Effects of topiramate on sodium-dependent action-potential firing by mouse spinal cord neurons in cell culture.

PURPOSE: The effects of topiramate (TPM) on sodium-dependent action potentials were studied by using cultured mouse spinal cord neurons. METHODS: The ability of TPM to limit (block) depolarization-induced spontaneous repetitive firing (SRF) was determined and compared with corresponding effects of phenytoin (PHT) and lamotrigine (LTG) in cultured mouse spinal neurons. RESULTS: Topiramate at concentrations of > or =3 microM caused a voltage-sensitive and time-dependent limitation of SRF that was associated with a decrease in the velocity of the upstroke of the action potential. At high concentrations (30-600 microM), TPM rapidly blocked SRF in about one third of the neurons tested and did not affect SRF in about one third. In some neurons, TPM caused an intermittent limitation (sputtering) of SRF (approximately 30% of the neurons) or blocked SRF only after a delay of several seconds (approximately 10%). This complex pattern of effects is distinctly different from that of PHT and LTG, in which the effect was always a rapid limitation or complete blockade of SRF. Another difference between TPM and the other anticonvulsants (AEDs) is that the effects of TPM were more dependent on the length of time the neurons were exposed to the compound and the intensity or duration of neuronal activity. CONCLUSIONS: The results of this study do not support the concept that Na+ channel blockade is the primary mechanism responsible for the anticonvulsant activity of TPM.

CM101-mediated recovery of walking ability in adult mice paralyzed by spinal cord injury.

CM101, an antiangiogenic polysaccharide derived from group B streptococcus, was administered by i.v. injection 1 hr post-spinal-cord crush injury in an effort to prevent inflammatory angiogenesis and gliosis (scarring) in a mouse model. We postulated that gliosis would sterically prevent the reestablishment of neuronal connectivity; thus, treatment with CM101 was repeated every other day for five more infusions for the purpose of facilitating regeneration of neuronal function. Twenty-five of 26 mice treated with CM101 survived 28 days after surgery, and 24 of 26 recovered walking ability within 2-12 days. Only 6 of 14 mice in the control groups survived 24 hr after spinal cord injury, and none recovered function in paralyzed limbs. MRI analysis of injured untreated and treated animals showed that CM101 reduced the area of damage at the site of spinal cord compression, which was corroborated by histological analysis of spinal cord sections from treated and control animals. Electrophysiologic measurements on isolated central nervous system and neurons in culture showed that CM101 protected axons from Wallerian degeneration; reversed gamma-aminobutyrate-mediated depolarization occurring in traumatized neurons; and improved recovery of neuronal conductivity of isolated central nervous system in culture.

Anesthetic-induced alteration of Ca2+ homeostasis in neural cells: a temperature-sensitive process that is enhanced by blockade of plasma membrane Ca2+-ATPase isoforms.

BACKGROUND: Many inhalation anesthetics at clinically relevant concentrations inhibit plasma membrane Ca2+-adenosine triphosphatase (PMCA) ion pumping in brain synaptic membranes and in cultured cells of neural origin. In this study, the authors investigated the effect of inhalation anesthetics on cytosolic calcium homeostasis in cortical neurons maintained at physiologic and room temperatures and on cortical neurons and pheochromocytoma cells with antisense blockade of specific PMCA isoforms. METHODS: Using Ca2+-specific confocal microfluorimetry, the anesthetic effects on Ca2+ dynamics were examined in mouse embryonic cortical neurons in association with ligand-stimulated Ca2+ influx. Studies were done at 21 degrees C and 37 degrees C. Mouse embryonic cortical neurons with oligodeoxyribonucleotide blockade of PMCA2 expression and transfected rat pheochromocytoma cells with blocked expression of PMCA1 were also examined. RESULTS: Baseline and poststimulation peak cytosolic calcium concentrations ([Ca2+]i) were increased, and Ca2+ clearance was delayed in cells exposed at 37 degrees C, but not at 21 degrees C, to concentrations < or = 1 minimum alveolar concentration (MAC)-equivalent of halothane, isoflurane, and sevoflurane. Neurons exposed to xenon solutions < or = 0.4, 0.6, and 0.8 MAC showed dose-related perturbations of cytosolic Ca2+. Calcium dynamics were altered in neural cells with blocked PMCA isoform production, but at much lower halothane concentrations: 0.5 MAC for cortical neurons and 0.1 MAC for pheochromocytoma cells. CONCLUSIONS: By extruding Ca2+ through the plasma membrane, PMCA maintains resting neuronal [Ca2+]i at low levels and clears physiologic loads of Ca2+ after influx through calcium channels. Inhalation anesthetics perturb this process and thus may interfere with neurotransmitter release, altering interneuronal signaling.

