tetrodotoxin and Seizures

Topics: Animals; Brain Chemistry; Cyclic AMP; Cyclic GMP; Humans; Phenytoin; Potassium; Seizures; Tetrodotoxin

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

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

Spreading depression (SD) is an intense and prolonged depolarization in the central nervous systems from insect to man. It is implicated in neurological disorders such as migraine and brain injury. Here, using an in vivo mouse model of focal neocortical seizures, we show that SD may be a fundamental defense against seizures. Seizures induced by topical 4-aminopyridine, penicillin or bicuculline, or systemic kainic acid, culminated in SDs at a variable rate. Greater seizure power and area of recruitment predicted SD. Once triggered, SD immediately suppressed the seizure. Optogenetic or KCl-induced SDs had similar antiseizure effect sustained for more than 30 min. Conversely, pharmacologically inhibiting SD occurrence during a focal seizure facilitated seizure generalization. Altogether, our data indicate that seizures trigger SD, which then terminates the seizure and prevents its generalization.

Topics: 4-Aminopyridine; Animals; Bicuculline; Brain Stem; Cortical Spreading Depression; Depression; Female; Gene Knock-In Techniques; Kainic Acid; Male; Mice; Nervous System; Optogenetics; Penicillins; Potassium Channel Blockers; Seizures; Tetrodotoxin

Tetrodotoxin (TTX) is an extremely toxic marine compound produced by different genera of bacteria that can reach humans through ingestion mainly of pufferfish but also of other contaminated fish species, marine gastropods or bivalves. TTX blocks voltage-gated sodium channels inhibiting neurotransmission, which in severe cases triggers cardiorespiratory failure. Although TTX has been responsible for many human intoxications limited toxicological data are available. The recent expansion of TTX from Asian to European waters and diversification of TTX-bearing organisms entail an emerging risk of food poisoning. This study is focused on the acute toxicity assessment of TTX administered to mice by oral gavage following macroscopic and microscopic studies. Necropsy revealed that TTX induced stomach swelling 2 h after administration, even though no ultrastructural alterations were further detected. However, transmission electron microscopy images showed an increase of lipid droplets in hepatocytes, swollen mitochondria in spleens, and alterations of rough endoplasmic reticulum in intestines as hallmarks of the cellular damage. These findings suggested that gastrointestinal effects should be considered when evaluating human TTX poisoning.

Topics: Administration, Oral; Animals; Brain; Endoplasmic Reticulum, Rough; Female; Intestines; Kidney; Liver; Lung; Mice; Microscopy, Electron, Transmission; Mitochondria; Myocardium; Neurotoxins; Paralysis; Seizures; Spleen; Stomach; Tetrodotoxin; Toxicity Tests, Acute

Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

Topics: Adenosine Triphosphate; Brain Waves; Calcium; Calmodulin; Cell Membrane; Cells, Cultured; Hippocampus; Humans; Magnesium; Patch-Clamp Techniques; Seizures; Tetrodotoxin; Voltage-Gated Sodium Channel Blockers; Voltage-Gated Sodium Channels

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

The antidepressant drug fluoxetine (FLX) has been shown to exert antiepileptic effects in several animal models, but mixed preclinical findings and occasional reports of proconvulsant effects have led to hesitation towards its use in epileptic people. Despite being developed as a selective serotonin reuptake inhibitor, FLX has numerous other targets in the brain. One of the proposed targets is the neuronal sodium channel, which is inhibited by many existing antiepileptic drugs. In this study, we used electrophysiological methods in a brain slice model of seizures to test for anticonvulsant and Na(+) channel-blocking effects of FLX. This approach allowed us to use a single biological system to study the effects of FLX on (1) epileptiform activity, (2) Na(+)-dependent action potential generation, and (3) the persistent Na(+) current (I(NaP)). We found that FLX was anticonvulsant in a dose- and time-dependent manner, and that this action was accompanied by strong I(NaP) inhibition and impairment of repetitive firing. These findings suggest that the effect of FLX on active membrane properties is similar to that of many antiepileptic drugs, and that this action may contribute to anticonvulsant effects.

Topics: Action Potentials; Animals; Anticonvulsants; Antidepressive Agents, Second-Generation; Dose-Response Relationship, Drug; Electrophysiological Phenomena; Fluoxetine; In Vitro Techniques; Male; Mice; Neurons; Olfactory Bulb; Seizures; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

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

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

Damage to the hippocampus (HPC) using the excitotoxin N-methyl-D-aspartate (NMDA) can cause retrograde amnesia for contextual fear memory. This amnesia is typically attributed to loss of cells in the HPC. However, NMDA is also known to cause intense neuronal discharge (seizure activity) during the hours that follow its injection. These seizures may have detrimental effects on retrieval of memories. Here we evaluate the possibility that retrograde amnesia is due to NMDA-induced seizure activity or cell damage per se. To assess the effects of NMDA induced activity on contextual memory, we developed a lesion technique that utilizes the neurotoxic effects of NMDA while at the same time suppressing possible associated seizure activity. NMDA and tetrodotoxin (TTX), a sodium channel blocker, are simultaneously infused into the rat HPC, resulting in extensive bilateral damage to the HPC. TTX, co-infused with NMDA, suppresses propagation of seizure activity. Rats received pairings of a novel context with foot shock, after which they received NMDA-induced, TTX+NMDA-induced, or no damage to the HPC at a recent (24 hours) or remote (5 weeks) time point. After recovery, the rats were placed into the shock context and freezing was scored as an index of fear memory. Rats with an intact HPC exhibited robust memory for the aversive context at both time points, whereas rats that received NMDA or NMDA+TTX lesions showed a significant reduction in learned fear of equal magnitude at both the recent and remote time points. Therefore, it is unlikely that observed retrograde amnesia in contextual fear conditioning are due to disruption of non-HPC networks by propagated seizure activity. Moreover, the memory deficit observed at both time points offers additional evidence supporting the proposition that the HPC has a continuing role in maintaining contextual memories.

