Werner Sieghart

GABAA receptors : Immunocytochemical distribution of 13 subunits in the adult rat brain

Abstract GABAA receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABAA receptor subunits deriving from 13 different genes (α1–α6, β1–β3, γ1–γ3 and δ) in the adult rat brain. Subunit α1-, β1-, β2-, β3- and γ2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit α2-, α3-, α4-, α5-, α6-, γ1- and δ-immunoreactivities were more confined to certain brain areas. Thus, α2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit α6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit γ1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The α5-subunit-immunoreactivity was strongest in Ammon’s horn, the olfactory bulb and hypothalamus. In contrast, α4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit α3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit γ3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit δ-immunoreactivity was frequently co-distributed with α4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit α2-, α4-, β1-, β3- and δ-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the α1-, β2-, γ1- and γ2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits α3, β1, β3 and γ2, most thalamic nuclei were rich in α1-, α4-, β2- and δ-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits α4, β2 and δ, the ventral lateral geniculate nucleus was predominantly labeled for subunits α2, α3, β1, β3 and γ2; subunit α1- and α5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits α1, α2, β1, β2 and β3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit α1, α2, β2, and γ2 antibodies was contrasted by perykarya labeled for α5-, β1- and δ-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit β2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit α1-, β2- and γ2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits β1, γ1 and γ2. In this study, a highly heterogeneous distribution of 13 different GABAA receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABAA receptor subunits support the assumption of multiple, differently assembled GABAA receptor subtypes and their heterogeneous distribution within the adult rat brain.

Segregation of Different GABAA Receptors to Synaptic and Extrasynaptic Membranes of Cerebellar Granule Cells

Two types of GABAA receptor-mediated inhibition (phasic and tonic) have been described in cerebellar granule cells, although these cells receive GABAergic input only from a single cell type, the Golgi cell. In adult rats, granule cells express six GABAAreceptor subunits abundantly (α1, α6, β2, β3, γ2, and δ), which are coassembled into at least four to six distinct GABAA receptor subtypes. We tested whether a differential distribution of GABAA receptors on the surface of granule cells could play a role in the different forms of inhibition, assuming that phasic inhibition originates from the activation of synaptic receptors, whereas tonic inhibition is provided mainly by extrasynaptic receptors. The α1, α6, β2/3, and γ2 subunits have been found by immunogold localizations to be concentrated in GABAergic Golgi synapses and also are present in the extrasynaptic membrane at a lower concentration. In contrast, immunoparticles for the δ subunit could not be detected in synaptic junctions, although they were abundantly present in the extrasynaptic dendritic and somatic membranes. Gold particles for the α6, γ2, and β2/3, but not the α1 and δ, subunits also were concentrated in some glutamatergic mossy fiber synapses, where their colocalization with AMPA-type glutamate receptors was demonstrated. The exclusive extrasynaptic presence of the δ subunit-containing receptors, together with their kinetic properties, suggests that tonic inhibition could be mediated mainly byextrasynaptic α6β2/3δ receptors, whereas phasic inhibition is attributable to the activation of synapticα1β2/3γ2, α6β2/3γ2, and α1α6β2/3γ2receptors.

Subunit Composition, Distribution and Function of GABA-A Receptor Subtypes

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. Depending on their subunit composition, these receptors exhibit distinct pharmacological and electrophysiological properties. Recent studies on recombinant and native GABA(A) receptors suggest the existence of far more receptor subtypes than previously assumed. Thus, receptors composed of one, two, three, four, or five different subunits might exist in the brain. Studies on the regional, cellular and subcellular distribution of GABA(A) receptor subunits, and on the co-localization of these subunits at the light and electron microscopic level for the first time provide information on the distribution of GABA(A) receptor subtypes in the brain. These studies will have to be complemented by electrophysiological and pharmacological studies on the respective recombinant and native receptors to finally identify the receptor subtypes present in the brain. The distinct cellular and subcellular location of individual receptor subtypes suggests that they exhibit specific functions in the brain that can be selectively modulated by subtype specific drugs. This conclusion is supported by the recent demonstration that different GABA(A) receptor subtypes mediate different effects of benzodiazepines. Together, these results should cause a revival of GABA(A) receptor research and strongly stimulate the development of drugs with a higher selectivity for alpha2-, alpha3-, or alpha5-subunit-containing receptor subtypes. Such drugs might exhibit quite selective clinical effects.

International Union of Pharmacology. LXX. Subtypes of γ-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update

In this review we attempt to summarize experimental evidence on the existence of defined native GABAA receptor subtypes and to produce a list of receptors that actually seem to exist according to current knowledge. This will serve to update the most recent classification of GABAA receptors (Pharmacol Rev 50:291–313, 1998) approved by the Nomenclature Committee of the International Union of Pharmacology. GABAA receptors are chloride channels that mediate the major form of fast inhibitory neurotransmission in the central nervous system. They are members of the Cys-loop pentameric ligand-gated ion channel (LGIC) superfamily and share struc-tural and functional homology with other members of that family. GABAA receptors are assembled from a family of 19 homologous subunit gene products and form numerous, mostly hetero-oligomeric, pentamers. Such receptor subtypes with properties that depend on subunit composition vary in topography and ontogeny, in cellular and subcellular localization, in their role in brain circuits and behaviors, in their mechanisms of regulation, and in their pharmacology. We propose several criteria, which can be applied to all the members of the LGIC superfamily, for including a receptor subtype on a list of native hetero-oligomeric subtypes. With these criteria, we develop a working GABAA receptor list, which currently includes 26 members, but will undoubtedly be modified and grow as information expands. The list is divided into three categories of native receptor subtypes: “identified,” “existence with high probability,” and “tentative.”

