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.     

Plasticity of synaptic inhibition in mouse spinal cord lamina II neurons during early postnatal development and after inactivation of the glycine receptor α3 subunit gene

Synaptic inhibition mediated by GABA_A receptors and glycine receptors (GlyRs) in the outer laminae of the spinal cord dorsal horn efficiently filters ascending nociceptive messages, controlling pathological pain symptoms. However, although many studies have utilized transgenic models to study spinal nociceptive processing, very little is known about the development of functional inhibitory synapses onto these interneurons in mice. Here we report that most interneurons in lamina II are placed under phasic control by both GABAergic and glycinergic synapses, a number of which exhibit dual GABA/glycine co‐release. A developmental switch is also apparent: a subpopulation of lamina II interneurons controlled exclusively by either GABAergic or glycinergic synapses becomes detectable only after postnatal days 15 and 21, respectively. Using mice older than postnatal day 21, we also characterized the plastic changes in glycinergic transmission resulting from the inactivation of the GlyR α3 subunit gene, a key player in inflammatory pain pathways. This allowed us to demonstrate that synapses containing GlyR α3 contribute in large part to synaptic inhibition in lamina II. In Glra3 knockout mice, we found that synaptic currents at the remaining glycinergic synapses, containing GlyR α1, showed faster decay kinetics with unchanged amplitudes but increased frequency. These findings explain the absence of any basal nociceptive hypersensitivity in Glra3 knockout mice, as GlyR α1 is still available for mediating synaptic inhibition at lamina II synapses, but cannot be modulated by the prostaglandin–E‐prostanoid type 2 (EP2) receptor–protein kinase A signalling cascade. Our results clearly demonstrate that presynaptic GABA/glycine release properties are influenced by the nature and complexity of postsynaptic inhibitory receptor subtypes.     

The Neuropeptide Orexin-A Inhibits the GABA<Subscript>A</Subscript> Receptor by PKC and Ca<Superscript>2+</Superscript>/CaMKII-Dependent Phosphorylation of Its β<Subscript>1</Subscript> Subunit

Orexin-A and orexin-B (Ox-A, Ox-B) are neuropeptides produced by a small number of neurons that originate in the hypothalamus and project widely in the brain. Only discovered in 1998, the orexins are already known to regulate several behaviours. Most prominently, they help to stabilise the waking state, a role with demonstrated significance in the clinical management of narcolepsy and insomnia. Orexins bind to G-protein-coupled receptors (predominantly postsynaptic) of two subtypes, OX₁R and OX₂R. The primary effect of Ox-OXR binding is a direct depolarising influence mediated by cell membrane cation channels, but a wide variety of secondary effects, both pre- and postsynaptic, are also emerging. Given that inhibitory GABAergic neurons also influence orexin-regulated behaviours, crosstalk between the two systems is expected, but at the cellular level, little is known and possible mechanisms remain unidentified. Here, we have used an expression system approach to examine the feasibility, and nature, of possible postsynaptic crosstalk between Ox-A and the GABA_A receptor (GABA_AR), the brain’s main inhibitory neuroreceptor. When HEK293 cells transfected with OX₁R and the α₁, β₁, and γ_2S subunits of GABA_AR were exposed to Ox-A, GABA-induced currents were inhibited, in a calcium-dependent manner. This inhibition was associated with increased phosphorylation of the β₁ subunit of GABA_AR, and the inhibition could itself be attenuated by (1) kinase inhibitors (of protein kinase C and CaM kinase II) and (2) the mutation, to alanine, of serine 409 of the β₁ subunit, a site previously identified in phosphorylation-dependent regulation in other pathways. These results are the first to directly support the feasibility of postsynaptic crosstalk between Ox-A and GABA_AR, indicating a process in which Ox-A could promote phosphorylation of the β₁ subunit, reducing the GABA-induced, hyperpolarising current. In this model, Ox-A/GABA_AR crosstalk would cause the depolarising influence of Ox-A to be boosted, a type of positive feedback that could, for example, facilitate the ability to abruptly awake.     

