GABA-mediated Inhibition Research Articles

Perinatal High Fat Diet and Acute Stress alters the Chloride Gradient in Dorsal Motor Nucleus of the Vagus Neurons Controlling Gastric Function

The US is currently facing an obesity epidemic, though the concurrent rise in maternal obesity rates receives less attention. Maternal obesity is an important factor that influences intrauterine and early postnatal environment and is associated with developmental changes in the offspring, including alterations in metabolic function and susceptibility to anxiety and depression, all of which are associated with abnormal gastrointestinal (GI) tract function. Previous work from this lab has shown that perinatal high fat diet (pHFD) leads to developmental changes to vagal neurocircuits, including changes to GABAA receptor subunits on dorsal motor nucleus of the vagus (DMV) neurons. However, the manner of neuronal recordings (whole‐cell patch clamp) disrupted internal chloride (Cl‐) concentration, preventing accurate assessment of the extent of GABA‐mediated inhibition. KCC2, a Cl‐ transporter, is important in determining GABAergic inhibition and stress outcomes in hypothalamic neurons, however its role in regulating these factors in DMV neurons is unknown. The current study was designed to test the hypothesis that pHFD reduces KCC2 levels in DMV neurons, subsequently collapsing the Cl‐ gradient and leading to altered gastric output.Sprague‐Dawley rats were fed either a control (14% kcal from fat) or high fat (60% kcal from fat) diet starting on embryonic day 13. After weaning on postnatal day 28, brainstem slices were made to conduct patch clamp recordings on DMV neurons. This was done in both naïve (unstressed) or stressed conditions, in which rats underwent a 10 minute forced swim (FS). To determine the Cl‐ reversal, perforated patch clamp recordings were performed in order to preserve the internal Cl‐ concentration. Then, DMV neurons were voltage clamped from ‐90 to ‐40 mV and the amplitude of the current response to picospritz of the GABAAagonist muscimol (100 μM) was measured. These events were plotted on an I‐V curve, with the x‐intercept representing the reversal of Cl‐. To determine a potential mediator in the shift in the Cl‐ reversal, changes in KCC2 levels following pHFD exposure were evaluated immunohistochemically. Lastly, gastric emptying (GE) assays were performed using the 13C octanoic acid breath assessment.In both pHFD and control diet + FS DMV neurons, there was a depolarizing shift in the Cl‐ reversal compared to naïve rats (‐43.05 mV and ‐44.58 mV respectively, compared to ‐64.56 mV in naïve conditions; p < 0.0001). It was also found that there is a significant reduction in KCC2 expression in pHFD DMV region compared to controls (2.237% area fluorescence in pHFD compared to 5.335% in control diet; p=0.0147). Lastly, pHFD rats had significantly delayed GE rates compared to those fed a control diet (83.7 minutes for half‐decay in pHFD compared to 66.7 minutes in controls; p < 0.05), resembling previously published data which showed a delay in GE following an acute stress. These results demonstrate that both a pHFD and acute stress lead to depolarizing shifts in the Cl‐ reversal and that this may be through a mechanism involving a reduction in KCC2 expression. As the gastric emptying rates demonstrate, these changes could lead to a maladaptive GI stress response in pHFD rats.Determining the mechanism in which pHFD and acute stress modulate DMV function, as well as the impact of their intersection on the gastric stress response, will better inform our understanding of the reduced stress resiliency seen in pHFD offspring.

