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Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles. Aβ oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aβ toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aβ-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-β has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration.

Allopregnanolone, a positive modulator of GABAA receptors with antiseizure activity, has potential in the treatment of seizure emergencies. Instillation of allopregnanolone in 40% sulfobutylether-β-cyclodextrin into the nose in mice rapidly elevated the seizure threshold in the timed intravenous pentylenetetrazol (ED50, 5.6 mg/kg), picrotoxin (ED50, 5.9 mg/kg), and bicuculline seizure tests. The effect peaked at 15 min, decayed over 1 h, and was still evident in some experiments at 6 h. Intranasal allopregnanolone also delayed the onset of seizures in the maximal PTZ test. At an allopregnanolone dose (16 mg/kg) that conferred comparable effects on seizure threshold as the benzodiazepines midazolam and diazepam (both at doses of 1 mg/kg), allopregnanolone caused minimal sedation or motor toxicity in the horizontal screen test whereas both benzodiazepines produced marked behavioral impairment. In addition, intranasal allopregnanolone failed to cause loss-of-righting reflex in most animals, but when the same dose was administered intramuscularly, all animals became impaired. Intranasal allopregnanolone (10 mg/kg) caused a rapid increase in brain allopregnanolone with a Tmax of ~5 min after initiation of the intranasal delivery. High levels of allopregnanolone were recovered in the olfactory bulb (Cmax, 16,000 ng/mg) whereas much lower levels (Cmax, 670 ng/mg) were present in the remainder of the brain. We conclude that the unique ability of intranasal allopregnanolone to protect against seizures without inducing behavioral adverse effects is due in part to direct nose-to-brain delivery, with preferential transport to brain regions relevant to seizures. Benzodiazepines are commonly administered intranasally for acute seizure therapy, including for the treatment of acute repetitive seizures, but are not transported from nose-to-brain. Intranasal allopregnanolone acts with greater speed, has less propensity for adverse effects, and has the ability to overcome benzodiazepine refractoriness. This is the first study demonstrating rapid functional central nervous system activity of a nose-to-brain-delivered steroid. Intranasal delivery circumvents the poor oral bioavailability of allopregnanolone providing a route of administration permitting its evaluation as a treatment for diverse neuropsychiatric indications.

Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 μM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2β3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.

Previous studies demonstrated that pentylenetetrazole (PTZ), a GABA type A receptor (GABAAR) antagonist, elicits seizure-like phenotypes in larval zebrafish (Danio rerio). Here, we determined whether the GABAAR antagonists, tetramethylenedisulfotetramine (TETS) and picrotoxin (PTX), both listed as credible chemical threat agents, similarly trigger seizures in zebrafish larvae. Larvae of three, routinely used laboratory zebrafish lines, Tropical 5D, NHGRI and Tupfel long fin, were exposed to varying concentrations of PTZ (used as a positive control), PTX or TETS for 20 min at 5 days post fertilization (dpf). Acute exposure to PTZ, PTX or TETS triggered seizure behavior in the absence of morbidity or mortality. While the concentration-effect relationship for seizure behavior was similar across zebrafish lines for each GABAAR antagonist, significantly less TETS was required to trigger seizures relative to PTX or PTZ. Recordings of extracellular field potentials in the optic tectum of 5 dpf Tropical 5D zebrafish confirmed that all three GABAAR antagonists elicited extracellular spiking patterns consistent with seizure activity, although the pattern varied between chemicals. Post-exposure treatment with the GABAAR positive allosteric modulators (PAMs), diazepam, midazolam or allopregnanolone, attenuated seizure behavior and activity but did not completely normalize electrical field recordings in the optic tectum. These data are consistent with observations of seizure responses in mammalian models exposed to these same GABAAR antagonists and PAMs, further validating larval zebrafish as a higher throughput-screening platform for antiseizure therapeutics, and demonstrating its appropriateness for identifying improved countermeasures for TETS and other convulsant chemical threat agents that trigger seizures via GABAAR antagonism.

The functional deficiency of the inhibitory system typically appears during development and can progress to psychiatric disorders or epilepsy, depending on its severity, in later years. It is known that interneurons, the major source of GABAergic inhibition in the cerebral cortex, can make direct connections with arterioles and participate in the regulation of vasomotion. The goal of this study was to mimic the functional deficiency of interneurons through the use of localized microinjections of the GABA antagonist, picrotoxin, in such a concentration that it did not elicit epileptiform neuronal activity. First, we recorded the dynamics of resting-state neuronal activity in response to picrotoxin injections in the somatosensory cortex of an awake rabbit; second, we assessed the altered neuronal and hemodynamic responses to whisker stimulation using BOLD fMRI and electrophysiology recordings; third, we evaluated brain tissue oxygen levels before and after picrotoxin injection. Our results showed that neuronal activity typically increased after picrotoxin administration, the BOLD responses to stimulation became negative, and the oxygen response was nearly abolished. Vasoconstriction during the resting baseline was not observed. These results indicate that picrotoxin provoked imbalanced hemodynamics either due to increased neuronal activity, decreased vascular response, or a combination of both. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Dalian University of Technology / Dalian Ligong Daxue Xuebao is the property of Journal of Dalian University of Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Introduction. Anticonvulsant drugs act on different molecular targets, selectively changing the excitability of neurons in such a way as to block the activity that leads to seizures without disrupting the normal activity required for the transmission of signals between neurons. The main mechanisms of reducing the excitability of neurons of the epielliptogenic focus are either inhibition of neuron activation or stimulation of inhibitory neurons. In general, these effects can be reduced to three main pharmaconeurophysiological mechanisms: enhancement of GABA or glycine transmission, reduction of excitatory (glutamate or aspartate) transmission, and nonspecific modulation of ion currents (sodium, calcium, and potassium channels). The most effective control of the tendency to convulsions is achieved precisely through the activation of GABA-ergic mechanisms. Propoxazepam, 7-bromo-5-(o-chlorophenyl)-3-propoxy-1,2-dihydro-3H-1,4-benzodiazepin-2-one, in the models of nociceptive and neuropathic pain showed significant analgesic activity. Similar to gabapentin and pregabalin, which are wellknown anti-epileptic drugs used in general medical practice in the treatment of neuropathic pain, propoxazepam also has an anticonvulsant effect, which explains the analgesic component of the pharmacological spectrum. The aim of the study was to find out and determine the mechanism of anticonvulsant action of propoxazepam on experimental animals using a wide range of convulsant substances. Materials and methods. Experiments were performed on outbred white mice of both sexes weighing 20-22 g. The use of different seizures inducers (picrotoxin (PCT), penthylenetetrazole (PTZ), strychnine (STR), thiosemicarbazide (TSC), bemegride (BMG), 4-aminopyridine (4-AP), and maximal electroshock (MES) allowed us to simulate different paroxyzmal manifestations and to suggest the propoxazepam mechanisms of antiseizure action. Results. The results indicate a high protective activity of propoxazepam based on the data of the dose-effect curves: PCT 1.67 ± 0.09, PTZ 0.9 ± 0.04, STR 14.24 ± 0.47, MES 0.57 ± 0.23, TSC 0.18 ± 0.09, 4-AP 37.3 ± 7.9 mg/kg. Depending on the type of сhemo convulsant, the protective dose of propoxazepam is significantly different (from 0.32 mg/kg for TSC up to 80 mg/kg for 4-AP), indicating a different degree of interaction with biotargets. Obviously, propoxazepam is more effective on the GABA-ergic dysfunction models, what is proved by a rise of the myoclonic component repre sentation in the seizures structrure with the simultaneous decrease of the tonic component. We have to mention that, on the BMG-induced seizures model, the relative quantity of the myoclonic component is only 32 %. This suggests the comparatively separate influence of benzdiazepine- and barbiturate-allosteric centers control. MES-induced seizures blocking demands the strong and steady cells hyperpolarization. Thus, the propoxazepam efficiency in this model is not very high. Moreover, even the relatively high doses (20 mg/kg) don't suppress the tonic component (68 %) possessing nevertheless, the protective action. The attention is attracted by the fact that in the model of 4-AP-induced seizures, propoxazepam not only shares the negligible effect (94 % of protective action is reached at a dose of 80 mg/kg), but also unusually redistributes the myoclonic and tonic components (reducing the firs and increasing the second component). Such peculiarities in the anticonvulsant action can be explained from the position of potassium ion channels functioning, which are inhibited by 4-AP. It is known that their activation leads to the membrane repolarization after the action potential development (participation in the potential restoring). Conclusion. Paying attention to such an «antiepileptic» effect, it is possible to suggest the division of propoxazepam and 4-AM actions -- first one acts predominantly on postsynaptic GABAA-receptors, causing the cell hyperpolarization, while the second interferes both pre- and postsynaptic potassium channels of the cell. Obviously, such an influence leads not only to the hyperpolarization efficiency reducing, but also increases the cell refractory period. Be cause of it, even the high doses of propoxazepam doesn′t share the 100 % efficiency in this model. [ABSTRACT FROM AUTHOR]

