Expression of distinct alpha subunits of GABAA receptor regulates inhibitory synaptic strength

Distinct alpha subunit subtypes in the molecular assembly of GABA(A) receptors are a critical determinant of the functional properties of inhibitory synapses and their modulation by a range of pharmacological agents. We investigated the contribution of these subunits to the developmental changes of inhibitory synapses in cerebellar granule neurons in primary cultures from wild-type and alpha1 subunit -/- mice. The decay time of miniature inhibitory postsynaptic currents (mIPSCs) halved between 6 days in vitro (DIV6) and DIV12. This was paralleled by the decrease of alpha2 and alpha3 subunits, the increase of alpha1 and alpha6 subunits expression at synapses, and changes in the action of selective alpha subunit modulators. A small but significant shortening of mIPSCs was observed with development in cells from -/- mice together with a decrease in the expression of alpha3 subunit. In contrast, the expression of alpha2 subunit at inhibitory synapses in -/- cells was significantly higher than in +/+ cells at DIV11-12. alpha5 subunit was not detected, and increased sensitivity to a selective alpha4/alpha6 subunit agonist suggests increased expression of extrasynaptic receptors in -/- mice. beta2/beta3 subunit expression and loreclezole sensitivity increased with development in +/+ but not in -/- cells, supporting the preferential association of the alpha1 with the beta2 subunit. Synaptic charge transfer strongly decreased with development but was not different between cells in the +/+ and -/- groups until DIV11-12. Our results uncover a pattern of sequential expression of alpha subunits underlying the changes in functional efficacy of GABAergic networks with development.     

Temporal and regional regulation of alpha1, beta2 and beta3, but not alpha2, alpha4, alpha5, alpha6, beta1 or gamma2 GABA(A) receptor subunit messenger RNAs following one-week oral flurazepam administration.

The effect of prolonged benzodiazepine administration on GABA(A) receptor subunit (alpha1-6, beta1-3, gamma2) messenger RNAs was investigated in the rat hippocampus and cortex, among other brain areas. Rats were orally administered flurazepam for one week, a protocol which results in benzodiazepine anticonvulsant tolerance in vivo, and in the hippocampus in vitro, in the absence of behavioral signs of withdrawal. Autoradiographs of brain sections, hybridized with [35S]oligoprobes in situ, were examined immediately (day 0) or two days after drug treatment, when rats were tolerant, or seven days after treatment, when tolerance had reversed, and were compared to sections from pair-handled, vehicle-treated controls. Alpha1 subunit messenger RNA level was significantly decreased in CA1 pyramidal cells and dentate granule cells at day 0, an effect which persisted only in CA1 neurons. Decreased "alpha1-specific" silver grain density over a subclass of interneurons at the pyramidal cell border suggested concomitant regulation of interneuron GABA(A) receptors. A reduction in beta3 subunit messenger RNA levels was more widespread among hippocampal cell groups (CA1, CA2, CA3 and dentate gyrus), immediately and two days after treatment, and was also detected in the frontal and parieto-occipital cortices. Changes in beta2 subunit messenger RNA levels in CA1, CA3 and dentate gyrus cells two days after ending flurazepam treatment suggested a concomitant up-regulation of beta2 messenger RNA. There was a trend toward an increased level of alpha5, beta3 and gamma2 subunit messenger RNAs in CA1, CA3 and dentate gyrus cells, which was significant for the beta3 and gamma2 subunit messenger RNAs in the frontal cortex seven days after ending flurazepam treatment. There were no flurazepam treatment-induced changes in any other GABA(A) receptor subunit messenger RNAs. The messenger RNA levels of three (alpha1, beta2 and beta3) of nine GABA(A) receptor subunits were discretely regulated as a function of time after ending one-week flurazepam treatment related to the presence of anticonvulsant tolerance, but not dependence. The findings suggested that a localized switch in the subunit composition of GABA(A) receptor subtypes involving these specific subunits may represent a minimal requirement for the changes in GABA(A) receptor-mediated function recorded previously at hippocampal CA1 GABAergic synapses, associated with benzodiazepine anticonvulsant tolerance.     

