tetrodotoxin and Epilepsy

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

2-Deoxy-d-glucose (2DG), a glucose analog that inhibits glycolysis, has acute and chronic antiepileptic effects. We evaluated 2DG's acute effects on synaptic and membrane properties of CA3 pyramidal neurons in vitro. 2DG (10 mM) had no effects on spontaneously occurring postsynaptic currents (PSCs) in 3.5 mM extracellular potassium concentration ([K

Topics: Animals; Bicuculline; CA3 Region, Hippocampal; Deoxyglucose; Epilepsy; Glycolysis; Neurons; Potassium; Rats; Rats, Sprague-Dawley; Synaptic Potentials; Tetrodotoxin

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

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

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

Hypersynchronous neuronal excitation manifests clinically as seizure (ictogenesis), and may recur spontaneously and repetitively after a variable latency period (epileptogenesis). Despite tremendous research efforts to describe molecular pathways and signatures of epileptogenesis, molecular pathomechanisms leading to chronic epilepsy remain to be clarified. We hypothesized that epigenetic modifications may form the basis for a cellular memory of epileptogenesis, and used a primary neuronal cell culture model of the rat hippocampus to study the translation of massive neuronal excitation into persisting changes of epigenetic signatures and pro-epileptogenic target gene expression. Increased spontaneous activation of cultured neurons was detected 3 and 7 days after stimulation with 10 μM glutamate when compared to sham-treated time-matched controls using calcium-imaging in vitro. Chromatin-immunoprecipitation experiments revealed short-term (3 h, 7 h, and 24 h) and long-term (3 d and 2 weeks) changes in histone modifications, which were directly linked to decreased expression of two selected epilepsy target genes, e.g. excitatory glutamate receptor genes Gria2 and Grin2a. Increased promoter methylation observed 4 weeks after glutamate stimulation at respective genes suggested long-term repression of Gria2 and Grin2a genes. Inhibition of glutamatergic activation or blocking the propagation of action potentials in cultured neurons rescued altered gene expression and regulatory epigenetic modifications. Our data support the concept of a cellular memory of epileptogenesis and persisting epigenetic modifications of epilepsy target genes, which are able to turn normal into pro-epileptic neurons and circuits.

Topics: Action Potentials; Animals; Animals, Newborn; Cells, Cultured; DNA Methylation; Epigenesis, Genetic; Epilepsy; Excitatory Amino Acid Agents; Gene Expression; Glutamic Acid; Hippocampus; Microtubule-Associated Proteins; Models, Biological; Neurons; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Tetrodotoxin; Valine

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

Studies have suggested that thalamus is involved in temporal lobe epilepsy, but the role of thalamus is still unclear. We obtained local filed potentials (LFPs) and single-unit activities from CA1 of hippocampus and parafascicular nucleus of thalamus during the development of epileptic seizures induced by pilocarpine in mice. Two measures, redundancy and directionality index, were used to analyze the electrophysiological characters of neuronal activities and the information flow between thalamus and hippocampus. We found that LFPs became more regular during the seizure in both hippocampus and thalamus, and in some cases LFPs showed a transient disorder at seizure onset. The variation tendency of the peak values of cross-correlation function between neurons matched the variation tendency of the redundancy of LFPs. The information tended to flow from thalamus to hippocampus during seizure initiation period no matter what the information flow direction was before the seizure. In some cases the information flow was symmetrically bidirectional, but none was found in which the information flowed from hippocampus to thalamus during the seizure initiation period. In addition, inactivation of thalamus by tetrodotoxin (TTX) resulted in a suppression of seizures. These results suggest that thalamus may play an important role in the initiation of epileptic seizures.

Topics: Algorithms; Animals; Atropine; CA1 Region, Hippocampal; Data Interpretation, Statistical; Electrodes, Implanted; Electroencephalography; Epilepsy; Male; Mice; Mice, Inbred C57BL; Muscarinic Agonists; Muscarinic Antagonists; Pilocarpine; Tetrodotoxin; Thalamus

During epileptogenesis a series of molecular and cellular events occur, culminating in an increase in neuronal excitability, leading to seizure initiation. The entorhinal cortex has been implicated in the generation of epileptic seizures in both humans and animal models of temporal lobe epilepsy. This hyperexcitability is due, in part, to proexcitatory changes in ion channel activity. Sodium channels play an important role in controlling neuronal excitability, and alterations in their activity could facilitate seizure initiation. We sought to investigate whether medial entorhinal cortex (mEC) layer II neurons become hyperexcitable and display proexcitatory behavior of Na channels during epileptogenesis. Experiments were conducted 7 days after electrical induction of status epilepticus (SE), a time point during the latent period of epileptogenesis and before the onset of seizures. mEC layer II stellate neurons from post-SE animals were hyperexcitable, eliciting action potentials at higher frequencies compared with control neurons. Na channel currents recorded from post-SE neurons revealed increases in Na current amplitudes, particularly persistent and resurgent currents, as well as depolarized shifts in inactivation parameters. Immunocytochemical studies revealed increases in voltage-gated Na (Nav) 1.6 isoform levels. The toxin 4,9-anhydro-tetrodotoxin, which has greater selectivity for Nav1.6 over other Na channel isoforms, suppressed neuronal hyperexcitability, reduced macroscopic Na currents, persistent and resurgent Na current densities, and abolished depolarized shifts in inactivation parameters in post-SE neurons. These studies support a potential role for Nav1.6 in facilitating the hyperexcitability of mEC layer II neurons during epileptogenesis.

Topics: Animals; Entorhinal Cortex; Epilepsy; In Vitro Techniques; Male; NAV1.6 Voltage-Gated Sodium Channel; Neurons; Rats; Rats, Sprague-Dawley; Sodium; Sodium Channel Blockers; Tetrodotoxin; Time Factors

Activation of CB(1) receptors on axon terminals by exogenous cannabinoids (eg, Δ(9)-tetrahydrocannabinol) and by endogenous cannabinoids (endocannabinoids) released by postsynaptic neurons leads to presynaptic inhibition of neurotransmission. The aim of this study was to characterize the effect of cannabinoids on GABAergic synaptic transmission in the human neocortex. Brain slices were prepared from neocortical tissues surgically removed to eliminate epileptogenic foci. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in putative pyramidal neurons using patch-clamp techniques. To enhance the activity of cannabinoid-sensitive presynaptic axons, muscarinic receptors were continuously stimulated by carbachol. The synthetic cannabinoid receptor agonist WIN55212-2 decreased the cumulative amplitude of sIPSCs. The CB(1) antagonist rimonabant prevented this effect, verifying the involvement of CB(1) receptors. WIN55212-2 decreased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, but did not change their amplitude, indicating that the neurotransmission was inhibited presynaptically. Depolarization of postsynaptic pyramidal neurons induced a suppression of sIPSCs. As rimonabant prevented this suppression, it is very likely that it was due to endocannabinods acting on CB(1) receptors. This is the first demonstration that an exogenous cannabinoid inhibits synaptic transmission in the human neocortex and that endocannabinoids released by postsynaptic neurons suppress synaptic transmission in the human brain. Interferences of cannabinoid agonists and antagonists with synaptic transmission in the cortex may explain the cognitive and memory deficits elicited by these drugs.

Topics: Adolescent; Adult; Benzoxazines; Bicuculline; Biophysics; Cannabinoid Receptor Modulators; Cannabinoids; Carbachol; Child; Child, Preschool; Cholinergic Agonists; Electric Stimulation; Epilepsy; Female; GABA-A Receptor Antagonists; Humans; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Male; Middle Aged; Morpholines; Naphthalenes; Neocortex; Neurons; Patch-Clamp Techniques; Sodium Channel Blockers; Statistics, Nonparametric; Synaptic Transmission; Tetrodotoxin; Young Adult

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

4-Aminopyridine (4-AP) is a convulsing agent that in vivo preferentially releases Glu, the most important excitatory amino acid neurotransmitter in the brain. Here the ionic dependence of 4-AP-induced Glu release and the effects of several of the most common antiepileptic drugs (AEDs) and of the new potential AED, vinpocetine on 4-AP-induced Glu release were characterized in hippocampus isolated nerve endings pre-loaded with labelled Glu ([3H]Glu). 4-AP-induced [3H]Glu release was composed by a tetrodotoxin (TTX) sensitive and external Ca2+ dependent fraction and a TTX insensitive fraction that was sensitive to the excitatory amino acid transporter inhibitor, TBOA. The AEDs: carbamazepine, phenytoin, lamotrigine and oxcarbazepine at the highest dose tested only reduced [3H]Glu release to 4-AP between 50-60%, and topiramate was ineffective. Vinpocetine at a much lower concentration than the above AEDs, abolished [3H]Glu release to 4-AP. We conclude that the decrease in [3H]Glu release linked to the direct blockade of presynaptic Na+ channels, may importantly contribute to the anticonvulsant actions of all the drugs tested here (except topiramate); and that the significantly greater vinpocetine effect in magnitude and potency on [3H]Glu release when excitability is exacerbated like during seizures, may involve the increase additionally exerted by vinpocetine in some K+ channels permeability.

Topics: 4-Aminopyridine; Animals; Anticonvulsants; Calcium; Carbamazepine; Drug Interactions; Epilepsy; Fructose; gamma-Aminobutyric Acid; Glutamic Acid; Hippocampus; In Vitro Techniques; Lamotrigine; Male; Nerve Endings; Oxcarbazepine; Phenytoin; Potassium Channel Blockers; Rats; Rats, Wistar; Sodium; Sodium Channel Blockers; Tetrodotoxin; Topiramate; Triazines; Tritium; Vinca Alkaloids

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

Functional expression of the K-Cl cotransporter KCC2 in developing central neurons is crucial for the maturation of Cl(-)-dependent, GABA(A) receptor-mediated inhibitory responses. In pyramidal neurons of the rodent hippocampus, GABAergic postsynaptic responses are typically depolarizing and often excitatory during the first postnatal week. Here, we show that a single neonatal seizure episode induced by kainate injection during postnatal days 5-7 results in a fast increase in the Cl(-) extrusion capacity of rat hippocampal CA1 neurons, with a consequent hyperpolarizing shift of the reversal potential of GABA(A)-mediated currents (E(GABA)). A significant increase in the surface expression of KCC2 as well as the alpha2 subunit of the Na-K-ATPase parallels the seizure-induced increase in the Cl(-) extrusion capacity. Exposing hippocampal slices to kainate resulted in a similar increase in the neuronal Cl(-) extrusion and in the surface expression of KCC2. Both effects were blocked by the kinase inhibitor K252a. Hence, in the neonatal hippocampus the overall KCC2 expression level is high enough to promote a rapid functional activation of K-Cl cotransport and a consequent negative shift in E(GABA) close to the adult level. The activity-dependent regulation of KCC2 function and its effect on GABAergic transmission may represent an intrinsic antiepileptogenic mechanism.