Songorine (SON) is a diterpenoid alkaloid from Aconitum plants. Preparations of Aconitum roots have been employed in traditional oriental herbal medicine, however, their mechanisms of action are still unclear. Since GABA-receptors are possible brain targets of SON, we investigated which subtypes of GABA-receptors contribute to the effects of SON, and how SON affects anxiety-like trait behavior and psychomotor cognitive performance of rats. First, we investigated the effects of microiontophoretically applied SON alone and combined with GABA-receptor agents picrotoxin and saclofen on neuronal firing activity in various brain areas. Next, putative anxiolytic effects of SON (1.0–3.0 mg/kg) were tested against the GABA-receptor positive allosteric modulator reference compound diazepam (1.0–5.0 mg/kg) in the elevated zero maze (EOM). Furthermore, basic cognitive effects were assessed in a rodent version of the psychomotor vigilance task (PVT). Local application of SON predominantly inhibited the firing activity of neurons. This inhibitory effect of SON was successfully blocked by GABA(A)-receptor antagonist picrotoxin but not by GABA(B)-receptor antagonist saclofen. Similar to GABA(A)-receptor positive allosteric modulator diazepam, SON increased the time spent by animals in the open quadrants of the EOM without any signs of adverse psychomotor and cognitive effects observed in the PVT. We showed that, under in vivo conditions, SON acts as a potent GABA(A)-receptor agonist and effectively decreases anxiety without observable side effects. The present findings facilitate the deeper understanding of the mechanism of action and the widespread pharmacological use of diterpene alkaloids in various CNS indications. [ABSTRACT FROM AUTHOR]

The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the United States Department of Homeland Security. TETS has previously been studied in vivo for toxicity and in vitro in binding assays, with the latter demonstrating it to be a non-competitive antagonist on GABAA receptors. To determine whether TETS exhibits subtype selectivity for a particular GABAA receptor combination, we used whole-cell patch-clamp to determine the potency of TETS on the major synaptic and extrasynaptic GABAA receptors associated with convulsant activity. The active component of picrotoxin, picrotoxinin, was used as a control. While picrotoxinin did not differentiate well between 13 GABAA receptors, TETS exhibited the highest activity on α2β3γ2 (IC50 480 nM, 95% CI 320-640 nM) and α6β3γ2 (IC50 400 nM, 95% CI 290-510 nM). Introducing β1 or β2 subunits into these receptor combinations reduced or abolished TETS sensitivity, suggesting that TETS preferentially affects receptors with α2/β3 or α6/β3 composition. Since α2β3γ2 receptors make up 15-20% of the GABAA receptors in the mammalian CNS, we suggest that α2β3γ2 is probably the most important GABAA receptor for the seizure-inducing activity of TETS.

Retinal prostheses partially restore vision in patients blinded by retinitis pigmentosa (RP) and age-related macular degeneration (AMD). One issue that limits the effectiveness of retinal stimulation is the desensitization of the retina response to repeated pulses. Rapid fading of percepts is reported in clinical studies. We studied the retinal output evoked by fixed pulse trains vs. pulse trains that have variable parameters pulse-to-pulse. We used the current clamp to record RGC spiking in the isolated mouse retina. Trains of biphasic current pulses at different frequencies and amplitudes were applied. The main results we report are: (1) RGC desensitization was induced by increasing stimulus frequency, but was unrelated to stimulus amplitude. Desensitization persisted when the 20 Hz stimulation pulses were applied to the retinal ganglion cells at 65 µA, 85 µA, and 105 µA. Subsequent pulses in the train evoked fewer spikes. There was no obvious desensitization when 2 Hz stimulation pulse trains were applied. (2) Blocking inhibitory GABAA receptor increased spontaneous activity but did not reduce desensitization. (3) Pulse trains with constant charge or excitation (based on strengthduration curves) but varying pulse width, amplitude, and shape increased the number of evoked spikes/pulse throughout the pulse train. This suggests that retinal desensitization can be partially overcome by introducing variability into each pulse. [ABSTRACT FROM AUTHOR]

