Reduced neurosteroid potentiation of GABAA receptors in epilepsy and depolarized hippocampal neurons

OBJECTIVE: Neurosteroids regulate neuronal excitability by potentiating γ-aminobutyric acid type-A receptors (GABARs). In animal models of temporal lobe epilepsy, the neurosteroid sensitivity of GABARs is diminished and GABAR subunit composition is altered. We tested whether similar changes occur in patients with epilepsy and if depolarization-induced increases in neuronal activity can replicate this effect. METHODS: We determined GABAR α4 subunit expression in cortical tissue resected from pediatric epilepsy patients. Modulation of human GABARs by allopregnanolone and Ro15-4513 was measured in Xenopus oocytes using whole-cell patch clamp. To extend the findings obtained using tissue from epilepsy patients, we evaluated GABAR expression and modulation by allopregnanolone and Ro15-4513 in cultured rat hippocampal neurons exposed to high extracellular potassium (HK) to increase neuronal activity. RESULTS: Expression of α4 subunits was increased in pediatric cortical epilepsy specimens encompassing multiple pathologies. The potentiation of GABA-evoked currents by the neurosteroid allopregnanolone was decreased in Xenopus oocytes expressing GABARs isolated from epilepsy patients. Furthermore, receptors isolated from epilepsy but not control tissue were sensitive to potentiation by Ro15-4513, indicating higher expression of α INTERPRETATION: These findings suggest that seizure activity-induced upregulation of

Phylogenetic and Functional Diversity of Faecal Microbiome of Pack Animals

The present chapter describes the microbial diversity of faecal microbiomes of pack animals. The sequencing data generated through ion semiconductor sequencing technology were analysed using EBI metagenomics and MG‐RAST server tools. Bacteria were the major domain in all the pack animals. At the phylogenetic level, Firmicutes was the major phylum. Clostridiales was the major order. Ruminococcus flavefaciens was the major species in camel, whereas the top‐most species existing in Equidae family was Streptococcus equinus. Among the 28 major functional categories, protein metabolism functionality was dominant in pack animals. The genes associated with protein processing and modification as well as for protein folding are higher in mules and in camel they are lowest. Central carbohydrate metabolism was the major functional group under carbohydrate metabolism in pack animals. Variation in the amino acids and its derivatives was seen in pack animals. Genes associated with proline and 4‐hydroxy prolines were present in Equidae family only. Clustering using ward with Bray‐Curtis distance matrix for the functional categories showed that donkey and mule are most closely related and clustered with the horse metagenome

Neurosteroid regulation of GABA(A) receptors: A role in catamenial epilepsy

The female reproductive hormones progesterone and estrogen regulate network excitability. Fluctuations in the circulating levels of these hormones during the menstrual cycle cause frequent seizures during certain phases of the cycle in women with epilepsy. This seizure exacerbation, called catamenial epilepsy, is a dominant form of drug-refractory epilepsy in women of reproductive age. Progesterone, through its neurosteroid derivative allopregnanolone, increases gamma-aminobutyric acid type-A receptor (GABAR)-mediated inhibition in the brain and keeps seizures under control. Catamenial seizures are believed to be a neurosteroid withdrawal symptom, and it was hypothesized that exogenous administration of progesterone to maintain its levels high during luteal phase will treat catamenial seizures. However, in a multicenter, double-blind, phase III clinical trial, progesterone treatment did not suppress catamenial seizures. The expression of GABARs with reduced neurosteroid sensitivity in epileptic animals may explain the failure of the progesterone clinical trial. The expression of neurosteroid-sensitive delta subunit containing GABARs is reduced, and the expression of alpha 4 gamma 2 subunit-containing GABARs is upregulated, which alters the inhibition of dentate granule cells in epilepsy. These changes reduce the endogenous neurosteroid control of seizures and contribute to catamenial seizures. (C) 2018 Elsevier B.V. All rights reserved

The Frail Male: Early Life Inflammation Promotes Chloride Loading in Adult Male Mice

