mg/kg) elevated the extracellular glutamate levels and caused considerable neuronal loss in the hippocampal CA3 area. These KA-induced alterations were attenuated by pretreatment with hesperidin (10 or 50 mg/kg, i.p.) before administering the KA. These results demonstrate that hesperidin inhibits evoked glutamate release in vitro and attenuates in vivo KA-induced neuronal death in the hippocampus. Our findings indicate that hesperidin may be a promising candidate for preventing or treating glutamate excitotoxicity related brain disorders such as neurodegenerative diseases.

Topics: 4-Aminopyridine; Animals; Calcium; Disease Models, Animal; Egtazic Acid; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Hesperidin; Hippocampus; Kainic Acid; Male; Membrane Potentials; Neuroprotective Agents; Neurotoxicity Syndromes; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Synaptosomes; Tetrodotoxin

Neuropathic pain is a debilitating condition for which the development of effective treatments has been limited by an incomplete understanding of its molecular basis. The cationic current Ih mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels plays an important role in pain by facilitating ectopic firing and hyperexcitability in DRG neurons, however little is known regarding the role of Ih in supraspinal pain pathways. The medial prefrontal cortex (mPFC), which is reported to be involved in the affective aspects of pain, exhibits high HCN channel expression. Using the spared nerve injury (SNI) model of neuropathic pain in Long-Evans rats and patch-clamp recordings in layer II/III pyramidal neurons of the contralateral mPFC, we observed a hyperpolarizing shift in the voltage-dependent activation of Ih in SNI neurons, whereas maximal Ih remained unchanged. Accordingly, SNI mPFC pyramidal neurons exhibited increased input resistance and excitability, as well as facilitated glutamatergic mGluR5-mediated persistent firing, compared with sham neurons. Moreover, intracellular application of bromo-cAMP abolished the hyperpolarizing shift in the voltage-dependent activation of Ih observed in SNI neurons, whereas protein kinase A (PKA) inhibition further promoted this shift in both SNI and sham neurons. Behaviorally, acute HCN channel blockade by local injection of ZD7288 in the mPFC of SNI rats induced a decrease in cold allodynia. These findings suggest that changes in the cAMP/PKA axis in mPFC neurons underlie alterations to HCN channel function, which can influence descending inhibition of pain pathways in neuropathic conditions. Significance statement: Recent studies investigating the role of the medial prefrontal cortex (mPFC) in neuropathic pain have led to an increased awareness of how affective and cognitive factors can influence pain perception. It is therefore imperative that we advance our understanding of the involvement of supraspinal pain pathways. Our electrophysiological and behavioral results support an important role for hyperpolarization-activated cyclic nucleotide-gated channels and the cAMP/protein kinase A signaling axis in promoting hyperexcitability and persistent firing in pyramidal neurons of the mPFC in neuropathic animals. These findings offer novel insights, with potential therapeutic implications, into pathophysiological mechanisms underlying the abnormal contribution of layer II/III prefrontal pyramidal neu

Topics: Animals; Biophysical Phenomena; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Hyperalgesia; In Vitro Techniques; Male; Membrane Potentials; Methoxyhydroxyphenylglycol; Neuralgia; Pain Measurement; Pain Threshold; Prefrontal Cortex; Pyramidal Cells; Pyrimidines; Rats; Rats, Long-Evans; Sodium Channel Blockers; Synaptic Potentials; Tetrodotoxin

Persistent muscle pain is a common and disabling symptom for which available treatments have limited efficacy. Since tetrodotoxin (TTX) displays a marked antinociceptive effect in models of persistent cutaneous pain, we tested its local antinociceptive effect in rat models of muscle pain induced by inflammation, ergonomic injury and chemotherapy-induced neuropathy. While local injection of TTX (0.03-1 μg) into the gastrocnemius muscle did not affect the mechanical nociceptive threshold in naïve rats, exposure to the inflammogen carrageenan produced a marked muscle mechanical hyperalgesia, which was dose-dependently inhibited by TTX. This antihyperalgesic effect was still significant at 24h. TTX also displayed a robust antinociceptive effect on eccentric exercise-induced mechanical hyperalgesia in the gastrocnemius muscle, a model of ergonomic pain. Finally, TTX produced a small but significant inhibition of neuropathic muscle pain induced by systemic administration of the cancer chemotherapeutic agent oxaliplatin. These results indicate that TTX-sensitive sodium currents in nociceptors play a central role in diverse states of skeletal muscle nociceptive sensitization, supporting the suggestion that therapeutic interventions based on TTX may prove useful in the treatment of muscle pain.

Topics: Analgesics; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Hyperalgesia; Male; Motor Activity; Muscle, Skeletal; Myalgia; Nociceptive Pain; Organoplatinum Compounds; Oxaliplatin; Pain Threshold; Rats, Sprague-Dawley; Tetrodotoxin; Touch

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy.. In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.

Topics: Action Potentials; Analysis of Variance; Animals; CA1 Region, Hippocampal; Dendrites; Disease Models, Animal; Down-Regulation; Humans; In Vitro Techniques; Male; Muscarinic Agonists; Pilocarpine; Rats; Rats, Wistar; RNA, Messenger; Sodium Channel Blockers; Sodium Channels; Spermine; Statistics, Nonparametric; Status Epilepticus; Synaptophysin; Tetrodotoxin; Up-Regulation

A series of imidazol-1-ylethylindazole sodium channel ligands were developed and optimized for sodium channel inhibition and in vitro neuroprotective activity. The molecules exhibited displacement of a radiolabeled sodium channel ligand and selectivity for blockade of the inactivated state of cloned neuronal Nav channels. Metabolically stable analogue 6 was able to protect retinal ganglion cells during optic neuritis in a mouse model of multiple sclerosis.

Topics: Animals; Disease Models, Animal; Female; Humans; Imidazoles; Lymph Nodes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Multiple Sclerosis; Neuroprotective Agents; Optic Neuritis; Retinal Ganglion Cells; Voltage-Gated Sodium Channels

Amitriptyline (AMI), a tricyclic antidepressant, has been widely used to prevent migraine attacks and alleviate other various chronic pain, but the underlying mechanism remains unclear. Accumulated evidence suggests that the efficacy of AMI is related to the blockade of voltage-gated sodium channels. The aim of the present study was to investigate the effect of AMI on Na(v)1.8 currents in nociceptive trigeminal neurons and trigeminovascular nociception induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus (SSS) in rats, as in the animal model of vascular headaches such as migraines. Using a whole-cell voltage recording technique, we showed that Na(v)1.8 currents were blocked by AMI in a concentration-dependent manner, with an IC50 value of 6.82 μM in acute isolated trigeminal ganglion neurons of the rats. AMI caused a hyperpolarizing shift in the voltage-dependent activation and steady-state inactivation and significantly blocked in a use-dependent manner and slowed the recovery from the inactivation of Na(v)1.8 currents. In addition, the systemic administration of AMI and A-803467 (a selective Na(v)1.8 channel blocker) potently alleviated the nociceptive behaviors (head flicks and grooming) induced by the electrical stimulation of the dura mater surrounding the SSS. Taken together, our data suggest that Na(v)1.8 currents in nociceptive trigeminal neurons are blocked by AMI through modulating the activation and inactivation kinetics, which may contribute to anti-nociceptive effect of AMI in animal models of migraines.

Topics: Afferent Pathways; Amitriptyline; Aniline Compounds; Animals; Blood Vessels; Disease Models, Animal; Dose-Response Relationship, Drug; Dura Mater; Electric Stimulation; Furans; Ion Channel Gating; Male; Migraine Disorders; NAV1.8 Voltage-Gated Sodium Channel; Nociception; Nociceptors; Rats; Rats, Sprague-Dawley; Single-Blind Method; Sodium; Sodium Channel Blockers; Superior Sagittal Sinus; Tetrodotoxin; Trigeminal Nerve

Although postsynaptic glycine receptors (GlyRs) as αβ heteromers attract considerable research attention, little is known about the role of presynaptic GlyRs, likely α homomers, in diseases. Here, we demonstrate that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, can rescue GlyR functional deficiency and exaggerated acoustic and tactile startle responses in mice bearing point mutations in α1 GlyRs that are responsible for a hereditary startle-hyperekplexia disease. The GlyRs expressed as α1 homomers either in HEK-293 cells or at presynaptic terminals of the calyceal synapses in the auditory brainstem are more vulnerable than heteromers to hyperekplexia mutation-induced impairment. Homomeric mutants are more sensitive to DH-CBD than are heteromers, suggesting presynaptic GlyRs as a primary target. Consistent with this idea, DH-CBD selectively rescues impaired presynaptic GlyR activity and diminished glycine release in the brainstem and spinal cord of hyperekplexic mutant mice. Thus, presynaptic α1 GlyRs emerge as a potential therapeutic target for dominant hyperekplexia disease and other diseases with GlyR deficiency.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain Stem; Disease Models, Animal; Excitatory Amino Acid Antagonists; Female; HEK293 Cells; Humans; In Vitro Techniques; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Mutation; Neurons; Presynaptic Terminals; Receptors, Glycine; Sodium Channel Blockers; Spinal Cord; Stiff-Person Syndrome; Tetrodotoxin; Valine

Diastolic dysfunction can lead to heart failure with preserved ejection fraction, for which there is no effective therapeutic. Ranolazine has been reported to reduce diastolic dysfunction, but the specific mechanisms of action are unclear. The effect of ranolazine on diastolic function was examined in spontaneously hypertensive rats (SHRs), where left ventricular relaxation is impaired and stiffness increased. The objective of this study was to determine whether ranolazine improves diastolic function in SHRs and identify the mechanism(s) by which improvement is achieved. Specifically, to test the hypothesis that ranolazine, by inhibiting late sodium current, reduces Ca(2+) overload and promotes ventricular relaxation and reduction in diastolic stiffness, the effects of ranolazine or vehicle on heart function and the response to dobutamine challenge were evaluated in aged male SHRs and Wistar-Kyoto rats by echocardiography and pressure-volume loop analysis. The effects of ranolazine and the more specific sodium channel inhibitor tetrodotoxin were determined on the late sodium current, sarcomere length, and intracellular calcium in isolated cardiomyocytes. Ranolazine reduced the end-diastolic pressure-volume relationship slope and improved diastolic function during dobutamine challenge in the SHR. Ranolazine and tetrodotoxin also enhanced cardiomyocyte relaxation and reduced myoplasmic free Ca(2+) during diastole at high-stimulus rates in the SHR. The density of the late sodium current was elevated in SHRs. In conclusion, ranolazine was effective in reducing diastolic dysfunction in the SHR. Its mechanism of action, at least in part, is consistent with inhibition of the increased late sodium current in the SHR leading to reduced Ca(2+) overload.

Topics: Acetanilides; Aging; Animals; Blood Pressure; Calcium; Cells, Cultured; Diastole; Disease Models, Animal; Dobutamine; Enzyme Inhibitors; Hypertension; In Vitro Techniques; Male; Myocytes, Cardiac; Piperazines; Ranolazine; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tetrodotoxin; Ventricular Dysfunction, Left

Functional alterations in the properties of Aβ afferent fibers may account for the increased pain sensitivity observed under peripheral chronic inflammation. Among the voltage-gated sodium channels involved in the pathophysiology of pain, Na(v)1.8 has been shown to participate in the peripheral sensitization of nociceptors. However, to date, there is no evidence for a role of Na(v)1.8 in controlling Aβ-fiber excitability following persistent inflammation.. Distribution and expression of Na(v)1.8 in dorsal root ganglia and sciatic nerves were qualitatively or quantitatively assessed by immunohistochemical staining and by real time-polymerase chain reaction at different time points following complete Freund's adjuvant (CFA) administration. Using a whole-cell patch-clamp configuration, we further determined both total INa and TTX-R Na(v)1.8 currents in large-soma dorsal root ganglia (DRG) neurons isolated from sham or CFA-treated rats. Finally, we analyzed the effects of ambroxol, a Na(v)1.8-preferring blocker on the electrophysiological properties of Nav1.8 currents and on the mechanical sensitivity and inflammation of the hind paw in CFA-treated rats.. Our findings revealed that Na(v)1.8 is up-regulated in NF200-positive large sensory neurons and is subsequently anterogradely transported from the DRG cell bodies along the axons toward the periphery after CFA-induced inflammation. We also demonstrated that both total INa and Na(v)1.8 peak current densities are enhanced in inflamed large myelinated Aβ-fiber neurons. Persistent inflammation leading to nociception also induced time-dependent changes in Aβ-fiber neuron excitability by shifting the voltage-dependent activation of Na(v)1.8 in the hyperpolarizing direction, thus decreasing the current threshold for triggering action potentials. Finally, we found that ambroxol significantly reduces the potentiation of Na(v)1.8 currents in Aβ-fiber neurons observed following intraplantar CFA injection and concomitantly blocks CFA-induced mechanical allodynia, suggesting that Na(v)1.8 regulation in Aβ-fibers contributes to inflammatory pain.. Collectively, these findings support a key role for Na(v)1.8 in controlling the excitability of Aβ-fibers and its potential contribution to the development of mechanical allodynia under persistent inflammation.

Topics: Ambroxol; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Freund's Adjuvant; Ganglia, Spinal; Gene Expression Regulation; Inflammation; Male; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Nerve Fibers, Myelinated; Neurons; Pain Threshold; Protein Transport; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Sodium Channel Blockers; Tetrodotoxin

Cognitive deficits in schizophrenia (SZ) reflect maturational disruptions within a neural system that includes the ventral hippocampus (VH), nucleus accumbens (NAc), basal forebrain, and prefrontal cortex (PFC). A better understanding of these changes may reveal drug targets for more efficacious cognition enhancers. We have utilized an animal model in which the above distributed system is altered, during a sensitive period of development, by transiently inactivating the VH and its efferent projections. We determined the ability of NAc shell activation to evoke prefrontal glutamate release in adult male Wistar rats that had received saline (Sal) or tetrodotoxin (TTX) as neonates (PD7) or as adolescents (PD32). The nucleus accumbens shell (NAcSh) was activated by NMDA infusions (0.05-0.30 μg/0.5 μL). Basal and evoked glutamate levels were measured amperometrically using a glutamate-sensitive microelectrode. There were no differences in basal glutamate levels among the groups tested (overall 1.41 ± 0.26 uM). However, the dose-related stimulation of prefrontal glutamate levels seen in control rats treated with saline on PD7 (4.31 ± 0.22 μM after 0.15 μg) was markedly attenuated in rats treated with TTX on PD7 (0.45 ± 0.12 μM after 0.15 μg). This effect was age-dependent as infusions of TTX on PD32 did not alter the NMDA-induced increases in glutamate release (4.10 ± 0.37 μM after 0.15 μg). Collectively, these findings reveal that transient inactivation of VH transmission, during a sensitive period of development, leads to a functional mesolimbic-cortical disconnection that produces neurochemical and ultimately cognitive impairments resembling those seen in SZ.

Topics: Animals; Animals, Newborn; Catheters, Indwelling; Disease Models, Animal; Electrodes, Implanted; Glutamic Acid; Hippocampus; Male; Microelectrodes; N-Methylaspartate; Nucleus Accumbens; Prefrontal Cortex; Rats, Wistar; Schizophrenia; Sodium Channel Blockers; Tetrodotoxin

The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

Topics: Animals; Calcium; Disease Models, Animal; Epilepsy, Temporal Lobe; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Male; Microdialysis; Muscarinic Agonists; Neurons; Parvalbumins; Pilocarpine; Potassium Chloride; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Somatostatin; Tetrodotoxin; Time Factors; Video Recording

Specific missense mutations in the CACNA1A gene, which encodes a subunit of voltage-gated CaV2.1 channels, are associated with familial hemiplegic migraine type 1 (FHM1), a rare monogenic subtype of common migraine with aura. We used transgenic knock-in (KI) mice harboring the human pathogenic FHM1 mutation S218L to study presynaptic Ca(2+) currents, EPSCs, and in vivo activity at the calyx of Held synapse. Whole-cell patch-clamp recordings of presynaptic terminals from S218L KI mice showed a strong shift of the calcium current I-V curve to more negative potentials, leading to an increase in basal [Ca(2+)]i, increased levels of spontaneous transmitter release, faster recovery from synaptic depression, and enhanced synaptic strength despite smaller action-potential-elicited Ca(2+) currents. The gain-of-function of transmitter release of the S218L mutant was reproduced in vivo, including evidence for an increased release probability, demonstrating its relevance for glutamatergic transmission. This synaptic phenotype may explain the misbalance between excitation and inhibition in neuronal circuits resulting in a persistent hyperexcitability state and other migraine-relevant mechanisms such as an increased susceptibility to cortical spreading depression.

Topics: Agatoxins; Animals; Brain Stem; Calcium; Calcium Channels, N-Type; Disease Models, Animal; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Migraine with Aura; Mutation; Neurotoxins; Sodium Channel Blockers; Synapses; Tetrodotoxin; Time Factors

Chronic pain caused by insults to the CNS (central neuropathic pain) is widely assumed to be maintained exclusively by central mechanisms. However, chronic hyperexcitablility occurs in primary nociceptors after spinal cord injury (SCI), suggesting that SCI pain also depends upon continuing activity of peripheral sensory neurons. The present study in rats (Rattus norvegicus) found persistent upregulation after SCI of protein, but not mRNA, for a voltage-gated Na(+) channel, Nav1.8, that is expressed almost exclusively in primary afferent neurons. Selectively knocking down Nav1.8 after SCI suppressed spontaneous activity in dissociated dorsal root ganglion neurons, reversed hypersensitivity of hindlimb withdrawal reflexes, and reduced ongoing pain assessed by a conditioned place preference test. These results show that activity in primary afferent neurons contributes to ongoing SCI pain.

Topics: Animals; Cells, Cultured; Conditioning, Operant; Disease Models, Animal; Ganglia, Spinal; Hindlimb; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Neurons; Oligodeoxyribonucleotides, Antisense; Pain; Rats; Reflex; Sodium Channel Blockers; Spinal Cord Injuries; Tetrodotoxin; Transduction, Genetic; Up-Regulation

Episodic ataxia type 2 (EA2) is an autosomal dominant disorder associated with attacks of ataxia that are typically precipitated by stress, ethanol, caffeine or exercise. EA2 is caused by loss-of-function mutations in the CACNA1A gene, which encodes the α1A subunit of the CaV2.1 voltage-gated Ca(2+) channel. To better understand the pathomechanisms of this disorder in vivo, we created the first genetic animal model of EA2 by engineering a mouse line carrying the EA2-causing c.4486T>G (p.F1406C) missense mutation in the orthologous mouse Cacna1a gene. Mice homozygous for the mutated allele exhibit a ~70% reduction in CaV2.1 current density in Purkinje cells, though surprisingly do not exhibit an overt motor phenotype. Mice hemizygous for the knockin allele (EA2/- mice) did exhibit motor dysfunction measurable by rotarod and pole test. Studies using Cre-flox conditional genetics explored the role of cerebellar Purkinje cells or cerebellar granule cells in the poor motor performance of EA2/- mice and demonstrate that manipulation of either cell type alone did not cause poor motor performance. Thus, it is possible that subtle dysfunction arising from multiple cell types is necessary for the expression of certain ataxia syndromes.

Topics: Animals; Ataxia; Calcium Channel Blockers; Calcium Channels, N-Type; Cerebellum; Disease Models, Animal; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Mutation, Missense; Neurons; Nystagmus, Pathologic; Patch-Clamp Techniques; Reaction Time; Sodium Channel Blockers; Tetrodotoxin

Neurodegeneration and synaptic dysfunction observed in Alzheimer's disease (AD) have been associated with progressive decrease in neuronal activity. Here, we investigated the effects of Notoginsenoside R1 (NTR1), a major saponin isolated from Panax notoginseng, on neuronal excitability and assessed the beneficial effects of NTR1 on synaptic and memory deficits under the Aβ-enriched conditions in vivo and in vitro. We assessed the effects of NTR1 on neuronal excitability, membrane ion channel activity, and synaptic plasticity in acute hippocampal slices by combining electrophysiological extracellular and intracellular recording techniques. We found that NTR1 increased the membrane excitability of CA1 pyramidal neurons in hippocampal slices by lowering the spike threshold possibly through a mechanism involving in the inhibition of voltage-gated K(+) currents. In addition, NTR1 reversed Aβ1-42 oligomers-induced impairments in long term potentiation (LTP). Reducing spontaneous firing activity with 10 nM tetrodotoxin (TTX) abolished the protective effect of NTR1 against Aβ-induced LTP impairment. Finally, oral administration of NTR1 improved the learning performance of the APP/PS1 mouse model of AD. Our work reveals a novel mechanism involving in modulation of cell strength, which contributes to the protective effects of NTR1 against Aβ neurotoxicity.

Topics: Amyloid; Amyloid beta-Peptides; Animals; Disease Models, Animal; Ginsenosides; Hippocampus; Long-Term Potentiation; Male; Memory Disorders; Mice; Mice, Inbred C57BL; Neuronal Plasticity; Neurons; Potassium; Tetrodotoxin

Long-term potentiation of glutamatergic transmission has been observed after physiological learning or pathological injuries in different brain regions, including the spinal cord, hippocampus, amygdala, and cortices. The insular cortex is a key cortical region that plays important roles in aversive learning and neuropathic pain. However, little is known about whether excitatory transmission in the insular cortex undergoes plastic changes after peripheral nerve injury. Here, we found that peripheral nerve ligation triggered the enhancement of AMPA receptor (AMPAR)-mediated excitatory synaptic transmission in the insular cortex. The synaptic GluA1 subunit of AMPAR, but not the GluA2/3 subunit, was increased after nerve ligation. Genetic knock-in mice lacking phosphorylation of the Ser845 site, but not that of the Ser831 site, blocked the enhancement of the synaptic GluA1 subunit, indicating that GluA1 phosphorylation at the Ser845 site by protein kinase A (PKA) was critical for this upregulation after nerve injury. Furthermore, A-kinase anchoring protein 79/150 (AKAP79/150) and PKA were translocated to the synapses after nerve injury. Genetic deletion of adenylyl cyclase subtype 1 (AC1) prevented the translocation of AKAP79/150 and PKA, as well as the upregulation of synaptic GluA1-containing AMPARs. Pharmacological inhibition of calcium-permeable AMPAR function in the insular cortex reduced behavioral sensitization caused by nerve injury. Our results suggest that the expression of AMPARs is enhanced in the insular cortex after nerve injury by a pathway involving AC1, AKAP79/150, and PKA, and such enhancement may at least in part contribute to behavioral sensitization together with other cortical regions, such as the anterior cingulate and the prefrontal cortices.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cerebral Cortex; Disease Models, Animal; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; In Vitro Techniques; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Neuralgia; Phosphorylation; Picrotoxin; Receptors, AMPA; Sodium Channel Blockers; Subcellular Fractions; Synaptic Transmission; Tetrodotoxin

Schizophrenia is a debilitating disorder that affects 1% of the US population. While the exogenous administration of cannabinoids such as tetrahydrocannabinol is reported to exacerbate psychosis in schizophrenia patients, augmenting the levels of endogenous cannabinoids has gained attention as a possible alternative therapy to schizophrenia due to clinical and preclinical observations. Thus, patients with schizophrenia demonstrate an inverse relationship between psychotic symptoms and levels of the endocannabinoid anandamide. In addition, increasing endocannabinoid levels (by blockade of enzymatic degradation) has been reported to attenuate social withdrawal in a preclinical model of schizophrenia. Here we examine the effects of increasing endogenous cannabinoids on dopamine neuron activity in the sub-chronic phencyclidine (PCP) model. Aberrant dopamine system function is thought to underlie the positive symptoms of schizophrenia.. Using in vivo extracellular recordings in chloral hydrate-anesthetized rats, we now demonstrate an increase in dopamine neuron population activity in PCP-treated rats.. Interestingly, endocannabinoid upregulation, induced by URB-597, was able to normalize this aberrant dopamine neuron activity. Furthermore, we provide evidence that the ventral pallidum is the site where URB-597 acts to restore ventral tegmental area activity.. Taken together, we provide preclinical evidence that augmenting endogenous cannabinoids may be an effective therapy for schizophrenia, acting in part to restore ventral pallidal activity.

Topics: Animals; Basal Forebrain; Benzamides; Carbamates; Central Nervous System Agents; Disease Models, Animal; Dopaminergic Neurons; Endocannabinoids; Hippocampus; Male; Microelectrodes; Phencyclidine; Rats, Sprague-Dawley; Schizophrenia; Sodium Channel Blockers; Tetrodotoxin; Ventral Tegmental Area

The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

Topics: 4-Aminopyridine; Adult Stem Cells; Animals; Cell Count; Cell Differentiation; Disease Models, Animal; Eye Proteins; Green Fluorescent Proteins; In Vitro Techniques; Infarction, Middle Cerebral Artery; Lateral Ventricles; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Degeneration; Nerve Tissue Proteins; Neurogenesis; Neurons; Patch-Clamp Techniques; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin

Neuropathic pain, a chronic pain due to neuronal lesion, remains unaltered even after the injury-induced spinal afferent discharges have declined, suggesting an involvement of supraspinal dysfunction. The midbrain ventrolateral periaqueductal gray (vlPAG) is known to be a crucial supraspinal region for initiating descending pain inhibition, but its role in neuropathic pain remains unclear. Therefore, here we examined neuroplastic changes in the vlPAG of midbrain slices isolated from neuropathic rats induced by L5/L6 spinal nerve ligation (SNL) via electrophysiological and neurochemical approaches. Significant mechanical hypersensitivity was induced in rats 2 d after SNL and lasted for >14 d. Compared with the sham-operated group, vlPAG slices from neuropathic rats 3 and 10 days after SNL displayed smaller EPSCs with prolonged latency, less frequent and smaller miniature EPSCs, higher paired-pulse ratio of EPSCs, smaller AMPAR-mediated EPSCs, smaller AMPA currents, greater NMDAR-mediated EPSCs, greater NMDA currents, lower AMPAR-mediated/NMDAR-mediated ratios, and upregulation of the NR1 and NR2B subunits, but not the NR2A, GluR1, or GluR2 subunits, of glutamate receptors. There were no significant differences between day 3 and day 10 neuropathic groups. These results suggest that SNL leads to hypoglutamatergic neurotransmission in the vlPAG resulting from both presynaptic and postsynaptic mechanisms. Upregulation of NMDARs might contribute to hypofunction of AMPARs via subcellular redistribution. Long-term hypoglutamatergic function in the vlPAG may lead to persistent reduction of descending pain inhibition, resulting in chronic neuropathic pain.

Topics: Animals; Bicuculline; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Agents; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Male; Membrane Potentials; Neuralgia; Neurons; Pain Measurement; Patch-Clamp Techniques; Periaqueductal Gray; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Spinal Nerves; Synaptic Transmission; Tetrodotoxin

Voltage-gated Na(+) channels (Nav) are the targets of a variety of scorpion toxins. Here, we investigated the effects of Amm VIII, a toxin isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus, on pain-related behaviours in mice. The effects of Amm VIII were compared with the classic scorpion α-toxin AaH II from Androctonus australis. Contrary to AaH II, intraplantar injection of Amm VIII at relatively high concentrations caused little nocifensive behaviours. However, Amm VIII induced rapid mechanical and thermal pain hypersensitivities. We evaluated the toxins' effects on Nav currents in nociceptive dorsal root ganglion (DRG) neurons and immortalized DRG neuron-derived F11 cells. Amm VIII and AaH II enhanced tetrodotoxin-sensitive (TTX-S) Nav currents in DRG and F11 cells. Both toxins impaired fast inactivation and negatively shifted activation. AaH II was more potent than Amm VIII at modulating TTX-S Nav currents with EC50 of 5 nM and 1 μM, respectively. AaH II and Amm VIII also impaired fast inactivation of Nav1.7, with EC50 of 6.8 nM and 1.76 μM, respectively. Neither Nav1.8 nor Nav1.9 was affected by the toxins. AaH II and Amm VIII reduced first spike latency and lowered action potential threshold. Amm VIII was less efficient than AaH II in increasing the gain of the firing frequency-stimulation relationship. In conclusion, our data show that Amm VIII, although less potent than AaH II, acts as a gating-modifier peptide reminiscent of classic α-toxins, and suggest that its hyperalgesic effects can be ascribed to gain-of-function of TTX-S Na(+) channels in nociceptors.

Topics: Animals; Biophysical Phenomena; Disease Models, Animal; Dose-Response Relationship, Drug; Ganglia, Spinal; Hyperalgesia; Hypersensitivity; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Neurons; Pain; Pain Threshold; Rats; Scorpion Venoms; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Brain ischemia triggers excessive release of neurotransmitters that mediate neuronal damage following ischemic injury. The striatum is one of the areas most sensitive to ischemia. Release of dopamine (DA) from ischemic neurons is neurotoxic and directly contributes to the cell death in affected areas. Astrocytes are known to be critically involved in the physiopathology of cerebrovascular disease. However, their response to ischemia and their role in neuroprotection in striatum are not completely understood. In this study, we used an in vitro model to evaluate the mechanisms of ischemia-induced DA release, and to study whether astrocytes modulate the release of DA in response to short-term ischemic conditions. Using slices of adult mouse brain exposed to oxygen and glucose deprivation (OGD), we measured the OGD-evoked DA efflux using fast cyclic voltammetry and also assessed metabolic impairment by 2,3,5-triphenyltetrazolium chloride (TTC) and tissue viability by propidium iodide (PI) staining. Our data indicate that ischemia induces massive release of DA by dual mechanisms: one which operates via vesicular exocytosis and is action potential dependent and another involving reverse transport by the dopamine transporter (DAT). Simultaneous blockade of astrocyte glutamate transporters and DAT prevented the massive release of dopamine and reduced the brain tissue damage. The present results provide the first experimental evidence that astrocytes function as a key cellular element of ischemia-induced DA release in striatum, constituting a novel and promising therapeutic target in ischemia.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Aspartic Acid; Astrocytes; Calcium; Corpus Striatum; Disease Models, Animal; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dopamine Uptake Inhibitors; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Exocytosis; Functional Laterality; Hypoxia-Ischemia, Brain; In Vitro Techniques; Mice; Mice, Inbred C57BL; Neurotoxicity Syndromes; Oxidopamine; Piperazines; Tetrodotoxin; Time Factors

The basolateral amygdala (BLA) and ventral hippocampus (vHPC) have both been implicated in mediating anxiety-related behaviors, but the functional contribution of BLA inputs to the vHPC has never been directly investigated. Here we show that activation of BLA-vHPC synapses acutely and robustly increased anxiety-related behaviors, while inhibition of BLA-vHPC synapses decreased anxiety-related behaviors. We combined optogenetic approaches with in vivo pharmacological manipulations and ex vivo whole-cell patch-clamp recordings to dissect the local circuit mechanisms, demonstrating that activation of BLA terminals in the vHPC provided monosynaptic, glutamatergic inputs to vHPC pyramidal neurons. Furthermore, BLA inputs exerted polysynaptic, inhibitory effects mediated by local interneurons in the vHPC that may serve to balance the circuit locally. These data establish a role for BLA-vHPC synapses in bidirectionally controlling anxiety-related behaviors in an immediate, yet reversible, manner and a model for the local circuit mechanism of BLA inputs in the vHPC.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Anxiety; Bacterial Proteins; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Channelrhodopsins; Disease Models, Animal; Excitatory Amino Acid Antagonists; Exploratory Behavior; Halorhodopsins; Hippocampus; In Vitro Techniques; Luminescent Proteins; Male; Maze Learning; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neural Pathways; Neurons; Sodium Channel Blockers; Synapses; Tetrodotoxin; Valine

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage-gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Nav 1.1, Nav 1.2, and Nav 1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS-induced NF-κB activation, expression of TNF-α, IL-1β and inducible nitric oxide synthase, and NO production. LPS-induced p38MAPK activation followed pro-nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS-induced JNK activation was not. TTX also inhibited caspase-3 activation and cell death of primary cortical neurons in neuron/microglia co-cultures by inhibiting LPS-induced microglia activation. Furthermore, TTX attenuated caspase-3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury.

Topics: Animals; Cell Death; Cells, Cultured; Disease Models, Animal; Inflammation; Microglia; Neurons; NF-kappa B; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Sodium Channels; Spinal Cord Injuries; Tetrodotoxin; Tumor Necrosis Factor-alpha

Dravet syndrome is a severe form of intractable pediatric epilepsy with a high incidence of SUDEP: Sudden Unexpected Death in epilepsy. Cardiac arrhythmias are a proposed cause for some cases of SUDEP, yet the susceptibility and potential mechanism of arrhythmogenesis in Dravet syndrome remain unknown. The majority of Dravet syndrome patients have de novo mutations in SCN1A, resulting in haploinsufficiency. We propose that, in addition to neuronal hyperexcitability, SCN1A haploinsufficiency alters cardiac electrical function and produces arrhythmias, providing a potential mechanism for SUDEP.. Postnatal day 15-21 heterozygous SCN1A-R1407X knock-in mice, expressing a human Dravet syndrome mutation, were used to investigate a possible cardiac phenotype. A combination of single cell electrophysiology and in vivo electrocardiogram (ECG) recordings were performed.. We observed a 2-fold increase in both transient and persistent Na(+) current density in isolated Dravet syndrome ventricular myocytes that resulted from increased activity of a tetrodotoxin-resistant Na(+) current, likely Nav1.5. Dravet syndrome myocytes exhibited increased excitability, action potential duration prolongation, and triggered activity. Continuous radiotelemetric ECG recordings showed QT prolongation, ventricular ectopic foci, idioventricular rhythms, beat-to-beat variability, ventricular fibrillation, and focal bradycardia. Spontaneous deaths were recorded in 2 DS mice, and a third became moribund and required euthanasia.. These data from single cell and whole animal experiments suggest that altered cardiac electrical function in Dravet syndrome may contribute to the susceptibility for arrhythmogenesis and SUDEP. These mechanistic insights may lead to critical risk assessment and intervention in human patients.