Topics: Amnesia, Retrograde; Animals; Fear; Female; Hippocampus; Memory; N-Methylaspartate; Neurotoxins; Rats; Rats, Long-Evans; Seizures; Tetrodotoxin; Time Factors

Inflammation influences several steps of adult neurogenesis, but whether it regulates the functional integration of the new neurons is unknown. Here, we explored, using confocal microscopy and whole-cell patch-clamp recordings, whether a chronic inflammatory environment affects the morphological and electrophysiological properties of new dentate gyrus granule cells, labeled with a retroviral vector encoding green fluorescent protein. Rats were exposed to intrahippocampal injection of lipopolysaccharide, which gave rise to long-lasting microglia activation. Inflammation caused no changes in intrinsic membrane properties, location, dendritic arborization, or spine density and morphology of the new cells. Excitatory synaptic drive increased to the same extent in new and mature cells in the inflammatory environment, suggesting increased network activity in hippocampal neural circuitries of lipopolysaccharide-treated animals. In contrast, inhibitory synaptic drive was more enhanced by inflammation in the new cells. Also, larger clusters of the postsynaptic GABA(A) receptor scaffolding protein gephyrin were found on dendrites of new cells born in the inflammatory environment. We demonstrate for the first time that inflammation influences the functional integration of adult-born hippocampal neurons. Our data indicate a high degree of synaptic plasticity of the new neurons in the inflammatory environment, which enables them to respond to the increase in excitatory input with a compensatory upregulation of activity and efficacy at their afferent inhibitory synapses.

Topics: Analysis of Variance; Animals; Calcium-Binding Proteins; Dendritic Spines; Dose-Response Relationship, Radiation; Ectodysplasins; Electric Stimulation; Electroencephalography; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Green Fluorescent Proteins; Hippocampus; In Vitro Techniques; Inflammation; Inhibitory Postsynaptic Potentials; Lipopolysaccharides; Lysine; Male; Microfilament Proteins; Microscopy, Confocal; Neurogenesis; Neurons; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Sprague-Dawley; Seizures; Tetrodotoxin; Time Factors

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

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

Neural networks operate robustly despite destabilizing factors, ranging from gene product turnover to circuit refinement, throughout life. Maintaining functional robustness of neuronal networks critically depends upon forms of homeostatic plasticity including synaptic scaling. Synaptic strength and intrinsic excitability have been shown to "scale" (up or down) in response to altered ambient activity levels, and this has led to the general idea that homeostatic plasticity operates along a continuum. After 48 hours of activity deprivation, cultured hippocampal networks exhibited a homeostatic-type reconfiguration that was discrete: a switch from spontaneous spiking to oscillatory bursting. Blockade of fast glutamatergic and GABAergic transmission abolished spontaneous network bursting, but the majority of neurons exhibited intrinsic bursting in response to current injection, which was not the case in control tissue. This de novo intrinsic bursting could be blocked by cadmium chloride, suggesting that this bursting involves calcium mechanisms. Immunohistochemistry confirmed that activity-deprived slice cultures exhibited a widespread upregulation of voltage-dependent calcium channels compared with controls. Calcium imaging studies from activity-deprived slices demonstrated that spontaneous bursting was not a local behavior, but rather a global, synchronous phenomenon, reminiscent of seizure activity. These data suggest that the input/output transformation of individual neurons undergoing homeostatic remodeling is more complex than simple scaling. Network consequences of this transformation include network destabilization of epileptic proportions. Spontaneous activity plays a critical role in actively maintaining homeostatic balance in networks, which is lost after activity deprivation.

Topics: Anesthetics, Local; Animals; Animals, Newborn; Calcium; Excitatory Postsynaptic Potentials; Hippocampus; Mice; Nerve Net; Neural Inhibition; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Seizures; Tetrodotoxin

Febrile (fever-induced) seizures (FS) are the most common form of seizures during childhood and have been associated with an increased risk of epilepsy later in life. The relationship of FS to subsequent epilepsy is, however, still controversial. Insights from animal models do indicate that especially complex FS are harmful to the developing brain and contribute to a hyperexcitable state that may persist for life. Here, we determined long-lasting changes in neuronal excitability of rat hippocampal CA1 pyramidal cells after prolonged (complex) FS induced by hyperthermia on postnatal day 10. We show that hyperthermia-induced seizures at postnatal day 10 induce a long-lasting increase in the hyperpolarization-activated current I(h). Furthermore, we show that a reduction in the amount of spike broadening and in the amplitude of the slow afterhyperpolarization following FS are also likely to contribute to the hyperexcitability of the hippocampus long term.

Topics: Action Potentials; Anesthetics, Local; Animals; Animals, Newborn; Dose-Response Relationship, Radiation; Electric Stimulation; Fever; Hippocampus; In Vitro Techniques; Male; Pyramidal Cells; Pyrimidines; Rats; Rats, Sprague-Dawley; Seizures; 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