Topics: Age Factors; Animals; Animals, Newborn; Biotinylation; Carbazoles; Enzyme Inhibitors; Epilepsy; Excitatory Amino Acid Agonists; Furosemide; Hippocampus; In Vitro Techniques; Indole Alkaloids; K Cl- Cotransporters; Kainic Acid; Membrane Potentials; Patch-Clamp Techniques; Protein Transport; Rats; Rats, Wistar; Sodium Channel Blockers; Sodium Potassium Chloride Symporter Inhibitors; Symporters; Tetrodotoxin

N-Methyl-D-aspartate (NMDA) receptors have been implicated in epileptogenesis, but how these receptors contribute to epilepsy remains unknown. In particular, their role is likely to be complicated because of their voltage-dependent behavior. Here, the authors investigate how activation of NMDA receptors can affect the intrinsic production of oscillation and the resonance properties of neocortical pyramidal neurons from children with intractable epilepsy. Intracellular whole-cell patch clamp recordings in cortical slices from these patients revealed that pyramidal neurons do not produce spontaneous oscillation under control conditions. However, they did exhibit resonance around 1.5 Hz. On NMDA receptor activation, with bath-applied NMDA (10 μM), the majority of neurons produced voltage-dependent intrinsic oscillation associated with a change in the stability of the neuronal system as reflected by the whole-cell I-V curve. Furthermore, the degree of resonance was amplified while the frequency of resonance was shifted to lower frequencies (∼1 Hz) in NMDA. These results suggest that NMDA receptors may both promote the production of low-frequency oscillation and sharpen the response of the cell to lower frequencies. Both these behaviors may be amplified in tissue from patients with epilepsy, resulting in an increased propensity to generate seizures.

Topics: Action Potentials; Adolescent; Anesthetics, Local; Biological Clocks; Biophysics; Child; Child, Preschool; Drug Interactions; Epilepsy; Excitatory Amino Acid Agonists; Female; Humans; In Vitro Techniques; Male; N-Methylaspartate; Neocortex; Patch-Clamp Techniques; Pyramidal Cells; Tetrodotoxin

Dendritic morphogenesis is an essential process for the establishment of proper neural circuitry. In the epileptic hippocampus, mature dentate granule cells (GCs) possess basal dendrites (BDs), which is abnormal and is assumed to contribute to seizure progression. However, there is a lack of direct time-lapse evidence showing that neuronal hyperactivity regulates the dendritic development of GCs. In the present study, we carried out time-lapse confocal analysis of the dendritic morphogenesis of GCs in hippocampal slice cultures that were prepared from postnatal 6-day-old (P6) rats. By electroporating membrane-targeted green fluorescent protein at 5 days in vitro (DIV), we found that most of the Prox1-positive and calbindin-negative immature GCs possessed several BDs and filopodia-rich apical dendrites at 7 DIV. BDs were gradually eliminated from 7 to 9 DIV, and they completely vanished at 14 DIV in all the GCs examined. However, most BDs failed to retract from 7 to 9 DIV, when the GABA(A) receptor antagonist picrotoxin was chronically applied to induce epileptic conditions in the cultures. These effects were blocked by coapplying tetrodotoxin, a sodium channel blocker, thus convincing us that neuronal hyperactivity contributes to the maintenance of BDs. Further, in the picrotoxin-treated cultures, most of the GCs persistently exhibited several BDs even after 14 DIV. In contrast, neither the progressive pruning of the filopodia nor the branch dynamics of the apical dendrites during the culture periods was affected by picrotoxin. These results, for the first time, provide us with direct evidence that neuronal hyperactivity contributes to the stability of pre-existing BDs.

Topics: Action Potentials; Analysis of Variance; Animals; Calbindins; Cell Shape; Dendrites; Electroporation; Epilepsy; Green Fluorescent Proteins; Hippocampus; Immunohistochemistry; In Vitro Techniques; Microscopy, Confocal; Neurons; Picrotoxin; Pseudopodia; Rats; Rats, Sprague-Dawley; S100 Calcium Binding Protein G; Sodium Channel Blockers; Tetrodotoxin

Epileptic seizures can induce pathological processes of plasticity in the brain that tend to promote the generation of further seizures. However, the immediate impact of epileptic seizures on cellular excitability remains poorly understood. In order to unravel such early mechanisms of epilepsy-induced plasticity, we studied synaptic transmission before and shortly after three ictal discharges induced by transient elevation of extracellular K(+) in mouse hippocampal slices. Discharges were initiated in the CA3 region and propagated via the Schaffer collaterals into CA1 where they were associated with sustained membrane depolarization and bursts of action potentials in CA1 pyramidal cells. Subsequently, discharges were followed by long-term potentiation (LTP) of Schaffer collateral-evoked field excitatory post-synaptic potentials (EPSPs) in the CA1. The ability to generate epileptiform activity in response to repetitive stimulation was enhanced during LTP. Changes in both inhibitory and excitatory synaptic transmission contributed to LTP in CA1 pyramidal cells. Discharges reduced gamma-aminobutyric acid-A receptor-mediated hyperpolarizing inhibitory post-synaptic potentials by shifting their reversal potentials in a positive direction. At the same time, the amplitudes of Schaffer collateral-evoked RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated EPSPs and action potential-independent miniature EPSPs were enhanced. However, N-methyl-d-aspartate receptor-mediated EPSPs remained unchanged. Paired-pulse stimulation revealed a reduced probability of glutamate release. Together, these changes in synaptic transmission produce a sustained increase in hippocampal excitability. We conclude that a few seizure-like ictal episodes are sufficient to cause fast and lasting changes in the excitation/inhibition balance in hippocampal networks, and therefore may contribute to early phases of progressive epileptogenesis.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Biophysics; Calcium; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; GABA-B Receptor Antagonists; Hippocampus; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Long-Term Potentiation; Magnesium; Male; Mice; Mice, Inbred C57BL; Neural Pathways; Neuronal Plasticity; Potassium; Pyridazines; Receptors, GABA-B; Receptors, N-Methyl-D-Aspartate; Sodium Channel Blockers; Synapses; Tetrodotoxin; Valine

This study showed that rat unilateral intracerebroventricular injection of BmK alphaIV, a sodium channel modulator derived from scorpion Buthus martensi Karsch, induced clusters of spikes, epileptic discharges and convulsion-related behavioral changes. BmK alphaIV potently promoted the release of endogenous glutamate from rat cerebrocortical synaptosomes. In vitro examination of the effect of BmK alphaIV on intrasynaptosomal free calcium concentration [Ca(2+)](i) and sodium concentration [Na(+)](i) revealed that BmK alphaIV-evoked glutamate release from synaptosomes was associated with an increase in Ca(2+) and Na(+) influx. Moreover, BmK alphaIV-mediated glutamate release and ion influx was completely blocked by tetrodotoxin, a blocker of sodium channel. Together, these results suggest that the induction of BmK alphaIV-evoked epileptic seizures may be involved in the modulation of BmK alphaIV on tetrodotoxin-sensitive sodium channels located on the nerve terminal, which subsequently enhances the Ca(2+) influx to cause an increase of glutamate release. These findings may provide some insight regarding the mechanism of neuronal action of BmK alphaIV in the central nervous system for understanding epileptogenesis involved in sodium channels.

Topics: Animals; Calcium; Cerebral Cortex; Dose-Response Relationship, Drug; Electroencephalography; Electrophysiology; Epilepsy; Fluorometry; Glutamic Acid; Injections, Intraventricular; Male; Potassium Chloride; Rats; Rats, Sprague-Dawley; Scorpion Venoms; Sodium; Sodium Channel Blockers; Sodium Channels; Synaptosomes; Tetrodotoxin; Time Factors

Hippocampal CA3 neurons of spontaneously epileptic rats (SER; zi/zi, tm/tm), which show both absence-like seizures and tonic convulsions, exhibit a long-lasting depolarization shift with repetitive firing with a single stimulation of mossy fibers. Therefore a whole-cell patch-clamp study using temporarily dissociated hippocampal CA3 neurons from SER was performed to elucidate whether such abnormal excitability was due to abnormalities in voltage-dependent Ca(2+) channels (VDCCs).. Hippocampal CA3 neurons were temporarily dissociated with enzymatic and mechanical treatments. In a voltage-clamp mode with whole-cell recording, depolarizing step pulses were applied to induce Ca(2+) currents in the presence of tetrodotoxin and tetraethylammonium.. The threshold level of the Ca(2+) current induced by depolarizing pulses was found to be lower in hippocampal CA3 neurons of SER compared with those of control Wistar rats. In addition, the Ca(2+) current peak amplitude was greater, and decay of the current was weaker in CA3 neurons of SER than in those of normal Wistar rats.. These findings suggest that enhancements of Ca(2+) influx into hippocampal CA3 neurons due to the easier activation properties of VDCCs, as well as a decrease in decay, are involved in SER epileptic seizures.

Topics: Action Potentials; Animals; Calcium; Calcium Channels; Epilepsy; Fluoresceins; Hippocampus; Membrane Potentials; Mossy Fibers, Hippocampal; Neurons; Organic Chemicals; Patch-Clamp Techniques; Rats; Rats, Mutant Strains; Rats, Wistar; Tetraethylammonium; Tetrodotoxin

Some enaminones are reported to have in vivo anticonvulsant activity. We asked if methyl 4-(4'-bromophenyl)aminocyclohex-3-en-6-methyl-2-oxo-1-oate (E139), one of such enaminones produced in vitro effects that may underlie or explain these in vivo anticonvulsant actions by testing if E139 suppressed in vitro seizures. In vitro seizures were generated chemically in hippocampal slices using picrotoxin and zero Mg(2+) buffer and electrically by high frequency stimulation (HFS). E139 (10 microM) depressed evoked field population spike (PS) amplitude by -28.6+/-4.5% (n=5), an effect that was blocked by 1 microM CGP55845 (2.7+/-5.5%, n=6). Picrotoxin (100 microM) transformed single PS into multiple PS (4.5+/-0.2, n=5) and E139 reversibly reduced the number of these multiple PS by -23.4+/-1.8% (n=5). Similarly, zero Mg(2+) buffer produced multiple spikes (3.6+/-0.6, n=5) that were suppressed by E139 (-54.8+/-9.7%, n=5). This effect was also blocked by CGP55845 (2.3+/-5.7%, n=6). Furthermore, E139 suppressed the frequency of spontaneous bursts (SB) that were recorded in zero Mg(2+) by -65.8+/-10.5% (n=12). CGP55845 significantly reduced this E139-induced SB suppression (-21.7+/-9.6%, n=6). In the electrical model, afterdischarges (AD) and SB recorded in area CA3 after a pattern of HFS (100Hz) were suppressed by E139 (-48.6+/-14.3% and -66.7+/-6.7%, respectively, n=6). These E139 effects on AD and SB were reduced, but not completely blocked, by CGP55845 (-32.1+/-5.3% and -44.4+/-9.7%, respectively, n=7). Finally, pretreatment of slices with E139 did not prevent zero Mg(2+)-induced multiple spikes and SB. We conclude that E139 suppresses in vitro seizures in the hippocampus by synaptic and non-synaptic mechanisms. These actions on network activity may underlie their reported in vivo anticonvulsant effects.