Topics: Action Potentials; Animals; Arrhythmias, Cardiac; Cardiac Electrophysiology; Death, Sudden, Cardiac; Disease Models, Animal; Epilepsies, Myoclonic; Heart Rate; Humans; Ion Channel Gating; Mice; Mice, Inbred C57BL; Mutation; Myocytes, Cardiac; NAV1.5 Voltage-Gated Sodium Channel; Pentylenetetrazole; Protein Biosynthesis; Telemetry; Tetrodotoxin; Transcription, Genetic

Aconitine, well-known for its high cardiotoxicity, causes severe arrhythmias, such as ventricular tachycardia and ventricular fibrillation, by opening membrane sodium channels. Tetrodotoxin, a membrane sodium-channel blocker, is thought to antagonize aconitine activity. Tetrodotoxin is a potent blocker of the skeletal muscle sodium-channel isoform Na(v)1.4 (IC50 10 nM), but micromolar concentrations of tetrodotoxin are required to inhibit the primary cardiac isoform Na(v)1.5. This suggests that substantial concentrations of tetrodotoxin are required to alleviate the cardiac toxicity caused by aconitine. To elucidate the interaction between aconitine and tetrodotoxin in the cardiovascular and respiratory systems, mixtures of aconitine and tetrodotoxin were simultaneously administered to mice, and the effects on electrocardiograms, breathing rates, and arterial oxygen saturation were examined. Compared with mice treated with aconitine alone, some mice treated with aconitine-tetrodotoxin mixtures showed lower mortality rates and delayed appearance of arrhythmia. The decreased breathing rates and arterial oxygen saturation observed in mice receiving aconitine alone were alleviated in mice that survived after receiving the aconitine-tetrodotoxin mixture; this result suggests that tetrodotoxin is antagonistic to aconitine. When the tetrodotoxin dose is greater than the dose that can block tetrodotoxin-sensitive sodium channels, which are excessively activated by aconitine, tetrodotoxin toxicity becomes prominent, and the mortality rate increases because of the respiratory effects of tetrodotoxin. In terms of cardiotoxicity, mice receiving the aconitine-tetrodotoxin mixture showed minor and shorter periods of change on electrocardiography. This finding can be explained by the recent discovery of tetrodotoxin-sensitive sodium-channel cardiac isoforms (Na(v)1.1, 1.2, 1.3, 1.4 and 1.6).

Topics: Aconitine; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Biomarkers; Disease Models, Animal; Electrocardiography; Heart Rate; Male; Mice; Mice, Inbred ICR; Myocardium; Oxygen; Respiratory Rate; Sodium Channel Blockers; Tetrodotoxin; Time Factors; Voltage-Gated Sodium Channels

Depression is usually associated with alterations in the monoaminergic system. However, new evidences suggest the involvement of the glutamatergic system in the aetiology of depression. Here we explored the glutamatergic system in a rat model of depression (i.e., the flinders sensitive line (FSL)) to reveal the mechanism underlying the emotional and cognitive aspects associated with the disease. We showed a dramatically elevated level of baseline glutamatergic synaptic transmission by whole-cell recordings as well as impairment in long-term potentiation induced by high-frequency stimulation in hippocampal slices from FSL rats compared with Sprague-Dawley rats. At behavioural level, FSL rats displayed recognition memory impairment in the novel object recognition test. Enantioselective chromatography analysis revealed lower levels of D-serine in the hippocampus of FSL rats and both synaptic plasticity and memory impairments were restored by administration of D-serine. We also observed dysfunctional astrocytic glutamate regulation including downregulation of the glia glutamate transporter GLAST as shown by western blot. One possibility is that the dysfunctional astrocytic glutamate reuptake triggers a succession of events, including the reduction of D-serine production as a safety mechanism to avoid NMDA receptor overactivation, which in turn causes the synaptic plasticity and memory impairments observed. These findings open up new brain targets for the development of more potent and efficient antidepressant drugs.

Topics: Animals; Astrocytes; Depression; Disease Models, Animal; Excitatory Amino Acid Agents; Excitatory Postsynaptic Potentials; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Hippocampus; Humans; In Vitro Techniques; Male; Nerve Tissue Proteins; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Recognition, Psychology; Serine; Sodium Channel Blockers; Statistics, Nonparametric; Swimming; Synaptic Transmission; Tetrodotoxin

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 μM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Calcium Channel Blockers; Calcium Channels, N-Type; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytophotometry; Disease Models, Animal; Electric Stimulation; Gene Expression Regulation; Humans; Membrane Potentials; Mice; Mice, Transgenic; Motor Neurons; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Sodium Channel Blockers; Superoxide Dismutase; Tetrodotoxin

Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

Topics: Animals; Base Sequence; Chronic Pain; CpG Islands; Cyclic AMP-Dependent Protein Kinases; Cystathionine beta-Synthase; Cysteine; Disease Models, Animal; DNA Methylation; Epigenomics; Ganglia, Spinal; Hyperalgesia; Inflammation; Isoquinolines; Molecular Sequence Data; Patch-Clamp Techniques; Promoter Regions, Genetic; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Sulfides; Sulfonamides; Tetrodotoxin

The over-expression of voltage-gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury contributes to neuropathic pain by generation of the ectopic discharges of action potentials. However, mechanisms underlying the change in VGSCs' expression are poorly understood. Our previous work has demonstrated that the pro-inflammatory cytokine TNF-α up-regulates VGSCs. In the present work we tested if anti-inflammatory cytokine IL-10, which had been proven to be effective for treating neuropathic pain, had the opposite effect. Western blot and immunofluorescence results showed that IL-10 receptor was localized in DRG neurons. Recombinant rat IL-10 (200 pg/ml) not only reduced the densities of TTX-sensitive and Nav1.8 currents in control DRG neurons, but also reversed the increase of the sodium currents induced by rat recombinant TNF-α (100 pg/ml), as revealed by patch-clamp recordings. Consistent with the electrophysiological results, real-time PCR and western blot revealed that IL-10 (200 pg/ml) down-regulated VGSCs in both mRNA and protein levels and reversed the up-regulation of VGSCs by TNF-α. Moreover, repetitive intrathecal administration of rrIL-10 for 3 days (4 times per day) attenuated mechanical allodynia in L5 spinal nerve ligation model and profoundly inhibited the excitability of DRG neurons. These results suggested that the down-regulation of the sodium channels in DRG neurons might contribute to the therapeutic effect of IL-10 on neuropathic pain.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Down-Regulation; Ganglia, Spinal; Interleukin-10; Ligation; Male; Membrane Potentials; Nerve Tissue Proteins; Neurons; Pain Measurement; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Receptors, Interleukin-10; Sodium Channel Blockers; Tetrodotoxin; Tumor Necrosis Factor-alpha; Voltage-Gated Sodium Channels

Acute Helicobacter pylori infection produces hypochlorhydria. The decrease in acid facilitates survival of the bacterium and its colonization of the stomach. The present study was designed to identify the pathways in oxyntic mucosa by which acute H. pylori infection inhibits acid secretion. In rat fundic sheets in an Ussing chamber, perfusion of the luminal surface with H. pylori in spent broth (10(3)-10(8) cfu/ml) or spent broth alone (1:10(5) to 1:10(0) final dilution) caused a concentration-dependent increase in somatostatin (SST; maximal: 200 ± 20 and 194 ± 9% above basal; P < 0.001) and decrease in histamine secretion (maximal: 45 ± 5 and 48 ± 2% below basal; P < 0.001); the latter was abolished by SST antibody, implying that changes in histamine secretion reflected changes in SST secretion. Both responses were abolished by the axonal blocker tetrodotoxin (TTX), the sensory neurotoxin capsaicin, or the CGRP antagonist CGRP8-37, implying that the reciprocal changes in SST and histamine secretion were due to release of CGRP from sensory neurons. In isolated rabbit oxyntic glands, H. pylori inhibited basal and histamine-stimulated acid secretion in a concentration-dependent manner; the responses were not affected by TTX or SST antibody, implying that H. pylori can directly inhibit parietal cell function. In conclusion, acute administration of H. pylori is capable of inhibiting acid secretion directly as well as indirectly by activating intramural CGRP sensory neurons coupled to stimulation of SST and inhibition of histamine secretion. Activation of neural pathways provides one explanation as to how initial patchy colonization of the superficial gastric mucosa by H. pylori can acutely inhibit acid secretion.

Topics: Achlorhydria; Animals; Calcitonin Gene-Related Peptide; Disease Models, Animal; Gastric Acid; Gastric Fundus; Gastric Mucosa; HeLa Cells; Helicobacter Infections; Helicobacter pylori; Histamine; Humans; Parietal Cells, Gastric; Peptide Fragments; Rabbits; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Somatostatin; Tetrodotoxin

Aconiti Brachypodi Radix, belonging to the genus of Aconitum (Family Ranunculaceae), are used clinically as anti-rheumatic, anti-inflammatory and anti-nociceptive in traditional medicine of China. However, its mechanism and influence on nociceptive threshold are unknown and need further investigation. The analgesic effects of ethanolic extract of Aconiti Brachypodi Radix (EABR) were thus studied in vivo and in vitro. Three pain models in mice were used to assess the effect of EABR on nociceptive threshold. In vitro study was conducted to clarify the modulation of the extract on the tetrodotoxin-sensitive (TTX-S) sodium currents in rat's dorsal root ganglion (DRG) neurons using whole-cell patch clamp technique. The results showed that EABR (5-20 mg/kg, i.g.) could produce dose-dependent analgesic effect on hot-plate tests as well as writhing response induced by acetic acid. In addition, administration of 2.5-10 mg/kg EABR (i.g.) caused significant decrease in pain responses in the first and second phases of formalin test without altering the PGE₂ production in the hind paw of the mice. Moreover, EABR (10 µg/ml -1 mg/ml) could suppress TTX-S voltage-gated sodium currents in a dose-dependent way, indicating the underlying electrophysiological mechanism of the analgesic effect of the folk plant medicine. Collectively, our results indicated that EABR has analgesic property in three pain models and useful influence on TTX-S sodium currents in DRG neurons, suggesting that the interference with pain messages caused by the modulation of EABR on TTX-S sodium currents in DRG neurones may explain some of its analgesic effect.

Topics: Acetic Acid; Aconitum; Analgesics; Animals; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Formaldehyde; Ganglia, Spinal; Hot Temperature; Male; Mice; Mice, Inbred Strains; Nociceptive Pain; Pain Threshold; Patch-Clamp Techniques; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Tetrodotoxin; Voltage-Gated Sodium Channels

Our previous study has shown that chronic heart failure (CHF) reduces expression and activation of voltage-gated sodium (Na(v)) channels in baroreceptor neurons, which are involved in the blunted baroreceptor neuron excitability and contribute to the impairment of baroreflex in the CHF state. The present study examined the role of mitochondria-derived superoxide in the reduced Na(v) channel function in coronary artery ligation-induced CHF rats. CHF decreased the protein expression and activity of mitochondrial complex enzymes and manganese SOD (MnSOD) and elevated the mitochondria-derived superoxide level in the nodose neurons compared with those in sham nodose neurons. Adenoviral MnSOD (Ad.MnSOD) gene transfection (50 multiplicity of infection) into the nodose neurons normalized the MnSOD expression and reduced the elevation of mitochondrial superoxide in the nodose neurons from CHF rats. Ad.MnSOD also partially reversed the reduced protein expression and current density of the Na(v) channels and the suppressed cell excitability (the number of action potential and the current threshold for inducing action potential) in aortic baroreceptor neurons from CHF rats. Data from the present study indicate that mitochondrial dysfunction, including decreased protein expression and activity of mitochondrial complex enzymes and MnSOD and elevated mitochondria-derived superoxide, contributes to the reduced Na(v) channel activation and cell excitability in the aortic baroreceptor neurons in CHF rats.

Topics: Action Potentials; Animals; Antimycin A; Biophysics; Bridged Bicyclo Compounds, Heterocyclic; Chronic Disease; Disease Models, Animal; Electric Stimulation; Enzyme Inhibitors; Gene Expression Regulation; Heart Failure; Hemodynamics; Humans; Lectins; Male; Mitochondria; Multienzyme Complexes; NADH Dehydrogenase; NAV1.7 Voltage-Gated Sodium Channel; Nodose Ganglion; Patch-Clamp Techniques; Phenanthridines; Pressoreceptors; Rats; Rats, Sprague-Dawley; Rotenone; Sodium Channel Blockers; Sodium Channels; Spectrophotometry; Succinate Dehydrogenase; Superoxide Dismutase; Tetrodotoxin; Transfection

Long-term memory requires fine-tuning regulation of gene expression in specific neural circuits of the brain. Transcriptional regulation of gene programs is a key mechanism for memory storage and its deregulation may contribute to synaptic and cognitive dysfunction in memory disorders. The molecular mechanisms underlying changes on activity-dependent gene expression in Alzheimer's disease (AD) are largely unknown.. We analyzed the expression of activity-dependent genes regulated by the cAMP response element binding protein (CREB) and activation of CREB and its coactivator CREB-regulated transcription coactivator 1 (CRTC1) in control and mutant β-amyloid precursor protein (APP(Sw,Ind); Swedish and Indiana mutations) transgenic mice.. Gene expression analyses revealed specific downregulation of a subset of well-known activity-induced CREB-dependent genes, including c-fos, Bdnf and Nr4a2, in the hippocampus of memory-impaired APP(Sw,Ind) transgenic mice. Activity-dependent CREB transcription induced by calcium/cAMP signals is disrupted through a mechanism involving deregulation of calcium/calcineurin-mediated dephosphorylation and activation of CRTC1. Expression of CRTC1 and pharmacological activation of L-type voltage-gated calcium channels reverse the deficits in CRTC1-mediated transcription in APP(Sw,Ind) neurons.. Our results suggest that CRTC1 dysfunction caused by Aβ accumulation underlies changes in gene expression required for hippocampal-dependent memory in AD transgenic mice.

Topics: Age Factors; Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Calcineurin; Calcium; Cells, Cultured; Cerebral Cortex; Cyclic AMP; Disease Models, Animal; Gene Expression Regulation; Hippocampus; Humans; Maze Learning; Memory Disorders; Mice; Mice, Transgenic; Mutation; Neurons; Peptide Fragments; Tetrodotoxin; Transcription Factors; Transfection

Chronic compression of rat dorsal root ganglion (CCD) produced tactile allodynia accompanied with hyperexcitability of the myelinated Aβ dorsal root ganglion (DRG) neurons. The Aβ DRG neuron hyperexcitability exhibits as bursting discharges in response to peripherally evoked action potentials (evoked bursting [EB]). The incidence of EB was significantly increased after chronic compression of DRG (CCD) (43.3%) vs control (13.3%). EB was maintained by oscillation of the membrane potential, and its duration was increased when the membrane potential was depolarized. EB was found to coexist in some neurons with spontaneous bursting (SB), but EB always occurred at a more negative membrane potential than SB. Afterdischarges of the wide dynamic range neurons of the dorsal horn in the spinal cord in response to electrical stimulation of Aβ afferent nerve fibers were suppressed by blocking EB of the DRG neurons. CCD neurons with EB exhibited increased current density of persistent sodium current (I(Nap)) and hyperpolarization-activated cation current (I(h)) and decreased α-dendrotoxin (α-DTX) sensitive current (I(DTX)). The increased I(h) activated by afterhyperpolarization of peripheral afferent action potential was necessary for EB generation and a balance between I(DTX) and I(Nap) might be necessary for EB maintenance. This study may suggest a role of EB of myelinated DRG neurons in development of allodynia after nerve injury and a potential pharmaceutical therapy in treating neuropathic allodynia.

Topics: Action Potentials; Animals; Biophysical Phenomena; Biophysics; Cardiovascular Agents; Chi-Square Distribution; Disease Models, Animal; Double-Blind Method; Elapid Venoms; Electric Stimulation; Female; Fourier Analysis; Ganglia, Spinal; Hyperalgesia; Male; Patch-Clamp Techniques; Pyrimidines; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Spinal Cord Compression; Tetrodotoxin; Time Factors

While infantile spasms is the most common catastrophic epilepsy of infancy and early-childhood, very little is known about the basic mechanisms responsible for this devastating disorder. In experiments reported here, spasms were induced in rats by the chronic infusion of TTX into the neocortex beginning on postnatal days 10-12. Studies of focal epilepsy suggest that high frequency EEG oscillations (HFOs) occur interictally at sites that are most likely responsible for seizure generation. Thus, our goal was to determine if HFOs occurred and where they occurred in cortex in the TTX model. We also undertook multiunit recordings to begin to analyze the basic mechanisms responsible for HFOs. Our results show that HFOs occur most frequently during hypsarrhythmia and NREM sleep and are most prominent contralateral to the TTX infusion site in the homotopic cortex and anterior to this region in frontal cortex. While HFOs were largest and most frequent in these contralateral regions, they were also commonly recorded synchronously across multiple and widely-spaced recordings sites. The amplitude and spatial distribution of interictal HFOs were found to be very similar to the high frequency bursts seen at seizure onset. However, the latter differed from the interictal events in that the high frequency activity was more intense at seizure onset. Microwire recordings showed that neuronal unit firing increased abruptly with the generation of HFOs. A similar increase in neuronal firing occurred at the onset of the ictal events. Taken together, results suggest that neocortical networks are abnormally excitable, particularly contralateral to TTX infusion, and that these abnormalities are not restricted to small areas of cortex. Multiunit firing coincident with HFOs is fully consistent with a neocortical hyperexcitability hypothesis particularly since they both occur at seizure onset.

Topics: Age Factors; Animals; Animals, Newborn; Disease Models, Animal; Electroencephalography; Epilepsy; Neocortex; Rats; Spasm; Tetrodotoxin

Emodin is traditionally used as a laxative and is found to increase or decrease the contractility of intestinal smooth muscle in low doses and high doses, respectively. In this study, we propose that bidirectional regulation (BR) on the contractility of jejunal smooth muscle (CJSM) is inducible by emodin in the absence of control by the central nervous system. The results indicated that emodin-induced BR had the following characteristics. A stimulatory effect on CJSM was induced by emodin at 7 low contractile states, and an inhibitory effect was induced on CJSM at 7 high contractile states. Emodin-induced BR on myosin phosphorylation was also observed. BR was not observed in the presence of tetrodotoxin, suggesting that enteric nervous system is required for producing BR. The stimulatory effect of emodin on CJSM was abolished by atropine and diphenhydramine, respectively, suggesting that BR was correlated with cholinergic and histamine system while jejunal smooth muscle was at low contractile state. The inhibitory effect of emodin on CJSM was abolished by phentolamine, propranolol, and L-NG-nitroarginine (L-NNA), respectively, suggesting that BR was related to adrenergic hyperactivity and with a nitric oxide relaxing mechanism while jejunal smooth muscle was in a high contractile state. The exact mechanism, however, needs further investigation.

Topics: Animals; Atropine; Constipation; Diarrhea; Diphenhydramine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Emodin; Enteric Nervous System; In Vitro Techniques; Jejunum; Laxatives; Muscle Contraction; Muscle, Smooth; Myosins; Nitroarginine; Phentolamine; Phosphorylation; Propranolol; Rats; Rats, Sprague-Dawley; Tetrodotoxin

Epigenetic upregulation of voltage-gated sodium channels (VGSCs) has been reported in a number of carcinoma cell lines and tissues. Furthermore, a large body of experimental evidence suggested that functional VGSC expression enhances various in vitro cell behaviours, such as directional motility, that would be involved in the metastatic cascade. However, it is not known if VGSC activity promotes metastasis in vivo. Here, using the Copenhagen rat model of prostate cancer and blocking VGSC activity in primary tumours with tetrodotoxin, we show (1) that the number of lung metastasis is reduced by >40% and (2) that lifespan is significantly improved.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; Male; NAV1.7 Voltage-Gated Sodium Channel; Neoplasm Invasiveness; Neoplasm Metastasis; Prostatic Neoplasms; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Growing evidence suggests schizophrenia may arise from abnormalities in early brain development. The prefrontal cortex (PFC) stands out as one of the main regions affected in schizophrenia. Latent inhibition, an interesting cognitive marker for schizophrenia, has been found in some studies to be reduced in acute patients. It is generally widely accepted that there is a dopaminergic dysfunctioning in schizophrenia. Moreover, several authors have reported that the psychostimulant, D-amphetamine (D-AMP), exacerbates symptoms in patients with schizophrenia. We explored in rats the effects in adulthood of neonatal transient inactivation of the PFC on behavioral and neurochemical anomalies associated with schizophrenia. Following tetrodotoxin (TTX) inactivation of the left PFC at postnatal day 8, latent inhibition-related dopaminergic responses and dopaminergic reactivity to D-AMP were monitored using in vivo voltammetry in the left core part of the nucleus accumbens in adult freely moving rats. Dopaminergic responses and behavioral responses were followed in parallel. Prefrontal neonatal inactivation resulted in disrupted behavioral responses of latent inhibition and latent inhibition-related dopaminergic responses in the core subregion. After D-AMP challenge, the highest dose (1.5 mg/kg i.p.) induced a greater dopamine increase in the core in rats microinjected with TTX, and a parallel increase in locomotor activity, suggesting that following prefrontal neonatal TTX inactivation animals display a greater behavioral and dopaminergic reactivity to D-AMP. Transitory inactivation of the PFC early in the postnatal developmental period leads to behavioral and neurochemical changes in adulthood that are meaningful for schizophrenia modeling. The data obtained may help our understanding of the pathophysiology of this disabling disorder.

Topics: Animals; Animals, Newborn; Dextroamphetamine; Disease Models, Animal; Dopamine; Dopamine Uptake Inhibitors; Inhibition, Psychological; Male; Motor Activity; Neural Inhibition; Nucleus Accumbens; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Schizophrenia; Sodium Channel Blockers; Tetrodotoxin

TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in-frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal-dominant, early-onset generalized DYT1 dystonia. Although only 30-40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE-torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild-type, heterozygous, and homozygous ΔE-torsinA knock-in mice using two approaches. In the first approach, patch-clamp electrophysiology was used to record glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA(A) receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild-type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild-type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild-type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE-torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect.

Topics: Animals; Disease Models, Animal; Dystonia Musculorum Deformans; Excitatory Postsynaptic Potentials; Exocytosis; Glutamic Acid; Heterozygote; Hippocampus; Homozygote; Inhibitory Postsynaptic Potentials; Mice; Mice, Transgenic; Miniature Postsynaptic Potentials; Molecular Chaperones; Mutation; Neurons; Receptors, GABA-A; Sodium Channels; Synaptic Vesicles; Tetrodotoxin; Vesicular Glutamate Transport Protein 1

Peripheral nerve hyperexcitability (PNH) is one of the distal peripheral neuropathy phenotypes often present in patients affected by type 2 diabetes mellitus (T2DM). Through in vivo and ex vivo electrophysiological recordings in db/db mice, a model of T2DM, we observed that, in addition to reduced nerve conduction velocity, db/db mice also develop PNH. By using pharmacological inhibitors, we demonstrated that the PNH is mediated by the decreased activity of K(v)1-channels. In agreement with these data, we observed that the diabetic condition led to a reduced presence of the K(v)1.2-subunits in juxtaparanodal regions of peripheral nerves in db/db mice and in nerve biopsies from T2DM patients. Together, these observations indicate that the T2DM condition leads to potassium channel-mediated PNH, thus identifying them as a potential drug target to treat some of the DPN related symptoms.

Topics: Action Potentials; Age Factors; Animals; Blood Glucose; Body Weight; Diabetes Mellitus, Type 2; Disease Models, Animal; Electric Stimulation; Humans; Kv1.2 Potassium Channel; Male; Mice; Mice, Mutant Strains; Mutation; Neural Conduction; Peripheral Nerves; Peripheral Nervous System Diseases; Potassium Channel Blockers; Protein Subunits; Ranvier's Nodes; Receptors, Leptin; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Animals; Antibodies; Disease Models, Animal; Excitatory Amino Acid Antagonists; Green Fluorescent Proteins; Humans; Hyperalgesia; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Injections, Spinal; Male; Mice; Mice, Transgenic; Motor Activity; Neuralgia; Oligodeoxyribonucleotides, Antisense; Pain Measurement; Pain Threshold; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Spinal Cord; Spinal Nerves; Tetrodotoxin; Thrombospondins; Up-Regulation; Valine

Cognitive deficits represent a core symptom cluster in schizophrenia that are thought to reflect developmental dysregulations within a neural system involving the ventral hippocampus (VH), nucleus accumbens (NAC), and prefrontal cortex (PFC). The present experiments determined the cognitive effects of transiently inactivating VH in rats during a sensitive period of development. Neonatal (postnatal day 7, PD7) and adolescent (PD32) male rats received a single bilateral infusion of saline or tetrodotoxin (TTX) within the VH to transiently inactivate local circuitry and efferent outflow. Rats were tested as adults on an attentional set-shifting task. Performance in this task depends upon the integrity of the PFC and NAC. TTX infusions did not affect the initial acquisition or ability to learn an intra-dimensional shift. However, TTX rats required a greater number of trials than did controls to acquire the first reversal and extra-dimensional shift (ED) stages. These impairments were age and region-specific as rats infused with TTX into the VH at PD32, or into the dorsal hippocampus at PD7, exhibited performance in the task similar to that of controls. Finally, acute systemic administration of the partial α7 nicotinic acetylcholine receptor (nAChR) agonist SSR 180711 (3.0 mg/kg) eliminated the TTX-induced performance deficits. Given that patients with schizophrenia exhibit hippocampal pathophysiology and deficits in the ED stages of set-shifting tasks, our results support the significance of transient hippocampal inactivation as an animal model for studying the cognitive impairments in schizophrenia as well as the pro-cognitive therapeutic potential of α7 nAChR agonists.

Topics: Age Factors; Analysis of Variance; Anesthetics, Local; Animals; Animals, Newborn; Attention Deficit Disorder with Hyperactivity; Bridged Bicyclo Compounds, Heterocyclic; Discrimination, Psychological; Disease Models, Animal; Hippocampus; Male; Nicotinic Agonists; Odorants; Rats; Rats, Wistar; Set, Psychology; Tetrodotoxin; Touch

Rat mesenteric arteries were maintained by both adrenergic vasoconstrictor nerves and calcitonin gene-related peptide (CGRP) vasodilator nerves. However, functions of these nerves in a pathophysiological state have not fully been analyzed. The use of disease models developed genetically in mice is expected to clarify neural function of perivascular nerves. Thus, we investigated basic mouse vascular responses. Mesenteric vascular beds isolated from male C57BL/6 mouse were perfused with Krebs solution and perfusion pressure was measured. Periarterial nerve stimulation (PNS, 8 - 24 Hz) induced frequency-dependent vasoconstriction, which increased flow rate-dependently. PNS-induced vasoconstriction was abolished by tetrodotoxin (neurotoxin) and guanethidine (adrenergic neuron blocker) and blunted by prazosin (α(1)-adrenoceptor antagonist). Injection of norepinephrine caused vasoconstriction, which was abolished by prazosin. In preparations with active tone, PNS (1 - 8 Hz) induced frequency-dependent vasodilation, which was inhibited by tetrodotoxin, capsaicin (CGRP depletor), and CGRP8-37 (CGRP-receptor antagonist). Injections of CGRP, acetylcholine, and sodium nitroprusside induced vasodilations. Vasodilator response to CGRP was inhibited by CGRP8-37. Immunohistochemical study showed innervation of tyrosine hydroxylase- and CGRP-immunopositive fibers in mesenteric arteries and veins. These results suggest that male mouse mesenteric vascular beds are useful for studying neural regulation of mesenteric arteries, which are innervated by adrenergic and CGRPergic nerves regulating vascular tone.

Topics: Acetylcholine; Animals; Calcitonin Gene-Related Peptide; Capsaicin; Disease Models, Animal; Guanethidine; Male; Mesenteric Arteries; Mice; Mice, Inbred C57BL; Nitroprusside; Norepinephrine; Peptide Fragments; Peripheral Nervous System; Prazosin; Tetrodotoxin; Tyrosine 3-Monooxygenase; Vasoconstriction; Vasodilation

To investigate the postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pig, and to provide method and evidence for forensic identification and clinical diagnosis and treatment.. Guinea pigs were intragastric administrated with 100, 50, 15 microg/kg tetrodotoxin, respectively. The poisoning symptoms were observed. The samples of heart, liver, spleen, lung, kidney, brain, stomach, intestines, bile, heart blood and urine were collected. The concentrations of tetrodotoxin in tissues and body fluids were measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS).. After administrated with tetrodotoxin, all guinea pigs came out poisoning signs including tachypnea, weary and dead finally. Tetrodotoxin concentrations in lung, stomach, intestines and urine were higher, followed by blood, heart and brain. The concentration in bile was the lowest.. Postmortem distribution of tetrodotoxin in guinea pig is uneven. The concentration in the lung, stomach, intestines, urine and heart blood are higher, those tissues could be used for diagnosis of tetrodotoxin poisoning.

Topics: Administration, Oral; Animals; Body Fluids; Brain Chemistry; Chromatography, Liquid; Disease Models, Animal; Forensic Toxicology; Guinea Pigs; Intestines; Kidney; Liver; Lung; Postmortem Changes; Stomach; Tandem Mass Spectrometry; Tetrodotoxin; Tissue Distribution

Marked hypersensitivity to heat and mechanical (pressure) stimuli develop after a burn injury, but the neural mechanisms underlying these effects are poorly understood. In this study, we establish a new mouse model of focal second-degree burn injury to investigate the molecular and cellular basis for burn injury-induced pain. This model features robust injury-induced behavioral effects and tissue-specific altered cytokine profile, but absence of glial activation in spinal dorsal horn. Three voltage-gated sodium channels, Na(v)1.7, Na(v)1.8, and Na(v)1.9, are preferentially expressed in peripheral somatosensory neurons of the dorsal root ganglia (DRGs) and have been implicated in injury-induced neuronal hyperexcitability. Using knock-out mice, we provide evidence that Na(v)1.7 selectively contributes to burn-induced hypersensitivity to heat, but not mechanical, stimuli. After burn model injury, wild-type mice display increased sensitivity to heat stimuli, and a normally non-noxious warm stimulus induces activity-dependent Fos expression in spinal dorsal horn neurons. Strikingly, both effects are absent in Na(v)1.7 conditional knock-out (cKO) mice. Furthermore, burn injury increases density and shifts activation of tetrodotoxin-sensitive currents in a hyperpolarized direction, both pro-excitatory properties, in DRG neurons from wild-type but not Na(v)1.7 cKO mice. We propose that, in sensory neurons damaged by burn injury to the hindpaw, Na(v)1.7 currents contribute to the hyperexcitability of sensory neurons, their communication with postsynaptic spinal pain pathways, and behavioral thresholds to heat stimuli. Our results offer insights into the molecular and cellular mechanisms of modality-specific pain signaling, and suggest Na(v)1.7-blocking drugs may be effective in burn patients.

Topics: Activating Transcription Factor 3; Analysis of Variance; Animals; Biophysics; Burns; Calcitonin Gene-Related Peptide; Calcium; Cells, Cultured; Cytokines; Disease Models, Animal; Edema; Electric Stimulation; Functional Laterality; Ganglia, Spinal; Glycoproteins; Hot Temperature; Hyperalgesia; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; NAV1.7 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; NAV1.9 Voltage-Gated Sodium Channel; Neuralgia; Neuroglia; Pain Threshold; Patch-Clamp Techniques; Proteins; RNA, Messenger; RNA, Untranslated; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Transfection

We have developed an approach to directly probe neuronal excitability during the period beginning with induction of global ischemia and extending after reperfusion using transgenic mice expressing channelrhodopsin-2 (ChR2) to activate deep layer cortical neurons independent of synaptic or sensory stimulation. Spontaneous, ChR2, or forepaw stimulation-evoked electroencephalogram (EEG) or local field potential (LFP) records were collected from the somatosensory cortex. Within 20 s of ischemia, a >90% depression of spontaneous 0.3-3 Hz EEG and LFP power was detected. Ischemic depolarization followed EEG depression with a ∼2 min delay. Surprisingly, neuronal excitability, as assessed by the ChR2-mediated EEG response, was intact during the period of strong spontaneous EEG suppression and actually increased before ischemic depolarization. In contrast, a decrease in the somatosensory-evoked potential (forepaw-evoked potential, reflecting cortical synaptic transmission) was coincident with the EEG suppression. After 5 min of ischemia, the animal was reperfused, and the ChR2-mediated response mostly recovered within 30 min (>80% of preischemia value). However, the recovery of the somatosensory-evoked potential was significantly delayed compared with the ChR2-mediated response (<40% of preischemia value at 60 min). By assessing intrinsic optical signals in combination with EEG, we found that neuronal excitability approached minimal values when the spreading ischemic depolarization wave propagated to the ChR2-stimulated cortex. Our results indicate that the ChR2-mediated EEG/LFP response recovers much faster than sensory-evoked EEG/LFP activity in vivo following ischemia and reperfusion, defining a period where excitable but synaptically silent neurons are present.

Topics: Anesthetics, Local; Animals; Bacterial Proteins; Carrier Proteins; Channelrhodopsins; Disease Models, Animal; Electroencephalography; Evoked Potentials; Excitatory Amino Acid Antagonists; Forelimb; Hyperalgesia; In Vitro Techniques; Ischemia; Luminescent Proteins; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Optogenetics; Physical Stimulation; Quinoxalines; Reperfusion Injury; Tetrodotoxin; Valine

To describe high frequency (HF) electrographic activity accompanying ictal discharges in the tetrodotoxin (TTX) model of infantile spasms. Previous studies of HF oscillations in humans and animals suggest that they arise at sites of seizure onset. We compared HF oscillations at several cortical sites to determine regional differences..   TTX was infused for 4 weeks into the neocortex of rats beginning on postnatal days 11 or 12. Electroencephalography (EEG) electrodes were implanted 2 weeks later and video-EEG recordings were analyzed between postnatal days 31 and 47. EEG recordings were digitally sampled at 2,048 Hz. HF EEG activity (20-900 Hz) was quantified using compressed spectral arrays and band-pass filtering..   Multiple seizures were analyzed in 10 rats. Ictal onset was associated with multiple bands of rhythmic HF activity that could extend to 700 Hz. The earliest and most intense discharging typically occurred contralaterally to where TTX was infused. HF activity continued to occur throughout the seizure (even during the electrodecrement that is recorded with more traditional filter settings), although there was a gradual decrease of the intensity of the highest frequency components as the amplitude of lower frequency oscillations increased. Higher frequencies sometimes reappeared in association with spike/sharp-waves at seizure termination.. The findings show that HF EEG activity accompanies ictal events in the TTX model. Results also suggest that the seizures in this model do not originate from the TTX infusion site. Instead HF discharges are usually most intense and occur earliest contralaterally, suggesting that these homologous regions may be involved in seizure generation.

Topics: Animals; Animals, Newborn; Disease Models, Animal; Electroencephalography; Humans; Infant, Newborn; Neocortex; Rats; Spasms, Infantile; Tetrodotoxin

The anticonvulsant activity of BmK AS, a sodium channel site 4-selective modulator purified from scorpion venom (Buthus martensi Karsch), was investigated in unanesthetized rats with acute pentylenetetrazole (PTZ)- and pilocarpine-induced seizures. Rats were microinjected in the CA1 region with either saline or BmK AS, followed by epileptogenic doses of PTZ or pilocarpine 30 minutes later. The anticonvulsant efficacy of BmK AS in PTZ- or pilocarpine-evoked seizure-like behavior and cortical epileptiform EEG activity was assessed. Intrahippocampal injections of BmK AS (0.05-1 μg in 1 μL) produced dose-dependent anticonvulsant activity in the PTZ model, suppressing seizure-associated behavior and reducing both the number and duration of high-amplitude, high-frequency discharges (HAFDs) on the EEG. In contrast, BmK AS did not affect the epileptiform EEG in the pilocarpine model over the same dose range, although it did increase the latency to status epilepticus onset and slightly, but significantly, reduced the seizure score. In summary, our results demonstrate that the sodium channel site 4-selective modulator BmK AS is an effective inhibitor of PTZ- but not pilocarpine-induced acute seizures. These results indicate that BmK AS may serve as a novel probe in exploring the role of different sodium channel subtypes in an epileptogenic setting and as a potential lead in developing antiepileptic drugs specifically for the therapy of sodium channel site 4-related epilepsy.