GABAA receptors: ligand-gated Cl− ion channels modulated by multiple drug-binding sites

GABAA receptors are ligand-gated Cl- ion channels and the site of action of a variety of pharmacologically and clinically important drugs. In this review evidence is summarized indicating that these drugs, by interacting with several distinct binding sites at these receptors, allosterically modulate GABA-induced Cl- ion flux. Other results indicate that the affinity, as well as the modulatory efficacy of drugs, changes with receptor composition. A though investigation of the pharmacological properties of the individual binding sites on different GABAA receptor subtypes could open new avenues for selective modulation of GABAA receptors in different brain regions.

GABAA receptor subunits in the rat hippocampus I: Immunocytochemical distribution of 13 subunits

The GABA(A) receptor is a ligand-operated chloride channel. It has a pentameric structure. In mammalian brain different subunits are recruited from four gene subfamilies. Using immunocytochemistry, we investigated the distribution of the 13 GABA(A) receptor subunits in the hippocampus of the rat. GABA(A) receptor subunits were heterogeneously distributed within different hippocampal subfields. High concentrations of alpha1-, alpha2-, alpha4-, beta3-, gamma2- and delta-immunoreactivities were observed within the molecular layer of the dentate gyrus, representing the dendritic area of the granule cells. In the hippocampus proper, the predominant GABA(A) receptor subunits were alpha1, alpha2, alpha5, beta3 and gamma2 that were located throughout the strata radiatum and oriens of CA1 to CA3. Immunocytochemical staining was there less prominent for alpha4-, beta1-, beta2- gamma3- and delta- subunits. In the hippocampus proper, the beta1 subunit was preferentially located in CA2. The alpha4- and delta-subunits were somewhat more abundant in CA1 than in CA3. Numerous local circuit neurons in the hippocampus proper and the hilus of the dentate gyrus contained alpha1-, beta2-, gamma2- and/or delta-subunits. Alpha3 and gamma1 were present only in minute amounts and no alpha6-IR was detected in the hippocampal formation. The distribution of the GABA(A) receptor subunits indicates the existence of heterogenously constituted GABA(A) receptor complexes within various hippocampal subfields, which may exert different physiological or pharmacological properties upon stimulation by GABA or its agonists.

Bidirectional Alterations of GABAA Receptor Subunit Peptide Levels in Rat Cortex During Chronic Ethanol Consumption and Withdrawal

Abstract: The pharmacological properties of γ‐aminobutyric acidA (GABAA) receptors are altered by prolonged exposure to ethanol both in vivo and in vitro. We have shown previously that prolonged ethanol exposure elicits selective alterations in various GABAA receptor subunit mRNA levels in rat cerebral cortex. Some of these effects are rapidly reversed during ethanol withdrawal. The present study was conducted to determine the effects of prolonged ethanol exposure (dependence) and ethanol withdrawal on cerebral cortical peptide expression for several subunits. GABAA receptor α1 subunit peptide levels were decreased by nearly 40%, whereas α4 subunit peptide levels were increased by 27% in both ethanol‐dependent and withdrawn rats. These changes correlate well with observed alterations in mRNA levels following prolonged ethanol exposure in dependent rats, but do not match the effects on mRNA levels during ethanol withdrawal. β2/3 subunit peptide levels increased by ∼32% in both ethanol‐dependent rats and rats undergoing ethanol withdrawal. We observed a 30–60% increase in γ1 subunit peptide levels in both dependent rats and those undergoing withdrawal, also correlating with the previous report on ethanol‐induced alterations in mRNA levels. Peptide levels for γ2 subunits did not differ from control values in either condition. These findings show that specific alterations in GABAA receptor subunit peptide levels are associated with ethanol dependence in rats. GABAA receptor subunit peptide expression is more stable than mRNA expression, and mRNA levels are not representative of peptide expression during ethanol withdrawal. These findings are consistent with the suggestion that alterations in GABAA receptor gene expression underlie the functional properties of GABAA receptors in ethanol‐dependent rats and those undergoing ethanol withdrawal.