Topics: Animals; Anticonvulsants; Cyclohexanes; Electric Stimulation; Electrophysiology; Epilepsy; Hippocampus; In Vitro Techniques; Magnesium; Rats; Tetrodotoxin

Mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, and SCN1B, and two GABAA receptor subunit genes, GABRG2 and GABRD, have been identified in families with generalized epilepsy with febrile seizures plus (GEFS+). A novel mutation, R859C, in the Nav1.1 sodium channel was identified in a four-generation, 33-member Caucasian family with a clinical presentation consistent with GEFS+. The mutation neutralizes a positively charged arginine in the domain 2 S4 voltage sensor of the Nav1.1 channel alpha subunit. This residue is conserved in mammalian sodium channels as well as in sodium channels from lower organisms. When the mutation was placed in the rat Nav1.1 channel and expressed in Xenopus oocytes, the mutant channel displayed a positive shift in the voltage dependence of sodium channel activation, slower recovery from slow inactivation, and lower levels of current compared with the wild-type channel. Computational analysis suggests that neurons expressing the mutant channel have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Therefore, this mutation should lead to decreased neuronal excitability, in contrast to most previous GEFS+ sodium channel mutations, which have changes predicted to increase neuronal firing.

Topics: Action Potentials; Animals; Arginine; Cell Line; Child; Cysteine; Dose-Response Relationship, Radiation; Epilepsy; Family Health; Female; Humans; Ion Channel Gating; Male; Membrane Potentials; Models, Molecular; Models, Neurological; Mutagenesis; Mutation; NAV1.1 Voltage-Gated Sodium Channel; Nerve Tissue Proteins; Oocytes; Patch-Clamp Techniques; Rats; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Xenopus

In order to further examine the role of spontaneous action potential (SAP) discharges in neocortical development, amino-acid-mediated synaptic transmission was selectively blocked in an improved organotypic neocortex culture preparation. Contralateral occipital cortex slices from neonatal rats were co-cultured for several weeks in a ventricle-to-ventricle orientation known to greatly enhance cyto-morphological and electrophysiological maturation. Such preparations are highly resistant to attempts to suppress neuronal firing by blocking ionotropic glutamate receptors: not only can kainate receptors partly substitute for NMDA- and AMPA-mediated neurotransmission when these receptors are pharmacologically blocked, but (muscarinic) cholinergic receptors also begin to drive SAP activity when the kainate receptors, too, are chronically blocked. Only tetrodotoxin proved able to eliminate SAPs altogether in these co-cultures, while GABAergic receptor blockade (using bicucculine) led to persistent epileptiform discharges. Treatment effects were assayed upon transfer to control medium by means of a quantitative analysis of spontaneously occurring polyneuronal spike trains. Total suppression of action potentials for several weeks (by tetrodotoxin treatment) led, as in earlier experiments, to strongly intensified burst firing upon transfer to control medium. Chronic glutamate receptor blocked cultures, on the other hand, showed only minor deviations from control firing levels and patterns when assayed in normal medium. Protection against the development of hyperactivity despite partial blockade of synaptic transmission was roughly proportional to the degree to which spontaneous firing during the treatment period approximated normal SAP levels. This homeostatic response to chronically reduced excitatory drive thus differs from earlier results using isolated organotypic cortex cultures, in which the restoration of SAP activity failed to prevent the development of network hyperactivity. Chronic bicucculine treatment, in contrast, had little or no homeostatic effect on SAP firing patterns; on the contrary, opposite to earlier findings using isolated occipital cortex explants, paroxysmal discharges persisted even after transfer to control medium.

Topics: Action Potentials; Animals; Animals, Newborn; Cell Differentiation; Cholinergic Antagonists; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Homeostasis; Neocortex; Nerve Net; Neural Inhibition; Neurons; Organ Culture Techniques; Rats; Receptors, GABA; Receptors, Glutamate; Receptors, Muscarinic; Receptors, Neurotransmitter; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

A paroxysmal depolarization shift (PDS) has been suggested to be a hallmark for epileptic activity in partial-onset seizures. By monitoring membrane potentials and currents in pairs of pyramidal neurons and astrocytes with dual patch-clamp recording and exocytosis of vesicles from astrocytes with two-photon laser scanning microscopy in hippocampal slices, we found that infusion of inositol 1,4,5-trisphosphate (IP(3)) into astrocytes by patch pipettes induced astrocytic glutamate release that triggered a transient depolarization (TD) and epileptiform discharges in CA1 pyramidal neurons. The TD is due to a tetrodotoxin (TTX)-insensitive slowly decaying transient inward current (STC). Astrocytic glutamate release simultaneously triggers both the STC in pyramidal neurons and a transport current (TC) in astrocytes. The neuronal STC is mediated by ionotropic glutamate receptors leading to the TD and epileptiform discharges; while the astrocytic TC is a glutamate reuptake current resulting from transporting released glutamate into the patched astrocyte. Fusion of a large vesicle in astrocytes was immediately followed by an astrocytic TC, suggesting that the fused vesicle contains glutamate. Both fusion of large vesicles and astrocytic TCs were blocked by tetanus toxin (TeNT), suggesting that astrocytic glutamate release is via SNARE-dependent exocytosis of glutamate-containing vesicles. In the presence of TTX, the epileptogenic reagent, 4-AP, also induced similar neuronal STCs and astrocytic TCs, suggesting that astrocytic glutamate release may play an epileptogenic role in initiation of epileptic seizures under pathological conditions. Our study provides a novel mechanism, astrocytic release of glutamate, for seizure initiation.

Topics: 2-Amino-5-phosphonovalerate; 4-Aminopyridine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Aspartic Acid; Astrocytes; Calcium; Diagnostic Imaging; Drug Interactions; Electric Stimulation; Epilepsy; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Microscopy, Confocal; Neurons; Neurotoxins; Patch-Clamp Techniques; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetanus Toxin; Tetrodotoxin; Time Factors

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

A-type currents powerfully modulate discharge behavior and have been described in a large number of different species and cell types. However, data on A-type currents in human brain tissue are scarce. Here we have examined the properties of a fast transient outward current in acutely dissociated human neocortical neurons from the temporal lobe of epilepsy patients by using the whole-cell voltage-clamp technique. The A-type current was isolated with a subtraction protocol. In addition, delayed potassium currents were reduced pharmacologically with 10 mM tetraethylammonium chloride. The current displayed an activation threshold of about -70 mV. The voltage-dependent activation was fitted with a Boltzmann function, with a half-maximal conductance at -14.8 +/- 1.8 mV (n = 5) and a slope factor of 17.0 +/- 0.5 mV (n = 5). The voltage of half-maximal steady-state inactivation was -98.9 +/- 8.3 mV (n = 5), with a slope factor of -6.6 +/- 1.9 mV (n = 5). Recovery from inactivation could be fitted monoexponentially with a time constant of 18.2 +/- 7.5 msec (n = 5). At a command potential of +30 mV, application of 5 mM 4-aminopyridine or 100 microM flecainide resulted in a reduction of A-type current amplitude by 35% or 22%, respectively. In addition, flecainide markedly accelerated inactivation. Current amplitude was reduced by 31% with application of 500 microM cadmium. All drug effects were reversible. In conclusion, neocortical neurons from epilepsy patients express an A-type current with properties similar to those described for animal tissues.

Topics: Adolescent; Adult; Anesthetics, Local; Anti-Arrhythmia Agents; Cadmium; Child, Preschool; Epilepsy; Female; Flecainide; Humans; In Vitro Techniques; Male; Membrane Potentials; Middle Aged; Neocortex; Neurons; Patch-Clamp Techniques; Potassium Channel Blockers; Potassium Channels; Tetraethylammonium; Tetrodotoxin; Valine

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

In epilepsy models, organic calcium antagonists regularly induce a transient activity increase before suppression of epileptiform discharges. This action was speculated to be mediated by a modulation of potassium currents. Since A-type currents potently regulate neuronal excitability, their modulation by calcium channel blockers was investigated in acutely isolated human neocortical temporal lobe neurons and CA1 neurons of guinea pigs using the whole-cell voltage-clamp technique. In human neurons, 40 microM nifedipine caused an amplitude reduction by 28% at a command potential of -6 mV and produced a biexponential, markedly accelerated current inactivation with time constants of 8.4 +/- 1.1 ms (n = 6) and 62.9 +/- 6.4 ms (n = 5). The time constant under control conditions was 50.1 +/- 8.5 ms (n = 6). Verapamil (40 microM) did not affect the current amplitude, but accelerated the monoexponential current inactivation from 40.2 +/- 7.1 ms to 13.3 +/- 0.8 ms (n = 9). Accordingly, verapamil accelerated the inactivation from 42.3 +/- 5.9 ms to 15.0 +/- 1.3 ms (n = 11) in guinea pig CA1 neurons, without affecting the current amplitude. In this preparation, it was shown that the two enantiomers of verapamil do not differ in their actions. The results show that the A-type current in human neocortical and in guinea pig hippocampal neurons is reduced by organic calcium channel blockers.

Topics: Adolescent; Adult; Aged; Anesthetics, Local; Animals; Calcium Channel Blockers; Child; Epilepsy; Female; Guinea Pigs; Humans; In Vitro Techniques; Male; Middle Aged; Neocortex; Neural Conduction; Neurons; Nifedipine; Potassium Channel Blockers; Pyramidal Cells; Stereoisomerism; Tetraethylammonium; Tetrodotoxin; Verapamil

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

Lateral amygdala (LA) activity during synchronized-epileptiform discharges in temporolimbic circuits was investigated in rat horizontal slices containing the amygdala, hippocampus (Hip), perirhinal (Prh) and lateral entorhinal (LEnt) cortex, through multiple-site extra- and intracellular recording techniques and measurement of the extracellular K+ concentration. Application of 4-aminopyridine (50 microm) induced epileptiform discharges in all regions under study. Slow interictal-like burst discharges persisted in the Prh/LEnt/LA after disconnection of the Hip, seemed to originate in the Prh as shown from time delay analyses, and often preceded the onset of ictal-like activity. Disconnection of the amygdala resulted in de-synchronization of epileptiform discharges in the LA from those in the Prh/LEnt. Interictal-like activity was intracellularly reflected in LA projection neurons as gamma-aminobutyric acid (GABA)A/B receptor-mediated synaptic responses, and depolarizing electrogenic events (spikelets) residing on the initial phase of the GABA response. Spikelets were considered antidromically conducted ectopic action potentials generated at axon terminals, as they were graded in amplitude, were not abolished through hyperpolarizing membrane responses (which effectively blocked evoked orthodromic action potentials), lacked a clear prepotential or synaptic potential, were not affected through blockers of gap junctions, and were blocked through remote application of tetrodotoxin at putative target areas of LA projection neurons. Remote application of a GABAB receptor antagonist facilitated spikelet generation. A transient elevation in the extracellular K+ level averaging 3 mm above baseline occurred in conjunction with interictal-like activity in all areas under study. We conclude that interictal-like discharges in the LA/LEnt/Prh spread in a predictable manner through the synaptic network with the Prh playing a leading role. The rise in extracellular K+ may provide a depolarizing mechanism for recruitment of interneurons and generation of ectopic action potentials at axon terminals of LA projection neurons. Antidromically conducted ectopic action potentials may provide a spreading mechanism of seizure activity mediated by diffuse axonal projections of LA neurons.