Topics: Analysis of Variance; Animals; Anticonvulsants; Behavior, Animal; Cells, Cultured; Convulsants; Disease Models, Animal; Dose-Response Relationship, Drug; Electroencephalography; Embryo, Mammalian; Exploratory Behavior; Male; Membrane Potentials; Motor Activity; Neurons; Patch-Clamp Techniques; Pentylenetetrazole; Peptides; Pilocarpine; Rats; Rats, Sprague-Dawley; Reaction Time; Scorpion Venoms; Seizures; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Valproic Acid

Application of phorbol 12,13-diacetate (PDA) caused marked enhancement of synaptic transmission of nociceptive parabrachio-amygdaloid (PBA) input onto neurons of the capsular central amygdaloid (CeAC) nucleus. The potentiation of PBA-CeAC EPSCs by PDA involved a presynaptic protein kinase C (PKC)-dependent component and a postsynaptic PKC-extracellular-regulated kinase (ERK)-dependent component. NMDA glutamatergic receptor (NMDAR)-dependent long-term potentiation (LTP) of PBA-CeAC EPSCs, which was also dependent on the PKC-ERK signaling pathway, was induced by tetanus stimulation at 100 Hz. In slices from mice subjected to acid-induced muscle pain (AIMP), phosphorylated ERK levels in the CeAC increased, and PBA-CeAC synaptic transmission was postsynaptically enhanced. The enhanced PBA-CeAC synaptic transmission in AIMP mice shared common mechanisms with the postsynaptic potentiation effect of PDA and induction of NMDAR-dependent LTP by high-frequency stimulation in normal slices, both of which required ERK activation. Since the CeAC plays an important role in the emotionality of pain, enhanced synaptic function of nociceptive (PBA) inputs onto CeAC neurons might partially account for the supraspinal mechanisms underlying central sensitization.

Topics: Acids; Afferent Pathways; Amygdala; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Agents; Excitatory Postsynaptic Potentials; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Enzymologic; In Vitro Techniques; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Pain; Pain Measurement; Patch-Clamp Techniques; Phorbol Esters; Phosphorylation; Sensory Receptor Cells; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin; Time Factors; Ventral Tegmental Area

In the mammalian retina, excitatory and inhibitory circuitries enable retinal ganglion cells (RGCs) to signal the occurrence of visual features to higher brain areas. This functionality disappears in certain diseases of retinal degeneration because of the progressive loss of photoreceptors. Recent work in a mouse model of retinal degeneration (rd1) found that, although some intraretinal circuitry is preserved and RGCs maintain characteristic physiological properties, they exhibit increased and aberrant rhythmic activity. Here, extracellular recordings were made to assess the degree of aberrant activity in adult rd1 retinas and to investigate the mechanism underlying such behavior. A multi-transistor array with thousands of densely packed sensors allowed for simultaneous recordings of spiking activity in populations of RGCs and of local field potentials (LFPs). The majority of identified RGCs displayed rhythmic (7-10 Hz) but asynchronous activity. The spiking activity correlated with the LFPs, which reflect an average synchronized excitatory input to the RGCs. LFPs initiated from random positions and propagated across the retina. They disappeared when ionotrophic glutamate receptors or electrical synapses were blocked. They persisted in the presence of other pharmacological blockers, including TTX and inhibitory receptor antagonists. Our results suggest that excitation-transmitted laterally through a network of electrically coupled interneurons-leads to large-scale retinal network oscillations, reflected in the rhythmic spiking of most rd1 RGCs. This result may explain forms of photopsias reported by blind patients, while the mechanism involved should be considered in future treatment strategies targeting the disease of retinitis pigmentosa.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Age Factors; Animals; Carbenoxolone; Cyclooxygenase Inhibitors; Disease Models, Animal; Evoked Potentials, Visual; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; Gap Junctions; Glutamic Acid; Glycine; In Vitro Techniques; Light; Male; Meclofenamic Acid; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Neurologic Mutants; Nerve Net; Neural Inhibition; Periodicity; Pyridazines; Quinoxalines; Retinal Degeneration; Retinal Rod Photoreceptor Cells; Sodium Channel Blockers; Statistics as Topic; Tetrodotoxin

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Topics: Acetanilides; Acrylates; Analysis of Variance; Animals; Animals, Newborn; Biophysics; Calcium Channels, T-Type; Cell Line, Transformed; Disease Models, Animal; Electric Stimulation; Ganglia, Spinal; Heart; Humans; Hyperalgesia; In Vitro Techniques; Ion Channels; Male; Membrane Transport Modulators; NAV1.7 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; Neural Inhibition; Neuralgia; Pain Measurement; Patch-Clamp Techniques; Piperazines; Rabbits; Rats; Rats, Wistar; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Spinal Cord; Spinal Nerves; Tetrodotoxin; Transfection

Noise can play a constructive role in the detection of weak signals in various kinds of peripheral receptors and neurons. What the mechanism underlying the effect of noise is remains unclear. Here, the perforated patch-clamp technique was used on isolated cells from chronic compression of the dorsal root ganglion (DRG) model. Our data provided new insight indicating that, under conditions without external signals, noise can enhance subthreshold oscillations, which was observed in a certain type of neurons with high-frequency (20-100 Hz) intrinsic resonance from injured DRG neurons. The occurrence of subthreshold oscillation considerably decreased the threshold potential for generating repetitive firing. The above effects of noise can be abolished by blocking the persistent sodium current (I(Na, P)). Utilizing a mathematical neuron model we further simulated the effect of noise on subthreshold oscillation and firing, and also found that noise can enhance the electrical activity through autonomous stochastic resonance. Accordingly, we propose a new concept of the effects of noise on neural intrinsic activity, which suggests that noise may be an important factor for modulating the excitability of neurons and generation of chronic pain signals.

Topics: Action Potentials; Animals; Biological Clocks; Cells, Cultured; Disease Models, Animal; Electric Stimulation; Ganglia, Spinal; Mathematics; Models, Neurological; Noise; Patch-Clamp Techniques; Radiculopathy; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Tetrodotoxin

Structural and functional plastic changes in the primary somatosensory cortex (S1) have been observed following peripheral nerve injury that often leads to neuropathic pain, which is characterized by tactile allodynia. However, remodeling of cortical connections following injury has been believed to take months or years; this is not temporally correlated with the rapid development of allodynia and S1 hyperexcitability. Here we first report, by using long-term two-photon imaging of postsynaptic dendritic spines in living adult mice, that synaptic connections in the S1 are rewired within days following sciatic nerve ligation through phase-specific and size-dependent spine survival/growth. Spine turnover in the S1 area corresponding to the injured paw markedly increased during an early phase of neuropathic pain and was restored in a late phase of neuropathic pain, which was prevented by immediate local blockade of the injured nerve throughout the early phase. New spines that generated before nerve injury showed volume decrease after injury, whereas more new spines that formed in the early phase of neuropathic pain became persistent and substantially increased their volume during the late phase. Further, preexisting stable spines survived less following injury than controls, and such lost persistent spines were smaller in size than the surviving ones, which displayed long-term potentiation-like enlargement over weeks. These results suggest that peripheral nerve injury induces rapid and selective remodeling of cortical synapses, which is associated with neuropathic pain development, probably underlying, at least partially, long-lasting sensory changes in neuropathic subjects.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Dendritic Spines; Diagnostic Imaging; Disease Models, Animal; Green Fluorescent Proteins; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuronal Plasticity; Pain Measurement; Pain Threshold; Polyvinyls; Pyramidal Cells; Sciatic Neuropathy; Somatosensory Cortex; Statistics, Nonparametric; Synapses; Tetrodotoxin

The reverse rate dependence (RRD) of actions of I(Kr)-blocking drugs to increase the action potential duration (APD) and beat-to-beat variability of repolarization (BVR) of APD is proarrhythmic. We determined whether inhibition of endogenous, physiological late Na(+) current (late I(Na)) attenuates the RRD and proarrhythmic effect of I(Kr) inhibition.. Duration of the monophasic APD (MAPD) was measured from female rabbit hearts paced at cycle lengths from 400 to 2000 milliseconds, and BVR was calculated. In the absence of a drug, duration of monophasic action potential at 90% completion of repolarization (MAPD(90)) and BVR increased as the cycle length was increased from 400 to 2000 milliseconds (n=36 and 26; P<0.01). Both E-4031 (20 nmol/L) and d-sotalol (10 μmol/L) increased MAPD(90) and BVR at all stimulation rates, and the increase was greater at slower than at faster pacing rates (n=19, 11, 12 and 7, respectively; P<0.01). Tetrodotoxin (1 μmol/L) and ranolazine significantly attenuated the RRD of MAPD(90,) reduced BVR (P<0.01), and abolished torsade de pointes in hearts treated with either 20 nmol/L E-4031 or 10 μmol/L d-sotalol. Endogenous late I(Na) in cardiomyocytes stimulated at cycle lengths from 500 to 4000 milliseconds was greater at slower than at faster stimulation rates, and rapidly decreased during the first several beats at faster but not at slower rates (n=8; P<0.01). In a computational model, simulated RRD of APD caused by E-4031 and d-sotalol was attenuated when late I(Na) was inhibited.. Endogenous late I(Na) contributes to the RRD of I(Kr) inhibitor-induced increases in APD and BVR and to bradycardia-related ventricular arrhythmias.

Topics: Acetanilides; Action Potentials; Animals; Anti-Arrhythmia Agents; Bradycardia; Disease Models, Animal; Enzyme Inhibitors; Female; Heart Rate; Long QT Syndrome; Models, Cardiovascular; Myocardial Contraction; Myocytes, Cardiac; Patch-Clamp Techniques; Piperazines; Piperidines; Pyridines; Rabbits; Ranolazine; Sodium; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Torsades de Pointes

Most processing of sensation involves the cortical hemisphere opposite (contralateral) to the stimulated limb. Stroke patients can exhibit changes in the interhemispheric balance of sensory signal processing. It is unclear whether these changes are the result of poststroke rewiring and experience, or whether they could result from the immediate effect of circuit loss. We evaluated the effect of mini-strokes over short timescales (<2 h) where cortical rewiring is unlikely by monitoring sensory-evoked activity throughout much of both cortical hemispheres using voltage-sensitive dye imaging. Blockade of a single pial arteriole within the C57BL6J mouse forelimb somatosensory cortex reduced the response evoked by stimulation of the limb contralateral to the stroke. However, after stroke, the ipsilateral (uncrossed) forelimb response within the unaffected hemisphere was spared and became independent of the contralateral forelimb cortex. Within the unaffected hemisphere, mini-strokes in the opposite hemisphere significantly enhanced sensory responses produced by stimulation of either contralateral or ipsilateral pathways within 30-50 min of stroke onset. Stroke-induced enhancement of responses within the spared hemisphere was not reproduced by inhibition of either cortex or thalamus using pharmacological agents in nonischemic animals. I/LnJ acallosal mice showed similar rapid interhemispheric redistribution of sensory processing after stroke, suggesting that subcortical connections and not transcallosal projections were mediating the novel activation patterns. Thalamic inactivation before stroke prevented the bilateral rearrangement of sensory responses. These findings suggest that acute stroke, and not merely loss of activity, activates unique pathways that can rapidly redistribute function within the spared cortical hemisphere.

Topics: Animals; Disease Models, Animal; Forelimb; Male; Mice; Mice, Inbred C57BL; Models, Biological; Neuronal Plasticity; Pia Mater; Somatosensory Cortex; Stroke; Tetrodotoxin; Thalamus; Voltage-Sensitive Dye Imaging

Approximately 20% of the adult population suffers from migraine. This debilitating pain disorder is three times more prevalent in women than in men. To begin to evaluate the underlying mechanisms that may contribute to this sex difference, we tested the hypothesis that there is a sex difference in the inflammatory mediator (IM)-induced sensitization of dural afferents. Acutely dissociated retrogradely labeled dural afferents from adult Sprague-Dawley rats were examined with whole cell patch-clamp recordings. Baseline passive and active electrophysiological properties of dural afferents from both sexes were comparable. However, while IM-induced increases in the excitability of dural afferents from male and female rats were also comparable, the proportion of dural afferents from female rats sensitized by IM (~100%) was significantly greater than that of dural afferents from male rats (~50%). This appeared to be due to differences downstream of IM receptors, as tetrodotoxin-resistant sodium current was increased by IM in a majority of male dural afferents (13/14). These data indicate that there are both quantitative and qualitative differences in the IM-induced sensitization of dural afferents that may contribute to the sex difference in the manifestation of migraine.

Topics: Action Potentials; Afferent Pathways; Animals; Blood Vessels; Bradykinin; Dinoprostone; Disease Models, Animal; Dura Mater; Electric Stimulation; Female; Histamine; Inflammation Mediators; Ion Channel Gating; Male; Migraine Disorders; Pain Perception; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptor, Serotonin, 5-HT1D; Sensory Receptor Cells; Sex Characteristics; Sodium; Tetrodotoxin; Trigeminal Ganglion

Dentate granule cells, at the gate of the hippocampus, use coincidence detection of synaptic inputs to code afferent information under a sparse firing regime. In both human patients and animal models of temporal lobe epilepsy, mossy fibers sprout to form an aberrant glutamatergic network between dentate granule cells. These new synapses operate via long-lasting kainate receptor-mediated events, which are not present in the naive condition. Here, we report that in chronic epileptic rat, aberrant kainate receptors in interplay with the persistent sodium current dramatically expand the temporal window for synaptic integration. This introduces a multiplicative gain change in the input-output operation of dentate granule cells. As a result, their sparse firing is switched to an abnormal sustained and rhythmic mode. We conclude that synaptic kainate receptors dramatically alter the fundamental coding properties of dentate granule cells in temporal lobe epilepsy.

Topics: Action Potentials; Animals; Biophysics; Dentate Gyrus; Disease Models, Animal; Electric Stimulation; Epilepsy, Temporal Lobe; Excitatory Amino Acid Agents; Excitatory Postsynaptic Potentials; Male; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, Kainic Acid; Sodium Channel Blockers; Sodium Channels; Synapses; Tetrodotoxin

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and the leading genetic cause of autism. It is associated with the lack of fragile X mental retardation protein (FMRP), a regulator of protein synthesis in axons and dendrites. Studies on FXS have extensively focused on the postsynaptic changes underlying dysfunctions in long-term plasticity. In contrast, the presynaptic mechanisms of FXS have garnered relatively little attention and are poorly understood. Activity-dependent presynaptic processes give rise to several forms of short-term plasticity (STP), which is believed to control some of essential neural functions, including information processing, working memory, and decision making. The extent of STP defects and their contributions to the pathophysiology of FXS remain essentially unknown, however. Here we report marked presynaptic abnormalities at excitatory hippocampal synapses in Fmr1 knock-out (KO) mice leading to defects in STP and information processing. Loss of FMRP led to enhanced responses to high-frequency stimulation. Fmr1 KO mice also exhibited abnormal synaptic processing of natural stimulus trains, specifically excessive enhancement during the high-frequency spike discharges associated with hippocampal place fields. Analysis of individual STP components revealed strongly increased augmentation and reduced short-term depression attributable to loss of FMRP. These changes were associated with exaggerated calcium influx in presynaptic neurons during high-frequency stimulation, enhanced synaptic vesicle recycling, and enlarged readily-releasable and reserved vesicle pools. These data suggest that loss of FMRP causes abnormal STP and information processing, which may represent a novel mechanism contributing to cognitive impairments in FXS.

Topics: Animals; Animals, Newborn; Calcium; Disease Models, Animal; Electric Stimulation; Excitatory Postsynaptic Potentials; Fragile X Mental Retardation Protein; Fragile X Syndrome; GABA Antagonists; Hippocampus; In Vitro Techniques; Mice; Mice, Knockout; Microscopy, Electron, Transmission; Neural Inhibition; Neuronal Plasticity; Patch-Clamp Techniques; Phosphinic Acids; Piperidines; Potassium Channel Blockers; Presynaptic Terminals; Propanolamines; Sodium Channel Blockers; Synapses; Tetraethylammonium; Tetrodotoxin; Time Factors

This study investigated the retinal adaptive mechanism in inner retinal dysfunction using the slow double-stimulation multifocal electroretinogram (mfERG) paradigm.. Slow double-stimulation mfERG responses were recorded from 15 eyes of 15 4-month-old Mongolian gerbils in control conditions and after suppression of inner retinal responses with injections of tetrodotoxin (TTX) and N-methyl-d-aspartic acid (NMDA). The stimulation consisted of five video frames: the two initial frames with multifocal flashes were triggered by two independent m-sequences, followed by three dark video frames. The results were compared with findings in humans: 7 subjects with glaucoma and 31 age-matched normal subjects were measured using the same mfERG protocol.. The stimulation generates two responses (M(1) and M(2)) from the two independent multifocal frames. The M(1):M(2) ratio showed a significant reduction after administration of TTX+NMDA in the animal study. This matched with the human glaucoma findings. Glaucoma subjects generally have a reduced M(1):M(2) ratio; this ratio showed a sensitivity of 86%, with a specificity of 84% for differentiating normal eyes from glaucomatous eyes.. This stimulation paradigm provides a method of measuring temporal visual characteristics. The M(1):M(2) ratio acts as an indirect functional indicator of retinal adaptation, which may be abnormal in the diseased retina. Further development of this method may help to describe the functional variation in the diseased retina and to predict the occurrence of a range of retinopathies.

Topics: Adaptation, Physiological; Adult; Animals; Case-Control Studies; Disease Models, Animal; Early Diagnosis; Electroretinography; Gerbillinae; Glaucoma, Open-Angle; Humans; Middle Aged; N-Methylaspartate; Photic Stimulation; Retina; Retinal Diseases; Tetrodotoxin; Visual Fields

To determine how the different stages of retinal processing change after photoreceptor degeneration in rabbits carrying the Pro347Leu rhodopsin mutation (Tg rabbits).. Cone electroretinograms (ERGs) were elicited by 150-ms duration stimuli from 13 Tg rabbits at 12 and 24 weeks of age. The ERG recordings were made before and after an intravitreal injection of tetrodotoxin citrate (TTX) plus N-methyl-dl-aspartic acid (NMDA), with the addition of 2-amino-4-phosphonobutyric acid (APB) and then cis-2,3-piperidine-dicarboxylic acid (PDA) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Digital subtraction of the ERG after the injection from the ERG before the injection was used to extract the components that were blocked by these drugs. Thirteen age-matched, wild-type (WT) rabbits were studied with the same protocol.. In Tg rabbits, the cone ERGs elicited by intermediate intensities had a depolarizing pattern. At 12 weeks of age, the photoreceptor and OFF-bipolar/horizontal cell responses reflected in the ERG in the Tg rabbits did not differ significantly from those in the WT rabbits. The ON-bipolar cells and the third-order neuronal responses recorded after pharmacologic blockade were significantly enhanced in the Tg rabbits compared with those recorded in the WT rabbits. At 24 weeks of age, the ERG waveforms representing the photoreceptors and OFF-bipolar/horizontal cell responses were significantly decreased, but those representing the ON-bipolar cell and third-order neuronal responses were still preserved in the Tg rabbits.. A depolarizing pattern of the cone ERG responses was seen in Pro347Leu Tg rabbits. The enhancement or preservation of the ON-bipolar cell response in the ERGs contributed to shaping the waveform in the Tg rabbits. In this model, the functional alterations in the ON-pathway took place before the deterioration of cone photoreceptor function.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Disease Models, Animal; Electroretinography; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Intravitreal Injections; Point Mutation; Rabbits; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Degeneration; Rhodopsin; Tetrodotoxin; Vision, Ocular

Chronic compression (CCD) of the dorsal root ganglion (DRG) is a model of human radicular pain produced by intraforaminal stenosis and other disorders affecting the DRG, spinal nerve, or root. Previously, we examined electrophysiological changes in small-diameter lumbar level 3 (L3) and L4 DRG neurons treated with CCD; the present study extends these observations to medium-sized DRG neurons, which mediate additional sensory modalities, both nociceptive and non-nociceptive. Whole-cell patch-clamp recordings were obtained from medium-sized somata in the intact DRG in vitro. Compared with neurons from unoperated control animals, CCD neurons exhibited a decrease in the current threshold for action potential generation. In the CCD group, current densities of TTX-resistant and TTX-sensitive Na(+) current were increased, whereas the density of delayed rectifier voltage-dependent K(+) current was decreased. No change was observed in the transient or "A" current after CCD. We conclude that CCD in the mouse produces hyperexcitability in medium-sized DRG neurons, and the hyperexcitability is associated with an increased density of Na(+) current and a decreased density of delayed rectifier voltage-dependent K(+) current.

Topics: Analysis of Variance; Animals; Biophysics; Disease Models, Animal; Electric Stimulation; Functional Laterality; Ganglia, Spinal; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Mice; Neurons; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels, Voltage-Gated; Radiculopathy; Sodium Channel Blockers; Sodium Channels; Tetraethylammonium; Tetrodotoxin

To determine retinal pathway origins of pattern electroretinogram (PERG) in macaque monkeys using pharmacologic dissections, uniform-field flashes, and PERG simulations.. Transient (2 Hz, 4 reversals/s) and steady state (8.3 Hz, 16.6 reversals/s) PERGs and uniform-field ERGs were recorded before and after intravitreal injections of L-AP4 (not APB) (2-amino-4-phosphonobutyric acid, 1.6-2.0 mM), to prevent ON pathway responses; PDA (cis-2,3-piperidinedicarboxylic acid, 3.3-3.8 mM), to block activity of hyperpolarizing second- and all third-order retinal neurons; and TTX (tetrodotoxin, 6 μM), to block Na+-dependent spiking. PERGs were also recorded from macaques with advanced unilateral experimental glaucoma, and were simulated by averaging ON and OFF responses to uniform-field flashes.. For 2-Hz stimulation, L-AP4 reduced both negative- and positive-going (N95 and P50) amplitudes in transient PERGs, and their counterparts, N2 and P1 in simulations, to half-amplitude. PDA eliminated N95 and N2, but increased P50 and P1 amplitudes, in that it enhanced b-waves. As previously reported, severe experimental glaucoma or TTX eliminated photopic negative responses, N95, and N2; glaucoma eliminated P50 and reduced P1 amplitude; TTX reduced P50 and hardly altered P1. For 8.3-Hz stimulation, L-AP4 eliminated the steady state PERG and reduced simulated PERG amplitude, whereas PDA enhanced both responses. TTX reduced PERG amplitude to less than half; simulations were less reduced. Blockade of all postreceptoral activity eliminated transient and steady state PERGs, but left small residual P1 in simulations.. Transient PERG receives nearly equal amplitude contributions from ON and OFF pathways. N95 reflects spiking activity of ganglion cells; P50 reflects nonspiking activity as well. Steady state PERG, in contrast, reflects mainly spike-related ON pathway activity.

Topics: Action Potentials; Animals; Computer Simulation; Disease Models, Animal; Electroretinography; Glaucoma; Intravitreal Injections; Macaca mulatta; Retina; Retinal Ganglion Cells; Sodium Channel Blockers; Tetrodotoxin; Visual Pathways

Spike timing precision is a fundamental aspect of neuronal information processing in the brain. Here we examined the temporal precision of input-output operation of dentate granule cells (DGCs) in an animal model of temporal lobe epilepsy (TLE). In TLE, mossy fibers sprout and establish recurrent synapses on DGCs that generate aberrant slow kainate receptor-mediated excitatory postsynaptic potentials (EPSP(KA)) not observed in controls. We report that, in contrast to time-locked spikes generated by EPSP(AMPA) in control DGCs, aberrant EPSP(KA) are associated with long-lasting plateaus and jittered spikes during single-spike mode firing. This is mediated by a selective voltage-dependent amplification of EPSP(KA) through persistent sodium current (I(NaP)) activation. In control DGCs, a current injection of a waveform mimicking the slow shape of EPSP(KA) activates I(NaP) and generates jittered spikes. Conversely in epileptic rats, blockade of EPSP(KA) or I(NaP) restores the temporal precision of EPSP-spike coupling. Importantly, EPSP(KA) not only decrease spike timing precision at recurrent mossy fiber synapses but also at perforant path synapses during synaptic integration through I(NaP) activation. We conclude that a selective interplay between aberrant EPSP(KA) and I(NaP) severely alters the temporal precision of EPSP-spike coupling in DGCs of chronic epileptic rats.

Topics: Action Potentials; Animals; Biophysics; Computer Simulation; Dentate Gyrus; Disease Models, Animal; Electric Stimulation; Epilepsy; Excitatory Amino Acid Agents; Excitatory Postsynaptic Potentials; In Vitro Techniques; Male; Models, Neurological; Mossy Fibers, Hippocampal; Neurons; Patch-Clamp Techniques; Pilocarpine; Rats; Rats, Wistar; Receptors, Kainic Acid; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

In this study, we examined the relationship between astrocyte activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. In addition, we also examined the effects of pre-emptive treatment with a number of drugs on astrocyte activation and hypersensitivity development in this model. Using immunohistochemistry and immunoblotting, little glial fibrillary acidic protein (GFAP; an astrocyte marker) immunoreactivity was detected in the CN of the normal rats. As early as 3 days after CCI, there was a significant increase in GFAP immunoreactivity in the lesion side of CN, and this reached a maximum at 7 days, and was followed by a decline. Counting of GFAP-immunoreactive astrocytes revealed that astrocytic hypertrophy, but not proliferation, contributes to increased GFAP immunoreactivity. Furthermore, microinjection of the glial activation inhibitor, fluorocitrate, into the CN at 3 days after CCI attenuated injury-induced behavioral hypersensitivity in a dose-dependent manner. These results suggest that median nerve injury-induced astrocytic activation in the CN modulated the development of behavioral hypersensitivity. Animals received MK-801 (glutamate N-methyl-d-aspartate (NMDA) receptor antagonist), clonidine (alpha(2)-adrenoreceptor agonist), tetrodotoxin (TTX, sodium channel blocker) or lidocaine (local anesthetic) 30 min prior to median nerve CCI. Pre-treatment with MK-801, TTX, and 2% lidocaine, but not clonidine, attenuated GFAP immunoreactivity and behavioral hypersensitivity following median nerve injury. In conclusion, suppressing reactions to injury, such as the generation of ectopic discharges and activation of NMDA receptors, can decrease astrocyte activation in the CN and attenuate neuropathic pain sensations.

Topics: Adrenergic alpha-Agonists; Animals; Astrocytes; Citrates; Clonidine; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glial Fibrillary Acidic Protein; Hyperalgesia; Lidocaine; Male; Median Neuropathy; Medulla Oblongata; Pain Threshold; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin; Time Factors; Up-Regulation

Many pro-inflammatory cytokines are involved in the process of inflammatory pain. Bi directional axonal transport of Tumor Necrosis Factor-alpha (TNF-alpha) occurs in case of neuropathic pain induced by nerve ligation. We used an in vivo preparation with injection of carrageenan and fluorescent TNF-alpha in the territory of the saphenous nerve of rats to study this transport. We have shown that retrograde transport of TNF-alpha occurs after an inflammatory insult caused by the injection of carrageenan. This transport was likely mediated by the TNF receptor 1. A nerve block with bupivacaine totally abolishes the expression of the receptor in the dorsal root ganglion and the retrograde transport of TNF-alpha. In addition, bupivacaine at low concentrations (1-10 microM) was able to stop the axonal transport ex vivo. Tetrodotoxin was less efficacious for inhibiting the TNF-alpha transport and the rise in receptor expression and for inhibiting the axonal transport ex vivo. This may partly explain the efficacy of nerve blocks with bupivacaine to decrease the neurogenic inflammation and in a lower extent the long-term inhibition of hyperalgesic phenomenon observed in animals and in humans.

Topics: Anesthetics, Local; Animals; Axonal Transport; Bupivacaine; Carrageenan; Disease Models, Animal; Edema; Femoral Nerve; Fluorescence; Foot; Ganglia, Spinal; Injections, Subcutaneous; Male; Nerve Block; Neurogenic Inflammation; Polymerase Chain Reaction; Polysaccharides; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor, Type I; Tetrodotoxin; Tumor Necrosis Factor-alpha

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

Topics: Action Potentials; Animals; Capsaicin; Disease Models, Animal; Guanosine Triphosphate; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Neurofibromatosis 1; Neurofibromin 1; Potassium Channels; Proto-Oncogene Proteins p21(ras); Sensory Receptor Cells; Sensory System Agents; Sodium Channels; Tetrodotoxin

Spinal cord injury can produce extensive long-term reorganization of the cerebral cortex. Little is known, however, about the sequence of cortical events starting immediately after the lesion. Here we show that a complete thoracic transection of the spinal cord produces immediate functional reorganization in the primary somatosensory cortex of anesthetized rats. Besides the obvious loss of cortical responses to hindpaw stimuli (below the level of the lesion), cortical responses evoked by forepaw stimuli (above the level of the lesion) markedly increase. Importantly, these increased responses correlate with a slower and overall more silent cortical spontaneous activity, representing a switch to a network state of slow-wave activity similar to that observed during slow-wave sleep. The same immediate cortical changes are observed after reversible pharmacological block of spinal cord conduction, but not after sham. We conclude that the deafferentation due to spinal cord injury can immediately (within minutes) change the state of large cortical networks, and that this state change plays a critical role in the early cortical reorganization after spinal cord injury.

Topics: Afferent Pathways; Analysis of Variance; Animals; Biophysics; Disease Models, Animal; Electric Stimulation; Electroencephalography; Evoked Potentials, Somatosensory; Lidocaine; Lower Extremity; Male; Rats; Rats, Wistar; Sodium Channel Blockers; Somatosensory Cortex; Spinal Cord Injuries; Statistics as Topic; Tetrodotoxin

While stressors are known to increase medial prefrontal cortex (PFC) glutamate (GLU) levels, the mechanism(s) subserving this response remain to be elucidated. We used microdialysis and local drug applications to investigate, in male Long-Evans rats, whether the PFC GLU stress response might reflect increased interhemispheric communication by callosal projection neurons. We report here that tail-pinch stress (20 min) elicited comparable increases in GLU in the left and right PFC that were sodium and calcium dependent and insensitive to local glial cystine-GLU exchanger blockade. Unilateral ibotenate-induced PFC lesions abolished the GLU stress response in the opposite hemisphere, as did contralateral mGlu(2/3) receptor activation. Local dopamine (DA) D(1) receptor blockade in the left PFC potently enhanced the right PFC GLU stress response, whereas the same treatment applied to the right PFC had a much weaker effect on the left PFC GLU response. Finally, the PFC GLU stress response was attenuated and potentiated, respectively, following alpha(1)-adrenoreceptor blockade and GABA(B) receptor activation in the opposite hemisphere. These findings indicate that the PFC GLU stress response reflects, at least in part, activation of callosal neurons located in the opposite hemisphere and that stress-induced activation of these neurons is regulated by GLU-, DA-, norepinephrine-, and GABA-sensitive mechanisms. In the case of DA, this control is asymmetrical, with a marked regulatory bias of the left PFC DA input over the right PFC GLU stress response. Together, these findings suggest that callosal neurons and their afferentation play an important role in the hemispheric specialization of PFC-mediated responses to stressors.

Topics: Adrenergic alpha-Antagonists; Amino Acids; Analysis of Variance; Animals; Baclofen; Benzazepines; Bridged Bicyclo Compounds, Heterocyclic; Chromatography, High Pressure Liquid; Disease Models, Animal; Dopamine Antagonists; Excitatory Amino Acid Agonists; Functional Laterality; GABA Agonists; Glutamic Acid; Ibotenic Acid; Male; Microdialysis; Neural Pathways; Oxathiins; Prefrontal Cortex; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Stress, Psychological; Tail; Tetrodotoxin

Tetanic stimulation of the sciatic nerve (TSS) produces long-lasting pain hypersensitivity in rats. Long-term potentiation (LTP) of C- and A-fiber-evoked field potentials in the spinal cord has been explored as contributing to central sensitization in pain pathways. However, the peripheral mechanism underlying TSS-induced pain hypersensitivity remains largely unknown. We investigated the effect of TSS on peripheral nerve and the expression of activating transcription factor 3 (ATF3) in dorsal root ganglion (DRG) as a marker of neuronal injury. TSS induced a mechanical allodynia for at least 35 days and induced ATF3 expression in the ipsilateral DRG. ATF3 is colocalized with NF200-labeled myelinated DRG neurons or CGRP- and IB4-labeled unmyelinated ones. Furthermore, we found that TSS induced Wallerian degeneration of sciatic nerve at the level of myelinisation by S100 protein (to label Schwann cells) immunohistochemistry, luxol fast blue staining, and electron microscopy. TSS also elicited the activation of satellite glial cells (SGCs) and enhanced the colocalization of GFAP and P2X7 receptors. Repeated local treatment with tetrodotoxin decreased GFAP expression in SGCs and behavioral allodynia induced by TSS. Furthermore, reactive microglia and astrocytes were found in the spinal dorsal horn after TSS. These results suggest that TSS-induced nerve injury and glial activation in the DRG and spinal dorsal horn may be involved in cellular mechanisms underlying the development of persistent pain after TSS and that TSS-induced nerve injury may be used as a novel neuropathic pain model.

Topics: Activating Transcription Factor 3; Analysis of Variance; Anesthetics, Local; Animals; Calcium-Binding Proteins; Disease Models, Animal; Electric Stimulation; Ganglia, Spinal; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Hyperalgesia; Microfilament Proteins; Microscopy, Immunoelectron; Neurofilament Proteins; Neuroglia; Pain Measurement; Pain Threshold; Plant Lectins; Rats; Receptors, Purinergic P2; S100 Proteins; Schwann Cells; Sciatic Neuropathy; Tetrodotoxin; Time Factors

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na(v)1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na(v)1.8 channel blockers. A-887826 exemplifies this series and potently (IC(50)=11nM) blocked recombinant human Na(v)1.8 channels. A-887826 was approximately 3 fold less potent to block Na(v)1.2, approximately 10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na(+)) currents and was >30 fold less potent to block Na(V)1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na(+)) currents (IC(50)=8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund's adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by approximately 4mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na(v)1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na(v)1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na(v)1.8 sodium channels in pathological pain states.