Renal failure has been viewed as a state of cellular calcium toxicity due to the retention of small fast-acting molecules. We have tested this hypothesis and identified potentially neuroexcitatory compounds among a number of putative uremic neurotoxins by examining the acute in vitro effects of these compounds on cultured central neurons. The in vitro neuroexcitatory and synergistic effects of guanidinosuccinate and spermine were also examined in vivo.. The acute effects of 17 candidate uremic neurotoxins on murine spinal cord neurons in primary dissociated cell culture were investigated using the tight-seal whole-cell recording technique. The compounds studied comprised low-molecular-weight solutes like urea, indoles, guanidino compounds, polyamines, purines and phenoles, homocysteine, orotate, and myoinositol. Currents evoked by these compounds were further examined using various ligand- and voltage-gated ion channel blockers. The acute in vivo effects of guanidinosuccinate and spermine were behaviorally assessed following their injection in mice.. It was shown that 3-indoxyl sulfate, guanidinosuccinate, spermine, and phenol evoked significant whole-cell currents. Inward whole-cell current evoked by 3-indoxyl sulfate was not blocked by any of the applied ligand- or voltage-gated ion channel blockers, and the compound appeared to influence miscellaneous membrane ionic conductances, probably involving voltage-gated Ca2+ channels as well. Phenol-evoked outward whole-cell currents were at least partly due to the activation of voltage-gated K+ channels, but may also involve a variety of other ionic conductances. On the other hand, inward whole-cell currents evoked by guanidinosuccinate and spermine were shown to be due to specific interaction with voltage- and ligand-gated Ca2+ channels. Guanidinosuccinate-evoked current was caused by activation of N-methyl-d-aspartate (NMDA) receptor-associated ion channels. Low (micromol/L) concentrations of spermine potentiated guanidinosuccinate-evoked current through the action of spermine on the polyamine binding site of the NMDA receptor complex, whereas current evoked by high (mmol/L) concentrations of spermine alone involved direct activation of voltage-gated Ca2+ channels. Finally, intracerebroventricular administration of 0.25 micromol/L spermine potentiated clonic convulsions induced by guanidinosuccinate. These neuroexcitatory and synergistic effects of guanidinosuccinate and spermine could take place at pathophysiologic concentrations.. The observed in vitro and in vivo effects of uremic retention solutes suggest that the identified compounds could play a significant role in uremic pathophysiology. Some of the compounds tested displayed in vitro and in vivo neuroexcitatory effects that were mediated by ligand- and voltage-gated Ca2+ channels. The findings suggest a mechanism for the involvement of calcium toxicity in the central nervous system complications in renal failure with particular reference to guanidinosuccinate and spermine.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Bicuculline; Calcium Channels; Cells, Cultured; Drug Synergism; Excitatory Amino Acid Antagonists; GABA Antagonists; Guanidines; Membrane Potentials; Mice; Neurons; Nickel; Piperidines; Potassium Channel Blockers; Seizures; Spermine; Spinal Cord; Succinates; Synapses; Tetraethylammonium; Tetrodotoxin; Uremia

Ras-GRF1 is a neuron-specific guanine nucleotide exchange factor for Ras proteins. Mice lacking Ras-GRF1 (-/-) are severely impaired in amygdala-dependent long-term synaptic plasticity and show higher basal synaptic activity at both amygdala and hippocampal synapses (Brambilla et al., 1997). In the present study we investigated the effects of Ras-GRF1 deletion on hippocampal neuronal excitability. Electrophysiological analysis of both primary cultured neurons and adult hippocampal slices indicated that Ras-GRF1-/- mice displayed neuronal hyperexcitability. Ras-GRF1-/- hippocampal neurons showed increased spontaneous activity and depolarized resting membrane potential, together with a higher firing rate in response to injected current. Changes in the intrinsic excitability of Ras-GRF1-/- neurons can entail these phenomena, suggesting that Ras-GRF1 deficiency might alter the balance between ionic conductances. In addition, we showed that mice lacking Ras-GRF1 displayed a higher seizure susceptibility following acute administration of convulsant drugs. Taken together, these results demonstrated a role for Ras-GRF1 in neuronal excitability.

Topics: Action Potentials; Animals; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; Genetic Predisposition to Disease; Glutamate Decarboxylase; Hippocampus; Isoenzymes; Male; Mice; Mice, Knockout; Nerve Net; Patch-Clamp Techniques; Pyramidal Cells; ras-GRF1; Seizures; Synaptic Transmission; Synaptophysin; Tetrodotoxin

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 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

In the present investigation, the rationale for the design, synthesis, and biological evaluation of potent inhibitors of neuronal Na+ channels is described. N,N'-diaryl- and N-aryl-N-aralkylguanidine templates were locked in conformations mimicking the permissible conformations of the flexible diarylguanidinium ion (AS+, AA+, SS+). The resulting set of constrained guanidines termed "lockamers" (cyclophane, quinazoline, aminopyrimidazolines, aminoimidazolines, azocino- and tetrahydroquinolinocarboximidamides) was examined for neuronal Na+ channel blockade properties. Inhibition of [14C]guanidinium ion influx in CHO cells expressing type IIA Na+ channels showed that the aminopyrimidazoline 9b and aminoimidazoline 9d, compounds proposed to lock the N,N'-diarylguanidinium in an SS+ conformation, were the most potent Na+ channel blockers with IC50's of 0.06 microM, a value 17 times lower than that of the parent flexible compound 18d. The rest of the restricted analogues with 4-p-alkyl substituents retained potency with IC50 values ranging between 0.46 and 2.9 microM. Evaluation in a synaptosomal 45Ca2+ influx assay showed that 9b did not exhibit high selectivity for neuronal Na+ vs Ca2+ channels. The retention of significant neuronal Na+ blockade in all types of semirigid conformers gives evidence for a multiple mode of binding in this class of compounds and can possibly be attributed to a poor structural specificity of the site(s) of action. Compound 9b was also found to be the most active compound in vivo based on the high level of inhibition of seizures exhibited in the DBA/2 mouse model. The pKa value of 9b indicates that 9b binds to the channel in its protonated form, and log D vs pH measurements suggest that ion-pair partitioning contributes to membrane transport. This compound stands out as an interesting lead for further development of neurotherapeutic agents.

Topics: Animals; Anticonvulsants; Biological Transport; Brain; Calcium; Calcium Channel Blockers; Calcium Channels; CHO Cells; Cricetinae; Drug Design; Female; Guanidine; Imidazoles; Male; Mice; Mice, Inbred DBA; Molecular Conformation; Neurons; Pyrimidines; Rats; Receptors, N-Methyl-D-Aspartate; Seizures; Sodium Channel Blockers; Sodium Channels; Structure-Activity Relationship; Synaptosomes

The VGF gene encodes a neuronal secretory-peptide precursor that is rapidly induced by neurotrophic growth factors and by depolarization in vitro. VGF expression in the animal peaks during critical periods in the developing peripheral and central nervous systems. To gain insight into the possible functions and regulation of VGF in vivo, we have used in situ hybridization to examine the regulation of VGF messenger RNA by experimental manipulations, and have found it to be regulated in the CNS by paradigms that affect electrical activity and by lesion. Inhibition of retinal electrical activity during the critical period of visual development rapidly repressed VGF messenger RNA in the dorsal lateral geniculate nucleus of the thalamus. In the adult, kainate-induced seizures transiently induced VGF messenger RNA in neurons of the dentate gyrus, hippocampus, and cerebral cortex within hours. Cortical lesion strongly induced VGF messenger RNA in ipsilateral cortex within hours, and strongly repressed expression in ipsilateral striatum. Ten days postlesion there was a delayed induction of VGF messenger RNA in a portion of deafferented striatum where compensatory cortical sprouting has been detected. Expression of the neuronal secretory-peptide precursor VGF is therefore modulated in vivo by monocular deprivation, seizure, and cortical lesion, paradigms which lead to neurotrophin induction, synaptic remodeling and axonal sprouting.

