strychnine and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

Assessment of neural activity in the specific brain area is critical for understanding the circuit mechanisms underlying altered brain function and behaviors. A number of immediate early genes (IEGs) that are rapidly transcribed in neuronal cells in response to synaptic activity have been used as markers for neuronal activity. However, protein detection of IEGs requires translation, and the amount of newly synthesized gene product is usually insufficient to detect using western blotting, limiting their utility in western blot analysis of brain tissues for comparison of basal activity between control and genetically modified animals. Here, we show that the phosphorylation status of eukaryotic elongation factor-2 (eEF2) rapidly changes in response to synaptic and neural activities. Intraperitoneal injections of the GABA A receptor (GABA

Topics: Animals; Brain; CA3 Region, Hippocampal; Eukaryotic Initiation Factor-2; Genes, Reporter; Mice; Muscimol; Nerve Tissue Proteins; Phosphorylation; Picrotoxin; Prosencephalon; Protein Processing, Post-Translational; Pyramidal Cells; Quinoxalines; Restraint, Physical; Stress, Physiological; Strychnine

Superficial laminae of the spinal cord possess a considerable number of neurons with spontaneous activity as reported in vivo and in vitro preparations of several species. Such neurons may play a role in the development of the nociceptive system and/or in the spinal coding of somatosensory signals. We have used electrophysiological techniques in a horizontal spinal cord slice preparation from adult mice to investigate how this activity is generated and what are the main patterns of activity that can be found. The results show the existence of neurons that fire regularly and irregularly. Within each of these main types, it was possible to distinguish patterns of spontaneous activity formed by single action potentials and different types of bursts according to intra-burst firing frequency. Activity in neurons with irregular patterns was blocked by a mixture of antagonists of the main neurotransmitter receptors present in the cord. Approximately 82% of neurons with a regular firing pattern were insensitive to synaptic antagonists but their activity was inhibited by specific ion channel blockers. It is suggested that these neurons generate endogenous activity due to the functional expression of hyperpolarisation-activated and persistent sodium currents driving the activity of irregular neurons.

Topics: Action Potentials; Animals; Chromosome Pairing; Membrane Potentials; Mice; Neurons; Picrotoxin; Posterior Horn Cells; Quinoxalines; Riluzole; Sodium; Strychnine; Tetrodotoxin

Sound localization critically depends on detection of differences in arrival time of sounds at the two ears (acoustic delay). The fundamental mechanisms are debated, but all proposals include a process of coincidence detection and a separate source of internal delay that offsets the acoustic delay and determines neural tuning. We used in vivo patch-clamp recordings of binaural neurons in the Mongolian gerbil and pharmacological manipulations to directly compare neuronal input to output and to separate excitation from inhibition. Our results cannot be accounted for by existing models and reveal that coincidence detection is not an instantaneous process, but is instead shaped by the interaction of intrinsic conductances with preceding synaptic activity. This interaction generates an internal delay as an intrinsic part of the process of coincidence detection. The multiplication and time-shifting stages thought to extract synchronous activity in many brain areas can therefore be combined in a single operation.

Topics: Acoustic Stimulation; Animals; Auditory Pathways; Brain; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Gerbillinae; Glycine Agents; In Vitro Techniques; Male; Neurons; Patch-Clamp Techniques; Psychoacoustics; Quinoxalines; Reaction Time; Signal Detection, Psychological; Sound Localization; Strychnine

The human fetus starts to hear and undergoes major developmental changes in the auditory system during the third trimester of pregnancy. Although there are significant data regarding development of the auditory system in rodents, changes in intrinsic properties and synaptic function of auditory neurons in developing primate brain at hearing onset are poorly understood. We performed whole-cell patch-clamp recordings of principal neurons in the medial nucleus of trapezoid body (MNTB) in preterm and term baboon brainstem slices to study the structural and functional maturation of auditory synapses. Each MNTB principal neuron received an excitatory input from a single calyx of Held terminal, and this one-to-one pattern of innervation was already formed in preterm baboons delivered at 67% of normal gestation. There was no difference in frequency or amplitude of spontaneous excitatory postsynaptic synaptic currents between preterm and term MNTB neurons. In contrast, the frequency of spontaneous GABA(A)/glycine receptor-mediated inhibitory postsynaptic synaptic currents, which were prevalent in preterm MNTB neurons, was significantly reduced in term MNTB neurons. Preterm MNTB neurons had a higher input resistance than term neurons and fired in bursts, whereas term MNTB neurons fired a single action potential in response to suprathreshold current injection. The maturation of intrinsic properties and dominance of excitatory inputs in the primate MNTB allow it to take on its mature role as a fast and reliable relay synapse.

Topics: Animals; Auditory Pathways; Bicuculline; Brain Stem; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA-A Receptor Antagonists; Gene Expression Regulation, Developmental; Glycine Agents; Male; Microtubule-Associated Proteins; Neurons; Papio; Premature Birth; Quinoxalines; Strychnine; Synapses; Synaptic Transmission; Vesicular Glutamate Transport Protein 1

Hypothermia can terminate epileptiform discharges in vitro and in vivo epilepsy models. Hypothermia is becoming a standard treatment for brain injury in infants with perinatal hypoxic ischemic encephalopathy, and it is gaining ground as a potential treatment in patients with drug resistant epilepsy. However, the exact mechanism of action of cooling the brain tissue is unclear. We have studied the 4-aminopyridine model of epilepsy in mice using single- and dual-patch clamp and perforated multi-electrode array recordings from the hippocampus and cortex. Cooling consistently terminated 4-aminopyridine induced epileptiform-like discharges in hippocampal neurons and increased input resistance that was not mimicked by transient receptor potential channel antagonists. Dual-patch clamp recordings showed significant synchrony between distant CA1 and CA3 pyramidal neurons, but less so between the pyramidal neurons and interneurons. In CA1 and CA3 neurons, hypothermia blocked rhythmic action potential discharges and disrupted their synchrony; however, in interneurons, hypothermia blocked rhythmic discharges without abolishing action potentials. In parallel, multi-electrode array recordings showed that synchronized discharges were disrupted by hypothermia, whereas multi-unit activity was unaffected. The differential effect of cooling on transmitting or secreting γ-aminobutyric acid interneurons might disrupt normal network synchrony, aborting the epileptiform discharges. Moreover, the persistence of action potential firing in interneurons would have additional antiepileptic effects through tonic γ-aminobutyric acid release.