Bile Duct Ligation Changes the Inhibitory Control of Vasopressin Neurons in Male Rats

Dilutional hyponatremia typically is caused by increased Arginine Vasopressin (AVP) secretion. This increases the risk of complications and mortality in patients with liver cirrhosis. Liver cirrhosis in male rats increases AVP release but the underlying mechanisms are still under investigation. In other models of chronic AVP release, changes in chloride homeostasis alters the inhibitory control of AVP neurons in the supraoptic nucleus (SON) causing GABA mediated excitation instead of inhibition. In the bile duct ligation (BDL) model of cirrhosis, changes in either intracellular chloride concentration or cell firing activity have not been directly tested. We hypothesize that liver cirrhosis increases intracellular chloride concentration leading to a decrease in GABA‐mediated inhibition of SON neurons from male rats.To test this hypothesis, we injected an adeno‐associated virus with a vasopressin promoter and a ratiometric chloride indicator, AAV2‐0VP1‐ClopHensorN into the SON of adult male Sprague‐Dawley rats. The rats either received BDL surgery, which was used to model liver cirrhosis, or a sham ligation surgery. All BDL rats had significantly higher liver weight to body weight ratio (n = 10‐20, p < 0.05), lower plasma osmolality (n = 10‐20, p < 0.001) and lower hematocrit (n = 10‐20, p < 0.05) as compared to sham rats. All rats were anesthetized with isoflurane (2‐3%), their brains were collected, and slices containing the SON were prepared. Loose‐cell patch recordings were made at 31±1oC using standard artificial cerebrospinal fluid. Labeled AVP cells and unlabeled SON cells were tested with focal application of muscimol (100µM), a GABAA receptor agonist.Cells from BDL rats showed impaired GABAA‐mediated inhibition in response to muscimol, as compared to those from sham rats. All putative AVP SON neurons from sham rats (4/4) showed decreased activity following muscimol while half of the putative AVP SON neurons (7/14) from BDL rats were excited (p < 0.001). Similar results were observed in experiments were the injections missed the SON. In these experiments, all SON neurons from sham rats were inhibited by muscimol (26/26) while thirty‐five percent of SON neurons from BDL rats (16/45) were excited by muscimol (p = 0.001). ClopHensorN based chloride imaging showed that muscimol application was associated with decreased intracellular chloride concentrations in putative AVP cells from BDL rats as compared to sham rats (n = 31‐32, p = 0.001). The results show that liver cirrhosis impairs the normal inhibitory response of SON neurons to GABAA receptor activation which could contribute to increased cell activity and dilutional hyponatremia in male rats.

Trophic factor BDNF inhibits GABAergic signaling by facilitating dendritic enrichment of SUMO E3 ligase PIAS3 and altering gephyrin scaffold

Posttranslational addition of a small ubiquitin-like modifier (SUMO) moiety (SUMOylation) has been implicated in pathologies such as brain ischemia, diabetic peripheral neuropathy, and neurodegeneration. However, nuclear enrichment of SUMO pathway proteins has made it difficult to ascertain how ion channels, proteins that are typically localized to and function at the plasma membrane, and mitochondria are SUMOylated. Here, we report that the trophic factor, brain-derived neurotrophic factor (BDNF) regulates SUMO proteins both spatially and temporally in neurons. We show that BDNF signaling via the receptor tropomyosin-related kinase B facilitates nuclear exodus of SUMO proteins and subsequent enrichment within dendrites. Of the various SUMO E3 ligases, we found that PIAS-3 dendrite enrichment in response to BDNF signaling specifically modulates subsequent ERK1/2 kinase pathway signaling. In addition, we found the PIAS-3 RING and Ser/Thr domains, albeit in opposing manners, functionally inhibit GABA-mediated inhibition. Finally, using oxygen–glucose deprivation as an in vitro model for ischemia, we show that BDNF–tropomyosin-related kinase B signaling negatively impairs clustering of the main scaffolding protein at GABAergic postsynapse, gephyrin, whereby reducing GABAergic neurotransmission postischemia. SUMOylation-defective gephyrin K148R/K724R mutant transgene expression reversed these ischemia-induced changes in gephyrin cluster density. Taken together, these data suggest that BDNF signaling facilitates the temporal relocation of nuclear-enriched SUMO proteins to dendrites to influence postsynaptic protein SUMOylation.