Trans-2-en-valproic acid limits action potential firing frequency in mouse central neurons in cell culture.

Effects of the trans-isomer of 2-en-valproate (trans-2-en-NaVP; E-delta2-en-valproate or 2-en-valproate), an unsaturated metabolite of valproic acid (VPA), on intracellularly recorded sodium-dependent action potentials of cultured mouse spinal cord and cortical neurons were compared with those of the anticonvulsant sodium valproate (NaVP). The maximal rate of rise of action potentials triggered by trains of 1-msec or 400-msec pulses declined progressively until failure to fire in both cell types during exposure to trans-2-en-NaVP or NaVP was observed. The limitation of firing by both drugs was concentration, voltage, rate and time dependent. The IC50 of trans-2-en-NaVP was 1.2 x 10(-3) at < or =1 hr and 4.8 x 10(-5) M at 24 to 48 hr. Trans-2-en-NaVP did not limit sustained repetitive firing in all cortical neurons. This may reflect slower rates of firing during 400-msec depolarizations in neurons of this type. In paired-pulse experiments, the absolute refractory period was 7 msec in control solution and 15 msec (P < .01 vs. control; n = 9) in solution containing 6 x 10(-4) M trans-2-en-NaVP. Firing was limited in all spinal cord neurons after exposure to 0.5 mM NaVP for 24 to 48 hr; 80% were limited by 1 mM NaVP at < or =1 hr. Coincubation of the spinal cord neurons with trans-2-en-NaVP and NaVP for 24 hr showed no hyperadditive effect of these two drugs in vitro. Limitation of sustained repetitive firing was reversed by hyperpolarization in the continuing presence of either drug and incubation in drug-free medium. Limitation of sodium-dependent action potential firing rates could contribute, at least in part, to the anticonvulsant effect of trans-2-en-NaVP.

Halothane alters electrical activity and calcium dynamics in cultured mouse cortical, spinal cord, and dorsal root ganglion neurons.

Halothane inhibits neural plasma membrane Ca(2+)-ATPase, a pump that ejects Ca2+ from the cell after influx through voltage- or ligand-activated channels. Intracellular microelectrode recordings in mouse embryonic cortical and spinal cord neurons showed that halothane and eosin, a pump inhibitor, prolonged repolarization associated with spontaneous bursts of depolarization. These agents also prolonged the repolarization phases of electrically induced action potentials and of capsaicin-mediated Ca(2+)-dependent depolarization in mouse adult dorsal root ganglion neurons. In keeping with these findings, confocal microfluorimetry showed that halothane delayed clearance of intracellular Ca2+ accumulated by N-methyl-D-aspartate stimulation of single neurons.

Remacemide HCl and its metabolite, FPL 12495AA, limit action potential firing frequency and block NMDA responses of mouse spinal cord neurons in cell culture.

The novel anticonvulsant, remacemide HCl [(+/-)-2-amino-N-(1-methyl-1,2-diphenylethyl)acetamide monohydrochloride; FPL12924AA], and a desglycinated metabolite [(+/-)-1-methyl-1,2-diphenylethylamine monohydrochloride; FPL 12495AA] reversibly limited sustained high-frequency repetitive firing (SRF) of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture. Limitation occurred with an IC50 of 7.9 X 10(-6) M for remacemide and 1.2 X 10(-6) M for FPL 12495AA (P < 0.05 vs. remacemide). Stereoisomers of the desglycinate limited SRF with IC50 values of 3.3 X 10(-6) M and 3.5 X 10(-6) M for the S(+) and R(-) compounds, respectively. The concentration of racemic desglycinate and of either stereoisomer that produced limitation in all neurons tested was 10(-4) M. Maximal rate of rise (Vmax) of action potentials decreased progressively until firing ceased during 400-ms depolarizing pulses. Efficacy of remacemide, but not of the desglycinate, increased with time (maximum at 16-36 h). The limitation was voltage dependent. In addition, reduction of Vmax and action potential failure occurred during stimulation with 400-ms pulses and trains of brief (1 ms) depolarizations at different frequencies. These findings suggest an effect on voltage-sensitive sodium current that generates the action potential upstroke. Remacemide and the desglycinate also significantly reduced the amplitude of neuronal responses to pressure application of NMDA in use-dependent manner at concentrations equal to the IC50 values for limitation of action potential firing. Resting potential and input resistance were not changed significantly by either drug. Limitation of high-frequency firing of action potentials by both remacemide HCl and FPL 12495AA may contribute to the anticonvulsant efficacy of these compounds at concentrations overlapping the range required to block glutamatergic hyperexcitability.