Topics: 4-Aminopyridine; Action Potentials; Amygdala; Anesthetics, Local; Animals; Anti-Ulcer Agents; Bicuculline; Carbenoxolone; Dissection; Electric Stimulation; Entorhinal Cortex; Epilepsy; Evoked Potentials; Extracellular Space; GABA Antagonists; Hippocampus; In Vitro Techniques; Male; Neurons; Phosphinic Acids; Potassium; Propanolamines; Rats; Rats, Wistar; Reaction Time; Synapses; Tetrodotoxin; Time Factors

Glial cells limit local K(+)-accumulation by K(+)-uptake through different mechanisms, sensitive to Ba(2+), ouabaine, furosemide, or DIDS. Since the relative contribution of these mechanisms has not yet been determined, we studied the effects of bath-applied barium (2 mM), ouabaine (9 microM), furosemide (2 mM), and DIDS (1 mM) on ionophoretically-induced rises in [K(+)](o) in the pyramidal layer of area CA1 from normal rat slices, in the presence of glutamate receptor (Glu-R) antagonists. We also investigated the effect of barium on ionophoretically-induced tetrapropylammonium (TPA(+))-signals in order to test for barium-induced changes of the extracellular space. Finally, we repeated the barium experiment on slices from human non-sclerotic and sclerotic hippocampal specimens to assess a reduced glial capability for barium-sensitive K(+)-uptake in sclerotic tissue from epilepsy patients. In normal rat slices barium augmented ionophoretically-induced rises in [K(+)](o) by approximately 120%, also in the presence of tetrodotoxin (TTX) (by approximately 150%), but did not significantly affect the TPA(+)-signal. Ouabaine also augmented the K(+)-signal, but only by 27%. Furosemide and DIDS had negligible effects. In slices from sclerotic human hippocampus an augmentation of the K(+)-signal by barium was absent. Thus barium augments ionophoretically-induced K(+)-signals to a similar extent as previously shown for stimulus-induced signals. We suggest that glial barium-sensitive K(+)-buffer mechanisms reduce fast local rises of [K(+)](o) by at least 50%. This capability of glial cells is extremely reduced in area CA1 of slices from human sclerotic hippocampal specimens.

Topics: 2-Amino-5-phosphonovalerate; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anesthetics, Local; Animals; Barium; Buffers; Diuretics; Enzyme Inhibitors; Epilepsy; Excitatory Amino Acid Antagonists; Extracellular Space; Furosemide; Hippocampus; Humans; In Vitro Techniques; Iontophoresis; Ouabain; Potassium; Quaternary Ammonium Compounds; Quinoxalines; Rats; Rats, Wistar; Sclerosis; Tetrodotoxin

Epileptic discharges propagate through apparently normal circuits, although it is still unclear how this recruitment takes place. To understand the role of different classes of neurons in neocortical epilepsy, we have developed a novel imaging assay that detects which neurons participate in epileptiform discharges. Using calcium imaging of neuronal populations during bicuculline-induced spontaneous epileptiform events in slices from juvenile mouse somatosensory cortex, we find that fast calcium transients correlate with epileptiform field potentials and intracellular depolarizing shifts and can be used as an optical signature that a given neuron has participated in an epileptiform event. Our results demonstrate a novel method to characterize epileptiform events with single-cell resolution. In addition, our data are consistent with an important role for layer 5 in generating neocortical seizures and indicate that subgroups of neurons are particularly prone to epileptiform recruitment.

Topics: Action Potentials; Animals; Bicuculline; Calcium; Cerebral Cortex; Convulsants; Electrophysiology; Epilepsy; Fura-2; Intracellular Membranes; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence; Neurons; Reaction Time; Tetrodotoxin

We investigated the pathophysiological mechanisms of glutamate-induced delayed neuronal damage in rat hippocampal slice cultures [Stoppini et al. (1991) J. Neurosci. Methods 37, 173-182], with propidium iodide as a marker of cell death. Exposure of the cultures to growth medium containing 10 mM glutamate for 30 min resulted in a slowly developing degeneration of hippocampal principal cells, starting from the medial end of the CA1 region and reaching the dentate gyrus by 48 h. By 24 h, most pyramidal cells in CA1 were damaged. An acute phase of degeneration preceded the delayed damage at 2-6 h, affecting cells in a spatially diffuse manner. When tetrodotoxin (0.5 microM) was present during the glutamate insult, a marked protection (mean 57%, P<0.001) of the CA1 damage was observed. Rather strikingly, when tetrodotoxin was applied immediately following or even with a delay of 30 min after the insult, a similar amount of protection was achieved. In field recordings carried out after the insult, the glutamate-treated slices exhibited spontaneously occurring negative shifts with a duration of 1-10 s and an amplitude of up to 400 microV in the CA3 region, whereas the control slices were always quiescent. Taken together, the results suggest that post-insult neuronal network activity, rather than the direct action of exogenous glutamate, is a major cause of delayed CA1 pyramidal cell death in the organotypic slices. These observations may have implications in the design of neuroprotective strategies for the treatment of brain traumas which are accompanied by delayed and/or distal neuronal damage.

Topics: Action Potentials; Animals; Brain Injuries; Brain Ischemia; Cell Death; Epilepsy; Glutamic Acid; Hippocampus; Nerve Degeneration; Nerve Net; Neurotoxins; Organ Culture Techniques; Pyramidal Cells; Rats; Tetrodotoxin; Time Factors

We have previously shown that presynaptic N-methyl-D-aspartate receptors (NMDARs) can facilitate glutamate release onto principal neurons in the entorhinal cortex (EC). In the present study, we have investigated the subunit composition of these presynaptic NMDARs. We recorded miniature alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (mEPSCs), from visually identified neurons in layers II and V of the EC in vitro. In both layers, bath application of the NR2A/B subunit-selective agonist, homoquinolinic acid (HQA), resulted in a marked facilitation of mEPSC frequency. Blockade of presynaptic Ca(2+) entry through either NMDARs or voltage-gated Ca(2+) channels with Co(2+) prevented the effects of HQA, confirming that Ca(2+) entry to the terminal was required for facilitation. When the NR2B-selective antagonist, ifenprodil, was applied prior to HQA, the increase in mEPSC frequency was greatly reduced. In addition, we found that an NMDAR antagonist blocked frequency-dependent facilitation of evoked release and reduced mEPSC frequency in layer V. Thus we have demonstrated that NMDA autoreceptors in layer V of the EC bear the NR2B subunit, and that NMDARs are also present at terminals onto superficial neurons.

Topics: 2-Amino-5-phosphonovalerate; Animals; Autoreceptors; Calcium; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Male; Neurons; Piperidines; Presynaptic Terminals; Quinolinic Acids; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

Repeated seizures induce mossy fiber axon sprouting, which reorganizes synaptic connectivity in the dentate gyrus. To examine the possibility that sprouted mossy fiber axons may form recurrent excitatory circuits, connectivity between granule cells in the dentate gyrus was examined in transverse hippocampal slices from normal rats and epileptic rats that experienced seizures induced by kindling and kainic acid. The experiments were designed to functionally assess seizure-induced development of recurrent circuitry by exploiting information available about the time course of seizure-induced synaptic reorganization in the kindling model and detailed anatomic characterization of sprouted fibers in the kainic acid model. When recurrent inhibitory circuits were blocked by the GABA(A) receptor antagonist bicuculline, focal application of glutamate microdrops at locations in the granule cell layer remote from the recorded granule cell evoked trains of excitatory postsynaptic potentials (EPSPs) and population burst discharges in epileptic rats, which were never observed in slices from normal rats. The EPSPs and burst discharges were blocked by bath application of 1 microM tetrodotoxin and were therefore dependent on network-driven synaptic events. Excitatory connections were detected between blades of the dentate gyrus in hippocampal slices from rats that experienced kainic acid-induced status epilepticus. Trains of EPSPs and burst discharges were also evoked in granule cells from kindled rats obtained after > or = 1 wk of kindled seizures, but were not evoked in slices examined 24 h after a single afterdischarge, before the development of sprouting. Excitatory connectivity between blades of the dentate gyrus was also assessed in slices deafferented by transection of the perforant path, and bathed in artificial cerebrospinal fluid (ACSF) containing bicuculline to block GABA(A) receptor-dependent recurrent inhibitory circuits and 10 mM [Ca(2+)](o) to suppress polysynaptic activity. Low-intensity electrical stimulation of the infrapyramidal blade under these conditions failed to evoke a response in suprapyramidal granule cells from normal rats (n = 15), but in slices from epileptic rats evoked an EPSP at a short latency (2.59 +/- 0.36 ms) in 5 of 18 suprapyramidal granule cells. The results are consistent with formation of monosynaptic excitatory connections between blades of the dentate gyrus. Recurrent excitatory circuits developed in the dentate gyrus of epilepti

Topics: Animals; Bicuculline; Dentate Gyrus; Electric Stimulation; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; GABA Antagonists; In Vitro Techniques; Kainic Acid; Kindling, Neurologic; Magnesium; Male; Mossy Fibers, Hippocampal; Rats; Rats, Sprague-Dawley; Tetrodotoxin

A number of mechanisms have been proposed to play a role in the regulation of activity-dependent variations in extracellular potassium concentration ([K(+)](o)). We tested possible regulatory mechanisms for [K(+)](o) during spontaneous recurrent epileptiform activity induced in the dentate gyrus of hippocampal slices from adult rats by perfusion with 8 mM potassium and 0-added calcium medium in an interface chamber. Local application of tetrodotoxin blocked local [K(+)](o) changes, suggesting that potassium is released and taken up locally. Perfusion with barium or cesium, blockers of the inward rectifying potassium channel, did not alter the baseline [K(+)](o), the ceiling level of [K(+)](o) reached during the burst, or the rate of [K(+)](o) recovery after termination of the bursts. Decreasing gap junctional conductance did not change the baseline [K(+)](o) or the half-time of recovery of the [K(+)](o) after the bursts but did cause a decrease in the ceiling level of [K(+)](o). Perfusion with furosemide, which will block cation/chloride cotransporters, or perfusion with low chloride did not change the baseline [K(+)](o) or the half-time of recovery of the [K(+)](o) after the bursts but did increase the ceiling level of [K(+)](o). Bath or local application of ouabain, a Na(+)/K(+)-ATPase inhibitor, increased the baseline [K(+)](o), slowed the rate of [K(+)](o) recovery, and induced spreading depression. These findings suggest that potassium redistribution by glia only plays a minor role in the regulation of [K(+)](o) in this model. The major regulator of [K(+)](o) in this model appears to be uptake via a Na(+)/K(+)-ATPase, most likely neuronal.

