The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor‐mediated inhibition

Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described ‘spillover’ mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ‐aminobutyric acid (GABA)_A receptors (GABA_ARs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABA_ARs to the thalamic effects of etomidate, we performed whole‐cell recordings from mouse TC neurons lacking synaptic (α1^0/0) or extrasynaptic (δ^0/0) GABA_ARs. Etomidate (3 μm ) significantly inhibited action‐potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABA_ARs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ‐GABA_ARs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate‐induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

Finasteride inhibits the disease-modifying activity of progesterone in the hippocampus kindling model of epileptogenesis

Progesterone (P) plays an important role in seizure susceptibility in women with epilepsy. Preclinical and experimental studies suggest that P appears to interrupt epileptogenesis, which is a process whereby a normal brain becomes progressively susceptible to recurrent, unprovoked seizures due to precipitating risk factors. Progesterone has not been investigated widely for its potential disease-modifying activity in epileptogenic models. Recently, P has been shown to exert disease-modifying effects in the kindling model of epileptogenesis. However, the mechanisms underlying the protective effects of P against epileptogenesis remain unclear. In this study, we investigated the role of P-derived neurosteroids in the disease-modifying activity of P. It is hypothesized that 5α-reductase converts P to allopregnanolone and related neurosteroids that retard epileptogenesis in the brain. To test this hypothesis, we utilized the mouse hippocampus kindling model of epileptogenesis and investigated the effect of finasteride, a 5α-reductase and neurosteroid synthesis inhibitor. Progesterone markedly retarded the development of epileptogenesis and inhibited the rate of kindling acquisition to elicit stage 5 seizures. Pretreatment with finasteride led to complete inhibition of the P-induced retardation of the limbic epileptogenesis in mice. Finasteride did not significantly influence the acute seizure expression in fully kindled mice expressing stage 5 seizures. Thus, neurosteroids that potentiate phasic and tonic inhibition in the hippocampus, such as allopregnanolone, may mediate the disease-modifying effect of P, indicating a new role of neurosteroids in acquired limbic epileptogenesis and temporal lobe epilepsy.     

Reduced tonic inhibition in the dentate gyrus contributes to chronic stress‐induced impairments in learning and memory

It is well established that stress impacts the underlying processes of learning and memory. The effects of stress on memory are thought to involve, at least in part, effects on the hippocampus, which is particularly vulnerable to stress. Chronic stress induces hippocampal alterations, including but not limited to dendritic atrophy and decreased neurogenesis, which are thought to contribute to chronic stress‐induced hippocampal dysfunction and deficits in learning and memory. Changes in synaptic transmission, including changes in GABAergic inhibition, have been documented following chronic stress. Recently, our laboratory demonstrated shifts in E_GABA in CA1 pyramidal neurons following chronic stress, compromising GABAergic transmission and increasing excitability of these neurons. Interestingly, here we demonstrate that these alterations are unique to CA1 pyramidal neurons, since we do not observe shifts in E_GABA following chronic stress in dentate gyrus granule cells. Following chronic stress, there is a decrease in the expression of the GABA_A receptor (GABA_AR) δ subunit and tonic GABAergic inhibition in dentate gyrus granule cells, whereas there is an increase in the phasic component of GABAergic inhibition, evident by an increase in the peak amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs). Given the numerous changes observed in the hippocampus following stress, it is difficult to pinpoint the pertinent contributing pathophysiological factors. Here we directly assess the impact of a reduction in tonic GABAergic inhibition of dentate gyrus granule cells on learning and memory using a mouse model with a decrease in GABA_AR δ subunit expression specifically in dentate gyrus granule cells (Gabrd/Pomc mice). Reduced GABA_AR δ subunit expression and function in dentate gyrus granule cells is sufficient to induce deficits in learning and memory. Collectively, these findings suggest that the reduction in GABA_AR δ subunit‐mediated tonic inhibition in dentate gyrus granule cells contributes, at least in part, to deficits in learning and memory associated with chronic stress. These findings have significant implications regarding the pathophysiological mechanisms underlying impairments in learning and memory associated with stress and suggest a role for GABA_AR δ subunit containing receptors in dentate gyrus granule cells. © 2016 Wiley Periodicals, Inc.     

