GABAergic Tonic Currents Research Articles

Reduction in hippocampal GABAergic transmission in a low birth weight rat model of depression.

Prenatal stress is believed to increase the risk of developing neuropsychiatric disorders, including major depression. Adverse genetic and environmental impacts during early development, such as glucocorticoid hyper-exposure, can lead to changes in the foetal brain, linked to mental illnesses developed in later life. Dysfunction in the GABAergic inhibitory system is associated with depressive disorders. However, the pathophysiology of GABAergic signalling is poorly understood in mood disorders. Here, we investigated GABAergic neurotransmission in the low birth weight (LBW) rat model of depression. Pregnant rats, exposed to dexamethasone, a synthetic glucocorticoid, during the last week of gestation, yielded LBW offspring showing anxiety- and depressive-like behaviour in adulthood. Patch-clamp recordings from dentate gyrus granule cells in brain slices were used to examine phasic and tonic GABAA receptor-mediated currents. The transcriptional levels of selected genes associated with synaptic vesicle proteins and GABAergic neurotransmission were investigated. The frequency of spontaneous inhibitory postsynaptic currents (sIPSC) was similar in control and LBW rats. Using a paired-pulse protocol to stimulate GABAergic fibres impinging onto granule cells, we found indications of decreased probability of GABA release in LBW rats. However, tonic GABAergic currents and miniature IPSCs, reflecting quantal vesicle release, appeared normal. Additionally, we found elevated expression levels of two presynaptic proteins, Snap-25 and Scamp2, components of the vesicle release machinery. The results suggest that altered GABA release may be an essential feature in the depressive-like phenotype of LBW rats.

Insulin decreases epileptiform activity in rat layer 5/6 prefrontal cortex in vitro.

Accumulating evidence indicates that insulin-mediated signaling in the brain may play important roles in regulating neuronal function. Alterations to insulin signaling are associated with the development of neurological disorders including Alzheimer's disease and Parkinson's disease. Also, hyperglycemia and insulin resistance have been associated with seizure activity and brain injury. In recent work, we found that insulin increased inhibitory GABAA -mediated tonic currents in the prefrontal cortex (PFC). In this work, we used local field potential recordings and calcium imaging to investigate the effect of insulin on seizure-like activity in PFC slices. Seizure-like events (SLEs) were induced by perfusing the slices with magnesium-free artificial cerebrospinal fluid (ACSF) containing the proconvulsive compound 4-aminopyridine (4-AP). We found that insulin decreased the frequency, amplitude, and duration of SLEs as well as the synchronic activity of PFC neurons evoked by 4-AP. These insulin effects were mediated by the PI3K/Akt signaling pathway and mimicked by gaboxadol (THIP), a δ GABAA receptor agonist. The effect of insulin on the number of SLEs was partially blocked by L-655,708, an inverse agonist with high selectivity for GABAA receptors containing the α5 subunit. Our results suggest that insulin reduces neuronal excitability by an increase of GABAergic tonic currents. The physiological relevance of these findings is discussed.

NKCC-1 mediated Cl\u2212 uptake in immature CA3 pyramidal neurons is sufficient to compensate phasic GABAergic inputs

Activation of GABAA receptors causes in immature neurons a functionally relevant decrease in the intracellular Cl− concentration ([Cl−]i), a process termed ionic plasticity. Amount and duration of ionic plasticity depends on kinetic properties of [Cl−]i homeostasis. In order to characterize the capacity of Cl− accumulation and to quantify the effect of persistent GABAergic activity on [Cl−]i, we performed gramicidin-perforated patch-clamp recordings from CA3 pyramidal neurons of immature (postnatal day 4–7) rat hippocampal slices. These experiments revealed that inhibition of NKCC1 decreased [Cl−]i toward passive distribution with a time constant of 381 s. In contrast, active Cl− accumulation occurred with a time constant of 155 s, corresponding to a rate of 15.4 µM/s. Inhibition of phasic GABAergic activity had no significant effect on steady state [Cl−]i. Inhibition of tonic GABAergic currents induced a significant [Cl−]i increase by 1.6 mM, while activation of tonic extrasynaptic GABAA receptors with THIP significantly reduced [Cl−]i.. Simulations of neuronal [Cl−]i homeostasis supported the observation, that basal levels of synaptic GABAergic activation do not affect [Cl−]i. In summary, these results indicate that active Cl−-uptake in immature hippocampal neurons is sufficient to maintain stable [Cl−]i at basal levels of phasic and to some extent also to compensate tonic GABAergic activity.

