GABA(B) receptors: synaptic functions and mechanisms of diversity

GABA(B) receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the mammalian central nervous system. They are implicated in a variety of neurological and psychiatric disorders. With the cloning of GABA(B) receptors ten years ago, substantial progress was made in our understanding of this receptor system. Here, we review current concepts of synaptic GABA(B) functions and present the evidence that points to specific roles for receptor subtypes. We discuss ultrastructural studies revealing that most GABA(B) receptors are located remote from GABAergic terminals, which raises questions as to when such receptors become activated. Finally, we provide possible explanations for the perplexing situation that GABA(B) receptor subtypes that have indistinguishable properties in vitro generate distinct GABA(B) responses in vivo.     

γ-Aminobutyric Acid Type A (GABAA) Receptor α Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting

GABA(A) receptors (GABA(A)-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of γ2 and δ subunits in synaptic versus extrasynaptic targeting of GABA(A)-Rs. Here, we demonstrate differential function of α2 and α6 subunits in guiding the localization of GABA(A)-Rs. To study the targeting of specific subtypes of GABA(A)-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA(A)-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by α2β3γ2 receptors were very fast (rise time ～2 ms), whereas events mediated by α6β3δ receptors were very slow (rise time ～20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by α6β3 or α6β3γ2 receptors were significantly slower than those mediated by α2β3 or α2β3γ2 receptors, suggesting a differential role of α subunit in receptor targeting. This was confirmed by differential targeting of the same δ-γ2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the α2 or α6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of α6 and δ subunits brought α6β3δ receptors closer to synaptic sites. Therefore, the α subunits, together with the γ2 and δ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA(A)-Rs, possibly through differential interactions with gephyrin.     

Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene

Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.     

Subunit composition and quantitative importance of GABA(A) receptor subtypes in the cerebellum of mouse and rat

In cerebellum, 13 different GABA(A) receptor subunits are expressed. The number of different receptor subtypes formed in this tissue, their subunit composition and their quantitative importance so far has not been determined. In the present study, immunodepletion by immunoaffinity chromatography, as well as immunoprecipitation and western blot analysis was performed using 13 different subunit-specific antibodies to provide an overview on the subunit composition and abundance of GABA(A) receptor subtypes in mouse and rat cerebellum. Results obtained indicate that alpha1betaxgamma2, alpha1alpha6betaxgamma2, alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta are the major GABA(A) receptor subtypes present in the cerebellum. In addition, small amounts of alpha1betaxdelta receptors and a series of minor receptor subtypes containing alpha2, alpha3, alpha4, alpha5, gamma1 or gamma3 subunits are also present in the cerebellum. Whereas the abundance of alpha1alpha6betaxgamma2, alpha6betaxdelta and alpha1alpha6betaxdelta receptors is different in mouse and rat cerebellum, that of other receptors is quite similar in these tissues. Data obtained for the first time provide an overview on the GABA(A) receptor subtypes present in the cerebellum and represent the basis for further studies investigating changes in receptor expression and composition under pathological conditions.     

GABA(A) receptors containing the α2 subunit are critical for direction-selective inhibition in the retina

Far from being a simple sensor, the retina actively participates in processing visual signals. One of the best understood aspects of this processing is the detection of motion direction. Direction-selective (DS) retinal circuits include several subtypes of ganglion cells (GCs) and inhibitory interneurons, such as starburst amacrine cells (SACs). Recent studies demonstrated a surprising complexity in the arrangement of synapses in the DS circuit, i.e. between SACs and DS ganglion cells. Thus, to fully understand retinal DS mechanisms, detailed knowledge of all synaptic elements involved, particularly the nature and localization of neurotransmitter receptors, is needed. Since inhibition from SACs onto DSGCs is crucial for generating retinal direction selectivity, we investigate here the nature of the GABA receptors mediating this interaction. We found that in the inner plexiform layer (IPL) of mouse and rabbit retina, GABA(A) receptor subunit α2 (GABA(A)R α2) aggregated in synaptic clusters along two bands overlapping the dendritic plexuses of both ON and OFF SACs. On distal dendrites of individually labeled SACs in rabbit, GABA(A)R α2 was aligned with the majority of varicosities, the cell's output structures, and found postsynaptically on DSGC dendrites, both in the ON and OFF portion of the IPL. In GABA(A)R α2 knock-out (KO) mice, light responses of retinal GCs recorded with two-photon calcium imaging revealed a significant impairment of DS responses compared to their wild-type littermates. We observed a dramatic drop in the proportion of cells exhibiting DS phenotype in both the ON and ON-OFF populations, which strongly supports our anatomical findings that α2-containing GABA(A)Rs are critical for mediating retinal DS inhibition. Our study reveals for the first time, to the best of our knowledge, the precise functional localization of a specific receptor subunit in the retinal DS circuit.     