Partial inactivation of GABAA receptors containing the α5 subunit affects the development of adult‐born dentate gyrus granule cells

Alterations of neuronal activity due to changes in GABA_A receptors (GABA_AR) mediating tonic inhibition influence different hippocampal functions. Gabra5‐null mice and α5 subunit^(H105R) knock‐in mice exhibit signs of hippocampal dysfunction, but are capable of improved performance in several learning and memory tasks. Accordingly, alleviating abnormal GABAergic tonic inhibition in the hippocampal formation by selective α5‐GABA_AR modulators represents a possible therapeutic approach for several intellectual deficit disorders. Adult neurogenesis in the dentate gyrus is an important facet of hippocampal plasticity; it is regulated by tonic GABAergic transmission, as shown by deficits in proliferation, migration and dendritic development of adult‐born neurons in Gabra4‐null mice. Here, we investigated the contribution of α5‐GABA_ARs to granule cell development, using retroviral vectors expressing eGFP for labeling precursor cells in the subgranular zone. Global α5‐GABA_AR knockout (α5‐KO) mice showed no alterations in migration and morphological development of eGFP‐positive granule cells. However, upregulation of α1 subunit‐immunoreactivity was observed in the hippocampal formation and cerebral cortex. In contrast, partial gene inactivation in α5‐heterozygous (α5‐het) mice, as well as single‐cell deletion of Gabra5 in newborn granule cells from α5‐floxed mice, caused severe alterations of migration and dendrite development. In α5‐het mice, retrovirally mediated overexpression of Cdk5 resulted in normal migration and dendritic branching, suggesting that Cdk5 cooperates with α5‐GABA_ARs to regulate neuronal development. These results show that minor imbalance of α5‐GABA_AR‐mediated transmission may have major consequences for neuronal plasticity; and call for caution upon chronic therapeutic use of negative allosteric modulators acting at these receptors.     

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABA_A receptor‐mediated extrasynaptic tonic inhibition. In the developing striatum, medium‐sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABA_A receptor‐mediated tonic conductance at postnatal days 3–35. The antagonist of the GABA transporter‐1, 1‐[2‐[[(diphenylmethylene)imino]oxy]ethyl]‐1,2,5,6‐tetrahydro‐3‐pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 μm ) and 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride, the δ‐subunit‐containing GABA_A receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridine‐3‐ol hydrochloride‐sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ‐subunit on sevoflurane‐enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.     

Ethanol-induced GABAA receptor alpha4 subunit plasticity involves phosphorylation and neuroactive steroids

GABA_A receptors containing α4 subunits are widely implicated in acute ethanol sensitivity, and their spatial and temporal regulation prominently contributes to ethanol-induced neuroplasticity in hippocampus and cortex. However, it is unknown if α4-containing GABA_A receptors in the thalamus, an area of high α4 expression, display similar regulatory patterns following ethanol administration, and if so, by which molecular mechanisms. In the current study, thalamic GABA_A receptor α4 subunit levels were increased following a 6-week-, but not a 2-week chronic ethanol diet. Following acute high-dose ethanol administration, thalamic GABA_A receptor α4 subunit levels were regulated in a temporal fashion, as a decrease was observed at 2 h followed by a delayed transient increase. PKCγ and PKCδ levels paralleled α4 temporal expression patterns following ethanol exposure. Initial decreases in α4 subunit expression were associated with reduced serine phosphorylation. Delayed increases in expression were not associated with a change in phosphorylation state, but were prevented by inhibiting neuroactive steroid production with the 5α-reductase inhibitor finasteride. Overall, these studies indicate that thalamic GABA_A receptor α4 subunit expression following acute and chronic ethanol administration exhibits similar regulatory patterns as other regions and that transient expression patterns following acute exposure in vivo are likely dependent on both subunit phosphorylation state and neuroactive steroids.     

Tonic GABAA Receptor-Mediated Currents of Human Cortical GABAergic Interneurons Vary Amongst Cell Types