Topics: Animals; Central Nervous System; Cerebral Cortex; Excitatory Amino Acid Antagonists; Eye; Geniculate Bodies; Image Processing, Computer-Assisted; In Situ Hybridization; Injections; Kainic Acid; Male; Neurons; Neuropeptides; Protein Biosynthesis; Proteins; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; RNA Probes; RNA, Messenger; Seizures; Tetrodotoxin

To elucidate molecular mechanisms underlying activity-dependent synaptic remodeling in the developing mammalian visual system, we screened for genes whose expression in the lateral geniculate nucleus (LGN) is regulated by spontaneously generated action potentials present prior to vision. Activity blockade did not alter expression in the LGN of 32 known genes. Differential mRNA display, however, revealed a decrease in mRNAs encoding class I major histocompatibility complex antigens (class I MHC). Postnatally, visually driven activity can regulate class I MHC in the LGN during the final remodeling of retinal ganglion cell axon terminals. Moreover, in the mature hippocampus, class I MHC mRNA levels are increased by kainic acid-induced seizures. Normal expression of class I MHC mRNA is correlated with times and regions of synaptic plasticity, and immunohistochemistry confirms that class I MHC is present in specific subsets of CNS neurons. Finally, beta2-microglobulin, a cosubunit of class I MHC, and CD3zeta, a component of a receptor complex for class I MHC, are also expressed by CNS neurons. These observations indicate that class I MHC molecules, classically thought to mediate cell-cell interactions exclusively in immune function, may play a novel role in neuronal signaling and activity-dependent changes in synaptic connectivity.

Topics: Aging; Animals; Brain; Cats; Cell Communication; Embryonic and Fetal Development; Fetus; Gene Expression Regulation; Gene Expression Regulation, Developmental; Genes, MHC Class I; Histocompatibility Antigens Class I; Kainic Acid; Neurons; Organ Specificity; Polymerase Chain Reaction; Rats; RNA, Messenger; Seizures; Synapses; Tetrodotoxin; Transcription, Genetic

Carbamazepine administration causes large increases in extracellular serotonin concentration and dose-related anticonvulsant effects in genetically epilepsy-prone rats (GEPRs). In order to determine the generality of the effect on serotonin, we determined the anticonvulsant ED50 for carbamazepine against maximal electroshock seizures in outbred, non-epileptic Sprague-Dawley rats. We then administered anticonvulsant carbamazepine doses to Sprague-Dawley rats and observed extracellular serotonin concentration in hippocampi by way of microdialysis. We found that administration of carbamazepine, either systemically or through the dialysis probe, resulted in significant and dose-related increases in extracellular serotonin concentration. Basal serotonin release was decreased by tetrodotoxin administration through the dialysis probe. Tetrodotoxin administration through the dialysis probe did not decrease the effect of systemically or focally administered carbamazepine on extracellular serotonin concentration. Similarly, elimination of Ca2+ from the dialysate did not alter the release of serotonin caused by carbamazepine. These findings suggest that the serotonin releasing effect of carbamazepine does not take place by exocytosis and does not require action potentials in the brain area in which the release takes place. Further they suggest that the effect is mediated by an action of carbamazepine directly on serotonergic nerve terminals.

Topics: Animals; Anticonvulsants; Calcium; Carbamazepine; Dose-Response Relationship, Drug; Electroshock; Extracellular Space; Female; Hippocampus; Microdialysis; Rats; Rats, Sprague-Dawley; Seizures; Serotonin; Tetrodotoxin

Cortical structures such as the hippocampus and cerebral cortex are considered to be particularly susceptible to seizure and epileptiform electrical activity and, as such, are the focus of intense investigation relative to hyperexcitability. To determine whether parallel glutamate-mediated hyperexcitability and seizure-like activity in the rat can be generated by neurons irrespective of their origin within the CNS, we maintained cells from the spinal cord,hippocampus, olfactory bulb, striatum, hypothalamus, and cortex in the long-term presence of glutamate receptor antagonists 2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2-3-dione. After removal of chronic (three to 11 weeks) glutamate receptor block, whole-cell patch-clamp recordings from current-clamped neurons (n = 94) revealed an immediate increase in large excitatory postsynaptic potentials and a depolarization of 20-35 mV that was often sustained for recording periods lasting 5 min (54% of 66 neurons from all six areas). The intense activity was not seen in age-matched control neurons not subjected to chronic glutamate receptor block. Selective blockade of ionotropic glutamate receptors showed that the hyperexcitability was due to an enhanced response through both AMPA/kainate and N-methyl-D-aspartate receptors. Relief from chronic glutamate receptor block also increased inhibitory activity, as revealed by an increase in inhibitory postsynaptic currents while neurons were voltage-clamped at -25 mV. These inhibitory postsynaptic currents could be blocked with bicuculline, indicating that they were mediated by an enhanced GABA release. This enhanced GABA activity reduced, but did not eliminate, the glutamate-mediated hyperactivity, shown by an increase in both intracellular Ca2+ and excitatory electrical activity when bicuculline was added. When the glutamate receptor block was removed, cells (n > 1000) from all six regions showed exaggerated Ca2+ activity, characterized by abnormally high increases in intracellular Ca2+, rising from basal levels of 50-100 nM up to 150-1600 nM. Cd2+ eliminated the hyperexcitability by blocking Ca2+ channels, and reducing excitatory transmitter release and response. Fura-2 digital imaging revealed Ca2+ oscillations with periods ranging from 4 to 60 s. Ca2+ peaks in oscillations in oscillations were synchronized among most neurons recorded simultaneously. That synchronization was dependent on a mechanism involving voltage-dependent Na+ channels was demonst

