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Current medical countermeasures for organophosphate (OP)-induced status epilepticus (SE) are not effective in preventing long-term morbidity and there is an urgent need for improved therapies. Rat models of acute intoxication with the OP, diisopropylfluorophosphate (DFP), are increasingly being used to evaluate therapeutic candidates for efficacy in mitigating the long-term neurologic effects associated with OP-induced SE. Many of these therapeutic candidates target neuroinflammation and oxidative stress because of their implication in the pathogenesis of persistent neurologic deficits associated with OP-induced SE. Critical to these efforts is the rigorous characterization of the rat DFP model with respect to outcomes associated with acute OP intoxication in humans, which include long-term electroencephalographic, neurobehavioral, and neuropathologic effects, and their temporal relationship to neuroinflammation and oxidative stress. To address these needs, we examined a range of outcomes at later times post-exposure than have previously been reported for this model. Adult male Sprague-Dawley rats were given pyridostigmine bromide (0.1 mg/kg, im) 30 min prior to administration of DFP (4 mg/kg, sc), which was immediately followed by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im). This exposure paradigm triggered robust electroencephalographic and behavioral seizures that rapidly progressed to SE lasting several hours in 90% of exposed animals. Animals that survived DFP-induced SE (~70%) exhibited spontaneous recurrent seizures and hyperreactive responses to tactile stimuli over the first 2 months post-exposure. Performance in the elevated plus maze, open field, and Pavlovian fear conditioning tests indicated that acute DFP intoxication reduced anxiety-like behavior and impaired learning and memory at 1 and 2 months post-exposure in the absence of effects on general locomotor behavior. Immunohistochemical analyses revealed significantly increased expression of biomarkers of reactive astrogliosis, microglial activation and oxidative stress in multiple brain regions at 1 and 2 months post-DFP, although there was significant spatiotemporal heterogeneity across these endpoints. Collectively, these data largely support the relevance of the rat model of acute DFP intoxication as a model for acute OP intoxication in the human, and support the hypothesis that neuroinflammation and/or oxidative stress represent potential therapeutic targets for mitigating the long-term neurologic sequelae of acute OP intoxication., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Benzodiazepines, including diazepam and midazolam, are the mainstay of treatment for seizure emergencies, including acute repetitive seizures. Nonparenteral dosage forms are used when parenteral (intravenous or intramuscular) dosing is not feasible. Currently available nonparenteral dosage forms have limitations in terms of usability, patient and caregiver acceptance, speed of action, and portability. Diazepam buccal film (DBF) is a compact, easily administered diazepam formulation. When placed onto the buccal mucosa inside the cheek, DBF adheres firmly and then rapidly dissolves, delivering diazepam transbucally and via the gastric route. In fasted healthy male volunteers, plasma levels were achieved rapidly after DBF placement in a linear dose-proportional fashion. Bioavailability in adult patients with epilepsy was not significantly different when DBF was applied interictally or periictally (within 5 min of a seizure). Diazepam buccal film was successfully placed and generally used without difficulty, even without patient cooperation immediately after a seizure. In a crossover comparative study with diazepam rectal gel (Diastat®) in adult patients with epilepsy, DBF performed equivalently to the rectal gel, but peak exposures were less variable. Diazepam buccal film is a convenient alternative for out-of-hospital treatment of seizure exacerbations. Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Rett syndrome (RTT) is a devastating neurodevelopmental disorder caused by loss-of-function mutations in the X-linked methyl-CpG binding protein 2 (Mecp2) gene. GABAergic dysfunction has been implicated contributing to the respiratory dysfunction, one major clinical feature of RTT. The nucleus tractus solitarius (NTS) is the first central site integrating respiratory sensory information that can change the nature of the reflex output. We hypothesized that deficiency in Mecp2 gene reduces GABAergic neurotransmission in the NTS. Using whole-cell patch-clamp recordings in NTS slices, we measured spontaneous inhibitory postsynaptic currents (sIPSCs), miniature IPSCs (mIPSCs), NTS-evoked IPSCs (eIPSCs), and GABA A receptor (GABA A -R) agonist-induced responses. Compared to those from wild-type mice, NTS neurons from Mecp2-null mice had significantly (p<0.05) reduced sIPSC amplitude, sIPSC frequency, and mIPSC amplitude but not mIPSC frequency. Mecp2-null mice also had decreased eIPSC amplitude with no change in paired-pulse ratio. The data suggest reduced synaptic receptor-mediated phasic GABA transmission in Mecp2-null mice. In contrast, muscimol (GABA A -R agonist, 0.3-100μM) and THIP (selective extrasynaptic GABA A -R agonist, 5μM) induced significantly greater current response in Mecp2-null mice, suggesting increased extrasynaptic receptors. Using qPCR, we found a 2.5 fold increase in the delta subunit of the GABA A -Rs in the NTS in Mecp2-null mice, consistent with increased extrasynaptic receptors. As the NTS was recently found required for respiratory pathology in RTT, our results provide a mechanism for NTS dysfunction which involves shifting the balance of synaptic/extrasynaptic receptors in favor of extrasynaptic site, providing a target for boosting GABAergic inhibition in RTT., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tetramethylenedisulfotetramine (TETS) is a potent convulsant poison for which there is currently no approved antidote. The convulsant action of TETS is thought to be mediated by inhibition of