Topics: Animals; Chlorides; Dentate Gyrus; Enzyme Inhibitors; Epilepsy; Evoked Potentials; Extracellular Space; Gap Junctions; In Vitro Techniques; Neuroglia; Ouabain; Potassium; Potassium Channels; Potassium Channels, Inwardly Rectifying; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin

Sipatrigine (BW619C89), a derivative of the antiepileptic agent lamotrigine, has potent neuroprotective properties in animal models of cerebral ischemia and head injury. In the present study we investigated the electrophysiological effects of sipatrigine utilizing intracellular current-clamp recordings obtained from striatal spiny neurons in rat corticostriatal slices and whole-cell patch-clamp recordings in isolated striatal neurons. The number of action potentials produced in response to a depolarizing current pulse in the recorded neurons was reduced by sipatrigine (EC(50) 4.5 microM). Although this drug preferentially blocked action potentials in the last part of the depolarizing current pulse, it also decreased the frequency of the first action potentials. Sipatrigine also inhibited tetrodotoxin-sensitive sodium (Na(+)) current recorded from isolated striatal neurons. The EC(50) for this inhibitory action was 7 microM at the holding potential (V(h)) of -65 mV, but 16 microM at V(h) = -105, suggesting a dependence of this pharmacological effect on the membrane potential. Moreover, although the inhibitory action of sipatrigine on Na(+) currents was maximal during high-frequency activation (20 Hz), it could also be detected at low frequencies. The amplitude of excitatory postsynaptic potentials (EPSPs), recorded following stimulation of the corticostriatal pathway, was depressed by sipatrigine (EC(50) 2 microM). This inhibitory action, however, was incomplete; in fact maximal concentrations of this drug reduced EPSP amplitude by only 45%. Sipatrigine produced no increase in paired-pulse facilitation, suggesting that the modulation of a postsynaptic site was the main pharmacological effect of this agent. The inhibition of voltage-dependent Na(+) channels exerted by sipatrigine might account for its depressant effects on both repetitive firing discharge and corticostriatal excitatory transmission. The modulation of Na(+) channels described here, as well as the previously observed inhibition of high-voltage-activated calcium currents, might contribute to the neuroprotective efficacy exerted by this compound in experimental models of in vitro and in vivo ischemia.

Topics: Action Potentials; Animals; Anticonvulsants; Brain Chemistry; Calcium Channels; Corpus Striatum; Epilepsy; Excitatory Postsynaptic Potentials; In Vitro Techniques; Lamotrigine; Male; Neurons; Neuroprotective Agents; Patch-Clamp Techniques; Piperazines; Pyrimidines; Rats; Rats, Wistar; Sodium; Stroke; Tetrodotoxin; Triazines

Postsynaptic metabotropic glutamate (mGlu) receptor-activated inward current mediated by Na(+)-Ca(2+) exchange was compared in basolateral amygdala (BLA) neurons from brain slices of control (naïve and sham-operated) and amygdala-kindled rats. In control neurons, the mGlu agonist, quisqualate (QUIS; 1-100 microM), evoked an inward current not associated with a significant change in membrane slope conductance, measured from current-voltage relationships between -110 and -60 mV, consistent with activation of the Na(+)-Ca(2+) exchanger. Application of the group I selective mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine [(S)-DHPG; 10-1000 microM] or the endogenous agonist, glutamate (10-1000 microM), elicited the exchange current. QUIS was more potent than either (S)-DHPG or glutamate (apparent EC(50) = 19 microM, 57 microM, and 0.6 mM, respectively) in activating the Na(+)-Ca(2+) exchange current. The selective mGlu5 agonist, (R, S)-2-chloro-5-hydroxyphenylglycine [(R,S)-CHPG; apparent EC(50) = 2. 6 mM] also induced the exchange current. The maximum response to (R, S)-DHPG was about half of that of the other agonists suggesting partial agonist action. Concentration-response relationships of agonist-evoked inward currents were compared in control neurons and in neurons from kindled animals. The maximum value for the concentration-response relationship of the partial agonist (S)-DHPG- (but not the full agonist- [QUIS or (R,S)-CHPG]) induced inward current was shifted upward suggesting enhanced efficacy of this agonist in kindled neurons. Altogether, these data are consistent with a kindling-induced up-regulation of a group I mGlu-, possibly mGlu5-, mediated responses coupled to Na(+)-Ca(2+) exchange in BLA neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Calcium; Dose-Response Relationship, Drug; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Kindling, Neurologic; Male; Membrane Potentials; Methoxyhydroxyphenylglycol; Phenylacetates; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Seizures; Sodium; Tetrodotoxin; Up-Regulation

Misplaced (heterotopic) cortical neurons are a common feature of developmental epilepsies. To better understand seizure disorders associated with cortical heterotopia, the sites of aberrant discharge activity were investigated in vivo and in vitro in a seizure-prone mutant rat (tish) exhibiting subcortical band heterotopia.. Depth electrode recordings and postmortem assessment of regional c-fos mRNA levels were used to characterize the distribution of aberrant discharge activity during spontaneous seizures in vivo. Electrophysiologic recordings of spontaneous and evoked activity also were performed by using in vitro brain slices from the tish rat treated with proconvulsant drugs (penicillin and 4-aminopyridine).. Depth electrode recordings demonstrate that seizure activity begins almost simultaneously in the normotopic and heterotopic areas of the tish neocortex. Spontaneous seizures induce c-fos mRNA in normotopic and heterotopic neocortical areas, and limbic regions. The threshold concentrations of proconvulsant drugs for inducing epileptiform spiking were similar in the normotopic and heterotopic areas of tish brain slices. Manipulations that blocked communication between the normotopic and heterotopic areas of the cortex inhibited spiking in the heterotopic, but not the normotopic, area of the cortex.. These findings indicate that aberrant discharge activity occurs in normotopic and heterotopic areas of the neocortex, and in certain limbic regions during spontaneous seizures in the tish rat. Normotopic neurons are more prone to exhibit epileptiform activity than are heterotopic neurons in the tish cortex, and heterotopic neurons are recruited into spiking by activity initiated in normotopic neurons. The findings indicate that seizures in the tish brain primarily involve telencephalic structures, and suggest that normotopic neurons are responsible for initiating seizures in the dysplastic neocortex.

Topics: Animals; Autoradiography; Brain; Cerebral Cortex; Electrodes, Implanted; Electrophysiology; Epilepsy; Evoked Potentials; Genes, fos; In Situ Hybridization; In Vitro Techniques; Penicillin G; Rats; Rats, Mutant Strains; RNA, Messenger; Seizures; Tetrodotoxin

Previous studies have shown that physiological stimulation of brain activity increases anaerobic glucose consumption, both in humans and in experimental animals. To investigate this phenomenon further, we measured extracellular lactate levels within different rat brain regions, using microdialysis. Experiments were performed comparing the effects of natural, physiological olfactory stimulation of the limbic system with experimental limbic seizures. Olfactory stimulation was carried out by using different odors (i.e. both conventional odors: 2-isobutyl-3-methoxypyrazine, green pepper essence; thymol; and 2-sec-butylthiazoline, a sexual pheromone). Limbic seizures were either induced by systemic injection of pilocarpine (200-400 mg/kg) or focally elicited by microinfusions of chemoconvulsants (bicuculline 118 pmol and cychlothiazide 1.2 nmol) within the anterior piriform cortex. Seizures induced by systemic pilocarpine tripled lactic acid within the hippocampus, whereas limbic seizures elicited by focal microinfusion of chemoconvulsants within the piriform cortex produced a less pronounced increase in extracellular lactic acid. Increases in extracellular lactate occurring during olfactory stimulation with the sexual pheromone (three times the baseline levels) were non-significantly different from those occurring after systemic pilocarpine. Increases in lactic acid following natural olfactory stimulation were abolished both by olfactory bulbectomy and by the focal microinfusion of tetrodotoxin, while they were significantly attenuated by the local application of the N-methyl-D-aspartate antagonist AP-5. Increases in hippocampal lactate induced by short-lasting stimuli (olfactory stimulation or microinfusion of subthreshold doses of chemoconvulsants, bicuculline 30 pmol) were reproducible after a short delay (1 h) and cumulated when applied sequentially. In contrast, limbic status epilepticus led to a long-lasting refractoriness to additional lactate-raising stimuli and there was no further increase in lactate levels when the olfactory stimulation was produced during status epilepticus. Increases in lactic acid following olfactory stimulation occurred with site specificity in the rhinencephalon (hippocampus, piriform and entorhinal cortex) but not in the dorsal striatum. Site specificity crucially relied on the quality of the stimulus. For instance, other natural stimuli (i.e. tail pinch) produced a similar increase in extracellular lactate in all brain areas

Topics: 2-Amino-5-phosphonovalerate; Animals; Convulsants; Denervation; Dose-Response Relationship, Drug; Epilepsy; Extracellular Space; Lactic Acid; Limbic System; Male; Neostriatum; Olfactory Bulb; Olfactory Pathways; Rats; Rats, Sprague-Dawley; Status Epilepticus; Stimulation, Chemical; Tetrodotoxin

The action of somatostatin on GABA-mediated transmission was investigated in cat and rat thalamocortical neurons of the dorsal lateral geniculate nucleus and ventrobasal thalamus in vitro. In the cat thalamus, somatostatin (10 microM) had no effect on the passive membrane properties of thalamocortical neurons and on the postsynaptic response elicited in these cells by bath or iontophoretic application of (+/-)baclofen (5-10 microM) or GABA, respectively. However, somatostatin (1-10 microM) decreased by a similar amount (45-55%) the amplitude of electrically evoked GABA(A) and GABA(B) inhibitory postsynaptic potentials in 71 and 50% of neurons in the lateral geniculate and ventrobasal nucleus, respectively. In addition, the neuropeptide abolished spontaneous bursts of GABA(A) inhibitory postsynaptic potentials in 85% of kitten lateral geniculate neurons, and decreased (40%) the amplitude of single spontaneous GABA(A) inhibitory postsynaptic potentials in 87% of neurons in the cat lateral geniculate nucleus. Similar results were obtained in the rat thalamus. Somatostatin (10 microM) had no effect on the passive membrane properties of thalamocortical neurons in this species, or on the outward current elicited by puff-application of (+/-)baclofen (5-10 microM). However, in 57 and 22% of neurons in the rat lateral geniculate and ventrobasal nuclei, respectively, somatostatin (1 microM) reduced the frequency, but not the amplitude, of miniature GABA(A) inhibitory postsynaptic currents by 31 and 37%, respectively. In addition, the neuropeptide (1 microM) decreased the amplitude of evoked GABA(A) inhibitory postsynaptic currents in 20 and 55% of rat ventrobasal neurons recorded in normal conditions and during enhanced excitability, respectively: this effect was stronger on bursts of inhibitory postsynaptic currents(100% decrease) than on single inhibitory postsynaptic currents (41% decrease). These results demonstrate that in the sensory thalamus somatostatin inhibits GABA(A)- and GABA(B)-mediated transmission via a presynaptic mechanism, and its action is more prominent on bursts of GABAergic synaptic currents/potentials.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Baclofen; Bicuculline; Cats; Cell Membrane; Epilepsy; Evoked Potentials; Excitatory Amino Acid Antagonists; GABA Agonists; GABA Antagonists; gamma-Aminobutyric Acid; Geniculate Bodies; Hormones; Male; Membrane Potentials; Neural Inhibition; Patch-Clamp Techniques; Presynaptic Terminals; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, GABA-B; Sleep; Somatostatin; Synaptic Transmission; Tetrodotoxin; Ventral Thalamic Nuclei

Mongolian gerbils are genetically predisposed to develop epileptic seizures in limbic structures. A species-specific property of the Mongolian gerbil is the expression of the calcium-binding protein parvalbumin in the perforant path where it is predominantly concentrated in nerve terminals. To test the hypothesis that this atypical expression of parvalbumin is induced by seizure-correlated hyperactivity in the entorhinohippocampal loop, we investigated whether it is dependent on extrinsic afferents to the entorhinal cortex. We cultivated organotypic slice cultures of neonate gerbil entorhinal cortex, isolated from all regions it is normally connected with in vivo. In these cultures, parvalbumin-expressing neurons demonstrated their characteristic features like in vivo. Blockade of spontaneous local activity with the sodium-channel blocker tetrodotoxin, however, considerably reduced the number of parvalbumin-expressing neurons in culture. These results indicate that spontaneous local activity, but not activity mediated by extrinsic afferents, is an essential factor for the expression of parvalbumin in the entorhinal cortex of the Mongolian gerbil.