Persistent anion conductances through GABA_A receptors (GABA_ARs) are important modulators of neuronal excitability. However, it is currently unknown how the amplitudes of these currents vary among different cell types in the human neocortex, particularly among diverse GABAergic interneurons. We have recorded 101 interneurons in and near layer 1 from cortical tissue surgically resected from both male and female patients, visualized 84 of them and measured tonic GABA_AR currents in 48 cells with an intracellular [Cl^–] of 65 mm and in the presence of 5 μm GABA. We compare these tonic currents among five groups of interneurons divided by firing properties and four types of interneuron defined by axonal distributions; rosehip, neurogliaform, stalked-bouton, layer 2–3 innervating and a pool of other cells. Interestingly, the rosehip cell, a type of interneuron only described thus far in human tissue, and layer 2–3 innervating cells exhibit larger tonic currents than other layer 1 interneurons, such as neurogliaform and stalked-bouton cells; the latter two groups showing no difference. The positive allosteric modulators of GABA_ARs allopregnanolone and DS2 also induced larger current shifts in the rosehip and layer 2–3 innervating cells, consistent with higher expression of the δ subunit of the GABA_AR in these neurons. We have also examined how patient parameters, such as age, seizures, type of cancer and anticonvulsant treatment may alter tonic inhibitory currents in human neurons. The cell type-specific differences in tonic inhibitory currents could potentially be used to selectively modulate cortical circuitry.SIGNIFICANCE STATEMENT Tonic currents through GABA_A receptors (GABA_ARs) are a potential therapeutic target for a number of neurologic and psychiatric conditions. Here, we show that these currents in human cerebral cortical GABAergic neurons display cell type-specific differences in their amplitudes which implies differential modulation of their excitability. Additionally, we examine whether the amplitudes of the tonic currents measured in our study show any differences between patient populations, finding some evidence that age, seizures, type of cancer, and anticonvulsant treatment may alter tonic inhibition in human tissue. These results advance our understanding of how pathology affects neuronal excitability and could potentially be used to selectively modulate cortical circuitry.     

Inhibition of thalamic excitability by 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridine-3-ol: a selective role for δ-GABAA receptors

The sedative and hypnotic agent 4,5,6,7‐tetrahydroisoxazolo[4,5‐c]pyridine‐3‐ol (THIP) is a GABA_A receptor (GABA_AR) agonist that preferentially activates δ‐subunit‐containing GABA_ARs (δ‐GABA_ARs). To clarify the role of δ‐GABA_ARs in mediating the sedative actions of THIP, we utilized mice lacking the α₁‐ or δ‐subunit in a combined electrophysiological and behavioural analysis. Whole‐cell patch‐clamp recordings were obtained from ventrobasal thalamic nucleus (VB) neurones at a holding potential of ?60 mV. Application of bicuculline to wild‐type (WT) VB neurones revealed a GABA_AR‐mediated tonic current of 92 ± 19 pA, which was greatly reduced (13 ± 5 pA) for VB neurones of δ^0/0 mice. Deletion of the δ‐ but not the α₁‐subunit dramatically reduced the THIP (1 μm )‐induced inward current in these neurones (WT, ?309 ± 23 pA; δ^0/0, ?18 ± 3 pA; α₁^0/0, ?377 ± 45 pA). Furthermore, THIP selectively decreased the excitability of WT and α₁^0/0 but not δ^0/0 VB neurones. THIP did not affect the properties of miniature inhibitory post‐synaptic currents in any of the genotypes. No differences in rotarod performance and locomotor activity were observed across the three genotypes. In WT mice, performance of these behaviours was impaired by THIP in a dose‐dependent manner. The effect of THIP on rotarod performance was blunted for δ^0/0 but not α₁^0/0 mice. We previously reported that deletion of the α₁‐subunit abolished synaptic GABA_A responses of VB neurones. Therefore, collectively, these findings suggest that extrasynaptic δ‐GABA_ARs vs. synaptic α₁‐subunit‐containing GABA_ARs of thalamocortical neurones represent an important molecular target underpinning the sedative actions of THIP.     

Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl^? reversal potential (E_Cl). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA_A receptors (GABA_ARs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2–4] then decreases dramatically by the second postnatal week (P7–10 and P30–40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA_AR α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E_Cl recorded from P2–5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.     

Elevated Neurosteroids in the Lateral Thalamus Relieve Neuropathic Pain in Rats with Spared Nerve Injury