Reduced anesthetic requirements in aged rats: association with altered brain synaptic plasma membrane Ca(2+)-ATPase pump and phospholipid methyltransferase I activities.

Aging is associated with a decrease in anesthetic requirements. Animal models of aging manifest alteration of brain Ca2+ homeostasis and increased methyltransferase I (PLMTI) activity. In this study we evaluated concurrently anesthetic requirements and brain plasma membrane Ca(2+)-ATPase (PMCA) and PLMTI activities in young and aged rats. Halothane, desflurane, isoflurane and xenon MEDs (lowest partial pressures that suppress a pain response) were measured in 2 and 25 month old, male Fisher-344 rats. Halothane MED was also measured in 2 and 30 month old F344/BNF1 rats, a strain that undergoes aging with less debilitation. PMCA pumping and PLMTI activities were measured in synaptic plasma membranes (SPM) prepared from the cortex and diencephalon-mesencephalon (DM). For aged Fisher-344 rats, MEDs for halothane, desflurane, isoflurane and xenon were reduced to 81%, 82%, 67% and 86%, respectively, of young controls; PMCA activity was diminished to 91% in cortical SPM and 82% in DM SPM; and cortical and DM PLMTI activities were increased to 131% and 114% of young control. For F344/BNF1 rats, MED for halothane was reduced to 87%, PMCA activity was diminished to 90% in cortical SPM and 72% DM SPM, and PLMTI activity was increased to 133% in cortical SPM and 112% in DM SPM. The strong association between age and reduced anesthetic requirements for inhalational agents on the one hand and altered PMCA and PLMTI activity on the other lends support to the underlying hypothesis that PMCA and PLMTI may be involved in the production of the anesthetic state.

Measurement and analysis of static magnetic fields that block action potentials in cultured neurons.

To characterize the properties of static magnetic fields on firing of action potentials (AP) by sensory neurons in cell culture, we developed a mathematical formalism based on the expression for the magnetic field of a single circular current loop. The calculated fields fit closely the field measurements taken with a Hall effect gaussmeter. The biological effect induced by different arrays of permanent magnets depended principally on the spatial variation of the fields, quantified by the value of the gradient of the field magnitude. Magnetic arrays of different sizes (macroarray: four center-charged neodymium magnets of approximately 14 mm diameter; microarray; four micromagnets of the same material but of approximately 0.4 mm diameter) allowed comparison of fields with similar gradients but different intensities at the cell position. These two arrays had a common gradient value of approximately 1 mT/mm and blocked > 70% of AP. Alternatively, cells placed in a field strength of approximately 0.2 mT and a gradient of approximately 0.02 mT/mm produced by the macroarray resulted in no significant reduction of firing; a microarray field of the same strength but with a higher gradient of approximately 1.5 mT/mm caused approximately 80% AP blockade. The experimental threshold gradient and the calculated threshold field intensity for blockade of action potentials by these arrays were estimated to be approximately 0.02 mT/mm and approximately 0.02 mT, respectively, In conclusion, these findings suggest that spatial variation of the magnetic field is the principal cause of AP blockade in dorsal root ganglia in vitro.

Blockade of sensory neuron action potentials by a static magnetic field in the 10 mT range.