GABAA receptor subtypes: from pharmacology to molecular biology.

GABAA receptors are GABA (gamma-aminobutyric acid)-gated chloride channels, which are major mediators of neuronal inhibition in the brain and are modulated by benzodiazepines, barbiturates, alcohol, and other important centrally acting drugs. Although previous pharmacological and biochemical data had suggested a degree of heterogeneity, recent cloning of at least 15 different receptor subunits, thought to be combined in groups of five, indicates that the brain may contain a truly astonishing variety of GABAA receptor subtypes. This review describes the little that is known about these subtypes, emphasizing possible molecular bases of receptor heterogeneity. We also discuss approaches to establishing the subunit composition of subtypes.     

GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition

GABA (gamma-aminobutyric acid) type A receptors (GABA(A)Rs) mediate most fast synaptic inhibition in the mammalian brain, controlling activity at both the network and the cellular levels. The diverse functions of GABA in the CNS are matched not just by the heterogeneity of GABA(A)Rs, but also by the complex trafficking mechanisms and protein-protein interactions that generate and maintain an appropriate receptor cell-surface localization. In this Review, we discuss recent progress in our understanding of the dynamic regulation of GABA(A)R composition, trafficking to and from the neuronal surface, and lateral movement of receptors between synaptic and extrasynaptic locations. Finally, we highlight a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABA(A)R trafficking occur.     

N-Acetylaspartylglutamate: the most abundant peptide neurotransmitter in the mammalian central nervous system

In the progress of science, as in life, timing is important. The acidic dipeptide, N-acetylaspartylglutamate (NAAG), was discovered in the mammalian nervous system in 1965, but initially was not considered to be a neurotransmitter candidate. In the mid-1980s, a few laboratories revisited the question of NAAG's role in the nervous system and pursued hypotheses regarding its function that ranged from a precursor for the transmitter pool of glutamate to a direct role as a peptide transmitter. Since that time, NAAG has been tested against nearly all of the established criteria for identification of a neurotransmitter. It successfully meets each of these tests, including a concentrated presence in neurons and synaptic vesicles, release from axon endings in a calcium-dependent manner following initiation of action potentials, and extracellular hydrolysis by membrane-bound peptidase activity. NAAG is the most prevalent and widely distributed neuropeptide in the mammalian nervous system. NAAG activates NMDA receptors with a low potency that may vary among receptor subtypes, and it is a highly selective agonist at the type 3 metabotropic glutamate receptor (mGluR3). Acting through this receptor, NAAG reduces cyclic AMP levels, decreases voltage-dependent calcium conductance, suppresses excitotoxicity, influences long-term potentiation and depression, regulates GABA(A) receptor subunit expression, and inhibits synaptic release of GABA from cortical neurons. Cloning of peptidase activities against NAAG provides opportunities to study the cellular and molecular mechanisms by which synaptic NAAG peptidase activity is controlled. Given the codistribution of this peptide with a spectrum of traditional transmitters and its ability to activate mGluR3, we speculate that one role for NAAG following synaptic release is the activation of metabotropic autoreceptors that inhibit subsequent transmitter release. A second role is the production of extracellular glutamate following NAAG hydrolysis.     

Cerebral GABAB receptor: Proposed mechanisms of action and purification procedures

The GABA_B receptor in brain is one of the GABA receptor subtypes, and has been found to be negatively coupled to adenylate cyclase and phosphatidylinositide turnover. This receptor easily solubilizes from cerebral synaptic membrane preparations by 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. GABA_B receptor solubilized from bovine cerebral cortex was purified using baclofen-coupled affinity beads (baclofen-coupled Toyopearl beads). Using these procedures, almost pure GABA_B receptor (80 KDa protein) was obtained in the affinity eluate. A monoclonal antibody has been also raised against the purified GABA_B receptor. The antibody recognized a protein of about 80 KDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove more than 90% of the baclofen suppressive GABA binding activity in the solubilized synaptic membrane, and this system was found to be useful for the immunoaffinity column chromatographic separation of GABA_B receptor. Preliminary studies of immunohistochemical visualization of GABA_B receptor in the rat cerebellum suggested that this receptor may be exclusively localized at the presynaptic site of GABAergic neurons.Special issue dedicated to Dr. Claude Baxter.     