Neurosteroids are synthesized in the nervous system from cholesterol or steroidal precursors imported from peripheral sources. These compounds are important allosteric modulators of c-aminobutyric acid A receptors(GABA_ARs), which play a vital role in pain modulation in the lateral thalamus, a main gate where somatosensory information enters the cerebral cortex. Using high-performance liquid chromatography/tandem mass spectrometry,we found increased levels of neurosteroids(pregnenolone,progesterone, deoxycorticosterone, allopregnanolone, and tetrahydrodeoxycorticosterone) in the chronic stage of neuropathic pain(28 days after spared nerve injury) in rats.The expression of the translocator protein TSPO, the upstream steroidogenesis rate-limiting enzyme, increased at the same time. In vivo stereotaxic microinjection of neurosteroids or the TSPO activator AC-5216 into the lateral thalamus(AP-3.0 mm, ML ±3.0 mm, DV 6.0 mm)alleviated the mechanical allodynia in neuropathic pain,while the TSPO inhibitor PK 11195 exacerbated it. The analgesic effects of AC-5216 and neurosteroids were significantly attenuated by the GABA_AR antagonist bicuculline. These results suggested that elevated neurosteroids in the lateral thalamus play a protective role in the chronic stage of neuropathic pain.     

GABAB receptor‐dependent modulation of network activity in the rat prefrontal cortex in vitro

GABA (γ‐aminobutyric acid) can mediate inhibition via pre‐ and post/extrasynaptic GABA receptors. In this paper we demonstrate potentially post/extrasynaptic GABA_B receptor‐dependent tonic inhibition in L2/3 pyramidal cells of rat medial prefrontal cortex (mPFC) in vitro. First, we show via voltage‐clamp experiments the presence of a tonic GABA_B receptor‐dependent outward current in these neurons. This GABA_Bergic current could be induced by ambient GABA when present at sufficient concentrations. To increase ambient GABA levels in the usually silent slice preparation, we amplified network activity and hence synaptic GABA release with a modified artificial cerebrospinal fluid. The amplitude of tonic GABA_B current was similar at different temperatures. In addition to the tonic GABA_B current, we found presynaptic GABA_B effects, GABA_B‐mediated inhibitory postsynaptic currents and tonic GABA_A currents. Second, we performed current‐clamp experiments to evaluate the functional impact of GABA_B receptor‐mediated inhibition in the mPFC. Activating or inactivating GABA_B receptors led to rightward (reduction of excitability) or leftward (increase of excitability) shifts, respectively, of the input–output function of mPFC L2/3 pyramidal cells without effects on the slope. Finally, we showed in electrophysiological recordings and epifluorescence Ca²⁺‐imaging that GABA_B receptor‐mediated tonic inhibition is capable of regulating network activity. Blocking GABA_B receptors increased the frequency of excitatory postsynaptic currents impinging on a neuron and prolonged network upstates. These results show that ambient GABA via GABA_B receptors is powerful enough to modulate neuronal excitability and the activity of neural networks.     

Étifoxine,neurostéroïdes et anxiété

Anxiety states are related to the hypofunctioning of GABAergic inhibitory synaptic transmission in certain regions of the encephalon. Benzodiazepines are currently used in the treatment of anxiety states, but they are associated with side effects (sedation, amnesia) and their use in long-term treatment poses problems of functional tolerance. Benzodiazepines bind to specific sites located on the GABA_A receptor channels that underlie rapid GABAergic transmission and potentiate their functioning. Neurosteroids such as allopregnanolone that are reduced in positions 3α and 5α (3α5α-NS) also potentiate GABA_A receptor function and may be considered as endogenous anxiolytics. Neurosteroids are distinguished from other steroids by the fact that they are synthesised de novo in the nervous system by neurons and glial cells independently of circulating steroids. This synthesis occurs from cholesterol imported into the mitochondrion by a multiprotein complex of which the « peripheral benzodiazepine receptor », recently renamed TSPO (translocator protein of 18 kDa), forms a part. The majority of benzodiazepines bind to this protein and stimulate the transfer of cholesterol and consequently neurosteroidogenesis. These effects can be blocked pharmacologically by PK11195, which acts as an antagonist at the mitochondrial binding site for benzodiazepines. Cholesterol is transformed in the mitochondrion to pregnenolone, the universal precursor of all steroids. Pregnenolone then leaves the mitochondrion and the nature of the steroids synthesised in the cytoplasm will depend on the type of neurosteroidogenesis enzymes expressed by the cell.Etifoxine does not possess a benzodiazepine-related structure but does exhibit anxiolytic effects. The objective of our study consequently was to understand the mechanism of action of this compound by studying its effects on the functional properties of GABA_A receptors and on GABAergic synaptic transmission. Binding tests on isolated membranes have revealed that etifoxine binds directly to the GABA_A receptor and the peripheral benzodiazepine receptor (TSPO). Electrophysiological experiments have shown that etifoxine dose-dependently potentiates membrane currents induced by the application of submaximal but not of saturating concentrations of GABA. Etifoxine also facilitates GABAergic transmission in hypothalamic neuronal cultures and increases the amplitude of a sustained GABAergic current due to the tonic activation of GABA_A receptors by a weak GABA concentration in the extracellular space. The effect of etifoxine on this current is partially antagonised by PK11195 but not by flumazenil, an antagonist of the benzodiazepine binding sites on the GABA_A receptor.All of our data indicate that etifoxine facilitates GABAergic inhibition by two types of action: [1] by directly potentiating the activity of GABA_A receptors via an allosteric modulatory different site from that of the benzodiazepines, and [2] by stimulating the production of 3α5α-NS, which in turn potentiate the activity of GABA_A receptors. These two effects are independent and additive. They therefore contribute to make GABAergic transmission more effective.     