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Brain; Cadmium; Calcium; Cell Survival; Cells, Cultured; Cerebral Cortex; Corpus Striatum; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Hypothalamus; Membrane Potentials; Neurons; Olfactory Bulb; Organ Specificity; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Seizures; Spinal Cord; Tetrodotoxin

Localized injections of 50 microM tetrodotoxin (TTX) in rat hippocampal slices blocked stimulus train-evoked electrographic seizures (EGSs) for several hours. Responses to single stimuli were minimally altered during TTX block of the EGSs. This selective reduction of epileptiform activity could result from general blockade of action potentials in an anatomically distinct group of neurons in the slice. To test this hypothesis, we systematically mapped TTX injection sites in the hippocampal slice, and found that TTX injections that blocked EGSs were nearly always located in or invaded CA2/3 stratum radiatum and/or stratum lacunosum-moleculare. A high degree of recurrent activity in this region contributes to both epileptiform activity and responses to single stimuli; hence our selective inhibition of EGSs suggests a more pharmacologically specific anticonvulsant effect of TTX. Consistent with this hypothesis, we found that low concentrations of TTX (5, 10, or 20 nM) in the perfusion medium blocked EGSs without decreasing the amplitude of extracellular responses to single stimuli. Polysynaptic activity and/or antidromic firing may be particularly vulnerable to TTX action on voltage-gated sodium channels, due to their lower the safety factor for action potential propagation. Selective reduction of this activity may disrupt the abnormal neuronal activity underlying EGSs.

Topics: Animals; Anticonvulsants; Brain Mapping; Dose-Response Relationship, Drug; Electric Stimulation; Hippocampus; In Vitro Techniques; Male; Microinjections; Rats; Rats, Sprague-Dawley; Seizures; Sodium Channels; Tetrodotoxin

Repetitive synchronized neuronal discharging that lasts for seconds and even minutes in in vitro brain slice preparations are important new models in experimental epilepsy. In hippocampal slices from 1-2-week-old rats, individual CA3 pyramidal cells undergo a sustained depolarization during such electrographic seizures, induced by GABAA receptor antagonists. In experiments reported here these events were produced in small isolated segments of the CA3 subfield, measuring only 400-500 microns along the cell body layer. In such minisclices local application of either kynurenic acid or 6-cyano-7-nitroquinoxaline-2-3-dione (CNQX) to the proximal basilar dendrites abolished the synchronized discharges of electrographic seizures. Interictal spikes appeared unaffected by this treatment. Application of these excitatory amino acid receptor antagonists to distal basilar dendrites or apical dendrites was ineffective. In "larger" minislices, measuring 700-1000 microns along the cell body layer, application of kynurenic acid, CNQX, or TTX to the proximal basilar dendrites did not abolish electrographic seizures but instead selectively suppressed the intracellularly recorded sustained depolarization and the coincident slow negative field potential recorded in proximal basilar dendrites. Results of several experiments suggest that electrographic seizures recorded under these conditions were produced by a remote network of "generator cells." Since the remote neurons were unaffected by local application of the drugs, it seemed likely that they continued to undergo a sustained depolarization. Simultaneous blockade of basilar dendritic synapses in the "generator" population abolished electrographic seizures throughout these larger minislices. These results suggest that the sustained depolarization plays a central role in seizure generation and that it does not have to be generated in every neuron, only in a critical number of "generator cells" for a seizure to occur. Taken together, results presented here suggest that the sustained depolarization of electrographic seizures is a separate physiological process from the more rapid repetitive depolarizations of the seizure discharges and is required if electrographic seizures are to occur. This slow depolarization appears to be synaptically mediated and generated exclusively in proximal basilar dendrites. Therefore, in addition to the excitatory synaptic potentials involved in paroxysmal depolarization shift generation, a second

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dendrites; Evoked Potentials; GABA Antagonists; Hippocampus; In Vitro Techniques; Kynurenic Acid; Membrane Potentials; Pyramidal Tracts; Quinoxalines; Rats; Seizures; Synapses; Tetrodotoxin

This study evaluates whether the rapid transient increases in glial fibrillary acidic protein (GFAP) mRNA in the hippocampus after electrolytic lesions of the entorhinal cortex (EC) are triggered by lesion-induced changes in hippocampal neuronal activity (either the decreases that result from loss of afferent drive or transient increases that occur during lesion production). To evaluate the role of activity, we carried out four experiments: (1) tetrodotoxin (TTX) was injected into the EC to mimic the decreases in afferent drive that occur after lesion; (2) TTX was injected into the EC or hippocampus before producing electrolytic lesions to block any abnormal activity induced during lesion production; (3) the EC was destroyed by aspiration, thus creating a lesion comparable in size to the electrolytic lesion, without passing direct current; (4) seizures were elicited by stimulating the EC of anesthetized rats, to examine whether electrographic seizures alone can induce the same type of increases in GFAP mRNA as lesions. Our results demonstrated that: (1) TTX injections into the EC did not induce the same increases in GFAP mRNA levels that occurred after EC lesions; (2) animals that received TTX injections into the EC prior to lesions exhibited increases in hippocampal GFAP mRNA that were nearly as great as following EC lesions alone; (3) aspiration lesions of the EC resulted in increases in GFAP mRNA that were comparable to those observed after electrolytic lesions; and (4) seizure-inducing stimulation of the EC resulted in 2-fold increases in GFAP mRNA in the hippocampus 24 hr after stimulation rather than the 5-13-fold increases observed after lesions. These results suggest that lesion-induced changes in hippocampal neuronal activity are not solely responsible for inducing the rapid transient increases in GFAP mRNA levels in the hippocampus ipsilateral to EC lesions.