Simple Summary: Gamma aminobutyric acid (GABA) a non-protein amino acid is an archaic signalling molecule for cellular communication across all kingdoms of life with distinct signalling roles in animals. Emerging research establishes GABA as a signalling molecule in planta. GABA modulates numerous developmental processes and negatively regulates root growth, stomatal aperture, and anion flux under abiotic stress. A putative GABA binding site with homology to the mammalian GABA binding site has been identified in a family of proteins (Aluminium Activated Malate Transporters -ALMT) involved in mediating anion flux and conferring plant aluminium tolerance. Studies have shown that ALMTs are regulated by GABA and transport both anions and GABA. To gain further insights into the mechanism of GABA regulation, it is essential to differentiate between the two transport modes. Pharmacological agents used to characterise mammalian GABA receptors have proved useful in gaining insights into GABA regulation of plant processes. In this study, picrotoxin an inhibitor of anion flux in mammalian GABA receptors was used to investigate the pathways for anion and GABA transport in ALMTs. Results suggest that picrotoxin inhibits anion flux but not GABA flux and can be used to gain further insights into mechanism of GABA regulation of plant proteins. Plant-derived pharmacological agents have been used extensively to dissect the structure–function relationships of mammalian GABA receptors and ion channels. Picrotoxin is a non-competitive antagonist of mammalian GABAA receptors. Here, we report that picrotoxin inhibits the anion (malate) efflux mediated by wheat (Triticum aestivum) ALMT1 but has no effect on GABA transport. The EC50 for inhibition was 0.14 nM and 0.18 nM when the ALMTs were expressed in tobacco BY2 cells and in Xenopus oocytes, respectively. Patch clamping of the oocyte plasma membrane expressing wheat ALMT1 showed that picrotoxin inhibited malate currents from both sides of the membrane. These results demonstrate that picrotoxin inhibits anion efflux effectively and can be used as a new inhibitor to study the ion fluxes mediated by ALMT proteins that allow either GABA or anion transport. [ABSTRACT FROM AUTHOR]

The spatial and temporal patterns of spontaneous synchronous Ca2+ oscillations (SCOs) regulate physiological pathways that influence neuronal development, excitability, and health. Hippocampal neuronal cultures (HN) and neuron/glia co-cultures (HNG) produced from neonatal mice were loaded with Fluo-4/AM and SCOs recorded in real-time using a Fluorescence Imaging Plate Reader at different developmental stages in vitro. HNG showed an earlier onset of SCOs, with low amplitude and low frequency SCOs at 4days in vitro (DIV), whereas HN were quiescent at this point. SCO amplitude peaked at 9 DIV for both cultures. SCO network frequency peaked at 12 DIV in HN, whereas in HNG the frequency peaked at 6 DIV. SCO patterns were associated with the temporal development of neuronal networks and their ratio of glutamatergic to GABAergic markers of excitatory/inhibitory balance. HN and HNG exhibited differential responses to the convulsant tetramethylenedisulfotetramine (TETS) and were highly dependent on DIV. In HN, TETS triggered an acute rise of intracellular Ca2+ (Phase I response) only in 14 DIV and a sustained decrease of SCO frequency with increased amplitude (Phase II response) at all developmental stages. In HNG, TETS decreased the SCO frequency and increased the amplitude at 6 and 14 but not 9 DIV. There was no acute Ca2+ rise (Phase I response) in any age of HNG tested with TETS. These data demonstrated the importance of glia and developmental stage in modulating neuronal responses to TETS. Our results illustrate the applicability of the model for investigating how caged convulsants elicit abnormal network activity during the development of HN and HNG cultures in vitro.

UnlabelledArginase 1 deficiency is a urea cycle disorder associated with hyperargininemia, spastic diplegia, loss of ambulation, intellectual disability, and seizures. To gain insight on how loss of arginase expression affects the excitability and synaptic connectivity of the cortical neurons in the developing brain, we used anatomical, ultrastructural, and electrophysiological techniques to determine how single-copy and double-copy arginase deletion affects cortical circuits in mice. We find that the loss of arginase 1 expression results in decreased dendritic complexity, decreased excitatory and inhibitory synapse numbers, decreased intrinsic excitability, and altered synaptic transmission in layer 5 motor cortical neurons. Hepatic arginase 1 gene therapy using adeno-associated virus rescued nearly all these abnormalities when administered to neonatal homozygous knock-out animals. Therefore, gene therapeutic strategies can reverse physiological and anatomical markers of arginase 1 deficiency and therefore may be of therapeutic benefit for the neurological disabilities in this syndrome.Significance statementThese studies are one of the few investigations to try to understand the underlying neurological dysfunction that occurs in urea cycle disorders and the only to examine arginase deficiency. We have demonstrated by multiple modalities that, in murine layer 5 cortical neurons, a gradation of abnormalities exists based on the functional copy number of arginase: intrinsic excitability is altered, there is decreased density in asymmetrical and perisomatic synapses, and analysis of the dendritic complexity is lowest in the homozygous knock-out. With neonatal administration of adeno-associated virus expressing arginase, there is near-total recovery of the abnormalities in neurons and cortical circuits, supporting the concept that neonatal gene therapy may prevent the functional abnormalities that occur in arginase deficiency.

Copyright of Iranian Journal of Veterinary Science & Technology is the property of Ferdowsi University of Mashhad Press and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Cancer patients sometimes have hiccups for the enforcement of chemotherapy or for tumor itself. Hiccups, which are rhythmical, clonic contractions of the diaphragm, are considered as a form of convulsions. For treatment of intractable hiccups, several major tranquilizer or anticonvulsant medications likes benzodiazepine or barbiturate have been used. The traditional Chinese medicine Shitei-To (SIT), which is a mixture of extracts from three medicinal herbs, Shitei (Persimoon; Kaki Calyx; calyx of Diospyros kaki L.f. ), Shokyo (Zinger; Zingiberis rhizome; rhizome of Zingiber officinale Roscoe) and Choji (Clove; Caryophylli flos; flower-bud of Syzygium aromaticum [L.] Merrill et. Perry) has long been used for the treatment of hiccups as natural herbal therapy in Japan and China. STT might be effective against convulsions since the anticonvulsants are effective against intractable hiccups. In this study, we examined the anticonvulsant effect of SIT and Shitei extract on experimental convulsion models, chemically induced convulsions (strychnine and picrotoxin) in mice. The animals (6-week-old male ddY mice) were pretreated STT, Shitei water extract, Shitei methanol extract and Shitei ethyl acetate extract (p.o.) at a specified time prior to injection of strychnine and picrotoxin (i.p.). In these experiments, the latency to clonic convulsions was measured and recorded in seconds from the beginning of injection of convulsant to the appearance of clonic convulsions. SIT and Shitei significantly prolonged the latency to strychnine and picrotoxin-induced clonic convulsions. Furthermore, Shitei extract also prolonged the latency to strychnine and picrotoxin-induced clonic convulsions. These findings suggest that STT has anticonvulsant effects. [ABSTRACT FROM AUTHOR]