Tonic GABAergic control of mouse dentate granule cells during postnatal development

The dentate gyrus is the main hippocampal input structure receiving strong excitatory cortical afferents via the perforant path. Therefore, inhibition at this ‘hippocampal gate’ is important, particularly during postnatal development, when the hippocampal network is prone to seizures. The present study describes the development of tonic GABAergic inhibition in mouse dentate gyrus. A prominent tonic GABAergic component was already present at early postnatal stages (postnatal day 3), in contrast to the slowly developing phasic postsynaptic GABAergic currents. Tonic currents were mediated by GABA_A receptors containing α₅‐ and δ‐subunits, which are sensitive to low ambient GABA concentrations. The extracellular GABA level was determined by synaptic GABA release and GABA uptake via the GABA transporter 1. The contribution of these main regulatory components was surprisingly stable during postnatal granule cell maturation. Throughout postnatal development, tonic GABAergic signals were inhibitory. They increased the action potential threshold of granule cells and reduced network excitability, starting as early as postnatal day 3. Thus, tonic inhibition is already functional at early developmental stages and plays a key role in regulating the excitation/inhibition balance of both the adult and the maturing dentate gyrus.     

Activation of extrasynaptic GABAA receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons

Extrasynaptic GABA_A receptors (GABA_ARs)-mediated tonic inhibition is reported to involve in the pathogenesis of epilepsy. In this study, we used cyclothiazide (CTZ)-induced in vitro brain slice seizure model to explore the effect of selective activation of extrasynaptic GABA_ARs by 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) on the CTZ-induced epileptiform activity in hippocampal neurons. Perfusion with CTZ dose-dependently induced multiple epileptiform peaks of evoked population spikes (PSs) in CA1 pyramidal neurons, and treatment with THIP (5 μmol/L) significantly reduced the multiple PS peaks induced by CTZ stimulation. Western blot showed that the δ-subunit of the GABA_AR, an extrasynaptic specific GABA_AR subunit, was also significantly down-regulated in the cell membrane 2 h after CTZ treatment. Our results suggest that the CTZ-induced epileptiform activity in hippocampal CA1 neurons is suppressed by the activation of extrasynaptic GABA_ARs, and further support the hypothesis that tonic inhibition mediated by extrasynaptic GABA_ARs plays a prominent role in seizure generation.     

Role of GABA<Subscript>A</Subscript> receptors in alcohol use disorders suggested by chronic intermittent ethanol (CIE) rodent model