Control of synaptic strength,a novel function of Akt

Akt (also known as PKB), a serine/threonine kinase involved in diverse signal-transduction pathways, is highly expressed in the brain. Akt is known to have a strong antiapoptotic action and thereby to be critically involved in neuronal survival, but its potential role in the dynamic modulation of synaptic transmission is unknown. Here we report that Akt phosphorylates, both in vitro and in vivo, the type A gamma-aminobutyric acid receptor (GABA(A)R), the principal receptor mediating fast inhibitory synaptic transmission in the mammalian brain. Akt-mediated phosphorylation increases the number of GABA(A)Rs on the plasma membrane surface, thereby increasing the receptor-mediated synaptic transmission in neurons. These results identify the GABA(A)R as a novel substrate of Akt, thereby linking Akt to the regulation of synaptic strength. This work also provides evidence for the rapid regulation of neurotransmitter receptor numbers in the postsynaptic domain by direct receptor phosphorylation as an important means of producing synaptic plasticity.     

Suppression of inhibitory synaptic potentiation by presynaptic activity through postsynaptic GABA(B) receptors in a Purkinje neuron

At inhibitory synapses on a cerebellar Purkinje neuron, the depolarization caused by heterosynaptic climbing fiber activation induces long-lasting potentiation accompanied by an increase in GABA(A) receptor responsiveness. Here we show that activation of a presynaptic inhibitory interneuron during the conditioning postsynaptic depolarization suppresses the potentiation. The suppression is due to postsynaptic GABA(B) receptor activation by GABA released from presynaptic terminals. The results suggest that GABA(B) receptor activation decreases the activity of cAMP-dependent protein kinase through the G(i)/G(o) proteins. The presynaptic activity-dependent suppression of synaptic plasticity is a novel regulatory mechanism of synaptic efficacy at individual synapses and may contribute to the learning and computational ability of the cerebellar cortex.     

Modulation of GABAA receptor activity by phosphorylation and receptor trafficking: implications for the efficacy of synaptic inhibition

Fast synaptic inhibition in the brain is largely mediated by GABA(A) receptors. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. It has become clear that the stability and activity of GABA(A) receptors at synapses can be dynamically modulated by receptor trafficking and phosphorylation. Here, we discuss these regulatory mechanisms, and their consequences for the efficacy of GABA(A) receptor mediated synaptic inhibition.     

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.     

Phosphorylation and chronic agonist treatment atypically modulate GABAB receptor cell surface stability

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. The dynamic control of the cell surface stability of GABA(B) receptors is likely to be of fundamental importance in the modulation of receptor signaling. Presently, however, this process is poorly understood. Here we demonstrate that GABA(B) receptors are remarkably stable at the plasma membrane showing little basal endocytosis in cultured cortical and hippocampal neurons. In addition, we show that exposure to baclofen, a well characterized GABA(B) receptor agonist, fails to enhance GABA(B) receptor endocytosis. Lack of receptor internalization in neurons correlates with an absence of agonist-induced phosphorylation and lack of arrestin recruitment in heterologous systems. We also demonstrate that chronic exposure to baclofen selectively promotes endocytosis-independent GABA(B) receptor degradation. The effect of baclofen can be attenuated by activation of cAMP-dependent protein kinase or co-stimulation of beta-adrenergic receptors. Furthermore, we show that increased degradation rates are correlated with reduced receptor phosphorylation at serine 892 in GABA(B)R2. Our results support a model in which GABA(B)R2 phosphorylation specifically stabilizes surface GABA(B) receptors in neurons. We propose that signaling pathways that regulate cAMP levels in neurons may have profound effects on the tonic synaptic inhibition by modulating the availability of GABA(B) receptors.     

Structure and subunit composition of GABA(A) receptors.

GABA(A) receptors are the major inhibitory neurotransmitter receptors in the brain and are the site of action of many clinically important drugs. These receptors are composed of five subunits that can belong to eight different subunit classes. If all GABA(A) receptor subunits could randomly combine with each other, an extremely large number of GABA(A) receptor subtypes with distinct subunit composition and arrangement would be formed. Depending on their subunit composition, these receptors would exhibit distinct pharmacological and electrophysiological properties. Recent evidence, however, indicates that not all subunits can assemble efficiently with each other and form functional homo- or hetero-oligomeric receptors. In addition, the efficiency of formation of hetero-oligomeric assembly intermediates determines the subunit stoichiometry and subunit arrangement for each receptor and thus further reduces the possible heterogeneity of GABA(A) receptors in the brain. Studies investigating the subunit composition of native GABA(A) receptors support this conclusion, but also indicate that receptors composed of one, two, three, four, or five different subunits might exist in the brain. Using a recently established immunodepletion technique, the subunit composition and quantitative importance of native GABA(A) receptor subtypes can be determined. This information, together with studies on the regional, cellular and subcellular distribution of these receptor subtypes, will be the basis for a rational development of drugs that specifically affect the GABAergic system.     

Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex

A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.     