Topics: Action Potentials; Aminoquinolines; Animals; Animals, Newborn; Bicuculline; Biophysics; Cerebral Cortex; Convulsants; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; GABA-A Receptor Antagonists; Glutamate Decarboxylase; Green Fluorescent Proteins; Hippocampus; Hypothermia, Induced; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neural Pathways; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Phorbol Esters; Quinaldines; Quinoxalines; Sodium Channel Blockers; Strychnine; Temperature; Tetrodotoxin

Retinal prosthetic devices are being developed to bypass degenerated retinal photoreceptors by directly activating retinal neurons with electrical stimulation. However, the retinal circuitry that is activated by epiretinal stimulation is not well characterized. Whole-cell patch clamp recordings were obtained from ganglion cells in normal and rd mice using flat-mount and retinal slice preparations. A stimulating electrode was positioned along the ganglion cell side of the preparation at different distances from the stimulated tissue. Pulses of cathodic current evoked action potentials in ganglion cells and less frequently evoked sustained inward currents that appeared synaptic in origin. Sustained currents reversed around E(Cl) and were inhibited by blockade of α-amino-3-hydroxyl-5-methyl-4-isoxazole-proprionate (AMPA)-type glutamate receptors with 2,3-dihydroxy-6-nitro-sulfamoyl-benzo(f)-quinoxaline-2,3-dione (NBQX), γ aminobutyric acid a/c (GABA(a/c)) receptors with picrotoxinin, or glycine receptors with strychnine. This suggests that epiretinal stimulation activates glutamate release from bipolar cell terminals, which in turn evokes release of GABA and glycine from amacrine cells. Synaptic current thresholds were lower in ON ganglion cells than OFF cells, but the modest difference did not attain statistical significance. Synaptic currents were rarely observed in rd mice lacking photoreceptors compared to normal retina. In addition, confocal calcium imaging experiments in normal mice retina slices revealed that epiretinal stimulation evoked calcium increases in the outer plexiform layer. These results imply a contribution from photoreceptor inputs to the synaptic currents observed in ganglion cells. The paucity of synaptic responses in rd mice retina slices suggests that it is better to target retinal ganglion cells directly rather than to attempt to engage the inner retinal circuitry.

Topics: Animals; Biophysics; Calcium; Disease Models, Animal; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA-A Receptor Antagonists; Glycine Agents; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Confocal; Patch-Clamp Techniques; Picrotoxin; Quinoxalines; Retina; Retinal Degeneration; Retinal Ganglion Cells; Sesterterpenes; Strychnine; Visual Pathways

To further understand the excitatory effects of motoneurons on spinal network function, we investigated the entrainment of disinhibited rhythms by ventral root (VR) stimulation in the neonatal mouse spinal cord. A brief train of stimuli applied to a VR triggered bursting reliably in 31/32 experiments. The same roots that entrained disinhibited bursting could also produce locomotor-like activity with a similar probability when the network was not disinhibited. The ability of VR stimulation to entrain the rhythm persisted in nicotinic and muscarinic cholinergic antagonists but was blocked by the AMPAR antagonist NBQX. Bath application of the type I mGluR1 receptor antagonist CPCCOEt reduced the ability of both dorsal root and VR stimulation to entrain the disinhibited rhythm and abolished the ability of either type of stimulation to evoke locomotor-like activity. Calcium imaging through the lateral aspect of the cord revealed that VR stimulation and spontaneously occurring bursts were accompanied by a wave of activity that originated ventrally and propagated dorsally. Imaging the cut transverse face of L(5) revealed that the earliest VR-evoked optical activity began ventrolaterally. The optical activity accompanying spontaneous bursts could originate ventrolaterally, ventromedially, or throughout the mediolateral extent of the ventral horn or very occasionally dorsally. Collectively, our data indicate that VR stimulation can entrain disinhibited spinal network activity and trigger locomotor-like activity through a mechanism dependent on activation of both ionotropic and metabotropic glutamate receptors. The effects of entrainment appear to be mediated by a ventrolaterally located network that is also active during spontaneously occurring bursts.

Topics: Action Potentials; Animals; Animals, Newborn; Benzothiadiazines; Bicuculline; Biophysics; Carbodiimides; Chromones; Electric Stimulation; Electroporation; Excitatory Amino Acid Antagonists; Functional Laterality; GABA Antagonists; Glycine Agents; In Vitro Techniques; Mice; Motor Neurons; Neural Inhibition; Neural Pathways; Organic Chemicals; Quinoxalines; Reaction Time; Spectrum Analysis; Spinal Cord; Spinal Nerve Roots; Strychnine; Time Factors

Adaptation or gain control allows sensory neurons to encode diverse stimuli using a limited range of output signals. Rod vision exemplifies a general challenge facing adaptational mechanisms-balancing the benefits of averaging to create a reliable signal for adaptation with the need to adapt rapidly and locally. The synapse between rod bipolar and AII amacrine cells dominates adaptation at low light levels. We find that adaptation occurs independently at each synapse and completes in <500 ms. This limited spatial and temporal integration suggests that the absorption of a single photon modulates gain. Indeed, responses to pairs of brief dim flashes showed directly that synaptic gain was depressed for 100-200 ms following transmission of a single-photon response. Presynaptic mechanisms mediated this synaptic depression. Thus, the division of light into discrete photons controls adaptation at this synapse, and gain varies with the irreducible statistical fluctuations in photon arrival.