Gain-of-function GABRB3 variants identified in vigabatrin-hypersensitive epileptic encephalopathies.

Variants in the GABRB3 gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with GABRB3 variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A GABRB3 variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of de novo variants in GABRB3 (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the GABRB3 p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the GABRB3 variant. The mRNA of these constructs was injected into Xenopus oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the GABRB3 p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of GABRB3 variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function GABRB3 variants.

Mice lacking spinal α2GABAA receptors: Altered GABAergic neurotransmission, diminished GABAergic antihyperalgesia, and potential compensatory mechanisms preventing a hyperalgesic phenotype

Diminished synaptic inhibition in the superficial spinal dorsal horn contributes to exaggerated pain responses that accompany peripheral inflammation and neuropathy. α2GABAA receptors (α2GABAAR) constitute the most abundant GABAAR subtype at this site and are the targets of recently identified antihyperalgesic compounds. Surprisingly, hoxb8-α2−/− mice that lack α2GABAAR from the spinal cord and peripheral sensory neurons exhibit unaltered sensitivity to acute painful stimuli and develop normal inflammatory and neuropathic hyperalgesia. Here, we provide a comprehensive analysis of GABAergic neurotransmission, of behavioral phenotypes and of possible compensatory mechanisms in hoxb8-α2−/− mice. Our results confirm that hoxb8-α2−/− mice show significantly diminished GABAergic inhibitory postsynaptic currents (IPSCs) in the superficial dorsal horn but no hyperalgesic phenotype. We also confirm that the potentiation of dorsal horn GABAergic IPSCs by the α2-preferring GABAAR modulator HZ-166 is reduced in hoxb8-α2−/− mice and that hoxb8-α2−/− mice are resistant to the analgesic effects of HZ-166. Tonic GABAergic currents, glycinergic IPSCs, and sensory afferent-evoked EPSCs did not show significant changes in hoxb8-α2−/− mice rendering a compensatory up-regulation of other GABAAR subtypes or of glycine receptors unlikely. Although expression of serotonin and of the serotonin producing enzyme tryptophan hydroxylase (TPH2) was significantly increased in the dorsal horn of hoxb8-α2−/− mice, ablation of serotonergic terminals from the lumbar spinal cord failed to unmask a nociceptive phenotype. Our results are consistent with an important contribution of α2GABAAR to spinal nociceptive control but their ablation early in development appears to activate yet-to-be identified compensatory mechanisms that protect hoxb8-α2−/− mice from hyperalgesia.

GABAA receptor currents in the dorsal motor nucleus of the vagus in females: influence of ovarian cycle and 5α-reductase inhibition.

The dorsal motor nucleus of the vagus (DMV) contains the preganglionic motor neurons important in the regulation of glucose homeostasis and gastrointestinal function. Despite the role of sex in the regulation of these processes, few studies examine the role of sex and/or ovarian cycle in the regulation of synaptic neurotransmission to the DMV. Since GABAergic neurotransmission is critical to normal DMV function, the present study used in vitro whole cell patch-clamping to investigate whether sex differences exist in GABAergic neurotransmission to DMV neurons. It additionally investigated whether the ovarian cycle plays a role in those sex differences. The frequency of phasic GABAA receptor-mediated inhibitory postsynaptic currents in DMV neurons from females was lower compared with males, and this effect was TTX sensitive and abolished by ovariectomy (OVX). Amplitudes of GABAergic currents (both phasic and tonic) were not different. However, females demonstrated significantly more variability in the amplitude of both phasic and tonic GABAA receptor currents. This difference was eliminated by OVX in females, suggesting that these differences were related to reproductive hormone levels. This was confirmed for GABAergic tonic currents by comparing females in two ovarian stages, estrus versus diestrus. Female mice in diestrus had larger tonic current amplitudes compared with those in estrus, and this increase was abolished after administration of a 5α-reductase inhibitor but not modulation of estrogen. Taken together, these findings demonstrate that DMV neurons undergo GABAA receptor activity plasticity as a function of sex and/or sex steroids.NEW & NOTEWORTHY Results show that GABAergic signaling in dorsal vagal motor neurons (DMV) demonstrates sex differences and fluctuates across the ovarian cycle in females. These findings are the first to demonstrate that female GABAA receptor activity in this brain region is modulated by 5α-reductase-dependent hormones. Since DMV activity is critical to both glucose and gastrointestinal homeostasis, these results suggest that sex hormones, including those synthesized by 5α-reductase, contribute to visceral, autonomic function related to these physiological processes.