GABA-mediated inhibition of cadmium uptake and accumulation in apples

GABA, a four-carbon non-protein amino acid, plays an important role in animals and plants. We previously found GABA could alleviate alkali stress in apple seedlings. However, its physiological mechanism under heavy metal cadmium (Cd) stress need to be further studied. Thus, we explored its biological role in response to Cd stress. It was verified that 0.5 mM GABA could effectively alleviate Cd toxicity. Using NMT technique, we found that exogenous GABA could significantly reduce the net Cd2+ fluxes in apple roots, and Cd content was significantly lower than that in roots under Cd stress. Further analysis indicated exogenous GABA could significantly reduce the expression of genes related to the uptake and transport of Cd in apples under Cd stress. In addition, exogenous GABA could significantly increase the content of amino acids in apple roots under Cd stress. GAD is a key enzyme in GABA synthesis, we obtained transgenic apple roots of overexpression MdGAD1. Compared with the control, transgenic roots accumulated less Cd, maintained lower Cd uptake by roots, and lower expression of related transport genes. These results showed that GABA could effectively alleviate Cd toxicity in apple seedlings and provide a new perspective of GABA to alleviate Cd stress.

Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome

A common feature of diverse brain disorders is the alteration of GABA-mediated inhibition because of aberrant, intracellular chloride homeostasis induced by changes in the expression and/or function of chloride transporters. Notably, pharmacological inhibition of the chloride importer NKCC1 is able to rescue brain-related core deficits in animal models of these pathologies and in some human clinical studies. Here, we show that reducing NKCC1 expression by RNA interference in the Ts65Dn mouse model of Down syndrome (DS) restores intracellular chloride concentration, efficacy of gamma-aminobutyric acid (GABA)-mediated inhibition, and neuronal network dynamics in vitro and ex vivo. Importantly, adeno-associated virus (AAV)-mediated, neuron-specific NKCC1 knockdown in vivo rescues cognitive deficits in diverse behavioral tasks in Ts65Dn animals. Our results highlight a mechanistic link between NKCC1 expression and behavioral abnormalities in DS mice and establish a molecular target for new therapeutic approaches, including gene therapy, to treat brain disorders characterized by neuronal chloride imbalance.

The Pathological Role of Astrocytic MAOB in Parkinsonism Revealed by Genetic Ablation and Over-expression of MAOB.

The cause of Parkinson’s disease has been traditionally believed to be the dopaminergic neuronal death in the substantia nigra pars compacta (SNpc). This traditional view has been recently challenged by the proposal that reactive astrocytes serve as key players in the pathology of Parkinson’s disease through excessive GABA release. This aberrant astrocytic GABA is synthesized by the enzymatic action of monoamine oxidase B (MAOB), whose pharmacological inhibition and gene-silencing are reported to significantly alleviate parkinsonian motor symptoms in animal models of Parkinson’s disease. However, whether genetic ablation and over-expression of MAOB can bidirectionally regulate parkinsonian motor symptoms has not been tested. Here we demonstrate that genetic ablation of MAOB blocks the MPTP-induced augmentation of astrocytic GABA-mediated tonic inhibition of neighboring dopaminergic neurons as well as parkinsonian motor symptoms, indicating the necessity of MAOB for parkinsonian motor symptoms. Furthermore, we demonstrate that GFAP-MAOB transgenic mice, in which MAOB is over-expressed under the GFAP promoter for astrocyte-specific over-expression, display exacerbated MPTP-induced tonic inhibition and parkinsonian motor symptoms compared to wild-type mice, indicating the importance of astrocytic MAOB for parkinsonian motor symptoms. Our study provides genetic pieces of evidence for the causal link between the pathological role of astrocytic MAOB-dependent tonic GABA synthesis and parkinsonian motor symptoms.