Topics: Animals; Cerebral Cortex; Electric Stimulation; Evoked Potentials; Glial Fibrillary Acidic Protein; Hippocampus; Kindling, Neurologic; Male; Nerve Degeneration; Neurons; Nucleic Acid Hybridization; Rats; Rats, Sprague-Dawley; RNA Probes; RNA, Messenger; Seizures; Tetrodotoxin; Up-Regulation

We used E1 mice, a ddY mouse-derived, autosomal mutant strain and a model of hereditary sensory-precipitated epilepsy, to test the hypothesis that epileptic susceptibility may be associated with the activity of voltage-dependent ion channels. We examined the saxitoxin binding capacity of the receptor site 1 of the Na+ channel alpha-subunit, the expression activity of the Na+ channel mRNA, the veratridine-induced 22Na+ influx in the brain synaptosomes, and the regional distribution of Na+ channels in the brain. Compared with control ddY mice, in E1 mice which have not experienced seizures, the number of Na+ channels in the brain synaptosomes increased by approximately 20% starting at the fourth postnatal week through the adult stage as determined by [3H]saxitoxin binding assay. Northern blot hybridization analysis showed excess expression of Na+ channel mRNA (by 30-40%) coincidentally with Na+ channel increases. Regional analysis using the saxitoxin binding assay demonstrated approximately 1.3-fold denser distribution of Na+ channels in the cortex and cerebellum but not the hippocampus and midbrain including thalamus of E1 mice compared to ddY mice. Scatchard plot analysis for saxitoxin binding in the cortex of E1 mouse brains revealed higher maximum binding capacity (Bmax) values (ddY, 4.43 +/- 0.28 pmol/mg protein; E1, 5.43 +/- 0.25 pmol/mg protein) without a change in Kd (ddY, 1.05 +/- 0.03 nM; E1, 1.03 +/- 0.01 nM). Lastly, veratridine-evoked 22Na+ influx, sensitive to tetrodotoxin, was increased approximately 45% in the cortical synaptosomes in six-week-old E1 mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aging; Animals; Blotting, Northern; Brain; Cerebral Cortex; Mice; Mice, Inbred Strains; Mice, Neurologic Mutants; Organ Specificity; Poly A; RNA; RNA, Messenger; Saxitoxin; Seizures; Sodium; Sodium Channels; Synaptic Membranes; Tetrodotoxin; Veratridine

Previously, a unique type of epileptiform discharge, recorded in the dentate gyrus, has been identified and termed maximal dentate activation. Maximal dentate activation is defined by the presence of bursts of large amplitude population spikes, associated with a secondary rise in the extracellular potassium and a negative shift of the dc potential. Prior work has linked maximal dentate activation to lengthening of afterdischarges when they are elicited in the hippocampus or outside of the hippocampus in the amygdala. The current study used two approaches to further examine the relationship of maximal dentate activation to seizures in limbic circuits in urethane-anesthetized rats. First, simultaneous recordings were employed to document that during maximal dentate activation, synchronous discharges occurred in the dentate gyrus, cornu Ammonis, subiculum, and entorhinal cortex. From anatomical work, these structures are known to be connected in a hippocampal-parahippocampal loop. The second approach used lesions of the entorhinal cortex to document the importance of this loop in the initiation and maintenance of maximal dentate activation. Both electrolytic and chemical (focal injections of tetrodotoxin) lesions of the entorhinal cortex blocked maximal dentate activation on the side of the lesion. However, maximal dentate activation was maintained on the opposite side, where the hippocampal-parahippocampal loop was intact. Altogether, these data support the hypothesis that maximal dentate activation is a marker for the presence of reverberatory, synchronized paroxysmal activity throughout the hippocampal-parahippocampal loop and that this loop behaves as a unit in epileptogenesis.

Topics: Animals; Differential Threshold; Electric Stimulation; Electrophysiology; Hippocampus; Limbic System; Male; Rats; Rats, Inbred Strains; Seizures; Tetrodotoxin

1. The effects of L-glutamate diethyl ester (GDEE) HCl, glutarate diethyl ester (GlrDEE) and glutarate dimethyl ester (GlrDME) on depolarizing responses to alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionate (AMPA), kainate (Kain), N-methyl-D-aspartate (NMDA) and quisqualate (Quis), and spontaneous paroxysmal discharges (SPDs) were examined. Experiments were performed on slices of rat cingulate cortex using the in vitro grease gap recording technique in nominally Mg(2+)-free Krebs medium. 2. GDEE HCl (3-14 mM) caused a concentration-dependent depolarization of the d.c. baseline potential. L-Glutamate (0.1-0.5 mM), HCl (15 mM) and sucrose (30 mM) also depolarized the baseline. GlrDEE (3-12 mM) and GlrDME (4-26 mM) had no consistent effect on baseline potential. 3. GDEE HCl (10 mM) had no effect on depolarizing responses to AMPA, Kain and NMDA, but caused potentiation of those to Quis with a dose-ratio of 0.53 (0.44-0.63) (n = 4). In two other experiments, where the depolarization of the baseline induced by GDEE HCl was large, a depression of Quis response amplitude was observed. 4. GlrDEE (10 mM) antagonized depolarizing responses to Kain, and to a lesser extent NMDA, with dose-ratios of 2.14 (1.92-2.38) and 1.61 (1.39-1.87), respectively. This concentration of GlrDEE had no effect on AMPA responses, but potentiated Quis responses, with a dose-ratio of 0.64 (0.58-0.71). 5. GlrDME (10 mM) antagonized depolarizing responses to Kain and to Quis, with dose-ratios of 1.66 (1.48-1.85) and 1.22 (1.15-1.29), respectively, and had no effect on responses to NMDA. 6. The SPDs were inhibited by GDEE HCI (IC50 6.7 +/- 0.37mM), GlrDEE (IC50 5.6 +/- 0.38 mM) and GlrDME (IC50 10.4 +/- 0.73 mM). 7. In conclusion, there is little evidence that GDEE HCI is an antagonist of the postsynaptic excitatory amino acid receptors in the rat neocortex, and its effects may result from its contamination with Lglutamate and increased osmolarity of the bathing medium at high concentrations. The deaminated analogues of GDEE are very weak Kain antagonists.