GABAergic inhibitory transmission is involved in the acute and chronic effects of ethanol on the brain and behavior. One-dose ethanol exposure induces transient plastic changes in GABA_A receptor subunit levels, composition, and regional and subcellular localization. Rapid down-regulation of early responder δ subunit-containing GABA_A receptor subtypes mediating ethanol-sensitive tonic inhibitory currents in critical neuronal circuits corresponds to rapid tolerance to ethanol’s behavioral responses. Slightly slower, α1 subunit-containing GABA_A receptor subtypes mediating ethanol-insensitive synaptic inhibition are down-regulated, corresponding to tolerance to additional ethanol behaviors plus cross-tolerance to other GABAergic drugs including benzodiazepines, anesthetics, and neurosteroids, especially sedative-hypnotic effects. Compensatory up-regulation of synaptically localized α4 and α2 subunit-containing GABA_A receptor subtypes, mediating ethanol-sensitive synaptic inhibitory currents follow, but exhibit altered physio-pharmacology, seizure susceptibility, hyperexcitability, anxiety, and tolerance to GABAergic positive allosteric modulators, corresponding to heightened alcohol withdrawal syndrome. All these changes (behavioral, physiological, and biochemical) induced by ethanol administration are transient and return to normal in a few days. After chronic intermittent ethanol (CIE) treatment the same changes are observed but they become persistent after 30 or more doses, lasting for at least 120 days in the rat, and probably for life. We conclude that the ethanol-induced changes in GABA_A receptors represent aberrant plasticity contributing critically to ethanol dependence and increased voluntary consumption. We suggest that the craving, drug-seeking, and increased consumption in the rat model are tied to ethanol-induced plastic changes in GABA_A receptors, importantly the development of ethanol-sensitive synaptic GABA_A receptor-mediating inhibitory currents that participate in maintained positive reward actions of ethanol on critical neuronal circuits. These probably disinhibit nerve endings of inhibitory GABAergic neurons on dopamine reward circuit cells, and limbic system circuits mediating anxiolysis in hippocampus and amygdala. We further suggest that the GABA_A receptors contributing to alcohol dependence in the rat and presumably in human alcohol use disorders (AUD) are the ethanol-induced up-regulated subtypes containing α4 and most importantly α2 subunits. These mediate critical aspects of the positive reinforcement of ethanol in the dependent chronic user while alleviating heightened withdrawal symptoms experienced whenever ethanol is absent. The speculative conclusions based on firm observations are readily testable.     

Bicuculline‐ and neurosteroid‐sensitive tonic chloride current in rat hypoglossal motoneurons and atypical dual effect of SR95531

Hypoglossal motoneurons (HMs) are known to be under ‘permanent’ bicuculline‐sensitive inhibition and to show ‘transient’ synaptic γ‐aminobutyric acid (GABA)_A and glycine inhibitory responses. The present paper describes a permanent bicuculline‐sensitive current that should contribute to their tonic inhibition. This current was recorded in brainstem slices superfused without any exogenous agonist and remained detectable with tetrodotoxin. It could also be blocked by the other GABA_A antagonists picrotoxin (PTX) and 2‐(3‐carboxypropyl)‐3‐amino‐6‐(4 methoxyphenyl)pyridazinium bromide) (SR95531; gabazine), but persisted in the presence of a specific blocker of α5‐containing GABA_A receptors. Addition of 2 μm 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol hydrochloride (THIP), known to preferentially activate GABA_A receptors devoid of a γ‐subunit, induced a sustained anionic current that could be further enhanced by neurosteroids such as allopregnanolone (100 nm ). Thus, HMs show a tonic inhibitory current carried by extrasynaptic γ‐free GABA_A receptors, highly sensitive to neurosteroids. A second result was obtained by using SR95531 at concentrations sufficiently high to rapidly block the tonic current above the chloride equilibrium potential (E_Cl). Surprisingly, below E_Cl, SR95531 (10–40 μm ) activated a sustained inward current, associated with a conductance increase, and resistant to bicuculline or PTX (100 μm ). Similarly, after blockade of the bicuculline‐sensitive current, SR95531 activated an outward current above E_Cl. The bicuculline‐resistant anionic current activated by SR95531 could be blocked by a GABA_C receptor antagonist. Thus, two types of inhibitory GABA receptors, belonging to the GABA_A and GABA_C families, are able to show a sustained activity in HMs and provide promising targets for neuroprotection under overexcitatory situations known to easily damage these particularly fragile neurons.     

Phasic GABAA -receptor activation is required to suppress epileptiform activity in the CA3 region of the immature rat hippocampus