Background: Ceiba pentandra (L.) Gaertn. (Malvaceae) is a plant documented folklorically to be used in the management of epilepsy but with no scientific evidence to support its efficacy. Aim: The aim of this study was to evaluate the anticonvulsant properties of a hydroethanolic leaf extract of Ceiba pentandra (CPLE). Methods: Mouse models of acute seizures [maximal electroshock (MES)-, 4-aminopyridine (4-AP)-, pentylenetetrazole (PTZ)-, strychnine- and picrotoxin (PIC)- induced seizure tests)] and status epilepticus (lithium/pilocarpine test) were employed to investigate the anticonvulsant properties of CPLE (30–300 mg/kg, p.o.). The effects of CPLE on motor coordination (rotarod test) and spontaneous locomotor activity (open field test) were also examined. The study further investigated the possible involvement of GABAergic mechanisms in the actions of the extract. Results: CPLE, given orally, delayed the onset and reduced the frequency and duration of PTZ- and PIC-induced convulsions. Additionally, CPLE delayed the onset of convulsions and prolonged survival in the 4-aminopyridine test. The extract also prolonged survival in the MES test. CPLE, however, failed to attenuate strychnine-induced convulsions. The extract reduced severity of convulsions and increased the latency to status epilepticus in the lithium-pilocarpine test. Pre-treatment of the animals with flumazenil (1 mg/kg, i.p.) reversed the anticonvulsant effect of the extract (100 mg/kg) against PTZ-induced convulsions. The extract (at 300 mg/kg) impaired motor coordination in the rotarod test and reduced locomotor activity in the open field arena. Conclusion: The present findings indicate that Ceiba pentandra leaf extract has significant anticonvulsant properties in mouse models. These observations justify the traditional use of Ceiba pentandra for the management of seizure conditions.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ-aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the “odorant binding” pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is characterized by impairments in social interaction and restricted/repetitive behaviors. The neurotransmitter γ - aminobutyric acid (GABA) through GABAA receptor signaling in the immature brain plays a key role in the development of neuronal circuits. Excitatory/inhibitory imbalance in the mature brain has been investigated as a pathophysiological mechanism of ASD. However, whether and how disturbances of GABA signaling in embryos that are caused by GABAA receptor inhibitors cause ASD-like pathophysiology are poorly understood. The present study examined whether exposure to the GABAA receptor antagonist picrotoxin causes ASD-like pathophysiology in offspring by conducting behavioral tests from the juvenile period to adulthood and performing gene expression analyses in mature mouse brains. Here, we found that male mice that were prenatally exposed to picrotoxin exhibited a reduction of active interaction time in the social interaction test in both adolescence and adulthood. The gene expression analyses showed that picrotoxin-exposed male mice exhibited a significant increase in the gene expression of odorant receptors. Weighted gene co-expression network analysis showed a strong correlation between social interaction and enrichment of the "odorant binding" pathway gene module. Our findings suggest that exposure to a GABAA receptor inhibitor during the embryonic period induces ASD-like behavior, and impairments in odorant function may contribute to social deficits in offspring. [ABSTRACT FROM AUTHOR]

The suprachiasmatic nucleus (SCN) of the hypothalamus is the brain structure that controls circadian rhythms in mammals. The SCN is formed by two neuroanatomical regions: the ventral and dorsal. Gamma-aminobutyric acid (GABA) neurotransmission is important for the regulation of circadian rhythms. Excitatory GABA effects have been described in both SCN regions displaying a circadian variation. Moreover, the GABAergic system transfers photic information from the ventral to the dorsal SCN. However, there is almost no knowledge about GABA neurotransmission during the prenatal or postnatal development of the SCN. Here, we used whole-cell patch-clamp recordings to study spontaneous inhibitory postsynaptic currents (IPSCs) in the two SCN regions, at two zeitgeber times (day or night), and at four postnatal (P) ages: P3-5, P7-9, P12-15, and P20-25. The results herein show that the three analyzed parameters of the IPSCs, frequency, amplitude, and decay time, were significantly affected by the postnatal age: mostly, the IPSC frequency increased with age, principally in the ventral SCN in both day and night recordings; similarly, the amplitude of IPSCs augmented with age, especially at night, whereas the IPSC decay time was reduced (it was faster) with postnatal age, mainly during the day. Our findings first reveal that parameters of GABA neurotransmission are modified by postnatal development, implying that synaptic adjustments are required for an appropriate maturation of the GABAergic system in the SCN. [ABSTRACT FROM AUTHOR]

AMPA-type glutamate receptors mediate fast, excitatory neurotransmission in the brain, and their concentrations at synapses are important determinants of synaptic strength. We investigated the post-transcriptional regulation of GluA2, the calcium-impermeable AMPA receptor subunit, by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in cultured mouse hippocampal neurons. Using computational approaches, we identified a conserved microRNA-124 (miR-124) binding site in the 3'UTR of GluA2 and demonstrated that miR-124 regulated the translation of GluA2 mRNA reporters in a sequence-specific manner in luciferase assays. While we hypothesized that this regulation might occur in dendrites, our biochemical and fluorescent in situ hybridization (FISH) data indicate that GluA2 mRNA does not localize to dendrites or synapses of mouse hippocampal neurons. In contrast, we detected significant concentrations of miR-124 in dendrites. Overexpression of miR-124 in dissociated neurons results in a 30% knockdown of GluA2 protein, as measured by immunoblot and quantitative immunocytochemistry, without producing any changes in GluA2 mRNA concentrations. While total GluA2 concentrations are reduced, we did not detect any changes in the concentration of synaptic GluA2. We conclude from these results that miR-124 interacts with GluA2 mRNA in the cell body to downregulate translation. Our data support a model in which GluA2 is translated in the cell body and subsequently transported to neuronal dendrites and synapses, and suggest that synaptic GluA2 concentrations are modified primarily by regulated protein trafficking rather than by regulated local translation.