Topics: Amino Acids; Animals; Cerebral Cortex; Deamination; Glutamates; Glutarates; In Vitro Techniques; Male; Membrane Potentials; Rats; Rats, Inbred Strains; Seizures; Tetrodotoxin

Sudden cooling of the isolated spinal cord of frogs results in characteristic seizure-like activity in the hind legs. In the present investigation, these spinal seizures induced by sudden cooling (SSSC) were studied to determine whether excitatory amino acids (EAAs) are involved in the mediation of this activity. The nonspecific EAA antagonist, L-glutamic acid diethyl ester and cis-2,3-piperidine dicarboxylic acid inhibited the clonic and tonic phase of SSSC after intralymphatic or intrathecal administration. The antagonist gamma-D-glutamylaminomethylsulfonic acid and gamma-D-glutamyltaurine also suppressed both phases after intrathecal injections. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid, DL-2-amino-7-phosphonoheptanoic acid, and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid were effective inhibitors of the tonic phase and actually prolonged the duration of the clonic phase, an effect similar to that observed after low doses of gamma-D-glutamylglycine. SSSC were resistant to spinal perfusion to tetrodotoxin (1 microM). The concentrations of glutamate, aspartate, and glycine were increased in the Ringer's solution surrounding rapidly cooled spinal cord slices, but only in cords from species that elicited some magnitude of SSSC, not in cords from species resistant to induction of SSSC. Our data support the hypothesis that EAAs play a role in SSSC via activation of quisqualate receptors.

Topics: Amino Acids; Animals; Anticonvulsants; Anura; Cold Temperature; Dipeptides; Glutamates; In Vitro Techniques; Muscle Contraction; Pipecolic Acids; Receptors, N-Methyl-D-Aspartate; Seizures; Spinal Cord; Tetrodotoxin

Topics: Animals; Brain Chemistry; Brain Injuries; Cycloheximide; Gene Expression Regulation; Hippocampus; Humans; Kindling, Neurologic; Neuronal Plasticity; Neurons; Proto-Oncogene Proteins; Proto-Oncogenes; Rats; Receptors, N-Methyl-D-Aspartate; Seizures; Signal Transduction; Sodium Channels; Synapses; Tetrodotoxin

Hippocampal neurons that were grown for prolonged periods in the continuous presence of agents that interfere with synaptic transmission, especially excitatory synaptic transmission, appeared to become seizure-prone. Washout of the synaptic blocking agents, that had been continuously present for several weeks to several months, caused the population of neurons to produce an abnormal and intense electrical activity. This consisted of two major components: spontaneously arising phasic responses that closely resembled paroxysmal depolarization shifts and, less frequently, slowly rising depolarizations similar to the sustained depolarizations observed during ictus-like episodes in intact cortex or cortical slices. We describe here observations on the role of the N-methyl-D-aspartate (NMDA) and non-NMDA types of glutamate receptors in the generation of these activities.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Electrophysiology; Glutamates; Hippocampus; Neurons; Quinoxalines; Receptors, Glutamate; Receptors, Neurotransmitter; Seizures; Tetrodotoxin

These studies compared the actions of apamin and nicotinic acetylcholine antagonists on seizure genesis within the inferior collicular cortex. In vitro alpha-bungarotoxin, d-tubocurarine and gallamine all competitively displaced [125I]apamin binding to brain sections through the inferior colliculus, while intracollicular microinjection of intermediate doses of apamin (21 pmol), alpha-bungarotoxin (0.3 nmol), d-tubocurarine (0.22 nmol) or gallamine (1.7 nmol) all significantly reduced the seizure initiation threshold current. However, higher doses of apamin did not cause spontaneous seizure activity, while higher doses of the nicotinic acetylcholine antagonists caused spontaneous seizures. Carbamylcholine also produced spontaneous seizures, but did not alter the seizure threshold current. N-Methyl-atropine caused a dose-related elevation of the seizure threshold current, yet microinjection of N-methyl-atropine (10 nmol) into the inferior collicular cortex reversed the effects of alpha-bungarotoxin on seizure threshold, partially opposed the effects of gallamine and d-tubocurarine, and had no effect on the ability of apamin to reduce the seizure threshold current. Thus, both the apamin-sensitive potassium channel and a variety of distinct cholinergic mechanisms contribute in vivo to seizure genesis within the inferior collicular cortex, but not through the same mechanisms.

Topics: Animals; Apamin; Binding, Competitive; Brain Chemistry; Electric Stimulation; Inferior Colliculi; Iodine Radioisotopes; Kinetics; Male; Microinjections; Parasympatholytics; Rats; Rats, Inbred Strains; Receptors, Nicotinic; Seizures; Tetrodotoxin

CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid) was found to be a potent, stereospecific inhibitor of N-methyl-D-aspartate (NMDA)-evoked, but not KCl-evoked, [3H] acetylcholine release from slices of the rat striatum. The concentration-response curve to NMDA was shifted to the right by CGS 19755 (pA2 = 5.94), suggesting a competitive interaction with NMDA-type receptors. CGS 19755 inhibited the binding of [3H]-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid to NMDA-type receptors with an IC50 of 50 nM, making it the most potent NMDA-type receptor antagonist reported to date. CGS 19755 failed to interact with 23 other receptor types as assessed by receptor binding, including the quisqualate- and kainate-type excitatory amino acid receptors. In crude P2 fractions, no evidence was obtained to suggest that CGS 19755 is taken up by an active transport system. Furthermore, CGS 19755 failed to affect the uptake of L-[3H]glutamate, or to interact with aconitine-induced inhibition of L-[3H]glutamate uptake, the latter finding suggesting a lack of membrane-stabilizing or local anesthetic properties. CGS 19755 selectively antagonized the excitatory effect of iontophoretically applied NMDA in the red nucleus of the rat without affecting the excitatory effects of quisqualate. CGS 19755 blocked the harmaline-induced increase in cerebellar cyclic GMP levels at a dose of 4 mg/kg i.p. with a duration of action exceeding 2 hr. CGS 19755 inhibited convulsions elicited by maximal electroshock in rat (ED50 = 3.8 mg/kg i.p. 1 hr after administration) and in mouse (ED50 = 2.0 mg/kg i.p. 0.5 hr after administration). Likewise, convulsions elicited by picrotoxin were inhibited by CGS 19755, whereas the compound was relatively weak in protecting against convulsions elicited by pentylenetetrazole or strychnine. CGS 19755 produced retention performance deficits in a dark avoidance task. However, CGS 19755 did not show a unique propensity for learning and memory disruption compared to other anticonvulsants.

