Synaptic clustering of GABAC receptor ρ‐subunits in the rat retina

Polyclonal antibodies which recognize the ρ-subunits of the GABA_C receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABA_C receptors at synaptic sites. During postnatal development diffuse ρ-immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the ρ-immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the ρ-antiserum were performed. The experiments showed that ρ-immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABA_C receptor ρ-subunits coassemble with GABA_A receptor subunits, double-labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABA_C receptor expressing puncta did not coincide with GABA_A receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABA_A and GABA_C receptor subunits are coassembled. Similar double-labelling experiments were also performed to find out whether GABA_C receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABA_C receptors consist of ρ-subunits and are not coassembled with GABA_A- or glycine-receptor subunits.     

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.     

Immunocytochemical localization of the GABAC receptor ρ subunits in the cat,goldfish, and chicken retina

Polyclonal antibodies against the N-terminus of the rat ρ 1 subunit were used to study the distribution of γ-aminobutyric acid C (GABA_C) receptors in the cat, goldfish, and chicken retina. Strong punctate immunoreactivity was present in the inner plexiform layer (IPL) of all three species. The punctate labelling suggests a clustering of the GABA_C receptors at synaptic sites. Weak label was also found in the outer plexiform layer (OPL) and over the cell bodies of bipolar cells. Double immunostaining of vertical sections with an antibody against protein kinase C (PKC) showed the punctate immunofluorescence to colocalize with bipolar cell axon terminals. In the goldfish retina, the axon terminals of Mb1 bipolar cells were enclosed by ρ-immunoreactive puncta. In the chicken retina, several distinct strata within the IPL showed a high density of ρ-immunoreactive puncta. The results suggest a high degree of sequence homology between the ρ subunits of different vertebrate species, and they show that the retinal localization of GABA_C receptors is similar across different species. J. Comp. Neurol. 380:520–532, 1997. © 1997 Wiley-Liss, Inc.     

Synapse-specific localization of NMDA and GABAA receptor subunits revealed by antigen-retrieval immunohistochemistry

Conventional immunohistochemistry provides little evidence for the synaptic localization of ionotropic neurotransmitter receptors, suggesting that their epitopes are not readily accessible in situ. Here, we have adapted antigen retrieval procedures based on microwave irradiation to enhance the immunohistochemical staining of γ-aminobutyric acid type A (GABA_A) and N-methyl-D-aspartate (NMDA) receptor subunits in rat brain tissue. Microwave irradiation of fixed tissue produced a marked reduction of nonspecific staining, allowing an improved detection of GABA_A receptor subunits. However, staining of NMDA receptor subunits remained suboptimal. In contrast, microwave irradiation of cryostat sections prepared from fresh tissue resulted in a major enhancement of both NMDA and GABA_A receptor subunit staining. The diffuse, partially intracellular signals were largely replaced by numerous, intensely immunoreactive puncta outlining neuronal somata and dendrites, highly suggestive of synaptic receptors. In hippocampus CA1–CA3 fields, the NR2A and NR2B subunit positive puncta exhibited an extensive colocalization in the stratum oriens and radiatum, whereas pyramidal cell bodies, which receive no excitatory synapses, were unstained. In addition, the NR2A subunit, but not the NR2B subunit, was selectively detected on pyramidal cell dendrites in the stratum lucidum of CA3, suggesting a selective targeting to sites of mossy fiber input. For the GABA_A receptor subunits, the most striking change induced by this protocol was the selective staining of the axon initial segment of cortical and hippocampal pyramidal cells. The α2 subunit immunoreactivity was particularly prominent in these synapses. In control experiments, the staining of cytoskeletal proteins (neurofilaments, glial fibrillary acid protein) was not influenced by prior microwave irradiation. The enhancement of cell-surface–associated staining is therefore strongly suggestive of an ‘unmasking’ of subunit epitopes by the microwave treatment. These results reveal a remarkable specificity in the synaptic targeting of NMDA and GABA_A receptor subunits in hippocampal and neocortical neurons, suggesting that individual neurons can express multiple receptor subtypes in functionally distinct synapses. J. Comp. Neurol. 390:194–210, 1998. © 1998 Wiley-Liss, Inc.     