Cone photoreceptors are the first neurons along the visual pathway that exhibit center-surround antagonistic receptive fields, the basic building blocks for spatial information processing in the visual system. The surround responses in cones are mediated by the horizontal cells (HCs) via multiple feedback synaptic mechanisms. It has been controversial on which mechanisms are responsible for the surround-elicited depolarizing responses in cones (ΔVCone(s)), and whether the surround responses of various types of cones are mediated by the same HC feedback mechanisms. In this report, we studied ΔVCone(s)) of four types of cones in the salamander retina, and found that they are mediated by feedback synapses from A-type, B-type or A- and B-type HCs. ΔVCone(s) are observable in the presence of concomitant center light spots, and surround + center light stimuli of various intensity, size and wavelength differentially activate the feedback synapses from A- and B-type HCs to cones. We found that ΔVCone(s) of the L-cones are mediated by both A- and B-type HCs, those of the P- and S-cones by B-type HCs, and those of the A-cones by the A-type HCs. Moreover, our results suggest that B-type HCs mediate ΔVCone(s) through both GABAergic and GluT-ClC feedback synaptic mechanisms, and A-type HCs mediate ΔVCone(s) via the GluT-ClC feedback mechanism. Feedback synaptic mechanisms that increase calcium influx in cone synaptic terminals play important roles in mediating the antagonistic surround responses in the postsynaptic bipolar cells, but they may not generate enough current to depolarize the cones and significantly contribute to ΔVCone(s). [ABSTRACT FROM AUTHOR]

The plant metabolite picrotoxinin (PXN) is a widely used tool in neuroscience for the identification of GABAergic signaling. Its hydrolysis in weakly alkaline media has been observed for over a century and the structure of the unstable hydrolysis intermediate was assigned by analogy to the degradation product picrotoxic acid. Here we show this assignment to be in error and we revise the structure of the hydrolysis product by spectroscopic characterization in situ. Counterintuitively, hydrolysis occurs at a lactone that remains closed in the major isolable degradation product, which accounts for the longstanding mistake in the literature. [ABSTRACT FROM AUTHOR]

GABA A receptors are pentameric ligand-gated ion channels that mediate most fast neuronal inhibition in the brain. In addition to their important physiological roles, they are noteworthy in their rich pharmacology; prominent drugs used for anxiety, insomnia, and general anesthesia act through positive modulation of GABA A receptors. Direct structural information for how these drugs work was absent until recently. Efforts in structural biology over the past few years have revealed how important drug classes and natural products interact with the GABA A receptor, providing a foundation for studies in dynamics and structure-guided drug design. Here, we review recent developments in GABA A receptor structural pharmacology, focusing on subunit assemblies of the receptor found at synapses. GABA A receptors are ion channels important in brain function and are the target of chemically diverse and clinically important drugs for insomnia, epilepsy, and anesthesia. Recent structures of GABA A receptors have revealed detailed interactions of the neurotransmitter GABA, benzodiazepines, general anesthetics, and antagonists. Intravenous anesthetics bind in both common and distinctive membrane sites. Electrophysiological and molecular dynamics studies build off the static structures, to probe mechanisms of potentiation and inhibition by chemically diverse compounds. [ABSTRACT FROM AUTHOR]

Tetramethylenedisulfotetramine (TETS) is a potent convulsant that is considered a chemical threat agent. We characterized TETS as an activator of spontaneous Ca²⁺ oscillations and electrical burst discharges in mouse hippocampal neuronal cultures at 13-17 days in vitro using FLIPR Fluo-4 fluorescence measurements and extracellular microelectrode array recording. Acute exposure to TETS (≥ 2 µM) reversibly altered the pattern of spontaneous neuronal discharges, producing clustered burst firing and an overall increase in discharge frequency. TETS also dramatically affected Ca²⁺ dynamics causing an immediate but transient elevation of neuronal intracellular Ca²⁺ followed by decreased frequency of Ca²⁺ oscillations but greater peak amplitude. The effect on Ca²⁺ dynamics was similar to that elicited by picrotoxin and bicuculline, supporting the view that TETS acts by inhibiting type A gamma-aminobutyric acid (GABA(A)) receptor function. The effect of TETS on Ca²⁺ dynamics requires activation of N-methyl-D-aspartic acid (NMDA) receptors, because the changes induced by TETS were prevented by MK-801 block of NMDA receptors, but not nifedipine block of L-type Ca²⁺ channels. Pretreatment with the GABA(A) receptor-positive modulators diazepam and allopregnanolone partially mitigated TETS-induced changes in Ca²⁺ dynamics. Moreover, low, minimally effective concentrations of diazepam (0.1 µM) and allopregnanolone (0.1 µM), when administered together, were highly effective in suppressing TETS-induced alterations in Ca²⁺ dynamics, suggesting that the combination of positive modulators of synaptic and extrasynaptic GABA(A) receptors may have therapeutic potential. These rapid throughput in vitro assays may assist in the identification of single agents or combinations that have utility in the treatment of TETS intoxication.

We found previously that acute ex vivo as well as repeated cycles of in vivo ethanol exposure and withdrawal, including excessive voluntary consumption of ethanol, produces a long-lasting increase in the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in the dorsomedial striatum (DMS) of rats (Wang et al., 2010a). Activation of NMDARs is required for the induction of long-term potentiation (LTP) of AMPA receptor (AMPAR)-mediated synaptic response. We therefore examined whether the ethanol-mediated upregulation of NMDAR activity alters the induction of LTP in the DMS. We found that ex vivo acute exposure of striatal slices to, and withdrawal from, ethanol facilitates the induction of LTP in DMS neurons, which is abolished by the inhibition of NR2B-NMDARs. We also report that repeated systemic administration of ethanol causes an NR2B-NMDAR-dependent facilitation of LTP in the DMS. LTP is mediated by the insertion of AMPAR subunits into the synaptic membrane, and we found that repeated systemic administration of ethanol, as well as cycles of excessive ethanol consumption and withdrawal, produced a long-lasting increase in synaptic localization of the GluR1 and GluR2 subunits of AMPARs in the DMS. Importantly, we report that inhibition of AMPARs in the DMS attenuates operant self-administration of ethanol, but not of sucrose. Together, our data suggest that aberrant synaptic plasticity in the DMS induced by repeated cycles of ethanol exposure and withdrawal contributes to the molecular mechanisms underlying the development and/or maintenance of excessive ethanol consumption.

Tetramethylenedisulfotetramine (tetramine; TETS) is a potent convulsant poison that is considered to be a chemical threat agent. To provide a basis for the investigation of antidotes for TETS-induced seizures, we characterized the convulsant activity of TETS in mice and rats when administered by the intraperitoneal, intravenous, oral, and intraventricular routes as a single acute dose and with repeated sublethal doses. In mice, parenteral and oral TETS caused immobility, myoclonic body jerks, clonic seizures of the forelimbs and/or hindlimbs, tonic seizures, and death. The CD₅₀ values for clonic and tonic seizures after oral administration were 0.11 and 0.22 mg/kg, respectively. Intraventricular administration of TETS (5-100 μg) in rats also caused clonic-tonic seizures and death. In mice, repeated sublethal doses of TETS at intervals of 2, 24, and 48 h failed to result in the development of persistent enhanced seizure responsivity ("kindling") as was observed with repeated pentylenetetrazol treatment. In mice, sublethal doses of TETS that produced clonic seizures did not cause observable structural brain damage as assessed with routine histology and Fluoro-Jade B staining 7 days after treatment. However, 1 to 3 days after a single convulsant dose of TETS the expression of glial fibrillary acidic protein, an astrocyte marker, and ionized calcium binding adaptor molecule 1, a microglia marker, were markedly increased in cortex and hippocampus. Although TETS doses that are compatible with survival are not associated with overt evidence of cellular injury or neurodegeneration, there is transient reactive astrocytosis and microglial activation, indicating that brain inflammatory responses are provoked.