Immunocytochemical Localization of the α1 and β2/3 Subunits of the GABAA Receptor in Relation to Specific GABAergic Synapses in the Dentate Gyrus

Dentate granule cells receive spatially segregated GABAergic innervation from at least five types of local circuit neurons, and express mRNA for at least 11 subunits of the GABA_A receptor. At most two to four different subunits are required to make a functional pentamer, raising the possibility that cells have on their surface several types of GABA_A receptor channel, which may not be uniformly distributed. In order to establish the subcellular location of GABA_A receptors on different parts of dentate neurons, the distribution of immunoreactivity for the α1 and β2/3 subunits of the receptor was studied using high-resolution immunocytochemistry. Light microscopic immunoperoxidase reactions revealed strong GABAA receptor immunoreactivity in the molecular layer of the dentate gyrus. Pre-embedding immunogold localization of the α1 and β2/3 subunits consistently showed extrasynaptic location of the GABA_A receptor on the somatic, dendritic and axon initial segment membrane of granule cells, but failed to show receptors in synaptic junctions. Using a postembedding immunogold technique on freeze-substituted, Lowicryl-embedded tissue, synaptic enrichment of immunoreactivity for these subunits was found on both granule and non-principal cells. Only the postembedding immunogold method is suitable for revealing relative differences in receptor density at the subcellular level, giving ～20 nm resolution. The immunolabelling for GABA_A receptor occupied the whole width of synaptic junctions, with a sharp decrease in labelling at the edge of the synaptic membrane specialization. Both subunits have been localized in the synaptic junctions between basket cell terminals and somata, and between axo-axonic cell terminals and axon initial segments of granule cells, with no qualitative difference in labelling. Receptor-immunopositive synapses were found at all depths of the molecular layer. Some of the boutons forming these dendritic synapses have been shown to contain GABA, providing evidence that some of the GABAergic cells that terminate only on the dendrites of granule cells also act through GABAA receptors. Double immunolabelling experiments demonstrated that a population of GABA-immunopositive neurons expresses a higher density of immunoreactive GABA_A receptor on their surface than principal cells. Interneurons were found to receive GABA_A receptor-positive synapses on their dendrites in the hilus, molecular and granule cell layers. Receptor-immunopositive synapses were also present throughout the hilus on presumed mossy cells. The results demonstrate that both granule cells and interneurons exhibit a compartmentalized distribution of the GABA_A receptor on their surface, the postjunctional membrane to GABAergic terminals having the highest concentration of receptor. The α1 and β2/3 subunits have a similar distribution in synapses on the axon initial segment, soma, proximal and distal dendrites of granule cells. The very strong immunoreactivity of a subpopulation of GABAergic interneurons for GABA_A receptors containing the α1 and β2/3 subunits predicts their high sensitivity to GABA and modulators of the receptor complex.     

Dopamine depletion induces neuron‐specific alterations of GABAergic transmission in the mouse striatum

Lack of dopamine (DA) in the striatum and the consequential dysregulation of thalamocortical circuits are major causes of motor impairments in Parkinson's disease. The striatum receives multiple cortical and subcortical afferents. Its role in movement control and motor skills learning is regulated by DA from the nigrostriatal pathway. In Parkinson's disease, DA loss affects striatal network activity and induces a functional imbalance of its output pathways, impairing thalamocortical function. Striatal projection neurons are GABAergic and form two functionally antagonistic pathways: the direct pathway, originating from DA receptor type 1‐expressing medium spiny neurons (D₁R‐MSN), and the indirect pathway, from D₂R‐MSN. Here, we investigated whether DA depletion in mouse striatum also affects GABAergic function. We recorded GABAergic miniature IPSCs (mIPSC) and tonic inhibition from D₁R‐ and D₂R‐MSN and used immunohistochemical labeling to study GABA_AR function and subcellular distribution in DA‐depleted and control mice. We observed slower decay kinetics and increased tonic inhibition in D₁R‐MSN, while D₂R‐MSN had increased mIPSC frequency after DA depletion. Perisomatic synapses containing the GABA_AR subunits α₁ or α₂ were not affected, but there was a strong decrease in non‐synaptic GABA_ARs containing these subunits, suggesting altered receptor trafficking. To broaden these findings, we also investigated GABA_ARs in GABAergic and cholinergic interneurons and found cell type‐specific alterations in receptor distribution, likely reflecting changes in connectivity. Our results reveal that chronic DA depletion alters striatal GABAergic transmission, thereby affecting cellular and circuit activity. These alterations either result from pathological changes or represent a compensatory mechanism to counteract imbalance of output pathways.     