GABAA receptor alpha1 and alpha6 subunits mediate cell surface anchoring in cultured cells

The clustering and immobility of gamma-aminobutyric acid type A receptors (GABAARs) at discrete and functionally significant domains on the nerve cell surface is an important determinant in the integration of synaptic inputs. To investigate the role that different GABAAR alpha subunit isoforms play in determining receptor mobility, alphaxbeta3gamma2s subunits (where x = subunit isoforms 1-6) were co-transfected into COS 7 and human embryonic kidney (HEK) 293 cells and the surface mobility of these recombinant complexes was measured by fluorescence photobleach recovery (FPR). In addition, the lateral mobility of endogenous GABAARs in cerebellar granule (CG) cells was measured. We show that the alpha1 and alpha6 subunits immobilize recombinant GABAAR in transfected cells. This is consistent with the immobility of native receptors in CG cells, which express alpha1 and alpha6.     

GABAergic and glutamatergic terminals differentially influence the organization of GABAergic synapses in rat cerebellar granule cells in vitro

Synapse formation in CNS neurons requires appropriate sorting and clustering of neurotransmitter receptors and associated proteins at postsynaptic sites. In GABAergic synapses, clustering of GABA(A) receptors requires gephyrin, but it is not known whether presynaptic signals are also involved in this process. To investigate this issue, we analyzed the subcellular distribution of GABA(A) receptors and gephyrin in primary cultures of cerebellar granule cells, by comparing cells receiving GABAergic input with cells devoid of such afferents. Using immunofluorescence staining, we show that the GABA(A) receptor alpha1 and gamma2 subunit, but not alpha6 or delta subunit, form clusters co-localized with gephyrin in granule cell neurites, irrespective of the presence of GABAergic axons. GABAergic terminals typically were surrounded by groups of gephyrin clusters, pointing to the presence of multiple synaptic sites. In contrast, in neurites devoid of GABAergic input, gephyrin clusters were distributed at random and apposed to glutamatergic terminals, suggesting the formation of mismatched synapses. Both populations of gephyrin clusters were co-localized with GABA(A) receptor subunits, indicating that these proteins are associated also in non-GABAergic synapses. To determine whether signaling mediated by GABA(A) receptors is required for the formation of appropriately matched gephyrin clusters, cultures were treated chronically with bicuculline, or with either muscimol or 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol. All these treatments failed to influence the distribution of gephyrin clusters. We conclude that although GABAergic presynaptic terminals have a preponderant influence on the distribution of gephyrin clusters in dendrites of cerebellar granule cells, GABA transmission is dispensable for postsynaptic clustering of gephyrin and GABA(A) receptors and for the formation of appropriately matched GABAergic synapses.     

Postnatal expression of alpha2 nicotinic acetylcholine receptor subunit mRNA in developing cortex and hippocampus

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated cation channels composed of alpha and beta subunits. nAChR subunit expression is highly regulated during development. Previous studies have revealed increased expression of alpha3, alpha5, alpha7, and beta4 subunit mRNAs and alpha7 binding sites during hippocampal and cortical development. Here, we examined the expression of alpha2 subunit mRNA in rat cortex and hippocampus using highly sensitive radioactive in situ hybridization. alpha2 Subunit mRNA expression was first detected at P3 in cortex and hippocampus. During postnatal development the distribution of alpha2 subunit mRNA expression was spatially similar to the one found in adult, exhibiting highly restricted expression in scattered cells mostly in cortical layer V and retrosplenial cortex, and in scattered cells in CA1/CA3 stratum oriens and CA3 stratum radiatum. However, the expression intensity and number of alpha2 positive cells strongly increased to reach peak levels in both cortex and hippocampus at P7 and decreased thereafter to moderate to low to levels. Double in situ hybridization revealed that most, but not all, alpha2 mRNA expression was located in non-pyramidal GAD-positive cortical and hippocampal interneurons. Thus, similar to other nAChR subunits, alpha2 mRNA expression is transiently upregulated during postnatal development and nAChRs containing alpha2 subunits could regulate GABAergic activity during a critical period of network formation.     

GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain

GABA(A) receptors are ligand-operated chloride channels assembled from five subunits in a heteropentameric manner. Using immunocytochemistry, we investigated the distribution of GABA(A) receptor subunits deriving from 13 different genes (alpha1-alpha6, beta1-beta3, gamma1-gamma3 and delta) in the adult rat brain. Subunit alpha1-, beta1-, beta2-, beta3- and gamma2-immunoreactivities were found throughout the brain, although differences in their distribution were observed. Subunit alpha2-, alpha3-, alpha4-, alpha5-, alpha6-, gamma1- and delta-immunoreactivities were more confined to certain brain areas. Thus, alpha2-subunit-immunoreactivity was preferentially located in forebrain areas and the cerebellum. Subunit alpha6-immunoreactivity was only present in granule cells of the cerebellum and the cochlear nucleus, and subunit gamma1-immunoreactivity was preferentially located in the central and medial amygdaloid nuclei, in pallidal areas, the substantia nigra pars reticulata and the inferior olive. The alpha5-subunit-immunoreactivity was strongest in Ammon's horn, the olfactory bulb and hypothalamus. In contrast, alpha4-subunit-immunoreactivity was detected in the thalamus, dentate gyrus, olfactory tubercle and basal ganglia. Subunit alpha3-immunoreactivity was observed in the glomerular and external plexiform layers of the olfactory bulb, in the inner layers of the cerebral cortex, the reticular thalamic nucleus, the zonal and superficial layers of the superior colliculus, the amygdala and cranial nerve nuclei. Only faint subunit gamma3-immunoreactivity was detected in most areas; it was darkest in midbrain and pontine nuclei. Subunit delta-immunoreactivity was frequently co-distributed with alpha4 subunit-immunoreactivity, e.g. in the thalamus, striatum, outer layers of the cortex and dentate molecular layer. Striking examples of complementary distribution of certain subunit-immunoreactivities were observed. Thus, subunit alpha2-, alpha4-, beta1-, beta3- and delta-immunoreactivities were considerably more concentrated in the neostriatum than in the pallidum and entopeduncular nucleus. In contrast, labeling for the alpha1-, beta2-, gamma1- and gamma2-subunits prevailed in the pallidum compared to the striatum. With the exception of the reticular thalamic nucleus, which was prominently stained for subunits alpha3, beta1, beta3 and gamma2, most thalamic nuclei were rich in alpha1-, alpha4-, beta2- and delta-immunoreactivities. Whereas the dorsal lateral geniculate nucleus was strongly immunoreactive for subunits alpha4, beta2 and delta, the ventral lateral geniculate nucleus was predominantly labeled for subunits alpha2, alpha3, beta1, beta3 and gamma2; subunit alpha1- and alpha5-immunoreactivities were about equally distributed in both areas. In most hypothalamic areas, immunoreactivities for subunits alpha1, alpha2, beta1, beta2 and beta3 were observed. In the supraoptic nucleus, staining of conspicuous dendritic networks with subunit alpha1, alpha2, beta2, and gamma2 antibodies was contrasted by perykarya labeled for alpha5-, beta1- and delta-immunoreactivities. Among all brain regions, the median emminence was most heavily labeled for subunit beta2-immunoreactivity. In most pontine and cranial nerve nuclei and in the medulla, only subunit alpha1-, beta2- and gamma2-immunoreactivities were strong, whereas the inferior olive was significantly labeled only for subunits beta1, gamma1 and gamma2. In this study, a highly heterogeneous distribution of 13 different GABA(A) receptor subunit-immunoreactivities was observed. This distribution and the apparently typical patterns of co-distribution of these GABA(A) receptor subunits support the assumption of multiple, differently assembled GABA(A) receptor subtypes and their heterogeneous distribution within the adult rat brain.     