The neurotransmitter glutamate is released by excitatory projection neurons throughout the brain. However, non-glutamatergic cells, including cholinergic and monoaminergic neurons, express markers that suggest that they are also capable of vesicular glutamate release. Striatal cholinergic interneurons (CINs) express the Type-3 vesicular glutamate transporter (VGluT3), although whether they form functional glutamatergic synapses is unclear. To examine this possibility, we utilized mice expressing Cre-recombinase under control of the endogenous choline acetyltransferase locus and conditionally expressed light-activated Channelrhodopsin2 in CINs. Optical stimulation evoked action potentials in CINs and produced postsynaptic responses in medium spiny neurons that were blocked by glutamate receptor antagonists. CIN-mediated glutamatergic responses exhibited a large contribution of NMDA-type glutamate receptors, distinguishing them from corticostriatal inputs. CIN-mediated glutamatergic responses were insensitive to antagonists of acetylcholine receptors and were not seen in mice lacking VGluT3. Our results indicate that CINs are capable of mediating fast glutamatergic transmission, suggesting a new role for these cells in regulating striatal activity.

Synaptic transmission is initiated via spontaneous or action-potential evoked fusion of synaptic vesicles. At excitatory synapses, glutamatergic receptors activated by spontaneous and evoked neurotransmission are segregated. Although inhibitory synapses also transmit signals spontaneously or in response to action potentials, they differ from excitatory synapses in both structure and function. Therefore, we hypothesized that inhibitory synapses may have different organizing principles. We report picrotoxin, a GABAAR antagonist, blocks neurotransmission in a use-dependent manner at rat hippocampal synapses and therefore can be used to interrogate synaptic properties. Using this tool, we uncovered partial segregation of inhibitory spontaneous and evoked neurotransmission. We found up to 40% of the evoked response is mediated through GABAARs which are only activated by evoked neurotransmission. These data indicate GABAergic spontaneous and evoked neurotransmission processes are partially non-overlapping, suggesting they may serve divergent roles in neuronal signaling.

Introduction: Epilepsy is a common neurological disease, although its etiology and pathophysiology are not yet fully understood. Oxidative stress plays a key role in the pathogenesis of many neurological diseases, including epilepsy, and there have been many studies reporting that antiepileptic medicines with neuroprotective and antioxidant activity inhibit free oxygen radicals. This study evaluates the effects of tempol on epileptic activity through behavioral parameters in acute picrotoxin (Ptx) models.Materials and Methods: This experimental study was conducted on 42 adult male Wistar Albino rats weighing 450-500 g. Ptx (2.5 mg/kg) was injected i.p. as a single dose and observed for one hour to establish the acute Ptx model. Following injection, the animals were observed for 30 min in glass observation cages measuring 35 cm x 35 cm x 35 cm.Results: In picrotoxin-induced epilepsy, the total number of seizures and the total duration of seizures were decreased significantly with Ptx + tempol 100 mg/kg and Ptx + Tempol 150 mg/kg. The seizure phases were reduced significantly by Ptx + tempol 150 mg/kg (P < 0.05).Conclusion: Tempol 100 mg/kg and tempol 150 mg/kg are found to be effective in epilepsy models caused by Ptx, with tempol 150 mg/kg found especially to be more effective. [ABSTRACT FROM AUTHOR]

Spodoptera litura, commonly known as taro caterpillar, is a major pest of several vegetables and economically important crops. The pest is reported to reduce the yield of the affected crop up to 10-30%. Hence, controlling this pest is one of the very crucial steps in increasing crop yield. The extensive use of broad-spectrum chemical pesticides to control S. litura has many negative impacts. The present study is an attempt to evaluate the efficacy of certain plants against S. litura. In this study, we investigated the anti-insect properties of leaf and bark extracts of Anamirta cocculus, Cardiospermum halicacabum, Cocculus laurifolius and Strychnos nux-vomica. All four plant extracts showed significant anti-feedant activity compared to control at different concentrations. The leaf extracts showed the activity in the order S. nux-vomica >C. halicacabum >C. laurifolius >A. cocculus at the exposure of maximum concentration. Anti-feedant activity of the bark extract was in the order C. laurifolius >S. nux-vomica >C. halicacabum. The leaf extracts of three plants A. cocculus, C. halicacabum and S. nux-vomica showed significant repellent activity. The repellent activity of the bark extracts was in the order, C. laurifolius > C. halicacabum >S. nux-vomica >A. cocculus. The GC-MS analysis of these plant extracts have shown many compounds with known anti-insect properties and specific molecule-based bio-assays might be required to ascertain the distinctive effects of these compounds. [ABSTRACT FROM AUTHOR]

Reduced inhibitory GABA function, so‐called neural disinhibition, has been implicated in cognitive disorders, including schizophrenia and age‐related cognitive decline. We previously showed in rats that hippocampal disinhibition by local microinfusion of the GABA‐A receptor antagonist picrotoxin disrupted memory and attention and enhanced hippocampal multi‐unit burst firing recorded around the infusion site under isoflurane anesthesia. Here, we analyzed the hippocampal local field potential (LFP) recorded alongside the multi‐unit data. We predicted frequency‐specific LFP changes, based on previous studies implicating GABA in hippocampal oscillations, with the weight of evidence suggesting that disinhibition would facilitate theta and disrupt gamma oscillations. Using a new semi‐automated method based on the kurtosis of the LFP peak‐amplitude distribution as well as on amplitude envelope thresholding, we separated three distinct hippocampal LFP states under isoflurane anesthesia: "burst" and "suppression" states—high‐amplitude LFP spike bursts and the interspersed low‐amplitudeperiods—and a medium‐amplitude "continuous" state. The burst state showed greater overall power than suppression and continuous states and higher relative delta/theta power, but lower relative beta/gamma power. The burst state also showed reduced functional connectivity across the hippocampal recording area, especially around theta and beta frequencies. Overall neuronal firing was higher in the burst than the other two states, whereas the proportion of burst firing was higher in burst and continuous states than the suppression state. Disinhibition caused state‐ and frequency‐dependent LFP changes, tending to increase power at lower frequencies (<20 Hz), but to decrease power and connectivity at higher frequencies (>20 Hz) in burst and suppression states. The disinhibition‐induced enhancement of multi‐unit bursting was also state‐dependent, tending to be more pronounced in burst and suppression states than the continuous state. Overall, we characterized three distinct hippocampal LFP states in isoflurane‐anesthetized rats. Disinhibition changed hippocampal LFP oscillations in a state‐ and frequency‐dependent way. Moreover, the disinhibition‐induced enhancement of multi‐unit bursting was also LFP state‐dependent. [ABSTRACT FROM AUTHOR]

