tetrodotoxin and Epilepsy--Temporal-Lobe

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

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

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

Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA(A) receptor (GABA(A)R) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA(A)R signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA(A)R responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA(A)R responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA(A)Rs, or by a change in the conductance of GABA(A)Rs. Rather, A(1)R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA(A)R currents. As depolarizing GABA(A)R signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA(A)R signaling may represent an important mechanism by which adenosine can limit seizure activity.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Animals; CA3 Region, Hippocampal; Chlorides; Epilepsy, Temporal Lobe; GABA Agonists; Gramicidin; In Vitro Techniques; Male; Membrane Potentials; Muscimol; Neural Conduction; Patch-Clamp Techniques; Pyramidal Cells; Rats; Rats, Wistar; Receptors, GABA-A; Seizures; Signal Transduction; Tetrodotoxin

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

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

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

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

One factor common to many neurological insults that can lead to acquired epilepsy is a loss of afferent neuronal input. Neuronal activity is one cellular mechanism implicated in transducing deafferentation into epileptogenesis. Therefore the effects of chronic activity blockade on seizure susceptibility and its underlying mechanisms were examined in organotypic hippocampal slice cultures treated chronically with the sodium channel blocker, tetrodotoxin (TTX), or the N-methyl-D-aspartate receptor (NMDAR) antagonist, D-2-amino-5-phosphonovaleric acid (D-APV). Granule cell field potential recordings in physiological buffer revealed spontaneous electrographic seizures in 83% of TTX-, 9% of D-APV-, but 0% of vehicle-treated cultures. TTX-induced seizures were not associated with membrane property alterations that would elicit granule cell hyperexcitability. Seizures were blocked by glutamate receptor antagonists, suggesting that plasticity in excitatory synaptic circuits contributed to seizures. The morphology of granule cells and their mossy fiber axons remained largely unchanged, and the number of synapses onto granule cells measured immunohistochemically was not increased in TTX- or D-APV-treated cultures. However, voltage-clamp recordings revealed that miniature excitatory postsynaptic current frequency and kinetics were increased and miniature inhibitory postsynaptic current kinetics were decreased in D-APV- and TTX-treated cultures compared with vehicle. Changes were more profound and qualitatively different in TTX- compared with D-APV-treated cultures, consistent with the dramatic effects of TTX treatment on seizure expression. We propose that chronic blockade of action potentials by TTX induces homeostatic responses including plasticity of both excitatory and inhibitory synapses. Removal of TTX unmasks the impact of these synaptic plasticities on local circuit excitability, resulting in spontaneous seizures.

Topics: 2-Amino-5-phosphonovalerate; Action Potentials; Anesthetics, Local; Animals; Epilepsy, Temporal Lobe; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Neuronal Plasticity; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Reaction Time; Receptors, N-Methyl-D-Aspartate; Seizures; Synapses; Tetrodotoxin

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

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

Mossy fiber sprouting is a common abnormality found in patients and models of temporal lobe epilepsy. The role of mossy fiber sprouting in epileptogenesis is unclear, and its blockade would be useful experimentally and perhaps therapeutically. Results from previous attempts to block mossy fiber sprouting have been disappointing or controversial. In some brain regions, prolonged application of the sodium channel blocker tetrodotoxin prevents axon sprouting and posttrauma epileptogenesis. The present study tested the hypothesis that prolonged, focal infusion of tetrodotoxin would block mossy fiber sprouting after an epileptogenic treatment.. Adult rats were treated with pilocarpine to induce status epilepticus. Several hours to 3 days after pilocarpine treatment, a pump with a cannula directed toward the dentate gyrus was implanted to deliver 10 microM tetrodotoxin or vehicle alone at 0.25 microl/h. This method blocks local EEG activity in the hippocampus (Galvan et al. J Neurosci 2000; 20:2904-16). After 28 days of continuous infusion, rats were perfused with fixative, and their hippocampi analyzed anatomically with stereologic techniques.. Tetrodotoxin infusion was verified immunocytochemically in tetrodotoxin-treated but not vehicle-treated hippocampi. Tetrodotoxin-infused and vehicle-infused hippocampi displayed similar levels of hilar neuron loss. The Timm stain revealed mossy fiber sprouting regardless of whether hippocampi were treated with tetrodotoxin infusion, vehicle infusion, or neither.. Prolonged infusion of tetrodotoxin did not block mossy fiber sprouting. This finding suggests that sodium channel-mediated neuronal activity is not necessary for mossy fiber sprouting after an epileptogenic treatment.

Topics: Animals; Cell Count; Dentate Gyrus; Electroencephalography; Epilepsy, Temporal Lobe; Immunohistochemistry; Infusions, Parenteral; Male; Mossy Fibers, Hippocampal; Pharmaceutical Vehicles; Pilocarpine; Rats; Rats, Sprague-Dawley; Sodium Channel Blockers; Staining and Labeling; Status Epilepticus; Tetrodotoxin