Allopregnanolone augments epileptiform activity of an in-vitro mouse hippocampal preparation in the first postnatal week

In the immature brain the neurotransmitter γ-amino butyric acid (GABA) mediates a membrane depolarization and can contribute to both, inhibition and excitation. Therefore the consequences of a positive modulation of GABA(A) receptors by neurosteroids on epileptiform activity are hard to predict. In order to analyze whether neurosteroids attenuate or exaggerate epileptiform activity in the immature brain, we investigated the effect of the neurosteroid allopregnanolone on epileptiform activity in an in-toto hippocampus preparation of early postnatal mice (postnatal days 4–7) using field potential recordings. These in-vitro experiments revealed that 0.5 μmol/L allopregnanolone had no effect on ictal-like epileptiform activity, but increased the occurrence of interictal epileptiform events. The allopregnanolone-induced enhancement of interictal epileptiform activity could be blocked by a selective inhibition of synaptic GABAA receptors. In contrast, allopregnanolone had no effect on interictal epileptiform activity upon enhanced extrasynaptic GABAergic activity. Patch-clamp experiments demonstrated that allopregnanolone prolonged the decay of GABAergic postsynaptic currents, but had no effect on tonic GABAergic currents. We conclude from these results that allopregnanolone can enhance excitability in the immature hippocampus viaprolonged synaptic GABAergic currents. This potential effect of neurosteroids on brain excitability should be considered if they are applied as anticonvulsants to premature or early postnatal babies.

Age- and Experience-Related Plasticity of ATP-Mediated Signaling in the Neocortex.

There is growing recognition of the important role of interaction between neurons and glial cells for brain longevity. The extracellular ATP have been shown to bring significant contribution into bi-directional glia-neuron communications, in particular into astrocyte-driven modulation of synaptic plasticity. To elucidate a putative impact of brain aging on neuron-glia networks, we explored the aging-related plasticity of the purinoreceptors-mediated signaling in cortical neurons and astrocytes. We investigated the age- and experience-related alterations in purinergic components of neuronal synaptic currents and astroglial calcium signaling in the layer2/3 of neocortex of mice exposed to the mild caloric restriction (CR) and environmental enrichment (EE) which included ad libitum physical exercise. We observed the considerable age-related decline in the neuronal P2X receptor-mediated miniature spontaneous currents which originated from the release of ATP from both synapses and astrocytes. We also found out that purinergic astrocytic Ca2+-signaling underwent the substantial age-related decline but EE and CR rescued astroglial signaling, in particular mediated by P2X1, P2X1/5, and P2Y1 receptors. Our data showed that age-related attenuation in the astroglial calcium signaling caused a substantial decrease in the exocytosis of ATP leading to impairment of astroglia-derived purinergic modulation of excitatory synaptic currents and GABAergic tonic inhibitory currents. On a contrary, exposure to EE and CR, which enhanced purinergic astrocytic calcium signaling, up-regulated the excitatory and down-regulated the inhibitory currents in neurons of old mice, thus counterbalancing the impact of aging on synaptic signaling. Combined, our results strongly support the physiological importance of ATP-mediated signaling for glia-neuron interactions and brain function. Our data also show that P2 purinoreceptor-mediated communication between astrocytes and neurons in the neocortex undergoes remodeling during brain aging and decrease in the ATP release may contribute to the age-related impairment of synaptic transmission.

Oxytocin Increases Phasic and Tonic GABAergic Transmission in CA1 Region of Mouse Hippocampus.