GABAA receptor changes in δ subunit-deficient mice: Altered expression of α4 and γ2 subunits in the forebrain

The δ subunit is a novel subunit of the pentameric γ‐aminobutyric acid (GABA)A receptor that conveys special pharmacological and functional properties to recombinant receptors and may be particularly important in mediating tonic inhibition. Mice that lack the δ subunit have been produced by gene‐targeting technology, and these mice were studied with immunohistochemical and immunoblot methods to determine whether changes in GABAA receptors were limited to deletion of the δ subunit or whether alterations in other GABAA receptor subunits were also present in the δ subunit knockout (δ–/–) mice. Immunohistochemical studies of wild‐type mice confirmed the restricted distribution of the δ subunit in the forebrain. Regions with moderate to high levels of δ subunit expression included thalamic relay nuclei, caudate‐putamen, molecular layer of the dentate gyrus, and outer layers of the cerebral cortex. Virtually no δ subunit labeling was evident in adjacent regions, such as the thalamic reticular nucleus, hypothalamus, and globus pallidus. Comparisons of the expression of other subunits in δ–/– and wild‐type mice demonstrated substantial changes in the α4 and γ2 subunits of the GABAA receptor in the δ–/– mice. γ2 Subunit expression was increased, whereas α4 subunit expression was decreased in δ–/– mice. Importantly, alterations of both the α4 and the γ2 subunits were confined primarily to brain regions that normally expressed the δ subunit. This suggests that the additional subunit changes are directly linked to loss of the δ subunit and could reflect local changes in subunit composition and function of GABAA receptors in δ–/– mice. J. Comp. Neurol. 446:179–197, 2002.

The γ2 Subunit of the GABAA Receptor is Concentrated in Synaptic Junctions Containing the α1 and β23 Subunits in Hippocampus, Cerebellum and Globus Pallidus

Abstract The γ2 subunit is necessary for the expression of the full benzodiazepine pharmacology of GABAA receptors and is one of the major subunits in the brain. In order to determine the location of channels containing the γ2 subunit in relation to GABA-releasing terminals on the surface of neurons, a new polyclonal antipeptide antiserum was developed to the γ2 subunit and used in high resolution, postembedding, immunoelectron-microscopic procedures. Dual immunogold labelling of the same section for two subunits, and up to three sections of the same synapse reacted for different subunits, were used to characterize the subunit composition of synaptic receptors. The γ2 subunit was present in type 2, “symmetrical” synapses in each of the brain areas studied, with the exception of the granule cell layer of the cerebellum. The γ2 subunit was frequently co-localized in the same synaptic junction with the α1 and β 2 3 subunits. The immunolabelling of synapses was coincident with the junctional membrane specialization of the active zone. Immunolabelling for the receptor often occurred in multiple clusters in the synapses. In the hippocampus, the γ2 subunit was present in basket cell synapses on the somata and proximal dendrites and in axo-axonic cell synapses on the axon initial segment of pyramidal and granule cells. Some synapses on the dendrites of GABAergic interneurones were densely labelled for the γ2, α1 and β 2 3 subunits. In the cerebellum, the γ2 subunit was present in both distal and proximal Purkinje cell dendritic synapses established by stellate and basket cells, respectively. On the soma of Purkinje cells, basket cell synapses were only weakly labelled. Synapses on interneuron dendrites were more densely labelled for the γ2, α1 and β 2 3 subunits than synapses on Purkinje or granule cells. Although immunoperoxidase and immunofluorescence methods show an abundance of the γ2 subunit in granule cells, the labelling of Golgi synapses was much weaker with the immunogold method than that of the other cell types. In the globus pallidus, many type 2 synapses were labelled for the γ2 subunit together with α1 and β 2 3 subunits. The results show that γ2 subunit-containing receptor channels are highly concentrated in GABAergic synapses that also contain the α1 and β 2 3 subunits. Channels containing the γ2 subunit are expressed in synapses on functionally distinct domains of the same neuron receiving GABA from different presynaptic sources. There are quantitative differences in the density of GABAA receptors at synapses on different cell types in the same brain area. Copyright

Antibodies Specific for GABAA Receptor α Subunits Reveal that Chronic Alcohol Treatment Down-Regulates α-Subunit Expression in Rat Brain Regions

Abstract— Chronic administration of ethanol results in the development of tolerance and dependence. The molecular mechanism underlying these behavioral actions of ethanol is poorly understood. Several lines of evidence have suggested that some of the pharmacological actions of ethanol are mediated via a potentiation of GABAergic transmission. Chronic ethanol administration results in a reduction in the GABAA receptor‐mediated 36Cl− uptake in cortical synaptoneurosomes and primary cultured neurons. We and others have shown that it also results in a 40‐50% reduction in GABAA receptor α‐subunit mRNA levels in the rat cerebral cortex. In the present study, we investigated the expression of α1, α2, and α3 subunits of the GABAA receptor in the cerebral cortex and the α1 subunit in the cerebellum by immunoblotting using polyclonal antibodies raised against α1‐, α2‐, and α3‐subunit polypeptides following chronic ethanol treatment. These results reveal that chronic ethanol administration to rats results in a 61 ± 4% reduction in level of the GABAA receptor α1subunit (51 kDa), 47 ± 8% reduction in level of the α2subunit (53 kDa), and 30 ± 7% reduction in level of the α3subunit (59 kDa) in the cerebral cortex and a 56 ± 5% reduction in content of the α1 subunit in the cerebellum. In summary, this ethanol‐induced reduction in content of the GABAA receptor α subunits may underlie alterations in the GABAA receptor function and could be related to cellular adaptation to the functional disturbance caused by ethanol.