Functional analysis of GABA(A) receptors in nucleus tractus solitarius neurons from neonatal rats.

To gain insight into specific GABA(A) receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1-14 day old rats using whole-cell patch clamp recordings. GABA(A-) receptor-mediated currents were detected in all neurons tested, with an average EC(50) of 22.2 microM. GABA currents were consistently stimulated by diazepam (EC(50)=63 nM), zolpidem (EC(50)=85 nM), loreclezole (EC(50)=10.1 microM) and the neurosteroid 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn(2+) (IC(50)=33.6 microM) picrotoxin (IC(50)=2.4 microM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10-1000 microM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant alpha subunit isoform present in GABA(A) receptors of the NTS appears to be the alpha1 and/or alpha2 subunit. beta2 and/or beta3 subunits are the major beta isoform, while the predominant gamma subunit is likely gamma2. Our data suggest the contribution to NTS GABA currents by alpha3-alpha6, beta1, gamma1 and delta subunits, if present, is minor by comparison.     

Differential expression of GABA(A) receptor subunits in the distinct nuclei of the rat amygdala

Detailed knowledge of the anatomical distribution of different GABA(A) receptor subunits is crucial for understanding the physiological actions of GABA in individual brain areas and for developing drugs acting through the individual GABA receptor subtypes. Since the amygdala is a key brain structure in the processing of emotional information with distinct functions in each nucleus, GABA(A) receptors in the amygdala are an important target of treatment for emotional disorders. In this study, we analyzed by quantitative RT-PCR the expression levels of all GABA(A) receptor subunits in distinct nuclei of the amygdala, the central (Ce) and the lateral/basolateral (LA/BLA) amygdala. We found the strongest expression of the gamma(2) subunit mRNA in both the Ce and LA/BLA, modest expressions of alpha(1), alpha(2) and alpha(3) mRNAs in the LA/BLA and alpha(2) and gamma(1) mRNAs in the Ce, and weak expressions of alpha(6), rho(2) and rho(3) mRNAs in both regions. We further revealed the significantly different expressions of alpha(1), alpha(3), alpha(5), gamma(1), gamma(2), delta, epsilon and theta subunit mRNAs in the Ce and LA/BLA. Differences in the expression levels of GABA(A) receptor subunits suggest different sensitivity to a variety of drugs including benzodiazepines and anesthetics in amygdala nuclei with distinct functions.     

Regional and cellular localisation of GABA(A) receptor subunits in the human basal ganglia: An autoradiographic and immunohistochemical study.

The regional and cellular localisation of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the human basal ganglia using receptor autoradiography and immunohistochemical staining for five GABA(A) receptor subunits (alpha(1), alpha(2), alpha(3), beta(2, 3), and gamma(2)) and other neurochemical markers. The results demonstrated that GABA(A) receptors in the striatum showed considerable subunit heterogeneity in their regional distribution and cellular localisation. High densities of GABA(A) receptors in the striosome compartment contained the alpha(2), alpha(3), beta(2, 3), and gamma(2) subunits, and lower densities of receptors in the matrix compartment contained the alpha(1), alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. Also, six different types of neurons were identified in the striatum on the basis of GABA(A) receptor subunit configuration, cellular and dendritic morphology, and chemical neuroanatomy. Three types of alpha(1) subunit immunoreactive neurons were identified: type 1, the most numerous (60%), were medium-sized aspiny neurons that were immunoreactive for parvalbumin and alpha(1), beta(2,3), and gamma(2) subunits; type 2 (38%) were medium-sized to large aspiny neurons immunoreactive for calretinin and alpha(1), alpha(3), beta(2,3), and gamma(2) subunits; and type 3 (2%) were large sparsely spiny neurons immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits. Type 4 neurons were calbindin-positive and immunoreactive for alpha(2), alpha(3), beta(2,3), and gamma(2) subunits. The remaining neurons were immunoreactive for choline acetyltransferase (ChAT) and alpha(3) subunit (type 5) or were neuropeptide Y-positive with no GABA(A) receptor subunit immunoreactivity (type 6). The globus pallidus contained three types of neurons: types 1 and 2 were large neurons and were immunoreactive for alpha(1), alpha(3), beta(2,3), and gamma(2) subunits and for parvalbumin alone (type 1) or for both parvalbumin and calretinin (type 2); type 3 neurons were medium-sized and immunoreactive for calretinin and alpha(1), beta(2, 3), and gamma(2) subunits. These results show that the subunit composition of GABA(A) receptors displays considerable regional and cellular variation in the human striatum but are more homogeneous in the globus pallidus.     

