Changes in hippocampal GABAA/cBZR density during limbic epileptogenesis: Relationship to cell loss and mossy fibre sprouting

Reduced GABA_A/central benzodiazepine receptor (GABA_A/cBZR) density, mossy fibre sprouting (MFS) and hippocampal cell loss are well described pathological features of human temporal lobe epilepsy (TLE), and animal models thereof. However, the temporal relationship of their development, and their roles in the emergence of the epilepsy, are uncertain. This was investigated in the kainic acid (KA)-induced post-status epilepticus (SE) model of TLE. Male Wistar rats (7 weeks, n = 53) were randomised into control and KA groups. At 24 h, 2, 4 or 6 weeks sham and KA post-SE animals were euthanised, brains extracted and GABA_A/cBZR density, neuronal loss and MFS measured in hippocampal sub-regions. GABA_A/cBZR density (B_max) was measured by saturation-binding analysis using [³H]-flumazenil. At 24 h post-SE GABA_A/cBZR density was increased in almost all hippocampal subregions, but was decreased at the later time points with the exception of the dentate gyrus. There was significant neuronal loss in the CA3 SPc region (?24±9.3%, p<0.05) at 24h, which remained stable at the later time points associated with an elevated GABA_A/cBZR density per surviving neuron at 24h post-SE (+56.4%; p<0.05) which returned to control levels by 6 weeks post-SE. MFS in the dentate gyrus progressively increased over the 6 weeks following SE (+ 70.6% at 6 weeks), at which time there was a significant inverse relationship with GABA_A/cBZR binding (r² = 0.87; p = 0.02). The temporal evolution of GABA_A/cBZR density changes post-KA-induced SE, and the relationship with decreases in hippocampal pyramidal cell numbers and MFS, may point to a key role for these changes in the pathogenesis of acquired limbic epileptogenesis.     

Evaluation of the anticonvulsant effect of Centella asiatica (gotu kola) in pentylenetetrazol-induced seizures with respect to cholinergic neurotransmission

The study described here was carried out to investigate the anticonvulsant effect of different extracts of Centella asiatica with respect to cholinergic activity on pentylenetetrazol (PTZ)-induced seizures. Rats were randomly divided into eight groups of six rats each: nonepileptic rats treated with saline; PTZ (60 mg/kg, IP)-induced seizure rats treated with saline; PTZ-induced seizure rats pretreated with n-hexane, chloroform, ethyl acetate, n-butanol, and water extracts of C. asiatica; and PTZ-induced seizure rats pretreated with diazepam (2 mg/kg body wt). The seized rats pretreated with different extracts were administered a dose of 200 mg/kg body wt orally for 1 week before induction of epilepsy. Increased acetylcholine content and decreased acetylcholinesterase activity were recorded in different brain regions during PTZ-induced seizures. Pretreatment with C. asiatica extracts caused recovery of the levels of acetylcholine and acetylcholinesterase. These findings suggest that C. asiatica causes perceptible changes in the cholinergic system as one of the facets of its anticonvulsant activity.     

Naringin ameliorates pentylenetetrazol-induced seizures and associated oxidative stress,inflammation, and cognitive impairment in rats: Possible mechanisms of neuroprotection

Oxidative stress and cognitive impairment are associated with PTZ-induced convulsions. Naringin is a bioflavonoid present in the grapefruit. It is a potent antioxidant, and we evaluated its effect on PTZ-induced convulsions. Rats were pretreated with normal saline, naringin (20, 40, and 80 mg/kg, i.p.), or diazepam (5 mg/kg, i.p.) 30 min prior to the administration of PTZ. The administration of PTZ induced myoclonic jerks and generalized tonic–clonic seizures (GTSs). We observed that naringin significantly prolonged the induction of myoclonic jerks dose-dependently. Naringin (80 mg/kg, i.p.) pretreatment protected all rats, and this protective effect was annulled by the GABA_A receptor antagonist, flumazenil. In addition, naringin reduced brain MDA and TNF-α levels and conserved GSH. The pretreatment also enhanced the performance of rats in the passive avoidance task. Our observations highlight the antioxidant, antiinflammatory, and anticonvulsant potential of naringin. Also, naringin modulates the GABA_A receptor to produce anticonvulsant effects and to ameliorate cognitive impairment.     