Topics: Animals; Biophysics; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Ganglia, Spinal; Humans; Hyperalgesia; Male; Membrane Potentials; Morpholines; NAV1.8 Voltage-Gated Sodium Channel; Neuralgia; Niacinamide; Pain Threshold; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Spinal Cord Injuries; Tetrodotoxin; Transfection

Hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN channels) have large influences upon neuronal excitability. However, the participation of spinal HCN channels in chronic pain states, where pathological conditions are related to altered neuronal excitability, has not been clarified. Intraperitoneally (i.p.) or intrathecally (i.t.) administered ZD7288, a selective blocker of Ih channels, reduced thermal and mechanical hypersensitivity in mice under neuropathic conditions induced by the partial ligation of the sciatic nerve, while no analgesic effect was observed in naïve animals. Moreover, in the mouse formalin test, ZD7288 (i.p. and i.t.) reduced the licking/biting behavior observed during the second phase without affecting the first phase. To further explore the pain-modulatory action of spinal HCN channels, whole-cell patch clamp recordings were made from the visually identified substantia gelatinosa neurons in adult mouse spinal cord slices with an attached dorsal root, and A-fiber- and/or C-fiber-mediated monosynaptic excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation of the L4 or L5 dorsal root using a suction electrode. Bath-applied ZD7288 reduced A-fiber- and C-fiber-mediated monosynaptic EPSCs more preferentially in slices prepared from mice after peripheral nerve injury. In addition, ZD7288 reduced the frequency of miniature EPSCs without affecting their amplitude in cells receiving monosynaptic afferent inputs, indicating that it inhibits EPSCs via presynaptic mechanisms. The present behavioral and electrophysiological data suggest that spinal HCN channels, most likely at the primary afferent terminals, contribute to the maintenance of chronic pain.

Topics: Animals; Cardiotonic Agents; Chronic Disease; Cyclic Nucleotide-Gated Cation Channels; Disease Models, Animal; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Hyperalgesia; In Vitro Techniques; Male; Membrane Potentials; Mice; Mice, Neurologic Mutants; Nerve Fibers; Pain Measurement; Patch-Clamp Techniques; Presynaptic Terminals; Pyrimidines; Sciatica; Sodium Channel Blockers; Spinal Cord; Tetrodotoxin

A large body of evidence has demonstrated that the ectopic discharges of action potentials in primary afferents, resulted from the abnormal expression of voltage gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury are important for the development of neuropathic pain. However, how nerve injury affects the expression of VGSCs is largely unknown. Here, we reported that selective injury of motor fibers by L5 ventral root transection (L5-VRT) up-regulated Nav1.3 and Nav1.8 at both mRNA and protein level and increased current densities of TTX-S and TTX-R channels in DRG neurons, suggesting that nerve injury may up-regulate functional VGSCs in sensory neurons indirectly. As the up-regulated Nav1.3 and Nav1.8 were highly co-localized with TNF-α, we tested the hypothesis that the increased TNF-α may lead to over-expression of the sodium channels. Indeed, we found that peri-sciatic administration of recombinant rat TNF-α (rrTNF) without any nerve injury, which produced lasting mechanical allodynia, also up-regulated Nav1.3 and Nav1.8 in DRG neurons in vivo and that rrTNF enhanced the expression of Nav1.3 and Nav1.8 in cultured adult rat DRG neurons in a dose-dependent manner. Furthermore, inhibition of TNF-α synthesis, which prevented neuropathic pain, strongly inhibited the up-regulation of Nav1.3 and Nav1.8. The up-regulation of the both channels following L5-VRT was significantly lower in TNF receptor 1 knockout mice than that in wild type mice. These data suggest that increased TNF-α may be responsible for up-regulation of Nav1.3 and Nav1.8 in uninjured DRG neurons following nerve injury.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Electric Stimulation; Functional Laterality; Ganglia, Spinal; Immunosuppressive Agents; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Motor Neurons; NAV1.3 Voltage-Gated Sodium Channel; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor, Type I; RNA, Messenger; Sciatica; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Thalidomide; Tumor Necrosis Factor-alpha; Up-Regulation

Thyroid hormone deficiency during a critical period of development severely affects cognitive functions, resulting in profound mental retardation. Despite the importance of the disorder, the cellular mechanisms underlying these deficits remain largely unexplored. The aim of this study was to examine the effects of the absence of thyroid hormone on the development of the intrinsic properties of CA1 hippocampal pyramidal cells. These cells are known to exhibit different firing patterns during development, being classified as either regular-spiking or burst-spiking cells. Patch-clamp experiments showed that hypothyroid rats presented a larger number of regular-spiking cells at early postnatal age (P9-11). This difference in firing-pattern distribution disappeared at the pre-weanling age (P17-19), when almost every cell displayed bursting behavior in both control and hypothyroid rats. However, when studied in detail, weanling hypothyroid rats presented a smaller number of spikes per burst than did control animals. One of the major factors behind bursting behavior is sustained depolarization following an action potential. In this study, we show that action potential afterdepolarizations of hypothyroid animals registered shorter half-durations than did controls, a fact which could explain the smaller number of action potentials per burst. Additionally, the afterdepolarizations observed on both hypothyroid and control neurons were highly sensitive to low concentrations of nickel, suggesting that a low-threshold Ca(2+) current is key in the generation of spike afterdepolarizations and in the control of the bursting pattern of firing of these neurons. In agreement with this, experiments performed on dissociated hippocampal neurons have shown that this current is significantly depressed in hypothyroid animals. Therefore, we conclude that an alteration of the low-threshold calcium current is the basic factor explaining the differences observed in the firing behavior of hypothyroid animals.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Age Factors; Animals; Animals, Newborn; Biophysics; Calcium; Calcium Channels; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; Hippocampus; Hypothyroidism; In Vitro Techniques; Methimazole; Neurons; Patch-Clamp Techniques; Picrotoxin; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Wistar; Sodium Channel Blockers; Tetrodotoxin

The anterior cingulate cortex (ACC) has been demonstrated to play an important role in the affective dimension of pain. Although much evidence has pointed to an increased excitatory synaptic transmission in the ACC in some of the pathological pain state, the inhibitory synaptic transmission in this process has not been well studied. Also, the overall changes of excitatory and inhibitory synaptic transmission have not been comparatively studied in an animal model displaying both long-term persistent nociception and hyperalgesia. Here we used patch clamp recordings in ACC brain slices to observe the changes in synaptic transmission in a pain model induced by peripheral bee venom injection. First, we show that, comparing with those of naive and saline controlled rats, there was a significant increase in spike frequency in ACC neurons harvested from rats after 2 h period of peripheral persistent painful stimuli. Second, it is further shown that the frequency, amplitude and half-width were all increased in spontaneous excitatory post-synaptic currents (sEPSCs), while the amplitude of spontaneous inhibitory post-synaptic currents (sIPSCs) was decreased. The recordings of miniature post-synaptic currents demonstrate an increase in frequency of miniature excitatory post-synaptic currents (mEPSCs) and a decrease in both frequency and amplitude of miniature inhibitory post-synaptic currents (mIPSCs) in rats' ACC slice of bee venom treatment. Taken together, the present results demonstrate an unparalleled change between excitatory and inhibitory synaptic transmission in the ACC under a state of peripheral persistent nociception that might be underlying mechanisms of the excessive excitability of the ACC neurons. We propose that the painful stimuli when lasts or becomes persistent may cause a disruption of the balance between excitatory and inhibitory synaptic transmission that can contribute to the functional change in the ACC.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Analysis of Variance; Animals; Bee Venoms; Bicuculline; Biophysics; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; Gyrus Cinguli; Male; Neural Inhibition; Neurons; Pain; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly(93)-->Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.

Topics: Amyotrophic Lateral Sclerosis; Animals; Biophysical Phenomena; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; Humans; Ion Channel Gating; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Patch-Clamp Techniques; Riluzole; Sodium Channel Blockers; Sodium Channels; Superoxide Dismutase; Tetrodotoxin

In human patients, cortical dysplasia produced by Doublecortin (DCX) mutations lead to mental retardation and intractable infantile epilepsies, but the underlying mechanisms are not known. DCX(-/-) mice have been generated to investigate this issue. However, they display no neocortical abnormality, lessening their impact on the field. In contrast, in utero knockdown of DCX RNA produces a morphologically relevant cortical band heterotopia in rodents. On this preparation we have now compared the neuronal and network properties of ectopic, overlying, and control neurons in an effort to identify how ectopic neurons generate adverse patterns that will impact cortical activity. We combined dynamic calcium imaging and anatomical and electrophysiological techniques and report now that DCX(-/-)EGFP(+)-labeled ectopic neurons that fail to migrate develop extensive axonal subcortical projections and retain immature properties, and most of them display a delayed maturation of GABA-mediated signaling. Cortical neurons overlying the heterotopia, in contrast, exhibit a massive increase of ongoing glutamatergic synaptic currents reflecting a strong reactive plasticity. Neurons in both experimental fields are more frequently coactive in coherent synchronized oscillations than control cortical neurons. In addition, both fields displayed network-driven oscillations during evoked epileptiform burst. These results show that migration disorders produce major alterations not only in neurons that fail to migrate but also in their programmed target areas. We suggest that this duality play a major role in cortical dysfunction of DCX brains.

Topics: Analysis of Variance; Animals; Animals, Genetically Modified; Animals, Newborn; Bicuculline; Cerebral Cortex; Disease Models, Animal; Doublecortin Domain Proteins; Doublecortin Protein; Electroporation; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Glutamic Acid; Green Fluorescent Proteins; Humans; In Vitro Techniques; Malformations of Cortical Development; Membrane Potentials; Microtubule-Associated Proteins; Mutation; Nerve Net; Neurons; Neuropeptides; Pregnancy; Quinoxalines; Rats; Rats, Wistar; RNA, Small Interfering; Sodium Channel Blockers; Tetrodotoxin; Valine

Tetrodotoxin (TTX)-resistant sodium channels are found in small diameter primary sensory neurons and are thought to be important in the maintenance of inflammatory pain. Here we examined bladder urodynamics of Nav1.9 voltage-gated sodium channel knock out (KO) mice, and the contribution of Nav1.9 to the development of inflammation-based bladder dysfunction. Basal urodynamics were not different between wildtype (WT) mice and those lacking Nav1.9. Peripheral nerve recordings from pelvic afferents in Nav1.9 KO mice revealed a lack of sensitization to intravesicularly applied prostaglandin E2 (PGE2). Consistent with this, cyclophosphamide treatment in vivo, which is associated with an enhancement of PGE2 production, evoked a reduction in bladder capacity of WT, but not Nav1.9 KO mice. We conclude that the Nav1.9 sodium channel provides an important link between inflammatory processes and changes in urodynamic properties that occur during urinary bladder inflammation.

Topics: Acetic Acid; Animals; Antirheumatic Agents; Cyclophosphamide; Cystitis; Dinoprostone; Disease Models, Animal; Female; In Vitro Techniques; Male; Mice; Mice, Knockout; NAV1.9 Voltage-Gated Sodium Channel; Nerve Fibers, Unmyelinated; Neuropeptides; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Urinary Bladder; Urination; Urodynamics

Large aspiny neurons and most of the GABAergic interneurons survive transient cerebral ischemia while medium spiny neurons degenerate in 24 h. Expression of a long-term enhancement of excitatory transmission in medium spiny neurons but not in large aspiny neurons has been indicated to contribute to this selective vulnerability. Because neuronal excitability is determined by the counterbalance of excitation and inhibition, the present study examined inhibitory synaptic transmission in large aspiny neurons after ischemia in rats. Transient cerebral ischemia was induced for 22 min using the four-vessel occlusion method and whole-cell voltage-clamp recording was performed on striatal slices. The amplitudes of evoked inhibitory postsynaptic currents in large aspiny neurons were significantly increased at 3 and 24 h after ischemia, which was mediated by the increase of presynaptic release. Postsynaptic responses were depressed at 24 h after ischemia. Inhibitory postsynaptic currents could be evoked in large aspiny neurons at 24 h after ischemia, suggesting that they receive GABAergic inputs from the survived GABAergic interneurons. Muscimol, a GABA(A) receptor agonist, presynaptically facilitated inhibitory synaptic transmission at 24 h after ischemia. Such facilitation was dependent on the extracellular calcium and voltage-gated sodium channels. The present study demonstrates an enhancement of inhibitory synaptic transmission in large aspiny neurons after ischemia, which might reduce excitotoxicity and contribute, at least in part, to the survival of large aspiny neurons. Our data also suggest that large aspiny neurons might receive inhibitory inputs from GABAergic interneurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Biophysics; Biotin; Calcium; Choline O-Acetyltransferase; Corpus Striatum; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Glutamate Decarboxylase; In Vitro Techniques; Ion Channel Gating; Ischemic Attack, Transient; Male; Membrane Potentials; Muscimol; Neurons; Ovulation Inhibition; Patch-Clamp Techniques; Rats; Rats, Wistar; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin; Time Factors

Satellite glial cells in the dorsal root ganglion (DRG), like the better-studied glia cells in the spinal cord, react to peripheral nerve injury or inflammation by activation, proliferation, and release of messengers that contribute importantly to pathological pain. It is not known how information about nerve injury or peripheral inflammation is conveyed to the satellite glial cells. Abnormal spontaneous activity of sensory neurons, observed in the very early phase of many pain models, is one plausible mechanism by which injured sensory neurons could activate neighboring satellite glial cells. We tested effects of locally inhibiting sensory neuron activity with sodium channel blockers on satellite glial cell activation in a rat spinal nerve ligation (SNL) model. SNL caused extensive satellite glial cell activation (as defined by glial fibrillary acidic protein [GFAP] immunoreactivity) which peaked on day 1 and was still observed on day 10. Perfusion of the axotomized DRG with the Na channel blocker tetrodotoxin (TTX) significantly reduced this activation at all time points. Similar findings were made with a more distal injury (spared nerve injury model), using a different sodium channel blocker (bupivacaine depot) at the injury site. Local DRG perfusion with TTX also reduced levels of nerve growth factor (NGF) in the SNL model on day 3 (when activated glia are an important source of NGF), without affecting the initial drop of NGF on day 1 (which has been attributed to loss of transport from target tissues). Local perfusion in the SNL model also significantly reduced microglia activation (OX-42 immunoreactivity) on day 3 and astrocyte activation (GFAP immunoreactivity) on day 10 in the corresponding dorsal spinal cord. The results indicate that early spontaneous activity in injured sensory neurons may play important roles in glia activation and pathological pain.

Topics: Animals; Biomarkers; Bupivacaine; CD11b Antigen; Disease Models, Animal; Ganglia, Spinal; Glial Fibrillary Acidic Protein; Gliosis; Ligation; Male; Microglia; Nerve Growth Factor; Neuralgia; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Rats; Rats, Sprague-Dawley; Satellite Cells, Perineuronal; Sensory Receptor Cells; Sodium Channel Blockers; Tetrodotoxin; Time Factors

Eugenol is widely used in dentistry as a local analgesic agent, because of its ability to allay tooth pain. Interestingly, eugenol shares several pharmacological actions with local anesthetics which include inhibition of voltage-gated sodium channel (VGSC) and activation of transient receptor potential vanilloid subtype 1 (TRPV1). In the present study, we investigated the effects of eugenol on pain behaviors in orofacial area, and as an attempt to elucidate its mechanism we characterized inhibitory effects of eugenol on VGSCs in trigeminal ganglion (TG) neurons. TG neurons were classified into four types on the basis of their neurochemical and electrophysiological properties such as cell size, shapes of action potential (AP), isolectin-B(4) (IB(4)) binding, and were analyzed for the association of their distinctive electrophysiological properties and mRNA expression of Na(v)1.8 and TRPV1 by using single-cell RT-PCR following whole-cell recordings. Subcutaneous injection of eugenol reduced the thermal nociception and capsaicin-induced thermal hyperalgesia in a dose-dependent manner. Eugenol also diminished digastric electromyogram evoked by noxious electrical stimulation to anterior tooth pulp, which was attributable to the blockade of AP conduction on inferior alveolar nerve. At cellular level, eugenol reversibly inhibited APs and VGSCs in IB(4)+/TRPV1+/Na(v)1.8+ nociceptive TG neurons (Type I-Type III) and IB(4)-/TRPV1-/Na(v)1.8- nociceptive TG neurons (Type IV). Both TTX-resistant I(Na) in Type I-Type III neurons and TTX-sensitive I(Na) in Type IV neurons were sensitive to eugenol. Taken together, these results suggest that eugenol may serve as local anesthetics for other pathological pain conditions in addition to its wide use in dental clinic.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Animals, Newborn; Capsaicin; Disease Models, Animal; Dose-Response Relationship, Drug; Electromyography; Eugenol; Gene Expression Regulation; Hyperalgesia; Jaw; Lectins; Male; Masticatory Muscles; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neural Inhibition; Neurons; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Reflex; Sodium Channels; Tetrodotoxin; Tooth; Trigeminal Ganglion; Trigeminal Nerve; TRPV Cation Channels

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

Topics: Animals; Animals, Newborn; Arginine; Biophysical Phenomena; Cells, Cultured; Chromosomes, Artificial, Bacterial; Disease Models, Animal; Dose-Response Relationship, Drug; Electroencephalography; Electromyography; Epilepsy, Generalized; Histidine; Kainic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; NAV1.1 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neurons; Patch-Clamp Techniques; RNA, Messenger; Seizures, Febrile; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

T-type Ca(2+) current-dependent burst firing of thalamic neurons is thought to be involved in the hyper-synchronous activity observed during absence seizures. Here we investigate the correlation between the expression of T-channel coding genes (alpha1G, -H, -I), T-type Ca(2+) current, and the T-current-dependent low threshold Ca(2+) spike in three functionally distinct thalamic nuclei (lateral geniculate nucleus; centrolateral nucleus; reticular nucleus) in a rat model of absence epilepsy, the WAG/Rij rats, and a non-epileptic control strain, the ACI rats. The lateral geniculate nucleus and centrolateral nucleus were found to primarily express alpha1G and alpha1I, while the reticular thalamic nucleus expressed alpha1H and alpha1I. Expression was higher in WAG/Rij when compared to ACI. The T-type Ca(2+) current properties matched the predictions derived from the expression pattern analysis. Current density was larger in all nuclei of WAG/Rij rats when compared to ACI and correlated with LTS size and the minimum LTS generating slope, while T-type Ca(2+) current voltage dependency correlated with the LTS onset potential.

Topics: Action Potentials; Animals; Calcium; Calcium Channels, T-Type; Disease Models, Animal; Epilepsy, Absence; Female; Humans; Ion Channel Gating; Male; Neurons; Patch-Clamp Techniques; Protein Isoforms; Rats; Rats, Inbred Strains; RNA, Messenger; Sodium Channel Blockers; Tetrodotoxin; Thalamus

We have previously shown that neurokinin-1 (NK1) receptors predominantly mediate substance P-induced secretion of the non-inflamed rat colonic mucosa in vitro with a gradient in the magnitude of these responses. The aim of this study was to examine the effects of chronic inflammation on the contributions of different neurokinin receptor subtypes to colonic mucosal secretion. Colitis was induced by the intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid in rats, reactivated 6 weeks later. Segments of proximal, mid- and distal colon were stripped of muscularis propria and mounted in Ussing chambers for measurement of short-circuit current. Use of selective agonists suggests that in the chronically inflamed rat colon NK1 receptors play a greater role in neurokinin-mediated mucosal secretion than do either NK2 or NK3. Selective antagonism implies that this is region-specific, with the inflammatory process altering the relative contribution of the neurokinin receptor subtypes within each region of the rat colon.

Topics: Anesthetics, Local; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antipsychotic Agents; Benzamides; Colitis; Disease Models, Animal; Indomethacin; Intestinal Mucosa; Male; Neurokinin A; Neurokinin-1 Receptor Antagonists; Neurotransmitter Agents; Piperidines; Quinuclidines; Rats; Rats, Sprague-Dawley; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Tachykinin; Stereoisomerism; Substance P; Tetrodotoxin; Trinitrobenzenesulfonic Acid

The brain is heavily dependant on glucose for its function and survival. Hypoglycemia can have severe, irreversible consequences, including seizures, coma and death. However, the in vivo content of brain glycogen, the storage form of glucose, is meager and is a function of both neuronal activity and glucose concentration. In the intact in vitro hippocampus isolated from mice aged postnatal days 8-13, we have recently characterized a novel model of hypoglycemic seizures, wherein seizures were abolished by various neuroprotective strategies. We had hypothesized that these strategies might act, in part, by increasing cerebral glycogen content. In the present experiments, it was found that neither decreasing temperature nor increasing glucose concentrations (above 2 mM) significantly increased hippocampal glycogen content. Preparations of isolated frontal neocortex in vitro do not produce hypoglycemic seizures yet it was found they contained significantly lower glycogen content as compared to the isolated intact hippocampus. Further, the application of either TTX, or a cocktail containing APV, CNQX and gabazine, to block synaptic activity, did not increase, but paradoxically decreased, hippocampal glycogen content in the isolated intact hippocampus. Significant decreases in glycogen were noted when neuronal activity was increased via incubation with l-aspartate (500 muM) or low Mg(2+). Lastly, we examined the incidence of hypoglycemic seizures in hippocampi isolated from mice aged 15-19 and 22-24 days, and compared it to the incidence of hypoglycemic seizures of hippocampi isolated from mice aged 8-13 days described previously (Abdelmalik et al., 2007 Neurobiol Dis 26(3):646-660). It was noted that hypoglycemic seizures were generated less frequently, and had less impact on synaptic transmission in hippocmpi from PD 22-24 as compared to hippocampi from mice PD 15-19 or PD 8-13. However, hippocampi from 8- to 13-day-old mice had significantly more glycogen than the other two age groups. The present data suggest that none of the interventions which abolish hypoglycemic seizures increases glycogen content, and that low glycogen content, per se, may not predispose to the generation of hypoglycemic seizures.

Topics: Age Factors; Analysis of Variance; Anesthetics, Local; Animals; Animals, Newborn; Aspartic Acid; Cerebellum; Disease Models, Animal; Drug Combinations; Excitatory Amino Acid Antagonists; Glucose; Glycogen; Hippocampus; Hypoglycemia; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Seizures; Synaptic Transmission; Tetrodotoxin

Infantile spasms is one of the most severe epileptic syndromes of infancy and early childhood. Progress toward understanding the pathophysiology of this disorder and the development of effective therapies has been hindered by the lack of a relevant animal model. We report here the creation of such a model.. The sodium channel blocker, tetrodotoxin (TTX), was chronically infused into the developing neocortex or hippocampus of infant rats by way of an osmotic minipump starting on postnatal day 10-12.. After a minimum of 10 days of infusion, approximately one-third of these rats began to display very brief (1-2 s) spasms, which consisted of symmetric or asymmetric flexion or extension of the trunk and sometimes involvement of one or both forelimbs. The typical ictal EEG pattern associated with the behavioral spasms consisted of an initial generalized, high amplitude, slow wave followed by an electrodecrement with superimposed fast activity. The interictal EEG revealed multifocal spikes and sharp waves, and in most animals that had spasms a hypsarrhythmic pattern was seen, at least intermittently, during NREM sleep. Like in humans, the spasms in the rat often occurred in clusters especially during sleep-wake transitions. Comparison of the ictal and interictal EEGs recorded in this model and those from humans with infantile spasms revealed that the patterns and the frequency components of both the ictal events and hypsarrhythmia were very similar.. The TTX model of infantile spasms should be of value in furthering an understanding of the pathophysiology of this seizure disorder.

Topics: Animals; Animals, Newborn; Behavior, Animal; Disease Models, Animal; Electrodes, Implanted; Electroencephalography; Hippocampus; Humans; Infant; Infusion Pumps, Implantable; Male; Neocortex; Rats; Rats, Wistar; Sodium Channel Blockers; Spasms, Infantile; Tetrodotoxin; Videotape Recording

Neural ectopic rewiring in retinal degeneration such as retinitis pigmentosa (RP) may form functional synapses between cones and rod bipolar cells that cause atypical signal processing. In this study, the multifocal electroretinograms (mfERGs) of a large animal model of RP, the rhodopsin P347L transgenic (Tg) pig, were measured to examine the sources and nature of altered signal processing.. mfERG responses from a 6-week-old Tg pig were recorded before and after sequential application of tetrodotoxin (TTX), N-methyl-D-aspartate (NMDA), 2-amino-4-phosphonobutyric acid (APB), and cis-2,3-piperidinedicarboylic acid (PDA), to identify contributions to the retinal signal from inner retinal neurons, the ON-pathway, the OFF-pathway, and photoreceptors. The mfERG response contributions from different retinal components of in the Tg eyes were estimated and compared with control data from eyes of age-matched wild-type (WT) pigs.. There was a prominent difference in the estimates of the inner retinal response and ON-bipolar cell pathway contribution between the Tg and WT mfERG responses. In particular, the early components of the inner retinal contribution were obviously altered in the Tg mfERG. The inner retinal components at approximately 24 and 40 ms appeared to be inverted. Differences in the estimates of OFF-bipolar cell pathway contributions were minimal. There was no change in cone cell responses in the Tg mfERG.. In Tg retinas, ectopic synapses formed between cones and rod bipolar cells probably altered signal processing of the ON-bipolar cell pathway. In response to the altered visual signal input from the outer retina, signal processing in inner retinal neurons was also modified.

Topics: Aminobutyrates; Animals; Animals, Genetically Modified; Computers, Handheld; Disease Models, Animal; Electroretinography; Mutation; N-Methylaspartate; Photoreceptor Cells, Vertebrate; Pipecolic Acids; Retinal Bipolar Cells; Retinitis Pigmentosa; Rhodopsin; Swine; Synapses; Synaptic Transmission; Tetrodotoxin; Vision, Ocular

The purpose of this study was to develop and characterize a model of orofacial inflammatory hyperalgesia. Injection of complete Freund's adjuvant (CFA) into the upper lip/whisker pad of the rat produced significant and long-lasting thermal (> or =14 days) and mechanical (> or =28 days) hyperalgesia in the area of CFA injection. Both indomethacin and morphine, given systemically, significantly attenuated thermal hyperalgesia; the effect of morphine was shown to be opioid receptor-mediated. We also examined the contribution of the tetrodotoxin-resistant voltage-gated sodium channel Na(v)1.8 in CFA-produced orofacial mechanical hypersensitivity. Na(v)1.8 mRNA was increased > or =2.5-fold in trigeminal ganglion neurons 1 and 2 weeks after CFA treatment, and Na(v)1.8 protein was increased in the infraorbital nerve over a similar time course. The changes observed were time-dependent and had returned to baseline when examined 2 months after inflammation; there were no changes in Na(v)1.9 mRNA in trigeminal ganglion neurons after CFA treatment. In support of this, Na(v)1.8 antisense oligodeoxynucleotide treatment significantly attenuated CFA-produced mechanical hypersensitivity. These results document development of a model of inflammatory orofacial hyperalgesia, which, consistent with other reports, indicate a contribution of tetrodotoxin-resistant, voltage-gated sodium channel Na(v)1.8.. Orofacial hypersensitivity develops postoperatively as a routine course of orofacial surgery, and mechanical allodynia is characteristic of temporomandibular joint disorder. The results described in this report are novel with respect to the duration of orofacial hypersensitivity produced and suggest that pharmacological targeting of the voltage-gated sodium channel Na(v)1.8 may be useful in managing hypersensitivity.

Topics: Analgesics, Opioid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Disease Models, Animal; Dose-Response Relationship, Radiation; Freund's Adjuvant; Gene Expression Regulation; Hot Temperature; Hyperalgesia; Indomethacin; Male; Morphine; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neurons; Oligodeoxyribonucleotides, Antisense; Rats; Rats, Sprague-Dawley; Reaction Time; RNA, Messenger; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Time Factors; Trigeminal Ganglion; Vibrissae

The congenital muscular dystrophies present in infancy with muscle weakness and are often associated with mental retardation. Many of these inherited disorders share a common etiology: defective O-glycosylation of alpha-dystroglycan, a component of the dystrophin complex. Protein-O-mannosyl transferase 1 (POMT1) is the first enzyme required for the glycosylation of alpha-dystroglycan, and mutations in the POMT1 gene can lead to both Walker-Warburg syndrome (WWS) and limb girdle muscular dystrophy type 2K (LGMD2K). WWS is associated with severe mental retardation and major structural abnormalities in the brain; however, LGMD2K patients display a more mild retardation with no obvious structural defects in the brain. In a screen for synaptic mutants in Drosophila, we identified mutations in the Drosophila ortholog of POMT1, dPOMT1. Because synaptic defects are a plausible cause of mental retardation, we investigated the molecular and physiological defects associated with loss of dPOMT1 in Drosophila. In dPOMT1 mutants, there is a decrease in the efficacy of synaptic transmission and a change in the subunit composition of the postsynaptic glutamate receptors at the neuromuscular junction. We demonstrate that dPOMT1 is required to glycosylate the Drosophila dystroglycan ortholog Dg in vivo, and that this is the likely cause of these synaptic defects because (1) mutations in Dg lead to similar synaptic defects and (2) genetic interaction studies suggest that dPOMT1 and Dg function in the same pathway. These results are consistent with the model that dPOMT1-dependent glycosylation of Dg is necessary for proper synaptic function and raise the possibility that similar synaptic defects occur in the congenital muscular dystrophies.

Topics: Action Potentials; Anesthetics, Local; Animals; Animals, Genetically Modified; Disease Models, Animal; Drosophila; Dystroglycans; Gene Expression Regulation; Glycosylation; Mannosyltransferases; Muscular Dystrophies, Limb-Girdle; Mutation; Neuromuscular Junction; Receptors, Glutamate; Synaptic Transmission; Tetrodotoxin

Topics: Analgesics, Non-Narcotic; Animals; Disease Models, Animal; Hyperalgesia; Injections, Subcutaneous; Male; Pain Measurement; Rats; Tetrodotoxin

Dysfunction of the spinal GABAergic system has been implicated in pain syndromes following spinal cord injury (SCI). Since lamina I is involved in nociceptive and thermal signaling, we characterized the effects of chronic SCI on the cellular properties of its GABAergic neurons fluorescently identified in spinal slices from GAD67-GFP transgenic mice. Whole cell recordings were obtained from the lumbar cord of 13- to 17-day-old mice, including those having had a thoracic segment (T8-11) removed 6-9 days prior to experiments. Following chronic SCI, the distribution, incidence, and firing classes of GFP+ cells remained similar to controls, and there were minimal changes in membrane properties in cells that responded to current injection with a single spike. In contrast, cells displaying tonic/initial burst firing had more depolarized membrane potentials, increased steady-state outward currents, and increased spike heights. Moreover, higher firing frequencies and spontaneous plateau potentials were much more prevalent after chronic SCI, and these changes occurred predominantly in cells displaying a tonic firing pattern. Persistent inward currents (PICs) were observed in a similar fraction of cells from spinal transects and may have contributed to these plateaus. Persistent Na+ and L-type Ca2+ channels likely contributed to the currents as both were identified pharmacologically. In conclusion, chronic SCI induces a plastic response in a subpopulation of lamina I GABAergic interneurons. Alterations are directed toward amplifying neuronal responsiveness. How these changes alter spinal sensory integration and whether they contribute to sensory dysfunction remains to be elucidated.

Topics: Animals; Animals, Newborn; Cadmium; Calcium Channel Blockers; Disease Models, Animal; Dose-Response Relationship, Radiation; Electric Stimulation; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Green Fluorescent Proteins; In Vitro Techniques; Interneurons; Membrane Potentials; Mice; Mice, Transgenic; Neuronal Plasticity; Patch-Clamp Techniques; Potassium Channel Blockers; Sodium Channel Blockers; Spinal Cord Injuries; Tetrodotoxin

Status epilepticus (SE) is a severe neurological condition that can result in brain damage. In animals, SE is associated with cell loss and aberrant synaptogenesis. These pathological processes appear to be activity-dependent and may continue after the SE has ended. We postulated that suppression of electrical activity following SE at the site of the epileptic focus will reduce seizure-induced damage. To achieve this goal, tetrodotoxin (TTX) was used to suppress electrical activity in the hippocampi bilaterally following SE. Adult rats experienced lithium-pilocarpine-induced SE for 2h while controls underwent sham-SE with saline injections. Starting 12h after the SE or sham-SE rats received either continuous TTX (1 microM) or saline infusions through cannulas implanted in the bilateral hippocampi for 5h daily for 4 days. TTX resulted in significant EEG suppression and reduction in spikes and sharp waves. Rats were sacrificed 2 weeks after SE and the brains examined for cell loss and sprouting. Rats receiving TTX following SE had significantly more cell loss as well as a trend toward more mossy fiber sprouting than saline-treated rats following SE. TTX injection in sham-SE rats caused no cell loss or mossy fiber sprouting. These results suggest that suppression of electrical activity following SE is detrimental.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Disease Models, Animal; Electroencephalography; Hippocampus; Lithium Chloride; Male; Pilocarpine; Rats; Rats, Sprague-Dawley; Staining and Labeling; Status Epilepticus; Tetrodotoxin

Recent experimental and modeling results demonstrated that surviving mossy cells in the dentate gyrus play key roles in the generation of network hyperexcitability. Here we examined if mossy cells exhibit long-term plasticity in the posttraumatic, hyperexcitable dentate gyrus. Mossy cells 1 wk after fluid percussion head injury did not show alterations in their current-firing frequency (I-F) and current-membrane voltage (I-V) relationships. In spite of the unchanged I-F and I-V curves, mossy cells showed extensive modifications in Na(+), K(+) and h-currents, indicating the coordinated nature of these opposing modifications. Computational experiments in a realistic large-scale model of the dentate gyrus demonstrated that individually, these perturbations could significantly affect network activity. Synaptic inputs also displayed systematic, opposing modifications. Miniature excitatory postsynaptic current (EPSC) amplitudes were decreased, whereas miniature inhibitory postsynaptic current (IPSC) amplitudes were increased as expected from a homeostatic response to network hyperexcitability. In addition, opposing alterations in miniature and spontaneous synaptic event frequencies and amplitudes were observed for both EPSCs and IPSCs. Despite extensive changes in synaptic inputs, cannabinoid-mediated depolarization-induced suppression of inhibition was not altered in posttraumatic mossy cells. These data demonstrate that many intrinsic and synaptic properties of mossy cells undergo highly specific, long-term alterations after traumatic brain injury. The systematic nature of such extensive and opposing alterations suggests that single-cell properties are significantly influenced by homeostatic mechanisms in hyperexcitable circuits.