Ca2+/calmodulin-dependent protein kinase II in the rat cerebellum: an immunohistochemical study with monoclonal antibodies specific to either alpha or beta subunit.

Monoclonal antibodies specific to either alpha or beta subunit of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) of the rat brain were produced and the distribution of each subunit in the rat cerebellum was examined immunohistochemically. Each antibody detected solely the corresponding subunit in immunoblot analysis of crude homogenates of the rat forebrain and cerebellum, and purified CaM kinase II from the rat forebrain. Immunoreactivity for alpha subunit was present selectively in Purkinje cells: perikarya, dendrites with their spines, axons and their terminal-like structures in the cerebellar cortex, cerebellar nuclei and lateral vestibular nucleus. Many of these alpha subunit-immunoreactive axons from the cerebellum were traced only through the inferior cerebellar peduncle. beta Subunit was detected in perikarya and dendrites of a limited number of Purkinje cells, many granule cells and neurons in the cerebellar nuclei. Thus, different distributions of alpha and beta subunits of CaM kinase II in the cerebellum were demonstrated.     

Expression of GABA(A) receptor subunits in rat brainstem auditory pathways: cochlear nuclei, superior olivary complex and nucleus of the lateral lemniscus

Inhibition by GABA is important for auditory processing, but any adaptations of the ionotropic type A receptors are unknown. Here we describe, using in situ hybridization, the subunit expression patterns of GABA(A) receptors in the rat cochlear nucleus, superior olivary complex, and dorsal and ventral nuclei of the lateral lemniscus. All neurons express the beta3 and gamma2L subunit messenger RNAs, but use different alpha subunits. In the dorsal cochlear nucleus, fusiform (pyramidal) and giant cells express alpha1, alpha3, beta3 and gamma2L. Dorsal cochlear nucleus interneurons, particularly vertical or tuberculoventral cells and cartwheel cells, express alpha3, beta3 and gamma2L. In the ventral cochlear nucleus, octopus cells express alpha1, beta3, gamma2L and delta. Spherical cells express alpha1, alpha3, alpha5, beta3 and gamma2L. In the superior olivary complex, the expression profile is alpha3, alpha5, beta3 and gamma2L. Both dorsal and ventral cochlear nucleus granule cells express alpha1, alpha6, beta3 and gamma2L; unlike their cerebellar granule cell counterparts, they do not express beta2, gamma2S or the delta subunit genes. The delta subunit's absence from cochlear nucleus granule cells may mean that tonic inhibition mediated by extrasynaptic GABA(A) receptors is less important for this cell type. In both the dorsal and ventral nuclei of the lateral lemniscus, alpha1, beta3 and gamma2L are the main subunit messenger RNAs; the ventral nucleus also expresses the delta subunit. We have mapped, using in situ hybridization, the subunit expression patterns of the GABA(A) receptor in the auditory brainstem nuclei. In contrast to many brain regions, the beta2 subunit gene and gamma2S splice forms are not highly expressed in auditory brainstem nuclei. GABA(A) receptors containing beta3 and gamma2L may be particularly well suited to auditory processing, possibly because of the unique phosphorylation profile of this subunit combination.     

Immunohistochemical localization of the alpha 1, alpha 2 and alpha 3 subunit of the GABAA receptor in the rat brain.

The immunohistochemical distribution of the alpha 1, alpha 2 and alpha 3 subunit of the gamma-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells.     

Spatial distribution and subunit composition of GABA(A) receptors in the inferior olivary nucleus

