Cell type-specific distribution of GABAA receptor subtypes in the mouse dorsal striatum

The striatum is the main input nucleus of the basal ganglia, mediating motor and cognitive functions. Striatal projection neurons are GABAergic medium spiny neurons (MSN), expressing either the dopamine receptor type 1 (D₁-R MSN) and forming the direct, movement-promoting pathway, or dopamine receptor type 2 (D₂-R MSN), forming the indirect movement-suppressing pathway. Locally, activity and synchronization of MSN are modulated by several subtypes of GABAergic and cholinergic interneurons. Overall, GABAergic circuits in the striatum remain poorly characterized, and little is known about the intrastriatal connectivity of interneurons and the distribution of GABA_A receptor (GABA_AR) subtypes, distinguished by their subunit composition, in striatal synapses. Here, by using immunofluorescence in mouse tissue, we investigated the distribution of GABA_ARs containing the α₁, α₂, or α₃ subunit in perisomatic synapses of striatal MSN and interneurons, as well as the innervation pattern of D₁R- and D₂R-MSN soma and axonal initial segment (AIS) by GABAergic and cholinergic interneurons. Our results show that perisomatic GABAergic synapses of D₁R- and D₂R-MSN contain the GABA_AR α₁ and/or α₂ subunits, but not the α₃ subunit; D₂R-MSN have significantly more α₁-GABA_ARs on their soma than D₁R-MSN. Further, interneurons have few perisomatic synapses containing α₂-GABA_ARs, whereas α₃-GABA_ARs (along with the α₁-GABA_ARs) are abundant in perisomatic synapses of CCK⁺, NPY⁺/SOM⁺, and vAChT⁺ interneurons. Each MSN and interneuron population analyzed received a distinct pattern of GABAergic and cholinergic innervation, complementing this postsynaptic heterogeneity. In conclusion, intra-striatal GABAergic circuits are distinguished by cell-type specific innervation patterns, differential expression and postsynaptic targeting of GABA_AR subtypes.     

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.     

Exercise modifies α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor expression in striatopallidal neurons in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐lesioned mouse

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic‐acid‐type glutamate receptor (AMPAR) plays a critical role in modulating experience‐dependent neuroplasticity, and alterations in AMPAR expression may underlie synaptic dysfunction and disease pathophysiology. Using the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of dopamine (DA) depletion, our previous work showed exercise increases total GluA2 subunit expression and the contribution of GluA2‐containing channels in MPTP mice. The purpose of this study was to determine whether exercise‐dependent changes in AMPAR expression after MPTP are specific to the striatopallidal (D₂R) or striatonigral (D₁R) medium spiny neuron (MSN) striatal projection pathways. Drd₂‐eGFP‐BAC transgenic mice were used to delineate differences in AMPAR expression between striatal D₂R‐MSNs and D₁R‐MSNs. Striatal AMPAR expression was assessed by immunohistochemical (IHC) staining, Western immunoblotting (WB) of preparations enriched for postsynaptic density (PSD), and alterations in the current–voltage relationship of MSNs. We found DA depletion results in the emergence of GluA2‐lacking AMPARs selectively in striatopallidal D₂R‐MSNs and that exercise reverses this effect in MPTP mice. Exercise‐induced changes in AMPAR channels observed after DA depletion were associated with alterations in GluA1 and GluA2 subunit expression in postsynaptic protein, D₂R‐MSN cell surface expression, and restoration of corticostriatal plasticity. Mechanisms regulating experience‐dependent changes in AMPAR expression may provide innovative therapeutic targets to increase the efficacy of treatments for basal ganglia disorders, including Parkinson's disease. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

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.     