Topics: Amacrine Cells; Animals; Dark Adaptation; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; GABA Antagonists; Light; Mice; Mice, Inbred C57BL; Neural Inhibition; Patch-Clamp Techniques; Photons; Pyridazines; Quinoxalines; Retina; Retinal Bipolar Cells; Retinal Rod Photoreceptor Cells; Strychnine; Synapses; Synaptic Transmission

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-Bötzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.

Topics: Animals; Cats; Excitatory Amino Acid Antagonists; Exhalation; GABA Antagonists; Glycine Agents; Inhalation; Medulla Oblongata; Motor Neurons; Nitrogen Mustard Compounds; Phrenic Nerve; Pyridazines; Quinoxalines; Receptors, Amino Acid; Receptors, GABA-A; Receptors, Glutamate; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Respiratory Center; Respiratory Mechanics; Strychnine; Time Factors

Electroretinograms (ERGs) were recorded from the giant danio (Danio aequipinnatus) to study glutamatergic input mechanisms onto bipolar cells. Glutamate analogs were applied to determine which receptor types mediate synaptic transmission from rods and cones to on and off bipolar cells. Picrotoxin, strychnine, and tetrodotoxin were used to isolate the effects of the glutamate analogs to the photoreceptor-bipolar cell synapse. Under photopic conditions, the group III metabotropic glutamate receptor (mGluR) antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) only slightly reduced the b-wave, whereas the excitatory amino acid transporter (EAAT) blocker dl-threo-beta-benzyl-oxyaspartate (TBOA) removed most of it. Complete elimination of the b-wave required both antagonists. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) blocked the d-wave. Under scotopic conditions, rod and cone inputs onto on bipolar cells were studied by comparing the sensitivities of the b-wave to photopically matched green and red stimuli. The b-wave was >1 log unit more sensitive to the green than to the red stimulus under control conditions. In CPPG or l-AP4 (l-(+)-2-amino-4-phosphonobutyric acid, a group III mGluR agonist), the sensitivity of the b-wave to the green stimulus was dramatically reduced and the b-waves elicited by the 2 stimuli became nearly matched. The d-wave elicited by dim green stimuli, which presumably could be detected only by the rods, was eliminated by NBQX.. 1) cone signals onto on bipolar cells involve mainly EAATs but also mGluRs (presumably mGluR6) to a lesser extent; 2) rods signal onto on bipolars by mainly mGluR6; 3) off bipolar cells receive signals from both photoreceptor types by AMPA/kainate receptors.

Topics: Aminobutyrates; Anesthetics, Local; Animals; Aspartic Acid; Cyclopentanes; Dose-Response Relationship, Radiation; Drug Interactions; Electroretinography; Excitatory Amino Acid Agonists; GABA Antagonists; Glycine; Glycine Agents; Neurons; Photic Stimulation; Picrotoxin; Quinoxalines; Receptors, Metabotropic Glutamate; Retina; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Strychnine; Synaptic Transmission; Tetrodotoxin; Visual Pathways; Zebrafish

Rod signals traverse several synapses en route to cone bipolar cells. In one pathway, rods communicate directly with cones via gap junctions. In a second pathway, signals flow rods-rod bipolars-AII amacrines-cone bipolars. The relative contribution of each pathway to retinal function is not well understood. Here we have examined this question from the perspective of the AII amacrine. AIIs form bidirectional electrical synapses with on cone bipolars. Consequently, as on cone bipolars are activated by outer plexiform inputs, they too should contribute to the AII response. Rod bipolar inputs to AIIs were blocked by AMPA receptor antagonists, revealing a smaller, non-AMPA component of the light response. This small residual response did not reverse between -70 and +70 mV and was blocked by carbenoxolone, suggesting that the current arose in on cone bipolars and was transmitted to AIIs via gap junctions. The residual component was evident for stimuli 2 log units below cone threshold and was prolonged for bright stimuli, demonstrating that it was rod driven. Because the rod bipolar-AII pathway was blocked, the rod-driven residual current likely was generated via the rod-cone pathway activation of on cone bipolars. Thus for a large range of intensities, rod signals reach the inner retina by both rod bipolar-AII and rod-cone coupling pathways.

Topics: Amacrine Cells; Animals; Benzodiazepines; Benzothiadiazines; Diagnostic Imaging; Dose-Response Relationship, Radiation; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Fluorescein; GABA Antagonists; Glycine Agents; In Vitro Techniques; Light; Male; Membrane Potentials; Models, Biological; Neurons; Patch-Clamp Techniques; Picrotoxin; Quinoxalines; Rabbits; Retina; Retinal Rod Photoreceptor Cells; Strychnine; Synapses; Synaptic Transmission; Visual Pathways