The persistent sodium current is a common target of anti-epileptic drugs and contributes to burst firing. Intrinsically burst firing subicular neurons are involved in the generation and spread of epileptic activity. We measured whole-cell sodium currents in pyramidal neurons isolated from the subiculum resected in drug-resistant epileptic patients and in rats. In half of the cells from both patients and rats, the sodium current inactivated within 500 ms at -30 mV. Others displayed a tetrodotoxin-sensitive slowly or non-inactivating sodium current of up to 53% of the total sodium current amplitude. Compared with the transient sodium current in the same cells, this persistent sodium current activated with normal kinetics but its voltage-dependent activation occurred 7 mV more hyperpolarized. Depolarizing voltage steps that lasted 10 s completely inactivated the persistent sodium current. Its voltage dependence did not differ from that of the transient sodium current but its slope was less steep. The voltage dependence and kinetics of the persistent sodium current in cells from patients were not different from that in subicular cells from rats. The current density and the relative amplitude contribution were 3-4 times greater in neurons from drug-resistant epilepsy patients. The abundant presence of persistent sodium current in half of the subicular neurons could lead to a larger number of neurons with intrinsic burst firing. The extraordinarily large amplitude of the persistent sodium current in this subset of subicular neurons might explain why these patients are susceptible to seizures and hard to treat pharmacologically.

Topics: Animals; Cells, Cultured; Dose-Response Relationship, Radiation; Electric Stimulation; Epilepsy, Temporal Lobe; Hippocampus; Humans; Ion Channel Gating; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Wistar; Sodium; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Functional properties of astrocytes were investigated with the patch-clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco-resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammon's horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to -130 mV. Comparative recordings were performed in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE. During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears reasonable to suggest that astrocytes in human sclerotic tissue return to an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release, and may thus contribute to seizure generation in this particular condition of human TLE.

Topics: Adult; Astrocytes; Calcium-Binding Proteins; Epilepsy, Temporal Lobe; Female; Gene Expression; Hippocampus; Humans; Kinetics; Male; Membrane Potentials; Nerve Growth Factors; Patch-Clamp Techniques; Potassium; Potassium Channels; Potassium Channels, Inwardly Rectifying; RNA, Messenger; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Sclerosis; Sodium; Tetrodotoxin

Glutamate receptor-mediated changes in light transmittance were imaged in the dentate gyri of the epileptic hippocampi, taken from patients with temporal lobe epilepsy and the rat pilocarpine model, to investigate epilepsy-associated alterations in activity-induced cell swelling. A static pattern of light transmittance corresponded to the layered structure of dentate gyrus and reflected epilepsy-associated alterations. Hypoosmotic stress produced more than 35% of dynamic changes in the increase of light transmittance as a reflection of osmotic swelling in the epileptic dentate gyri. This degree of increase was not different from the increase observed in control dentate gyri, suggesting that the capability of osmotically regulating cell volume was preserved in the epileptic dentate gyri. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) induced activity-dependent swelling and an increase in light transmittance by 60.5% in the control dentate gyri, whereas the degree of increase in the epileptic dentate gyri remained 17.9% in response to AMPA. Selective attenuation of light transmittance in response to AMPA in the epileptic but not control dentate gyri suggested a possible alteration in the swelling properties of the epileptic dentate gyri that are linked to the AMPA receptor activation. Surviving cells in the epileptic hippocampus may have a mechanism of downregulating neuronal activity-dependent swelling to maintain optimal cell volume during repeated network hyperexcitation in epilepsy.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adult; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Brain Edema; Dentate Gyrus; Epilepsy, Temporal Lobe; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Humans; Light; Male; Membrane Potentials; Neurons; Optics and Photonics; Organ Culture Techniques; Osmotic Pressure; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Tetrodotoxin

1. Properties of voltage-dependent Na+ currents were investigated in forty-two dentate granule cells (DGCs) acutely isolated from the resected hippocampus of twenty patients with therapy-refractory temporal lobe epilepsy (TLE) using the whole-cell patch-clamp technique. 2. Depolarizing voltage commands elicited large, rapidly activating and inactivating Na+ currents (140 pS microm-2; 163 mM extracellular Na+) that were reduced in amplitude by lowering the Na+ gradient (43 mM extracellular Na+). At low temperatures (8-12 C), the time course of Na+ currents slowed and could be well described by the model of Hodgkin & Huxley. 3. Na+ currents were reversibly blocked by tetrodotoxin (TTX) and saxitoxin (STX) with a half-maximal block of 4.7 and 2.6 nM, respectively. In order to reduce series resistance errors, the Na+ current was partially blocked by low toxin concentrations (10-15 nM) in the experiments described below. Under these conditions, Na+ currents showed a threshold of activation of about -50 mV, and the voltages of half-maximal activation and inactivation were -29 and -55 mV, respectively. 4. The time course of recovery from inactivation could be described with a double-exponential function (time constants, 3-20 and 60-200 ms). The rapid and slow time constants showed a distinct voltage dependence with maximal values around -55 and -80 mV, respectively. These properties contributed to a reduction of the Na+ currents during repetitive stimulation that was more pronounced with higher stimulation frequencies and also showed a dependence on the holding potential. 5. In summary, the most striking features of DGC Na+ currents were the large current density and the presence of a current component showing a slow recovery from inactivation. Our data provide a basis for comparison with properties of Na+ currents in animal models of epilepsy, and for the study of drug actions in therapy-refractory epilepsy.

Topics: Adult; Algorithms; Dentate Gyrus; Electric Stimulation; Electrophysiology; Epilepsy, Temporal Lobe; Humans; In Vitro Techniques; Membrane Potentials; Microelectrodes; Patch-Clamp Techniques; Saxitoxin; Sodium Channels; Tetrodotoxin