Distribution of immunoreactivity for the β2 and β3 subunits of the GABAA receptor in the mammalian spinal cord

The localization of GABA_A receptors in cat and rat spinal cord was analyzed using two monoclonal antibodies specific for an epitope shared by the β₂ and β₃ subunits of the receptor. β₂/β₃-subunit immunoreactivity was the most intense in inner lamina II, lamina III, and lamina X, and it was the least intense in lamina IX. In laminae I–III, generally, the staining had a rather diffuse appearance, but the surfaces of small cell bodies in these laminae were outlined clearly by discrete labeling, as were many cell bodies and dendrites in deeper laminae. Rhizotomy experiments and ultrastructural observations indicated that β₂/β₃-subunit immunoreactivity in the dorsal horn was largely localized in intrinsic neuropil elements rather than in the terminals of primary afferent fibers, even though labeling overlapped with the terminal fields of different types of primary afferents and was also detected on the membranes of dorsal root ganglion neurons. With few exceptions (most notably, a highly immunoreactive group of dorsolaterally located cells in the cat lumbar ventral horn), motoneurons expressed low levels of β₂/β₃-subunit immunoreactivity. Labeling of neuronal membranes was fairly continuous, but focal accumulations of β₂/β₃-subunit immunoreactivity were also detected using immunofluorescence. Focal “hot spots” correlated ultrastructurally with the presence of synaptic junctions. Dual-color immunofluorescence revealed that focal accumulations of β₂/β₃-subunit immunoreactivity were frequently apposed by glutamic acid decarboxylase (GAD)-immunoreactive terminals. However, the density of continuous-membrane β₂/β₃ immunolabeling and GAD terminal density were not correlated in many individual neurons. The results suggest the existence of “classical” (synaptic) and “nonclassical” (paracrine) actions mediated via spinal cord GABA_A receptors. The study also revealed the relative paucity of β₂/β₃-subunit immunoreactivity postsynaptic to certain GABAergic terminals, particularly those presynaptic to motoneurons or primary afferent terminals. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of the GABAA receptor in the cerebral cortex of the lizard Psammodromus algirus

This study examined the distribution and localization of the γ-aminobutyric acid (GABA)_A receptor in the brain cortex of a reptile by light and electron microscopy, to test whether cortical GABA inhibition is mainly mediated through the GABA_A receptro complex. We used preembedding immunocytochemistry and a monoclonal antibody, raised against the receptor complex, that recognizes the β2 and β3 subunits of the receptor. GABA_A receptors were distributed throughout the entire cerebral restricted to the plexiform layers of the cortex as seen by light microscopy. This granular aspect of the immunoreactivity most likely corresponds to the immunopositive dendritic and axonal profiles observed under the electron microscope. Some neurons in the medial and lateral cortices displayed pathches of immunoreactivity along the cell body and processes, and as a result their morphology was outlined. We discuss the possibility that these neurons were GABAergic as well. The immunocytochemical data demonstrate that the distribution and localization of GABA_A receptors in discrete regions of the reptilian cerebral cortex resemble that of parts of the hippocampal formation of humans and rats, suggesting that the basic configuration of the GABA system in these regions is conserved. © 1994 Wiley-Liss, Inc.     

Immunocytochemical localization of the β2 subunit of the gamma-aminobutyric acidA receptor in the rat brain

An antiserum to the β₂ subunit of the rat gamma-aminobutyric acid (GABA_A) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the β₂ subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABA_A receptors. The anti-β₂ antibodies were affinity purified on immobilized β₂ intracellular loop peptide. The antibodies recognized a 55–57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABA_A receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the β₂ subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-β₂ antibody and the monoclonal antibody 62-3G1 (which recognizes both β₂ and β₃ subunits) is presented. The procedure described for generating and preparing specific anti-β₂ subunit antibodies that are valuable for immunocytochemistry could be extended to other GABA_A receptor subunits. © 1994 Wiley-Liss, Inc.     

Immunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABAA receptor complex

The distribution of the γ-aminobutyric acid (GABA)_A receptor/benzodizepine receptor/CI⁻ channel complex in the rat brain was examined immunohistochemically using the specific antibody against purified GABA_A receptor complex. The immunization of white albino rabbit with purified GABA_A receptor complex resulted in the formation of specific antibody as indicated by the immunoprecipitation test. Immunohistochemical examinations using the antiserum on rat brain slices by the peroxidase-antiperoxidase method revealed the presence of the following immunoreactive sites which coincided with a previous report using antibody againstl-glutamic acid decar☐ylase; ventromedial nucleus of hypothalamus, red nucleus, globus pallidus, zona compacta and zona reticulata of substantia nigra, layers of Purkinje cells and granular cells of cerebellum, layers III–V of cerebral cortex and stratum radiatum of hippocampus. These results strongly suggest that immunohistochemical application of the antibody against the purified GABA_A receptor complex is a useful tool for identifying GABAergic neurons having GABA_A receptor complex-mediated synapses.     

GABAA-receptor heterogeneity in the adult rat brain: Differential regional and cellular distribution of seven major subunits

GABA_A-receptors display an extensive structural heterogeneity based on the differential assembly of a family of at least 15 subunits (α1–6, β1–3, γ1–3, θ, ρl–2) into distinct heteromeric receptor complexes. The subunit composition of receptor subtypes is expected to determine their physiological properties andipharmacological profiles, thereby contributing to flexibility in signal transduction and allosteric modulation. In heterologous expression systems, functional receptors require a combination of α-, β-, and γ-subunit variants, the γ2-subunit being essential to convey a classical benzodiazepine site to the receptor. The subunit composition and stoichiometry of native GABA_A-receptor subtypes remain unknown. The aim of this study was to identify immunohistochemically the main subunit combinations expressed in the adult rat brain and to allocate them to identified neurons. The regional and cellular distribution of seven major subunits (α1, α2, α3, α5, β2,3, γ2, δ) was visualized by immunoperoxidase staining with subunit-specific antibodies (the β2- and β3-subunits were covisualized with the monoclonal antibody bd-17). Putative receptor subtypes were identified on the basis of colocalization of subunits within individual neurons, as analyzed by confocal laser microscopy in double- and triple-immunofluoreseence staining expeximents. The results reveal an extraordinary heterogeneity in the distribution of GABA_A-receptor subunits, as evidenced by abrupt changes in immunoreactivity along well-defined cytoarchitectonic boundaries and by pronounced differences in the cellular distribution of subunits among various types of neurons. Thus, functionally and morphologically diverse neurons were characterized by a distinct GABA_A-receptor subunit repertoire. The pultiple staining experiments identified 12 subunit combinations in defined neurons. The most prevalent combination was the triplet α1/β2,3/γ2, detected in numerous cell types throughout the brain. An additional subunit (α2, α3, or δ) sometimes was associated with this triplet, pointing to the existence of receptors containing four subunits. The triplets α2/β2,3/γ2, α3/β2,3/γ2, and α5/β2,3/γ2 were also identified in discrete cell populations. The prevalence of these seven combinations suggest that they represent major GABAA-receptor subtypes. Five combinations also apparently lacked the β2,3-subunits, including one devoid of γ2-subunit (α1/α2/γ2, α2/γ2, α3/γ2, α2/α3/γ2, α2/α5/δ). These combinations were selectively associated with small neuron populations, thereby representing minor GABA_A receptor subtypes. These results provide the basis for a functional analysis of GABA_A-receptor subtypes of known subunit composition and may open the way for unproved therapeutic approaches based on the development of subtype-selective drugs. © 1995 Wiley-Liss, Inc.     

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.     

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA_A receptor binding and GABA_A receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA_A receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA_A receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA_A receptor bindings were analyzed by quantitative autoradiography using [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 μl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [³H]flunitrazepam binding and [³⁵S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of β2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of β3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of α6- and δ-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [³H]muscimol binding throughout the brain, increases regional [³H]flunitrazepam and [³⁵S]TBPS binding, and alters GABA_A receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA_A receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.     