βCCT,an antagonist selective for α1GABAA receptors,reverses diazepam withdrawal-induced anxiety in rats

The abrupt discontinuation of prolonged benzodiazepine treatment elicits a withdrawal syndrome with increased anxiety as a major symptom. The neural mechanisms underlying benzodiazepine physical dependence are still insufficiently understood. Flumazenil, the non-selective antagonist of the benzodiazepine binding site of GABA_A receptors was capable of preventing and reversing the increased anxiety during benzodiazepine withdrawal in animals and humans in some, but not all studies. On the other hand, a number of data suggest that GABA_A receptors containing α₁ subunits are critically involved in processes developing during prolonged use of benzodiazepines, such are tolerance to sedative effects, liability to physical dependence and addiction. Hence, we investigated in the elevated plus maze the level of anxiety 24 h following 21 days of diazepam treatment and the influence of flumazenil or a preferential α₁-subunit selective antagonist βCCt on diazepam withdrawal syndrome in rats. Abrupt cessation of protracted once-daily intraperitoneal administration of 2 mg/kg diazepam induced a withdrawal syndrome, measured by increased anxiety-like behavior in the elevated plus maze 24 h after treatment cessation. Acute challenge with either flumazenil (10 mg/kg) or βCCt (1.25, 5 and 20 mg/kg) alleviated the diazepam withdrawal-induced anxiety. Moreover, both antagonists induced an anxiolytic-like response close, though not identical, to that seen with acute administration of diazepam. These findings imply that the mechanism by which antagonism at GABA_A receptors may reverse the withdrawal-induced anxiety involves the α₁ subunit and prompt further studies aimed at linking the changes in behavior with possible adaptive changes in subunit expression and function of GABA_A receptors.     

Development of NMDA-induced theta rhythm in hippocampal formation slices

During the past 20 years experimental evidence has accumulated demonstrating that the appearance of theta rhythm requires a certain level of excitation of local neuronal networks. In this study we extended our earlier in vitro observations concerning the involvement of cholinergic and GABAergic neurotransmission in hippocampal theta production. Specifically, we investigated whether the hippocampal neuronal network is capable of generating theta oscillations in the presence of N-methyl-d-aspartic acid (NMDA) in a brain slice preparation. To answer this question, the effect of different concentrations of NMDA (Experiment I) and the effect of interaction between NMDA and GABA_A/_B agonists and antagonists on field potentials recorded in the CA3c region of hippocampal formation (HPC) slice preparations (Experiment II) was examined. We demonstrated for the first time that apart from the epileptiform activity recorded in almost all series of Experiments I and II, only the perfusion of HPC slices with NMDA in doses of 30 and 50 μM, as well as the perfusion of HPC slices with NMDA and GABA_B agonist baclofen (50 μM NMDA + 50 μM BACL), resulted in the appearance of individual theta epochs. The best synchronized theta oscillations obtained after administration of 50 μM NMDA + 50 μM BACL resembled theta activity induced by a bath perfusion of 50 μM carbachol. In light of the obtained results we conclude that besides the cholinergic and GABAergic input, NMDA glutamatergic drive is also important for the appearance of theta oscillations in HPC in vitro.     

Comparison of pediatric patients with status epilepticus lasting 5–29 min versus ≥ 30 min

The most common thresholds for considering prolonged seizures as status epilepticus (SE) are 5 and 30 min. It is unknown whether these different thresholds (5 or 30 min) identify patient populations with different electroclinical characteristics. We compared the characteristics of patients with SE lasting 5–29 min (SE_5–29) with those with SE lasting ≥ 30 min (SE_≥ 30). Inclusion criteria were the following: 1) 1 month to 21 years of age at the time of SE, 2) convulsive seizures, and 3) seizure duration ≥ 5 min. Exclusion criteria were the following: 1) exclusively neonatal seizures, 2) psychogenic nonepileptic seizures, or 3) incomplete information about seizure duration. Four hundred forty-five patients (50.1% male) with a median (p₂₅–p₇₅) age at SE of 5.5 (2.8–10.5) years were enrolled. Status epilepticus lasted for 5–29 min in 296 (66.5%) of subjects and for ≥ 30 min in 149 (33.5%). Patients with SE_≥ 30 were younger than the patients with SE_5–29 at the time of seizure onset (median: 1 versus 2.1 years, p = 0.0007). Status epilepticus as the first seizure presentation was more frequent in patients with SE_≥ 30 (24.2% versus 12.2%, p = 0.002). There was a tendency towards a higher rate of abnormalities in the magnetic resonance imaging at baseline in patients with SE_≥ 30 (70.5% versus 57.1%, p = 0.061). Differences were not detected in seizure frequency, seizure types, presence of developmental delay, and electroencephalogram abnormalities at baseline. In the pediatric population, SE thresholds of either 5 or 30 min identify groups of patients with very similar electroclinical characteristics, which may influence future definitions of pediatric SE.     