Estrous Cycle Plasticity in the Central Clock Output to Kisspeptin Neurons: Implications for the Preovulatory Surge.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3VKiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCNAVP neurons innervate approximately half of the RP3VKiss1 neurons. Optogenetic activation of SCNAVP projections in brain slices caused an AVP-mediated stimulation of RP3VKiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCNAVP projections resulted in GABA-mediated inhibition of RP3VKiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCNAVP neuron output that drives opposing effects on RP3VKiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

Mechanisms of GABA-mediated inhibition of the angiotensin II-induced cytosolic Ca2+ increase in rat subfornical organ neurons

Neurons in the subfornical organ (SFO) sense both neurotransmitters and circulating humoral factors such as angiotensin II (AII) and atrial natriuretic peptide (ANP), and regulate multiple physiological functions including drinking behavior. We recently reported that AII at nanomolar concentrations induced a persistent [Ca2+]i increase in acutely dissociated SFO neurons and that this effect of AII was reversibly inhibited by GABA. In the present study, we studied the inhibitory mechanism of GABA using Ca2+ imaging and patch-clamp electrophysiology. The AII-induced persistent [Ca2+]i increase was inhibited by GABA in more than 90% of AII-responsive neurons and by other two SFO inhibitory ligands, ANP and galanin, in about 60 and 30% of neurons respectively. The inhibition by GABA was mimicked by the GABAA and GABAB receptor agonists muscimol and baclofen. The involvement of both GABA receptor subtypes was confirmed by reversal of the GABA-mediated inhibition only when the GABAA and GABAB receptors antagonists bicuculline methiodide and CGP55845 were both present. The GABAB agonist baclofen rapidly and reversibly inhibited voltage-gated Ca2+ channel (VGCC) currents recorded in response to depolarizing pulses in voltage-clamp electrophysiology using Ba2+ as a charge carrier (IBa). Baclofen inhibition of IBa was antagonized by CGP55845, confirming GABAB receptor involvement; was reduced by N-ethylmaleimide, suggesting downstream Gi-mediated actions; and was partially removed by a large prepulse, indicating voltage-dependency. The magnitude of IBa inhibition by baclofen was reduced by the application of selective blockers for N-, P/Q-, and L-type VGCCs (ω-conotoxin GVIA, ω-agatoxin IVA, and nifedipine respectively). Overall, our study indicates that GABA inhibition of the AII-induced [Ca2+]i increase is mediated by both GABAA and GABAB receptors, and that GABAB receptors associated with Gi proteins suppress Ca2+ entry through VGCCs in SFO neurons.

The safety and efficacy of metformin in fragile X syndrome: An open-label study

Fragile X syndrome (FXS) is a rare genetic disorder characterized by a deficit of the fragile X mental retardation protein (FMRP), encoded by the fragile X mental retardation gene (FMR1) on the X chromosome. It has been hypothesized that the absence of FRMP leads to higher levels of Insulin-like Growth Factor 1 (IGF-1) in the brain, possibly contributing to the intellectual impairment characteristic of the disorder. Preclinical studies have shown that metformin downregulates the insulin/IGF-1 signaling pathway, corrects dendritic defects, and improves repetitive behavior in Fmr1 knockout mice. Here, we conducted an open-label study to evaluate: (1) the safety of metformin in normoglycemic individuals with FXS; and (2) the efficacy of metformin to improve aberrant behavior, attention, and to modulate cortical functioning. Fifteen patients with FXS, aged from 17 to 44, received 500 mg of metformin twice/daily over a 9-week treatment period. The primary outcome measures were: (1) the incidence of adverse events (AE); (2) the decrease in IGF-1 levels; and (3) the global score of the Aberrant Behavior Checklist-Community, Fragile X. The secondary outcomes were: (1) the Test of Attentional Performance for children (KiTAP); and (2) the Transcranial Magnetic Stimulation (TMS) parameters measuring cortical excitability. The metformin treatment was well tolerated, with no significant related AE. The TMS data showed an increase in corticospinal inhibition mediated by GABAA and GABAB mechanisms. This study demonstrates the safety of metformin in normoglycemic patients with FXS, and suggests the potential of this medication in modifying GABA-mediated inhibition, a hallmark of FXS pathophysiology. Implications for future clinical trials are discussed.

Visual cortex cTBS increases mixed percept duration while a-tDCS has no effect on binocular rivalry.