Topics: Animals; Animals, Newborn; Computer Simulation; Craniocerebral Trauma; Disease Models, Animal; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; In Vitro Techniques; Membrane Potentials; Models, Neurological; Mossy Fibers, Hippocampal; Nerve Net; Neurons; Patch-Clamp Techniques; Piperidines; Potassium Channel Blockers; Pyrazoles; Pyrimidines; Rats; Sodium Channel Blockers; Tetraethylammonium; Tetrodotoxin

Some chronic pain conditions are maintained or enhanced by sympathetic activity. In animal models of pathological pain, abnormal sprouting of sympathetic fibers around large- and medium-sized sensory neurons is observed in dorsal root ganglia (DRGs). Large- and medium-sized cells are also more likely to be spontaneously active, suggesting that sprouting may be related to neuron activity. We previously showed that sprouting could be reduced by systemic or locally applied lidocaine. In the complete sciatic nerve transection model in rats, spontaneous activity initially originates in the injury site; later, the DRGs become the major source of spontaneous activity. In this study, spontaneous activity reaching the DRG soma was reduced by early nerve blockade (local perfusion of the transected nerve with TTX for the 1st 7 days after injury). This significantly reduced sympathetic sprouting. Conversely, increasing spontaneous activity by local nerve perfusion with K(+) channel blockers increased sprouting. The hyperexcitability and spontaneous activity of DRG neurons observed in this model were also significantly reduced by early nerve blockade. These effects of early nerve blockade on sprouting, excitability, and spontaneous activity were all observed 4-5 wk after the end of early nerve blockade, indicating that the early period of spontaneous activity in the injured nerve is critical for establishing the more long-lasting pathologies observed in the DRG. Individual spontaneously active neurons, labeled with fluorescent dye, were five to six times more likely than quiescent cells to be co-localized with sympathetic fibers, suggesting a highly localized correlation of activity and sprouting.

Topics: Action Potentials; Animals; Autonomic Nerve Block; Axotomy; Disease Models, Animal; Female; Fluorescent Dyes; Ganglia, Spinal; Growth Cones; Nerve Regeneration; Neural Conduction; Neuralgia; Neurons, Afferent; Organ Culture Techniques; Peripheral Nerve Injuries; Peripheral Nerves; Peripheral Nervous System Diseases; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Sodium Channel Blockers; Sympathetic Fibers, Postganglionic; Tetrodotoxin; Time Factors

Late Na(+) current (I(NaL)) in human and dog hearts has been implicated in abnormal repolarization associated with heart failure (HF). HF slows inactivation gating of late Na(+) channels, which could contribute to these abnormalities.. To test how altered gating affects I(NaL) time course, Na(+) influx, and action potential (AP) repolarization.. I(NaL) and AP were measured by patch clamp in left ventricular cardiomyocytes from normal and failing hearts of humans and dogs. Canine HF was induced by coronary microembolization.. I(NaL) decay was slower and I(NaL) density was greater in failing hearts than in normal hearts at 24 degrees C (human hearts: tau=659+/-16 vs. 529+/-21 ms; n=16 and 4 hearts, respectively; mean+/-SEM; p<0.002; dog hearts: 561+/-13 vs. 420+/-17 ms; and 0.307+/-0.014 vs. 0.235+/-0.019 pA/pF; n=25 and 14 hearts, respectively; p<0.005) and at 37 degrees C this difference tended to increase. These I(NaL) changes resulted in much greater (53.6%) total Na(+) influx in failing cardiomyocytes. I(NaL) was sensitive to cadmium but not to cyanide and exhibited low sensitivity to saxitoxin (IC(50)=62 nM) or tetrodotoxin (IC(50)=1.2 muM), tested in dogs. A 50% I(NaL) inhibition by toxins or passing current opposite to I(NaL), decreased beat-to-beat AP variability and eliminated early afterdepolarizations in failing cardiomyocytes.. Chronic HF leads to larger and slower I(NaL) generated mainly by the cardiac-type Na(+) channel isoform, contributing to larger Na(+) influx and AP duration variability. Interventions designed to reduce/normalize I(NaL) represent a potential cardioprotective mechanism in HF via reduction of related Na(+) and Ca(2+) overload and improvement of repolarization.

Topics: Action Potentials; Adult; Animals; Cadmium; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Female; Heart; Heart Failure; Heart Ventricles; Humans; Ion Channel Gating; Ion Transport; Male; Middle Aged; Myocytes, Cardiac; Patch-Clamp Techniques; Saxitoxin; Sodium; Sodium Channels; Tetrodotoxin

The effects of serosally added 5-hydroxytryptamine (5-HT, 100 microM) on the short circuit-current (Isc) across jejunum and ileum taken from fed, starved and undernourished (Gerbillus cheesmani) were investigated. The effects of the neurotoxin, tetrodotoxin (TTX, 10 microM) on the basal Isc as well as on the maximum increase in Isc induced by 5-HT were also studied. There were regional variations in the basal Isc as well as in the way by which the small intestine responds to 5-HT. The basal Isc was greater in jejunum than in ileum and such differences were TTX-sensitive. The maximum increase in Isc, which results from addition of 5-HT, was higher in jejunum than in ileum under all three feeding conditions. TTX reduced the maximum increase in Isc induced by 5-HT across stripped and intact intestine of the two regions in the three nutritional states. The 5-HT-induced Isc in the jejunum of both starved and undernourished gerbils and in the ileum of starved animals was the function of both submucosal and myenteric plexus. In jejunum and ileum taken from starved and undernourished gerbils the 5-HT-induced Isc was both chloride- and bicarbonate-dependent. Thus the results indicated that both starvation and undernourishment increase that response and such increases were TTX-sensitive and both chloride- and bicarbonate-dependent.

Topics: Animal Nutritional Physiological Phenomena; Animals; Bicarbonates; Chlorides; Disease Models, Animal; Gerbillinae; Gluconates; Ileum; Jejunum; Malnutrition; Membrane Potentials; Neurotoxins; Serotonin; Starvation; Tetrodotoxin

Most cochlear implant systems available today provide the user with information about the envelope of the speech signal. The goal of this study was to explore the feasibility of recording electrically evoked auditory steady-state response (ESSR) and in particular to evaluate the degree to which the response recorded using electrical stimulation could be separated from stimulus artifact. Sinusoidally amplitude-modulated electrical stimuli with alternating polarities were used to elicit the response in adult guinea pigs. Separation of the stimulus artifact from evoked neural responses was achieved by summing alternating polarity responses or by using spectral analysis techniques. The recorded response exhibited physiological response properties including a pattern of nonlinear growth and their abolishment following euthanasia or administration of tetrodotoxin. These findings demonstrate that the ESSR is a response generated by the auditory system and can be separated from electrical stimulus artifact. As it is evoked by a stimulus that shares important features of cochlear implant stimulation, this evoked potential may be useful in either clinical or basic research efforts.

Topics: Acoustic Stimulation; Anesthetics, Local; Animals; Artifacts; Auditory Perception; Cochlear Implants; Disease Models, Animal; Electric Stimulation; Evoked Potentials, Auditory; Guinea Pigs; Hearing Loss, Sensorineural; Tetrodotoxin

The recovery of persistent inward currents (PICs) and motoneuron excitability after chronic spinal cord transection is mediated, in part, by the development of supersensitivity to residual serotonin (5HT) below the lesion. The purpose of this paper is to investigate if, like 5HT, endogenous sources of norepinephrine (NE) facilitate motoneuron PICs after chronic spinal transection. Cutaneous-evoked reflex responses in tail muscles of awake chronic spinal rats were measured after increasing presynaptic release of NE by administration of amphetamine. An increase in long-lasting reflexes, known to be mediated by the calcium component of the PIC (CaPIC), was observed even at low doses (0.1-0.2 mg/kg) of amphetamine. These findings were repeated in a reduced S2 in vitro preparation, demonstrating that the increased long-lasting reflexes by amphetamine were neural. Under intracellular voltage clamp, amphetamine application led to a large facilitation of the motoneuron CaPIC. This indicates that the increases in long-lasting reflexes induced by amphetamine in the awake animal were, in part, due to actions directly on the motoneuron. Reflex responses in acutely spinal animals were facilitated by amphetamine similar to chronic animals but only at doses that were ten times greater than that required in chronic animals (0.2 mg/kg chronic vs. 2.0 mg/kg acute), pointing to a development of supersensitivity to endogenous NE in chronic animals. In summary, the increases in long-lasting reflexes and associated motoneuron CaPICs by amphetamine are likely due to an increased release of endogenous NE, which motoneurons become supersensitive to in the chronic stages of spinal cord injury.

Topics: Adrenergic Uptake Inhibitors; Amphetamine; Anesthetics, Local; Animals; Anterior Horn Cells; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Electromyography; Female; In Vitro Techniques; Membrane Potentials; Muscle Contraction; Norepinephrine; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Skin; Spasm; Spinal Cord Injuries; Tetrodotoxin

The biophysical properties of a tetrodotoxin resistant (TTXr) sodium channel, Na(V)1.8, and its restricted expression to the peripheral sensory neurons suggest that blocking this channel might have therapeutic potential in various pain states and may offer improved tolerability compared with existing sodium channel blockers. However, the role of Na(V)1.8 in nociception cannot be tested using a traditional pharmacological approach with small molecules because currently available sodium channel blockers do not distinguish between sodium channel subtypes. We sought to determine whether small interfering RNAs (siRNAs) might be capable of achieving the desired selectivity. Using Northern blot analysis and membrane potential measurement, several siRNAs were identified that were capable of a highly-selective attenuation of Na(V)1.8 message as well as functional expression in clonal ND7/23 cells which were stably transfected with the rat Na(V)1.8 gene. Functional knockdown of the channel was confirmed using whole-cell voltage-clamp electrophysiology. One of the siRNA probes showing a robust knockdown of Na(V)1.8 current was evaluated for in vivo efficacy in reversing an established tactile allodynia in the rat chronic constriction nerve-injury (CCI) model. The siRNA, which was delivered to lumbar dorsal root ganglia (DRG) via an indwelling epidural cannula, caused a significant reduction of Na(V)1.8 mRNA expression in lumbar 4 and 5 (L4-L5) DRG neurons and consequently reversed mechanical allodynia in CCI rats. We conclude that silencing of Na(V)1.8 channel using a siRNA approach is capable of producing pain relief in the CCI model and further support a role for Na(V)1.8 in pathological sensory dysfunction.

Topics: Anesthetics, Local; Animals; Blotting, Northern; Cell Line, Tumor; Disease Models, Animal; Drug Interactions; Electric Stimulation; Hyperalgesia; Male; Membrane Potentials; NAV1.8 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Neuroblastoma; Patch-Clamp Techniques; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sciatica; Sodium Channels; Tetrodotoxin; Time Factors; Transfection

This study used the Matching Pursuit (MP) method, a time-frequency analysis, to identify and characterize oscillatory potentials (OPs) in the primate electroretinogram (ERG). When the slow-sequence mfERG from the macular region of the retina was matched with Gabor functions, OPs were identified in two distinct bands: a high-frequency band peaking around 150 Hz that contributes to early OPs, and a low-frequency band peaking around 80 Hz that contributes to both early and late OPs. Pharmacological blockade and experimental glaucoma studies showed that the high-frequency OPs depend upon sodium-dependent spiking activity of retinal ganglion cells, whereas the low-frequency OPs depend primarily upon non-spiking activity of amacrine cells, and more distal retinal activity.

Topics: Animals; Disease Models, Animal; Electroretinography; gamma-Aminobutyric Acid; Glaucoma; Macaca; N-Methylaspartate; Photic Stimulation; Pipecolic Acids; Retina; Retinal Ganglion Cells; Signal Processing, Computer-Assisted; Tetrodotoxin

Recent studies suggest that altered neural regulation of the gastrointestinal microvasculature contributes to the pathogenesis of inflammatory bowel disease. Therefore, we employed video microscopy techniques to monitor nerve-evoked vasoconstrictor responses in mouse colonic submucosal arterioles in vitro and examined the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis. Nerve stimulation (2-20 Hz) caused frequency-dependent vasoconstrictor responses that were abolished by tetrodotoxin (300 nm) and guanethidine (10 microm). The P2 receptor antagonist suramin (100 microm) or the alpha(1)-adrenoceptor antagonist prazosin (100 nm) reduced the vasoconstriction and the combination of suramin and prazosin completely abolished responses. Nerve-evoked constrictions of submucosal arterioles from mice with TNBS colitis were inhibited by prazosin but not suramin. Superfusion of ATP (10 microm) resulted in large vasoconstrictions in control mice but had no effect in mice with colitis whereas constrictions to phenylephrine (3 microm) were unaffected. P2X(1) receptor immunohistochemistry did not suggest any alteration in receptor expression following colitis. However, Western blotting revealed that submucosal P2X(1) receptor expression was increased during colitis. In contrast to ATP, alphabeta-methylene-ATP (1 microm), which is resistant to catabolism by nucleotidases, constricted control and TNBS arterioles. This indicates that reduced purinergic transmission to submucosal arterioles may be due to increased degradation of ATP during colitis. These data comprise the first description of the neural regulation of mouse submucosal arterioles and identify a defect in sympathetic regulation of the GI vasculature during colitis due to reduced purinergic neurotransmission.

Topics: Adenosine Triphosphate; Adrenergic Agents; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Arterioles; Colitis; Colon; Disease Models, Animal; Electric Stimulation; Enteric Nervous System; Guanethidine; Intestinal Mucosa; Male; Mice; Microscopy, Video; Norepinephrine; Phenylephrine; Prazosin; Purinergic P2 Receptor Antagonists; Receptors, Adrenergic, alpha-1; Receptors, Purinergic P2; Receptors, Purinergic P2X; Suramin; Sympathetic Nervous System; Tetrodotoxin; Time Factors; Trinitrobenzenesulfonic Acid; Up-Regulation; Vasoconstriction

We investigated the responses of morphologically identified myenteric neurons of the guinea-pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin-resistant component, and a prolonged after-hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca(2+)-dependent K(+) channel blocker TRAM-34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin-resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (I(h)) antagonist Cs(+). It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

Topics: Action Potentials; Animals; Disease Models, Animal; Guinea Pigs; Ileitis; Ileum; Intermediate-Conductance Calcium-Activated Potassium Channels; Myenteric Plexus; Neurons; Phenotype; Potassium Channel Blockers; Pyrazoles; Synaptic Transmission; Tetrodotoxin; Time Factors; Trinitrobenzenesulfonic Acid

Chronic pain conditions may result from peripheral nerve injury, chronic peripheral inflammation, or sensory ganglia inflammation. However, inflammatory processes may also contribute to peripheral nerve injury responses. To isolate the contribution of local inflammation of sensory ganglia to chronic pain states, the authors previously developed a rat model in which long-lasting pain is induced by inflaming sensory ganglia without injuring the neurons. This results in prolonged mechanical pain, local increases in proinflammatory cytokines, increased neuronal hyperexcitability, and abnormal spontaneous activity.. The authors used whole cell patch clamp in acutely isolated small-diameter neurons to determine how localized inflammation (3-5 days) of L4 and L5 ganglia altered voltage-gated K and Na currents.. Tetrodotoxin-sensitive Na currents increased twofold to threefold in neurons from inflamed ganglia. Tetrodotoxin-resistant Na currents increased more than twofold, but only in cells that bound isolectin B4. These increases occurred without shifts in voltage dependence of activation and inactivation. Similar results are seen in models of peripheral inflammation, except for the large magnitudes. Unlike most pain models, localized inflammation increased rather than decreased voltage-gated K currents, due to increased amplitudes of the sustained (delayed rectifier) and fast-inactivating transient components. The overall effect in current clamp experiments was an increase in excitability as indicated by decreased rheobase and lower action potential threshold.. Neuronal inflammation per se, in the absence of nerve injury, causes large increases in Na channel density and enhanced excitability. The unusual finding of increased K current may reflect regulation of excitability in the face of such large increases in Na current.

Topics: Action Potentials; Anesthetics, Local; Animals; Cells, Cultured; Disease Models, Animal; Electric Conductivity; Electrophysiology; Female; Ganglia, Spinal; Inflammation; Ion Channel Gating; Membrane Potentials; Neural Conduction; Neurons, Afferent; Patch-Clamp Techniques; Potassium Channels, Voltage-Gated; Rats; Rats, Sprague-Dawley; Sodium Channels; Tetrodotoxin

There is evidence that the dorsal midline thalamus is involved in the seizures of limbic epilepsy. However, little is known about the inhibitory synaptic function in this region. In the present study, inhibitory postsynaptic currents (IPSCs) mediated by GABA(A) receptors were recorded from the mediodorsal (MD) and paraventricular (PV) nuclei from control and epileptic animals. In the MD, the spontaneous (s)IPSCs for epileptic animals had a lower frequency, prolonged rise time, prolonged decay, but unaltered net charge transfer compared with controls. The miniature (m)IPSC parameters were unaltered in the epileptic animals. In contrast, in the PV, both sIPSCs and mIPSCs in the epileptic animals were more frequent with larger amplitudes and there was an increase in the net charge transfer compared with controls. The rise times of the sIPSCs of the PV neurons were significantly prolonged, whereas the weighted decay time of the mIPSC was significantly shortened in epileptic animals. These findings suggest that the changes associated with inhibitory synaptic transmission in limbic epilepsy are not uniform across regions in the thalamus that are part of the seizure circuit.

Topics: Anesthetics, Local; Animals; Bicuculline; Disease Models, Animal; Electric Stimulation; Epilepsy, Temporal Lobe; GABA Antagonists; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Lysine; Male; Midline Thalamic Nuclei; Neurons; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Tetrodotoxin

Colitis induced by Trichinella spiralis in rat induces alterations in the spontaneous motor pattern displayed by circular colonic muscle [Auli, M., Fernandez, E., 2005. Characterization of functional and morphological changes in a rat model of colitis induced by T. spiralis. Digestive Diseases and Sciences 50(8), 1432-1443]. We examined the temporal relationship between the severity of inflammation and the altered contractility of the underlying circular muscle as well as the role of NANC inhibitory pathways in the disruption of the motility pattern. Colitis was induced by intrarectal administration of T. spiralis larvae. Responses to acetylcholine (ACh) and increased extracellular potassium as well as the effect of tetrodotoxin (TTX, 1 microM), N-nitro-l-arginine (L-NOARG, 1 mM) and apamin (1 microM) were determined in vitro in the organ bath with circular muscle strips from sham-infected and infected rats at days 2-30 postinfection (PI). Microelectrode recordings were performed to study the putative changes in electrical activity of colonic smooth muscle cells. Responses to ACh and KCl were decreased at all days PI compared to sham. Intracellular calcium depletion had a greater inhibitory effect in inflamed tissue (6-14 PI). The effect of TTX, L-NOARG and apamin on the spontaneous contractions was found to be altered in all infected rats, i.e. their effects were transient and milder. Inflamed tissue showed lower resting membrane potential and a decreased duration of inhibitory junction potentials induced by electrical stimulation. These data suggest that the decreased contractility of colonic circular smooth muscle induced by the intrarectal T. spiralis infection results from the impairment of the excitation-contraction coupling, from a persistent hyperpolarization of smooth muscle cells and from impaired NANC inhibitory neurotransmission.

Topics: Acetylcholine; Animals; Apamin; Colitis; Disease Models, Animal; Disease Progression; Electric Stimulation; Enteric Nervous System; Gastrointestinal Motility; Inflammation; Intestines; Male; Muscle Contraction; Muscle, Smooth; Nitroarginine; Rats; Rats, Sprague-Dawley; Signal Transduction; Synaptic Transmission; Tetrodotoxin; Time Factors; Trichinella spiralis; Trichinellosis

Rett syndrome (RTT) is caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Although MeCP2 is thought to act as a transcriptional repressor of brain-derived neurotrophic factor (BDNF), Mecp2 null mice, which develop an RTT-like phenotype, exhibit progressive deficits in BDNF expression. These deficits are particularly significant in the brainstem and nodose cranial sensory ganglia (NGs), structures critical for cardiorespiratory homeostasis, and may be linked to the severe respiratory abnormalities characteristic of RTT. Therefore, the present study used Mecp2 null mice to further define the role of MeCP2 in regulation of BDNF expression and neural function, focusing on NG neurons and respiratory control. We find that mutant neurons express significantly lower levels of BDNF than wild-type cells in vitro, as in vivo, under both depolarizing and nondepolarizing conditions. However, BDNF levels in mutant NG cells can be increased by chronic depolarization in vitro or by treatment of Mecp2 null mice with CX546, an ampakine drug that facilitates activation of glutamatergic AMPA receptors. Ampakine-treated Mecp2 null mice also exhibit marked functional improvement, characterized by restoration of normal breathing frequency and minute volume. These data demonstrate that BDNF expression remains plastic in Mecp2 null mice and raise the possibility that ampakine compounds could be of therapeutic value in the treatment of RTT.

Topics: Analysis of Variance; Anesthetics, Local; Animals; Animals, Newborn; Brain-Derived Neurotrophic Factor; Cells, Cultured; Depsipeptides; Dioxoles; Disease Models, Animal; Gene Expression Regulation; Methyl-CpG-Binding Protein 2; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons; Nodose Ganglion; Piperidines; Plethysmography; Rett Syndrome; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrodotoxin

Cognitive coordination refers to processes that organize the timing of activity among neurons without altering individual discharge properties. Coordinating processes allow neural networks to coactivate related representations and prevent the coactivation of unrelated representations. Impaired cognitive coordination, also called cognitive disorganization, is hypothesized to be the core deficit in the disorganized syndrome of schizophrenia (Phillips and Silverstein, 2003), a condition characterized by hallucinations, disorganization, and thought disorder. This disorganization hypothesis is based on the observation that schizophrenic subjects are impaired at segregating relevant and irrelevant stimuli and selectively using associations between relevant cues. We report that injecting the neural activity blocker tetrodotoxin (TTX) into one hippocampus persistently coactivated pyramidal cells in the uninjected hippocampus that initially discharged independently. In accord with the definition of cognitive disorganization, pyramidal cell firing rates only changed for 15 min and did not accompany the coactivation. The TTX-induced coactivity was maximal at gamma periods, consistent with altered gamma oscillations and disorganization in schizophrenia. A network model confirmed that increasing the coupling of weakly associated cells impairs the selective activation and inhibition of stored spatial representations. This TTX-induced cognitive disorganization correctly predicted that the same TTX injection selectively impaired the ability of rats to segregate relevant associations among distal spatial stimuli from irrelevant local stimuli (Wesierska et al., 2005). The TTX-induced coactivity of hippocampal pyramidal cell discharge has construct and predictive validity as a physiological model of psychosis-related disorganization.

Topics: Action Potentials; Animals; Cognition Disorders; Disease Models, Animal; Hippocampus; Psychotic Disorders; Rats; Tetrodotoxin

Tetrodotoxin (TTX) is a powerful sodium channel blocker extracted from the puffer fish. The analgesic effects of TTX were investigated in different animal pain models.. Wistar rats were submitted to the formalin test and to partial ligation of the sciatic nerve (Seltzer's model). Swiss Webster mice were used in the writhing test. Rodents were divided into six groups receiving a s.c. injection of either 0.9% NaCl, TTX 0.3, 1, 3, or 6 microg kg(-1), or morphine (5 mg kg(-1)). Substances were injected 30 min before 2.5% formalin injection into the hind paw, acetic acid administration intraperitoneally or neuropathic pain testing consisting of mechanical allodynia (von Frey filament) and thermal hyperalgesia (Plantar test).. TTX decreased pain behaviour in the formalin test at the highest dose and in the writhing test at 3 and 6 microg kg(-1). It also diminished mechanical allodynia and thermal hyperalgesia with an ED(50) of 1.08 (0.89) and 0.62 (0.33) microg kg(-1), respectively. Observation of the rats after TTX injection did not show any motor deficit, respiratory distress or sedation. Morphine was also effective in relieving pain in all three tests but with signs of considerable sedation.. Systemic injections of TTX diminished pain behaviour in a dose-dependent manner in models of inflammatory, visceral and neuropathic pain without causing adverse events, whereas morphine analgesia was associated with heavy sedation. TTX is a very promising substance for the treatment of various types of pain but needs further evaluation.

Topics: Acetic Acid; Analgesics; Analgesics, Opioid; Animals; Disease Models, Animal; Dose-Response Relationship, Drug; Formaldehyde; Hot Temperature; Hyperalgesia; Male; Mice; Morphine; Pain; Pain Measurement; Physical Stimulation; Rats; Rats, Wistar; Tetrodotoxin

An increase in Rho kinase (ROK) activity has been associated with agonist-induced sustained contraction of the smooth muscle, but its role in the pathophysiology of spontaneously tonic smooth muscle is not known.. Present studies examined the effects of ROK inhibitor Y-27632 in the tonic smooth muscle of the rat internal anal sphincter (IAS) versus in the flanking phasic smooth muscle of the rectum. In addition, studies were performed to determine the relationship between the decreases in the basal IAS tone and the ROK activity. Confocal microscopic studies determined the cellular distribution of the smooth muscle-predominant isoform of ROK (ROCK-II) in the smooth muscle cells (SMCs).. In in vitro studies using neurohumoral inhibitors and tetrodotoxin and the use of SMCs demonstrate direct relaxation of the IAS SMCs by Y-27632. The ROK inhibitor was more potent in the IAS than in the rectal smooth muscle. The IAS relaxation by Y-27632 correlated specifically with the decrease in ROK activity. Confocal microscopy revealed high levels of ROCK-II toward the periphery of the IAS SMCs. In in vivo studies, the lower doses of Y-27632 caused a potent and selective decrease in the IAS pressures without any adverse cardiovascular systemic effects. The ROK inhibitor also caused potent relaxation of the hypertensive IAS.. RhoA/ROK play a crucial role in the maintenance of the basal tone in the IAS, and ROK inhibitors have a therapeutic potential in the IAS dysfunction characterized by the hypertensive IAS.

Topics: Amides; Anal Canal; Animals; Disease Models, Animal; Enzyme Inhibitors; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Muscle Hypertonia; Muscle Relaxation; Muscle, Smooth; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Tetrodotoxin; Treatment Outcome

Hemiballism (HB) is a quite rare disorder, generally secondary to stroke, neoplasms or demyelinating plaques, classically considered as almost pathognomonic of a lesion in the subthalamic nucleus (STN). This alteration causes involuntary movements in the chorea-ballism spectrum. One theory is that the output nuclei of the basal ganglia are overinhibited in HB, while little is known about the physiological state of the striatum, the major input structure of the basal ganglia. In the present study, we recorded spontaneous and miniature excitatory and inhibitory postsynaptic currents (sEPSCs, mEPSCs, sIPSCs, mIPSCs) from projection neurons of the striatum of experimental HB. We found a selective reduction of striatal sEPSC and mEPSC frequency following chemical lesion of the STN of the rat, suggesting that reduced synaptic excitation of the input structure of the basal ganglia represents a physiological correlate of HB.

Topics: Anesthetics, Local; Animals; Corpus Striatum; Disease Models, Animal; Dopamine Antagonists; Dyskinesias; Electric Stimulation; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sulpiride; Synaptic Transmission; Tetrodotoxin; Triazines; Triazoles

To determine the effect of the spatial frequency of a small grating stimulus centered on the macula on the focal macular ERGs (fmacERGs) of monkeys.. fmacERGs were recorded from eight eyes of four adult monkeys (Macaca fuscata). The spatial frequency of the stimulus was changed from 0.25 to 8 cycles/degree. The luminance of the light bars was 10 cd/m(2), and the contrast was 95%. The stimulus was flashed on and off with an on duration of 100 ms and an off duration of 150 ms (4 Hz). The stimulus was centered on the fovea and subtended 12.7 degrees at the cornea. The luminance of the steady light-adapting background was 3.5 cd/m(2). The location of the stimulus on the retina was monitored throughout the recordings. The effects of the spatial frequency of the stimulus on the amplitudes and implicit times of the a-waves, b-waves, and oscillatory potentials (OPs) were determined. fmacERGs were also recorded following intravitreal tetrodotoxin (TTX).. The amplitudes of the a- and b-waves did not change with changes in the spatial frequency of the stimulus. The OPs, on the other hand, responded best to the lowest spatial frequency, and the OPs after the first two were attenuated at intermediate and higher frequencies (Wilcoxon signed-rank test: P < 0.05). TTX reduced all OP wavelets in monkeys.. The OPs of the photopic macular ERGs are affected by the spatial frequency of the stimulus and are reduced by TTX, consistent with their being generated by inner retinal neurons.

Topics: Adaptation, Ocular; Anesthetics, Local; Animals; Disease Models, Animal; Electroretinography; Evoked Potentials, Visual; Injections; Macaca mulatta; Macula Lutea; Male; Photic Stimulation; Tetrodotoxin; Vitreous Body

Loss of axons is a major contributor to nonremitting deficits in the inflammatory demyelinating disease multiple sclerosis (MS). Based on biophysical studies showing that activity of axonal sodium channels can trigger axonal degeneration, recent studies have tested sodium channel-blocking drugs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and have demonstrated a protective effect on axons. However, it is possible that, in addition to a direct effect on axons, sodium channel blockers may also interfere with inflammatory mechanisms. We therefore examined the novel hypothesis that sodium channels contribute to activation of microglia and macrophages in EAE and acute MS lesions. In this study, we demonstrate a robust increase of sodium channel Nav1.6 expression in activated microglia and macrophages in EAE and MS. We further demonstrate that treatment with the sodium channel blocker phenytoin ameliorates the inflammatory cell infiltrate in EAE by 75%. Supporting a role for sodium channels in microglial activation, we show that tetrodotoxin, a specific sodium channel blocker, reduces the phagocytic function of activated rat microglia by 40%. To further confirm a role of Nav1.6 in microglial activation, we examined the phagocytic capacity of microglia from med mice, which lack Nav1.6 channels, and show a 65% reduction in phagocytic capacity compared with microglia from wildtype mice. Our findings indicate that sodium channels are important for activation and phagocytosis of microglia and macrophages in EAE and MS and suggest that, in addition to a direct neuroprotective effect on axons, sodium channel blockade may ameliorate neuroinflammatory disorders via anti-inflammatory mechanisms.

Topics: Animals; Axons; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Gliosis; Macrophages; Male; Mice; Mice, Inbred C57BL; Microglia; Multiple Sclerosis; NAV1.6 Voltage-Gated Sodium Channel; Nerve Degeneration; Nerve Tissue Proteins; Neuroprotective Agents; Phagocytosis; Phenytoin; RNA, Messenger; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Up-Regulation

The hippocampus is critical for navigation in an open field. One component of this navigation requires the subject to recognize the target place using distal cues. The experiments presented in this report tested whether blocking hippocampal function would impair open field place recognition. Hungry rats were trained to press a lever on a feeder for food. In Experiment 1, they were passively transported with the feeder along a circular trajectory. Lever pressing was reinforced only if the feeder was passing through a 60 degrees -wide sector. Thus, rats preferentially lever pressed in the vicinity of the reward sector indicating that they recognized its location. Tetrodotoxin (TTX) infusions aimed at the dorsal hippocampi caused rats to substantially increase lever pressing with no preference for any region. The aim of Experiment 2 was to determine whether the TTX injections caused a loss of place recognition or a general increase of lever pressing. A separate group of rats was conditioned in a stationary apparatus to press the lever in response to a light. The TTX injections did not abolish preferential lever pressing in response to light. Lever pressing increased less than half as much as the TTX-induced increase in Experiment 1. When these animals with functional hippocampi could not determine the rewarded period because the light was always off, lever pressing increased much more and was similar to the TTX-induced increase in Experiment 1. We conclude that the TTX inactivation of the hippocampi impaired the ability to recognize the reward place.

Topics: Anesthetics, Local; Animals; Behavior, Animal; Disease Models, Animal; Feeding Behavior; Hippocampus; Male; Memory; Memory Disorders; Orientation; Rats; Rats, Long-Evans; Space Perception; Tetrodotoxin

We sought to identify the inhibitory interneurons of the rat hippocampal CA1 region selectively vulnerable in the kainic acid (KA) model of temporal lobe epilepsy and to determine whether their selective vulnerability could be due to differential short-term KA effects.. We quantified vulnerable interneurons in stratum oriens-alveus (O/A) by using immunohistochemistry for glutamic acid decarboxylase (GAD), parvalbumin (PV), and somatostatin (SS) after KA injections in rats, and then compared in normal slices the effects of KA on interneurons either in O/A (vulnerable to KA) or in strata radiatum and lacunosum-moleculare (R/LM) (resistant to KA) by using whole-cell recording and calcium imaging.. GAD-, PV- and SS-positive cells in O/A were decreased after KA treatment in P20 and P30 rats. Both short (1-min) and long (10-min) applications of KA produced similar tetrodotoxin (TTX)-insensitive membrane depolarization and decrease in input resistance in O/A and R/LM interneurons. KA responses were antagonized by CNQX and GYKI52466, suggesting AMPA receptor activation. KA also generated a similar increase in intracellular Ca2+ in O/A and R/LM interneurons, which was antagonized by CNQX and GYKI52466.. The selective vulnerability of GAD-, PV-, and SS-immunopositive O/A interneurons in the KA model may not arise from cell-specific short-term membrane effects or calcium responses induced by KA, but from other glutamate receptor-mediated excitotoxic processes.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Disease Models, Animal; Epilepsy, Temporal Lobe; Excitatory Amino Acid Agonists; Glutamate Decarboxylase; Hippocampus; Immunohistochemistry; In Vitro Techniques; Interneurons; Kainic Acid; Male; Neural Inhibition; Parvalbumins; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Somatostatin; Tetrodotoxin

Intractable neuropathic pain often results from nerve injury. One immediate event in damaged nerve is a sustained increase in spontaneous afferent activity, which has a well-established role in ongoing pain. Using two rat models of neuropathic pain, the CCI and SNI models, we show that local, temporary nerve blockade of this afferent activity permanently inhibits the subsequent development of both thermal hyperalgesia and mechanical allodynia. Timing is critical-the nerve blockade must last at least 3-5 days and is effective if started immediately after nerve injury, but not if started at 10 days after injury when neuropathic pain is already established. Effective nerve blockade also prevents subsequent development of spontaneous afferent activity measured electrophysiologically. Similar results were obtained in both pain models, and with two blockade methods (200mg of a depot form bupivacaine at the injury site, or perfusion of the injured nerve just proximal to the injury site with TTX). These results indicate that early spontaneous afferent fiber activity is the key trigger for the development of pain behaviors, and suggest that spontaneous activity may be required for many of the later changes in the sensory neurons, spinal cord, and brain observed in neuropathic pain models. Many pre-clinical and clinical studies of pre-emptive analgesia have used much shorter duration of blockade, or have not started immediately after the injury. Our results suggest that effective pre-emptive analgesia can be achieved only when nerve block is administered early after injury and lasts several days.