Homeostatic maintenance of widespread functions is critically dependent on the activity of the sympathetic nervous system. This activity is generated by the CNS acting on the sole output cells in the spinal cord, sympathetic preganglionic neurons (SPNs). SPNs are subject to control from both supraspinal and spinal inputs that exert effects through activation of direct or indirect pathways. A high proportion of indirect control is attributable to activation of spinal interneurons in a number of locations. However, little is known about the different groups of interneurons with respect to their neurochemistry or function. In this study, we report on a novel group of GABAergic interneurons located in the spinal central autonomic area (CAA) that directly inhibit SPN activity. In situ hybridization studies demonstrated a group of neurons that contained mRNA for glutamic acid decarboxylase (GAD)65 and GAD67 within the CAA. Combining in situ hybridization with trans-synaptic labeling from the adrenal gland using pseudorabies virus identified presympathetic GABAergic neurons in the CAA. Electrical stimulation of the CAA elicited monosynaptic IPSPs in SPNs located laterally in the intermediolateral cell column. IPSPs were GABAergic, because they reversed at the chloride reversal potential and were blocked by bicuculline. Chemical activation of neurons in the CAA hyperpolarized SPNs, an effect that was also bicuculline sensitive. We conclude that the CAA contains GABAergic interneurons that impinge directly onto SPNs to inhibit their activity and suggest that these newly identified interneurons may play an essential role in the regulation of sympathetic activity and thus homeostasis.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Autonomic Fibers, Preganglionic; Axonal Transport; Bicuculline; Chlorides; Electric Stimulation; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Glutamic Acid; Herpesvirus 1, Suid; Homeostasis; In Situ Hybridization; Interneurons; Isoenzymes; Kynurenic Acid; Male; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Wistar; RNA, Messenger; Spinal Cord; Strychnine

Previous studies have demonstrated that microinjection of the putative group III metabotropic glutamate receptor (mGluR) agonist, l(+)-2-amino-4-phosphonobutyric acid (L-AP4), into the nucleus tractus solitarius (NTS) produces depressor and sympathoinhibitory responses. These responses are significantly attenuated by a group III mGluR antagonist and may involve ionotropic glutamatergic transmission. Alternatively, a previous report in vitro suggests that preparations of L-AP4 may nonspecifically activate NMDA channels due to glycine contamination (Contractor A, Gereau RW, Green T, and Heinemann SF. Proc Natl Acad Sci USA 95: 8969-8974, 1998). Therefore, the present study tested whether responses to L-AP4 specifically require the N-methyl-D-aspartate (NMDA) receptor and whether they are due to actions at the glycine site on the NMDA channel. To test these possibilities in vivo, we performed unilateral microinjections of L-AP4, glycine, and selective antagonists into the NTS of urethane-anesthetized rats. L-AP4 (10 mM, 30 nl) produced sympathoinhibitory responses that were abolished by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP-5, 10 mM) but were unaffected by the non-NMDA antagonist 6-nitro-7-sulfamobenzoquinoxaline-2,3-dione (NBQX, 2 mM). Microinjection of glycine (0.02-20 mM) failed to mimic sympathoinhibitory responses to L-AP4, even in the presence of the inhibitory glycine antagonist, strychnine (3 mM). Strychnine blocked pressor and sympathoexcitatory actions of glycine (20 mM) but failed to reveal a sympathoinhibitory component due to presumed activation of NMDA receptors. The results of these experiments suggest that responses to L-AP4 require NMDA receptors and are independent of non-NMDA receptors. Furthermore, although it is possible that glycine contamination or other nonspecific actions are responsible for the sympathoinhibitory actions of L-AP4, our data and data in the literature argue against this possibility. Thus we conclude that responses to L-AP4 in the NTS are mediated by an interaction between group III mGluRs and NMDA receptors. Finally, we also caution that nonselective actions of L-AP4 should be considered in future studies.

Topics: 2-Amino-5-phosphonovalerate; Aminobutyrates; Animals; Cardiovascular System; Excitatory Amino Acid Agonists; Glycine; Glycine Agents; Male; Microinjections; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate; Solitary Nucleus; Strychnine

In neonatal spinal cord, we previously reported that exogenous angiotensin II (ANG II) acts at postsynaptic AT1 receptors to depolarize neonatal rat spinal ventral horn neurons in vitro. This study evaluated an associated increase in synaptic activity. Patch clamp recordings revealed that 38/81 thoracolumbar (T7-L5) motoneurons responded to bath applied ANG II (0.3-1 microM; 30 s) with a prolonged (5-10 min) and reversible increase in spontaneous postsynaptic activity, selectively blockable with Losartan (n = 5) but not PD123319 (n = 5). ANG-II-induced events included both spontaneous inhibitory (IPSCs; n = 6) and excitatory postsynaptic currents (EPSCs; n = 5). While most ANG induced events were tetrodotoxin-sensitive, ANG induced a significant tetrodotoxin-resistant increase in frequency but not amplitude of miniature IPSCs (n = 7/13 cells) and EPSCs (n = 2/7 cells). In 35/77 unidentified neurons, ANG II also induced a tetrodotoxin-sensitive and prolonged increase in their spontaneous synaptic activity that featured both IPSCs (n = 5) and EPSCs (n = 4) when tested in the presence of selective amino acid receptor antagonists. When tested in the presence of tetrodotoxin, ANG II was noted to induce a significant increase in the frequency but not the amplitude of mIPSCs (n = 9) and mEPSCs (n = 8). ANG also increased spontaneous motor activity from isolated mouse lumbar ventral rootlets. Collectively, these observations support the existence of a wide pre- and postsynaptic distribution of ANG II AT1 receptors in neonatal ventral spinal cord that are capable of influencing both inhibitory and excitatory neurotransmission.