Neuromodulation of the primary visual cortex using anodal transcranial direct current stimulation (a-tDCS) can alter visual perception and enhance neuroplasticity. However, the mechanisms that underpin these effects are currently unknown. When applied to the motor cortex, a-tDCS reduces the concentration of the inhibitory neurotransmitter gamma aminobutyric acid (GABA), an effect that has been linked to increased neuroplasticity. The aim of this study was to assess whether a-tDCS also reduces GABA-mediated inhibition when applied to the human visual cortex. Changes in visual cortex inhibition were measured using the mixed percept duration in binocular rivalry. Binocular rivalry mixed percept duration has recently been advocated as a direct and sensitive measure of visual cortex inhibition whereby GABA agonists decrease mixed percept durations and agonists of the excitatory neurotransmitter acetylcholine (ACH) increase them. Our hypothesis was that visual cortex a-tDCS would increase mixed percept duration by reducing GABA-mediated inhibition and increasing cortical excitation. In addition, we measured the effect of continuous theta-burst transcranial magnetic stimulation (cTBS) of the visual cortex on binocular rivalry dynamics. When applied to the motor or visual cortex, cTBS increases GABA concentration and we therefore hypothesized that visual cortex cTBS would decrease the mixed percept duration. Binocular rivalry dynamics were recorded before and after active and sham a-tDCS (N = 15) or cTBS (N = 15). Contrary to our hypotheses, a-tDCS had no effect, whereas cTBS increased mixed percepts during rivalry. These results suggest that the neurochemical mechanisms of a-tDCS may differ between the motor and visual cortices.

GABA-Mediated Inhibition of Cadmium Uptake and Accumulation in Apples

GABA-Mediated Inhibition of Cadmium Uptake and Accumulation in Apples

Synthesis and investigation of anti-inflammatory and anticonvulsant activities of novel coumarin-diacylated hydrazide derivatives

A number of novel coumarin derivatives synthesized by the reaction of 3-carbonyl chloride coumarin with some substituted aryl acid hydrazides to investigate their anti-inflammatory and anticonvulsant activities. Carrageenan (0.1 ml of 1%, w/v) was injected subplantarly in the right paw of rats to induce an acute model of inflammation. Anti-inflammatory efficacy was evaluated for 5 hours at 3 different dosages 5, 10, 25 mg/kg. After that, the changes in the level of paw edema volumes and percentage inhibition of all groups were observed and the most effective coumarin derivative was found as N'-(2-hyroxybenzoyl)-2-oxo-2H-chromene-3-carbohydrazide. In addition, N’-(2-oxo-2H-chromene-3-carbonyl)nicotinohydrazide, (E)-N’-(3-(4-hydroxyphenyl)acryloyl)-2-oxo-2H-chromene-3-carbohydrazide, and N’-(5-amino-2-hydroxybenzoyl)-2-oxo-2H-chromone-3-carbohydrazide showed their anti-inflammatory effects in a dose-dependent manner. On the other hand, pentylenetetrazole (PTZ, 80 mg/kg, i.p.)-induced seizure model was used to investigate the anticonvulsant activities of six newly synthesized coumarin derivatives in mice. Hybrid compound of salicylic acid hydrazide and 3-carbonyl chloride coumarin (8d) was found the most promising anticonvulsant agent among all treatment groups according to the onset of seizure and survival rate. Moreover, (E)-N'-cinnamoyl-2-oxo-2H-chromene-3-carbohyrazide (8b) and (E)-N'-(3-(4-hyroxyphenyl)acryloyl)-2-oxo-2H-chromene-3-carbohydrazide (8c) has potential anticonvulsant efficiency in low doses (30 mg/kg). The anticonvulsant effect of these coumarin derivatives may be through enhanced GABA-mediated inhibition in the brain.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

Differential Coassembly of α1-GABAARs Associated with Epileptic Encephalopathy.