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.     

Selective Allocation of GABAA Receptors Containing the α1 Subunit to Neurochemically Distinct Subpopulations of Rat Hippocampal Interneurons

The identification of a large variety of GABA_A receptor subunits by molecular cloning suggests the existence of multiple receptor subtypes differing in localization and functional properties. In the present study we analysed immunohistochemically the cellular distribution of GABA_A receptors containing the α1 subunit in the rat hippocampus with a subunit-specific antiserum. Prominent staining of numerous interneurons was evident in Ammon's horn and the dentate gyrus, which contrasted with moderate and diffuse immunoreactivity in the dendritic layers of pyramidal and granule cells. Double immunofluorescence staining with antibodies to GABA revealed that a subset of GABAergic neurons in the hippocampus were immunoreactive for the α1 subunit. To determine whether these cells represent distinct subpopulations of interneurons, we analysed the co-localization of the GABA_A receptor α1 subunit with selective markers of hippocampal interneurons (selected calcium-binding proteins and neuropeptides). In both Ammon's horn and the dentate gyrus, all parvalbumin-positive neurons and 50% of calretinin-positive neurons were double-labelled, whereas interneurons containing calbindin-D_28k were devoid of α1 subunit staining. Similarly, most neurons positive for neuropeptide Y and a subset of somatostatin-positive cells were double-labelled, in contrast to cholecystokinin- and vasoactive intestinal peptide-containing cells, which lacked the α1 subunit staining. These results demonstrate cell-specific expression of GABA_A receptors containing the α1 subunit among subsets of hippocampal interneurons, pointing to a pronounced functional specialization of these cells. Furthermore, the prominent expression of GABA_A receptors by interneurons suggests that disinhibition may be of major functional relevance in regulating the balance between excitation and inhibition in hippocampal circuits.     

Different pools of postsynaptic GABAA receptors mediate inhibition evoked by low‐ and high‐frequency presynaptic stimulation at hippocampal synapses

Patterns of short‐term synaptic plasticity could considerably differ between synapses of the same axon. This may lead to separation of synaptic receptors transmitting either low‐ or high‐frequency signals and, therefore, may have functional consequences for the information transfer in the brain. Here, we estimated a degree of such separation at hippocampal GABAergic synapses using a use‐dependent GABA_A receptor antagonist, picrotoxin, to selectively suppress a pool of GABA_A receptors monosynaptically activated during the low‐frequency stimulation. The relative changes in postsynaptic responses evoked by the high‐frequency stimulation before and after such block were used to estimate the contribution of this GABA_A receptor pool to synaptic transmission at high frequencies. Using this approach, we have shown that IPSCs evoked by low‐frequency (0.2 Hz) stimulation and asynchronous currents evoked by high‐frequency (20–40 Hz) stimulation are mediated by different pools of postsynaptic GABA_A receptors. Thus, our findings suggest that inhibition produced by a single hippocampal interneuron may be selectively routed to different postsynaptic targets depending on the presynaptic discharge frequency. Synapse 68:344–354, 2014 . © 2014 Wiley Periodicals, Inc.     

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.     

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.     