Topics: 2-Amino-5-phosphonovalerate; Angiotensin II; Animals; Animals, Newborn; Bicuculline; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; In Vitro Techniques; Laminectomy; Male; Motor Neurons; Neural Inhibition; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Spinal Cord; Strychnine; Synaptic Transmission; Tetrodotoxin

Cultures of neurons can be grown on microelectrode arrays (MEAs), so that their spike and burst activity can be monitored. These activity patterns are quite sensitive to changes in the environment, such as chemical exposure, and hence the cultures can be used as biosensors. One key issue in analyzing the data from neuronal networks is how to quantify the level of synchronization among different units, which represent different neurons in the network. In this paper, we propose a synchronization metric, based on the statistical distribution of unit-to-unit correlation coefficients. We show that this synchronization metric changes significantly when the networks are exposed to bicuculline, strychnine, or 2,3-dioxo-6-nitro-l,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX). For that reason, this metric can be used to characterize pharmacologically induced changes in a network, either for research or for biosensor applications.

Topics: Action Potentials; Algorithms; Animals; Bicuculline; Biosensing Techniques; Cells, Cultured; Dose-Response Relationship, Drug; Mice; Mice, Inbred ICR; Microelectrodes; Models, Neurological; Models, Statistical; Nerve Net; Neurons; Quinoxalines; Reproducibility of Results; Sensitivity and Specificity; Spinal Cord; Statistics as Topic; Strychnine

Nitric oxide (NO) synthesis by prepositus hypoglossi (PH) neurons is necessary for the normal performance of horizontal eye movements. We have previously shown that unilateral injections of NO synthase (NOS) inhibitors into the PH nucleus of alert cats produce velocity imbalance without alteration of the eye position control, both during spontaneous eye movements and the vestibulo-ocular reflex (VOR). This NO effect is exerted on the dorsal PH neuropil, whose fibres increase their cGMP content when stimulated by NO. In an attempt to determine whether NO acts by modulation of a specific neurotransmission system, we have now compared the oculomotor effects of NOS inhibition with those produced by local blockade of glutamatergic, GABAergic or glycinergic receptors in the PH nucleus of alert cats. Both glutamatergic antagonists used, 2-amino-5-phosphonovaleric acid (APV) and 2,3-dihydro-6-nitro-7-sulphamoyl-benzo quinoxaline (NBQX), induced a nystagmus contralateral to that observed upon NOS inhibition, and caused exponential eye position drift. In contrast, bicuculline and strychnine induced eye velocity alterations similar to those produced by NOS inhibitors, suggesting that NO oculomotor effects were due to facilitation of some inhibitory input to the PH nucleus. To investigate the anatomical location of the putative NO target neurons, the retrograde tracer Fast Blue was injected in one PH nucleus, and the brainstem sections containing Fast Blue-positive neurons were stained with double immunohistochemistry for NO-sensitive cGMP and glutamic acid decarboxylase. GABAergic neurons projecting to the PH nucleus and containing NO-sensitive cGMP were found almost exclusively in the ipsilateral medial vestibular nucleus and marginal zone. The results suggest that the nitrergic PH neurons control their own firing rate by a NO-mediated facilitation of GABAergic afferents from the ipsilateral medial vestibular nucleus. This self-control mechanism could play an important role in the maintenance of the vestibular balance necessary to generate a stable and adequate eye position signal.

Topics: Abducens Nerve; Animals; Bicuculline; Brain Stem; Cats; Excitatory Amino Acid Antagonists; Eye Movements; Female; GABA Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Glycine Agents; Nitric Oxide; Nitric Oxide Synthase; Oculomotor Nerve; Quinoxalines; Reflex, Vestibulo-Ocular; Strychnine; Synaptic Transmission

Prominent arginine-vasopressin (AVP) binding and AVP V(1) type receptors are expressed early in the developing rat spinal cord. We sought to characterize their influence on neural excitability by using patch-clamp techniques to record AVP-induced responses from a population of motoneurons and interneurons in neonatal (5-18 days) rat spinal cord slices. Data were obtained from 58 thoracolumbar (T(7)-L(5)) motoneurons and 166 local interneurons. A majority (>90%) of neurons responded to bath applied AVP (10 nM to 3 microM) and (Phe(2), Orn(8))-vasotocin, a V(1) receptor agonist, but not V(2) or oxytocin receptor agonists. In voltage-clamp, postsynaptic responses in motoneurons were characterized by slowly rising, prolonged (7-10 min) and tetrodotoxin-resistant inward currents associated with a 25% reduction in a membrane potassium conductance that reversed near -100 mV. In interneurons, net AVP-induced inward currents displayed three patterns: decreasing membrane conductance with reversal near -100 mV, i.e., similar to that in motoneurons (24 cells); increasing conductance with reversal near -40 mV (21 cells); small reduction in conductance with no reversal within the current range tested (41 cells). A presynaptic component recorded in most neurons was evident as an increase in the frequency but not amplitude (in motoneurons) of inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs), in large part due to AVP-induced firing in inhibitory (mainly glycinergic) and excitatory (glutamatergic) neurons synapsing on the recorded cells. An increase in frequency but not amplitude of miniature IPSCs and EPSCs also indicated an AVP enhancement of neurotransmitter release from axon terminals of inhibitory and excitatory interneurons. These observations provide support for a broad presynaptic and postsynaptic distribution of AVP V(1) type receptors and indicate that their activation can enhance the excitability of a majority of neurons in neonatal ventral spinal cord.