Propylparaben applied after pilocarpine-induced status epilepticus modifies hippocampal excitability and glutamate release in rats

Propylparaben (PPB) induces cardioprotection after ischemia–reperfusion injury by inhibiting voltage-dependent Na⁺ channels. The present study focuses on investigating whether the i.p. application of 178 mg/kg PPB after pilocarpine-induced status epilepticus (SE) reduces the acute and long-term consequences of seizure activity. Initially, we investigated the effects of a single administration of PPB after SE. Our results revealed that compared to rats receiving diazepam (DZP) plus vehicle after 2 h of SE, animals receiving a single dose of PPB 1 h after DZP injection presented 126% (p < 0.001) lower extracellular levels of glutamate in the hippocampus. This effect was associated with an increased potency of low-frequency oscillations (0.1–13 Hz bands, p < 0.001), a reduced potency of 30–250 Hz bands (p < 0.001) and less neuronal damage in the hippocampus. The second experiment examined whether the subchronic administration of PPB during the post-SE period is able to prevent the long-term consequences of seizure activity. In comparison to animals that were treated subchronically with vehicle after SE, rats administered with PPB for 5 days presented lower hippocampal excitability and interictal glutamate release, astrogliosis, and neuroprotection in the dentate gyrus. Our data indicate that PPB, when applied after SE, can be used as a therapeutic strategy to reduce the consequences of seizure activity.     

Assessing the role of serotonergic receptors in cannabidiol's anticonvulsant efficacy

Cannabidiol (CBD) is a phytocannabinoid that has demonstrated anticonvulsant efficacy in several animal models of seizure. The current experiment validated CBD's anticonvulsant effect using the acute pentylenetetrazol (PTZ) model. Furthermore, it tested whether CBD reduces seizure activity by interacting with either the serotonergic 5HT1A or 5HT2A receptor. 120 male adolescent Wistar-Kyoto rats were randomly assigned to 8 treatment groups in two consecutive experiments. In both experiments, subjects received either CBD (100 mg/kg) or vehicle 60 min prior to seizure testing. In Experiment 1, subjects received either WAY-100635 (1 mg/kg), a 5HT1A antagonist, or saline vehicle injection 80 min prior to seizure testing. In Experiment 2, subjects received either MDL-100907 (0.3 mg/kg), a specific 5HT2A antagonist, or 40% DMSO vehicle 80 min prior to seizure testing. 85 mg/kg of PTZ was administered to induce seizure, and behavior was recorded for 30 min. Seizure behaviors were subsequently coded using a 5-point scale of severity. Across both experiments, subjects in the vehicle control groups exhibited high levels of seizure activity and mortality. In both experiments, CBD treatment significantly attenuated seizure activity. Pre-treatment with either WAY-100635 or MDL-100907 did not block CBD's anticonvulsant effect. WAY-100635 administration, by itself, also led to a significant attenuation of seizure activity. These results do not support the hypothesis that CBD attenuates seizure activity through activation of the 5HT1A or 5HT2A receptor. While this work further confirms the anticonvulsant efficacy of CBD and supports its application in the treatment of human seizure disorders, additional research on CBD's mechanism of action must be conducted.     