GABAA receptors (GABAARs) are profoundly important for controlling neuronal excitability. Spontaneous and familial mutations to these receptors feature prominently in excitability disorders and neurodevelopmental deficits following disruption to GABA-mediated inhibition. Recent genotyping of an individual with severe epilepsy and Williams-Beuren syndrome identified a frameshifting de novo variant in a major GABAAR gene, GABRA1 This truncated the α1 subunit between the third and fourth transmembrane domains and introduced 24 new residues forming the mature protein, α1Lys374Serfs*25 Cell surface expression of mutant murine GABAARs is severely impaired compared with WT, due to retention in the endoplasmic reticulum. Mutant receptors were differentially coexpressed with β3, but not with β2, subunits in mammalian cells. Reduced surface expression was reflected by smaller IPSCs, which may underlie the induction of seizures. The mutant does not have a dominant-negative effect on native neuronal GABAAR expression since GABA current density was unaffected in hippocampal neurons, although mutant receptors exhibited limited GABA sensitivity. To date, the underlying mechanism is unique for epileptogenic variants and involves differential β subunit expression of GABAAR populations, which profoundly affected receptor function and synaptic inhibition.SIGNIFICANCE STATEMENT GABAARs are critical for controlling neural network excitability. They are ubiquitously distributed throughout the brain, and their dysfunction underlies many neurologic disorders, especially epilepsy. Here we report the characterization of an α1-GABAAR variant that results in severe epilepsy. The underlying mechanism is structurally unusual, with the loss of part of the α1 subunit transmembrane domain and part-replacement with nonsense residues. This led to compromised and differential α1 subunit cell surface expression with β subunits resulting in severely reduced synaptic inhibition. Our study reveals that disease-inducing variants can affect GABAAR structure, and consequently subunit assembly and cell surface expression, critically impacting on the efficacy of synaptic inhibition, a property that will orchestrate the extent and duration of neuronal excitability.

Optopharmacology reveals a differential contribution of native GABAA receptors to dendritic and somatic inhibition using azogabazine

γ-aminobutyric acid type-A receptors (GABAARs) are inhibitory ligand-gated ion channels in the brain that are crucial for controlling neuronal excitation. To explore their physiological roles in cellular and neural network activity, it is important to understand why specific GABAAR isoforms are distributed not only to various brain regions and cell types, but also to specific areas of the membrane in individual neurons. To address this aim we have developed a novel photosensitive compound, azogabazine, that targets and reversibly inhibits GABAARs. The receptor selectivity of the compound is based on the competitive antagonist, gabazine, and photosensitivity is conferred by a photoisomerisable azobenzene group. Azogabazine can exist in either cis or trans conformations that are controlled by UV and blue light respectively, to affect receptor inhibition. We report that the trans-isomer preferentially binds and inhibits GABAAR function, whilst promotion of the cis-isomer caused unbinding of azogabazine from GABAARs. Using cultured cerebellar granule cells, azogabazine in conjunction with UV light applied to defined membrane domains, revealed higher densities of GABAARs at somatic inhibitory synapses compared to those populating proximal dendritic zones, even though the latter displayed a higher number of synapses per unit area of membrane. Azogabazine also revealed more pronounced GABA-mediated inhibition of action potential firing in proximal dendrites compared to the soma. Overall, azogabazine is a valuable addition to the photochemical toolkit that can be used to interrogate GABAAR function and inhibition.

Nest Carbon Dioxide Masks GABA-Dependent Seizure Susceptibility in the Naked Mole-Rat.