Topics: 2-Amino-5-phosphonovalerate; Animals; Animals, Newborn; Anterior Horn Cells; Arginine Vasopressin; Bicuculline; Deamino Arginine Vasopressin; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; GABA Antagonists; Glycine Agents; Hemosiderin; Hormone Antagonists; Interneurons; Male; Oxytocin; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Vasopressin; Strychnine; Vasotocin

Cultured spinal cord networks grown on microelectrode arrays display complex patterns of spontaneous burst and spike activity. During disinhibition with bicuculline and strychnine, synchronized burst patterns routinely emerge. However, the variability of both intra- and interculture burst periods and durations are typically large under these conditions. As a further step in simplification of synaptic interactions, we blocked excitatory AMPA synapses with 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzoquinoxaline-7-sulphonamide (NBQX), resulting in network activity mediated through the N-methyl-D-aspartate (NMDA) receptor (NMDA(ONLY)). This activity was APV sensitive. The oscillation under NMDA(ONLY) conditions at 37 degrees C was characterized by a period of 2.9 +/- 0.3 s (16 separate cultures). More than 98% of all neurons recorded participated in this highly rhythmic activity. The temporal coefficients of variation, reflecting the rhythmic nature of the oscillation, were 3.7, 4.7, and 4.9% for burst rate, burst duration, and interburst interval, respectively [mean coefficients of variation (CVs) for 16 cultures]. The oscillation persisted for at least 12 h without change (maximum observation time). Once established, it was not perturbed by agents that block mGlu receptors, GABA(B) receptors, cholinergic receptors, purinergic receptors, tachykinin receptors, serotonin (5-HT) receptors, dopamine receptors, electrical synapses, burst afterhyperpolarization, NMDA receptor desensitization, or the hyperpolarization-activated current. However, the oscillation was destroyed by bath application of NMDA (20-50 microM). These results suggest a presynaptic mechanism underlying this periodic rhythm that is solely dependent on the NMDA synapse. When the AMPA/kainate synapse was the sole driving force (n = 6), the resulting burst patterns showed much higher variability and did not develop the highly periodic, synchronized nature of the NMDA(ONLY) activity. Network size or age did not appear to influence the reliability of expression of the NMDA(ONLY) activity pattern. For this reason, we suggest that the NMDA(ONLY) condition unmasks a fundamental rhythmogenic mechanism of possible functional importance during periods of NMDA receptor-dominated activity, such as embryonic and early postnatal development.

Topics: Animals; Bicuculline; Cells, Cultured; Electrophysiology; GABA Antagonists; Glycine Agents; Membrane Potentials; Mice; Mice, Inbred ICR; Microelectrodes; Nerve Net; Neurotransmitter Agents; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Strychnine

Synchronized spontaneous rhythmic activity is a feature common to many parts of the developing nervous system. In the early visual system, before vision, developing circuits in the retina generate synchronized patterns of bursting activity that contain information useful for patterning connections between retinal ganglion cells and their central targets. However, how developing retinal circuits generate and regulate these spontaneous activity patterns is still incompletely understood. Here we show that in developing retinal circuits, the nature of excitatory neurotransmission driving correlated bursting activity in ganglion cells is not fixed but undergoes a developmental shift from cholinergic to glutamatergic transmission. In addition, we show that this shift occurs as presynaptic glutamatergic bipolar cells form functional connections onto the ganglion cells, implicating the role of bipolar cells in providing endogenous drive to bursting activity later in development. This transition coincides with the period when subsets of ganglion cells (On and Off cells) develop distinct activity patterns that are thought to underlie the refinement of their connectivity with their central targets. Here, our results suggest that the differences in activity patterns of On and Off ganglion cells may be conferred by differential synaptic drive from On and Off bipolar cells, respectively. Taken together, our results suggest that the regulation of patterned spontaneous activity by neurotransmitters undergoes systematic change as new cellular elements are added to developing circuits and also that these new elements can help specify distinct activity patterns appropriate for shaping connectivity patterns at later ages.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Bicuculline; Cholinergic Fibers; Excitatory Amino Acid Antagonists; Ferrets; GABA Antagonists; gamma-Aminobutyric Acid; Glutamic Acid; Glycine; Glycine Agents; Interneurons; Periodicity; Quinoxalines; Retina; Retinal Ganglion Cells; Strychnine; Synaptic Transmission; Vision, Ocular; Visual Pathways

The relative contribution of phasic and tonic excitatory synaptic drives to the augmenting discharge patterns of inspiratory (I) neurons within the ventral respiratory group (VRG) was studied in anesthetized, ventilated, paralyzed, and vagotomized dogs. Multibarrel micropipettes were used to record simultaneously single-unit neuronal activity and pressure microejected antagonists of GABAergic, glycinergic, N-methyl-D-aspartate (NMDA) and non-NMDA glutamatergic, and cholinergic receptors. The discharge patterns were quantified via cycle-trigger histograms. The findings suggest that two-thirds of the excitatory drive to caudal VRG I neurons is tonic and mediated by NMDA receptors and the other third is ramp-like phasic and mediated by non-NMDA receptors. Cholinergic receptors do not appear to be involved. The silent expiratory phase is produced by phasic inhibition of the tonic activity, and approximately 80% of this inhibition is mediated by gamma-aminobutyric acid receptors (GABA(A)) and approximately 20% by glycine receptors. Phasic I inhibition by the I decrementing neurons does not appear to contribute to the predominantly step-ramp patterns of these I neurons. However, this decrementing inhibition may be very prominent in controlling the rate of augmentation in late-onset I neurons and those with ramp patterns lacking the step component.