Topics: Action Potentials; Analysis of Variance; Anesthetics, Local; Animals; Axotomy; Behavior, Animal; Bupivacaine; Disease Models, Animal; Dose-Response Relationship, Drug; Functional Laterality; Hot Temperature; Hyperalgesia; Ligation; Male; Nerve Block; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Reaction Time; Sciatic Neuropathy; Tetrodotoxin; Time Factors; Touch

With the increasing use of the rat as an animal model for glaucoma and for the evaluation of neuroprotective treatments, there is a need for a sensitive test of retinal ganglion cell (RGC) function in this species. The aims of this study were to detect functional abnormalities of the inner retina in a rat model of high intraocular pressure (IOP) using the pattern electroretinogram (PERG), and to correlate them with morphometric analysis of RGC survival and the functional integrity of the inner retina. Unilateral ocular hypertension was induced in 17 Lewis rats through laser photocoagulation. Pattern ERGs were recorded prior to lasering and 3 weeks later, using a series of shifting patterns of decreasing spatial frequency projected directly onto the animals' fundus. IOP was measured at the same intervals, and the number of surviving RGCs estimated. Low amplitude PERG signals could be recorded in response to a narrow grating of 0.368 cycles per degree (cpd), and increased with stimulus size. Lasering caused mean (+/-s.d.) IOP to increase significantly from 18.3+/-4.5 (baseline) to 29.8+/-8.8 mmHg within 3 weeks (p<0.0001). At this time, PERG amplitudes were significantly reduced (p<0.05), declining an average of 45% compared to the normotensive, control eyes. No outer retinal damage was observed, but the mean number of RGCs decreased significantly (p<0.001), from 2 525.0+/-372.4 to 1 542.8+/-333.8 cells per mm2. This decrease in RGC number was significantly (p=0.03) correlated the decrease in PERG amplitude. The correlation between functional integrity of the inner retina and the rat PERG was further demonstrated by intravitreal tetrodotoxin injections, which temporarily abolished the PERG but did not affect outer retinal activity, reflected in the flash ERG. The evidence for early functional deficits, combined with tonometry and documentation of correlated ganglion cells loss, confirms the sensitivity of this diagnostic tool and the validity and importance of this animal model in glaucoma research.

Topics: Animals; Cell Survival; Disease Models, Animal; Electroretinography; Glaucoma; Male; Rats; Rats, Inbred Lew; Retinal Diseases; Retinal Ganglion Cells; Tetrodotoxin; Tonometry, Ocular

Persistent activation of GABAA receptors by extracellular GABA (tonic inhibition) plays a critical role in signal processing and network excitability in the brain. In hippocampal principal cells, tonic inhibition has been reported to be mediated by alpha5-subunit-containing GABAA receptors (alpha5GABAARs). Pharmacological or genetic disruption of these receptors improves cognitive performance, suggesting that tonic inhibition has an adverse effect on information processing. Here, we show that alpha5GABAARs contribute to tonic currents in pyramidal cells only when ambient GABA concentrations increase (as may occur during increased brain activity). At low ambient GABA concentrations, activation of delta-subunit-containing GABAA receptors predominates. In epileptic tissue, alpha5GABAARs are downregulated and no longer contribute to tonic currents under conditions of raised extracellular GABA concentrations. Under these conditions, however, the tonic current is greater in pyramidal cells from epileptic tissue than in pyramidal cells from nonepileptic tissue, implying substitution of alpha5GABAARs by other GABAA receptor subtypes. These results reveal multiple components of tonic GABAA receptor-mediated conductance that are activated by low GABA concentrations. The relative contribution of these components changes after the induction of epilepsy, implying an adaptive plasticity of the tonic current in the presence of spontaneous seizures.

Topics: Animals; Behavior, Animal; Disease Models, Animal; Drug Interactions; Epilepsy; GABA Agents; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; Imidazoles; Immunohistochemistry; In Vitro Techniques; Male; Membrane Potentials; Neural Inhibition; Neuronal Plasticity; Neurons; Nipecotic Acids; Patch-Clamp Techniques; Picrotoxin; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Tetrodotoxin

Whole cell patch-clamp measurements were made in neurons enzymatically dispersed from the nucleus of the solitary tract (NTS) to determine if alterations occur in voltage-dependent potassium channels from rats made hypertensive (HT) by unilateral nephrectomy/renal wrap for 4 wk. Some rats had the fluorescent tracer DiA applied to the aortic nerve before the experiment to identify NTS neurons receiving monosynaptic baroreceptor afferent inputs. Mean arterial pressure (MAP) was greater in 4-wk HT (165 +/- 5 mmHg, n = 26, P < 0.001) rats compared with normotensive (NT) rats (109 +/- 3 mmHg measured in 10 of 69 rats). Transient outward currents (TOCs) were observed in 67-82% of NTS neurons from NT and HT rats. At activation voltages from -10 to +10 mV, TOCs were significantly less in HT neurons compared with those observed in NT neurons (P < 0.001). There were no differences in the voltage-dependent activation kinetics, the voltage dependence of steady-state inactivation, and the rise and decay time constants of the TOCs comparing neurons isolated from NT and HT rats. The 4-aminopyridine-sensitive component of the TOC was significantly less in neurons from HT compared with NT rats (P < 0.001), whereas steady-state outward currents, whether or not sensitive to 4-aminopyridine or tetraethylammonium, were not different. Delayed excitation, studied under current clamp, was observed in 60-80% of NTS neurons from NT and HT rats and was not different comparing neurons from NT and HT rats. However, examination of the subset of NTS neurons exhibiting somatic DiA fluorescence revealed that DiA-labeled neurons from HT rats had a significantly shorter duration delayed excitation (n = 8 cells, P = 0.022) than DiA-labeled neurons from NT rats (n = 7 cells). Neurons with delayed excitation from HT rats had a significantly broader first action potential (AP) and a slower maximal downstroke velocity of repolarization compared with NT neurons with delayed excitation (P = 0.016 and P = 0.014, respectively). The number of APs in the first 200 ms of a sustained depolarization was greater in HT than NT neurons (P = 0.012). These results suggest that HT of 4-wk duration reduces TOCs in NTS neurons, and this contributes to reduced delayed excitation and increased AP responses to depolarizing inputs. Such changes could alter baroreflex function in hypertension.

Topics: 4-Aminopyridine; Animals; Blood Pressure; Disease Models, Animal; Dose-Response Relationship, Radiation; Electric Stimulation; Hypertension, Renal; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Potassium Channels; Pyridinium Compounds; Rats; Rats, Sprague-Dawley; Reaction Time; Sodium Channel Blockers; Solitary Nucleus; Tetraethylammonium; Tetrodotoxin; Time Factors

This study aimed to establish an animal model of reversible bilateral vestibular disorders that is suitable for examining the mechanisms of vestibular plasticity, and to observe the changes in the plasticity of vestibular efferent systems. Tetrodotoxin (TTX) was infused continuously for 7 days into the bilateral perilymph of guinea pig cochlea. We assessed the vestibulo-ocular reflex (VOR) for evaluating the vestibular function. We also investigated the changes in calcitonin gene-related peptide (CGRP) immunoreactivity in vestibular end organs to observe the changes in the plasticity of vestibular systems. The VOR was completely eliminated by TTX administration and returned to the preoperative levels within 120 h after TTX discontinuation. An obvious increase in the number of CGRP-immunoreactive fibers was observed within the neurosensory epithelia of the maculae and cristae. An animal model of reversible bilateral vestibular disorders was established and used for investigating the plasticity of the vestibular nervous system.

Topics: Anesthetics, Local; Animals; Calcitonin Gene-Related Peptide; Disease Models, Animal; Guinea Pigs; Immunohistochemistry; Male; Reflex, Vestibulo-Ocular; Tetrodotoxin; Vestibular Diseases; Vestibular Function Tests; Vestibule, Labyrinth

To investigate the contribution to the photopic negative response (PhNR) of the electroretinogram (ERG) by retinal ganglion cells (RGCs). The PhNR was assessed longitudinally following optic nerve transection (ONTx).. Photopic ERGs were recorded from each eye of an anesthetized (ketamine/xylazine, 60 mg/kg and 5 mg/kg) Brown Norway rat using custom made electrodes (PT-IR Tef., A-M System Inc). ERGs were elicited using green Ganzfeld flashes (11.38 scd/m(2), 22.76 cds/m(2)) and a rod suppressing green-background (40 cd/m(2)). PhNRs were compared before and after optic nerves were transected. Cresyl violet stained retinal flatmounts were used to estimate cell loss in the ganglion cell layer 3 and 15 weeks after optic nerve transection. The pharmacological effect of 1.3 microM intravitreal TTX on the PhNR was also evaluated.. There was a significant loss (p <0.05) in the PhNR of 20, 36, 34, 35, 48, 48 and 56% for ONTx eye versus the contralateral eye, at post ONTx times of 24 h, 1, 2, 3, 4, 8 and 15 weeks. B-wave amplitudes of ONTx eyes were not significantly different from the control eyes. In ONTx eyes, mean cell loss in the retinal ganglion cell layer was 27 and 55% at the 3 week and 15 week time periods. In the eyes with ONTx, the decline of PhNR amplitudes was correlated positively with RGC loss (r = 0.98; p < 0.01). Thirty minutes after intravitreal TTX injection, the PhNR was significantly reduced (57%, p<0.01).. There was a time-dependent decline in the PhNR after ONTx, as exemplified by a 35% reduction from 1-3 weeks, a 48% decline for 4-8 weeks and a 56% decline after 15 weeks. The correlation between the decline in the PhNR and retinal ganglion cell loss suggests that the PhNR depends on inner retina integrity and the PhNR may be important biological signal or detecting glaucomatous damage and the monitoring of RGC function changes in early glaucoma.

Topics: Anesthetics, Local; Animals; Disease Models, Animal; Electroretinography; Follow-Up Studies; Injections; Male; Optic Nerve Injuries; Photic Stimulation; Rats; Rats, Inbred BN; Retinal Ganglion Cells; Tetrodotoxin; Vitreous Body

Nociceptive neurons within dorsal root ganglia (DRG) express multiple voltage-gated sodium channels, of which the tetrodotoxin-resistant (TTX-R) channel Na(v)1.8 has been suggested to play a major role in inflammatory pain. Previous work has shown that acute administration of inflammatory mediators, including prostaglandin E2 (PGE2), serotonin, and adenosine, modulates TTX-R current in DRG neurons, producing increased current amplitude and a hyperpolarizing shift of its activation curve. In addition, 4 days following injection of carrageenan into the hind paw, an established model of inflammatory pain, Na(v)1.8 mRNA and slowly-inactivating TTX-R current are increased in DRG neurons projecting to the affected paw. In the present study, the expression of sodium channels Na(v)1.1-Na(v)1.9 in small (< or = 25 micromdiameter) DRG neurons was examined with in situ hybridization, immunocytochemistry, Western blot and whole-cell patch-clamp methods following carrageenan injection into the peripheral projection fields of these cells. The results demonstrate that, following carrageenan injection, there is increased expression of TTX-S channels Na(v)1.3 and Na(v)1.7 and a parallel increase in TTX-S currents. The previously reported upregulation of Na(v)1.8 and slowly-inactivating TTX-R current is not accompanied by upregulation of mRNA or protein for Na(v)1.9, an additional TTX-R channel that is expressed in some DRG neurons. These observations demonstrate that chronic inflammation results in an upregulation in the expression of both TTX-S and TTX-R sodium channels, and suggest that TTX-S sodium channels may also contribute, at least in part, to pain associated with inflammation.

Topics: Anesthetics, Local; Animals; Blotting, Western; Carrageenan; Cells, Cultured; Disease Models, Animal; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Immunohistochemistry; In Situ Hybridization; Inflammation; Male; Membrane Potentials; Neurons; Pain; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Channels; Tetrodotoxin

Spectral analysis of electrical noise recorded from the round window (RW) of the cochlea is referred to as the ensemble spontaneous activity (ESA) of the cochlear nerve. The ESA is considered to represent the summed spontaneous activity of single fibers of the auditory nerve and changes in the spectral characteristics of the ESA have been observed in humans with tinnitus. Experiments were undertaken to determine the relationship of the ESA to auditory neurotransmission. The ESA consisted of energy centered at approximately 900 Hz, similar to the spectral peak of single auditory neuron discharges. The amplitude of the ESA was correlated with good auditory sensitivity in the 12-30 kHz region of the cochlea. Constant pure tones of 12-22 kHz suppressed the ESA reducing its amplitude in a frequency and intensity dependent manner implying that the ESA recorded at the RW is generated or dominated by neurons in the basal region of the cochlea. The ESA was significantly suppressed by round window perfusion of the P2X receptor agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) (10 mM) the glutamate receptor antagonist 6-7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM), and the sodium channel antagonist tetrodotoxin (TTX) (20 microM). Following intravenous furosemide injection (40 mg/kg) reduction and recovery of the ESA correlated with similar changes in the endocochlear potential (EP). Following DNQX and ATPgammaS an additional spectral peak at 200 Hz was often observed. This peak has been postulated to be a correlate of tinnitus in humans but had not previously been observed in a guinea-pig model of tinnitus. These data confirm the spectral characteristics of the ESA in guinea-pigs and show it is dependent on the sensitivity of the auditory nerve and intact auditory neurotransmission. In addition these experiments support the view that the ESA represents summed spontaneous neural activity in the cochlea and provide a platform for studies of the influence of ototoxic compounds on the spontaneous neural outflow of the cochlea as a model of tinnitus.

Topics: Acoustic Stimulation; Adenosine Triphosphate; Animals; Cochlear Nerve; Disease Models, Animal; Electrophysiology; Excitatory Amino Acid Antagonists; Female; Furosemide; Guinea Pigs; Humans; Male; Nerve Fibers; Purinergic P2 Receptor Agonists; Quinoxalines; Receptors, Purinergic P2X; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin; Tinnitus

Penetrating cortical trauma frequently results in delayed development of epilepsy. In the rat undercut model of neocortical posttraumatic hyperexcitability, suppression of neuronal activity by exposing the injured cortex to tetrodotoxin (TTX) in vivo for approximately 2 weeks prevents the expression of abnormal hypersynchronous discharges in neocortical slices. We examined the relationship between neuronal activity during the latent period after trauma and subsequent expression of hyperexcitability by varying the timing of TTX treatment. Partially isolated islands of rat sensorimotor cortex were treated with Elvax polymer containing TTX to suppress cortical activity and slices obtained for in vitro experiments 10 to 15 days later. TTX treatment was either started immediately after injury and discontinued after a variable number of days or delayed for a variable time after the lesion was placed. Immediate treatment lasting only 2 to 3 days and treatment delayed up to 3 days prevented hyperexcitability. Thus, there is a critical period for development of hyperexcitability in this model that depends on cortical activity. We propose that the hyperexcitability caused by partial cortical isolation may represent an early stage of posttraumatic epileptogenesis. A hypothetical cascade of events leading to subsequent pathophysiological activity is likely initiated at the time of injury but remains plastic during this critical period.

Topics: Anesthetics, Local; Animals; Animals, Newborn; Behavior, Animal; Critical Period, Psychological; Disease Models, Animal; Drug Administration Schedule; Electroencephalography; Electrophysiology; Epilepsy; Evoked Potentials, Somatosensory; Immunohistochemistry; In Vitro Techniques; Male; Neocortex; Polyvinyls; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

Critical illness myopathy is a disorder in which skeletal muscle becomes electrically inexcitable. We previously demonstrated that a shift in the voltage dependence of fast inactivation of sodium currents contributes to inexcitability of affected fibres in an animal model of critical illness myopathy in which denervated rat skeletal muscle is treated with corticosteroids (steroid-denervated; SD). In the current study we examined whether expression of Nav1.5 contributes to the altered voltage dependence of sodium channel inactivation in SD muscle. We used TTX and mu-conotoxin GIIIB to selectively block Nav1.4 in SD muscle and found that the level of Nav1.5 did not correlate closely with the shift in fast inactivation. Surprisingly, we found that the voltage dependence of inactivation of Nav1.4 was similar to that of Nav1.5 in skeletal muscle in vivo. In severely affected fibres, inactivation of both Nav1.4 and Nav1.5 was shifted towards hyperpolarized potentials. We examined the role of denervation and steroid treatment in the shift of the voltage dependence of inactivation and found that both denervation and steroid treatment contribute to the shift in inactivation. Our results suggest that modulation of the voltage dependence of inactivation of both Nav1.4 and Nav1.5 in vivo contributes to loss of electrical excitability in SD muscle.

Topics: Adrenal Cortex Hormones; Animals; Disease Models, Animal; Muscle Denervation; Muscle Proteins; Muscular Diseases; NAV1.4 Voltage-Gated Sodium Channel; NAV1.5 Voltage-Gated Sodium Channel; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Sodium channel blockers are used clinically to treat a number of neuropathic pain conditions, but more potent and selective agents should improve on the therapeutic index of currently used drugs. In a high-throughput functional assay, a novel sodium channel (Na(V)) blocker, N-[[2'-(aminosulfonyl)biphenyl-4-yl]methyl]-N'-(2,2'-bithien-5-ylmethyl)succinamide (BPBTS), was discovered. BPBTS is 2 orders of magnitude more potent than anticonvulsant and antiarrhythmic sodium channel blockers currently used to treat neuropathic pain. Resembling block by these agents, block of Na(V)1.2, Na(V)1.5, and Na(V)1.7 by BPBTS was found to be voltage- and use-dependent. BPBTS appeared to bind preferentially to open and inactivated states and caused a dose-dependent hyperpolarizing shift in the steady-state availability curves for all sodium channel subtypes tested. The affinity of BPBTS for the resting and inactivated states of Na(V)1.2 was 1.2 and 0.14 microM, respectively. BPBTS blocked Na(V)1.7 and Na(V)1.2 with similar potency, whereas block of Na(V)1.5 was slightly more potent. The slow tetrodotoxin-resistant Na(+) current in small-diameter DRG neurons was also potently blocked by BPBTS. [(3)H]BPBTS bound with high affinity to a single class of sites present in rat brain synaptosomal membranes (K(d) = 6.1 nM), and in membranes derived from HEK cells stably expressing Na(V)1.5 (K(d) = 0.9 nM). BPBTS dose-dependently attenuated nociceptive behavior in the formalin test, a rat model of tonic pain. On the basis of these findings, BPBTS represents a structurally novel and potent sodium channel blocker that may be used as a template for the development of analgesic agents.

Topics: Amides; Analgesics; Animals; Binding Sites; Biphenyl Compounds; Brain; Cell Line; Disease Models, Animal; Formaldehyde; Ganglia, Spinal; Humans; Mice; Muscle Proteins; NAV1.2 Voltage-Gated Sodium Channel; NAV1.5 Voltage-Gated Sodium Channel; NAV1.7 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Pain Measurement; Patch-Clamp Techniques; Rats; Recombinant Proteins; Sodium Channel Blockers; Sodium Channels; Succinates; Synaptosomes; Tetrodotoxin

The neurotoxicity of the AMPA/kainate receptor agonist kainate was investigated in motor and cortical neurones from mice over-expressing the wild-type and G93A mutant form of Cu/Zn superoxide dismutase (SOD1) human gene, a mouse model of familial amyotrophic lateral sclerosis. G93A mutant motor neurones were more vulnerable and wild-type SOD1 motor neurones were more resistant to kainate toxicity than were controls. Voltage-gated Na channels blockage prevented G93A mutant SOD1 motor neurone death. Cortical cultures exhibited fewer differences in their vulnerability to kainate toxicity. These results demonstrate that SOD1 over-expression selectively affects the sensitivity to kainate excitotoxicity of motor neurones but not neocortical neurones, and that wild-type SOD1 expression increases the resistance to excitotoxicity of motor neurones.

Topics: Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Asparagine; Calcium Channel Blockers; Cell Count; Cell Survival; Cells, Cultured; Cerebral Cortex; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Female; Glial Fibrillary Acidic Protein; Glutamic Acid; Immunohistochemistry; Kainic Acid; Male; Mice; Mice, Transgenic; Motor Neurons; Neurotoxins; Nifedipine; Phosphopyruvate Hydratase; Sodium Channel Blockers; Spinal Cord; Superoxide Dismutase; Tetrodotoxin; Time Factors

Alterations in the corticostriatal pathway may precede symptomatology and striatal cell death in Huntington's disease (HD) patients. Here we examined spontaneous EPSCs in striatal medium-sized spiny neurons in slices from a mouse model of HD (R6/2). Spontaneous EPSC frequency was similar in young (3-4 weeks) transgenics and controls but decreased significantly in transgenics when overt behavioral symptoms began (5-7 weeks) and was most pronounced in severely impaired transgenics (11-15 weeks). These differences were maintained after bicuculline or tetrodotoxin, indicating they were specific to glutamatergic input and likely presynaptic in origin. Decreases in presynaptic and postsynaptic protein markers, synaptophysin and postsynaptic density-95, occurred in 11-15 week R6/2 mice, supporting the electrophysiological results. Furthermore, isolated, large-amplitude synaptic events (>100 pA) occurred more frequently in transgenic animals, particularly at 5-7 weeks, suggesting additional dysregulation of cortical inputs. Large events were blocked by tetrodotoxin, indicating a possible cortical origin. Addition of bicuculline and 4-aminopyridine facilitated the occurrence of large events. Riluzole, a compound that decreases glutamate release, reduced these events. Together, these observations indicate that both progressive and transient alterations occur along the corticostriatal pathway in experimental HD. These alterations are likely to contribute to the selective vulnerability of striatal medium-sized spiny neurons.

Topics: Animals; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Disease Progression; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Glutamic Acid; Huntington Disease; In Vitro Techniques; Mice; Neural Pathways; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Potassium Channel Blockers; Riluzole; Tetrodotoxin

In the adult cerebellum, the glutamate receptor delta2 subunit (GluRdelta2) is selectively targeted to the spines of the distal Purkinje cell dendrites, the spiny branchlets, that are innervated by the parallel fibers. Although GluRdelta2 has no known channel function, it is presumed to be involved in the formation and stabilization of these synapses. After block of electrical activity by tetrodotoxin, GluRdelta2s appear in the postsynaptic densities of the proximal dendritic spines, which then lose their contact with climbing fibers and become ectopically innervated by parallel fibers. This phenomenon suggests that climbing fiber activity prevents GluRdelta2 targeting to proximal dendrites and that GluRdelta2s admitted to the postsynaptic density of the spine cause withdrawal of the silent climbing fiber. To test this hypothesis, we studied the distribution of GluRdelta2s in the rat cerebellum by immunoelectron microscopy during the recovery period that follows removal of the electrical block, and during the sprouting of climbing fibers that follows subtotal deletion of the parent inferior olivary neurons by administration of the drug 3-acetylpyridine. We found that after removal of the electrical block, the climbing fibers reinnervate proximal spines that bear GluRdelta2s and these subunits are successively repressed. Similarly, after subtotal lesion of the inferior olive, reinnervation of denervated Purkinje cells occurs on spines bearing GluRdelta2s. Thus, GluRdelta2s are not responsible for displacing silent climbing fibers. We propose instead that GluRdelta2s are associated with climbing fiber-to-Purkinje cell synapses, during development or at early stages of climbing fiber regeneration or sprouting, and are downregulated during the process of synapse maturation.

Topics: Animals; Cell Surface Extensions; Cerebellar Ataxia; Cerebellum; Dendrites; Disease Models, Animal; Drug Administration Routes; Enzyme Inhibitors; Neurons, Afferent; Neurotoxins; Olivary Nucleus; Presynaptic Terminals; Protein Subunits; Purkinje Cells; Pyridines; Rats; Rats, Wistar; Receptors, Glutamate; Recovery of Function; Synapses; Tetrodotoxin

Bladder overactivity associated with outflow obstruction is a common human condition recapitulated in the female rat by narrowing the diameter of the urethra. The goal of these studies was to evaluate the role of intercellular communication through connexin43 (Cx43)-derived gap junction channels to bladder overactivity following partial urethral outflow obstruction of 3-day to 6-wk duration. Cx43 mRNA and protein expression were barely detectable by Northern or Western blots, respectively, in the detrusor layer of normal bladders, but bands were found with both techniques after 6 wk of obstruction. Linear regression analysis of the RT-PCR data revealed a statistically significant positive correlation between the duration of obstruction (again, ranging from 3-day to 6-wk duration) and Cx43 mRNA transcript levels, such that after 6 wk of obstruction, Cx43 transcript levels were approximately 15-fold greater than initial control values. When taking into account the approximately fivefold increase in bladder weight over this same time frame, the absolute amount of Cx43 mRNA in the bladder apparently increased by approximately 75-fold. In that regard, as anticipated, and consistent with previous observations, 6 wk of obstruction was also associated with a significant increase in spontaneous bladder contractions between micturitions. The amplitude of these contractions was significantly reduced by heptanol given intravesically. Furthermore, carbachol-precontracted bladder strips from obstructed animals were more sensitive to heptanol-induced relaxation (100 microM) than their unobstructed counterparts (n = 6; P < 0.01). When bladder strips were equivalently precontracted via electrical field stimulation (EFS; 20 Hz), similar heptanol-induced relaxation responses were observed. However, the tetrodotoxin-resistant portion of the EFS-induced contraction was greater in the obstructed than in the unobstructed animals, and this portion of the contractile response was more sensitive to heptanol-induced relaxation in obstructed than unobstructed bladders (n = 7; P < 0.01). Taken together, these observations indicate that partial outlet obstruction produces an overactive bladder that may be more dependent on intercellular communication through gap junctions for modulation of contractile responses than its normal counterpart.

Topics: Animals; Carbachol; Cell Communication; Connexin 43; Disease Models, Animal; Female; Gap Junctions; Gene Expression; Heptanol; Muscle Contraction; Muscle Relaxation; Organ Size; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tetrodotoxin; Time Factors; Urinary Bladder; Urinary Incontinence

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.

Topics: Acetylcholine; Animals; Calcium; Choline; Choline O-Acetyltransferase; Corpus Striatum; Disease Models, Animal; Dopamine; Electric Stimulation; Exons; Extracellular Space; Female; Humans; Huntington Disease; In Vitro Techniques; Male; Mice; Mice, Transgenic; Microdialysis; Neurotransmitter Agents; Presynaptic Terminals; Receptor, Muscarinic M2; Receptors, Dopamine D2; Receptors, Muscarinic; Tetrodotoxin

(1) Increased vascular resistance in chronic heart failure (CHF) has been attributed to stimulated neurohumoral systems. However, local mechanisms may also importantly contribute to set arterial tone. Our aim, therefore, was to test whether pressure-induced myogenic constriction of resistance arteries in vitro--devoid of acute effects of circulating factors--is increased in CHF and to explore underlying mechanisms. (2) At 12 weeks after coronary ligation-induced myocardial infarction or SHAM-operations in rats, we studied isolated mesenteric arteries for myogenic constriction, determined as the active constriction (% of passive diameter) in response to stepwise increase in intraluminal pressure (20 - 160 mmHg), in the absence and presence of inhibitors of potentially involved modulators of myogenic constriction. (3) We found that myogenic constriction in mesenteric arteries from CHF rats was markedly increased compared to SHAM over the whole pressure range, the difference being most pronounced at 60 mmHg (24+/-2 versus 4+/-3%, respectively, P<0.001). (4) Both removal of the endothelium as well as inhibition of NO production (L-N(G)-monomethylarginine, 100 micro M) significantly increased myogenic constriction (+16 and +25%, respectively), the increase being similar in CHF- and SHAM-arteries (P=NS). Neither endothelin type A (ET(A))-receptor blockade (BQ123, 1 micro M) nor inhibition of perivascular (sympathetic) nerve conduction (tetrodotoxin, 100 nM) affected the myogenic response in either group. (5) Interestingly, increased myogenic constriction in CHF was fully reversed after angiotensin II type I (AT(1))-receptor blockade (candesartan, 100 nM; losartan, 10 micro M), which was without effect in SHAM. In contrast, neither angiotensin-converting enzyme (ACE) inhibition (lisinopril, 1 micro M; captopril, 10 micro M) or AT(2)-receptor blockade (PD123319, 1 micro M), nor inhibition of superoxide production (superoxide dismutase, 50 U ml(-1)), TXA(2)-receptor blockade (SQ29,548, 1 micro M) or inhibition of cyclooxygenase-derived prostaglandins (indomethacin, 10 micro M) affected myogenic constriction. (6) Sensitivity of mesenteric arteries to angiotensin II (10 nM - 100 micro M) was increased (P<0.05) in CHF (pD(2) 7.1+/-0.4) compared to SHAM (pD(2) 6.2+/-0.3), while the sensitivity to KCl and phenylephrine was not different. (7) Our results demonstrate increased myogenic constriction in small mesenteric arteries of rats with CHF, potentially making it an i

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Captopril; Chronic Disease; Coronary Vessels; Disease Models, Animal; Endothelium-Dependent Relaxing Factors; Endothelium, Vascular; Fatty Acids, Unsaturated; Heart; Heart Failure; Hydrazines; Imidazoles; Indomethacin; Lisinopril; Losartan; Male; Mesenteric Arteries; Nitric Oxide; omega-N-Methylarginine; Pyridines; Rats; Rats, Wistar; Receptor, Angiotensin, Type 1; Renin-Angiotensin System; Superoxide Dismutase; Sympathetic Nervous System; Tetrazoles; Tetrodotoxin; Vascular Resistance

Release of serotonin (5-HT) from dorsal raphe nucleus (DRN) neurons projecting to the ventromedial hypothalamus (VMH) has a modulatory effect on the neural pathway involved in feeding, hunger, and satiety. The obese Zucker rat, an animal model of genetic obesity, exhibits differences in serotonin signaling as well as a mutated leptin receptor. To evaluate possible mechanisms underlying this difference in serotonin signaling, we have compared electrophysiological responses of DRN neurons from 14- to 25-day-old male lean (Fa/Fa) and obese (fa/fa) Zucker rats using the whole-cell patch clamp technique on cells in brain slices from these animals. We found that the resting properties of these neurons are not different, but the DRN neurons from obese rats are hyperexcitable in response to current injection. This hyperexcitability is not accompanied by an increase in the depolarization caused by current injection or by changes in the threshold for spiking. However, the hyperexcitability is accompanied by reduction in the size and time course of the afterhyperpolarization (AHP) following an action potential. DRN neurons of obese rats recover from the AHP faster due to a smaller amplitude AHP and a faster time constant (tau) of decay of the AHP. These deficits are not due to changes in the spike waveform, as the spike amplitude and duration do not differ between lean and obese animals. In summary, we provide evidence that serotonergic DRN neurons from obese Zucker rats are intrinsically hyperexcitable compared with those from lean rats. These results suggest a potential mechanism for the reported increase in 5-HT release at the VMH of obese rats during feeding, and provide the first direct evidence of changes in the intrinsic activity of serotonergic neurons, which are crucial regulators of feeding behavior, in a genetic model of obesity.

Topics: Action Potentials; Animals; Disease Models, Animal; Genotype; Male; Membrane Potentials; Neurons; Obesity; Patch-Clamp Techniques; Raphe Nuclei; Rats; Rats, Zucker; Serotonin; Sodium Channel Blockers; Tetrodotoxin

Neuronal activity is thought to play an important role in refining patterns of synaptic connectivity during development and in the molecular maturation of synapses. In experiments reported here, a 2-week infusion of tetrodotoxin (TTX) into rat hippocampus beginning on postnatal day 12 produced abnormal synchronized network discharges in in vitro slices. Discharges recorded upon TTX washout were called 'minibursts', owing to their small amplitude. They were routinely recorded in area CA3 and abolished by CNQX, an AMPA receptor antagonist. Because recurrent excitatory axon collaterals remodel and glutamate receptor subunit composition changes after postnatal day 12, experiments examined possible TTX-induced alterations in recurrent excitation that could be responsible for network hyperexcitability. In biocytin-labelled pyramidal cells, recurrent axon arbors were neither longer nor more highly branched in the TTX infusion site compared with saline-infused controls. However, varicosity size and density were increased. Whereas most varicosities contained synaptophysin and synaptic vesicles, many were not adjacent to postsynaptic specializations, and thus failed to form anatomically identifiable synapses. An increased pattern of excitatory connectivity does not appear to explain network hyperexcitability. Quantitative immunoblots also indicated that presynaptic markers were unaltered in the TTX infusion site. However, the postsynaptic AMPA and NMDA receptor subunits, GluR1, NR1 and NR2B, were increased. In electrophysiological studies EPSPs recorded in slices from TTX-infused hippocampus had an enhanced sensitivity to the NR2B containing NMDA receptor antagonist, ifenprodil. Thus, increases in subunit protein result in alterations in the composition of synaptic NMDA receptors. Postsynaptic changes are likely to be the major contributors to the hippocampal network hyperexcitability and should enhance both excitatory synaptic efficacy and plasticity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Anesthetics, Local; Animals; Animals, Newborn; Axons; Disease Models, Animal; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Immunoblotting; Immunohistochemistry; In Vitro Techniques; Lysine; Membrane Potentials; Microscopy, Confocal; Microscopy, Electron; Nerve Net; Patch-Clamp Techniques; Piperidines; Pyramidal Cells; Rats; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptophysin; Tetrodotoxin; Time Factors

Ca2+-sensitive K+ (K(Ca)) channels play an important role in mediating perinatal pulmonary vasodilation. We hypothesized that lung K(Ca) channel function may be decreased in persistent pulmonary hypertension of the newborn (PPHN). To test this hypothesis, pulmonary artery smooth muscle cells (PASMC) were isolated from fetal lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for at least 7 days. Age-matched, sham-operated animals were used as controls. PASMC K+ currents and membrane potentials were recorded using amphotericin B-perforated patch-clamp techniques. The increase in whole cell current normally seen in response to normoxia was decreased (333.9 +/- 63.6% in control vs. 133.1 +/- 16.0% in hypertensive fetuses). The contribution of the K(Ca) channel to the whole cell current was diminished in hypertensive, compared with control, fetal PASMC. In PASMC from hypertensive fetuses, a change from hypoxia to normoxia caused no change in membrane potential compared with a -14.6 +/- 2.8 mV decrease in membrane potential in PASMC from control animals. In PASMC from animals with pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger depolarization than iberiotoxin, whereas in PASMC from control animals, iberiotoxin caused a larger depolarization than 4-AP. These data confirm the hypothesis that the contribution of the K(Ca) channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the abnormal perinatal pulmonary vasoreactivity associated with PPHN.

Topics: 4-Aminopyridine; Animals; Animals, Newborn; Disease Models, Animal; Female; Gestational Age; Hypertension, Pulmonary; Membrane Potentials; Muscle, Smooth, Vascular; Peptides; Potassium Channels, Calcium-Activated; Pregnancy; Pulmonary Artery; Pulmonary Circulation; Sheep; Tetrodotoxin; Vasodilation

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na(+) or Ca(2+) was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca(2+) overload that follows a reduction in ATP levels in the face of continued influx of Na(+) through voltage-dependent channels.