African naked mole-rats were likely the first mammals to evolve eusociality, and thus required adaptations to conserve energy and tolerate the low oxygen (O2) and high carbon dioxide (CO2) of a densely populated fossorial nest. As hypercapnia is known to suppress neuronal activity, we studied whether naked mole-rats might demonstrate energy savings in GABAergic inhibition. Using whole-colony behavioral monitoring of captive naked mole-rats, we found a durable nest, characterized by high CO2 levels, where all colony members spent the majority of their time. Analysis of the naked mole-rat genome revealed, uniquely among mammals, a histidine point variation in the neuronal potassium-chloride cotransporter 2 (KCC2). A histidine missense substitution mutation at this locus in the human ortholog of KCC2, found previously in patients with febrile seizures and epilepsy, has been demonstrated to diminish neuronal Cl- extrusion capacity, and thus impairs GABAergic inhibition. Seizures were observed, without pharmacological intervention, in adult naked mole-rats exposed to a simulated hyperthermic surface environment, causing systemic hypocapnic alkalosis. Consistent with the diminished function of KCC2, adult naked mole-rats demonstrate a reduced efficacy of inhibition that manifests as triggering of seizures at room temperature by the GABAA receptor (GABAAR) positive allosteric modulator diazepam. These seizures are blocked in the presence of nest-like levels of CO2 and likely to be mediated through GABAAR activity, based on invitro recordings. Thus, altered GABAergic inhibition adds to a growing listofadaptations in the naked mole-rat and provides a plausible proximate mechanism for nesting behavior, where a return to the colony nest restores GABA-mediated inhibition.

Primary motor cortex function and motor skill acquisition: insights from threshold-hunting TMS.

Modulation of GABA-mediated inhibition in primary motor cortex (M1) is important for the induction of training-induced plasticity. The downregulation of inhibition during acquisition may promote cortical reorganization, whereas an upregulation once performance has plateaued may promote consolidation of the newly acquired skill. GABA-related inhibition in human M1 is routinely assessed using the paired-pulse transcranial magnetic stimulation (TMS) paradigm of short-interval intracortical inhibition (SICI). However, modulation of SICI with motor skill learning is not a consistent finding and may be influenced by TMS parameters. The aim of this study was to compare the modulation of SICI by motor skill learning between conventional and adaptive threshold-hunting techniques with an anterior-posterior and posterior-anterior induced current. Sixteen participants (21-33years) trained with their dominant (right) hand on a sequential visual isometric pinch task. Electromyographic recordings were obtained from the right first dorsal interosseous muscle. Corticomotor excitability and SICI were examined before and immediately after 12 blocks of training. Skill increased throughout the training, with performance plateauing before completion. Corticomotor excitability increased after motor training for both current directions. The amount of SICI was greater with anterior-posterior stimulation than posterior-anterior for both conventional and adaptive threshold-hunting techniques. SICI increased after motor training, but only for adaptive threshold-hunting with an anterior-posterior-induced current. The increased GABA-mediated inhibition evident after motor skill learning may promote consolidation of the newly acquired skill. The findings also support the notion that adaptive threshold-hunting SICI using an anterior-posterior current provides an effective assessment in interventional studies.

Novel 3-{4-[2-Amino-4-(Substitutedphenyl)-2H-[1, 3] Oxazin/Thiazin-6-Yl} -2-Phenyl-3H-Quinazolin-4-One Derivatives as Enhancer of GABA Mediated Inhibition: Synthesis, Molecular Modeling and Pharmacological Studies