GABAergic inhibitory feedback from the cerebellum onto the inferior olivary (IO) nucleus plays an important role in olivo-cerebellar function. In this study we characterized the physiology, subunit composition, and spatial distribution of gamma-aminobutyric acid-A (GABA(A)) receptors in the IO nucleus. Using brain stem slices, we identified two types of IO neuron response to local pressure application of GABA, depending on the site of application: a slow desensitizing response at the soma and a fast desensitizing response at the dendrites. The dendritic response had a more negative reversal potential than did the somatic response, which confirmed their spatial origin. Both responses showed voltage dependence characterized by an abrupt decrease in conductance at negative potentials. Interestingly, this change in conductance occurred in the range of potentials wherein subthreshold membrane potential oscillations usually occur in IO neurons. Immunostaining IO sections with antibodies for GABA(A) receptor subunits alpha 1, alpha 2, alpha 3, alpha 5, beta 2/3, and gamma 2 and against the postsynaptic anchoring protein gephyrin complemented the electrophysiological observation by showing a differential distribution of GABA(A) receptor subtypes in IO neurons. A receptor complex containing alpha 2 beta 2/3 gamma 2 subunits is clustered with gephyrin at presumptive synaptic sites, predominantly on distal dendrites. In addition, diffuse alpha 3, beta 2/3, and gamma 2 subunit staining on somata and in the neuropil presumably represents extrasynaptic receptors. Combining electrophysiology with immunocytochemistry, we concluded that alpha 2 beta 2/3 gamma 2 synaptic receptors generated the fast desensitizing (dendritic) response at synaptic sites whereas the slow desensitizing (somatic) response was generated by extrasynaptic alpha 3 beta 2/3 gamma 2 receptors.     

The expression of GABAA beta subunit isoforms in synaptic and extrasynaptic receptor populations of mouse dentate gyrus granule cells

The subunit composition of GABA(A) receptors influences their biophysical and pharmacological properties, dictates neuronal location and the interaction with associated proteins, and markedly influences the impact of intracellular biochemistry. The focus has been on alpha and gamma subunits, with little attention given to beta subunits. Dentate gyrus granule cells (DGGCs) express all three beta subunit isoforms and exhibit both synaptic and extrasynaptic receptors that mediate 'phasic' and 'tonic' transmission, respectively. To investigate the subcellular distribution of the beta subunits we have utilized the patch-clamp technique to compare the properties of 'tonic' and miniature inhibitory postsynaptic currents (mIPSCs) recorded from DGGCs of hippocampal slices of P20-26 wild-type (WT), beta(2)(-/-), beta(2N265S) (etomidate-insensitive), alpha(1)(-/-) and delta(-/-) mice. Deletion of either the beta(2) or the delta subunit produced a significant reduction of the tonic current and attenuated the increase of this current induced by the delta subunit-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). By contrast, mIPSCs were not influenced by deletion of these genes. Enhancement of the tonic current by the beta(2/3) subunit-selective agent etomidate was significantly reduced for DGGCs derived from beta(2N265S) mice, whereas this manipulation had no effect on the prolongation of mIPSCs produced by this anaesthetic. Collectively, these observations, together with previous studies on alpha(4)(-/-) mice, identify a population of extrasynaptic alpha(4)beta(2)delta receptors, whereas synaptic GABA(A) receptors appear to primarily incorporate the beta(3) subunit. A component of the tonic current is diazepam sensitive and is mediated by extrasynaptic receptors incorporating alpha(5) and gamma(2) subunits. Deletion of the beta(2) subunit had no effect on the diazepam-induced current and therefore these extrasynaptic receptors do not contain this subunit. The unambiguous identification of these distinct pools of synaptic and extrasynaptic GABA(A) receptors should aid our understanding of how they act in harmony, to regulate hippocampal signalling in health and disease.     

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.     

GABA(A) receptor beta3 subunit deletion decreases alpha2/3 subunits and IPSC duration

Deletion of the beta3 subunit of the GABA(A) receptor produces severe behavioral deficits and epilepsy. GABA(A) receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in cortical neurons in cultures from beta3 -/- mice were significantly faster than those in beta3 +/+ mice and were more prolonged by zolpidem. Surface staining revealed that the number of beta2/3, alpha2, and alpha3 (but not of alpha1) subunit-expressing neurons and the intensity of subunit clusters were significantly reduced in beta3 -/- mice. Transfection of beta3 -/- neurons with beta3 cDNA restored beta2/3, alpha2, and alpha3 subunits immunostaining and slowed mIPSCs decay. We show that the deletion of the beta3 subunit causes the loss of a subset of GABA(A) receptors with alpha2 and alpha3 subunits while leaving a receptor population containing predominantly alpha1 subunit with fast spontaneous IPSC decay and increased zolpidem sensitivity.     