Differential distribution of GABAA receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial γ-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA_A receptor α1 and α3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam^TM soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the α1 subunit, or to serotonin and the α3 subunit of the GABA_A receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the α1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the α3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No α1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the α3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the α3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA_A receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA_A receptor antagonists in the VMM. J. Comp. Neurol. 384:337–348, 1997. © 1997 Wiley-Liss, Inc.     

Distinct properties of murine α5 γ‐aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

γ‐Aminobutyric acid type A receptors (GABA_ARs) that contain the α5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABA_ARs containing the α5 subunit (α5 GABA_ARs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of α5 GABA_ARs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate α5 GABA_ARs preferentially from the mouse hippocampus and to identify the α5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the α5 subunits was distinct from that of α1 and α2 subunits, and the relative distribution of the α5 subunits in Triton X‐100‐soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the α5 subunit and showed that this subunit associates with multiple α, β, and γ subunits, but most frequently the β3 subunit. Thus, the α5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of α5 GABA_ARs in hippocampal neurons. © 2009 Wiley‐Liss, Inc.     

Diversity of glycine receptors in the mouse retina: localization of the alpha4 subunit

Glycine and gamma-aminobutyric acid (GABA) are the major inhibitory neurotransmitters in the retina. Approximately half of the amacrine cells release glycine at their synapses with bipolar, other amacrine, and ganglion cells. Whereas the retinal distributions of glycine receptor (GlyR) subunits alpha1, alpha2, and alpha3 have been mapped, the role of the alpha4 subunit in retinal circuitry remains unclear. A rabbit polyclonal antiserum was raised against a peptide that comprises the C-terminal 14 amino acids of the mouse GlyR alpha4 subunit. Using immunocytochemistry, we localized the alpha4 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by double-labeling sections for GlyR alpha4 and synaptic markers (bassoon, gephyrin). Double-labeling sections for GlyR alpha4 and the other GlyR alpha subunits shows that they are mostly clustered at different synapses; however, approximately 30% of the alpha4-containing synapses also express the alpha2 subunit. We also studied the pre- and postsynaptic partners at GlyR alpha4-containing synapses and found that displaced (ON-) cholinergic amacrine cells prominently expressed the alpha4 subunit. The density of GlyR alpha4-expressing synapses in wildtype, Glra1(ot/ot), and Glra3(-/-) mouse retinas did not differ significantly. Thus, there is no apparent compensation of the loss of alpha1 or alpha3 subunits by an upregulation of alpha4 subunit gene expression; however, the alpha2 subunit is moderately upregulated.     

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.     

Localization of the clustering protein gephyrin at GABAergic synapses in the main olfactory bulb of the rat

The tubulin-binding protein gephyrin is essential for the formation of postsynaptic glycine-receptor clusters in cultured spinal neurons. In addition, there is increasing evidence that gephyrin can also be present at nonglycinergic synapses. Here we analyzed immunocytochemically the subcellular localization of gephyrin in the main olfactory bulb of the rat and compared its distribution with that of γ-aminobutyric acid (GABA) and of two major GABA_A-receptor subunits. Gephyrin was selectively localized to the postsynaptic side of symmetric synaptic junctions, where the presynaptic terminals contained GABA. Moreover, gephyrin colocalized extensively with the α1 and γ2 subunits of the GABA_A receptor. In contrast, gephyrin was not detected at presumed glutamatergic synapses. These results indicate that gephyrin is not uniquely associated with glycine receptors, but can also be found at distinct GABAergic synapses. Thus, they raise the possibility that gephyrin is involved in anchoring certain GABA_A-receptor subtypes in the postsynaptic membrane. J. Comp. Neurol. 395:231–244, 1998. © 1998 Wiley-Liss, Inc.     