To characterize the inhibitory effect of a static magnetic field, action potentials (AP) were elicited by intracellular application of 1 ms depolarizing current pulses of constant amplitude to the somata of adult mouse dorsal root ganglion neurons in monolayer dissociated cell culture. During the control period, < 5% of stimuli failed to elicit AP. During exposure to an approximately 11 mT static magnetic field at the cell position produced by an array of four permanent center-charged neodymium magnets of alternating polarity (MAG-4A), 66% of stimuli failed to elicit AP. The number of failures was maximal after about 200-250 s in the field and returned gradually to baseline over 400-600 s. A direct or indirect effect on the conformation of AP generating sodium channels could account for these results because 1) failure was preceded often by reduction of maximal rate of rise, an indirect measure of sodium current; 2) recovery was significantly prolonged in more than one-half of neurons that were not stimulated during exposure to the MAG-4A field; and 3) resting membrane potential, input resistance, and chronaxie were unaffected by the field. The effect was diminished or prevented by moving the MAG-4A array along the X or Z axis away from the neuron under study and by increasing the distance between magnets in the XY plane. Reduction of AP firing during exposure to the approximately 0.1 mT field produced by a MAG-4A array of micromagnets was about the same as that produced by a MAG-4A array of the large magnets above. The approximately 28 mT field produced at cell position by two magnets of alternating polarity and the approximately 88 mT field produced by a single magnet had no significant effect on AP firing. These findings suggest that field strength alone cannot account for AP blockade.

Effects of oxcarbazepine and 10-hydroxycarbamazepine on action potential firing and generalized seizures.

The anticonvulsant compound oxcarbazepine and its principal 10-monohydroxy metabolite protected potently against electroshock-induced tonic hindlimb extension. Maximal plasma concentrations depended on dose and were reached < or = 1 h after an oral dose of oxcarbazepine and < 2 h after monohydroxy derivative. In mice, the ED50 was 14 mg/kg for oxcarbazepine and 20.5 mg/kg for the monohydroxy derivative, p.o. In rats, the ED50 was 13.5 mg/kg for oxcarbazepine and 17.0 mg/kg for monohydroxy derivative, p.o. This protective effect compared favorably with the efficacy of carbamazepine, phenytoin, phenobarbital and diazepam in the same test. As observed previously, valproate and ethosuximide were markedly less potent. The effect of oxcarbazepine and its monohydroxy derivative on sustained high frequency repetitive firing of sodium-dependent action potentials of mouse spinal cord neurons in cell culture was also examined using intracellular recording techniques. Both compounds reduced the percentage of neurons capable of sustained action potential firing in concentration-dependent manner. The EC50 for oxcarbazepine was 5 x 10(-8) M and that for monohydroxy derivative was 2 x 10(-8) M (P > 0.05 vs. oxcarbazepine). For comparison, the EC50 for carbamazepine was significantly higher (6 x 10(-7) M, P < 0.001 vs. oxcarbazepine and monohydroxy derivative). Limitation of firing by oxcarbazepine and the monohydroxy derivative depended on firing frequency and membrane potential and was enhanced by depolarization. Input resistance and resting membrane potential were not altered by either drug. The in vitro effect on action potential firing frequency occurred at concentrations below plasma levels of oxcarbazepine and monohydroxy derivative which protected animals against electroshock and were therapeutically effective in patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide for the treatment of chronic paroxysmal hemicrania.

A 25-year-old patient presented with clinical characteristics of chronic paroxysmal hemicrania which failed to respond to indomethacin 300 mg daily. Total relief of headaches was obtained with acetazolamide 250 mg t.i.d.

Oxcarbazepine: mechanisms of action.

The antiepileptic drug (AED) oxcarbazepine (OCBZ) and its rapidly formed 10-monohydroxy metabolite (MHD) protect against electroshock-induced tonic hindlimb extension in rodents (ED50 14-21 mg/kg p.o.). Both stereoisomers of MHD also protect. As with carbamazepine (CBZ), these findings suggest clinical efficacy against generalized tonic-clonic and, to some extent, partial seizures. OCBZ (IC50 5 x 10(-8) M), MHD (IC50 2 x 10 (-8) M), and CBZ (IC50 6 x 10(-7) M) limit the frequency of firing of sodium-dependent action potentials by cultured mouse central neurons and reduce Vmax progressively in a use-dependent manner at concentrations below therapeutic plasma concentrations in OCBZ-treated patients. This suggests that blockade of voltage-sensitive sodium channels could contribute to the antiepileptic efficacy of OCBZ. Blockade of penicillin-induced epileptiform discharges in hippocampal slices by MHD and its stereoisomers was diminished when the potassium channel blocker 4-aminopyridine was added to the bath fluid. This indicates that additional mechanisms of action, e.g., an effect on potassium channels, might be clinically important. In addition, both stereoisomers are equally responsible for the antiepileptic activity of the racemate, i.e., MHD, and are therefore likely to play a therapeutic role. Such actions could confer broad clinical utility on OCBZ.