Properties of nicotinic receptors underlying Renshaw cell excitation by alpha-motor neurons in neonatal rat spinal cord

We used anatomical and physiological approaches to characterize nicotinic receptors (AChRs) on Renshaw cells of the neonatal rat spinal cord. Confocal imaging of Renshaw cells, identified by their characteristic pattern of gephyrin immunoreactivity, revealed that these neurons are immuno-positive for the alpha4 and beta2 AChR subunits but not for the alpha7 subunit. We used whole cell recording in spinal cord slices to characterize synaptic transmission from alpha-motor neurons to Renshaw cells, which could be identified pharmacologically by the sensitivity of transmission to d-tubocurarine. alpha-Motor neuron-to-Renshaw cell synapses were blocked by 10 microM dihydro-beta-erythroidine (dHbetaE), but not 50 nM methyllycaconitine (MLA), a selective alpha7 antagonist. These findings support a role for alpha4beta2-like AChRs, but not alpha7 AChRs, in rapid excitatory transmission between alpha-motor neurons and Renshaw cells in rat spinal cord.     

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.     

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.     

Synaptic and extrasynaptic GABA-A and GABA-B receptors in the globus pallidus: an electron microscopic immunogold analysis in monkeys

GABA-A and GABA-B receptors mediate differential effects in the CNS. To better understand the role of these receptors in regulating pallidal functions, we compared their subcellular and subsynaptic localization in the external and internal segments of the globus pallidus (GPe and GPi) in monkeys, using pre- and post-embedding immunocytochemistry with antibodies against GABA-A (alpha1, beta2/3 subunits) and GABA-BR1 receptor subtype. Our results demonstrate that GABA-A and GABA-B receptors display a differential pattern of subcellular and subsynaptic localization in both segments of the globus pallidus. The majority of GABA-BR1 immunolabeling is intracellular, whereas immunoreactivity for GABA-A receptor subunits is mostly bound to the plasma membrane. A significant proportion of both GABA-BR1 and GABA-A receptor immunolabeling is extrasynaptic, but GABA-A receptor subunits also aggregate in the main body of putative GABAergic symmetric synapses established by striatal- and pallidal-like terminals. GABA-BR1 immunoreactivity is expressed presynaptically in putative glutamatergic terminals, while GABA-A alpha1 and beta2/3 receptor subunits are exclusively post-synaptic and often coexist at individual symmetric synapses in both GPe and GPi. In conclusion, our findings corroborate the concept that ionotropic and metabotropic GABA receptors are located to subserve different effects in pallidal neurons. Although the aggregation of GABA-A receptors at symmetric synapses is consistent with their role in fast inhibitory synaptic transmission, the extrasynaptic distribution of both GABA-A and GABA-B receptors provides a substrate for complex modulatory functions that rely predominantly on the spillover of GABA.     

GABAergic signaling to newborn neurons in dentate gyrus

Neurogenesis in the dentate gyrus begins before birth but then continues into adulthood. Consequently, many newborn granule cells must integrate into a preexisting hippocampal network. Little is known about the timing of this process or the characteristics of the first established synapses. We used mice that transiently express enhanced green fluorescent protein in newborn granule cells to examine their synaptic input. Although newborn granule cells had functional glutamate receptors, evoked and spontaneous synaptic currents were exclusively GABAergic with immature characteristics including slow rise and decay phases and depolarized reversal potentials. Synaptic currents in newborn granule cells were relatively insensitive to the GABA(A) receptor modulator zolpidem compared with neighboring mature granule cells. Consistent with the kinetics and pharmacology, newborn granule cells isolated by fluorescent cell sorting lacked the alpha1 GABA(A) receptor subunit. Our results indicate that newborn granule cells initially receive only GABAergic synapses even in the adult.