Propofol facilitated excitatory postsynaptic currents frequency on nucleus tractus solitarii (NTS) neurons

Propofol, an intravenous anesthetic, is broadly used for general anesthesia and diagnostic sedations due to its fast onset and recovery. Propofol depresses respiratory and cardiovascular reflex responses, however, their underlying mechanisms are not well known. Cardiorespiratory information from visceral afferent vagus nerves is integrated in the nucleus tractus solitarii (NTS). Cardiac and respiratory signals transducing vagal afferent neurons release the excitatory neurotransmitter glutamate onto NTS neurons in an activity dependent manner and trigger negative feedback reflex responses. In this experiment, the effects of propofol on glutamatergic synaptic responses at NTS neurons was tested using patch clamp methods. Glutamatergic excitatory postsynaptic currents (EPSC) were recorded at chloride reversal potential (? 49 mV) without γ-aminobutyric acid type A (GABA_A) receptor antagonists. Propofol (≥ 3 μM) facilitated frequency of the spontaneous EPSCs in a concentration dependent manner without altering amplitude and decay time. The GABA_A receptor selective antagonist, gabazine (6 μM), attenuated propofol effects on glutamate release. Propofol (10 μM) evoked glutamate release was also blocked in the presence of the voltage dependent Na⁺ and Ca²⁺ channel blockers TTX (0.3 μM) and Cd²⁺ (0.2 mM), respectively. In addition, the Na⁺–K⁺–Cl^? cotransporter type 1 antagonist bumetanide (10 μM) also inhibited propofol evoked increase in sEPSC frequency. These results suggest that propofol evoked glutamate release onto NTS neurons by GABA_A receptor-mediated depolarization of the presynaptic excitatory terminals.     

Electrical stimulation of left anterior thalamic nucleus with high-frequency and low-intensity currents reduces the rate of pilocarpine-induced epilepsy in rats

PurposeBilateral electrical stimulation of anterior nuclei of thalamus (ANT) has shown promising effects on epileptic seizures. However, bilateral implantation increases the risk of surgical complications and side effects. This study was undertaken to access the effectiveness of a stimulation paradigm involving high frequency and low intensity currents to stimulate the left ANT in rats.MethodsMale Sprague-Dawley rats were implanted with electroencephalogram (EEG) electrodes, and an additional concentric bipolar stimulation electrode into either the left or right ANT. The stimulus was a train of pulses (90 μs duration each) delivered with a frequency of 200 Hz and a current intensity of 50 μA. Thalamic stimuli were started 1 h before the first intraperitoneal pilocarpine injection (i.p., 300 mg/kg), and were applied for 5 h.ResultsEEG documented seizure activity and status epilepticus (SE) developed in 87.5% of rats treated with no ANT stimulation after a single dose of pilocarpine. Left ANT stimulation significantly increased the tolerance threshold for pilocarpine-induced EEG seizure activity; 20% of rats developed their EEG documented seizure activity after receiving the first dose, whereas 50%, 10% and 20% of rats did not develop seizure activity until they had received the 2nd, 3rd and 4th pilocarpine injection at 1-h intervals. Moreover, left thalamic stimulation reduced the occurrences of both EEG documented seizure activity and SE induced by single-dose pilocarpine to 25%. However, our result demonstrated that little effect on the occurrence rate of seizures and SE was found when rats received right ANT stimulation.ConclusionsThese results suggest that continuously 5-h left ANT stimulation with high frequency and low intensity currents, beginning from 1 h before the pilocarpine administration, may successfully reduce the occurrence rate of EEG documented seizure activity and SE development in rats.     

Positive modulation of α5 GABAA receptors in preadolescence prevents reduced locomotor response to amphetamine in adult female but not male rats prenatally exposed to lipopolysaccharide

We previously demonstrated that lipopolysaccharide (LPS) administered intraperitoneally (i.p.) to pregnant Wistar rat dams, at embryonic days 15 and 16 (E15/16), induced a decrease of baseline locomotor activity and diminished reactivity to amphetamine in adult female offspring. In the present study we aimed to assess the duration of LPS-induced maternal immune activation (MIA) and investigate possible changes in levels of main neurotransmitters in fetal brain during MIA. We hypothesized that the observed behavioral changes may be linked with MIA-induced disturbance of prenatal GABAergic system development, especially with α5 GABA_A receptors (α5GABA_ARs), expression of which takes place between E14 and E17. Thereafter, we set to investigate if later potentiation of α5GABA_ARs in offspring’s preadolescence (from postnatal day 22–28) could prevent the deficit in locomotor reactivity to amphetamine observed in adulthood, at postnatal day P60. The elevation of IL-6 in amniotic fluid 6 h after LPS treatment (100 μg/kg, i.p.) at E15 was concurrent with a significant increase of GABA and decrease of glutamate concentration in fetal brain. Moreover, repeated administration of MP-III-022, a selective positive allosteric modulator of α5GABA_ARs, at a dose (2 mg/kg daily, i.p.) derived from a separate pharmacokinetic study, prevented the LPS-induced decrease in locomotor reactivity to amphetamine (0.5 mg/kg, i.p.) in adult females. These results were not mirrored in the parallel set of experiments with male offspring from LPS-treated rats. The results suggest that pharmacological potentiation of α5GABA_ARs activity in preadolescence may ameliorate at least some of adverse consequences of exposure to MIA in utero.     