Limitation by gabapentin of high frequency action potential firing by mouse central neurons in cell culture.

The investigational anticonvulsant drug, gabapentin (GP; 1-(aminomethyl) cyclohexaneacetic acid) limited repetitive firing of sodium-dependent action potentials of mouse spinal cord and neocortical neurons in monolayer dissociated cell culture. The effect developed slowly over time with sustained exposure. The IC50 was 1.3 x 10(-4) M for exposure times < or = 60 s, 1.9 x 10(-5) M for 10-60 min, and 4.0 x 10(-6) M for 12-48 h. Hyperpolarization restored sustained firing in the continuing presence of GP. Blockade of action potential firing by GP was frequency (use)-dependent. After preincubation with 2.9 x 10(-5) M GP (5 micrograms/ml), trains of brief stimuli at > or = 50 Hz elicited fewer action potentials than in control solution. Also, at 150 Hz, maximal rate of rise of action potentials decreased progressively with repetitive firing in GP-containing, but not control, solution. After overnight incubation in 2.9 x 10(-5) M GP, the absolute refractory period was prolonged from 2.4 +/- 0.6 ms in control solution (n = 11) to 4.7 +/- 0.3 ms (n = 10; P = 0.02 vs. control), and complete recovery from inactivation was prolonged from 8.0 +/- 1.3 ms to 17.0 +/- 2.6 ms (P < 0.001 vs. control). These findings suggest that GP may alter function of voltage-activated sodium channels, but the mechanism is unproven and may be indirect. Limitation of firing was observed in > or = 50% of neurons at concentrations in the range of those found in plasma and cerebrospinal fluid of patients treated successfully with GP. These results suggest that limiting the rate of firing of sodium-dependent action potentials may contribute to the anticonvulsant efficacy of gabapentin.

Phenytoin blocks N-methyl-D-aspartate responses of mouse central neurons.

Intracellularly recorded depolarizing responses of mouse spinal cord neurons in cell culture to N-methyl-D-aspartate (NMDA) applied by pressure ejection at 37 degrees C had a reversal potential of about -13 mV. Amplitude increased when [Mg++]o was less than 1.0 mM or glycine was added to the buffer. Desensitization was complete within 30 pressure applications of NMDA (P30) at 2-s inter-response intervals (IRI; timed from return of one response to resting potential until next application) in bicarbonate buffer and was glycine-sensitive. Desensitization was insignificant in phosphate buffer. In both buffers, 8 x 10(-6) M phenytoin (PT) blocked responses reversibly by P10 of 10(-5) M NMDA at 0.2 Hz (overlapping responses) and at short 2-s IRI (responses not overlapping). At frequencies < or = 0.1 Hz or IRI > or = 5 s, desensitization and block were less prominent or inapparent. Block by PT was observed 1) in single isolated neurons; 2) in 7 mM [Mg++]o-, 150 mM [K+]o-, or tetrodotoxin (TTX)-containing buffer to suppress spontaneous synaptic activity and action potentials and 3) when voltage-dependent Mg++ block was removed by depolarization or in 0.1 mM Mg++, with or without glycine supplementation. The block was not competitive. The PT metabolite, 5-(4-hydroxyphenyl)-5-phenylhydantoin (80 microM), did not block responses to NMDA. Use- and frequency-dependent block of NMDA responses may contribute to clinical effects of PT, e.g., during sustained rapid activity along pathways excited by NMDA-preferring glutamate receptors.

Effect of temperature on limitation by MK-801 of firing of action potentials by spinal cord neurons in cell culture.