Topics: 2-Amino-5-phosphonovalerate; Acetylcholine; Animals; Dogs; Excitatory Amino Acid Antagonists; Female; GABA Antagonists; Glycine Agents; Male; Medulla Oblongata; Neurons; Picrotoxin; Quinoxalines; Respiratory Physiological Phenomena; Strychnine; Synapses

Glial-neuronal communication was studied by monitoring the effect of intercellular glial Ca2+ waves on the electrical activity of neighboring neurons in the eyecup preparation of the rat. Calcium waves in astrocytes and Müller cells were initiated with a mechanical stimulus applied to the retinal surface. Changes in the light-evoked spike activity of neurons within the ganglion cell layer occurred when, and only when, these Ca2+ waves reached the neurons. Inhibition of activity was observed in 25 of 53 neurons (mean decrease in spike frequency, 28 +/- 2%). Excitation occurred in another five neurons (mean increase, 27 +/- 5%). Larger amplitude Ca2+ waves were associated with greater modulation of neuronal activity. Thapsigargin, which reduced the amplitude of the glial Ca2+ increases, also reduced the magnitude of neuronal modulation. Bicuculline and strychnine, inhibitory neurotransmitter antagonists, as well as 6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) and D(-)-2-amino-7-phosphonoheptanoic acid (D-AP7), glutamate antagonists, reduced the inhibition of neuronal activity associated with glial Ca2+ waves, suggesting that inhibition is mediated by inhibitory interneurons stimulated by glutamate release from glial cells. The results suggest that glial cells are capable of modulating the electrical activity of neurons within the retina and thus, may directly participate in information processing in the CNS.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Astrocytes; Bicuculline; Calcium; Cell Communication; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; GABA Antagonists; Glycine Agents; Male; Neurotransmitter Agents; Photic Stimulation; Quinoxalines; Rats; Rats, Inbred Strains; Retinal Ganglion Cells; Strychnine; Thapsigargin

1. Recent studies have revealed that the excitatory synaptic input to spinal motoneurones during fictive swimming in Xenopus tadpoles has three main components: glutamatergic (Glu) from premotor excitatory interneurones, nicotinic cholinergic (nACh) from more rostral motoneurones, and electrotonic coupling from neighbouring motoneurones. During swimming, these components sum to produce two kinds of excitation: phasic excitation (EPSPs) underlying spikes, and tonic depolarization. 2. We have investigated the longitudinal distribution of these excitatory synaptic inputs to presumed motoneurones at different positions along the spinal cord using intracellular recording techniques. Different antagonists (10 microM dihydro-beta-erythroidine (DHbetaE) for nicotinic ACh receptors (nAChRs), 2 mM kynurenate (Kyn) for glutamate receptors (GluRs), and 100 microM Cd2+ for all chemical synapses) were microperfused very locally to unmask the relative contributions of these components to the total excitatory drive, and their distribution along the spinal cord during swimming. 3. If the potentials remaining when all chemical components were blocked by Cd2+ were subtracted from potentials recorded after blocking nAChRs and GluRs with DHbetaE plus Kyn, a small unidentified component was observed. This component was blocked by the specific AMPA antagonist 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX, 5 microM), so is glutamate mediated. 4. We used the potential measurements to calculate the relative synaptic conductances of the different synaptic inputs, and conclude that: (a) there is a rostral-caudal gradient in input during EPSPs and tonic depolarization; (b) the glutamatergic component accounts for most of the excitation, and decreases caudally; (c) cholinergic and electrotonic components are relatively constant in different positions along the spinal cord; and (d) these two components provide an increasing proportion of the input in more caudal neurones. 5. We propose that the glutamate components of excitation are fundamental to rhythm generation in the brainstem and rostral cord, while the electrotonic and cholinergic components ensure that the central pattern generator activates motoneurones effectively in all parts of the spinal cord.

Topics: Animals; Cadmium; Convulsants; Dihydro-beta-Erythroidine; Electric Conductivity; Electrophysiology; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Kynurenic Acid; Larva; Motor Neurons; Quinoxalines; Receptors, Nicotinic; Spinal Cord; Strychnine; Swimming; Synapses; Xenopus

When the quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline), a KA/AMPA antagonist, is bath applied to the tiger salamander retina, a paradoxical action is evident in the light-evoked synaptic responses of ganglion cells: NBQX enhances excitatory synaptic currents at light onset observed under whole-cell voltage-clamp conditions in a perfused retinal slice preparation. This observation was surprising because synaptic inputs into ganglion cells that are mediated by KA/AMPA receptors are entirely blocked by NBQX. Thus, the NBQX-enhanced current is entirely mediated by NMDA receptors. The purpose of this study was to determine the mechanism(s) by which blocking KA/AMPA receptors appears to enhance NMDA currents. Using hyperosmotic sucrose stimulation to activate neurotransmitter release from the inner retina, we observed that NBQX augmented the sucrose-evoked response, suggesting that at least a component of this enhancement may reside in the inner retina. NBQX does not enhance NMDA currents activated by bath applied NMDA, demonstrating that the NBQX-induced enhancement does not result from modulation of NMDA receptors. Voltage-clamp studies, carried out at the appropriate holding potential, indicate that NBQX enhances glutamatergic transmission and reduces inhibitory inputs onto ganglion cells. In the presence of strychnine and picrotoxin, the NBQX-induced enhancement of NMDA currents is eliminated, suggesting that NBQX facilitates the expression of NMDA currents by a selective and partial reduction of inhibitory mechanisms. Additional studies suggest that part of the NMDA enhancement by NBQX is evident at the postsynaptic level, but a presynaptic component probably also participates, perhaps at the level of bipolar cell terminals. One way to account for this observation is to assume that a subpopulation of inhibitory amacrine cells requires KA/AMPA receptors exclusively for their synaptic activation: previous studies of sustained amacrine cells support this interpretation. Thus the NBQX-induced enhancement phenomenon may reflect a network-selective distribution of NMDA and KA/AMPA receptors among third-order neurons.