The Galphimia gracilis Bartl. is used traditionally in conditions of anxiety, fear, stress, phobia and to bring a calming effect on the nerves. This study assesses the central nervous system depressant and muscle relaxant effects of G. gracilis stem methanol extract (GgSME). The GgSME was administered in Swiss albino mice in one day to animal models like Sodium pentobarbital induced sleep test, Open field test, Hole-board test, Picrotoxin induced convulsions, Pentylenetetrazole induced convulsions, Rota rod test and Grip strengthening test. The LD50 value of the GgSME was noticed to be > 2000 mg/kg. Mice treated with GgSME at 100, 200 and 400 mg/kg doses lengthened the duration of sleep induced by sodium pentobarbital (40 mg/kg, i.p.). The GgSME at 400 mg/kg dose produced a significant (P ≤ 0.001) sedative behavior in the hole-board test and drop off spontaneous activity in animals and also slowed the outset of seizures induced by Pentylenetetrazole (90 mg/kg, i.p) & by Picrotoxin (10 mg/kg, i.p). In open field test, there was a drop of the rearing and voluntary movements (P ≤ 0.001) in animals treated with GgSME. The GgSME also displayed significant (P ≤ 0.05) effects on muscle relaxant activity in both Rota rod test and Grip strengthening test. The overall results conclude that the GgSME has significant CNS depressant actions and muscle relaxant effects. Abbreviations: Gg, Galphimia gracilis; IAEC, Institutional Animal Ethics Committee; GgSME, Galphimia gracilis stem methanol extract; CPCSEA, Committee for the Purpose of Control and Supervision of Experimental Animals; b.w, Body weight; i.p, Intraperitoneal; per os, by mouth or orally; OECD, The Organization for Economic Co-operation and Development; AGI, Anurag Group of Institutions. [ABSTRACT FROM AUTHOR]

By using the multi-electrode array (MEA) recording technique in conjunction with white-noise checkerboard stimuli and reverse correlation methods, we studied modulatory actions of glycinergic and GABAergic interneurons on spatiotemporal profiles of ganglion cells (GCs) in dark-adapted mouse retinas. We found that application of 2 µM strychnine decreased receptive field center radii of GCs by a mean value of 11%, and shifted the GC receptive field (RF) centers by a mean distance of 28.3 µm. On the other hand, 200 µM picrotoxin + 100 µM bicuculline + 50 µM TPMPA increased GC receptive field center radii by a mean value of 19%, and shifted the GC RF centers by a mean distance of 53.7 µm. Glycinergic neurons in the mouse retina are narrow-field amacrine cells that have been shown to mediate ON-OFF crossover inhibitory synapses within the RGs' RF center, therefore they may increase the size and shift the location of GC RF center by synergistic addition to bipolar cell inputs to GCs. GABAergic neurons are wide-field amacrine cells and horizontal cells that are known to mediate antagonistic surround responses of GCs, and thus they decrease the GCs' RF center size. Our results suggest that a major global function of glycinergic and GABAergic interneurons in the mammalian retina is to provide the flexibility for adjusting the size and location of GCs' RF centers. The apparent shifts of GC RF centers suggest that the synergistic addition by GlyACs and the surround inhibition by GABAergic interneurons are not spatially symmetrical within GC RFs. [ABSTRACT FROM AUTHOR]

Picrotoxin (PTX), a convulsant of plant origin, has been used in many studies as research tool. PTX is the open channel blocker of the GABA A receptor (GABA A R). Being in the pore, PTX initiates transfer of the channel to the closed state and thus it falls into the "trap". The consequence of this PTX trapping is so-called aftereffect, i.e. continuation of the blockade of the GABA-induced chloride current (I GABA) after removal of PTX from the external solution. The present work shows that the positive allosteric modulators (PAMs) of the GABA A receptor, allopregnanolone (Allo) and zolpidem (Zolp) as well as a high concentration of GABA shortened the PTX aftereffect. Experiments were carried out on isolated Purkinje neurons of the rat cerebellum using the whole-cell patch-clamp method. I GABA was induced by applications of 5 μM GABA (EC 30) for 1 s with 30 s intervals. 50 μM PTX completely blocked I GABA , and recovery upon PTX washout occurred with a time constant (τ rec) of 20.2 min. 1 μM Allo reduced the blocking effect of PTX by 30% and accelerated the recovery of I GABA by almost 10 times (τ rec = 2.4 min). 0.5 μM Zolp did not change the I GABA block in the presence of PTX but accelerated the recovery of I GABA by more than 3 times (τ rec = 5.6 min). Increasing the GABA concentration to 20 μM did not change the blocking effect of PTX, but accelerated recovery by 6 times (τ rec = 3.3 min). The mechanism of the shortening of the PTX aftereffect is presumably the expansion of the GABA A R pore in the presence of PAMs and a high concentration of the agonist and, as a consequence, the escape of PTX from the "trap". The work describes new pharmacological properties of Allo and Zolp. • GABA-activated chloride current (I GABA) was recorded on isolated Purkinje cells. • Blockade of I GABA by picrotoxin (PTX) was accompanied by slow recovery of the current. • Increasing GABA concentration accelerated the recovery of the I GABA. • Zolpidem accelerated the recovery of the I GABA after PTX block by 3 times. • Allopregnanolone accelerated the recovery of the I GABA after PTX block by 10 times. [ABSTRACT FROM AUTHOR]

Mapping the fine-scale neural activity that underlies epilepsy is key to identifying potential control targets of this frequently intractable disease. Yet, the detailed in vivo dynamics of seizure progression in cortical microcircuits remain poorly understood. We combine fast (30-Hz) two-photon calcium imaging with local field potential (LFP) recordings to map, cell by cell, the spread of locally induced (4-AP or picrotoxin) seizures in anesthetized and awake mice. Using single-layer and microprism-assisted multilayer imaging in different cortical areas, we uncover reliable recruitment of local neural populations within and across cortical layers, and we find layer-specific temporal delays, suggesting an initial supra-granular invasion followed by deep-layer recruitment during lateral seizure spread. Intriguingly, despite consistent progression pathways, successive seizures show pronounced temporal variability that critically depends on GABAergic inhibition. We propose an epilepsy circuit model resembling an elastic meshwork, wherein ictal progression faithfully follows preexistent pathways but varies flexibly in time, depending on the local inhibitory restraint.