Increased expression of GABA(A) receptor beta-subunits in the hippocampus of patients with temporal lobe epilepsy

It has been postulated that dysfunction of the GABA-ergic transmission is causatively related to the development of epilepsy. Animal models of temporal lobe epilepsy (TLE) revealed considerable changes in the expression of GABA(A) receptor subunits in the hippocampus. Using immunocytochemistry, we investigated the expression of GABA(A) receptor subunits alpha1, alpha3, beta1-3, and gamma2 in hippocampal specimens obtained at surgery from TLE patients with and without hippocampal sclerosis and in autopsy controls. Consistent with the severe neurodegeneration in the CA1 sector, significant decreases in alpha1-, alpha3-, beta3-, and gamma2-subunit immunoreactivity (IR) were detected in sclerotic but not in nonsclerosic specimens. In contrast, pronounced increases in IR of all 3 beta-subunits were observed in most sectors of the hippocampal formation both in sclerotic and nonsclerotic specimens, being especially pronounced in the dentate molecular layer and in the subiculum where subunit alpha3- and gamma2-IR were also elevated. Using in situ hybridization for subunits beta2 and beta3, increased expression of the respective mRNAs was detected in dentate granule cells of patients with and without hippocampal sclerosis. Beta-subunits are important constituents of the GABA(A) receptor and contribute to the binding site of GABA. Our data indicate pronounced adaptive changes in the expression of these GABA(A) receptor subunits related to seizure activity and indicate altered assembly of GABA(A) receptors in TLE.     

Pharmacology of recombinant human GABA(A) receptor subtypes measured using a novel pH-based high-throughput functional efficacy assay

To facilitate the discovery of novel compounds that modulate human GABA(A) receptor function, we have developed a high throughput functional assay using a fluorescence imaging system. L(tk-) cells expressing combinations of human GABA(A) receptor subunits were incubated with the pH-sensitive dye 2',7'bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, then washed and placed in a 96-well real-time fluorescence plate reader. In buffer adjusted to pH 6.9 there was a robust and persisting acidification response to addition of GABA, which was antagonised by the GABA(A) receptor antagonist bicuculline. The concentration-response relationship for GABA was modulated by allosteric ligands, including benzodiazepine (BZ) site agonists and inverse agonists. The effects of BZ site ligands on the pH response to GABA for receptors containing alpha1beta3gamma2, alpha3beta3gamma2 or alpha5beta3gamma2 subunits were well correlated with results from electrophysiological studies on the same receptor subunit combinations expressed in Xenopus oocytes. Most modulatory compounds tested were found to be relatively unselective across the three subunit combinations tested; however, some showed subtype-dependent efficacy, such as diazepam, which had highest agonist effects on the alpha3beta3gamma2 subtype, substantial but lesser agonism on alpha1beta3gamma2 and still substantial but the least agonism on alpha5beta3gamma2. This indicates that the alpha subunit within the recombinant receptor expressed in L(tk-) cells can affect the efficacy of the response to some BZ compounds. Inhibitors of Na(+)/Cl(-) cotransport, anion/anion exchange and the gastric type of H(+)/K(+) ATPase potently inhibited GABA-evoked acidification, indicating that multiple transporters are involved in the GABA-evoked pH change. This novel fluorescence-based high throughput functional assay allows the rapid characterization of allosteric ligands acting on human GABA(A) receptors.     