Developmental profile of GABAA-receptors in the marmoset monkey: Expression of distinct subtypes in pre- and postnatal brain

Gamma aminobutyric acid (GABA)_A-receptors are expressed in fetal mammalian brain before the onset of synaptic inhibition, suggesting their involvement in brain development. In this study, we have analyzed the maturation of the GABA_A-receptor in the marmoset monkey forebrain to determine whether distinct receptor subtypes are expressed at particular stages of pre- and postnatal ontogeny. The distribution of the subunits α1, α2, and β2,3 was investigated immunohistochemically between embryonic day 100 (6 weeks before birth) and adulthood. Prenatally, the α2- and β2,3-subunit-immunoreactivity (-IR) was prominent throughout the forebrain, whereas the α1-subunit-IR appeared in selected regions shortly before birth. The α2-subunit-IR disappeared gradually to become restricted to a few regions in adult forebrain. By contrast, the α1-subunit-IR increased dramatically after birth and replaced the α2-subunit in the basal forebrain, pallidum, thalamus, and most of the cerebral cortex. Staining for the β2,3-subunits was ubiquitous at every age examined, indicating their association with either the α1- or the α2-subunit in distinct receptor subtypes. In neocortex, the α1-subunit-IR was first located selectively to layers IV and VI of primary somatosensory and visual areas. Postnatally, it increased throughout the cortex, with the adult pattern being established only during the second year. The switch in expression of the α1- and α2-subunits indicates that the subunit composition of major GABA_A-receptor subtypes changes during ontogeny. This change coincides with synaptogenesis, suggesting that the emergence of α1-GABA_A-receptors parallels the formation of inhibitory circuits. A similar pattern has been reported in rat, indicating that the developmental regulation of GABA_A-receptors is conserved across species, possibly including man. However, the marmoset brain is more mature than the rat brain at the onset of α1-subunit expression, suggesting that α1-GABA_A-receptors are largely dispensable in utero, but may be required for information processing after birth. © 1996 Wiley-Liss, Inc.     

Selective distribution of GABA(A) receptor subtypes in mouse spinal dorsal horn neurons and primary afferents

In the spinal cord dorsal horn, presynaptic GABA_A receptors (GABA_ARs) in the terminals of nociceptors as well as postsynaptic receptors in spinal neurons regulate the transmission of nociceptive and somatosensory signals from the periphery. GABA_ARs are heterogeneous and distinguished functionally and pharmacologically by the type of α subunit variant they contain. This heterogeneity raises the possibility that GABA_AR subtypes differentially regulate specific pain modalities. Here, we characterized the subcellular distribution of GABA_AR subtypes in nociceptive circuits by using immunohistochemistry with subunit‐specific antibodies combined with markers of primary afferents and dorsal horn neurons. Confocal laser scanning microscopy analysis revealed a distinct, partially overlapping laminar distribution of α1–3 and α5 subunit immunoreactivity in laminae I–V. Likewise, a layer‐specific pattern was evident for their distribution among glutamatergic, γ‐aminobutyric acid (GABA)ergic, and glycinergic neurons (detected in transgenic mice expressing vesicular glutamate transporter 2–enhanced green fluorescent protein [vGluT2–eGFP], glutamic acid decarboxylase [GAD]67–eGFP, and glycine transporter 2 (GlyT2)–eGFP, respectively). Finally, all four subunits could be detected within primary afferent terminals. C‐fibers predominantly contained either α2 or α3 subunit immunoreactivity; terminals from myelinated (Aβ/Aδ) fibers were colabeled in roughly equal proportion with each subunit. The presence of axoaxonic GABAergic synapses was determined by costaining with gephyrin and vesicular inhibitory amino acid transporter to label GABAergic postsynaptic densities and terminals, respectively. Colocalization of the α2 or α3 subunit with these markers was observed in a subset of C‐fiber synapses. Furthermore, gephyrin mRNA and protein expression was detected in dorsal root ganglia. Collectively, these results show that differential GABA_AR distribution in primary afferent terminals and dorsal horn neurons allows for multiple, circuit‐specific modes of regulation of nociceptive circuits. J. Comp. Neurol. 520:3895–3911, 2012. © 2012 Wiley Periodicals, Inc.     

