GABAA receptor subunits in the human amygdala and hippocampus: Immunohistochemical distribution of 7 subunits

GABAergic neurotransmission in the amygdala contributes to the regulation of emotional processes in anxiety, stress, reward, mnestic functions, addiction, and epilepsy. Species‐specific differences in the distribution and composition of GABA_A receptors may account for distinct effects and side‐effects of GABAergic agents. However, data on the distribution and composition of GABA_A receptors in the human amygdala are lacking. Here, the expression of GABA_A receptor subunits α1, α2, α3, α5, β2, β2/3, and γ2 was studied in the human amygdala using immunohistochemistry. Hippocampi were evaluated as a reference structure. Neuronal counts and field fraction analyses were performed, and subcellular expression of GABA_A receptor subunits was analyzed semiquantitatively. In the amygdala, field fraction analyses showed the highest α1 expression in the lateral nucleus (La), whereas α3 was prominent in intercalated nuclei (IC), and α5 and γ2 in the cortical nuclei, and amygdalo‐hippocampal/parahippocampal‐amygdala transition areas. In the hippocampus, α1 and α3 were accentuated in the dentate gyrus, CA1 region, and subiculum, whereas α5 expression was rather uniform. In both regions, α2 was homogenously distributed, and the two β subunits and γ2 showed faint immunostaining. The intensity of subunit expression also varied in the neuropil, neuronal somata, and/or cellular processes in the subregions. GABA_A receptors containing subunit α1, showing the strongest expression in the La, and α3, with the strongest expression in the IC and subiculum, could be targets for treating amygdala‐related disorders. Differences in GABA_A receptor subunit expression between the human and rodent amygdala should be taken into consideration when developing subunit‐selective drugs.     

Differential localization of γ‐aminobutyric acid type a and glycine receptor subunits and gephyrin in the human pons,medulla oblongata and uppermost cervical segment of the spinal cord: An immunohistochemical study

Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and γ‐aminobutyric acid type A receptors (GABA_AR). GlyR and GABA_AR are heteropentameric chloride ion channels that facilitate fast‐response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA_AR and GlyR subunits in the human light microscopically in the rostral and caudal one‐thirds of the pons, in the middle and caudal one‐thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA_AR subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA_AR α₁ and GlyR α₁ and β subunits show high levels of immunoreactivity in these nuclei. The GABA_AR subunits α₂, α₃, β_2,3, and γ₂ present weaker levels of immunoreactivity. Exceptions are intense levels of GABA_AR α₂ subunit immunoreactivity in the inferior olivary complex and high levels of GABA_AR α₃ subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors. J. Comp. Neurol. 518:305–328, 2010. © 2009 Wiley‐Liss, Inc.     

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 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.     

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.     

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.     

Orexin a phosphorylates the γ‐Aminobutyric acid type A receptor β2 subunit on a serine residue and changes the surface expression of the receptor in SH‐SY5Y cells exposed to propofol

Propofol activates the γ‐aminobutyric acid type A receptor (GABA_AR) and causes a reversible neurite retraction, leaving a thin, thread‐like structure behind; it also reverses the transport of vesicles in rat cortical neurons. The awakening peptide orexin A (OA) inhibits this retraction via phospholipase D (PLD) and protein kinase C? (PKC?). The human SH‐SY5Y cells express both GABA_ARs and orexin 1 and 2 receptors. These cells are used to examine the interaction between OA and the GABA_AR. The effects of OA are studied with flow cytometry and immunoblotting. This study shows that OA stimulates phosphorylation on the serine residues of the GABA_AR β₂ subunit and that the phosphorylation is caused by the activation of PLD and PKC?. OA administration followed by propofol reduces the cell surface expression of the GABA_AR, whereas propofol stimulation before OA increases the surface expression. The GABA_AR β₂ subunit is important for receptor recirculation, and the effect of OA on propofol‐stimulated cells may be due to a disturbed recirculation of the GABA_AR. © 2015 Wiley Periodicals, Inc.     

GABAA receptors in the primate basal ganglia: An autoradiographic and a light and electron microscopic immunohistochemical study of the α1 and β2,3 subunits in the baboon brain

The distribution of gamma-aminobutyric acid_A (GABA_A) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the α₁ and β_2,3 subunits of the GABA_A receptor by light and electron microscopy. In the caudate-putamen, the α₁ subunit was distributed in high densities in the matrix compartment, and the β_2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the α₁ and β_2,3 subunits. Four types of α₁ subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, α₁ and β_2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, α₁ and β_2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA_A receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus. J. Comp. Neurol. 397:297–325, 1998. © 1998 Wiley-Liss, Inc.     