type A gamma-aminobutyric acid receptor (GABAAR) function. We, therefore, investigated the effects of post-exposure administration of diazepam, a GABAAR positive allosteric modulator, on seizure activity, death and neuroinflammation in adult male Swiss mice injected with a lethal dose of TETS (0.15mg/kg, ip). Administration of a high dose of diazepam (5mg/kg, ip) immediately following the second clonic seizure (approximately 20min post-TETS injection) effectively prevented progression to tonic seizures and death. However, this treatment did not prevent persistent reactive astrogliosis and microglial activation, as determined by GFAP and Iba-1 immunoreactivity and microglial cell morphology. Inhibition of soluble epoxide hydrolase (sEH) has been shown to exert potent anti-inflammatory effects and to increase survival in mice intoxicated with other GABAAR antagonists. The sEH inhibitor TUPS (1mg/kg, ip) administered immediately after the second clonic seizure did not protect TETS-intoxicated animals from tonic seizures or death. Combined administration of diazepam (5mg/kg, ip) and TUPS (1mg/kg, ip, starting 1h after diazepam and repeated every 24h) prevented TETS-induced lethality and influenced signs of neuroinflammation in some brain regions. Significantly decreased microglial activation and enhanced reactive astrogliosis were observed in the hippocampus, with no changes in the cortex. Combining an agent that targets specific anti-inflammatory mechanisms with a traditional antiseizure drug may enhance treatment outcome in TETS intoxication., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ion channels, specifically voltage-gated sodium channels (Na(v)s), are common culprits in inheritable seizure disorders. Some Na(v) isoforms are particularly susceptible, while others are only weakly associated with neuronal hyperexcitability. Representative of the latter group is Na(v)1.2 (gene name SCN2A): despite its abundance in the brain, Na(v)1.2-related epilepsy is rare and only few studies have been conducted as to the pathophysiological basis of Na(v)1.2 in neuronal hyperexcitability. We here present a detailed functional analysis of Na(v)1.2 mutant, R1312T, which was originally found in a child with Dravet syndrome (formerly known as severe myoclonic epilepsy of infancy or SMEI). Whole-cell voltage clamp analysis revealed clearly compromised function: the mutant channels fast- and slow-inactivated at markedly more negative potentials and recovered from fast inactivation more slowly, which resulted in a use-dependent current reduction to less than 50% of wildtype levels. We also noted a small hyperpolarizing shift in the voltage dependence of activation. Our findings expand the spectrum of abnormal Na(v) channel behavior in epilepsy and raise the question as to how loss-of-function in a sodium channel predominantly expressed in excitatory neurons can lead to hyperexcitability., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Neurosteroids modulate seizure susceptibility, but their role in the regulation of epileptogenesis is unknown. Status epilepticus (SE) induces temporal lobe epileptogenesis following a latent period in which glial cells are activated. Here, we found that P450scc, the rate-limiting enzyme in steroid synthesis, is upregulated in hippocampal glia during the latent period after pilocarpine-induced SE in rats. More prolonged SE was associated with greater P450scc expression and longer latencies to the development of seizures, suggesting that enhanced steroid synthesis retards epileptogenesis. The 5alpha-reductase inhibitor finasteride, which blocks neurosteroid synthesis, reduced the latent period, indicating that glia-derived neurosteroids may be antiepileptogenic.

Deoxycorticosterone (DOC) is a mineralocorticoid precursor that has anticonvulsant properties in animals and possibly also in humans. Studies indicate that the anticonvulsant activity of DOC requires its enzymatic conversion to 5 alpha,3 alpha-tetrahydrodeoxycorticosterone (THDOC), a neurosteroid that lacks classical hormonal properties but acts as a powerful positive allosteric modulator of GABAA receptors. DOC can be considered a stress hormone because its synthesis is under the control of ACTH. Therefore, stress-induced fluctuations in seizure susceptibility could in part result from alterations in DOC availability. Also, the therapeutic activity of ACTH in infantile spasms could partially relate to its stimulatory effects on the synthesis of DOC, which then undergoes biotransformation to neurosteroids. The recent demonstration that the synthetic neurosteroid analog ganaxolone reduces spasm frequency in children with intractable infantile spasms suggests that neurosteroid-related anticonvulsants may offer a potential new nonhormonal approach for the treatment of infantile spasms and other developmental epilepsies. In addition, it further confirms the utility of pharmacological enhancement of GABA-mediated inhibition in the control of infantile spasms.

Biochemical and immunological studies have shown that mice infected with LP-BM5 virus develop antibodies to ionotropic glutamate receptors. Here, IgG isolated from brain of infected mice has been tested electrophysiologically on cultured rat cortical and hippocampal neurons. The IgG elicited glycine-independent currents that reversed at approximately 0 mV. Equivalent concentrations of IgG from uninfected mice were inactive. The glycine-independent currents were less influenced by DNQX and GYKI-52466 than currents elicited by AMPA and KA. The IgG also elicited glycine-dependent currents that reversed at -10 mV and were blocked by dl-AP5, 5,7-DCKA, and polyamine amides. Glycine-dependent and -independent currents were unaffected by tetrodotoxin, strychnine, the transmembrane Cl- gradient or d-tubocurare. Although part of the glycine-independent current remains uncharacterized, these results confirm that a virus-induced immunopathology produces IgG clones that activate ionotropic glutamate receptors and that could, thereby, contribute to the excitotoxic neurological syndrome observed in LP-BM5-infected mice.