Purpose: Despite the consistent observation that γ‐aminobutyric acid A (GABA_A) receptors mediate excitatory responses at perinatal stages, the role of the GABAergic system in the generation of neonatal epileptiform activity remains controversial. Therefore, we analyzed whether tonic and phasic GABAergic transmission had differential effects on neuronal excitability during early development. Methods: We performed whole cell patch‐clamp and field potential recordings in the CA3 region of hippocampal slices from immature (postnatal day 4–7) rats to analyze the effect of specific antagonists and modulators of tonic and phasic GABAergic components on neuronal excitability. Key Findings: The GABAergic antagonists gabazine (3 μm ) and picrotoxin (100 μm ) induced epileptiform discharges, whereas activation of GABA_A receptors attenuated epileptiform discharges. Under low‐Mg²⁺ conditions, 100 nm gabazine and 1 μm picrotoxin were sufficient to provoke epileptiform activity in 63.2% (n = 19) and 53.8% (n = 26) of the slices, respectively. Whole‐cell patch‐clamp experiments revealed that these concentrations significantly reduced the amplitude of phasic GABAergic postsynaptic currents but had no effect on tonic currents. In contrast, 1‐μm 4,5,6,7‐tetrahydroisoxaz‐olo[5,4‐c]‐pyridin‐3‐ol (THIP) induced a tonic current of ?12 ± 2.5 pA (n = 6) and provoked epileptiform discharges in 57% (n = 21) of the slices. Significance: We conclude from these results that in the early postnatal rat hippocampus a constant phasic synaptic activity is required to control excitability and prevent epileptiform activity, whereas tonic GABAergic currents can mediate excitatory responses. Pharmacologic intervention at comparable human developmental stages should consider these ambivalent GABAergic actions.     

The Neuroactive Steroid 5α-Tetrahydrodeoxycorticosterone Increases GABAergic Postsynaptic Inhibition in Rat Neocortical Neurons in vitro

The neuroactive steroid 5α-pregnane-3α,21-diol-20-one (5α-tetrahydrodeoxycorticosterone; 5α-THDOC) has been shown to potentiate GABA-induced chloride currents in cell cultures and subcellular preparations. In this study, we recorded from pyramidal neurons in an in vitro slice preparation of the adult rat frontal neocortex using intracellular microelectrodes. 5α-THDOC (10 μM) increased and prolonged the inhibitory postsynaptic potential (IPSP). The mean maximal synaptic conductance of the early, GABA_A receptor-mediated, IPSP was enhanced to more than 700%, the one at the maximum of the late, partially GABA_A receptor-mediated, IPSP to approximately 400%. The progesterone/glucocorticoid receptor antagonist RU 38486 did not prevent the IPSP increase. At a concentration of 1 μM 5α-THDOC increased only the early IPSP to about 125%. Responses to the iontophoretically applied specific GABA_A receptor agonist muscimol but not to the specific GABA_B receptor agonist L-baclofen were enhanced by 5α-THDOC (10 μM). In the giga-seal whole-cell configuration when the GABA_B receptor-mediated IPSP component was absent due to intracellular perfusion, 5α-THDOC (10 μM) increased IPSPs to a similar extent as in the conventional microelectrode recordings. Excitatory postsynaptic potentials, resting membrane potential, input resistance and action potential amplitude were not affected by 5α-THDOC (10 μM). These data demonstrate that in neocortical tissue of the rat 5α-THDOC enhances GABAergic inhibition by interacting with postsynaptic GABA_A receptors while synaptic excitation and parameters of electric excitability remain unchanged.     

GluN2B‐containing N‐methyl‐D‐aspartate receptors compensate for the inhibitory control of synaptic plasticity during the early critical period in the rat visual cortex

In the visual cortex, synaptic plasticity is very high during the early developmental stage known as the critical period and declines with development after the critical period. Changes in the properties of N‐methyl‐D‐aspartate receptor (NMDAR) and γ‐aminobutyric acid type A receptor (GABA_AR) have been suggested to underlie the changes in the characteristics of plasticity. However, it is largely unknown how the changes in the two receptors interact to regulate synaptic plasticity. The present study investigates the changes in the properties of NMDAR and GABA_AR from 3 to 5 weeks of age in layer 2/3 pyramidal neurons of the rat visual cortex. The impact of these changes on the characteristics of long‐term potentiation (LTP) is also investigated. The amplitude and decay time constant of GABA_AR‐mediated currents increased during this period. However, the decay time constant of NMDAR‐mediated currents decreased as a result of the decrease in the proportion of the GluN2B subunit‐mediated component. Induction of NMDAR‐dependent LTP at 3 weeks depended on the GluN2B subunit, but LTP at 5 weeks did not. Enhancement of GABA_AR‐mediated inhibition suppressed the induction of LTP only at 5 weeks. However, partial inhibition of the GluN2B subunit with a low concentration of ifenprodil allowed the GABA_AR‐mediated suppression of LTP at 3 weeks. These results suggest that changes in the properties of NMDAR‐ and GABA_AR‐mediated synaptic transmission interact to determine the characteristics of synaptic plasticity during the critical period in the visual cortex. © 2015 Wiley Periodicals, Inc.