γ‐Aminobutyric acid‐ρ expression in ependymal glial cells of the mouse cerebellum

The ependymal glial cells (EGCs) from the periventricular zone of the cerebellum were studied to determine their distribution and the functional properties of their γ‐aminobutyric acid type A (GABA_A) receptors. EGCs were identified by the presence of ciliated structures on their ventricular surface and their expression of glial fibrillary acidic protein (GFAP). Interestingly, diverse cell types, including neurons, astrocytes, and other types of glia, were identified in the subventricular zone by their current profiles. Electron microscopy showed ciliated cells and myelinated axons in this zone, but we found no collateral connections to suggest the presence of functional synapses. GABA‐mediated currents were recorded from EGCs in cerebellar slices from postnatal days 13 to 35 (PN13–PN35). These currents were blocked by TPMPA (a highly specific GABA_Aρ subunit antagonist) and bicuculline (a selective antagonist for classic GABA_A receptors). Pentobarbital failed to modulate GABA_A‐mediated currents despite the expression of GABAα1 and GABAγ2 subunits. In situ hybridization, RT‐PCR, and immunofluorescence studies confirmed GABAρ1 expression in EGCs of the cerebellum. We conclude that cerebellar EGCs express GABAρ1, which is functionally involved in GABA_A receptor‐mediated responses that are unique among glial cells of the brain. © 2013 Wiley Periodicals, Inc.     

GABAA/benzodiazepine receptor α6 subunit mRNA in granule cells of the cerebellar cortex and cochlear nuclei: Expression in developing and mutant mice

The gamma aminobutyric acid_A/benzodiazepine (GABA_A/BZ) receptor is a multisubunit (α, β, γ, δ, and ρ) ligand-gated chloride channel; there are several variants of the α, β, and γ subunits, each of which has been localized throughout the central nervous system. A large number of GABA_A/BZ subunit variants are expressed within the cerebellar cortex. In previous studies from other laboratories, α₆ subunit mRNA has been reported to be present exclusively in cerebellar granule cells. The developmental expression of α₆ mRNA in cerebellar and cochlear granule cells is of interest because it has been suggested that each of these cell types is derived from a common precursor pool. The polymerase chain reaction was used to generate a cDNA fragment encoding a portion of the M3–M4 intracellular loop of the α₆ subunit of the GABA_A/BZ receptor. A [³⁵S] riboprobe, transcribed from this cDNA fragment, was used to examine the expression of the α₆ subunit mRNA by in situ hybridization in developing normal mice and in adult mutant mice with known deficits in synaptic circuitry. A strong hybridization signal was observed over the granule cell layers of both the cerebellum and cochlear nuclei in adult mice. The signal over the cochlear nuclei appeared after birth toward the end of postnatal week 1, coinciding with the appearance of labeling in the cerebellar cortex. The intensity of the hybridization signal in both regions increased rapidly until postnatal day 14, after which it increased more gradually, reaching adult levels during postnatal week 3. In the weaver mutant, α₆ labeling was detected in surviving granule cells, but not in cerebellar regions devoid of granule cells. Significant levels of the α₆ hybridization signal were also present in cerebellar granule cells of Purkinje cell degeneration, lurcher, and staggerer mutants, suggesting that aberrations in synaptic circuitry do not prevent α₆ subunit gene expression. Our results demonstrate that α₆ subunit mRNA is not limited to the cerebellum, but is expressed in other neurons which share a common cellular precursor pool. These data also suggest that these granule cell precursors may be intrinsically programmed to acquire a specific form of the GABA_A/BZ receptor, irrespective of their final location and lack of connections with target neurons. © 1994 Wiley-Liss, Inc.     