Regional distribution of GABAA receptor binding sites in cat somatosensory and motor cortex

Inhibition in primary sensory cortex plays a role in neuronal response to periperal stimuli. For many neurons in cat primary somatosensory cortex, blockade of GABA_A receptors by bicuculline results in receptive field enlargement. The magnitude of this effect varies with the neuron's adaption characteristics and its location in particular laminae and submodality regions. To test whether these variation are correlated with the distribution of GABA_A receptors, we analyzed [³H] muscimol binding in cat primary somatosensory and motor cortical areas. The highest levels of binding were in layers IV was distinguished by highest levels were in layers V–VI. In somatosensory cortical areas, layer IV, levels of binding were significantly higher in posterior area 3b than in anterior area 3b. These difference may correspond to the rapidly adaption and slowly adapting submodality regions which have been described in this area. The laminar distribution of [³H] muscimol binding differed from that of [³H]flunitrazepam, and neither resembled the distribution of magnitude of bicuculline's effects on receptive field size. The laminar distribution of [³H] muscimol binding was highly correlated with the area density of GABA-immunoreactive neurons described in a companion study. © 1994 Wiley-Liss, Inc.     

Synaptogenesis in the rat retina: subcellular localization of glycine receptors,GABAA receptors,and the anchoring protein gephyrin

The mechanisms by which neurotransmitter receptors are clustered at postsynaptic sites of neurons are largely unknown. The 93-kDa peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic glycine receptor clusters, and there is now evidence that gephyrin can also be found at gamma-aminobutyric acid (GABA)ergic synapses. In this study, we have analyzed the synaptic localization of glycine receptors, GABA_A receptors, and the anchoring protein gephyrin in the inner plexiform layer of the developing rat retina, by using immunofluorescence with subunit specific antibodies. At early postnatal stages, the antibodies produced a diffuse staining, suggesting that early retinal neurons can express glycine and GABA_A receptors. A clustered distribution of the subunits in “hot spots” was also observed. The number of “hot spots” increased during development and reached adult levels in about 2 weeks. Electron microscopy showed that synapses of the conventional type are present in the inner plexiform layer of the postnatal retina and that the hot spots correspond to an aggregation of receptors at postsynaptic sites. Gephyrin was also localized to “hot spots,” and double immunofluorescence revealed a colocalization of gephyrin with the α2 subunit of the GABA_A receptor. These results indicate that clustering of receptor subunits occurs in parallel with the formation of morphologically identifiable synaptic specializations and suggest that gephyrin may be involved in clustering of GABA_A receptors at postsynaptic sites.J.Comp. Neurol. 381:158-174, 1997. © 1997 Wiley-Liss, Inc.     

Formation of synaptic specializations in the inner plexiform layer of the developing chick retina

The development of synapse-like specializations was investigated in the inner plexiform layer of the developing chick retina by using light and electron microscopy. Six monoclonal antibodies, directed against glycine and γ-aminobutyric acid (GABA)_A receptor subunits, the intracellular receptor-associated protein gephyrin, synaptotagmin, and synaptophysin were used to determine the initial appearance and distribution of their antigens. Synaptophysin and synaptotagmin immunoreactivity was detected in the retina concurrent with the formation of the inner plexiform layer at embryonic day 7. This early appearance before synaptic differentiation, together with the transient expression of synaptotagmin immunoreactivity in the synapse-free optic fiber layer, suggests that in the developing central nervous system (CNS) these proteins are not confined to synapses. The first immunofluorescence signal detected with specific antibodies against the β₂ and β₃-subunits of the GABA_A receptor, the glycine receptor, and gephyrin appeared at embryonic day 12. In contrast, the α₁-subunit of the adult-type glycine receptor heteromeric complex was detectable only at later stages of development, after embryonic day 16, suggesting a change in the subunit composition of some glycine receptor complexes. The staining was clearly punctate, indicating the clustering of the α₁-subunit at synapses. Electron microscopic investigation revealed the first postsynaptic densities and active zones in the inner plexiform layer of the retina at embryonic day 12. These results reveal different patterns of development for the investigated pre- and postsynaptic proteins and indicate a parallel appearance of gephyrin, glycine receptor, and the β₂ and β₃-subunits of the GABA_A receptor with the first synaptic specializations in the inner plexiform layer of the developing chick retina. © 1996 Wiley-Liss, Inc.     

Deciphering the native GABAA receptor: Is there hope?