Adenosine A1 receptors measured with 11C‐MPDX PET in early Parkinson's disease

Adenosine A₁ receptors (A₁Rs) interact negatively with dopamine D₁ receptors (D₁Rs) in neurons of the basal ganglia's direct pathway, while adenosine A_2A receptors (A_2ARs) negatively interact with dopamine D₂ receptors (D₂Rs) in indirect‐pathway neurons. The aim of this study was to investigate the cerebral density of A₁Rs in Parkinson's disease (PD) in its early stages, using PET scans with the radioligand 8‐dicyclopropylmethyl‐1‐¹¹C‐methyl‐3‐propylxanthine (¹¹C‐MPDX). We studied 10 drug‐naïve patients with early PD. Each patient was also examined for dopamine transporters (DATs) and D₂Rs by PET using ¹¹C‐2‐β‐carbomethoxy‐3‐β‐(4‐fluorophenyl)‐tropane (¹¹C‐CFT) and ¹¹C‐raclopride (¹¹C‐RAC), respectively. Ten elderly, healthy volunteers were recruited as controls for ¹¹C‐MPDX PET scanning and eight elderly volunteers were recruited as controls for ¹¹C‐CFT and ¹¹C‐RAC PET scanning. The PET scans revealed a decrease in the uptake ratio index (URI) of ¹¹C‐CFT and an increase in the URI of ¹¹C‐RAC in patients. In the temporal lobe, the binding potential for ¹¹C‐MPDX was higher in the patient group than in healthy subjects, but not in the other regions examined, including the striatum. In patients, we observed motor‐symptom asymmetry and a relationship between parkinsonism and the striatal density of DATs, but not A₁R density. In the putamen of early PD, asymmetrical down‐regulation of A_2ARs is likely a compensatory mechanism in response to a decrease in dopamine. However, our study suggests that A₁Rs are unaltered in the putamen of early PD.     

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.     

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.     

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.     

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.     

Adenosine–Dopamine Interactions in the Pathophysiology and Treatment of CNS Disorders

Adenosine–dopamine interactions in the central nervous system (CNS) have been studied for many years in view of their relevance for disorders of the CNS and their treatments. The discovery of adenosine and dopamine receptor containing receptor mosaics (RM, higher‐order receptor heteromers) in the striatum opened up a new understanding of these interactions. Initial findings indicated the existence of A_2AR‐D₂R heterodimers and A₁R‐D₁R heterodimers in the striatum that were followed by indications for the existence of striatal A_2AR‐D₃R and A_2AR‐D₄R heterodimers. Of particular interest was the demonstration that antagonistic allosteric A_2A‐D₂ and A₁‐D₁ receptor–receptor interactions take place in striatal A_2AR‐D₂R and A₁R‐D₁R heteromers. As a consequence, additional characterization of these heterodimers led to new aspects on the pathophysiology of Parkinson's disease (PD), schizophrenia, drug addiction, and l ‐DOPA‐induced dyskinesias relevant for their treatments. In fact, A_2AR antagonists were introduced in the symptomatic treatment of PD in view of the discovery of the antagonistic A_2AR–D₂R interaction in the dorsal striatum that leads to reduced D₂R recognition and G_i/o coupling in striato‐pallidal GABAergic neurons. In recent years, indications have been obtained that A_2AR‐D₂R and A₁R‐D₁R heteromers do not exist as heterodimers, rather as RM. In fact, A_2A‐CB₁‐D₂ RM and A_2A‐D₂‐mGlu₅ RM have been discovered using a sequential BRET‐FRET technique and by using the BRET technique in combination with bimolecular fluorescence complementation. Thus, other pathogenic mechanisms beside the well‐known alterations in the release and/or decoding of dopamine in the basal ganglia and limbic system are involved in PD, schizophrenia and drug addiction. In fact, alterations in the stoichiometry and/or topology of A_2A‐CB₁‐D₂ and A_2A‐D₂‐mGlu5 RM may play a role. Thus, the integrative receptor–receptor interactions in these RM give novel aspects on the pathophysiology and treatment strategies, based on combined treatments, for PD, schizophrenia, and drug addiction.     

GABAA receptors can initiate the formation of functional inhibitory GABAergic synapses

The mechanisms that underlie the selection of an inhibitory GABAergic axon's postsynaptic targets and the formation of the first contacts are currently unknown. To determine whether expression of GABA_A receptors (GABA_ARs) themselves – the essential functional postsynaptic components of GABAergic synapses – can be sufficient to initiate formation of synaptic contacts, a novel co‐culture system was devised. In this system, the presynaptic GABAergic axons originated from embryonic rat basal ganglia medium spiny neurones, whereas their most prevalent postsynaptic targets, i.e. α1/β2/γ2‐GABA_ARs, were expressed constitutively in a stably transfected human embryonic kidney 293 (HEK293) cell line. The first synapse‐like contacts in these co‐cultures were detected by colocalization of presynaptic and postsynaptic markers within 2 h. The number of contacts reached a plateau at 24 h. These contacts were stable, as assessed by live cell imaging; they were active, as determined by uptake of a fluorescently labelled synaptotagmin vesicle‐luminal domain‐specific antibody; and they supported spontaneous and action potential‐driven postsynaptic GABAergic currents. Ultrastructural analysis confirmed the presence of characteristics typical of active synapses. Synapse formation was not observed with control or N‐methyl‐d ‐aspartate receptor‐expressing HEK293 cells. A prominent increase in synapse formation and strength was observed when neuroligin‐2 was co‐expressed with GABA_ARs, suggesting a cooperative relationship between these proteins. Thus, in addition to fulfilling an essential functional role, postsynaptic GABA_ARs can promote the adhesion of inhibitory axons and the development of functional synapses.     