Topics: Animals; Chlorides; Electric Conductivity; Excitatory Amino Acid Antagonists; Glutamates; In Vitro Techniques; Light; N-Methylaspartate; Neurotransmitter Agents; Picrotoxin; Quinoxalines; Receptors, Amino Acid; Receptors, N-Methyl-D-Aspartate; Retinal Ganglion Cells; Strychnine; Sucrose; Synaptic Transmission; Urodela

Touch-evoked allodynia, an important symptom of clinical neural injury pain, can be modelled acutely and reversibly in the urethane-anesthetized rat using intrathecal (i.t.) strychnine (STR). Allodynia, after i.t. STR (40 micrograms), is manifest as a significant enhancement of cardiovascular and motor responses evoked by normally innocuous brushing of the hair (hair deflection), as compared to responses evoked by either hair deflection after i.t. saline (SAL), or to i.t. STR (40 micrograms) with no tactile stimulus. The present study investigated: (1) the pharmacology of afferent neural inputs involved in STR-dependent allodynia using neonatal capsaicin and the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX); and (2) the effect of i.t. STR on responses evoked by peripheral noxious stimulation. Neonatal capsaicin (25 mg/kg, s.c., post-natal day (PND) 1, and 50 mg/kg, s.c., PND 2, 3, 4, 11, 25, 55 and 85) significantly attenuated the responses evoked by noxious mechanical, thermal or chemical stimuli, but had no effect on STR-dependent allodynia. All hair deflection-evoked, STR-dependent responses were dose-dependently inhibited by i.t. NBQX. The ED50 values and 95% confidence intervals were 10.4 micrograms (5.5-19.6) for the motor withdrawal response, 14.4 micrograms (8.6-24.0) for changes in MAP and 12.2 micrograms (6.8-21.8) for changes in HR. Cortical EEG synchrony was unchanged by i.t. NBQX confirming its spinal locus of action. Intrathecal STR neither reduced nor enhanced the responses elicited by noxious stimuli in capsaicin- or vehicle-pretreated rats. These results indicate that STR-dependent allodynia is initiated by primary afferents not normally involved in nociception (possibly A beta-fibers), and that STR-sensitive modulation in the spinal cord is selective for non-noxious sensory input. The sensitivity of STR-dependent allodynia to non-NMDA receptor antagonists, and the failure of i.t. STR to produce hyperalgesia to mechanical, thermal or chemical noxious stimuli, confirm the independence of nociceptive pathways and STR-sensitive afferent inputs in this model.

Topics: Anesthesia, General; Animals; Animals, Newborn; Blood Pressure; Capsaicin; Central Nervous System Stimulants; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Nerve Fibers; Pain; Physical Stimulation; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Strychnine

A method for evoking neurotransmitter release without light stimulation has been developed and applied to a retinal slice preparation of the tiger salamander (Ambystoma Tigrum). This method utilizes a micropipette containing hyperosmotic levels of sucrose in Ringer, positioned within the inner plexiform layer (IPL) under visual control. Intermittent pressure (between 0.1 and 2 bars) applied to the pipette evoked release of neurotransmitters which were evaluated with whole-cell recording (WCR) technique applied to cells in the ganglion cell layer. Pharmacological studies were used to characterize the properties of the hyperosmotic sucrose-evoked response (HSER) and in some cases, we compared the HSER with synaptic currents evoked by light stimulation. The HSER typically consisted of both inhibitory and excitatory components with a reversal potential in between that for chloride (approximately -60 mV) and non-specific cation channels (approximately 0 mV). Relatively pure inhibition or excitation could be revealed through pharmacological techniques by blocking the inhibition with picrotoxin/strychnine or by blocking the glutamatergic neurotransmission with D-AP7 (D-2-amino-7-phosphonoheptanoate) and NBQX (2,3-dihydroxy-6-nitro-sulfamoyl -benzo (F) quinoxaline). A comparison of light-evoked responses (LER) and the HSER suggested that they activate the same pool of releasable neurotransmitter.

Topics: 2-Amino-5-phosphonovalerate; Ambystomatidae; Amino Acids; Animals; Anticonvulsants; Cadmium; Cobalt; Convulsants; Excitatory Amino Acid Antagonists; Light; Membrane Potentials; Neurotransmitter Agents; Organ Culture Techniques; Osmotic Pressure; Patch-Clamp Techniques; Picrotoxin; Presynaptic Terminals; Quinoxalines; Retinal Ganglion Cells; Strychnine; Sucrose

We report the presence of excitatory and inhibitory spontaneous and evoked synaptic currents in the dorsal motor nucleus of the vagus (DMV) in the rat upon vagal and perivagal stimulation. Whole cell current-clamp recordings from anatomically identified DMV neurons in rat brain stem slices show that these neurons are capable of sustained slow-frequency action potential firing probably because of the presence of pacemaker current. Spontaneously occurring, tetrodotoxin-resistant miniature inhibitory and excitatory synaptic potentials were observed. Stimulation of the vagus mostly induced antidromic action potentials in DMV neurons. However, careful positioning of the stimulating electrode in the tissue surrounding the recording neuron, and sometimes in the vagus itself, was capable of evoking orthodromic-evoked mixed inhibitory-excitatory postsynaptic potentials, and eventually, action potentials. Whole cell voltage-clamp recordings of the synaptic currents corresponding to these synaptic potentials in the presence of pharmacological antagonists of the neurotransmitters gamma-aminobutyric acid (GABA), glutamate, and glycine receptor subtypes indicate that the inhibitory synaptic currents are mediated by GABA-activated Cl- channels, while the excitatory synaptic currents are due to activation of ionotropic glutamate receptors of the N-methyl-D-aspartic acid (NMDA) and non-NMDA subtypes.

Topics: Action Potentials; Animals; Brain Stem; Electric Stimulation; Electrophysiology; Evoked Potentials; gamma-Aminobutyric Acid; Glutamates; Glutamic Acid; In Vitro Techniques; Membrane Potentials; Neurons; Picrotoxin; Quinoxalines; Rats; Rats, Inbred Strains; Strychnine; Synapses; Tetrodotoxin; Vagus Nerve