GABAA receptor expression and white matter disruption in intrauterine growth restricted piglets

Intrauterine growth restriction (IUGR) is one of the most common causes of perinatal mortality and morbidity. White matter and neuronal injury are major pathophysiological features of the IUGR neonatal brain. GABA_A (γ-aminobutyric acid type A) receptors have been shown to play a role in oligodendrocyte differentiation and proliferation in the neonatal brain and may be a key factor in white matter injury and myelination in IUGR neonates. Whether there are impairments to the GABAergic system and neuronal cytoskeleton in IUGR brain has yet to be elucidated. This study aims to examine GABA_A receptor α₁ and α₃ subunit protein expression and distribution in parietal cortex and hippocampus of the IUGR piglet at four different ages (term = 115 d – days gestational age), 100 d, 104 d, birth (postnatal day 0–P0) and P7 and to examine neuronal and myelination patterns. Significant alterations to GABA_A receptor α₁ and α₃ protein expression levels were observed in the IUGR piglet brain of P7 IUGR piglets with significantly greater α₃ expression compared to α₁ expression in the hippocampus while there was virtually no difference between the two subunits in the parietal cortex. However a significantly lower α₁/α₃ ratio was evident in P7 IUGR cortex when compared with P7 NG cortex. Neuronal somatodendrites studied using MAP2 immunohistochemistry showed reduced and disrupted somatodendrites while MBP immunolabelling showed loss of axonal fibres from gestational day 104 d through to P7. These findings provide insights into the effects of IUGR on the development of the GABA system, altered developmental maturation of GABA_A receptor subunit expression in the IUGR brain may influence myelination and may partly explain the cognitive disabilities observed in IUGR. Understanding the mechanisms behind grey and white matter injury in the IUGR infant is essential to identifying targets for treatments to improve long-term outcomes for IUGR infants.     

Comparison of risk factors for pediatric convulsive status epilepticus when defined as seizures ≥5 min versus seizures ≥30 min

PurposeTo identify risk factors (RF) of pediatric convulsive status epilepticus (SE) and to determine whether defining SE as seizures ≥5 min (SE₅) or seizures ≥30 min (SE₃₀) would modify the risk factors identified.MethodsRetrospective case–control study. We included patients 1 month to 21 years of age at the time of convulsive SE. We compared the characteristics of patients with SE (cases) versus those without SE (controls) using two different seizure duration thresholds: 5 min and 30 min.Results1062 patients (54% males) were enrolled. The median (p₂₅–p₇₅) age at the episode was 6.4 (2.8–11.8) years. 444 (41.8%) patients had SE₅ and 149 (14%) patients had SE₃₀. On univariate analysis, risk factors for SE were not markedly different when considering a 5 or 30 min threshold. Compared to their respective controls patients with both SE₅ and SE₃₀ were younger at the age of seizure onset and at the age of SE, were on more antiepileptic drugs (AEDs) at baseline, had a higher rate of changes in AEDs in the three months prior to the episode, were more likely to have developmental delay at baseline, and a higher mortality rate. A higher baseline seizure frequency, and a higher increase in seizure frequency prior to the index episode were seen only in SE₅.ConclusionThis series identifies RF which predict convulsive SE in pediatric patients. These RF are similar when considering a 5 min or a 30 min threshold for the definition of SE.     