GABAA and GABAB receptor dysregulation in superior frontal cortex of subjects with schizophrenia and bipolar disorder

Schizophrenia and bipolar disorder are complex psychiatric disorders that affect millions of people worldwide. Evidence from gene association and postmortem studies has identified abnormalities of the gamma‐aminobutyric acid (GABA) signaling system in both disorders. Abnormal GABAergic signaling and transmission could contribute to the symptomatology of these disorders, potentially through impaired gamma oscillations which normally occur during cognitive processing. In the current study, we examined the protein expression of 14 GABA_A and two GABA_B receptor subunits in the superior frontal cortex of subjects with schizophrenia, bipolar disorder, and healthy controls. Analyses of Variance (ANOVAs) identified significant group effects for protein levels for the α1, α6, β1, β3, δ, ?, and π GABA_A receptor subunits and R1 and R2 GABA_B receptor subunits. Follow‐up t tests confirmed changes for these subunits in subjects with schizophrenia, subjects with bipolar disorder, or both groups. Alterations in stoichiometry of GABA receptor subunits could result in altered ligand binding, transmission, and pharmacology of GABA receptors in superior frontal cortex. Thus, impaired GABAergic transmission may negatively contribute to symptoms such as anxiety or panic as well as impaired learning and information processing, all of which are disrupted in schizophrenia and bipolar disorder. Taken together, these results provide additional evidence of GABAergic receptor abnormalities in these disorders.     

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.     

Diversity of glycine receptors in the mouse retina: localization of the alpha2 subunit

Gamma-aminobutyric acid (GABA) and glycine are the major inhibitory neurotransmitters in the retina, glycine being produced in approximately half of all amacrine cells. Whereas retinal cell types expressing the glycine receptor (GlyR) alpha1 and alpha3 subunits have been mapped, the role of the alpha2 subunit in retinal circuitry remains unclear. By using immunocytochemistry, we localized the alpha2 subunit in the inner plexiform layer (IPL) in brightly fluorescent puncta, which represent postsynaptically clustered GlyRs. This was shown by doubly labeling sections for GlyR alpha2 and bassoon (a presynaptic marker) or gephyrin (a postsynaptic marker). Synapses containing GlyR alpha2 were rarely found on ganglion cell dendrites but were observed on bipolar cell axon terminals and on amacrine cell processes. Recently, an amacrine cell type has been described that is immunopositive for glycine and for the vesicular glutamate transporter vGluT3. The processes of this cell type were presynaptic to GlyR alpha2 puncta, suggesting that vGluT3 amacrine cells release glycine. Double labeling of sections for GlyR alpha1 and GlyR alpha2 subunits showed that they are clustered at different synapses. In sections doubly labeled for GlyR alpha2 and GlyR alpha3, approximately one-third of the puncta were colocalized. The most abundant GlyR subtype in retina contains alpha3 subunits, followed by those containing GlyR alpha2 and GlyR alpha1 subunits.     

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.     

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.     

Protein kinase C regulates tonic GABAA receptor‐mediated inhibition in the hippocampus and thalamus

Tonic inhibition mediated by extrasynaptic GABA_A receptors (GABA_ARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA_ARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole‐cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC‐mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA_AR‐mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA_AR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA_ARs, which represent a key extrasynaptic GABA_AR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA_ARs. The inhibitory effects of PKC activation on α4β2δ GABA_AR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC‐mediated phosphorylation can be an important physiological regulator of tonic GABA_AR‐mediated inhibition.     

γ-Aminobutyric acidA receptors on a bistratified amacrine cell type in the rabbit retina