Topics: Adenosine Triphosphate; Animals; Axons; Calcium; Central Nervous System; Demyelinating Diseases; Disease Models, Animal; Dose-Response Relationship, Drug; Membrane Potentials; Nerve Degeneration; Nerve Fibers, Myelinated; Neuroglia; Neurotoxins; Nitric Oxide; Nitric Oxide Donors; Optic Nerve; Organ Culture Techniques; Rats; Rats, Wistar; Sodium; Tetrodotoxin

Voltage-dependent Na+ currents are important determinants of excitability. We hypothesized that gastric inflammation alters Na+ current properties in primary sensory neurons.. The stomach was surgically exposed in rats to inject the retrograde tracer 1.1'-dioctadecyl-3,3,3,'3-tetramethylindocarbocyanine methanesulfonate and saline (control) or 20% acetic acid (ulcer group) into the gastric wall. Nodose or thoracic dorsal root ganglia (DRG) were harvested after 7 days to culture neurons and record Na+ currents using patch clamp techniques.. There were no lesions in the control and 3 +/- 1 ulcers in the ulcer group. Na+ currents recovered significantly more rapidly from inactivation in nodose and DRG neurons obtained from animals in the ulcer group compared with controls. This was partially a result of an increase in the relative contribution of the tetrodotoxin-resistant to the peak sodium current. In addition, the recovery kinetics of the tetrodotoxin-sensitive current were faster. In DRG neurons, gastric inflammation shifted the voltage-dependence of activation of the tetrodotoxin-resistant current to more hyperpolarized potentials.. Gastric injury alters the properties of Na+ currents in gastric sensory neurons. This may enhance excitability, thereby contributing to the development of dyspeptic symptoms.

Topics: Acetic Acid; Anesthetics, Local; Animals; Disease Models, Animal; Ganglia, Spinal; Gastritis; Hyperalgesia; Ion Channel Gating; Male; Membrane Potentials; Neurons, Afferent; Nodose Ganglion; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channels; Stomach; Stomach Ulcer; Tetrodotoxin

Rats with neonatal excitotoxic damage of the ventral hippocampus display in adulthood a variety of abnormalities reminiscent of schizophrenia and are used as an animal model of this disorder. In the present study, we hypothesized that transient inactivation of ventral hippocampal activity during a critical developmental period may be sufficient to disrupt normal maturation of relevant brain systems and produce similar lasting behavioral changes. We infused tetrodotoxin (TTX) or artificial CSF into the ventral hippocampus on postnatal day 7 (P7) and assessed behavioral changes in response to stress, amphetamine, and (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate in juvenile (P35) and young adult (P56) rats. In adulthood, rats infused neonatally with TTX displayed motor hyperactivity after pharmacological stimulation and after stress compared with sham controls. Analogous TTX infusions in adult animals did not alter these behaviors later in life. These data suggest that transient loss of ventral hippocampal function during a critical time in maturation of intracortical connections permanently changes the development of neural circuits mediating certain dopamine- and NMDA-related behaviors. These results represent a potential new model of aspects of schizophrenia without involving a gross anatomic lesion.

Topics: Age Factors; Aging; Amphetamine; Analysis of Variance; Animals; Animals, Newborn; Behavior, Animal; Central Nervous System Stimulants; Cohort Studies; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Hippocampus; Interpersonal Relations; Male; Microinjections; Motor Activity; Psychomotor Agitation; Rats; Rats, Sprague-Dawley; Schizophrenia; Tetrodotoxin

1. Neuronal cholinergic input is an important regulator of epithelial electrolyte transport and hence water movement in the gut. 2. In this study, colitis was induced by treating mice with 4% (w v(-1)) dextran sodium-sulphate (DSS)-water for 5 days followed by 3 days of normal water. Mid-colonic segments were mounted in Ussing chambers and short-circuit current (Isc, indicates net ion movement) responses to the cholinergic agonist, carbachol (CCh; 10(-4) M)+/-tetrodotoxin, atropine (ATR), hexamethonium (HEX), naloxone or phenoxybenzamine were assessed. 3. Tissues from mice with DSS-induced colitis displayed a drop in Isc in response to CCh (-11.3+/-3.3 microA/cm(2)), while those from control mice showed a transient increase in Isc (76.3+/-13.0 microA/cm(2)). The DeltaIsc in colon from DSS-treated mice was tetrodotoxin-sensitive, atropine-insensitive and was reversed by hexamethonium (HEX+CCh=16.7+/-7.8 microA/cm(2)), indicating involvement of a nicotinic receptor. 4. CCh induced a drop in Isc in tissues from controls only when they were pretreated with the cholinergic muscarinic receptor blocker, atropine: ATR+CCh=-21.3+/-7.0 microA/cm(2). Nicotine elicited a drop in Isc in Ussing-chambered colon from both control and DSS-treated mice that was TTX-sensitive. 5. The drop in Isc evoked by CCh challenge of colonic tissue from DSS-treated mice or ATR+CCh challenge of control tissue was not significantly affected by blockade of opiate or alpha-adrenergic receptors by naloxone or phenoxybenzamine, respectively. 6. The data indicate that DSS-colitis reveals a nicotinic receptor that becomes important in cholinergic regulation of ion transport.

Topics: Animals; Atropine; Carbachol; Cholinergic Agonists; Colitis; Dextran Sulfate; Disease Models, Animal; Hexamethonium; Intestinal Mucosa; Ion Transport; Male; Mice; Mice, Inbred BALB C; Narcotics; Nicotine; Receptors, Adrenergic, alpha; Receptors, Cholinergic; Receptors, Muscarinic; Tetrodotoxin

Functional neuroimaging in humans with acute brain damage often reveals decreases in blood flow and metabolism in areas unaffected by the lesion. This phenomenon, termed diaschisis, is presumably caused by disruption of afferent excitatory input from the lesioned area to other brain regions. By characterizing its neurophysiological basis, we used cerebellar diaschisis to study the relationship between electrical activity and blood flow during decreased neuronal activity. Here we show that focal cerebral ischemia in rats causes diaschisis in the cerebellar cortex characterized by pronounced decreases in Purkinje cell spiking activity and small decreases in cerebellar blood flow. The findings were explained by decreased excitatory input to the cerebellar cortex, i.e., deactivation, as cerebellar neuronal excitability and vascular reactivity were preserved. Functional ablation of the cerebral cortex by either spreading depression or tetrodotoxin reproduced the changes in cerebellar function with complete recovery of Purkinje cell activity and cerebellar blood flow concomitant with recovery of neocortical function. Decreases of activity involving the contralateral frontal cortex produced the largest decrease in cerebellar electrical activity and blood flow. Our data suggest that deactivation explains the decreases in blood flow and metabolism in cerebellar diaschisis observed in human neuroimaging studies. Decreases in spiking activity were 3-7 times larger than the respective decreases in flow. Therefore, under pathological conditions, neuroimaging methods based on hemodynamic signals may only show small changes, although the underlying decrease in neuronal activity is much larger.

Topics: Animals; Cerebellum; Cerebral Arteries; Disease Models, Animal; Functional Laterality; Humans; Ischemic Attack, Transient; Male; Neocortex; Neurons; Rats; Rats, Wistar; Regional Blood Flow; Regression Analysis; Tetrodotoxin

Intracellular sodium concentration ([Na(+)](i)) modulates cardiac contractile and electrical activity through Na/Ca exchange (NCX). Upregulation of NCX in heart failure (HF) may magnify the functional impact of altered [Na(+)](i).. We measured [Na(+)](i) by using sodium binding benzofuran isophthalate in control and HF rabbit ventricular myocytes (HF induced by aortic insufficiency and constriction). Resting [Na(+)](i) was 9.7+/-0.7 versus 6.6+/-0.5 mmol/L in HF versus control. In both cases, [Na(+)](i) increased by approximately 2 mmol/L when myocytes were stimulated (0.5 to 3 Hz). To identify the mechanisms responsible for [Na(+)](i) elevation in HF, we measured the [Na(+)](i) dependence of Na/K pump-mediated Na(+) extrusion. There was no difference in V(max) (8.3+/-0.7 versus 8.0+/-0.8 mmol/L/min) or K(m) (9.2+/-1.0 versus 9.9+/-0.8 mmol/L in HF and control, respectively). Therefore, at measured [Na(+)](i) levels, the Na/K pump rate is actually higher in HF. However, resting Na(+) influx was twice as high in HF versus control (2.3+/-0.3 versus 1.1+/-0.2 mmol/L/min), primarily the result of a tetrodotoxin-sensitive pathway.. Myocyte [Na(+)](i) is elevated in HF as a result of higher diastolic Na(+) influx (with unaltered Na/K-ATPase characteristics). In HF, the combined increased [Na(+)](i), decreased Ca(2+) transient, and prolonged action potential all profoundly affect cellular Ca(2+) regulation, promoting greater Ca(2+) influx through NCX during action potentials. Notably, the elevated [Na(+)](i) may be critical in limiting the contractile dysfunction observed in HF.

Topics: Action Potentials; Animals; Calcium; Cell Separation; Diastole; Disease Models, Animal; Electric Stimulation; Heart Failure; In Vitro Techniques; Intracellular Fluid; Membrane Potentials; Myocardial Contraction; Myocardium; Nickel; Patch-Clamp Techniques; Rabbits; Sodium; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; Strophanthidin; Tetrodotoxin

The mechanisms by which Dai-kenchu-to (TJ-100), a kampo medicine, enhances gastrointestinal motility was investigated using isolated guinea pig ileum. TJ-100 induced contractions accompanied by autonomous contraction at a concentration of more than 3 x 10(-4) g/ml in a dose-related manner. The TJ-100-induced ileal contraction was suppressed by atropine and tetrodotoxin, but not by hexamethonium. This effect was partially suppressed in the presence of high concentrations of ICS 205-930, a serotonin 4 (5-HT4) receptor antagonist. In addition, TJ-100 showed an acetylcholine (ACh)-releasing action in the smooth muscle tissues of ileum. These results suggest that contractile response induced by TJ-100 is partially mediated by ACh released from the cholinergic nerve endings and that 5-HT4 receptors would be involved in the effect of TJ-100.

Topics: Acetylcholine; Animals; Atropine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drugs, Chinese Herbal; Gastrointestinal Motility; Guinea Pigs; Hexamethonium; Ileum; Indoles; Intestinal Pseudo-Obstruction; Male; Medicine, Kampo; Muscle Contraction; Muscle, Smooth; Nicotinic Antagonists; Panax; Parasympatholytics; Pharmaceutical Preparations; Plant Extracts; Serotonin Antagonists; Tetrodotoxin; Tropisetron; Zanthoxylum; Zingiberaceae

In inflammatory bowel disease, smooth muscle function reportedly varies with disease duration. The aim of these studies was to determine changes in the control of spontaneous contractions in a model of experimental colitis that included reinflammation of the healed area. The amplitude and frequency of spontaneous contractions in circular smooth muscle were determined after intrarectal administration of trinitrobenzenesulfonic acid in rat distal colon. With the use of a novel paradigm, rats were studied 4 h (acute) or 28 days (healed) after the initial inflammation. At 28 days, rats were studied 4 h after a second inflammation (reinflamed) of the colon. Colitis induced transient increases in the amplitude of spontaneous contractions coincident with a loss of nitric oxide synthase activity. The frequency of contractions was controlled by constitutive nitric oxide in controls. Frequency was increased in healed and reinflamed colon and was associated with a shift in the dominance of neural constitutive nitric oxide synthase control to that of inducible nitric oxide synthase (iNOS). The initial colitis induced a remodeling of the neural control of spontaneous contractions reflecting changes in their regulation by constitutive nitric oxide synthase and iNOS.

Topics: Animals; Colitis; Colon; Disease Models, Animal; Gastrointestinal Motility; Guanidines; In Vitro Techniques; Inflammation; Inflammatory Bowel Diseases; Male; Muscle Contraction; Muscle, Smooth; NADPH Dehydrogenase; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors; Trinitrobenzenesulfonic Acid

Chronic renal failure is associated with delayed puberty and hypogonadism. To investigate the mechanisms subserving the reported reduced pulsatile release of gonadotropin-releasing hormone (GnRH) in chronic renal failure, this study examined the amino acid neurotransmitter milieu in the medial preoptic area (MPOA), the hypothalamic region where the GnRH-secreting neurons reside, in 5/6-nephrectomized male rats and in ad libitum-fed or pair-fed controls. All rats were castrated and received either a testosterone or a vehicle implant to evaluate additional effects of the prevailing sex steroid milieu. Local excitatory (essential amino acids: aspartate, glutamate) and inhibitory (gamma-aminobutyric acid [GABA], taurine) amino acid transmitter outflow in the MPOA was measured by microdialysis via stereotactically implanted cannulae in the awake, freely moving rats. In addition to basal extracellular concentrations, the neurosecretory capacity was assessed by the addition of 100 mM KCl to the dialysis fluid. The mechanisms of neurosecretion were evaluated further by inhibition of vesicular release with the use of Ca(2+)-free, Mg(2+)-enriched dialysis fluid and by local perfusion with inhibitors of voltage-dependent synaptic release (1 microM tetrodotoxin) and of GABA reuptake (0.5 mM nipecotic acid). In the uremic rats, basal outflow of GABA, glutamate and aspartate, and K(+)-stimulated aspartate outflow were increased. K(+)-stimulated GABA and glutamate release was less sensitive to Ca(2+) depletion in the uremic than in the control rats. The elevated basal GABA and essential amino acid outflow in the uremic rats was due to a voltage- and Ca(2+)-independent mechanism. GABA reuptake was inhibited proportionately by nipecotic acid in uremic and pair-fed control rats. Testosterone supplementation had no independent effects on neurotransmitter outflow. In summary, the amino acid neurotransmitter milieu is altered in the MPOA of uremic rats by a nonsynaptic, nonvesicular mechanism. These abnormalities may contribute to the impaired function of the GnRH pulse generator.

Topics: Amino Acids; Analysis of Variance; Animals; Disease Models, Animal; gamma-Aminobutyric Acid; Gonadotropin-Releasing Hormone; Hypothalamus; Male; Microdialysis; Neurotransmitter Agents; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Uremia

The sodium channel blocker tetrodotoxin (TTX) is an effective tool for blockade of action potentials. Unilateral transtympanic administration of 3 mM TTX produced behavioral symptoms similar to those following unilateral peripheral vestibular ablation. Complete resolution of visible symptoms occurred between 48 and 72 h post-TTX. Eye-coil recordings indicated a spontaneous nystagmus and a decrease in the VOR in TTX-treated animals. Neuronal activity in the central vestibular complex (VC), as monitored with Fos immunocytochemistry, revealed an asymmetric pattern of Fos labeling in the medial, inferior and superior vestibular nuclei and the prepositus hypoglossal nucleus. Although the spatio-temporal pattern of Fos labeling was consistent and reproducible at each time-point, changes were noted among time-points. Transient blockade with TTX may be useful for studying the central vestibular response to recurrent or episodic vestibular disruption in the intact system.

Topics: Animals; Behavior, Animal; Disease Models, Animal; Drug Administration Routes; Ear, Inner; Functional Laterality; Immunohistochemistry; Labyrinth Diseases; Male; Neurons; Nystagmus, Pathologic; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Reflex, Vestibulo-Ocular; Tetrodotoxin; Time Factors; Tympanic Membrane; Vestibular Nuclei

The tetrodotoxin-resistant voltage-gated sodium channel Nav 1.8 is expressed only in nociceptive sensory neurons. This channel has been proposed to contribute significantly to the sensitization of primary sensory neurons after injury. We have studied the nociceptive behaviours of mice carrying a null mutation in the Nav 1.8 gene (Nav 1.8 -/-) in models of peripheral inflammation as well as a model of neuropathic pain. The results from the present studies reveal that Nav 1.8 is a necessary mediator of NGF-induced thermal hyperalgesia but is not essential for PGE2-evoked hypersensitivity. Neuropathic pain behaviours were unchanged in Nav 1.8 -/- mice indicating that this channel is not involved in the alteration of sensory thresholds following peripheral nerve injury.

Topics: Animals; Dinoprostone; Disease Models, Animal; Female; Ganglia, Spinal; Hyperalgesia; Ligation; Male; Mice; Mice, Knockout; Nerve Growth Factor; Neuralgia; Neurons, Afferent; Nociceptors; Peripheral Nervous System Diseases; Sciatic Nerve; Sodium Channels; Tetrodotoxin

The present study investigated the effect of inhibiting the expression of Na(v)1.8 (PN3/SNS) sodium channels by an antisense oligodeoxynucleotide (ODN) on bladder nociceptive responses induced by intravesical acetic acid infusion in rats. Animals were injected intrathecally with either Na(v)1.8 antisense or mismatch ODN. Control cystometrograms under urethane anesthesia during intravesical saline infusion exhibited intercontraction intervals (ICIs) that were significantly longer in antisense-treated rats than in mismatch ODN-treated rats. Intravesical infusion of 0.1% acetic acid induced bladder hyperactivity as reflected by a 68% reduction in ICIs in mismatch ODN-treated rats but did not significantly reduce ICIs in antisense-treated rats. The number of Fos-positive cells after acetic acid administration were significantly reduced in the L6 spinal cord from antisense-treated animals, compared with mismatch ODN-treated animals. In addition, Na(v)1.8 immunoreactivity was reduced in L6 dorsal root ganglion neurons in the antisense-treated rat. In patch-clamp recordings, the conductance density of TTX-resistant sodium currents in dissociated bladder afferent neurons that were labeled by axonal transport of a fluorescent dye, Fast Blue, injected into the bladder wall was also smaller in antisense-treated rats than in mismatch ODN-treated rats, whereas no changes were observed in TTX-sensitive currents. These results indicate that the Na(v)1.8 TTX-resistant sodium channels are involved in the activation of afferent nerves after chemical irritation of the bladder. These channels represent a new target for the treatment of inflammatory pain from visceral organs such as the urinary bladder.

Topics: Acetic Acid; Administration, Intravesical; Animals; Disease Models, Animal; Female; Ganglia, Spinal; Injections, Spinal; NAV1.8 Voltage-Gated Sodium Channel; Neurons, Afferent; Neuropeptides; Oligonucleotides, Antisense; Pain; Pain Measurement; Patch-Clamp Techniques; Proto-Oncogene Proteins c-fos; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Sodium Channels; Spinal Cord; Tetrodotoxin; Urinary Bladder; Visceral Afferents

In normal rats maintained in the dark, very few cells in the primary visual centers, including the superior colliculus, show Fos-like immunoreactivity. By contrast, in rats presented with flashing lights many Fos-like immunoreactivity cells are observed distributed throughout the visual centers. In the dystrophic Royal College of Surgeons rat, in which there is major loss of photoreceptors over the first 3 months of life, similar numbers of Fos-like immunoreactivity cells are seen on light presentation, but in marked contrast, cell densities in the rats maintained in the dark are many times higher than in non-dystrophic rats maintained under similar conditions. Here we show that this elevated dark response can be abolished by intravitreal injection of the sodium channel blocker tetrodotoxin, indicating that this effect results from changed retinal activity, rather than being centrally generated. We suggest that since Fos-like immunoreactivity is not usually elicited by steady state conditions, the elevated levels in the superior colliculus in these animals reflect the return of waves of activity, first seen in development coursing across the retina, but lost with photoreceptor maturation.

Topics: Action Potentials; Animals; Cell Count; Darkness; Disease Models, Animal; Female; Functional Laterality; Immunohistochemistry; Male; Nerve Degeneration; Neuronal Plasticity; Photoreceptor Cells, Vertebrate; Proto-Oncogene Proteins c-fos; Rats; Rats, Mutant Strains; Retinal Degeneration; Retinal Ganglion Cells; Sodium Channel Blockers; Sodium Channels; Superior Colliculi; Tetrodotoxin; Up-Regulation; Visual Pathways

The neural substrates underlying relapse to drug-seeking behavior after chronic drug abuse may differ from those underlying immediate drug-taking behavior. In a model of relapse to drug-seeking behavior following chronic cocaine self-administration and prolonged extinction, we have previously shown that rats will significantly reinstate lever responding for either primary reward (cocaine) or secondary reward (tone + light stimulus previously paired with cocaine). In the present study, we utilized reversible inactivation of discrete brain nuclei with tetrodotoxin (TTX) in order to examine the neural substrates mediating primary and secondary cocaine reward in rats allowed two weeks of cocaine self-administration. After one week of daily extinction sessions, bilateral inactivation of the basolateral amygdala resulted in significant attenuation of lever pressing for a cocaine-conditioned reward (tone + light). Following three more days of extinction, bilateral TTX inactivation of the basolateral amygdala had no effect on the reinstatement of cocaine self-administration. In contrast, TTX inactivation of the nucleus accumbens produced the exact opposite effects, with significant blockade of primary reward (cocaine alone), but not secondary reward (tone + light). Thus, cocaine-conditioned reward is neuroanatomically dissociated from primary cocaine reward.

Topics: Amygdala; Animals; Cocaine; Cocaine-Related Disorders; Disease Models, Animal; Dopamine Uptake Inhibitors; Infusion Pumps, Implantable; Limbic System; Male; Nucleus Accumbens; Rats; Rats, Sprague-Dawley; Recurrence; Reward; Self Administration; Sodium Channels; Tetrodotoxin

Mitochondrial inhibition by 3-nitropropionic acid (3-NPA) causes striatal degeneration reminiscent of Huntington's disease. We studied 3-NPA neurotoxicity and possible indirect excitotoxicity in organotypic striatal and corticostriatal slice cultures. Neurotoxicity was quantified by assay of lactate dehydrogenase in the medium and glutamic acid decarboxylase in tissue homogenates. 3-NPA toxicity (25-100 microM in 5 mM glucose, 24-48 h) appeared to be highly dependent on culture medium glucose levels. 3-NPA treatment caused also a dose-dependent lactate increase, reaching a maximum of threefold increase above control at 100 microM. Both a high dose of glutamate (5 mM) and glutamate uptake blockade by dl-threo-beta-hydroxyaspartate potentiated 3-NPA neurotoxicity in corticostriatal slice cultures. Furthermore, striatum from corticostriatal cocultures was more sensitive to 3-NPA than striatum without cortex and tetrodotoxin, MK-801, and d-2-amino-5-phosphonopentanoic acid prevented or attenuated 3-NPA neurotoxicity, suggesting that membrane depolarization and/or neuronal activity of the glutamatergic corticostriatal pathway contributes to striatal pathology. The results indicate that in vivo characteristics of 3-NPA toxicity can be reproduced in organotypic corticostriatal slice cultures.

Topics: Animals; Aspartic Acid; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Excitatory Amino Acid Antagonists; Glucose; Glutamate Decarboxylase; Glutamic Acid; Huntington Disease; In Vitro Techniques; Mitochondria; Nitro Compounds; Propionates; Rats; Succinate Dehydrogenase; Tetrodotoxin

We hypothesize that the accumulation of tetrodotoxin (TTX) sensitive sodium channels in injured dorsal root ganglion (DRG) neurons plays a critically important role in the generation of ectopic discharges and mechanical allodynia after peripheral nerve injury. Using the segmental spinal nerve (L5) ligation model of neuropathic pain, this hypothesis was tested by examining the effect of TTX on the mechanical sensitivity of the affected hind paw. Various concentrations of TTX were applied topically to the L5 DRG by using chronically implanted polyethylene tubing. The data showed that application of TTX at low doses (12.5-50 nM), which are far less than those needed for blocking action potential conduction, produced a significant elevation of mechanical threshold in the paw for foot withdrawals, a sign of reduced allodynic behaviors. The data suggest that TTX-sensitive subtypes of sodium channels play an important role in maintaining allodynic behaviors in an animal model of neuropathic pain.

Topics: Action Potentials; Animals; Disease Models, Animal; Electric Stimulation; Ganglia, Spinal; Hindlimb; Male; Neurons; Pain; Pain Threshold; Physical Stimulation; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Skin; Sodium Channel Blockers; Tetrodotoxin

To determine whether the uniform field and pattern ERGs that are reduced in macaque eyes with experimental glaucoma have the same inner-retinal origins.. ERGs were recorded from 14 anesthetized adult macaques using DTL electrodes. Six monkeys had laser-induced experimental glaucoma, and two others received intravitreal injections of tetrodotoxin (TTX, 6 microM) to block spiking activity of inner-retinal neurons. The remaining 6 animals were normal. Uniform fields and grating patterns (0.1-3 cpd) were square-wave modulated at 1.7 Hz (transient) and 8 Hz (steady state). The test field (42 degrees x 32 degrees) had a mean luminance of 44 cd/m2 and a contrast of 10% to 82%.. In normal eyes transient ERGs to uniform fields contained photopic negative responses (PhNR) after the b-wave and after the d-wave. Transient pattern electroretinograms (PERGs) at each contrast reversal showed positive (P50) potentials followed by negative (N95) potentials of time course similar to that of the PhNR. The PhNR and N95 were greatly reduced or eliminated by experimental glaucoma and by TTX. Summing responses to luminance increments and decrements of the uniform field could simulate the PERG to low spatial frequency stimuli. Further, the PERG responses to high spatial frequencies were similar to the simulation in shape but slightly delayed in time. Experimental glaucoma and TTX had similar effects on the N95 of the simulated PERG as to those on the actual PERG. However, P50 was more reduced by experimental glaucoma than by TTX, indicating a nonspiking contribution to P50. For the steady state condition, the uniform field ERG, the simulated PERG, and the actual PERG all were affected by experimental glaucoma and TTX, indicating that they contained contributions from the spiking activity of ganglion cells.. The changes in the uniform field and PERG responses produced by experimental glaucoma are related and are largely a consequence of reduced spiking activity of ganglion cells and their axons. These findings raise the possibility that the uniform field ERG could serve as a useful alternative to the PERG in the assessment of clinical glaucomatous neuropathy.

Topics: Animals; Disease Models, Animal; Electroretinography; Glaucoma; Intraocular Pressure; Laser Therapy; Macaca mulatta; Microelectrodes; Pattern Recognition, Visual; Photic Stimulation; Retinal Ganglion Cells; Tetrodotoxin; Trabecular Meshwork; Visual Field Tests; Visual Fields

Primary cultured human coronary myocytes, derived from patients with end-stage heart failure (NYHA, classes III and IV) caused by an ischemic disease and undergoing heart transplantation, express a voltage-gated tetrodotoxin-sensitive sodium current (INa). This current has atypical electrophysiological and pharmacological properties and regulates intracellular sodium ([Na+]i) and calcium ([Ca2+]i). Our work is aimed at identifying its role and regulation of expression during pathophysiology. We currently investigate whether INa is expressed in vascular smooth muscles cells (VSMCs) isolated from either healthy or diseased (atheromatous) arteries in human and, in parallel, in pig, rabbit and rat. Cells were enzymatically isolated, primary cultured and macroscopic INa were recorded using the whole cell patch clamp technique. We found that INa is expressed in VSMCs grown from human aortic (90%; n = 48) and pulmonary (44%; n = 16) arteries and in the human aortic cell line HAVSMC (94%; n = 27). INa was also detected in pig coronary (60%; n = 25) and rabbit aortic (47%; n = 15) VSMCs, but not in rat aortic myocytes (n = 30). These different INa were activated at similar range of potentials (approximately -45 mV), had similar sensitivity to tetrodotoxin (IC50 around 5 nM) and similar density (2 to 4 pA/pF). Their expression was related to cell dedifferentiation in vitro. However, INa was observed more frequently in human myocytes derived from diseased arteries (ischemic cardiopathy) than in those derived from healthy tissues (dilated cardiopathy). In conclusion, INa may contribute to increase the basal arterial contractility and play a role in pathological situations including hypertension.

Topics: Action Potentials; Animals; Aorta; Aortic Diseases; Arterial Occlusive Diseases; Arteriosclerosis; Cardiomyopathy, Dilated; Cell Differentiation; Cells, Cultured; Coronary Artery Disease; Coronary Vessels; Disease Models, Animal; Humans; Hypertension; Ion Channel Gating; Muscle, Smooth, Vascular; Myocardial Ischemia; Patch-Clamp Techniques; Pulmonary Artery; Rabbits; Rats; Rats, Wistar; Sodium Channels; Swine; Tetrodotoxin; Vasomotor System

Ibutilide fumarate is indicated for the termination of atrial fibrillation and atrial flutter. It's mechanism of action is unclear but may involve activation of a late inward Na(+) current.. Twenty seven experiments were performed using an isolated perfused rabbit right ventricle preparation. In each experiment effective refractory periods (ERP) and transmembrane 90% action potential durations (APD) were measured. In 8 experiments ERP and APD were measured at baseline, in the presence of ibutilide (0. 1[emsp4 ]uM), and in the presence of both ibutilide and tetrodotoxin (TTX, 2[emsp4 ]uM). In 8 experiments lidocaine (10[emsp4 ]uM) was used in place of TTX. Measures were made at 200, 400, and 800[emsp4 ]msec paced cycle lengths under each condition. The baseline values for APD at 200, 400 and 800[emsp4 ]msec cycle lengths for the experiments treated with ibutilide and TTX were 111+/-8, 140+/-14 and 159+/-22[emsp4 ]msec, respectively. In the presence of ibutilide, APD increased to 130+/-19, 192+/-26 and 217+/-35[emsp4 ]msec at 200, 400 and 800[emsp4 ]msec cycle lengths, respectively (all p< or =0.03). After the addition of TTX there was no shortening of APD or ERP compared to treatment with ibutilide alone at any cycle length (all p> or =0.062). Similarly, in the presence of ibutilide and lidocaine there were no changes in APD or ERP compared to treatment with ibutilide alone (all p > or =0.41). In 11 control experiments, there were no changes in APD or ERP on serial measures after placebo and TTX or lidocaine.. Ibutilide induced prolongation of ventricular repolarization is not affected by Na(+) channel blockade with lidocaine or TTX in the isolated rabbit heart. These findings suggest that the effects of ibutilide are not mediated by a Na(+) channel dependent late current or that this mechanism contributes minimally to its action in this model.

Topics: Action Potentials; Analysis of Variance; Animals; Anti-Arrhythmia Agents; Culture Techniques; Disease Models, Animal; Electric Conductivity; Female; Heart Ventricles; Lidocaine; Male; Probability; Rabbits; Reference Values; Sodium Channel Blockers; Sulfonamides; Tetrodotoxin; Ventricular Function

Cortical dysplasia has a strong association with epilepsy in humans, but the underlying mechanisms for this are poorly understood. In utero irradiation of rats produces diffuse cortical dysplasia and neuronal heterotopia in the neocortex and hippocampus. Using in vitro neocortical slices, whole-cell patch-clamp recordings were obtained from pyramidal neurons in dysplastic cortex and control neocortex. Spontaneous IPSCs were reduced in amplitude (35%) and frequency (70%) in pyramidal cells from dysplastic cortex. Miniature IPSCs were reduced in frequency (66%) in dysplastic cortex. Two additional measures of cortical inhibition, monosynaptic evoked IPSCs and paired pulse depression of evoked EPSCs, were also impaired in dysplastic cortex. Spontaneous EPSCs were increased in amplitude (42%) and frequency (77%) in dysplastic cortex, but miniature EPSCs were not different between the two groups. These data demonstrate significant physiological impairment in inhibitory synaptic transmission in experimental cortical dysplasia. This supports previous immunohistochemical findings in this model and observations in humans of a reduction in the density of inhibitory interneurons in dysplastic cortex.

Topics: Abnormalities, Radiation-Induced; Animals; Cell Membrane; Choristoma; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA-A Receptor Antagonists; Gamma Rays; In Vitro Techniques; Maternal Exposure; Neocortex; Neural Inhibition; Patch-Clamp Techniques; Pregnancy; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Tetrodotoxin

Multifocal ERGs (MERGs) of 5 adult monkeys (Macaca mulatta) with inner retinal defects caused by laser-induced glaucoma were compared to MERGs from 3 monkeys with inner retinal activity suppressed pharmacologically. MERGs were recorded with DTL fiber electrodes from anesthetized monkeys. Stimuli consisted of 103 equal size hexagons within 17 degrees of the fovea. Stimuli at each location passed through a typical VERIS m-sequence of white (200 cd/m2) and black (12 cd/m2) presentations. In animals with laser-induced glaucoma, visual field sensitivity was assessed by static perimetry using the Humphrey C24-2 full-threshold program modified for animal behavior. Inner retinal (amacrine and ganglion cell) activity was suppressed by intravitreal injection of TTX (4.7-7.6 microM) and NMDA (1.6-5 mM). In normal eyes the first order response (1st order kernel) was larger and more complex, with more distinct oscillations (>60 Hz) in central than in peripheral locations. The 2nd order kernel also was dominated by oscillatory activity. There were naso-temporal variations in both kernels. Pharmacological suppression of inner retinal activity reduced or eliminated the oscillatory behavior, and naso-temporal variations. The 1st order kernel amplitude was increased most and was largest at the fovea. Removed inner retinal responses also were largest at the fovea. The 2nd order kernel was greatly reduced at all locations. In eyes with advanced glaucoma, the effects were similar to those produced by suppressing inner retinal activity, but the later portion of the 1st order kernel waveform was different, lacking a dip after the large positive wave. Visual sensitivity losses and MERG changes both increased over the timecourse of glaucoma, with changes in the MERG being more diffusely distributed across the visual field. We conclude that 1st and 2nd order responses of the primate MERG can be identified that originate from inner retina and are sensitive indicators of glaucomatous neuropathy.

Topics: Animals; Disease Models, Animal; Electroretinography; Glaucoma; Injections; Intraocular Pressure; Macaca mulatta; N-Methylaspartate; Retina; Tetrodotoxin; Visual Field Tests; Vitreous Body

We previously demonstrated improved myocardial preservation with polarized (tetrodotoxin-induced), compared with depolarized (hyperkalemia-induced), arrest and hypothermic storage. This study was undertaken to determine whether polarized arrest reduced ionic imbalance during ischemic storage and whether this was influenced by Na+/K +/2Cl- cotransport inhibition.. We used the isolated crystalloid perfused working rat heart preparation (1) to measure extracellular K+ accumulation (using a K+-sensitive intramyocardial electrode) during ischemic (control), depolarized (K+ 16 mmol/L), and polarized (tetrodotoxin, 22 micromol/L) arrest and hypothermic (7.5 degrees C) storage (5 hours), (2) to determine dose-dependent (0.1, 1.0, 10 and 100 micromol/L) effects of the Na +/K+/2Cl- cotransport inhibitor, furosemide, on extracellular K+ accumulation during polarized arrest and 7.5 degrees C storage, and (3) to correlate extracellular K+ accumulation to postischemic recovery of cardiac function.. Characteristic triphasic profiles of extracellular K+ accumulation were observed in control and depolarized arrested hearts; a significantly attenuated profile with polarized arrested hearts demonstrated reduced extracellular K+ accumulation, correlating with higher postischemic function (recovery of aortic flow was 54% +/-4% [P =.01] compared with 39% +/-3% and 32% +/-3% in depolarized and control hearts, respectively). Furosemide (0.1, 1.0, 10, and 100 micromol/L) modified extracellular K+ accumulation by -18%, -38%, -0.2%, and +9%, respectively, after 30 minutes and by -4%, -27%, +31%, and +42%, respectively, after 5 hours of polarized storage. Recovery of aortic flow was 53% +/-4% (polarized arrest alone), 56% +/-8%, 70% +/-2% (P =.04 vs control), 69% +/-4% (P =.04 vs control), and 65% +/-3% ( P =. 04 vs control), respectively.. Polarized arrest was associated with a reduced ionic imbalance (demonstrated by reduced extracellular K+ accumulation) and improved recovery of cardiac function. Further attenuation of extracellular K + accumulation (by furosemide) resulted in additional recovery.