Early expression of GABA(A) receptor delta subunit in the neonatal rat hippocampus

The cDNA library screening strategy was used to identify the genes encoding for GABA(A) receptor subunits in the rat hippocampus during development. With this technique, genes encoding eleven GABA(A) receptor subunits were identified. The alpha5 subunit was by far the most highly expressed, followed by the gamma2, alpha2 and alpha4 subunits respectively. The expression of the beta2, alpha1, gamma1, beta1 and beta3 subunits was moderate, although that of the alpha3 and delta subunits was weak. In situ hybridization experiments, using digoxigenin-labeled cRNA probes, confirmed that the delta subunit was expressed in the neonatal as well as in the adult hippocampus, and is likely to form functional receptors in association with other subunits of the GABA(A) receptor. When the more sensitive RT-PCR approach was used, the gamma3 subunit was also detected, suggesting that this subunit is present in the hippocampus during development but at low levels of expression. The insertion of the delta subunit into functional GABA(A) receptors may enhance the efficacy of GABA in the immediate postnatal period when this amino acid is still exerting a depolarizing and excitatory action.     

Expression of GABA(A) receptor subunits in the rat central nucleus of the inferior colliculus.

Expression of GABA(A) receptor (GABA(A)R) alpha(1), alpha(2), beta(2), gamma(1), gamma(2L) and gamma(2S) subunit mRNA was examined in three cell classes in the central nucleus of the rat inferior colliculus (CNIC). GABA(A)R alpha(1) and gamma(2L) subunit mRNA expression was greatest in large cells (over 25 microm long diameter), intermediate in medium sized cells (15 to 25 microm long diameter) and lowest in small cells (10 to 15 microm long diameter). GABA(A)R gamma(2S) and alpha(2) subunits had the opposite pattern, highest in the small cells, intermediate in medium cells and lowest in large cells. GABA(A)R beta(2) was significantly lower in small cells than the two other classes, while differences between large and medium cells were not significant. GABA(A)R gamma(1) subunit mRNAs expression was not above background in any of the three cell types assessed. The expression of GABA(A)R subunits suggests that cell classes in the rat CNIC may differ in their response to GABA and GABAergic drugs.     

Synchronous postnatal increase in alpha1 and gamma2L GABA(A) receptor mRNAs and high affinity zolpidem binding across three regions of rat brain

The objective of this study was to correlate postnatal changes in levels of mRNAs encoding predominant GABA(A) receptor subunits with a functional index of receptor development. This study is the first to quantify the temporal relationship between postnatal changes in predominant GABA(A) receptor mRNAs and zolpidem-sensitive GABA(A) receptor subtypes. In Experiment 1, we measured zolpidem displacement of 3H-flunitrazepam from rat cerebral cortex, hippocampus, and cerebellum at 0, 6, 14, 21, 29, and 90 postnatal days. Three independent 3H-flunitrazepam sites with high (K(i)=2. 7+/-0.6 nM), low (K(i)=67+/-4.8 nM), and very low (K(i)=4.1+/-0.9 mM) affinities for zolpidem varied in regional and developmental expression. In Experiment 2, we used RNAse protection assays to quantify levels of alpha1, alpha2, beta1, beta2, gamma2S and gamma2L mRNAs in the above regions at the same postnatal ages. Although there was a high degree of regional variation in the developmental expression of zolpidem-sensitive GABA(A) receptors and subunit mRNAs, a dramatic increase in high affinity zolpidem binding sites and alpha1 mRNA levels occurred within all three regions during the second postnatal week. Furthermore, a temporal overlap was observed between the rise in alpha1 mRNA and high affinity zolpidem binding and a more prolonged increase in gamma2L in each region. These results point to the inclusion of the alpha1 and gamma2L subunits in a GABA(A) receptor subtype with a high zolpidem affinity and suggest that a global signal may influence the emergence of this subtype in early postnatal life.     

Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.     