Striatal efferents preferentially innervate neurons in the ventral pallidum containing GABAA receptor α 1 subunit-like immunoreactivity

The γ-aminobutyric acid-A receptor consists of several subunits. In this immunohistochemical study we investigated the regional distribution of the α1 and α2 subunits with subunit-specific antibodies in the ventral pallidum, and compared the staining patterns to those of substance P (SP). α1 subunit antigenic sites were found to be localized to pallidal neurons, varicosities, and varicose fibers. α1 immunopositive fibers mainly appeared “tubulus-like” due to the intense staining of the membranes of the long pallidal dendrites. Double labelling of α1 subunit and substance P revealed that α1 subunit-like immunoreactive (IR) dendrites and somata of the pallidal neurons were often invested by SP-IR striatal efferents. Subcellularly, the dendritic and somatic membranes of pallidal neurons were strongly immunopositive for the α1 subunit, whereas only a few axon terminals exhibited α1-IR. α2-IR was restricted to a low number of ventral pallidal neurons. The distributional patterns obtained for the α1 and α2 subunits suggest that striatal efferent neurons directly influence pallidal neurons displaying a distinct GABA_A subunit composition, which may be of pharmacological importance since the α1βxγ2-subunits containing receptors have mainly a benzodiazepine type I pharmacology. © 1996 Wiley-Liss, Inc.     

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.     

Chronic pentobarbital administration alters γ-aminobutyric acidA receptor α6-subunit mRNA levels and diazepam-insensitive [3H]Ro15-4513 binding

In order to study the chronic effects of pentobarbital, a positive GABA_A receptor modulator, on the inverse agonist binding of the benzodiazepine site, binding of [³H]Ro15-4513 and levels of GABA_A receptor α₆-subunit mRNA were investigated in the brains of pentobarbital-tolerant/dependent animals, using receptor autoradiography and in situ hybridization histochemistry in consecutive brain sections. Pentobarbital was administered to rats either 60 mg/kg, i.p., once, for acute treatment, or 300 μg/10μl/h i.c.v. continuously for 6 days via osmotic minipumps to render rats tolerant to pentobarbital. Rats assigned to the dependent group were sacrificed 24 h after discontinuance of pentobarbital infusion, while those assigned to the tolerant group were sacrificed at the end of infusion. The α₆ subunit mRNA was increased in the tolerant group only. Diazepam-insensitive [³H]Ro15-4513 binding was increased in the cerebellar granule layer of pentobarbital-tolerant and -dependent rats. No alterations in these parameters were observed in acutely treated animals. These data suggest that chronic pentobarbital treatment induced expression of α₆-subunit mRNA. This was in contrast to α₁- and γ₂-subunit mRNA, which in tolerant animals are unchanged, but for which withdrawal triggers a surge in levels. Because the α₆-subunit is a major component of the diazepam-insensitive [³H]Ro15-4513 binding site, the increased diazepam-insensitive [³H]Ro15-4513 binding implied de novo synthesis of the receptor subunit protein. © 1996 Wiley-Liss, Inc.     

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.     

Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAAR α1/GABAAR α2 ratio

Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ‐aminobutyric acid receptor (GABA_AR) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABA_AR α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose–response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7‐day‐old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP‐ribose) polymerase‐1 protein (PARP‐1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP‐1 and GABA_AR α1/GABA_AR α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP‐1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP‐1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABA_AR surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7‐day‐old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABA_AR α2 to GABA_AR α1.     

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.     

Expression of the GABAA receptor α6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABAA receptors

Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABA_A receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the α6 GABA_A receptor subunit. Membranes prepared from these cultures were photolabeled with the imidazobenzodiazepine [³H]Ro15-4513. In THIP-treated cultures at 4 days in vitro (DIV), photolabeled [³H]Ro15-4513 binding in membranes was significantly increased for both the 51 kilodalton, kDa, (α1 subunit) and 56-kDa (α6 subunit) radioactive peaks in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). In contrast, THIP-treated granule cells at 8 DIV demonstrated a small but significant decrease from control cultures in the photoincorporation of [³H]Ro15-4513 in the 51-kDa peak; however, no significant change in [³H]Ro15-4513 binding was observed for the 56-kDa polypeptide. Immunolabeling of the α6 subunit using silver-enhanced, immuno-gold staining of granule cells showed a significant effect with THIP treatment only at 4 DIV and not at 8 DIV. Examination by light microscopy demonstrated that the major effect of THIP was to increase α6 subunit clustering on granule cell bodies as well as neurites, 15-fold and sixfold, respectively. Using in situ hybridization, a small THIP-induced increase in α6 mRNA was detected at 4 DIV; however, no effect was apparent at 8 DIV. These data suggest that THIP has a trophic effect on α6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABA_A agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABA_A receptor subunits in the developing cerebellum. J. Neurosci. Res. 50:1053–1062, 1997. © 1997 Wiley-Liss, Inc.