Oxytocin is a neuropeptide that plays important peripheral and central neuromodulatory functions. Our data show that, following activation of oxytocin receptors (OtRs) with the selective agonist TGOT (Thr4,Gly7-oxytocin), a significant increase in frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSC) occurred in hippocampal CA1 pyramidal neurons (PYR) in mice. TGOT affected also sIPSC deactivation kinetics, suggesting the involvement of perisynaptic GABAA receptors (GABAARs) as well. By contrast, TGOT did not cause significant changes in frequency, amplitude or deactivation kinetics of miniature IPSC, suggesting that the effects elicited by the agonist are strictly dependent on the firing activity of presynaptic neurons. Moreover, TGOT was able to modulate tonic GABAergic current mediated by extrasynaptic GABAARs expressed by PYRs. Consistently, at spike threshold TGOT induced in most PYRs a significant membrane hyperpolarization and a decrease in firing rate. The source of increased inhibition onto PYRs was represented by stuttering fast-spiking GABAergic interneurons (INs) that directly respond to TGOT with a depolarization and an increase in their firing rate. One putative ionic mechanism underlying this effect could be represented by OtR activation-induced up-modulation of L-type Ca2+ channels. In conclusion, our results indicate that oxytocin can influence the activity of a subclass of hippocampal GABAergic INs and therefore regulate the operational modes of the downstream PYRs by increasing phasic and tonic GABAergic transmission in CA1 region of mouse hippocampus.

Cyfip1 Haploinsufficiency Does Not Alter GABAA Receptor δ-Subunit Expression and Tonic Inhibition in Dentate Gyrus PV+ Interneurons and Granule Cells.

Copy number variation (CNV) at chromosomal region 15q11.2 is linked to increased risk of neurodevelopmental disorders including autism and schizophrenia. A significant gene at this locus is cytoplasmic fragile X mental retardation protein (FMRP) interacting protein 1 (CYFIP1). CYFIP1 protein interacts with FMRP, whose monogenic absence causes fragile X syndrome (FXS). Fmrp knock-out has been shown to reduce tonic GABAergic inhibition by interacting with the δ-subunit of the GABAA receptor (GABAAR). Using in situ hybridization (ISH), qPCR, Western blotting techniques, and patch clamp electrophysiology in brain slices from a Cyfip1 haploinsufficient mouse, we examined δ-subunit mediated tonic inhibition in the dentate gyrus (DG). In wild-type (WT) mice, DG granule cells (DGGCs) responded to the δ-subunit-selective agonist THIP with significantly increased tonic currents. In heterozygous mice, no significant difference was observed in THIP-evoked currents in DGGCs. Phasic GABAergic inhibition in DGGC was also unaltered with no difference in properties of spontaneous IPSCs (sIPSCs). Additionally, we demonstrate that DG granule cell layer (GCL) parvalbumin-positive interneurons (PV+-INs) have functional δ-subunit-mediated tonic GABAergic currents which, unlike DGGC, are also modulated by the α1-selective drug zolpidem. Similar to DGGC, both IPSCs and THIP-evoked currents in PV+-INs were not different between Cyfip1 heterozygous and WT mice. Supporting our electrophysiological data, we found no significant change in hippocampal δ-subunit mRNA expression or protein level and no change in α1/α4-subunit mRNA expression. Thus, Cyfip1 haploinsufficiency, mimicking human 15q11.2 microdeletion syndrome, does not alter hippocampal phasic or tonic GABAergic inhibition, substantially differing from the Fmrp knock-out mouse model.

Spontaneously opening GABAA receptors play a significant role in neuronal signal filtering and integration