γ-Aminobutyric acid (GABA) is considered to be a major inhibitory neurotransmitter in the inner plexiform layer of the retinas of all vertebrate species. It is contained in and released from nearly 40% of the amacrine cells and is known to play a major role in many aspects of visual processing. By using well-characterized antibodies to several subunits of the GABA_A receptor, we have analyzed their localization on the cell bodies and dendritic trees of two amacrine cell populations in the rabbit retina, which have been either filled intracellularly with Lucifer yellow or stained immunohistochemically. Both populations are selectively stained by intravitreal injection of the fluorescent nuclear dye 4′,6-diaminidin-2-phenylindoldihydrochloride (DAPI). We have found that the most significant concentration of the α₁ and β_2/3 GABA_A receptor subunits is localized to the DAPI-3 type amacrine cell. The perikarya of the DAPI-3 cells are found in the proximal inner nuclear layer and send their processes into two sublayers in sublaminae a and b of the inner plexiform layer. These processes abut but do not directly overlap those of the two mirror-symmetric populations of starburst amacrine cells. Because the cell bodies of the DAPI-3 cells are the only ones in the inner nuclear layer that stain strongly for either the α₁ or β_2/3 subunits, such staining is a diagnostic feature of these cells. Their processes also constitute the most strongly staining ones found within the inner plexiform layer. The dendritic trees of DAPI-3 cells, which range from about 150 μm up to about 300 μm, exhibit recurvate looping processes reminiscent of those described for directionally selective ganglion cells. In contrast to the DAPI-3 cell, we have also shown that the starburst amacrine cells exhibit no immunoreactivity for the α₁ GABA_A receptor subunit and very little for the β_2/3 subunit. Thus, we have shown that the DAPI-3 cells contain the highest concentrations of the α₁ and β_2/3 GABA_A receptor subunits in the rabbit retina. These cells, which costratify near the processes of both the starburst amacrine cells and the ON-OFF directionally selective ganglion cells, thus, are situated both anatomically and by virtue of their receptor content to potentially interact. J. Comp. Neurol. 393:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Mesencephalic trigeminal neuron responses to γ-aminobutyric acid

Mesencephalic trigeminal neurons are primary sensory neurons which have cell somata located within the brain stem. In spite of the presence of synaptic terminals on and around the cell somata, applications of a variety of neurotransmitter substances in earlier studies have failed to demonstrate responses. Using intracellular recording in a brain slice preparation, we have observed prominent depolarizations and decreases in input resistance in response to applications of γ-aminobutyric acid (GABA) in most recorded mesencephalic trigeminal neurons. Those cells failing to respond were located deeply within the slice, and the low responsiveness was shown to be related to uptake of GABA in the slice. The responses were direct, since they remained during perfusion with a low calcium, high magnesium solution that blocks synaptic transmission. The responses were mimicked by the GABA_A receptor agonist isoguvacine, and blocked by GABA_A receptor antagonists. The GABA_B receptor agonist baclofen evoked no changes in membrane potential or input resistance in neurons exhibiting depolarizations with GABA application. Tests of neuronal excitability during GABA applications indicated that the excitatory effects of the depolarization prevail over the depressant effects of the increase in membrane conductance. In situ hybridization histochemistry indicated that the GABA_A receptors in Me5 cells are comprised of α₂, β₂ and γ₂ subunits.© 1997 Elsevier Science B.V. All rights reserved.     

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.     

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.     

Experimental early‐life febrile seizures induce changes in GABAAR‐mediated neurotransmission in the dentate gyrus

Purpose: Febrile seizures (FS), the most frequent seizure type during childhood, have been linked to temporal lobe epilepsy (TLE) in adulthood. Yet, underlying mechanisms are still largely unknown. Altered γ‐aminobutyric acid (GABA)ergic neurotransmission in the dentate gyrus (DG) circuit has been hypothesized to be involved. This study aims at analyzing whether experimental FS change inhibitory synaptic input and postsynaptic GABA_AR function in dentate granule cells. Methods: We applied an immature rat model of hyperthermia (HT)–induced FS. GABA_AR‐mediated neurotransmission was studied using whole‐cell patch‐clamp recordings from dentate granule neurons in hippocampal slices within 6–9 days post‐HT. Key Findings: Frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs) were reduced in HT rats that had experienced seizures, whereas sIPSC amplitudes were enhanced. Whole‐cell GABA responses revealed a doubled GABA_AR sensitivity in dentate granule cells from HT animals, compared to that of normothermic (NT) controls. Analysis of sIPSCs and whole‐cell GABA responses showed similar kinetics in postsynaptic GABA_ARs of HT and NT rats. quantitative real‐time polymerase chain reaction (qPCR) experiments indicated changes in DG GABA_AR subunit expression, which was most pronounced for the α3 subunit. Significance: The data support the hypothesis that FS persistently alter neuronal excitability.