Topics: Afferent Pathways; Age Factors; Animals; Entorhinal Cortex; Epilepsy; Gene Expression; Gerbillinae; In Situ Hybridization; Organ Culture Techniques; Parvalbumins; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sodium Channel Blockers; Species Specificity; Tetrodotoxin

Excitotoxic injury of the dendrites of inhibitory interneurons could lead to decreases in their synaptic activation and explain subsequent local circuit hyperexcitability and epilepsy. A hallmark of dendrotoxicity, at least in principal neurons of the hippocampus and cortex, is focal or varicose swellings of dendritic arbors. In experiments reported here, transient (1h) exposure of hippocampal explant cultures to kainic acid produced marked focal swellings of the dendrites of parvalbumin-immunoreactive pyramidal basket cells in a highly reproducible and dose-dependent manner. At 5mM kainic acid, more than half of the immunopositive apical dendrites in area CA(1) had a beaded appearance. However, the somal volumes of these cells were unaltered by the same treatment. The presence of focal swellings was reversible with kainate washout and was not accompanied by interneuronal cell death. In contrast, exposure to much higher concentrations (300mM) of kainic acid resulted in the total loss of parvalbumin-positive interneurons from explants. Surprisingly, kainic acid-induced dendritic beading does not appear to be mediated by extracellular calcium. Beading was unaltered in the presence of N-methyl-D-aspartate receptor antagonists, the L-type calcium channel antagonist, nimodipine, cadmium, or by removing extracellular calcium. However, blockade of voltage-gated sodium channels by either tetrodotoxin or lidocaine abolished dendritic beading, while the activation of existing voltage-gated sodium channels by veratridine mimicked the kainic acid-induced dendritic beading. Finally, the removal of extracellular chloride prevented the kainic acid-induced dendritic beading.Thus, we suggest that the movement of Na(+) and Cl(-), rather than Ca(2+), into cells underlies the focal swellings of interneuron dendrites in hippocampus.

Topics: Animals; Calcium; Chloride Channels; Chlorides; Dendrites; Dose-Response Relationship, Drug; Epilepsy; Extracellular Space; Hippocampus; Interneurons; Kainic Acid; Lidocaine; Neural Inhibition; Neurotoxins; Parvalbumins; Rats; Rats, Wistar; Sodium Channels; Tetrodotoxin

Chlormethiazole has sedative, hypnotic, anticonvulsant and neuroprotective properties. Using in vitro grease-gap recordings, we show that it inhibits epileptiform activity in neocortical slices superfused with Mg(2+)-free medium (IC(50) approximately 200 microM). At an antiepileptic concentration (300 microM), chlormethiazole potentiated the action of exogenously applied GABA (1 mM) but did not affect responses to the glutamate receptor agonists N-methyl-D-aspartate (10 microM) or L-quisqualic acid (3 microM). The GABA(A) receptor antagonist N-methyl-bicuculline (50 microM) reduced chlormethiazole's potency to inhibit the epileptiform activity. These results indicate that chlormethiazole's anticonvulsant action is likely mediated by potentiating GABA(A)ergic inhibition rather than by antagonising glutamatergic excitation.

Topics: Action Potentials; Animals; Bicuculline; Cerebral Cortex; Chlormethiazole; Dose-Response Relationship, Drug; Epilepsy; gamma-Aminobutyric Acid; Male; N-Methylaspartate; Neurons; Neuroprotective Agents; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin; Valine

Previous work suggested a role for the voltage-dependent persistent sodium current, I(Na,P), in the generation of seizures and spreading depression (SD). Ordinarily, I(Na,P) is small in hippocampal neurons. We investigated the effect of raising external K(+) concentration, [K(+)](o), on whole-cell persistent inward current in freshly isolated hippocampal CA1 pyramidal neurons. I(Na,P) was identified by TTX-sensitivity and dependence on external Na(+) concentration. When none of the ion channels were blocked, I(Na,P) was not usually detectable, probably because competing K(+) current masked it, but after raising [K(+)](o) I(Na,P) appeared, while K(+) currents diminished. With K(+) channels blocked, I(Na,P) could usually be evoked in control solution and raising [K(+)](o) caused its reversible increase in most cells. The increase did not depend on external calcium [Ca(2+)](o). In CA1 pyramidal neurons in hippocampal slices a TTX-sensitive persistent inward current was always recorded and when [K(+)](o) was raised, it was reversibly enhanced. Strong depolarization evoked irregular current fluctuations, which were also augmented in high [K(+)](o). The findings support a role of potassium-mediated positive feedback in the generation of seizures and spreading depression.

Topics: Animals; Calcium; Cortical Spreading Depression; Epilepsy; Feedback; Hippocampus; Lasers; Membrane Potentials; Microscopy, Fluorescence; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Potassium; Rats; Seizures; Sodium; Sodium Channels; Tetrodotoxin

The hippocampal mossy fibers, which originate from the dentate granule cells, develop mainly in the early postnatal period and are involved in numerous pathological processes. In this study, hippocampal slices prepared from premature rats were cultivated in the presence of convulsants to evaluate the influences of epileptiform activities on mossy fiber ontogeny. Electrophysiological and histochemical analyses revealed that prolonged hyperexcitability inhibited proper growth of the mossy fibers and caused ectopic innervation to the stratum oriens and the dentate molecular layer. These phenomena were prevented by pharmacological blockade of L-type Ca2+ channels, which did not affect convulsant-evoked ictal bursts. After single-pulse stimulation of the stratum granulosum in the slices cultured under paroxysmal conditions, the dentate gyrus displayed excessive excitation, but synaptic transmission to the CA3 region was hypoactive. However, brief repetitive stimulation elicited delayed epileptiform discharges in the CA3 region that were inhibited by an NMDA receptor antagonist. Chronic treatment with an L-type Ca2+ channel blocker ameliorated such aberrant neurotransmissions. These results suggest that ictal neuron activities at the developmental stage of the mossy fibers bring about the errant maturation associated with hippocampal dysfunction, which may form a cellular basis for the sequelae of childhood epilepsy, including chronic epilepsy or cognitive deficits. Thus I propose that L-type Ca2+ channel blockers can ameliorate the aversive prognosis of childhood epilepsy.

Topics: Animals; Animals, Newborn; Calcium Channel Blockers; Calcium Channels; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Hippocampus; In Vitro Techniques; Kainic Acid; Microscopy, Confocal; Mossy Fibers, Hippocampal; Picrotoxin; Rats; Rats, Wistar; Synaptic Transmission; Tetrodotoxin

Severe cortical trauma frequently causes epilepsy that develops after a long latency. We hypothesized that plastic changes in excitability during this latent period might be initiated or sustained by the level of neuronal activity in the injured cortex. We therefore studied effects of action potential blockade by application of tetrodotoxin (TTX) to areas of cortical injury in a model of chronic epileptogenesis. Partially isolated islands of sensorimotor cortex were made in 28- to 30-day-old male Sprague-Dawley rats and thin sheets of Elvax polymer containing TTX or control vehicle were implanted over lesions. Ten to 15 days later neocortical slices were obtained through isolates for electrophysiological studies. Slices from all animals (n = 12) with lesions contacted by control-Elvax (58% of 36 slices) exhibited evoked epileptiform field potentials, and those from 4 rats had spontaneous epileptiform events. Only 2 of 11 lesioned animals and 6% of slices from cortex exposed to TTX in vivo exhibited evoked epileptiform potentials, and no spontaneous epileptiform events were observed. There was no evidence of residual TTX during recordings. TTX-Elvax was ineffective in reversing epileptogenesis when implanted 11 days after cortical injury. These data suggest that development of antiepileptogenic drugs for humans may be possible.

Topics: Action Potentials; Animals; Brain; Brain Injuries; Epilepsy; Male; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

Incubation of hippocampal slices in zero-Ca(2+) medium blocks synaptic transmission and results in spontaneous burst discharges. This seizure-like activity is characterized by negative shifts (bursts) in the extracellular field potential and a K(+) wave that propagates across the hippocampus. To isolate factors related to seizure initiation, propagation, and termination, a number of pharmacological agents were tested. K(+) influx and efflux mechanisms where blocked with cesium, barium, tetraethylammonium (TEA), and 4-aminopyridine (4-AP). The effect of the gap junction blockers, heptanol and octanol, on zero-Ca(2+) bursting was evaluated. Neuronal excitability was modulated with tetrodotoxin (TTX), charge screening, and applied electric fields. Glial cell function was examined with a metabolism antagonist (fluroacetate). Neuronal hyperpolarization by cation screening or applied fields decreased burst frequency but did not affect burst amplitude or duration. Heptanol attenuated burst amplitude and duration at low concentration (0.2 mM), and blocked bursting at higher concentration (0.5 mM). CsCl(2) (1 mM) had no effect, whereas high concentrations (1 mM) of BaCl(2) blocked bursting. TEA (25 mM) and low concentration of BaCl(2) (300 microM) resulted in a two- to sixfold increase in burst duration. Fluroacetate also blocked burst activity but only during prolonged application (>3 h). Our results demonstrate that burst frequency, amplitude, and duration can be independently modulated and suggest that neuronal excitability plays a central role in burst initiation, whereas potassium dynamics establish burst amplitude and duration.

Topics: 4-Aminopyridine; Animals; Barium; Calcium; Calibration; Cesium; Electric Stimulation; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Membrane Potentials; Models, Neurological; Neurons; Potassium; Rats; Rats, Sprague-Dawley; Tetraethylammonium; Tetrodotoxin

In vivo studies suggest that ontogenesis of limbic seizures is determined by the development of the limbic circuit. We have now used the newly-developed in vitro intact interconnected neonatal rat limbic structures preparation to determine the developmental profile of kainate-induced epileptiform activity in the hippocampus and its propagation to other limbic structures. We report gradual alterations in the effects of kainate during the first postnatal week on an almost daily basis; from no epileptiform activity at birth, through interictal seizures around postnatal day (P) 2 and ictal seizures by the end of the first week. The developmental profile of kainate-induced hippocampal seizures is paralleled by the expression of postsynaptic kainate receptor-mediated currents in CA3 pyramidal cells. Intralimbic propagation of the hippocampal seizures is also age-dependent: whereas seizures readily propagate to the septum and to the contralateral hippocampus via the commissures on P2, propagation to the entorhinal cortex only takes place from P4 onwards. Finally, repeated brief applications of kainate to the hippocampus induce recurrent spontaneous glutamatergic ictal and interictal discharges which persist for several hours after the kainate is washed away and which replace the physiological pattern of network activity. Paroxysmal activities are thus generated by kainate in the hippocampus at an early developmental stage and are initially restricted to this structure. Before the end of the first week of postnatal life, kainate generates the epileptiform activities that may perturb activity-dependent mechanisms that modulate neuronal development. Although at this stage neurons are relatively resistant to the pathological effects of kainate, the epileptiform activities that it generates will perturb activity-dependent mechanisms that modulate neuronal development.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Benzodiazepines; Calcium; Electrophysiology; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Kainic Acid; Limbic System; Male; Organ Culture Techniques; Potassium; Rats; Rats, Wistar; Septal Nuclei; Synapses; Tetrodotoxin