Étifoxine : études cliniques récentes

Etifoxine is a compound that is unrelated to the benzodiazepines and that has demonstrated anxiolytic and anticonvulsant properties in animals. Currently, etifoxine is indicated in humans in the treatment of stress-induced anxiety and its somatic manifestations.Randomised, controlled, double-blind studies by Servant, Graziani et al. [22] and Nguyen, Fakra et al. [16] versus a reference treatment have shown that daily administration of etifoxine for 28 days at a dose of 150 mg in patients suffering from adjustment disorder with anxiety (ADA) caused a reduction in anxiety and a global clinical improvement from day 7 of treatment. These studies suggest that etifoxine is more effective than buspirone and produces a favourable clinical response in a larger number of patients than lorazepam. In addition, the therapeutic index appears to be in favour of etifoxine. On discontinuation fewer patients exhibited withdrawal reactions with etifoxine than with lorazepam.The study by Micallef, Soubrouillard et al. [15] in healthy subjects randomised to receive a single dose of etifoxine (50 or 100 mg), lorazepam (2 mg) or placebo confirmed that, in contrast to lorazepam, etifoxine had little sedative effect and preserved psychomotor, attention and memory performance. Etifoxine facilitates GABAergic transmission by binding to the GABA_A receptor/chloride channel complex, a property that it shares with benzodiazepines. However, the binding sites are different, a feature that appears to be the origin of the observed clinical differences.The rapid onset of clinical efficacy and the safety profile of etifoxine might be of major interest in the management of ADA as an alternative to conventional anxiolytic treatments.     

Anticonvulsant effect of dextrometrophan on pentylenetetrazole-induced seizures in mice: Involvement of nitric oxide and N-methyl-d-aspartate receptors

Dextrometrophan (DM), widely used as an antitussive, has recently generated interest as an anticonvulsant drug. Some effects of dextrometrophan are associated with alterations in several pathways, such as inhibition of nitric oxide synthase (NOS) enzyme and N-methyl d-aspartate (NMDA) receptors. In this study, we aimed to investigate the anticonvulsant effect of acute administration of dextrometrophan on pentylenetetrazole (PTZ)-induced seizures and the probable involvement of the nitric oxide (NO) pathway and NMDA receptors in this effect. For this purpose, seizures were induced by intravenous PTZ infusion. All drugs were administrated by intraperitoneal (i.p.) route before PTZ injection. Our results demonstrate that acute DM treatment (10–100 mg/kg) increased the seizure threshold. In addition, the nonselective NOS inhibitor L-NAME (10 mg/kg) and the neural NOS inhibitor, 7-nitroindazole (40 mg/kg), at doses that had no effect on seizure threshold, augmented the anticonvulsant effect of DM (3 mg/kg), while the inducible NOS inhibitor, aminoguanidine (100 mg/kg), did not affect the anticonvulsant effect of DM. Moreover, the NOS substrate l-arginine (60 mg/kg) blunted the anticonvulsant effect of DM (100 mg/kg). Also, NMDA antagonists, ketamine (0.5 mg/kg) and MK-801 (0.05 mg/kg), augmented the anticonvulsant effect of DM (3 mg/kg). In conclusion, we demonstrated that the anticonvulsant effect of DM is mediated by a decline in neural nitric oxide activity and inhibition of NMDA receptors.     

Antiepileptic effects of quinine in the pentylenetetrazole model of seizure

Quinine, is an anti-malarial drug that specifically blocks connexin 36 (Cx36) at gap junction channels. Quinine has suppressed ictal epileptiform activity in vitro without decreasing neuronal excitability. Thus, we considered the possible anticonvulsant effects of quinine in the pentylenetetrazole (PTZ) model of seizure. Moreover, we studied the hypnotic effect and locomotor activity of quinine in mice. In the PTZ model, quinine at the dose of 60 mg/kg increased the latency of seizure. However, quinine at 40–60 mg/kg decreased the duration of seizure, dose dependently. In the potentiation of pentobarbitone sleep test, quinine significantly increased sleeping time and decreased latency to fall asleep at doses of 50 and 60 mg/kg in mice. Also, quinine decreased total locomotion in the present study. It can be concluded that quinine possesses anticonvulsant and hypnotic effects, which could contribute to the control of seizure.     