Topics: Animals; Chloride Channels; Disease Models, Animal; Diuretics; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Extracellular Space; Furosemide; Glucose; Heart Arrest, Induced; Heart Transplantation; Hyperkalemia; Male; Myocardial Reperfusion Injury; Myocardium; Organ Preservation; Rats; Rats, Wistar; Sodium Channels; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin; Time Factors; Tromethamine

Neuronal Na(+)/Ca(2+) exchanger plays a relevant role in maintaining intracellular Ca(2+) and Na(+) levels under physiological and pathological conditions. However, the role of this exchanger in excitotoxicity and ischemia-induced neuronal injury is still controversial and has never been studied in the same neuronal subtypes.. We investigated the effects of bepridil and 3',4'-dichlorobenzamil (DCB), 2 blockers of the Na(+)/Ca(2+) exchanger, in rat striatal spiny neurons by utilizing intracellular recordings in brain slice preparations to compare the action of these drugs on the membrane potential changes induced either by oxygen and glucose deprivation (OGD) or by excitatory amino acids (EAAs).. Bepridil (3 to 100 micromol/L) and DCB (3 to 100 micromol/L) caused a dose-dependent enhancement of the OGD-induced depolarization measured in striatal neurons. The EC(50) values for these effects were 31 micromol/L and 29 micromol/L, respectively. At these concentrations neither bepridil nor DCB altered the resting membrane properties of the recorded cells (membrane potential, input resistance, and current-voltage relationship). The effects of bepridil and DCB on the OGD-induced membrane depolarization persisted in the presence of D-2-amino-5-phosphonovalerate (50 micromol/L) plus 6-cyano-7-nitroquinoxaline-2,3-dione (20 micromol/L), which suggests that they were not mediated by an enhanced release of EAAs. Neither tetrodotoxin (1 micromol/L) nor nifedipine (10 micromol/L) affect the actions of these 2 blockers of the Na(+)/Ca(2+) exchanger, which indicates that voltage-dependent Na(+) channels and L-type Ca(2+) channels were not involved in the enhancement of the OGD-induced depolarization. Conversely, the OGD-induced membrane depolarization was not altered by 5-(N, N-hexamethylene) amiloride (1 to 3 micromol/L), an inhibitor of the Na(+)/H(+) exchanger, which suggests that this antiporter did not play a prominent role in the OGD-induced membrane depolarization recorded from striatal neurons. Bepridil (3 to 100 micromol/L) and DCB (3 to 100 micromol/L) did not modify the amplitude of the excitatory postsynaptic potentials evoked by cortical stimulation. Moreover, these blockers did not affect membrane depolarizations caused by brief applications of glutamate (0.3 to 1 mmol/L), AMPA (0. 3 to 1 micromol/L), and NMDA (10 to 30 micromol/L).. These results provide pharmacological evidence that the activation of the Na(+)/Ca(2+) exchanger exerts a protective role during the early phase of OGD in striatal neurons, although it does not shape the amplitude and the duration of the electrophysiological responses of these cells to EAA.

Topics: Amiloride; Animals; Bepridil; Calcium Channel Blockers; Corpus Striatum; Disease Models, Animal; Excitatory Amino Acids; Glucose; Hypoxia, Brain; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Sodium-Calcium Exchanger; Tetrodotoxin

The extent to which cardiorespiratory infirmity and other sublethal effects of saxitoxin (STX) and tetrodotoxin (TTX) can be reversed by 4-aminopyridine (4-AP) was investigated in guinea pigs chronically instrumented for the concurrent electrophysiological recordings of electrocorticogram (ECoG), diaphragmatic electromyogram (DEMG), Lead II electrocardiogram, and neck skeletal muscle electromyogram. Animals were intoxicated with either STX or TTX (2 and 3 microg/kg, im) to produce a state of progressive cardiorespiratory depression (depicted by decreasing DEMG amplitude, bradypnea, and bradycardia). At the point where cardiorespiratory performance was most seriously compromised (approximately 30 min posttoxin), 4-AP (1 or 2 mg/kg, im) was administered. The therapeutic effect of 4-AP was striking in that, within minutes, the toxin-induced diaphragmatic blockade, bradypnea, bradycardia, and depressed cortical activity were all restored to a level either comparable to, or surpassing, that of control. The optimal 4-AP dose level was determined to be 2 mg/kg (im) based on analyses of cardiorespiratory activity profiles throughout the course of intoxication and 4-AP treatment. At the dose levels (either 1 or 2 mg/kg) used to restore ventilatory function and cardiovascular performance, 4-AP produced no sign of seizures and convulsions. Although less serious secondary effects such as cortical excitant/arousal effect (indicated by ECoG power spectral analysis) and transient periods of skeletal muscle fasciculation were observed, these events were of minor concern particularly in view of the remarkable therapeutic effects of 4-AP.

Topics: 4-Aminopyridine; Animals; Bradycardia; Diaphragm; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Electrocardiography; Electroencephalography; Electromyography; Electrophysiology; Guinea Pigs; Heart Rate; Male; Neck Muscles; Respiration; Saxitoxin; Tetrodotoxin

To determine the cardiovascular, autonomic, and neuromuscular effects of an IV infusion of tetrodotoxin (TTX) when ventilation is supported.. TTX was infused in 18 anesthetized beagles during conventional mechanical ventilation. TTX infusion continued at a rate of 9.3 micrograms/kg/hr until apnea occurred with 1 minute of ventilator disconnection. Measurements included intravascular pressures, heart rate (HR), cardiac output, blood gases, displacements of the rib cage and abdomen, O2 delivery, and responses to train-of-four and tetanic peripheral nerve stimulation. Results are expressed as mean +/- SD.. During TTX infusion, all the dogs had discoordinate movements of the rib cage, abdomen, and limbs. Vomiting, urination, defecation, and increased salivation occurred. Nicotinic and muscarinic effects, neuromuscular blockade, and cardiovascular depression were produced by TTX. Apnea occurred in 72.0 +/- 17.0 minutes when a total of 119.0 +/- 17.4 micrograms of TTX was infused. At apnea, decreases in arterial pressure, cardiac index, HR, O2 delivery, and systemic vascular resistance occurred, while pulmonary artery pressure and pulmonary vascular resistance increased. Loss of response to tetanic stimulation was closely correlated with the dose of TTX that produced apnea.. The clinical symptoms and signs of TTX poisoning are similar to those of anticholinesterase poisons, and TTX dosing as described by this model may serve as a surrogate for organophosphorus poisoning. The model may be useful to determine optimum therapies for TTX poisoning and, since TTX prevents sodium influx into cells, to investigate enhanced survival in animals suffering from ischemia.

Topics: Animals; Apnea; Autonomic Nervous System; Cholinesterase Inhibitors; Disease Models, Animal; Dogs; Drug Evaluation, Preclinical; Hemodynamics; Infusions, Intravenous; Muscles; Respiration, Artificial; Tetrodotoxin

Achalasia is characterized by loss of myenteric neurons and incomplete relaxation of the lower esophageal sphincter (LES). The aim of this study was to develop an achalasia model in the opossum using the surfactant benzyldimethyltetradecylammonium chloride (BAC). This study further characterizes the achalasia model.. BAC or saline was injected circumferentially into the LES of 14 adult opossums. Eight months after injection, manometry, isolated muscle bath studies, electrical field stimulation, and histochemical analysis were performed.. Manometrically, the LES of BAC-treated opossums showed higher pressures (38.7 +/- 12 mm Hg vs. 17 +/- 3.0 mm Hg) and reduced esophageal body contraction amplitudes (4.2 +/- 3 mm Hg vs. 27.4 +/- 12 mm Hg). Isolated muscle strips challenged with carbachol and sodium nitroprusside contracted and relaxed similarly to controls. Electrical field stimulation failed to induce relaxation in BAC-treated tissue but did induce contraction. Contractile responses were markedly reduced by tetrodotoxin and atropine in BAC-treated animals and controls. An altered nitric oxide system was shown by the lack of response to L-arginine and N omega-nitro-L-arginine. Histology showed loss of myenteric neurons and increased cholinergic nerve bundles.. Loss of NO inhibitory myenteric neurons markedly reduces the relaxation of the LES, and histology and pharmacological responses suggest a proliferation of cholinergic nerves into the LES contributing to the static elevated pressures of the amyenteric LES.

Topics: Analysis of Variance; Animals; Arginine; Atropine; Benzalkonium Compounds; Cholinergic Fibers; Denervation; Disease Models, Animal; Electric Stimulation; Electrophysiology; Esophageal Achalasia; Esophagogastric Junction; Female; In Vitro Techniques; Male; Manometry; Muscle Contraction; Muscle Relaxation; Myenteric Plexus; Neurons; Nitric Oxide; Opossums; Pressure; Tetrodotoxin

Some controversy exists as to whether alpha-motoneurons adapt their oxidative metabolism to changes in chronic activity levels and to altered status of their end organs, as occurs in other neuron types in the central nervous system. We measured, using a personal computer-based image analysis system, succinate dehydrogenase (SDH) activity in rat hindlimb motoneurons under conditions of increased activity (daily voluntary exercise plus treadmill endurance training, the latter 2 h/day, 4 days/wk, for 12 wk) and in a condition of muscle disuse (tetrodotoxin-induced paralysis for 2 wk) in which muscle oxidative enzymes are reduced. Although exercise-trained medial gastrocnemius showed significant adaptations (increased mean SDH activity of type I and increased proportion and total SDH activity of type I and combined I + IIa fibers), SDH activity of innervating motoneurons (identified by retrograde tracing using fast blue) was unchanged. In addition, tetrodotoxin-induced disuse, which results in hindlimb atrophy and SDH decreases (30% decrease measured in medial gastrocnemius muscle homogenates), failed to alter soma SDH or size in unspecified lumbar motoneurons. These results, obtained over a wider range of activity levels than in previous reports, suggest that the oxidative enzymes of motoneurons do not change despite clear adaptations in the muscles they innervate.

Topics: Adaptation, Physiological; Animals; Disease Models, Animal; Male; Motor Neurons; Muscle Fibers, Skeletal; Muscle, Skeletal; Paralysis; Physical Exertion; Rats; Rats, Sprague-Dawley; Spectrophotometry; Succinate Dehydrogenase; Tetrodotoxin

Fluoxetine (15 mg/kg i.p.) decreased the audiogenic seizure intensity in 33% of severe seizure genetically epilepsy-prone rats (GEPR-9s). 5-Hydroxytryptophan (5-HTP, 12.5 mg/kg i.p.) produced no anticonvulsant effect in GEPR-9s. When GEPR-9s were treated with a combination of these two drugs, the combination treatment decreased the audiogenic seizure intensity in 83% of the animals tested. Brain microdialysis studies showed that the same combination of 5-HTP and fluoxetine also produced a marked potentiation of the increase in the extracellular serotonin concentration in the thalamus of freely-moving GEPR-9s when compared with administration of either drug alone. A negative correlation between audiogenic seizure intensity and extracellular serotonin concentration existed after either fluoxetine alone or the combination treatment. No significant changes in extracellular norepinephrine concentrations were observed after the combination treatment. These results coupled with our earlier reports strongly suggest that a serotonergic mechanism is involved in the anticonvulsant effects of fluoxetine in GEPRs.

Topics: 5-Hydroxytryptophan; Animals; Anticonvulsants; Disease Models, Animal; Drug Synergism; Epilepsy; Female; Fluoxetine; Injections, Intraperitoneal; Male; Norepinephrine; Rats; Serotonin; Tetrodotoxin; Thalamus

The hippocampus demonstrates a regional pattern of vulnerability to ischemic injury that depends on its characteristic differentiation and intrinsic connections. We now describe a model of ischemic injury using organotypic hippocampal culture, which preserves the anatomic differentiation of the hippocampus in long-term tissue culture.. Ischemic conditions were modeled by metabolic inhibition. Cultures were briefly exposed to potassium cyanide to block oxidative phosphorylation and 2-deoxyglucose to block glycolysis. The fluorescent dye propidium iodide was used to observe membrane damage in living cultures during recovery.. 2-Deoxyglucose/potassium cyanide incubation resulted in dose-dependent, regionally selective neuronal injury in CA1 and the dentate hilus, which began slowly after 2 to 6 hours of recovery. Subsequent histological examination of cultures after 1 to 7 days of recovery demonstrated neuronal pyknosis that was correlated with the early, direct observation of membrane damage with propidium. Both propidium staining and histological degeneration were prevented by the noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 when administered 30 minutes after the end of the exposure to 2-deoxyglucose and potassium cyanide. Tetrodotoxin, which blocks voltage-dependent sodium channels, had protective effects that were greatest during the period of 2-deoxyglucose and potassium cyanide incubation but also produced protection against the mildest conditions of metabolic inhibition when administered after 30 minutes of recovery.. This in vitro model reproduced elements of the time course, regional vulnerability, and pharmacologic sensitivities of in vivo ischemic hippocampal injury. Inhibition of metabolism in organotypic culture provides a rapid, easily controlled injury and reproduces the in vitro pattern of hippocampal regional vulnerability to ischemia. It is the first in vitro model of ischemia to exhibit complete protection by delayed administration of an NMDA receptor antagonist during recovery from a brief insult. The protective effects of tetrodotoxin suggest that an early period of sodium entry into cells during and after ATP depletion may be responsible for the more prolonged period of toxic NMDA receptor activation.

Topics: Animals; Brain Ischemia; Deoxyglucose; Disease Models, Animal; Dizocilpine Maleate; Glycolysis; Hippocampus; Neurons; Organ Culture Techniques; Oxidative Phosphorylation; Potassium Cyanide; Pyramidal Tracts; Rats; Tetrodotoxin; Time Factors

Mouse trisomy 16 is an animal model for Down's syndrome (human trisomy 21). The whole-cell patch-clamp technique was used to compare passive and active electrical properties of trisomy 16 and diploid mouse 16 fetal hippocampal neurons maintained in culture for 2-5 weeks. There was no significant difference in any mean passive property, including resting potential, membrane resistance, capacitance and time constant. However, in trisomic neurons, the action potential had a 20% significantly slower rising phase and a 20% significantly smaller inward sodium current and inward sodium conductance than did control neurons. The outward conductance was not altered. The ratio of maximum inward conductance to maximum outward conductance was 30% less in the trisomy 16 cells. These results indicate that trisomy 16 hippocampal neurons have abnormal active electrical properties, most likely reflecting reduced sodium channel membrane density. Such subtle differences may influence elaboration of the hippocampus during development.

Topics: Action Potentials; Animals; Cells, Cultured; Chromosome Banding; Disease Models, Animal; Down Syndrome; Electric Conductivity; Female; Fetus; Hippocampus; Karyotyping; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Neurologic Mutants; Neurons; Pregnancy; Reference Values; Sodium Channels; Tetraethylammonium; Tetraethylammonium Compounds; Tetrodotoxin; Trisomy

The present in vitro study was conducted to investigate possible alterations in the control of colonic electrolyte transport in an experimental model of colitis. Intrarectal administration of trinitrobenzenesulfonic acid induced a colitis-like inflammation in the rabbit distal colon. Responses to amiloride and residual short-circuit current after this treatment were unchanged, suggesting that the absorptive and secretory mechanisms remained intact. Electrical field stimulation and vasoactive intestinal polypeptide, a candidate secretomotor neurotransmitter, both elicited similar responses in control and colitic tissue. This suggests that communication at the neuroepithelial junction was unimpaired. In untreated tissue, the effects of prostaglandin E2 (PGE2) and of acetylcholine were attenuated by tetrodotoxin, suggesting, therefore, that both play a role in the modulation of secretomotor neurons. In addition, PGE2 had an appreciable direct epithelial effect. Responses to both of these agonists were absent in colitis. The effects of N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate were unchanged in colitis, suggesting that altered PGE2 responsiveness may involve changes in epithelial receptor number, affinity, or in their ability to mediate an increase in adenosine 3',5'-cyclic monophosphate levels. It is concluded that this rabbit model of colitis exhibits 1) defects in the modulation of secretomotor neurons by acetylcholine and PGE2 and 2) an attenuated epithelial response to PGE2.

Topics: Acetylcholine; Amiloride; Animals; Bucladesine; Colitis; Colon; Dinoprostone; Disease Models, Animal; Epithelium; Inflammation; Male; Membrane Potentials; Motor Neurons; Muscle, Smooth; Rabbits; Tetrodotoxin; Trinitrobenzenesulfonic Acid; Vasoactive Intestinal Peptide

[3H]dopamine ([3H]DA) release was measured from rat striatal slices under normoxic and hypoxic conditions. In some experiments hypoxia was combined with glucose withdrawal. Hypoxia increased the evoked release of dopamine without affecting resting release. Hypoglycemia itself increased only the resting release of [3H]DA. In the absence of glucose hypoxia provoked a dramatic rise in both resting and stimulation-evoked release of dopamine. This effect was partly reduced by Ca2+ withdrawal, and was abolished in the presence of tetrodotoxin (1 microM). The NMDA-receptor antagonist MK-801 (3 microM) attenuated the effect of hypoxia and hypoglycemia on [3H]DA release. It was suggested that activation of NMDA receptors is involved in dopamine release during hypoxia and energy deprivation.

Topics: Animals; Brain Ischemia; Cell Hypoxia; Corpus Striatum; Disease Models, Animal; Dizocilpine Maleate; Dopamine; Glucose; Male; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

This study examined the function in vitro of aganglionic colon musculature in mice with hereditary aganglionosis--a strain of animals used as a model of Hirschsprung's disease. Double sucrose gap recordings from the muscle strips of both normal and aganglionic colon showed bursts of spike potentials with muscle contraction. Intracellular recordings of the membrane potentials of the circular muscle cells of normal, aganglionic and oligo-ganglionic colon had no statistical difference. Microelectrode recordings from the circular muscle cells of normal siblings, in the presence of nifedipine, irregular ongoing fluctuations in membrane potential, which were abolished by tetrodotoxin and reduced by d-tubocurarine or apamin. The fluctuations were less effected by atropine. These observations suggest that there is ongoing inhibitory neural activity to the circular smooth muscle of normal colon. These ongoing fluctuations were not recorded from the cells of aganglionic and oligo-ganglionic colon of affected animals. Although transmural stimulation of the intrinsic nerves produced cholinergic excitatory and inhibitory junction potentials in normal colon, no junction potentials were evoked by transmural stimulation in aganglionic colon. It was concluded that the ongoing tonic inhibitory activity may contribute to the compliance of the normal mouse colon and lack of the compliance may affect functional intestinal obstruction of the aganglionic colon in Hirschsprung's disease.

Topics: Animals; Atropine; Colon; Disease Models, Animal; Female; Ganglia; Hirschsprung Disease; In Vitro Techniques; Male; Membrane Potentials; Mice; Muscle Denervation; Muscle, Smooth; Tetrodotoxin; Tubocurarine

The effects of progressive starvation for up to three days on the basal and secretagogue stimulated secretory functions of the rat ileum were investigated in vitro and in vivo. The secretagogues used included agents acting via cyclic AMP (dibutyryl cyclic AMP, theophylline, forskolin, and PGE2) and those acting via Ca++ (acetylcholine, bethanecol, carbachol, 5-hydroxytryptamine, and A23187). Starving rats for 24 h (day 1) had no effect on the basal electrogenic secretion (measured as the short circuit current, Isc muamps/cm2) or on the stimulated maximum electrogenic secretion (measured as the delta Isc where delta Isc = maxIsc-basal Isc). By day 2 of starvation, however, both the basal Isc and the delta Isc induced by all the secretagogues were significantly greater than in the fed and increased even more on day 3. Replacement of all the chloride ions and inhibition by furosemide indicated that the enhanced secretion was due mainly to chloride ions. Cholinergic stimulation was blocked by atropine, indicating the stimulation was via muscarinic receptors while cholinergic dose - delta Isc response curves for fed and starved ilea showed significantly increased maximum electrogenic secretory response in the latter but no evidence of any change in the affinity (ED50) of the receptors mediating the response. The basal secretion and the secretory response to acetylcholine in both fed and starved ilea was unaffected by tetrodotoxin, revealing that the enhanced secretory response could be expressed via the muscarinic receptors on the enterocytes without the enteric neural network. Measurement of ileal fluid movement in vivo showed that in fed and day 1 starved rats the basal, unstimulated 'tone' of the ileum was absorptive. On day 2, however, the basal 'tone' had reversed to one of secretion which increased further on day 3. Stimulation of fluid secretion in vivo by bethanecol, carbachol, or PGE2 induced larger increases in the starved ilea by day 2 which increased even further on day 3. Lumenal chloride and bicarbonate concentrations were greater in the starved ileal fluid than in the fed. The studies in rat ileum confirm and extend those on rat jejunum and indicate that starvation creates a hypersensitive small bowel that responds to secretagogues and cholinergic neurotransmitters with a greatly enhanced secretory response.

Topics: Acetylcholine; Animals; Atropine; Bethanechol Compounds; Bicarbonates; Chlorides; Diarrhea; Disease Models, Animal; Glucose; Ileum; Male; Rats; Starvation; Tetrodotoxin; Theophylline

Many Class I anti-arrhythmic drugs not only block the cardiac sodium channel but also block the calcium and/or potassium channels. The hypothesis tested in this study was that sodium channel blockade without blockade of calcium or potassium channels produced anti-arrhythmic activity in the treatment of malignant ventricular arrhythmias. The arrhythmia model consists of ventricular fibrillation induced by critically timed single extrastimuli at twice diastolic pacing threshold following 15 minutes of ischaemic injury in a rabbit heart perfused in vitro. Preparations were randomly assigned to either tetrodotoxin (a selective sodium channel blocking toxin) or vehicle. Ventricular fibrillation occurred in all vehicle treated preparations in response to single extrastimuli following ischaemic injury. Treatment with tetrodotoxin at concentrations of 0.1 to 1.0 micromolar protected some hearts from fibrillation, while at concentrations above 3 micromolar ventricular fibrillation was not inducible. Tetrodotoxin produced concentration dependent increases in ventricular effective refractory period and conduction time in the infarct zone which were associated with anti-arrhythmic activity. No concentration dependent change in action potential duration was seen with tetrodotoxin. Thus the electrophysiological and anti-arrhythmic activities of tetrodotoxin in this model demonstrate that the property of selective sodium channel blockade is sufficient to produce anti-arrhythmic activity.

Topics: Action Potentials; Animals; Anti-Arrhythmia Agents; Cardiac Pacing, Artificial; Disease Models, Animal; Heart; In Vitro Techniques; Myocardial Infarction; Myocardium; Rabbits; Sodium Channels; Tetrodotoxin; Ventricular Fibrillation

The anomalously active (AA) subendocardial Purkinje's fibers (PF) in the ischemic zone 24 hours after occlusion of the left descending coronary artery (LDCA) in dogs are proposed to be divided into 2 groups according to their reaction to the changes in the ionic composition of perfusion solution. The AAPF frequency in the first group decreased by increase in [Ca2+]o, increased by fall in [Ca2+]o and was proportional to [Na+]o. In the second group AAPF frequency rose under actions increasing [Ca2+]i (increase in [Ca+]o, decrease in [Na+]o), and fell under actions reducing [Ca2+]i (decrease in [Ca2+]o, increase in [Na+]o). After short-term (10 s) high frequency stimulation the AAPF frequency in the group I fibers either remained unchanged or insignificant transitory slowing down was registered. In the group II fibers the AA frequency after high frequency stimulation either remained unchanged or insignificant transitory elevation was observed. The anomalous activity in the group I fibers could be blocked by tetrodotoxin, while verapamil was practically ineffective. In the second group anomalous activity was blocked by both drugs. We conclude thus that one day after the occlusion of LDCA in the ischemic zone PF with anomalous activity caused by anomalous automaticity as well as by trigger mechanism may be present. The pacemaker current inducing anomalous automaticity is possibly caused by Na+ intake through ischemically modified natrium channels.

Topics: Action Potentials; Animals; Calcium; Disease Models, Animal; Dogs; Electrocardiography; Heart Conduction System; Membrane Potentials; Microelectrodes; Myocardial Infarction; Purkinje Fibers; Sodium; Tetrodotoxin; Time Factors; Verapamil

Topics: Animals; Cells, Cultured; Disease Models, Animal; Mice; Mice, Mutant Strains; Microscopy, Electron; Muscles; Muscular Diseases; Tetrodotoxin

Pathogenesis of coronary artery spasm induced by histamine in miniature pigs was studied angiographically in in vivo and in vitro conditions. Endothelial balloon denudation was performed and the animals were fed laboratory chow for 3 months, after which coronary artery spasm was repeatedly provoked by histamine given intracoronarily. Regional hypercontraction of the coronary artery was documented by selective coronary arteriography, and the resulting myocardial ischemia was confirmed by ECG-ST changes. To evaluate coronary artery spasm without the influence of blood constituents and neural control and to quantitate the pharmacophysiological characteristics of histamine-induced coronary constriction in the coronary spasm, the same heart was isolated and perfused with Krebs-Henseleit solution under a constant perfusion pressure of 90 mm Hg. Histamine (10(-5) M) reduced the diameter of the coronary artery of the isolated heart by 29 +/- 4 and 67 +/- 3% (p less than 0.001) in nondenuded and denuded areas, respectively. These figures were similar to data obtained angiographically in vivo after the administration of histamine 10 micrograms/kg. The constriction of the denuded areas in response to histamine was topologically the same in vivo and in vitro. The degree of focal constriction induced by histamine, defined as a percent of stenoses from the mean diameter of the areas of proximal and distal to the spastic site, was similar in in vivo (10 micrograms/kg i.c.) and in vitro (10(-5) M) conditions. KCl (40 mM) reduced both the denuded and nondenuded coronary artery diameter by 67 +/- 3% and 68 +/- 3% (NS), respectively. The dose-response relation of the coronary diameter to histamine was not influenced by pretreatment with the nerve transmitter blockers guanethidine (3 X 10(-6) M), atropine (10(-6) M), and tetrodotoxin (3 X 10(-7) M). Phenylephrine (10(-5) M) did not potentiate constriction of the denuded areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Atropine; Calcium; Coronary Vasospasm; Coronary Vessels; Disease Models, Animal; Endothelium; Guanethidine; Histamine; Pyrilamine; Swine; Tetrodotoxin

This study examined the projections in vitro of excitatory and inhibitory colonic motor nerves in the unaffected siblings of mice with hereditary aganglionic colon--a strain of animals used as a model of Hirschsprung's disease. Microelectrode recordings from the circular muscle layer showed ongoing fluctuations in membrane potential (presumably junction potentials) that were sensitive to tetrodotoxin. Transmural electrical stimulation of the intrinsic nerves produced cholinergic excitatory and inhibitory junction potentials. The latter responses were more prolonged when recorded aboral to the stimulating electrodes. Apamin reduced, but did not block, these inhibitory potentials. Cholinergic excitatory junction potentials were abolished by d-tubocurarine when recording 2 mm from the stimulating electrodes; inhibitory junction potentials were recorded up to 10 mm in the presence of this drug. Thus, the intrinsic excitatory cholinergic motor neurons have relatively short projections to the circular smooth muscle compared with the projections of the inhibitory motor neurons.

Topics: Animals; Apamin; Atropine; Colon; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Hirschsprung Disease; Membrane Potentials; Mice; Mice, Mutant Strains; Microelectrodes; Motor Neurons; Muscle, Smooth; Neuromuscular Junction; Nifedipine; Tetrodotoxin; Tubocurarine

These studies were directed toward better characterization of the abnormalities of motor function in the large intestine of mutant mice with congenital aganglionosis and megacolon. Analysis of pressure-volume relations in the megacolon and aganglionic terminal segment showed increased intestinal wall compliance in the dilated colon and reduced wall compliance in the aganglionic region as compared to normal littermates. Migrating contractile complexes occurred spontaneously in ganglionated regions of the large intestine of both normal and mutant mice, but never propagated into the aganglionic segment of the abnormal bowel. Tetrodotoxin eliminated the migrating complexes and increased random spontaneous contractions in all areas except the aganglionic region. Circular muscle tension was reduced by electrical field stimulation, and poststimulus rebound contractions occurred in all ganglionated regions of the intestine of both normal and mutant mice. No responses to electrical stimulation occurred in the aganglionic segments of the preparations from mutant mice. The poststimulus responses "fatigued" at a faster rate in the megacolonic region of the abnormal bowel than in the equivalent region of the normal bowel, when evoked repetitively over prolonged time periods. There were no differences between the intestines of normal and mutant mice in latency, amplitude, duration, or area under the contractile curves of the poststimulus responses. Intracellular electrical recording from circular muscle fibers revealed slow depolarizing potentials with action potentials at the crests in all regions of the large bowel from both normal and abnormal mice. It also showed excitatory and inhibitory junction potentials in response to electrical stimulation. Inhibitory junction potentials summated during repetitive stimulation and postinhibitory rebound excitation occurred after offset of the stimulation. Stimulus-evoked junction potentials were recorded in all regions of the large intestine except in the aganglionic segment of the mutant mice. We concluded that most of the electrical and mechanical behavior of the aganglionic terminal segment reflected the absence of inhibitory innervation of the musculature in this region.

Topics: Animals; Disease Models, Animal; Electric Stimulation; Epinephrine; Hirschsprung Disease; Intestine, Large; Lidocaine; Membrane Potentials; Mice; Mice, Mutant Strains; Microelectrodes; Muscle Contraction; Tetrodotoxin; Time Factors; Transducers, Pressure

Intestinal secretion was produced in anesthetized cats and rats by exposing isolated intestinal segments to cholera enterotoxin. Giving, for example, tetrodotoxin, a nerve-conduction-blocking agent, or adding lidocaine, a local anesthetic agent, to the solution in the intestinal segments markedly inhibited the rate of choleraic secretion, and in most experiments a net absorption of fluid was observed. The results suggest that intramural nervous mechanisms are involved in the pathogenesis of choleraic secretion.

Topics: Anesthetics, Local; Animals; Cats; Celiac Plexus; Cholera; Cholera Toxin; Disease Models, Animal; Female; Intestinal Secretions; Jejunum; Lidocaine; Male; Poisons; Rats; Rats, Sprague-Dawley; Tetrodotoxin

1 Electrical and mechanical properties of smooth muscle cells or of neuro-effector transmission in the smooth muscle layer of the dog trachea, were studied after treatment with indomethacin, by means of the double sucrose gap, microelectrode or tension recording methods. 2 After several subcutaneous injections of indomethacin (1.0 mg/kg daily), 6 out of 12 dogs were coughing and wheezing. 3 Smooth muscle tissues dissected from the trachea of the coughing dog showed spontaneous electrical and mechanical activities at the frequency of 8-15 per min. These spontaneous electrical and mechanical activities were completely suppressed by treatment with atropine (10(-6) M), isoprenaline (5 X 10(-7) M) or prostaglandin E2 (10(-9) M) but not by tetrodotoxin (1.5 X 10(-6) M). 4 Direct muscle stimulation induced oscillatory potential changes followed by tension development in the trachea of the indomethacin-treated dog. 5 In the indomethacin-treated dog, mean membrane potential of the tracheal smooth muscle cells was -52.4 mV, and in the control trachea, the potential was -59.0 mV. 6 In the trachea from control dogs, the amplitude of test e.j.ps after conditioning e.j.ps was always smaller than the conditioning e.j.p., at any time interval between the two stimuli. In the trachea from indomethacin-treated dogs, facilitation phenomena were observed. 7 In the trachea from the indomethacin-treated dog, prostaglandin E1 (PGE1) or PGE2 (10(-10)-10(-9) M) markedly suppressed the amplitude of the e.j.p. but did not affect the facilitation phenomenon. 8 These results indicate that endogenous prostaglandins play important physiological roles in the feed-back inhibitory mechanisms for acetylcholine release from the nerve terminals during the resting and active states. 9 The results are also discussed in relation to the genesis of aspirin-induced asthma in man.

Topics: Animals; Aspirin; Asthma; Atropine; Disease Models, Animal; Dogs; Female; Indomethacin; Male; Muscle Contraction; Muscle, Smooth; Neuroeffector Junction; Tetrodotoxin; Trachea

Topics: Action Potentials; Aging; Animals; Disease Models, Animal; Electric Conductivity; Goats; Muscle Development; Muscles; Myotonia; Tetrodotoxin

Topics: Action Potentials; Animals; Carbachol; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Electrocardiography; Heart Atria; Heart Block; Heart Conduction System; Heart Rate; Membrane Potentials; Rabbits; Tachycardia, Paroxysmal; Temperature; Tetrodotoxin

Topics: Acetylcholine; Animals; Cricetinae; Disease Models, Animal; Electrophysiology; Membrane Potentials; Motor Endplate; Muscles; Muscular Dystrophies; Regeneration; Tetrodotoxin; Time Factors

Topics: Action Potentials; Animals; Disease Models, Animal; Electric Stimulation; Female; Hindlimb; Male; Membrane Potentials; Mice; Motor Neurons; Muscles; Myofibrils; Nerve Endings; Neuromuscular Diseases; Tetrodotoxin

Intracoronary injection of 14 mcg. of tetrodotoxin into the ischemic isolated rat heart resulted in immediate cessation of mechanical activity. Upon reperfusion with oxygenated, modified Krebs-Henseleit bicarbonate buffer in a modified Langendorff apparatus, all hearts recovered normal rate, rtythm, and contractile vigor after up to 60 minutes of ischemia. In contrast, all hearts not administered tetrodotoxin showed bradycardia, irregular rhythm, and weak contraction upon reperfusion after 30 and 45 minutes of ischemia; after 60 minutes, no mechanical activity was evident. The improved cardiac function following ischemia in the tetrodotoxin-treated hearts was associated with persistence of normal adenosine triphosphate (ATP) levels after up to 30 minutes of ischemia and normal or elevated creatine phosphate (CP) levels after up to 60 minutes of ischemia. On the other hand, ATP and CP levels progressively declined to reach 50 per cent of normal values after 30 minutes in the ischemic hearts without tetrodotoxin. These findings indicate that postarrest ATP and CP levels play an important role in myocardial recovery after ischemic arrest.

Topics: Adenosine Triphosphate; Animals; Bicarbonates; Buffers; Coronary Disease; Disease Models, Animal; Heart; Heart Arrest; Heart Conduction System; Heart Rate; Myocardium; Perfusion; Phosphocreatine; Rats; Tetrodotoxin; Time Factors

Topics: Acetylcholine; Action Potentials; Animals; Chickens; Disease Models, Animal; Electric Stimulation; Female; Male; Membrane Potentials; Muscle Denervation; Muscles; Muscular Dystrophies; Neuromuscular Junction; Synaptic Transmission; Tetrodotoxin

Topics: Action Potentials; Animals; Disease Models, Animal; Electric Stimulation; Female; In Vitro Techniques; Male; Membrane Potentials; Mice; Muscle Denervation; Muscles; Muscular Dystrophies; Nerve Degeneration; Tetrodotoxin