Early Life Inflammation Increases CA1 Pyramidal Neuron Excitability in a Sex and Age-Dependent Manner Through a Chloride Homeostasis Disruption Gomez CD, Read J, Acharjee S, Pittman QJ. J Neurosci . 2019;39(37):7244. Early life, systemic inflammation causes long-lasting changes in behavior. To unmask possible mechanisms associated with this phenomenon, we asked whether the intrinsic membrane properties in hippocampal neurons were altered as a consequence of early life inflammation. C57BL/6 mice were bred in-house and both male and female pups from multiple litters were injected with lipopolysaccharide (LPS; 100 g/kg, intraperitoneally.) or vehicle at postnatal day (P)14, and kept until adolescence (P35-P45) or adulthood (P60-P70), when brain slices were prepared for whole-cell and perforated-patch recordings from CA1 hippocampal pyramidal neurons. In neurons of adult male mice pretreated with LPS, the number of action potentials elicited by depolarizing current pulses was significantly increased compared with control neurons, concomitant with increased input resistance, and a lower action potential threshold. Although these changes were not associated with changes in relevant sodium channel expression or differences in capacitance or dendritic architecture, they were linked to a mechanism involving intracellular chloride overload, revealed through a depolarized γ aminobutyric acid reversal potential and increased expression of the chloride transporter, NKCC1. In contrast, no significant changes were observed in neurons of adult female mice pretreated with LPS, nor in adolescent mice of either sex. These data uncover a potential mechanism involving neonatal inflammation induced plasticity in chloride homeostasis, which may contribute to early life inflammation-induced behavioral alterations

Progesterone modulates neuronal excitability bidirectionally

•Progesterone regulation of neuronal activity and seizures.•Progesterone receptor activation exerts seizure-promoting effects.•Progesterone receptor activation could contribute to catamenial seizure exacerbation.•Allopregnanolone suppresses seizure activity through potentiation of GABAA receptors. Progesterone acts on neurons directly by activating its receptor and through metabolic conversion to neurosteroids. There is emerging evidence that progesterone exerts excitatory effects by activating its cognate receptors (progesterone receptors, PRs) through enhanced expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs). Progesterone metabolite 5α,3α-tetrahydro-progesterone (allopregnanolone, THP) mediates its anxiolytic and sedative actions through the potentiation of synaptic and extrasynaptic γ-aminobutyric acid type-A receptors (GABAARs). Here, we review progesterone's neuromodulatory actions exerted through PRs and THP and their opposing role in regulating seizures, catamenial epilepsy, and seizure exacerbation associated with progesterone withdrawal

GABAergic transmission in temporal lobe epilepsy: The role of neurosteroids

Modification of GABAergic inhibition is an intensely investigated hypothesis guiding research into mechanisms underlying temporal lobe epilepsy (TLE). Seizures can be initiated by blocking gamma amino butyric acid type A (GABAA receptors, GABARs), which mediate fast synaptic inhibition in the brain, and controlled by drugs that enhance their function. Derivatives of steroid hormones called neurosteroids are natural substances that physiologically enhance GABAR function and suppress seizures. GABAR structure, function, expression, assembly, and pharmacological properties are changed in the hippocampus of epileptic animals. These alterations render GABARs less sensitive to neurosteroid modulation, which may contribute to seizure susceptibility. Plasticity of GABARs could play a role in periodic exacerbation of seizures experienced by women with epilepsy, commonly referred to as catamenial epilepsy. (C) 2011 Elsevier Inc. All rights reserved

Characterization of goat plasma vitronectin

186-193Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4ºC resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca2+ caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity in the protein

Neurosteroid‐sensitive δ‐GABAA receptors: A role in epileptogenesis?

Summary Objective We determined the role of the neurosteroid‐sensitive δ subunit–containing γ‐aminobutyric acid A receptors (δ‐GABARs) in epileptogenesis. Methods Status epilepticus (SE) was induced via lithium pilocarpine in adult rats, and seizures were assessed by continuous video–electroencephalography (EEG) monitoring. Finasteride was administered to inhibit neurosteroid synthesis. The total and surface protein expression of hippocampal δ, α4, and γ2 GABAR subunits was studied using biotinylation assays and Western blotting. Neurosteroid potentiation of the tonic currents of dentate granule cells (DGCs) was measured by whole‐cell patch‐clamp technique. Finally, the effects of inhibiting N‐methyl‐d‐aspartate receptors (NMDARs) during SE on the long‐term plasticity of δ‐GABARs, neurosteroid‐induced modulation of tonic current, and epileptogenesis were studied. Results The inhibition of neurosteroid synthesis 4 days after SE triggered acute seizures and accelerated the onset of chronic recurrent spontaneous seizures (epilepsy). The down‐regulation of neurosteroid‐sensitive δ‐GABARs occurred prior to the onset of epilepsy, whereas an increased expression of the γ2‐GABAR subunits occurred after seizure onset. MK801 blockade of NMDARs during SE preserved the expression of neurosteroid‐sensitive δ‐GABARs. NMDAR blockade during SE also prevented the onset of spontaneous seizures. Significance Changes in neurosteroid‐sensitive δ‐GABAR expression correlated temporally with epileptogenesis. These findings raise the possibility that δ‐GABAR plasticity may play a role in epileptogenesis