The entorhinal cortex (EC) is a major gateway for sensory information into the hippocampus and receives a cholinergic input from the forebrain. Therefore, we studied muscarinic effects on excitability and intracellular Ca2+ signalling in layer II stellate and layer III pyramidal projection neurons of the EC. In both classes of neurons, local pressure-pulse application of carbachol (1 mM) caused small, atropine-sensitive membrane depolarizations that were not accompanied by any detectable changes in [Ca2+]i. At a higher concentration (10 mM), carbachol induced a larger membrane depolarization associated with synaptic oscillations and epileptiform activity in both classes of neurons. In contrast to the intrinsic theta rhythm in stellate cells with one dominant peak frequency at approximately 7 Hz, the synaptically mediated oscillation induced by carbachol showed three characteristic peaks in the theta and gamma frequency range at approximately 11, 23 and 40 Hz. Although carbachol-induced epileptiform activity was associated with increases in intracellular free Ca2+ in both layer II and III cells, the observed [Ca2+]i accumulation was significantly larger in layer III than in layer II cells. Responses to intracellular current injections showed differences in Ca2+ accumulation in layer II and III cells at the same membrane potentials, suggesting a dominant expression of low- and high-voltage-activated Ca2+ channels in these layer II and III cells, respectively. In conclusion, we present evidence for significant differences in the [Ca2+]i regulation between layer II stellate and layer III pyramidal cells of the medial EC.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Afferent Pathways; Animals; Calcium; Calcium Signaling; Carbachol; Cholinergic Agonists; Electrophysiology; Entorhinal Cortex; Epilepsy; Excitatory Amino Acid Antagonists; Organ Culture Techniques; Periodicity; Pyramidal Cells; Quinoxalines; Rats; Rats, Wistar; Synaptic Transmission; Tetrodotoxin

Despite the susceptibility of immature neurons to seizures, there are few models of epilepsy in the developing brain. By taking advantage of activity-dependent developmental changes in young neurons, we have developed a novel model of chronic epilepsy in cultured hippocampal slices. Incubating slices in tetrodotoxin (TTX) for at least 1 week produced significant changes in the electrical activity and appearance of CA1 pyramidal neurons. Extracellular recordings revealed multiple population spikes, and, in whole-cell recordings, evoked synaptic potentials lasting hundreds of milliseconds with many superimposed action potentials were present. Spontaneous firing with burst-like discharges was also evident. These changes were secondary to increased AMPA-receptor-mediated responses and decreased GABA(A) receptor events. Altered membrane properties involved increased expression of T-type Ca(2+) channels which are normally down-regulated in these neurons. TTX-treated neurons also showed abnormal dendritic branching. This model of chronic epilepsy in developing hippocampal neurons demonstrated many changes at the membrane, cellular and synaptic level that may provide new insights into the nature of epileptogenesis in the young brain.

Topics: Animals; Animals, Newborn; Calcium Channels, T-Type; Chronic Disease; Electrophysiology; Epilepsy; Hippocampus; In Vitro Techniques; Neurons; Rats; Rats, Wistar; Reaction Time; Receptors, AMPA; Receptors, GABA-A; Synaptic Transmission; Tetrodotoxin

Cellular excitability of CA1 neurons from a kindled focus in the rat hippocampus is persistently increased. The changes in the underlying voltage-dependent sodium current were characterized under whole-cell voltage-clamp conditions. We compared sodium currents in acutely isolated CA1 neurons from kindled rats with those in matched controls, one day and five weeks after cessation of kindling stimulations. The sodium current in CA1 neurons was tetrodotoxin sensitive and inactivated completely with two time-constants. In 97 cells from control rats, the current evoked at -20 mV consisted of a fast-inactivating component of 3.8 +/- 0.2 nA which decayed with a time-constant of 1.0 +/- 0.1 ms, and a slow-inactivating component of 1.2 +/- 0.1 nA with a time-constant of 3.6 +/- 0.1 ms. The potential of half-maximal inactivation was -72.2 +/- 1.0 mV for the fast-inactivating component and -63.2 +/- 1.0 mV for the slow-inactivating component. The time-constant of recovery at -80 mV was 14.1 +/- 0.4 ms for the fast-inactivating component and 9.3 +/- 0.4 ms for the slow-inactivating component. One day after kindling, the voltage dependence of inactivation of the slow-inactivating and the fast-inactivating component was shifted in the depolarizing direction (3.2 +/- 1.3 and 3.0 +/- 1.3 mV, respectively). The voltage dependence of recovery from inactivation was shifted in the same direction. Five weeks after kindling, the shift in voltage dependence of inactivation was (3.3 +/- 1.2 and 2.9 +/- 1.2 mV, respectively) and was accompanied by a 20% increase in sodium current amplitude. The voltage-dependent activation was not different after kindling. The changes in sodium current inactivation will increase the number of channels available for activation and may enhance the maximum firing rate. This implies that the changes in sodium current inactivation will contribute to the enhanced excitability of pyramidal neurons observed after kindling.

Topics: Animals; Epilepsy; Hippocampus; Kindling, Neurologic; Male; Membrane Potentials; Pyramidal Cells; Rats; Rats, Wistar; Reaction Time; Sodium Channels; Tetrodotoxin; Time Factors

To elucidate the mechanism underlying epileptiform discharges in kindled rats, synaptic responses in kindled basolateral amygdala neurons in vitro were compared with those from control rats by using intracellular and whole cell patch-clamp recordings. In kindled neurons, electrical stimulation of the stria terminalis induced epileptiform discharges. The resting potential, apparent input resistance, current-voltage relationship of the membrane, and the threshold, amplitude, and duration of action potentials in kindled neurons were not different from those in control neurons. The electrical stimulation of stria terminalis elicited excitatory postsynaptic potentials (EPSPs) and DL-2-amino-5-phosphonopentanoic acid (AP5)-sensitive and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive excitatory postsynaptic currents (EPSCs). The amplitude of evoked EPSPs and of evoked AP5-sensitive and CNQX-sensitive EPSCs were enhanced markedly, whereas fast and slow inhibitory postsynaptic potentials (IPSPs) induced by electrical stimulation of lateral amygdaloid nucleus were not significantly different. The rise time and the decay time constant of the evoked CNQX-sensitive EPSCs were shortened, whereas the rise time of the evoked AP5-sensitive EPSCs was shortened, but the decay time constants were not significantly different. In both tetrodotoxin (TTX)-containing medium and low Ca2+ and TTX-containing medium, the frequency and amplitude of spontaneous EPSCs were increased in kindled neurons. These increases are presumably due to nearly synchronous multiquantal events resulted from the increased probability of Glu release at the nerve terminals. The rise time of evoked CNQX- and AP5-sensitive EPSCs and the decay time constant of evoked CNQX-sensitive EPSCs were shortened, suggesting that excitatory synapses at the proximal dendrite and/or the soma in kindled neurons may contribute more effectively to generate evoked EPSCs than those at distal dendrites. In conclusion, the increases in the amplitudes of spontaneous and evoked EPSCs and in the frequency of spontaneous EPSCs may contribute to the epileptiform discharges in kindled neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Amygdala; Animals; Baclofen; Bicuculline; Epilepsy; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Kindling, Neurologic; Male; Neural Inhibition; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Tetrodotoxin

Using microcultured neurons and hippocampal slices, we found that under conditions that completely block AMPA receptors, kainate induces a reduction in the effectiveness of GABAergic synaptic inhibition. Evoked inhibitory postsynaptic currents (IPSCs) were decreased by kainate by up to 90%, showing a bell-shaped dose-response curve similar to that of native kainate-selective receptors. The down-regulation of GABAergic inhibition was not affected by antagonism of metabotropic receptors, while it was attenuated by CNQX. Kainate increased synaptic failures and reduced the frequency of miniature IPSCs, indicating a presynaptic locus of action. In vivo experiments using brain dialysis demonstrated that kainate reversibly abolished recurrent inhibition and induced an epileptic-like electroencephalogram (EEG) activity. These results indicate that kainate receptor activation down-regulates GABAergic inhibition by modulating the reliability of GABA synapses.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Cells, Cultured; Electric Stimulation; Electroencephalography; Embryo, Mammalian; Epilepsy; Evoked Potentials; Female; Functional Laterality; GABA Antagonists; gamma-Aminobutyric Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Neurons; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Kainic Acid; Receptors, Presynaptic; Synaptic Transmission; Tetrodotoxin

The effects of nicardipine, a Ca2+ channel antagonist, on the abnormal excitability of hippocampal CA3 neurons in spontaneously epileptic rats (SER), a double mutant (zi/zi, tm/tm), were examined to elucidate whether or not the abnormality was due to that of Ca2+ channels. An intracellular recording study was performed using brain slice preparations of SER 12-15 weeks of age, when SER showed both tonic convulsions and absence-like seizures. Bath application of nicardipine (10 nM) completely inhibited the depolarizing shifts lasting for 60-120 ms and accompanying repetitive firings on mossy fiber stimulation in SER. However, this drug did not affect the single action potential induced by the mossy fiber stimulation in CA3 neurons of SER and normal Wistar rats. In the CA3 pyramidal neurons of SER, the Ca2+ spikes induced by the depolarizing pulse applied in the cell in the presence of tetrodotoxin and tetraethylammonium had a different configuration from that in normal Wistar rats. Nicardipine also inhibited the Ca2+ spikes in SER CA3 neurons at a concentration (1 nM) that had no effect on those in normal Wistar rats, while the Ca2+ spikes in Wistar rat CA3 neurons were inhibited by 10 nM nicardipine. These findings suggest that the abnormal excitability of CA3 pyramidal neurons in SER might be attributed to abnormalities of the Ca2+ channels, and that the Ca2+ channel antagonist may be effective as an antiepileptic drug.

Topics: Animals; Calcium; Calcium Channel Blockers; Endoplasmic Reticulum, Smooth; Epilepsy; Hippocampus; Membrane Potentials; Nerve Fibers; Nicardipine; Pyramidal Cells; Rats; Rats, Wistar; Tetraethylammonium Compounds; Tetrodotoxin

Layer II of the entorhinal cortex (EC) provides the first step in the hippocampal trisynaptic loop via the perforant path projection to the dentate gyrus. While a great deal is known about this projection and the properties of the dentate granule cells, much less information is available concerning the properties of and synaptic inputs to the cells of origin of the pathway in layer II. The present experiments have employed a slice preparation of the rat EC to study the intrinsic membrane properties and synaptic organization of layer II neurons. Two types of neurons could be identified electrophysiologically. The majority were designated type I and displayed a pronounced time-dependent inward rectification in the hyperpolarizing direction. Type II displayed little evidence of this characteristic. However, morphological examination suggested that both types were spiny stellate neurons projecting via the perforant path. Synaptic responses of both types displayed evidence of excitatory inputs mediated by both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors. In general, however, at low frequencies the responses were dominated by inhibitory inputs mediated by both GABAA and GABAB receptors. At higher frequencies the bias was shifted much more toward excitation. The contribution of synaptic and intrinsic properties of layer II neurons to the processing capabilities of the EC is discussed.