Topics: Acetylcholine; Aconitine; Animals; Anticonvulsants; Aspartic Acid; Avoidance Learning; Binding, Competitive; Darkness; Glutamates; Glutamic Acid; Male; N-Methylaspartate; Pentylenetetrazole; Picrotoxin; Pipecolic Acids; Piperidines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Seizures; Structure-Activity Relationship; Tetrodotoxin

Seizures, neuronal damage and extracellular Ca2+ concentration were studied in rats unilaterally injected in the dorsal hippocampus with quinolinic acid, a brain metabolite with excitotoxic properties. In freely moving animals, in the first 2 h after the injection of a convulsant and neurotoxic dose (156 nmol), quinolinic acid induced a tetrodotoxin-insensitive decrease in the extracellular Ca2+ concentration (nadir 40%) in the injected area, as assessed by brain dialysis coupled to a fluorimetric method for Ca2+ detection. Blockade of quinolinic acid-induced decrements in Ca2+ by 15.6 nmol D-(-)2-amino-7-phosphonoheptanoic acid indicated that this effect was receptor-mediated. Dose-response relationships showed a close association between seizure activity (measured by EEG) and extracellular Ca2+ changes in the injected area. Changes in Ca2+ were apparent at the site of injection prior to the onset of focal seizures and they were not found in the homotypic structure where seizures were conducted. Drugs effective in blocking seizures (carbamazepine and flunarizine) prevented the fall in extracellular Ca2+, while drugs without anticonvulsant activity (ethosuximide and nifedipine) did not. Destruction of nerve cells by quinolinic acid was not prevented by treatment with carbamazepine and flunarizine. The results suggest that the fall in extracellular Ca2+ observed in the first 2 h after quinolinic acid, probably reflecting the ion influx into neurons, is involved in triggering focal seizures but is not related to the occurrence of nerve cell death.

Topics: 2-Amino-5-phosphonovalerate; Amino Acids; Animals; Calcium; Dialysis; Dose-Response Relationship, Drug; Electroencephalography; Hippocampus; Male; Pyridines; Quinolinic Acid; Quinolinic Acids; Rats; Rats, Inbred Strains; Seizures; Tetrodotoxin; Veratridine

The effect of systemic administration of grayanotoxin (GTX)-III, a constituent in leaves of Pieris japonica D. Don with an ability to activate the voltage-sensitive sodium channels in excitable tissues, on general behaviors of animals was studied using Std-ddy mice. Intraperitoneal administration of the toxin (0.1-0.25 mg/kg b. wt.) resulted in a dose-dependent manner in a significant and reversible muscle relaxation, and a profound and long lasting (greater than or equal to 60 min) depression of locomotor activity. Pretreatment with GTX-III caused a profound potentiation of the duration of loss of righting reflex by pentobarbital with a concomitant delay of the onset of convulsive seizures by various convulsants such as strychnine, picrotoxin and pentetrazol. Neither tetrodotoxin (1-5 micrograms/kg, i.p.) nor Ro15-1788 (1-5 mg/kg, i.p.) prevented the GTX-III-induced suppression of locomotor activity. These results suggest that GTX-III may elicit a central depressant action in mice through a molecular mechanism other than activation of the voltage-sensitive sodium channels in the brain.

Topics: Animals; Brain; Depression, Chemical; Diterpenes; Electrophysiology; Ion Channels; Male; Mice; Motor Activity; Muscle Relaxation; Reflex; Seizures; Tetrodotoxin

The ionic dependence and the nature of conductance was examined at slowly inactivating inward current in metacerebral giant cells of Helix pomatia, induced by 50 mM pentylenetetrazol. Ramp and square wave depolarizations in voltage clamp mode revealed, that withdrawal of sodium ions prevented this current to flow. While TTX was ineffective, Mn, Co and Ni-ions and verapamil blocked the current. It is concluded that PTZ, especially in presence of TEA impairs calcium channels, which loose their specificity and transmit sodium ions, with very slow kinetics.

Topics: Animals; Cobalt; Electrophysiology; Helix, Snails; Ions; Manganese; Nickel; Pentylenetetrazole; Seizures; Tetraethylammonium Compounds; Tetrodotoxin; Verapamil

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

Topics: Animals; Bicuculline; Culture Techniques; Evoked Potentials; GABA Antagonists; gamma-Aminobutyric Acid; Guinea Pigs; Hippocampus; Neural Inhibition; Neurons; Picrotoxin; Receptors, Adrenergic, beta; Seizures; Synapses; Synaptic Transmission; Tetrodotoxin; Tubocurarine

Topics: Amino Acids; Animals; Brain; Calcium; Cerebral Cortex; Electric Stimulation; Potassium; Seizures; Tetrodotoxin

1. Extra- and intracellular potentials were recorded from neurons and glia during spreading depression (SD) in cerebral cortex of cats. The glial membrane depolarized during SD and the time course of depolarization was concurrent with the surface DC change of SD. The glial depolarization evoked by 20-Hz repetitive cortical stimulation disappeared during the negative DC shift of SD. Simultaneous recording of the extra- and intracellular potentials from a single glial cell with a coaxial microelectrode showed that the extracellular DC potential change was of opposite polarity to the glial intracellular potential, which suggests that the slow glial depolarization concurrent with SD is not the field potential. In contrast to glial cells, the neuronal burst discharges as well as the neuronal membrane depolarization associated with SD did not show a close relationship to SD: the neuronal membrane depolarization and discharge were frequently delayed by 10-3- s from the onset of the SD slow wave. Sometimes SD was observed without accompanying neuronal depolarization. The degree of neuronal depolarization was not always correlated with the amplitude of the negative wave of SD. 2. The effect of tetrodotoxin (TTX) on the negative DC potential of SD was examined. Simultaneous recording of glial membrane potential and the neuronal unit activity as well as extracellular DC potential and surface DC potential during SD was performed and the TTX-treated cortex was compared with the normal state. TTX did not change the DC level of the cerebral cortex. SD could be evoked by KCl when neuronal discharge was completely abolished by TTX application...

Topics: Animals; Cats; Cerebral Cortex; Cortical Spreading Depression; Electric Stimulation; Electrophysiology; Evoked Potentials; Extracellular Space; Membrane Potentials; Neuroglia; Neurons; Penicillin G; Potassium; Seizures; Tetrodotoxin