Developmental Changes in Serotonergic Modulation of GABAergic Synaptic Transmission and Postsynaptic GABA<Subscript>A</Subscript> Receptor Composition in the Cerebellar Nuclei

Outputs from the cerebellar nuclei (CN) are important for generating and controlling movement. The activity of CN neurons is controlled not only by excitatory inputs from mossy and climbing fibers and by γ-aminobutyric acid (GABA)-based inhibitory transmission from Purkinje cells in the cerebellar cortex but is also modulated by inputs from other brain regions, including serotonergic fibers that originate in the dorsal raphe nuclei. We examined the modulatory effects of serotonin (5-HT) on GABAergic synapses during development, using rat cerebellar slices. As previously reported, 5-HT presynaptically decreased the amplitudes of stimulation-evoked inhibitory postsynaptic currents (IPSCs) in CN neurons, with this effect being stronger in slices from younger animals (postnatal days [P] 11–13) than in slices from older animals (P19–21). GABA release probabilities accordingly exhibited significant decreases from P11–13 to P19–21. Although there was a strong correlation between the GABA release probability and the magnitude of 5-HT-induced inhibition, manipulating the release probability by changing extracellular Ca²⁺ concentrations failed to control the extent of 5-HT-induced inhibition. We also found that the IPSCs exhibited slower kinetics at P11–13 than at P19–21. Pharmacological and molecular biological tests revealed that IPSC kinetics were largely determined by the prevalence of α₁ subunits within GABA_A receptors. In summary, pre- and postsynaptic developmental changes in serotonergic modulation and GABAergic synaptic transmission occur during the second to third postnatal weeks and may significantly contribute to the formation of normal adult cerebellar function.     

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.     

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.     

Direct binding of GABAA receptor β2 and β3 subunits to gephyrin

GABAergic transmission is essential to brain function, and a large repertoire of GABA type A receptor (GABA_AR) subunits is at a neuron's disposition to serve this function. The glycine receptor (GlyR)‐associated protein gephyrin has been shown to be essential for the clustering of a subset of GABA_AR. Despite recent progress in the field of gephyrin‐dependent mechanisms of postsynaptic GABA_AR stabilisation, the role of gephyrin in synaptic GABA_AR localisation has remained a complex matter with many open questions. Here, we analysed comparatively the interaction of purified rat gephyrin and mouse brain gephyrin with the large cytoplasmic loops of GABA_AR α1, α2, β2 and β3 subunits. Binding affinities were determined using surface plasmon resonance spectroscopy, and showed an ~ 20‐fold lower affinity of the β2 loop to gephyrin as compared to the GlyR β loop–gephyrin interaction. We also probed in vivo binding in primary cortical neurons by the well‐established use of chimaeras of GlyR α1 that harbour respective gephyrin‐binding motifs derived from the different GABA_AR subunits. These studies identify a novel gephyrin‐binding motif in GABA_AR β2 and β3 large cytoplasmic loops.     

Methylmercury induces an initial increase in GABA-evoked currents in Xenopus oocytes expressing α1 and α6 subunit-containing GABAA receptors