The bewildering number of GABA_A receptor subunits, their regionally dependent expression in the brain, and their supernumerary expression in single cells present major challenges in studying the function of native GABA_A receptors. Which subunit combinations actually exist in native neurons? In this mini-review, GABA_A receptor subunit diversity is considered in light of using the wealth of “structure-function” information gained from studying recombinant receptor to predict the subunit composition and functional properties of native GABA_A receptors. © 1995 Wiley-Liss, Inc.     

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.     

Inhibitory neurotransmission in rat spinal cord: co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining

Synaptic inhibition in rat spinal cord is mediated by the amino acids γ-aminobutyric acid (GABA) and glycine. Most spinal cord neurons respond to both neurotransmitters, suggesting co-expression of GABA_A- and strychnine-sensitive glycine-receptors in individual cells. While the distribution of glycine-receptors has been extensively characterized, much less is known about the cellular localization of GABA_A-receptors in spinal cord neurons. In the present study, the distribution of GABA_A-receptors was analyzed immunohistochemically with a subunit-specific antiserum recognizing the α1-subunit. Their co-localization with glycine-receptors and their apposition to GABAergic axon terminals were assessed by confocal laser microscopy in sections processed for double- and triple-immunofluorescence staining, using a monoclonal antibody against the 93 kDa glycine-receptor-associated protein, gephyrin, and an antiserum to glutamic acid decar☐ylase. Staining for the GABA_A-receptor α1-subunit decorated the soma and dendrites of numerous neurons in laminae III–VIII and X of the spinal cord, revealing their morphology in clear detail. By contrast, laminae II and IX contained little immunoreactivity for these GABA_A-receptors. Double-immunofluorescence staining showed that most GABA_A-receptor-positive cells in layers III–VIII and X also exhibited a prominent glycine-receptor immunoreactivity. Both types of receptors had very similar distribution patterns in the cell membrane and were frequently co-localized in sites apposed to GABAergic axon terminals. These results indicate that GABA_A- and glycine-receptors may co-exist within single postsynaptic densities, suggesting a possible synergism in the action of GABA and glycine in spinal cord neurons.     

Localization of GABAB receptors in midbrain monoamine containing neurons in the rat

The localization of γ-aminobutyric acid (GABA)_B receptors in the midbrain of the rat was examined in multiple labeling studies using antibodies directed against the GABA_B receptor and either tryptophan hydroxylase or tyrosine hydroxylase. Almost all of the serotonergic and dopaminergic cell bodies in the midbrain displayed GABA_B receptor-like immunoreacrtivity. Conversely, most neurons in the raphe nuclei and ventral tegmentum which exhibited intense immunoreactivity for GABA_B receptors were also immunopositive for serotonergic or dopaminergic markers. These results demonstrate directly that GABA_B receptors are present in monoaminergic neurons in certain regions of the midbrain.     

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta

GABA_A receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABA_A receptors in the mammalian brain consist of two α-, two β- and one γ- or δ-subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABA_A receptors. Using immunohistochemistry, we investigated the distribution of 10 GABA_A receptor subunits (α1, α2, α3, α4, α5, β1, β2, β3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, β2, β3, and γ2 were found in all layers, α2, α3, and β1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three β-subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, β1, β2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, β2, β3, and γ2, whereas α1, α3, β1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, β1, β3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, β1, β3, and δ, and in the central amygdala α1, α2, β3, and γ2 were most abundant. Interestingly, subunit α3-IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, β2, β3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, β1, β2, β3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABA_A receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.     

GABAA Receptor subunits have differential distributions in the rat retinae: In situ hybridization and immunohistochemistry

The distributions of nine different subunits of the gamma-aminobutyric acid_A (GABA_A) receptor (α₁, α₂, α₃, ₅; β₁, β₂, β₃; γ₂; δ) were investigated in the rat retina using immunocytochemistry and in situ hybridization. With the exception of the α₅ subunit, all subunits could be localized. Each subunit was expressed in characteristic strata within the inner plexiform layer (IPL). Some subunits (e.g., γ₂) showed a ubiquitous distribution, while others (e.g., δ) were restricted to narrow sublayers. Double labeling experiments using different combinations of the subunit-specific antibodies revealed colocalizations of subunits within individual neurons. Additionally, GABA_A receptor subunits were mapped to distinct populations of retinal neurons by coapplication of defined immunocytochemical markers and subunit-specific antibodies. Cholinergic amacrine cells were found to express the α₂, β1, β_2/3 and δ subunits, while dopaminergic amacrine cells express the α₂, α₃ and γ₂ subunits. Dissociated rod bipolar cells express the γ₂ subunits. In summary, this study provides evidence for the existence of multiple GABA_A receptor subtypes in the retina. The distinct stratification pattern of the subunits in the IPL suggests that different functional circuits involve specific subtypes of GABAA receptors. © 1995 Wiley-Liss, Inc.     