Continuous (tonic) charge transfer through ionotropic receptors of γ-aminobutyric acid (GABAARs) is an important mechanism of inhibitory signalling in the brain. The conventional view has been that tonic GABA-ergic inhibitory currents are mediated by low concentrations of ambient GABA. Recently, however, it was shown that the GABA-independent, spontaneously opening GABAARs (s-GABAARs), may contribute significantly to the tonic GABAAR current. One of the common approaches to temporal lobe epilepsy (TLE) therapy is an increase of GABA concentration in the cerebrospinal fluid to augment tonic current through GABAARs. Such an increase, however, generates multiple side effects, which impose significant limitations on the use of correspondent drugs. In contrast, activation/deactivation of s-GABAARs in a GABA-independent manner may provide a mechanism of regulation of tonic conductance without modification of extracellular GABA concentration, thus avoiding connected side effects. Although s-GABAARs have been detected in our earlier work, it is unclear whether they modulate neural signalling, or, due to their independence from the neurotransmitter, they provide just a stable background effect without much impact on neural crosstalk dynamics. Here, we focused on the causal relationship between s-GABAAR activity and signal integration in the rat’s dentate gyrus granule cells to find that s-GABAARs play an important role in neural signal transduction. s-GABAARs shape the dynamics of phasic inhibitory responses, regulate the action potential generation machinery and control the coincidence detection window pertinent to excitatory input summation. Our results demonstrate that tonic inhibition delivered by s-GABAARs contributes to the key mechanisms that ensure implementation of neural signal filtering and integration, in a GABA-independent manner. This makes s-GABAAR a new and important actor in the regulation of long-term neural plasticity and a perspective target for TLE therapy.

Heteromeric α/βglycine receptors regulate excitability in parvalbumin-expressing dorsal horn neurons through phasic and tonic glycinergic inhibition.

Key points Spinal parvalbumin‐expressing interneurons have been identified as a critical source of inhibition to regulate sensory thresholds by gating mechanical inputs in the dorsal horn.This study assessed the inhibitory regulation of the parvalbumin‐expressing interneurons, showing that synaptic and tonic glycinergic currents dominate, blocking neuronal or glial glycine transporters enhances tonic glycinergic currents, and these manipulations reduce excitability.Synaptically released glycine also enhanced tonic glycinergic currents and resulted in decreased parvalbumin‐expressing interneuron excitability.Analysis of the glycine receptor properties mediating inhibition of parvalbumin neurons, as well as single channel recordings, indicates that heteromeric α/β subunit‐containing receptors underlie both synaptic and tonic glycinergic currents.Our findings indicate that glycinergic inhibition provides critical control of excitability in parvalbumin‐expressing interneurons in the dorsal horn and represents a pharmacological target to manipulate spinal sensory processing. The dorsal horn (DH) of the spinal cord is an important site for modality‐specific processing of sensory information and is essential for contextually relevant sensory experience. Parvalbumin‐expressing inhibitory interneurons (PV+ INs) have functional properties and connectivity that enables them to segregate tactile and nociceptive information. Here we examine inhibitory drive to PV+ INs using targeted patch‐clamp recording in spinal cord slices from adult transgenic mice that express enhanced green fluorescent protein in PV+ INs. Analysis of inhibitory synaptic currents showed glycinergic transmission is the dominant form of phasic inhibition to PV+ INs. In addition, PV+ INs expressed robust glycine‐mediated tonic currents; however, we found no evidence for tonic GABAergic currents. Manipulation of extracellular glycine by blocking either, or both, the glial and neuronal glycine transporters markedly decreased PV+ IN excitability, as assessed by action potential discharge. This decreased excitability was replicated when tonic glycinergic currents were increased by electrically activating glycinergic synapses. Finally, we show that both phasic and tonic forms of glycinergic inhibition are mediated by heteromeric α/β glycine receptors. This differs from GABAA receptors in the dorsal horn, where different receptor stoichiometries underlie phasic and tonic inhibition. Together these data suggest both phasic and tonic glycinergic inhibition regulate the output of PV+ INs and contribute to the processing and segregation of tactile and nociceptive information. The shared stoichiometry for phasic and tonic glycine receptors suggests pharmacology is unlikely to be able to selectively target each form of inhibition in PV+ INs.

Spike Timing-Dependent Plasticity in the Mouse Barrel Cortex Is Strongly Modulated by Sensory Learning and Depends on Activity of Matrix Metalloproteinase 9