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.     

GABAA,NMDA and mGlu2 receptors tonically regulate inhibition and excitation in the thalamic reticular nucleus

Traditionally, neurotransmitters are associated with a fast, or phasic, type of action on neurons in the central nervous system (CNS). However, accumulating evidence indicates that γ‐aminobutyric acid (GABA) and glutamate can also have a continual, or tonic, influence on these cells. Here, in voltage‐ and current‐clamp recordings in rat brain slices, we identify three types of tonically active receptors in a single CNS structure, the thalamic reticular nucleus (TRN). Thus, TRN contains constitutively active GABA_A receptors (GABA_ARs), which are located on TRN neurons and generate a persistent outward Cl^? current. When TRN neurons are depolarized, blockade of this current increases their action potential output in response to current injection. Furthermore, TRN contains tonically active GluN2B‐containing N‐methyl‐D‐aspartate receptors (NMDARs). These are located on reticuloreticular GABAergic terminals in TRN and generate a persistent facilitation of vesicular GABA release from these terminals. In addition, TRN contains tonically active metabotropic glutamate type 2 receptors (mGlu2Rs). These are located on glutamatergic cortical terminals in TRN and generate a persistent reduction of vesicular glutamate release from these terminals. Although tonically active GABA_ARs, NMDARs and mGlu2Rs operate through different mechanisms, we propose that the continual and combined activity of these three receptor types ultimately serves to hyperpolarize TRN neurons, which will differentially affect the output of these cells depending upon the current state of their membrane potential. Thus, when TRN cells are relatively depolarized, their firing in single‐spike tonic mode will be reduced, whereas when these cells are relatively hyperpolarized, their ability to fire in multispike burst mode will be facilitated.     

GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity

GABA_BRs have a well-established role in controlling neuronal excitability and presynaptic neurotransmitter release. We examined the role of GABA_BR activity in modulating the number and lateral diffusion of GABA_ARs at inhibitory synapses. Changes in diffusion of GABA_ARs at synapses were observed when subunit heterogeneity was taken into account. While α1-GABA_ARs were unaffected, α2- and α5-GABA_ARs showed inverse changes in enrichment and diffusion. The intracellular TM3-4 loop of α2 was sufficient to observe the changes in diffusion by GABA_BR activity, whereas the loop of α5 was not. The opposing effect on α2- and α5-GABA_ARs was caused by a competition between GABA_ARs for binding slots at synapses. Receptor immobilization by cross-linking revealed that α5-GABA_AR trapping at synapses is regulated by modulation of α2-GABA_AR mobility. Finally, PKC activity was determined to be part of the signaling pathway through which GABA_BR activity modulates α2-GABA_AR diffusion at synapses. These results outline a novel mechanism for tuning inhibitory transmission in a subunit-specific manner, and for the first time describe competition between GABA_ARs with different subunit compositions for binding slots at synapses.     

Molecular mechanisms of antiseizure drug activity at GABAA receptors

The GABA_A receptor (GABA_AR) is a major target of antiseizure drugs (ASDs). A variety of agents that act at GABA_ARs s are used to terminate or prevent seizures. Many act at distinct receptor sites determined by the subunit composition of the holoreceptor. For the benzodiazepines, barbiturates, and loreclezole, actions at the GABA_AR are the primary or only known mechanism of antiseizure action. For topiramate, felbamate, retigabine, losigamone and stiripentol, GABA_AR modulation is one of several possible antiseizure mechanisms. Allopregnanolone, a progesterone metabolite that enhances GABA_AR function, led to the development of ganaxolone. Other agents modulate GABAergic “tone” by regulating the synthesis, transport or breakdown of GABA. GABA_AR efficacy is also affected by the transmembrane chloride gradient, which changes during development and in chronic epilepsy. This may provide an additional target for “GABAergic” ASDs. GABA_AR subunit changes occur both acutely during status epilepticus and in chronic epilepsy, which alter both intrinsic GABA_AR function and the response to GABA_AR-acting ASDs. Manipulation of subunit expression patterns or novel ASDs targeting the altered receptors may provide a novel approach for seizure prevention.     