A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by cAMP-dependent protein kinase via selective interaction with receptor beta subunits

GABA(A) receptors, the key mediators of fast synaptic inhibition in the brain, are predominantly constructed from alpha(1-6), beta(1-3), gamma(1-3), and delta subunit classes. Phosphorylation by cAMP-dependent protein kinase (PKA) differentially regulates receptor function dependent upon beta subunit identity, but how this kinase is selectively targeted to GABA(A) receptor subtypes remains unresolved. Here we establish that the A-kinase anchoring protein 150 (AKAP150), directly binds to the receptor beta1 and beta3, but not to alpha1, alpha2, alpha3, alpha6, beta2, gamma2, or delta subunits. Furthermore, AKAP79/150 is critical for PKA-mediated phosphorylation of the receptor beta3 subunit. Together, our observations suggest a mechanism for the selective targeting of PKA to GABA(A) receptor subtypes containing the beta1 or beta3 subunits dependent upon AKAP150. Therefore, the selective interaction of beta subunits with AKAP150 may facilitate GABA(A) receptor subtype-specific functional modulation by PKA activity which may have profound local effects on neuronal excitation.     

Molecular basis underlying GABA(A) responses in rat mesencephalic trigeminal neurons

We studied the molecular basis of GABA(A) receptor (GABA(A)R) expressed in the rat mesencephalic trigeminal (Vmes) sensory neuron using the immunohistochemical and single-cell RT-PCR methods. Using anti-GABA(A)R alpha2 subunit antibody, abundant GABA(A)Rs were visualized in Vmes neurons. A single-cell RT-PCR clarified that GABA(A)Rs expressed in Vmes neurons were predominantly composed of alpha2, alpha5, beta1, gamma1 and gamma2 subunits. Novel splicing variants in both alpha5 and beta1 were found invariably, and they lacked multiple amino acid sequence in the extracellular N-terminal portion. Known functional roles of both beta and gamma subunits in regulating the expression at the cell surface suggest that the unique subunit composition of GABA(A)Rs may be involved in the characteristics of GABA(A) response in Vmes neurons.     

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.     

Synaptic localization of GABA(A) receptor subunits in the striatum of the rat

The inhibitory amino acid gamma-aminobutyric acid (GABA) is widely distributed in the basal ganglia. It plays a critical role in the functioning of the striatum as it is the transmitter of projection neurons and sub-populations of interneurons, as well as afferents from the globus pallidus. Some of the factors controlling GABA transmission are the type(s) of GABA receptor expressed at the site of transmission, their subunit composition, and their location in relation to GABA release sites. To address these issues, we examined the sub-cellular localization of subunits of the GABA(A) receptor in the striatum of the rat. Sections of freeze-substituted, Lowicryl-embedded striatum were immunolabelled by the post-embedding immunogold technique with antibodies specific for subunits of the GABA(A) receptor. Immunolabelling for alpha1, beta2/3, and gamma2 GABA(A) receptor subunits was primarily located at symmetrical synapses on perikarya, dendrites, and spines. Quantitative analysis of the distribution of immunolabelling for the beta2/3 subunits revealed that the majority of membrane associated immunogold particles were at synapses and that, on average for the whole population, they were evenly distributed across the synapse. Double labelling for the beta2/3 subunits and for GABA itself revealed that receptor-positive synapses were formed by at least two populations of terminals. One population (59.3%) of terminals forming receptor-positive synapses was positive for GABA, whereas the other (40.7%) had low or undetectable levels of GABA. Furthermore, the post-synaptic neurons were characterised on neurochemical and morphological grounds as both medium spiny neurons and GABA interneurons. Triple immunolabelling revealed the co-localization of alpha1, beta2/3, and gamma2 subunits at some symmetrical axodendritic synapse. It is concluded that fast GABA(A)-mediated transmission occurs primarily at symmetrical synapses within the striatum, that the populations of boutons giving rise to receptor-positive synapses are heterogeneous, and that previously reported co-existence of different subunits of the GABA(A) receptor at the cellular level also occurs at the level of individual synapses.     