Early onset effects of methylmercury (MeHg) on recombinant α₁β₂γ_2S or α₆β₂γ_2S subunit-containing GABA_A receptors were examined. These are two of the most prevalent receptor types found in cerebellum–a consistent target of MeHg-induced neurotoxicity. Heterologously expressed receptors were used in order to: (1) isolate receptor-mediated events from extraneous effects of MeHg due to stimulation of the receptor secondary to increased release of GABA seen with MeHg in neurons in situ and (2) limit the phenotypes of GABA_A receptors present at one time. Initial changes in I_GABA in Xenopus laevis oocytes expressing either α₁β₂γ_2S or α₆β₂γ_2S receptors were compared during continuous bath application of MeHg. A time-dependent increase in I_GABA mediated by both receptor subtypes occurred following the first 25–30 min of MeHg (5 μM) exposure. In α₆β₂γ_2S containing receptors, the MeHg-induced increase in I_GABA was less pronounced compared to that mediated by α₁β₂γ_2S containing receptors, although the pattern of effects was generally similar. Washing with MeHg-free solution reversed the increase in current amplitude. Application of bicuculline at the time of peak potentiation of I_GABA rapidly and completely reversed the MeHg-induced currents. Therefore these MeHg-increased inward currents are mediated specifically by the two subtypes of GABA_A receptors and appear to entail direct actions of MeHg on the receptor. However bicuculline did not affect stimulation by MeHg of oocyte endogenous Cl⁻ -mediated current, which presumably results from increased [Ca²⁺]_i. Thus, MeHg initially potentiates I_GABA in oocytes expressing either α₁β₂γ_2S or α₆β₂γ_2S receptors prior to its more defined later effects, suggesting that MeHg may initially interact directly with GABA_A receptors in a reversible manner to cause this potentiation.     

Developmental and degenerative modulation of GABAergic transmission in the mouse hippocampus

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter involved in synaptic plasticity. GABAergic transmission is also implicated in developmental and degenerative processes in the brain. The goal of the present study was to understand the developmental and degenerative regulation of GABAergic transmission in the mouse hippocampus by examining changes in GABA receptor subunit mRNA levels and GABA-related protein expression during postnatal development of the hippocampus and trimethyltin (TMT)-induced neurodegeneration in the juvenile (postnatal day [PD] 24) and adult hippocampus (PD 56). During postnatal development, the mRNA levels of GABA A receptor (GABA_AR) subunits, including α1, α4, β1, β2, and δ; GABA B receptor (GABA_BR) subunit 2; and the expression of GABA-related proteins, including glutamic acid decarboxylase, vesicular GABA transporter (VGAT), and potassium chloride cotransporter 2 increased gradually in the mouse hippocampus. The results of seizure scoring and histopathological findings in the hippocampus revealed a more pronounced response to the same administered TMT dose in juvenile mice, compared with that in adult mice. The mRNA levels of most GABA receptor subunits in the juvenile hippocampus, excluding GABA_AR subunit β3, were dynamically altered after TMT treatment. The mRNA levels of GABA_AR subunits γ2 and δ decreased significantly in the adult hippocampus following TMT treatment, whereas the level of GABA_BR subunit 1 mRNA increased significantly. Among the GABA-related proteins, only VGAT decreased significantly in the juvenile and adult mouse hippocampus after TMT treatment. In conclusion, regulation of GABAergic signaling in the mouse hippocampus may be related to maturation of the central nervous system and the degree of neurodegeneration during postnatal development and TMT-induced neurodegeneration in the experimental animals.     

Antisense oligonucleotide to GABAA receptor γ2 subunit induces limbic status epilepticus

γ-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. A deficiency of GABAergic inhibition mediated via the GABA_A receptor complex has for a long time been suspected to be a central factor in epileptogenesis. Status epilepticus is a condition of sustained and prolonged excitation of neuronal circuits, as detected by epileptiform discharges in the electroencephalogram (EEG). Reduction of GABA_A receptor-mediated hippocampal inhibition has been implicated in the development of status epilepticus. The present study provides direct evidence of a link between the GABA_A receptor and epilepsy. We show that selective inhibition of the expression of the GABA_A receptor γ2 subunit in the rat hippocampus by means of antisense oligonucleotides leads to spontaneous electrographic seizures that evolve into profound limbic status epilepticus, ultimately resulting in severe neurodegenerative changes. Concurrent treatment with diazepam prevents the development of status epilepticus and markedly reduces neuronal cell loss. These findings strongly support the hypothesis that the GABA_A receptor is critically involved in the pathogenesis of seizures and status epilepticus. J. Neurosci. Res. 54:863–869, 1998. © 1998 Wiley-Liss, Inc.     

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.