Background: According to WHO, the 50 million people worldwide are suffering from epilepsy, making it one of the most common neurological diseases globally. Epilepsy is often characterized by neurobiological, cognitive, psychological and behavioral changes and that may enhance the susceptibility to seizures and affect the quality of life. Objective: The aim of the present work was to develop 2, 3 disubstituted 4-(3H)-quinazolinone derivatives in order to find an effective and highly lipophilic compound with lesser side effects and to evaluate them for anticonvulsant and neurotoxic activity. Methods: A novel series of 3-4-[2-amino-4-(substitutedphenyl)-2H-[1.3] oxazin/thiazin-6-yl 2- phenyl-3H-quinazolin-4-one derivatives were synthesized and evaluated for their anticonvulsant activity. The structures of the compound have been confirmed by spectral analysis. The molecular docking study was performed for finding the binding affinity with GABAA receptor in order to rationalize their anticonvulsant activities in a qualitative way. Quantitative estimate of drug-likeness was also performed which calculate the molecular properties and screen the molecules based on drug-likeness rules. Anticonvulsant activities of synthesized compounds were done by using (Maximal electroshock) MES induced seizures and subcutaneous pentylenetetrazole (scPTZ) induced seizure models in Wistar rats of either sex. None of the compounds demonstrated any sign of neurotoxicity. Results: Compounds 3-4-[2-amino-4-(fluorophenyl)-2H- [1, 3] oxazin-6-yl 2-phenyl-3H quinazolin-4-one (5i) and 3-4-[2-amino-4-(fluorophenyl)-2H- [1, 3] thiazin -6-yl 2-phenyl-3H quinazolin-4-one (5n) have shown significant activity against tonic seizure by the MES model and clonic seizure by scPTZ induced seizure model. Conclusion: These ten novels synthesized compounds had significant anticonvulsant activity. As a result, the compound (5i) and (5n) emerged out as the pilot molecule with a better anticonvulsant activity without any neurotoxicity, while the other compounds have moderate activity. QED analysis of compounds (5i) and (5n) also indicated that these compounds will have good oral absorption. The proposed work is to make efforts towards the development and identification of novel molecules as anticonvulsant agents by the synthesis of some novel quinazolinone derivatives with improved biological activity.

GABA-mediated tonic inhibition differentially modulates gain in functional subtypes of cortical interneurons

The binding of GABA (γ-aminobutyric acid) to extrasynaptic GABAA receptors generates tonic inhibition that acts as a powerful modulator of cortical network activity. Despite GABA being present throughout the extracellular space of the brain, previous work has shown that GABA may differentially modulate the excitability of neuron subtypes according to variation in chloride gradient. Here, using biophysically detailed neuron models, we predict that tonic inhibition can differentially modulate the excitability of neuron subtypes according to variation in electrophysiological properties. Surprisingly, tonic inhibition increased the responsiveness (or gain) in models with features typical for somatostatin interneurons but decreased gain in models with features typical for parvalbumin interneurons. Patch-clamp recordings from cortical interneurons supported these predictions, and further in silico analysis was then performed to seek a putative mechanism underlying gain modulation. We found that gain modulation in models was dependent upon the magnitude of tonic current generated at depolarized membrane potential-a property associated with outward rectifying GABAA receptors. Furthermore, tonic inhibition produced two biophysical changes in models of relevance to neuronal excitability: 1) enhanced action potential repolarization via increased current flow into the dendritic compartment, and 2) reduced activation of voltage-dependent potassium channels. Finally, we show theoretically that reduced potassium channel activation selectively increases gain in models possessing action potential dynamics typical for somatostatin interneurons. Potassium channels in parvalbumin-type models deactivate rapidly and are unavailable for further modulation. These findings show that GABA can differentially modulate interneuron excitability and suggest a mechanism through which this occurs in silico via differences of intrinsic electrophysiological properties.

Altered motor system function in post-concussion syndrome as assessed via transcranial magnetic stimulation

ObjectiveIt is unclear why specific individuals incur chronic symptoms following a concussion. This exploratory research aims to identify and characterize any neurophysiological differences that may exist in motor cortex function in post-concussion syndrome (PCS). MethodsFifteen adults with PCS and 13 healthy, non-injured adults were tested. All participants completed symptom questionnaires, and transcranial magnetic stimulation (TMS) was used to measure intracortical and transcallosal excitability and inhibition in the dominant motor cortex. ResultsCortical silent period (p = 0.02, g = 0.96) and ipsilateral silent period (p = 0.04, g = 0.78) were shorter in the PCS group compared to the control group which may reflect reduced GABA-mediated inhibition in PCS. Furthermore, increased corticomotor excitability was observed in the left hemisphere but not the right hemisphere. ConclusionsThese data suggest that persistent neurophysiological differences are present in those with PCS. The exact contributing factors to such changes remain to be investigated by future studies. SignificanceThis study provides novel evidence of lasting neurophysiological changes in PCS.