Experience and learning in adult primary somatosensory cortex are known to affect neuronal circuits by modifying both excitatory and inhibitory transmission. Synaptic plasticity phenomena provide a key substrate for cognitive processes, but precise description of the cellular and molecular correlates of learning is hampered by multiplicity of these mechanisms in various projections and in different types of neurons. Herein, we investigated the impact of associative learning on neuronal plasticity in distinct types of postsynaptic neurons by checking the impact of classical conditioning (pairing whisker stroking with tail shock) on the spike timing-dependent plasticity (t-LTP and t-LTD) in the layer IV to II/III vertical pathway of the mouse barrel cortex. Learning in this paradigm practically prevented t-LTP measured in pyramidal neurons but had no effect on t-LTD. Since classical conditioning is known to affect inhibition in the barrel cortex, we examined its effect on tonic GABAergic currents and found a strong downregulation of these currents in the layer II/III interneurons but not in pyramidal cells. Matrix metalloproteinases emerged as crucial players in synaptic plasticity and learning. We report that the blockade of MMP-9 (but not MMP-3) abolished t-LTP having no effect on t-LTD. Moreover, associative learning resulted in an upregulation of gelatinolytic activity within the “trained” barrel. We conclude that LTP induced by spike timing-dependent plasticity (STDP) paradigm is strongly correlated with associative learning and critically depends on the activity of MMP-9.

2-Deoxy-d-glucose enhances tonic inhibition through the neurosteroid-mediated activation of extrasynaptic GABAA receptors.

The inhibition of glycolysis exerts potent antiseizure effects, as demonstrated by the efficacy of ketogenic and low-glucose/nonketogenic diets in the treatment of drug-resistant epilepsy. ATP-sensitive potassium (KATP ) channels have been initially identified as the main determinant of the reduction of neuronal hyperexcitability. However, a plethora of other mechanisms have been proposed. Herein, we report the ability of 2-deoxy-d-glucose (2-DG), a glucose analog that inhibits glycolytic enzymes, of potentiating γ-aminobutyric acid (GABA)ergic tonic inhibition via neurosteroid-mediated activation of extrasynaptic GABAA receptors. Acute effects of 2-DG on the ATP-sensitive potassium currents, GABAergic tonic inhibition, firing activity, and interictal events were assessed in hippocampal slices by whole-cell patch-clamp and local field potential recordings of dentate gyrus granule cells. Acute application of 2-DG activates two distinct outward conductances: a KATP channel-mediated current and a bicuculline-sensitive tonic current. The effect of 2-DG on such GABAergic tonic currents was fully prevented by either finasteride or PK11195, which are specific inhibitors of the neurosteroidogenesis pathway acting via different mechanisms. Moreover, the oxidized form of vitamin C, dehydroascorbic acid, known for its ability to induce neurosteroidogenesis, also activated a bicuculline-sensitive tonic current in a manner indistinguishable from that of 2-DG. Finally, we found that the enhancement of KATP current by 2-DG primarily regulates intrinsic firing rate of granule cells, whereas the increase of the GABAergic tonic current plays a key role in reducing the frequency of interictal events evoked by treatment of hippocampal slices with the convulsive agent 4-aminopyridine. We demonstrated, for the first time, that 2-DG potentiates the extrasynaptic tonic GABAergic current through activation of neurosteroidogenesis. Such tonic inhibition represents the main conductance responsible for the antiseizure action of this glycolytic inhibitor.

Hydrogen peroxide modulates neuronal excitability and membrane properties in ventral horn neurons of the rat spinal cord

Hydrogen peroxide (H2O2), a reactive oxygen species, is an important signaling molecule for synaptic and neuronal activity in the central nervous system; it is produced excessively in brain ischemia and spinal cord injury. Although H2O2-mediated modulations of synaptic transmission have been reported in ventral horn (VH) neurons of the rat spinal cord, the effects of H2O2 on neuronal excitability and membrane properties remain poorly understood. Accordingly, the present study investigated such effects using a whole-cell patch-clamp technique. The bath-application of H2O2 decreased neuronal excitability accompanied by decreased input resistance, firing frequency, and action potential amplitude and by increased rheobase. These H2O2-mediated changes were induced by activation of extrasynaptic, but not synaptic, GABAA receptors. Indeed, GABAergic tonic currents were enhanced by H2O2. On the other hand, the amplitude of medium and slow afterhyperpolarization (mAHP and sAHP), which plays important roles in controlling neuronal excitability and is mediated by small-conductance calcium-activated potassium (SK) channels, was significantly decreased by H2O2. When extrasynaptic GABAA receptors were completely blocked, these decreases of mAHP and sAHP persisted, and H2O2 increased excitability, suggesting that H2O2 per se might have the potential to increase neuronal excitability via decreased SK channel conductance. These findings indicate that activating extrasynaptic GABAA receptors or SK channels may attenuate acute neuronal damage caused by H2O2-induced hyperexcitability and therefore represent a novel therapeutic target for the prevention and treatment of H2O2-induced motor neuron disorders.