Desensitization and binding properties determine distinct alpha1beta2gamma2 and alpha3beta2gamma2 GABA(A) receptor-channel kinetic behavior

GABA(A) receptor subtypes comprising the alpha1 and alpha3 subunits change with development and have a specific anatomical localization in the adult brain. These receptor subtypes have been previously demonstrated to greatly differ in deactivation kinetics but the underlying gating mechanisms have not been fully elucidated. Therefore, we expressed rat alpha1beta2gamma2 and alpha3beta2gamma2 receptors in human embryonic kidney 293 cells and recorded current responses to ultrafast GABA applications at macroscopic and single-channel levels. We found that the slow deactivation of alpha3beta2gamma2-mediated currents is associated with a relatively small rate and extent of apparent desensitization. In contrast, responses mediated by alpha1beta2gamma2 receptors had faster deactivation and stronger desensitization. Alpha3beta2gamma2 receptors had faster recovery in the paired-pulse agonist applications than alpha1beta2gamma2 channels. The onset of currents mediated by alpha3beta2gamma2 receptors was slower than that of alpha1beta2gamma2 for a wide range of GABA concentrations. Single-channel analysis did not reveal differences in the opening/closing kinetics of alpha1beta2gamma2 and alpha3beta2gamma2 channels but burst durations were longer in alpha3beta2gamma2 receptors. Simulation with a previously reported kinetic model was used to explore the differences in respective rate constants. Reproduction of major kinetic differences required a smaller desensitization rate as well as smaller binding and unbinding rates in alpha3beta2gamma2 compared with alpha1beta2gamma2 receptors. Our work describes the mechanisms underlying the kinetic differences between two major GABA(A) receptor subtypes and provides a framework to interpret data from native GABA receptors.     

Antidepressant effects on GABA-stimulated 36Cl(-) influx in rat cerebral cortex are altered after treatment with GABA(A) receptor antisense oligodeoxynucleotides

Antidepressants act at the GABA(A) receptor to inhibit GABA-stimulated 36Cl(-) influx and GABA reduction of [35S]TBPS binding. This study examined how selective knock-down (via antisense oligodeoxynucleotides, aODNs) of GABA(A) receptor subunits modified antidepressant activity. The specific aODNs used were for the alpha1, beta1, beta2 or gamma2 subunits of the GABA(A) receptor. The aODN microinjections reduced corresponding GABA(A) receptor subunit mRNA levels by 30-40% as assessed by RT-PCR. The inhibitory effect of the antidepressants amitriptyline and mianserin on GABA-stimulated 36Cl(-) influx was decreased after microinjections of alpha1, beta1, or beta2 subunit aODNs but potentiated after microinjections of gamma2 subunit aODNs. This pattern of aODNs effect on amitriptyline and mianserin modulation of GABA-stimulated 36Cl(-) influx was the same for both antidepressants and similar to GABA but different than that of diazepam and bicuculline. We conclude that multiple subunits of the GABA(A) receptor regulate the effect of amitriptyline and mianserin on the GABA(A) receptor chloride ionophore complex. However, the exact identity of the subunit mediating the direct or allosteric modulation of the antidepressant effect on GABA-stimulated 36Cl(-) influx remains unclear.     

Co-localization of PKCepsilon with various GABA(A) receptor subunits in the mouse limbic system

The distribution of PKCepsilon and its co-localization with various GABA(A) receptor subunits within limbic structures of the mouse brain was examined by fluorescence immunohistochemistry. Levels of PKCepsilon immunoreactivity were highest in the cingulate cortex and dentate gyrus, moderate in the nucleus accumbens, and lowest in the prelimbic cortex and basolateral amygdala. Co-localization of PKCepsilon immunoreactivity with the GABA(A) receptor alpha1, beta 2/3, and gamma2 subunits varied by subunit and brain region examined, with the majority of co-localization occuring in the dentate gyrus, nucleus accumbens and basolateral amygdala. These results demonstrate that PKCepsilon may interact with GABA(A) receptors in a subunit- and region-specific manner, and provide a potential anatomical basis for recent behavioral and biochemical evidence that PKCepsilon modulates GABA(A) receptor function.