Neuronal activity influences the growth of barrels in developing rat primary somatosensory cortex without affecting the expression pattern of four major GABAA receptor α subunits

Thalamic innervation plays a major role in parcellation of neocortex and maturation of cortical circuits. While the underlying mechanisms are unknown, lesion studies have identified GABA_A receptors in neocortex as molecular targets of thalamic regulation [J. Paysan, A. Kossel, J. Bolz, J.M. Fritschy, Area-specific regulation of γ-aminobutyric acid A receptor subtypes by thalamic afferents in developing rat neocortex, Proc. Natl. Acad. Sci. USA 94 (1997) 6995–7000]. To determine the factors regulating the expression of GABA_A receptors, the overall level of neuronal activity was chronically modulated in neonatal rat cortex. Slices of Elvax polymer loaded with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 or with brain derived neurotrophic factor (BDNF) were placed unilaterally over the left parietal cortex in newborn animals. Unlike thalamic lesions (Paysan et al., 1997), these chronic drug treatments did not alter the laminar distribution or the expression level of the four major GABA_A receptor α subunit isoforms (α1, α2, α3, α5) in primary somatosensory cortex (S1), as assessed immunohistochemically after one week. In particular, the staining of the barrel field in layers III–IV, which is very prominent with the α1-subunit, was preserved in the drug-treated hemisphere. Even systemic administration of MK-801 at birth, which resulted in pronounced retardation of cortical development, had no effect on the laminar distribution and staining intensity of the four GABA_A receptor α subunit variants. However, the size of barrels in S1, as measured in tangential sections stained for the GABA_A receptor α1 subunit, was enlarged upon chronic, topical blockade of NMDA receptors with MK-801 and was reduced to the same extent upon chronic exposure to BDNF. Thus, these pharmacological treatments modulated cortical growth, possibly by exerting opposite effects on neuronal activity in S1. The results suggest that the parcellation of somatosensory cortex and the laminar distribution of GABA_A receptor subtypes are governed primarily by factors independent of thalamocortical activity.     

GABAA and GABAB receptor subunit localization on neurochemically identified neurons of the human subthalamic nucleus

The subthalamic nucleus (STN) is a critical excitatory signaling center within the basal ganglia circuitry. The activity of subthalamic neurons is tightly controlled by upstream inhibitory signaling centers in the basal ganglia. In this study, we used immunohistochemical techniques to firstly, visualize and quantify the STN neurochemical organization based on neuronal markers including parvalbumin (PV), calretinin (CR), SMI‐32, and GAD_65/67. Secondly, we characterized the detailed regional, cellular and subcellular expression of GABA_A (α₁, α₂, α₃, β_2/3, and γ₂) and GABA_B (R1 and R2) receptor subunits within the normal human STN. Overall, we found seven neurochemically distinct populations of principal neurons in the human STN. The three main populations detected were: (a) triple‐labeled PV⁺/CR⁺/SMI32⁺; (b) double‐labeled PV⁺/CR⁺; and (c) single‐labeled CR⁺ neurons. Subthalamic principal neurons were found to express GABA_A receptor subunits α₁, α₃, β_2/3, γ₂, and GABA_B receptor subunits R1 and R2. However, no expression of GABA_A receptor α₂ subunit was detected. We also found a trend of increasing regional staining intensity for all positive GABA_A receptor subunits from the dorsolateral pole to ventromedial extremities. The GAD⁺ interneurons showed relatively low expression of GABA_A receptor subunits. These results provide the morphological basis of GABAergic transmission within the normal human subthalamic nucleus and evidence of GABA innervation through both GABA_A and GABA_B receptors on subthalamic principal neurons.     

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.