Isolation Rearing Reduces Neuronal Excitability in Dentate Gyrus Granule Cells of Adolescent C57BL/6J Mice: Role of GABAergic Tonic Currents and Neurosteroids.

Early-life exposure to stress, by impacting on a brain still under development, is considered a critical factor for the increased vulnerability to psychiatric disorders and abuse of psychotropic substances during adulthood. As previously reported, rearing C57BL/6J weanling mice in social isolation (SI) from their peers for several weeks, a model of prolonged stress, is associated with a decreased plasma and brain levels of neuroactive steroids such as 3α,5α-THP, with a parallel up-regulation of extrasynaptic GABAA receptors (GABAAR) in dentate gyrus (DG) granule cells compared to group-housed (GH) mice. In the present study, together with the SI-induced decrease in plasma concentration of both progesterone and 3α,5α-THP, and an increase in THIP-stimulated GABAergic tonic currents, patch-clamp analysis of DG granule cells revealed a significant decrease in membrane input resistance and action potential (AP) firing rate, in SI compared to GH mice, suggesting that SI exerts an inhibitory action on neuronal excitability of these neurons. Voltage-clamp recordings of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) revealed a SI-associated decrease in frequency as well as a shift from paired-pulse (PP) depression to PP facilitation (PPF) of evoked EPSCs, indicative of a reduced probability of glutamate release. Daily administration of progesterone during isolation reverted the changes in plasma 3α,5α-THP as well as in GABAergic tonic currents and neuronal excitability caused by SI, but it had only a limited effect on the changes in the probability of presynaptic glutamate release. Overall, the results obtained in this work, together with those previously published, indicate that exposure of mice to SI during adolescence reduces neuronal excitability of DG granule cells, an effect that may be linked to the increased GABAergic tonic currents as a consequence of the sustained decrease in plasma and hippocampal levels of neurosteroids. All these changes may be consistent with cognitive deficits observed in animals exposed to such type of prolonged stress.

Reductions in tonic GABAergic current in substantia gelatinosa neurons and GABAA receptor δ subunit expression after chronic constriction injury of the sciatic nerve in mice.

Decreased Gamma-aminobutyric acid (GABA)-ergic phasic inhibitory transmission in the spinal cord is thought to be responsible for the development of neuropathic pain. However, the role of GABAergic tonic current in substantia gelatinosa (SG) neurons in neuropathic pain remains to be fully elucidated. In this study, we assessed GABAergic tonic currents of SG neurons in a sciatic nerve chronic constriction injury (CCI) mouse. Whole-cell patch clamp recordings form lumbar spinal cord slices was performed to evaluate GABAergic currents. We also investigated the expression changes of GABAA receptor subunits which are considered to mediate tonic currents. The percentage of SG neurons receiving GABAergic tonic currents decreased in CCI mice compared with Naïve mice. No significant change was observed in the mean amplitude of GABAergic tonic currents. RT-PCR and Western blot revealed that the expression of GABAA receptor δ subunits decreased following CCI. A reduction in the expression the δ subunit of the GABAA receptor and diminished GABAergic tonic current in SG neurons were observed after CCI in mice. GABAergic tonic current plays a key role in neuropathic pain. The GABAA receptor δ subunit may be a therapeutic target in neuropathic pain. WHAT DOES THIS STUDY ADD?: In spinal SG neurons, GABAergic inhibitory transmission operates through both phasic and tonic currents, but physiological role is largely unknown. In this study, we report dysregulation of GABAA receptor δ subunit-mediated tonic current in SG neurons may result in spinal disinhibition resulting in neuropathic pain in CCI mice.

Sensitivity of GABAergic Tonic Currents to Acute Ethanol in Cerebellar Granule Neurons is Not Age- or δ Subunit-Dependent in Developing Rats.