The anticonvulsant, MK-801, limited sustained high frequency repetitive firing of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture. Limitation was voltage- and temperature-dependent and was accompanied by decreasing rate of rise of action potentials until firing ceased during the 400 ms depolarizations. The IC50 for limitation was 2 x 10(-7) M at 37 degrees C, 6.4 x 10(-7) M at 35 degrees C, and 4 x 10(-5) M at 23 degrees C. The relationship between the percentage of neurons capable of sustained repetitive firing and MK-801 concentration at 33 degrees C was biphasic. The first phase (about 50%) of limitation had IC50a = 1.5 x 10(-7) M, and the second had IC50b = 2 x 10(-4) M; the midpoint of the connecting plateau was 10(-5) M. At temperatures below 37 degrees C, the current needed to achieve maximal firing increased. The maximal rate of rise, maximal firing frequency and sensitivity to MK-801 of action potentials elicited by 1 ms stimuli decreased at temperatures below 37 degrees C. Passive membrane properties were unchanged. Slow firing and a temperature-sensitive conformational change in voltage-activated sodium channels could account for the higher concentrations of MK-801 required to block sodium-dependent action potentials at temperatures below 37 degrees C.

Use-, concentration- and voltage-dependent limitation by MK-801 of action potential firing frequency in mouse central neurons in cell culture.

The anticonvulsant, (+/-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine (MK-801), blocked single postsynaptic responses of mouse spinal cord neurons in cell culture to N-methyl-D-aspartate (NMDA). The block was concentration dependent with IC50 = 10(-7) M against 10(-5) M NMDA and 2 x 10(-7) M against 10(-3) M NMDA. Serial responses were blocked in use-dependent manner by 10 times lower doses of MK-801, depending on rate of NMDA application. MK-801 (approximate IC50, 8 x 10(-8) M) also limited sustained high-frequency repetitive firing of sodium-dependent action potentials (AP) elicited by long (400 msec) depolarizing pulses. Without changing resting membrane potential, single AP elicited by short (0.5-1 msec) depolarizing current pulses were blocked in voltage-, use- and frequency-dependent manner. Maximal rate of rise of individual AP elicited by short or long pulses decreased progressively until failure to fire. The time constant of recovery of AP from inactivation in a paired pulse protocol was prolonged from about 1 msec in control solution to 12 msec in solution containing 3 x 10(-7) M MK-801. These characteristics suggest that MK-801 blocks voltage-sensitive sodium current, which generates the upstroke of the AP. Overlap of concentrations blocking NMDA responses and sustained repetitive firing suggests that both actions may contribute to anticonvulsant efficacy of MK-801.

Prophylactic and therapeutic efficacy of memantine against seizures produced by soman in the rat.

Male Sprague-Dawley rats injected sc with a single sublethal dose of the organophosphate nerve agent, soman (100 micrograms/kg), had motor limbic seizures within 5-15 min. Pretreatment with a single dose of memantine HCl (MEM, 18 mg/kg, sc), alone or in combination with atropine sulfate (ATS, 16 mg/kg, sc), before soman prevented seizures without sedation or ataxia. Rats appeared normal or demonstrated increased exploratory activity. Excessive salivation, a peripheral manifestation of soman intoxication, was decreased by ATS, but pretreatment with ATS alone did not prevent seizures. After seizure onset, MEM +/- ATS, but not ATS, abolished seizures. Acetylcholinesterase (AChE) activity in several brain regions (cortex, stem, striatum, and hippocampus) was markedly reduced by soman, but not by MEM, ATS, or MEM + ATS. Preadministration of MEM + ATS in vivo significantly protected AChE from inhibition by soman. Memantine reduced inhibition of AChE activity in crude brain homogenates by soman, but not by edrophonium (anionic site inhibitor) or decamethonium (peripheral site inhibitor). Thus, MEM may bind to a different modulatory site, not yet characterized, to protect AChE. When given after onset of soman-induced seizures, treatment with MEM +/- ATS did not reactivate AChE although seizures were controlled, suggesting additional anticonvulsant mechanisms of action. At concentrations (10(-4) to 5 x 10(-4) M) which did not significantly alter the spontaneous firing of action potentials (APs), MEM limited sustained high frequency repetitive firing (SRF) induced by depolarization of spinal cord (mouse and rat) and neocortical (mouse) neurons in monolayer-dissociated cell culture. In the same range of concentrations, ATS both limited SRF and suppressed spontaneous activity, suggesting toxicity. In addition, MEM and ATS reversibly produced use-dependent block of depolarizing responses to acetylcholine (ACh) applied by pressure ejection to spinal cord neurons. Thus, the anticonvulsant efficacy of MEM, with or without ATS, may have resulted from a combination of actions, including protection of AChE from inhibition by soman, limitation of high frequency firing of APs, and blockade of excitatory postsynaptic responses to ACh.