Modulation of allopregnanolone on excitatory transmitters release from single glutamatergic terminal

Neurosteroids such as allopregnanolone (Allo) are widely distributed in the brain and may modulate neuronal excitability under physiological or pathological states. Allo modulates GABA_A receptor responses, and in this study we investigated the functional effects of Allo on presynaptic GABA_A receptors on single glutamatergic nerve terminal projecting on CA3 neurons. In the present study, we measured spontaneous and evoked excitatory postsynaptic currents (sEPSCs and eEPSCs), the latter was elicited with single or paired-pulse focal electrical stimulation, using mechanically isolated ‘synaptic bouton’ preparation. Allo (10 nM) increased significantly eEPSC amplitude while decreasing the failure rate (R_f) and the paired-pulse response ratio (PPR). Conversely high concentration (100 nM) of Allo decreased eEPSC amplitude and increased R_f and PPR. Allo also increased significantly the frequency and amplitude of sEPSCs at low concentrations (10–30 nM) but at high concentration (100 nM) it had no effect on current amplitude but modestly decreased sEPSC frequency. Application of Allo at nanomolar concentrations facilitated exogenous muscimol-induced outward postsynaptic currents but had no effect on glutamate-induced inward postsynaptic currents. Our results demonstrate that Allo modulates glutamate release via presynaptic GABA_A receptors, in addition to its better characterized effects to modulate postsynaptic GABA_A responses. Both pre- and postsynaptic GABA_A receptor modulation is likely to contribute to the physiological actions of neurosteroids.     

Combined diazepam and MK-801 therapy provides synergistic protection from tetramethylenedisulfotetramine-induced tonic–clonic seizures and lethality in mice

The synthetic rodenticide, tetramethylenedisulfotetramine (TMDT), is a persistent and highly lethal GABA-gated Cl⁻ channel blocker. TMDT is clandestinely produced, remains popular in mainland China, and causes numerous unintentional and deliberate poisonings worldwide. TMDT is odorless, tasteless, and easy to manufacture, features that make it a potential weapon of terrorism. There is no effective treatment. We previously characterized the effects of TMDT in C57BL/6 mice and surveyed efficacies of GABAergic and glutamatergic anticonvulsant treatments. At 0.4 mg/kg i.p., TMDT produced neurotoxic symptomatology consisting of twitches, clonic and tonic–clonic seizures, often progressing to status epilepticus and death. If administered immediately after the occurrence of the first clonic seizure, the benzodiazepine diazepam (DZP) effectively prevented all subsequent seizure symptoms, whereas the NMDA receptor antagonist dizocilpine (MK-801) primarily prevented tonic–clonic seizures. The latter agent, however, appeared to be more effective at preventing delayed death. The present study further explored these phenomena, and characterized the therapeutic actions of DZP and MK-801 as combinations. Joint treatment with both DZP and MK-801 displayed synergistic protection against tonic–clonic seizures and 24 h lethality as determined by isobolographic analysis. Clonic seizures, however, remained poorly controlled. A modification of the treatment regimen, where DZP was followed 10 min later by MK-801, yielded a reduction in both types of seizures and improved overall outcome. Simultaneous monitoring of subjects via EEG and videography confirmed effectiveness of this sequential regimen. We conclude that TMDT blockage at GABA_A receptors involves early activation of NMDA receptors, which contribute to persistent ictogenic activity. Our data predict that a sequential combination treatment with DZP followed by MK-801 will be superior to either individual therapy with, or simultaneous administration of, these two agents in treating TMDT poisoning.     

Microinjection of GABAergic agents into the anterior nucleus of the thalamus modulates pilocarpine-induced seizures and status epilepticus

The anterior nucleus of the thalamus (AN) has been suggested as a potential target for seizure modulation in animal models and patients with refractory epilepsy. We investigate whether microinjections of GABAergic agonists into the AN were protective against pilocarpine-induced generalized seizures and status epilepticus (SE). Rats were treated with bilateral AN injections of muscimol (160 or 80 nmol), bicuculline (15 nmol), or saline (controls) 20 min prior to pilocarpine administration (350 mg/kg i.p.). Electrographic recordings were used to confirm seizure activity. We found that pretreatment with AN muscimol 160 nmol increased the latency to seizures and SE by 2.5–3.0-fold. This dose however was associated with side effects, particularly hypotonia. AN bicuculline was proconvulsant, whereas no major effect was observed after muscimol 80 nmol injections. The percentage of animals that developed SE was similar across groups. Overall, microinjection of high doses of muscimol into the AN delayed the occurrence of pilocarpine-induced seizures and SE but was not able to prevent these events.