The age of first exposure to ethanol (EtOH), as well as reduced sensitivity to its motor-impairing effects, are associated with a future predisposition to abuse EtOH. In adolescence, acute EtOH potentiates GABA transmission, including tonic inhibition mediated by δ-containing extrasynaptic GABAA receptors (GABAA Rs) in cerebellar granule neurons (CGNs), an effect that likely contributes to EtOH-induced motor impairment. Prenatal EtOH exposure is strikingly prevalent and is associated with increased EtOH abuse later in life; however, the acute effects of EtOH on GABA transmission in developing CGNs are unknown. Using whole-cell patch-clamp electrophysiological techniques in acute brain slices, we examined the acute effects of EtOH on GABA transmission and functionally assessed the role of δ-containing GABAA Rs in CGNs of preweanling (postnatal day [P] 12 to 14) and postweanling (P28 to 30) male Sprague-Dawley rats. The magnitude of basal tonic currents were similar at both ages. However, 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol hydrochloride, an agonist with preferential affinity for δ-containing GABAA Rs, significantly potentiated tonic currents to a larger magnitude in CGNs from postweanlings compared to preweanlings. Conversely, acute application of EtOH (80mM) significantly increased tonic currents and the frequency of spontaneous inhibitory postsynaptic currents to a similar extent in CGNs from pre- and postweanlings. These findings highlight the sensitivity of the developing cerebellum to EtOH. Furthermore, this study demonstrates age-dependent functional changes in a well-characterized circuitry that may contribute to the short- and long-term effects of prenatal exposure to EtOH.

Increased Voluntary Ethanol Consumption and Changes in Hippocampal Synaptic Plasticity in Isolated C57BL/6J Mice

Social isolation (SI) is a notable model of prolonged mild stress, characterized by multiple neurochemical and behavioral alterations, that appears particularly suitable for studying different aspects of the interplay between stress and ethanol (EtOH) consumption in order to characterize potential molecular mechanisms, including changes in the function of inhibitory GABAergic synapses, underlying such interaction. In C57BL/6J mice, SI is associated with an altered hippocampal concentration of the neuroactive steroids 3α-hydroxy-5α-pregnan-20-one (3α-5α-THP), an increased expression of the α4 and δ subunit of γ-aminobutyric acid type A receptors (GABAARs) in the dentate gyrus (DG), and a parallel enhancement of the stimulatory action of 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) on GABAergic tonic currents recorded in voltage-clamped DG granule cells (DGGCs). In addition, SI in C57BL/6J mice determines an increase in voluntary EtOH consumption and EtOH preference when compared to group-housed (GH) control animals. Furthermore, in hippocampal slices of SI mice we also observed a marked reduction of both cellular excitability and long term potentiation (LTP) in pyramidal neurons of the CA1 hippocampal sub-region, effects that were prevented by the long term treatment of SI mice with the neuroactive steroid precursor progesterone. In this article, we summarize some of our recent findings on the effects of SI in C57BL/6J mice on voluntary EtOH intake, regulation of GABAARs gene expression and function and hippocampal long term synaptic plasticity.

Stiripentol in refractory status epilepticus

Benzodiazepines (BZDs), which enhance γ-aminobutyric acid (GABAA ) receptor-mediated inhibition, are the first-line therapy for treatment of status epilepticus (SE). However, pharmacoresistance to BZDs develops rapidly after SE initiation. This is due to an activity-dependent internalization of BZD-sensitive GABAA receptors during SE. Stiripentol (STP) is a positive allosteric modulator of GABAA receptors with a unique subunit selectivity profile. We report that in a rodent model of SE, STP terminates behavioral seizures and remains effective in established SE when seizures have become BZD resistant. The anticonvulsant effects of STP are age dependent, with greater potency in juvenile animals. Whole cell recordings from dentate granule cells in hippocampal slices reveal that STP potentiates GABAergic inhibitory postsynaptic currents (IPSCs) and tonic GABAergic currents by acting at a site on the GABAA receptor that is separate from the benzodiazepine binding site. This potentiation persists in established SE, whereas potentiation of GABAergic inhibition by BZDs is lost. STP potentiates IPSCs in juvenile animals with greater potency than in adult animals. We suggest that STP, either alone or as add-on therapy, may prove useful in treating established and BZD-resistant status epilepticus. Furthermore, STP may be particularly effective in terminating SE in children when SE is most prevalent.