Benzodiazepine-dependent stabilization of GABAA receptors at synapses

GABA_A receptors constitutively enter and exit synapses by lateral diffusion in the plane of the neuronal membrane. They are trapped at synapses through their interactions with gephyrin, the main scaffolding protein at inhibitory post-synaptic densities. Previous work has shown that the synaptic accumulation and diffusion dynamics of GABA_ARs are controlled via excitatory synaptic activity. However, it remains unknown whether GABA_AR activity can itself impact the surface trafficking of the receptors. Here we report the effects of GABA_AR agonists, antagonists and allosteric modulators on the receptor's surface dynamics. Using immunocytochemistry and single particle tracking experiments on mouse hippocampal neurons, we show that the agonist muscimol decreases GABA_AR and gephyrin levels at synapses and accelerates the receptor's lateral diffusion within 30–120 min of treatment. In contrast, the GABA_AR antagonist gabazine increased GABA_AR amounts and slowed down GABA_AR diffusion at synapses. The response to GABA_AR activation or inhibition appears to be an adaptative regulation of GABAergic synapses. Surprisingly, the positive allosteric modulator diazepam abolished the regulation induced by muscimol, and this effect was observed on α1, α2, α5 and γ2 GABA_AR subunits. Altogether these results indicate that diazepam stabilizes synaptic GABA_ARs and thus prevents the agonist-induced regulation of GABA_AR levels at synapses. This occurred independently of neuronal activity and intracellular calcium and involved GABA_AR–gephyrin interactions, suggesting that the changes in GABA_AR diffusion depend on conformational changes of the receptor. Our study provides a new molecular mechanism involved in the adaptative response to changes in GABA_AR activity and benzodiazepine treatments.     

Nucleus-specific modulation of phasic and tonic inhibition by endogenous neurosteroidogenesis in the murine thalamus

Neurosteroids are potent allosteric modulators of GABA_A receptors (GABA_ARs). Although the effects of exogenous neurosteroids on GABA_AR function are well documented, less is known about effects of neurosteroids produced by local endogenous biosynthesis. The neurosteroidogenic enzymes 5α-reductase and 3α-hydroxysteroid dehydrogenase are expressed in two nuclei of somatosensory thalamus, the thalamic reticular nucleus (nRT) and ventrobasal nucleus (VB). Here, the effects of acute blockade of neurosteroidogenesis by the 5α-reductase inhibitor finasteride on phasic and tonic GABA_AR-mediated currents were examined in nRT and VB of mice. In nRT, finasteride altered the decay and amplitude, but not the frequency, of phasic currents, with no effect on tonic inhibition. In VB neurons, by contrast, finasteride reduced both the size and frequency of phasic currents, and also reduced the degree of tonic inhibition. These studies thus provide novel evidence for endogenous modulation of GABA_AR function by 5α-reduced neurosteroids in the mature thalamus.     

Altered behavioral responses to gamma‐aminobutyric acid pharmacological agents in a mouse model of Huntington's disease

Background : Disruptions in gamma‐aminobutyric (GABA) acid signaling are believed to be involved in Huntington's disease pathogenesis, but the regulation of GABAergic signaling remains elusive. Here we evaluated GABAergic signaling by examining the function of GABAergic drugs in Huntington's disease and the expression of GABAergic molecules using mouse models and human brain tissues from Huntington's disease. Methods : We treated wild‐type and R6/2 mice (a transgenic Huntington's disease mouse model) acutely with vehicle, diazepam, or gaboxadol (drugs that selectively target synaptic or extrasynaptic GABA_A receptors) and monitored their locomotor activity. The expression levels of GABA_A receptors and a major neuron‐specific chloride extruder (potassium‐chloride cotransporter‐2) were analyzed by real‐time quantitative polymerase chain reaction, Western blot, and immunocytochemistry. Results : The R6/2 mice were less sensitive to the sedative effects of both drugs, suggesting reduced function of GABA_A receptors. Consistently, the expression levels of α1/α2 and δ subunits were lower in the cortex and striatum of R6/2 mice. Similar results were also found in 2 other mouse models of Huntington's disease and in Huntington's disease patients. Moreover, the interaction and expression levels of potassium‐chloride cotransporter‐2 and its activator (brain‐type creatine kinase) were decreased in Huntington's disease neurons. These findings collectively suggest impaired chloride homeostasis, which further dampens GABA_A receptor‐mediated inhibitory signaling in Huntington's disease brains. Conclusions : The dysregulated GABAergic responses and altered expression levels of GABA_A receptors and potassium‐chloride cotransporter‐2 in Huntington's disease mice appear to be authentic and may contribute to the clinical manifestations of Huntington's disease patients. © 2017 International Parkinson and Movement Disorder Society