Topics: Animals; Cell Membrane; Dendrites; Electrophysiology; Entorhinal Cortex; Epilepsy; Evoked Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Interneurons; Neurons; Rats; Rats, Wistar; Receptors, AMPA; Receptors, GABA; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Synapses; Tetrodotoxin

We investigated the effect of in vivo administration of an antiepileptic drug, phenytoin, on the saxitoxin binding capacity of receptor site 1 of the Na+ channel alpha-subunit, and the expression activity of the channel messenger RNA in epileptic El mouse brains, as compared with parental ddY mice. Subchronic treatment with phenytoin (25 mg/kg per day) for 14 days increased the [3H]saxitoxin binding to brain-derived synaptic membranes of both El and control ddY mice in a time dependent manner. This increase plateaued at 21 +/- 4% in El mice and 28 +/- 3% in ddY control mice after administration of phenytoin for seven days. After cessation of treatment with phenytoin, [3H]saxitoxin binding capacity returned to the basal level within two weeks in both ddY and El brains. Scatchard plot analysis revealed that the phenytoin treatment caused a 20-30% increase in maximum binding capacity of [3H]saxitoxin binding without any change in equilibrium dissociation constant in the brain cortical synaptic membranes of both epileptic El and control ddY mice. A single injection of phenytoin (25 mg/kg) elevated the level of Na+ channel messenger RNA within 1 h in ddY mouse brains. The increase in Na+ channel messenger RNA reached a peak (about 80% increase) after 5 h of phenytoin administration in a concentration-dependent manner (6.25-50 mg/kg). On the other hand, in El mouse brains, Na+ channel messenger RNA was not elevated until more than 5 h after phenytoin injection, and was increased by only about 33%.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amphibian Proteins; Animals; Carrier Proteins; Cerebral Cortex; Epilepsy; Mice; Mice, Neurologic Mutants; Nerve Tissue Proteins; Phenytoin; RNA, Messenger; Saxitoxin; Sodium Channels; Synaptosomes; Tetrodotoxin; Up-Regulation; Veratridine

Pentylenetetrazol is a chemical convulsant, often used to generate experimental seizures, that is thought to act as a GABA antagonist. In the urethane anesthetized rat, pentylenetetrazol produces a characteristic epileptiform discharge in the dentate gyrus. The hypothesis that this discharge is generated by activity in the entorhinal cortex was examined in this study. Recording electrodes were placed in the dentate gyrus bilaterally and neuronal activity was recorded after administration of pentylenetetrazol. A laminar analysis of the convulsant-induced activity was compared to responses evoked by angular bundle stimulation (n = 6). Both convulsant-induced and evoked activity were negative-going in the molecular layer of the dentate gyrus. In other animals tetrodotoxin (TTX) was injected into the right entorhinal cortex before the onset of epileptiform activity (n = 5). The TTX prevented epileptiform activity in the right dentate gyrus. Injection of TTX after the onset of epileptiform activity caused the epileptiform activity to cease on the side of injection (n = 5). These experiments support the hypothesis that pentylenetetrazol specifically activates the entorhinal cortex to produce the epileptiform activity recorded in the dentate gyrus. This selective activation suggests that the mechanism of action of pentylenetetrazol, as a convulsant, is not simply as an antagonist of GABA receptors.

Topics: Anesthesia; Animals; Cerebral Cortex; Electric Stimulation; Epilepsy; Hippocampus; Male; Neurons, Efferent; Pentylenetetrazole; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Urethane

Conventional intracellular recordings were made from neurons located in the superficial/middle layers of human temporal neocortical slices obtained from patients undergoing neurosurgical procedures for the treatment of epilepsy or brain tumour. In most of the neurons, inward membrane rectification was observed when the cell was depolarized or hyperpolarized from rest by intracellular injection of positive or negative current pulses. Bath application of tetrodotoxin abolished the depolarizing inward rectification, but not the "anomalous rectification" in the hyperpolarizing direction. Single action potential firing was followed by a fast afterhyperpolarization, a depolarizing afterpotential and a medium afterhyperpolarization, while a slower afterhyperpolarization was seen following repetitive firing. Blockade of Ca2+ channels with Cd2+ diminished all three types of afterhyperpolarization. Although the repetitive firing pattern in all cells indicated that they discharge in a regular-spiking fashion, 63% of the cells fired tonically in the initial part of discharge, while the remaining 37% of the cells fired phasically. Frequency-current plot for the initial interspike intervals during long depolarizing pulses revealed primary and secondary ranges of firing. Spike frequency adaptation was also observed. In conclusion, our experiments indicate that human neocortical cells in the superficial/middle layers display electrophysiological characteristics that are similar to those described in rodent and feline neocortices.

Topics: Action Potentials; Cadmium; Cerebral Cortex; Electric Stimulation; Electrophysiology; Epilepsy; Humans; In Vitro Techniques; Membrane Potentials; Microelectrodes; Neurons; Temporal Lobe; 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

Recordings from the CA3 region of hippocampal slices indicate a developmental change in the divalent cation sensitivity of the response elicited by N-methyl-D-aspartate (NMDA) application. In parallel experiments a developmental difference is demonstrated in the capacity of extracellular calcium to modulate electrographic seizure generation. Calcium modulation of the NMDA-elicited response may contribute to the pronounced capacity of immature hippocampus to generate electrographic seizures. Under these conditions activity dependent changes in extracellular calcium could have a greater influence on ion flow produced by activation of the NMDA receptor. The possibility that changes in the receptor isoform may occur during development would have widespread implications for normal cognitive functions and dysfunctions during brain maturation.

Topics: Age Factors; Animals; Calcium; Calcium Channels; Drug Interactions; Epilepsy; GABA Antagonists; Hippocampus; Ion Channel Gating; N-Methylaspartate; Penicillins; Picrotoxin; Quisqualic Acid; Rats; Receptors, N-Methyl-D-Aspartate; Tetrodotoxin

In order to analyze the elementary mechanisms underlying caffeine-induced epileptiform discharges, hippocampal slices of guinea pigs were exposed to this drug. When the bath concentration of caffeine exceeded 0.2 mM, periodically occurring paroxysmal depolarizations (PD) in CA3 neurons appeared. They were accompanied by declines of extracellular free calcium concentration and were suppressed by the organic calcium antagonists verapamil and flunarizine. PD-like fluctuations of the membrane potential could be evoked also in CA3 neurons functionally isolated by tetrodotoxin (TTX). The observations indicate that caffeine-induced PD are generated endogenously and that transmembranous calcium currents contribute to these mechanisms.

Topics: Animals; Caffeine; Calcium; Epilepsy; Flunarizine; Guinea Pigs; Hippocampus; In Vitro Techniques; Membrane Potentials; Tetrodotoxin

We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculine-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 microM) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50-150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.

Topics: Action Potentials; Animals; Calcium Channels; Electric Stimulation; Epilepsy; Female; Hippocampus; In Vitro Techniques; Neurons; Pyramidal Tracts; Rats; Rats, Inbred Strains; Sodium Channels; Tetrodotoxin; Verapamil

Organotypic cultures of rat hippocampal slices were maintained for periods of up to 12 weeks in vitro. Cultures adopted a two-dimensional architecture whilst retaining the subfields characteristic of intact hippocampal slices. Coventional intracellular onset of spontaneous long-lasting epileptiform activity. Epileptiform activity characteristic of both interictal and ictal events (paroxysmal depolarising shifts, tonic/clonic phases and afterdischarges) was observed in the absence of pharmacological manipulation or of orthodromic stimulation. Epileptiform activity was abolished in the presence of high Mg2+ concentration or tetrodotoxin, agents known to block synaptic transmission. In addition, the frequency of epileptiform events was independent of membrane potential and the amplitude of the paroxysmal depolarising shift (PDS) displayed a near linear relationship with membrane potential. The PDS could be reversed at potentials approaching synaptic equilibrium potential. The N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphonovalerate (DL-APV) dose-dependently reduced both the amplitude and duration of the spontaneous paroxysmal shift, having no effect on the initiation of the event or the resting membrane parameters of the neurone. DL-APV also attenuated a late component of the synaptically evoked excitatory postsynaptic potentials (epsp) not observed in non-epileptiform neurones. Application of GABAA receptor antagonists bicuculline or picrotoxin converted interictal events to ictus. In the presence of these agents, ictal events were up to 90 s in duration. These results suggest that long-term culturing of hippocampal explants leads to an alteration in the balance of excitatory and inhibitory synaptic activity. This allows the expression of an excitatory amino acid depolarisation acting through NMDA receptors which contributes to the generation and maintenance of spontaneous epileptiform activity which is synaptic in origin.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Animals; Culture Techniques; Epilepsy; Hippocampus; Magnesium; Membrane Potentials; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Tetrodotoxin; Time Factors; Valine

The epileptogenic properties of the mast cell degranulating peptide (MCD) have been investigated in the CA3 region of the hippocampal slice preparation. Brief (3-5 min) bath application of MCD (0.5-2 microM) to CA3 hippocampal neurones produced an enhancement of the spontaneous synaptic activity and the appearance of spontaneous bursts that persisted for several hours. These bursts were network driven and the underlying paroxysmal depolarizing shift met the criteria for a giant excitatory postsynaptic potential (EPSP), with a reversal potential close to 0 mV. Furthermore following the application of MCD, stimulation of the mossy fibres, commissural or temporo-ammonic pathway evoked an EPSP followed by an evoked network burst. The bursts which could be elicited for several hours were reversibly blocked by a brief application of tetrodotoxin (TTX; 1 microM) or cobalt (2 mM). In contrast, prior and concomitant treatment with TTX or cobalt prevented the occurrence of the bursts induced by MCD. The effects of MCD were not due to a blockade of GABAergic inhibition since the toxin did not reduce the fast and slow IPSP. Furthermore, the N-methyl-D-aspartate (NMDA) antagonists D-2-amino-phosphonovalerate (D-APV; 30 microM) or DL-amino-phosphoheptanoic acid (AP-7, 30 microM) did not block the action of MCD, suggesting that the activation of NMDA receptors are neither necessary nor sufficient for MCD-induced bursts. It is concluded that MCD induces in the CA3 region long-lasting changes in the synaptic responses which may be mediated through a presynaptic mechanism.

Topics: Action Potentials; Animals; Bee Venoms; Epilepsy; Evoked Potentials; Hippocampus; In Vitro Techniques; Male; Neurons; Peptides; Pyramidal Tracts; Rats; Rats, Inbred Strains; Tetrodotoxin

Topics: Amino Acids; Animals; Aspartic Acid; Bicuculline; Calcium; Epilepsy; Ethanolamines; Extracellular Space; Glutamates; Hippocampus; Kainic Acid; N-Methylaspartate; Rabbits; Synaptic Membranes; Synaptosomes; Taurine; Tetrodotoxin

Intracellular recordings were obtained in the vitro slice preparation from neurons of lateral and mesial temporal cortex removed from human epileptics suffering from intractable temporal lobe seizures. Spontaneous rhythmic synaptic events, which were capable of triggering action potential discharge, were observed in many neurons, particularly in mesial tissue slices. Such activity may reflect the epileptogenic capacity of this human cortex.

Topics: Action Potentials; Cerebral Cortex; Epilepsy; Hippocampus; Humans; Neurons; Seizures; Temporal Lobe; Tetrodotoxin