Activation of picrotoxin-resistant GABA receptors by GABA and related compounds induces modulation of cockroach dorsal paired median (DPM) neuron firing

Activation of gamma-aminobutyric acid (GABA) receptors in insect dorsal paired median (DPM) neurons induced two types of response which appeared to be mediated by two different GABA receptor subtypes. When activated by bath application of GABA, one receptor subtype, insensitive to picrotoxin (PTX), mediated a drastic reduction in the firing frequency, leading to a blockade of the spontaneous electrical activity. These effects were accompanied by decreases in the amplitude and duration of the plateau action potential (AP) and the spike after-hyperpolarization (AHP). In most cases, a slight depolarization of the resting membrane potential occurred. Bath application of the vertebrate GABA(B) receptor agonists 3-aminopropyl(methyl)phosphinic acid (SKF 97541) and 3-aminopropylphosphinic acid (CGA 147823/CGP 27492) induced similar responses. Another GABA receptor subtype, less sensitive to GABA, mediated a chloride dependent hyperpolarization that was suppressed by bath application of PTX. The approximate locations of these two GABA receptor subtypes were determined by local pressure microapplications of GABA and vertebrate GABAergic agonists. The PTX-sensitive receptors were located predominantly on the surface of the ganglion where the apical pole of the soma is situated, while the PTX-resistant receptors appeared to be located deeper within the ganglion.These results reveal the existence of two GABA receptor subtypes on the DPM neurons and provide evidence for a functional role for PTX-resistant GABA receptors in the regulation of spontaneous firing.     

GABAergic modulation of primary gustatory afferent synaptic efficacy

Modulation of synaptic transmission at the primary sensory afferent synapse is well documented for the somatosensory and olfactory systems. The present study was undertaken to test whether GABA impacts on transmission of gustatory information at the primary afferent synapse. In goldfish, the vagal gustatory input terminates in a laminated structure, the vagal lobes, whose sensory layers are homologous to the mammalian nucleus of the solitary tract. We relied on immunoreactivity for the GABA-transporter, GAT-1, to determine the distribution of GABAergic synapses in the vagal lobe. Immunocytochemistry showed dense, punctate GAT-1 immunoreactivity coincident with the layers of termination of primary afferent fibers. The laminar nature and polarized dendritic structure of the vagal lobe make it amenable to an in vitro slice preparation to study early synaptic events in the transmission of gustatory input. Electrical stimulation of the gustatory nerves in vitro produces synaptic field potentials (fEPSPs) predominantly mediated by ionotropic glutamate receptors. Bath application of either the GABA(A) receptor agonist muscimol or the GABA(B) receptor agonist baclofen caused a nearly complete suppression of the primary fEPSP. Coapplication of the appropriate GABA(A) or GABA(B) receptor antagonist bicuculline or CGP-55845 significantly reversed the effects of the agonists. These data indicate that GABAergic terminals situated in proximity to primary gustatory afferent terminals can modulate primary afferent input via both GABA(A) and GABA(B) receptors. The mechanism of action of GABA(B) receptors suggests a presynaptic locus of action for that receptor.     

Neurosteroid modulation of GABAA receptors: molecular determinants and significance in health and disease

Over the past 20 years it has become apparent that certain steroids, synthesised de novo in the brain, hence named neurosteroids, produce immediate changes (within seconds) in neuronal excitability, a time scale that precludes a genomic locus of action. Identified molecular targets underlying modulation of brain excitability include both the inhibitory GABA(A) and the excitatory NMDA receptor. Of particular interest is the interaction of certain neurosteroids with the GABA(A) receptor, the major inhibitory receptor in mammalian brain. During the last decade, compelling evidence has accrued to reveal that locally produced neurosteroids may selectively "fine tune" neuronal inhibition. A range of molecular mechanisms including the subunit composition of the receptor(s), phosphorylation and local steroid metabolism, underpin the region- and neuronal selectivity of action of neurosteroids at synaptic and extrasynaptic GABA(A) receptors. The relative contribution played by each of these mechanisms in a variety of physiological and pathophysiological scenarios is currently being scrutinised at a cellular and molecular level. However, it is not known how such mechanisms may act in concert to influence behavioural profiles in health and disease. An important question concerns the identification of the anatomical substrates mediating the repertoire of behaviours produced by neurosteroids. "Knock-in" mice expressing mutant GABA(A) subunits engineered to be insensitive to benzodiazepines or general anaesthetics have proved invaluable in evaluating the role of GABA(A) receptor subtypes in complex behaviours such as sedation, cognition and anxiety [Rudolph, U., Mohler, H., 2006. GABA-based therapeutic approaches: GABA(A) receptor subtype functions. Curr. Opin. Pharmacol. 6, 18-23]. However, the development of a similar approach for neurosteroids has been hampered by the limited knowledge that, until recently, has surrounded the identity of the amino acid residues contributing to the neurosteroid binding pocket. Here, we will review recent progress in identifying the neurosteroid binding site on the GABA(A) receptor, and discuss how these discoveries will impact on our understanding of the role of neurosteroids in health and disease.