Introduction. In rats, long-term ovariectomy results in low concentrations of steroid hormones and reproduces anxiety- and depression-like behavior after surgical menopause in women. Progesterone produces antidepressant-like effects two weeks post-ovariectomy (i.e., early post-ovariectomy) through actions on γ-aminobutyric acid-A (GABAA) receptors, but its antidepressant-like effects and mechanism of action in rats eight weeks post-ovariectomy (i.e., late post-ovariectomy, considered a model of surgical menopause) remain unknown. Objective. To explore the antidepressant-like effects of progesterone and the participation of GABAA receptors in rats eight weeks post-ovariectomy. Method. Long-term ovariectomized female Wistar rats were treated sub-acutely with vehicle or progesterone (.5, 1, and 2 mg/kg) and subjected to the open field and forced swim tests, and behavior was compared with cycling or fluoxetine-treated rats. The rats were then pretreated with picrotoxin (1 mg/kg) followed by progesterone (1 mg/kg) to explore the role of GABAA receptors in long-terminduced depression-like behavior. Results. Long-term ovariectomized rats exhibited depression-like behavior in the forced swim test compared with intact rats, an effect that was not observed in progesterone- and fluoxetine-treated long-term ovariectomized rats. These effects were not attributable to psychomotor alterations. In the open field test, the time spent rearing and grooming was lower in ovariectomized rats compared with intact rats, which was not observed in progesterone- and fluoxetine-treated rats. Picrotoxin blocked the effects of progesterone in both behavioral tests. Discussion and conclusion. These results indicated that sub-acute progesterone treatment reduced depression-like behavior through actions on GABAA receptors in a rat model of surgical menopause. [ABSTRACT FROM AUTHOR]

Studies have indicated that epilepsy, an important neurological disease, can generate oxidative stress and mitochondrial dysfunction, among other damages to the brain. In this context, the use of antioxidant compounds could provide neuroprotection and help to reduce the damage caused by epileptic seizures and thereby the use of anticonvulsant drugs. Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenylactic acid that prevents cell damage caused by free radicals, acting as an antioxidant. It also presents anti-inflammatory, antimutagenic, and antiapoptotic properties. In this work, we used two models of acute seizure, 4-aminopyridine (4-AP) and picrotoxin (PTX)-induced seizures in mice, to investigate the anticonvulsant, antioxidant, and neuroprotective profile of RA. Diazepam and valproic acid, antiepileptic drugs already used in the treatment of epilepsy, were used as positive controls. Although RA could not prevent seizures in the models used in this study, neither enhance the latency time to first seizure at the tested doses, it exhibited an antioxidant and neuroprotective effect. RA (8 and 16 mg/kg) decreased reactive oxygen species production, superoxide dismutase activity, and DNA damage, measured in hippocampus, after seizures induced by PTX and 4-AP. Catalase activity was decreased by RA only after seizures induced by 4-AP. The activity of the mitochondrial complex II was increased by RA in hippocampus samples after both seizure models. The results obtained in this study suggest that RA is able to reduce cell damage generated by the 4-AP and PTX seizures and therefore could represent a potential candidate in reducing pathophysiological processes involved in epilepsy. [ABSTRACT FROM AUTHOR]

Introduction. Propoxazepam (3-alcoxyderivative of 1.4-benzodiazepine), in the tail-flick test and picrotoxin-induced convulsions showed significant analgesic and antiepileptic activity. Flumazenil (GABA antagonist) reduced its analgesic action, although antiseizure activity was changed slightly. As specific propoxazepam actions are anticonvulsant (1 subtype GABAA-R) and analgesic (2 subtype GABAA-R in the spinal cord), it can be suggested that the substance has no abuse-related side-effects. Materials and method. The possible involvement of the GABA system in the antinociceptive effect of propoxazepam was examined by administering flumazenil (1 and 10 mg/kg, i.p.), a selective GABA- receptor antagonist, 30 min prior to propoxazepam (1.8 and 10 mg/kg, orally) in rats. Picrotoxin solution (6.5 mg/kg, 95 % of lethality effect in mice) was injected subcutaneously 30 min after propoxazepam administration. Flumazenil was administered intraperitoneally 0.5 hour prior to propoxazepam administration. Time counting was starting from the convulsant injection and during the supervision time, the number of myoclonic convulsions and tonic extensia, time of their onset, as well as time to lethal effect (survival time), were registered. Results. Flumazenil and propoxazepam antiseizure effect in the picrotoxin-induced seizures test were estimated as 5.24±2.38 and 0.37±0.07 mg/kg. Antiseizure effect of propoxazepam was reduced with flumazenil administration. Flumazenil (1 mg/ kg) had no effect in tail-flick test but abolished the antinociceptive action of propoxazepam (tail flick latency period changes were -1.6% and +55.1, compared to control and propoxazepam alone administration, respectively). Conclusions. The mechanism of propoxazepam antiseizure and antinociceptive actions are GABA-R determined. Analgesic action mechanism assumes additional biotargets involvement. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the anticonvulsant effect of the essential oil of Myrothamnus moschatus (M. moschatus) in convulsion induced by pentylenetetrazole and picrotoxin in rodent models. Methods: The essential oil of the aerial parts of M. moschatus was extracted by steam distillation. Thereafter, it was injected subcutaneously to rats and mice at escalating doses (0.1–0.8 mL/kg). Ten minutes after drug injection, pentylenetetrazole was injected intraperitoneally to rats and picrotoxin was administered to mice by the same route. Diazepam served as the positive control. Every single animal was placed into transparent cage and observed for convulsive behavior for 30 min by using ordinary security cameras connected to a video recorder. Death occurring for a period of 24 h was also recorded. Results: The essential oil at 0.8 mL/kg completely arrested the pentylenetetrazole-induced convulsion without any sedative effect and delayed its appearance at lower doses, but showed moderate activities on picrotoxin-induced convulsion. For the rats treated with pentylenetetrazole alone, the mortality was 100% within 1 h, but for the rats pre-treated with the essential oil, the mortality was 0%. For the mice treated with picrotoxin, the mortality rate was also 100%, while 20%–100% died in those that had been pre-treated with the oil. Conclusions: The results confirmed at least partly the traditional uses of the smoke of M. moschatus for the management of convulsion, and implied that the essential oil may inhibit the convulsion by GABAergic neuromodulation.