Compensatory alteration of inhibitory synaptic circuits in cerebellum and thalamus of gamma-aminobutyric acid type A receptor alpha1 subunit knockout mice

Targeted deletion of the alpha1 subunit gene results in a profound loss of gamma-aminobutyric acid type A (GABA(A)) receptors in adult mouse brain but has only moderate behavioral consequences. Mutant mice exhibit several adaptations in GABA(A) receptor subunit expression, as measured by Western blotting. By using immunohistochemistry, we investigated here whether these adaptations serve to replace the missing alpha1 subunit or represent compensatory changes in neurons that normally express these subunits. We focused on cerebellum and thalamus and distinguished postsynaptic GABA(A) receptor clusters by their colocalization with gephyrin. In the molecular layer of the cerebellum, alpha1 subunit clusters colocalized with gephyrin disappeared from Purkinje cell dendrites of mutant mice, whereas alpha3 subunit/gephyrin clusters, presumably located on dendrites of Golgi interneurons, increased sevenfold, suggesting profound network reorganization in the absence of the alpha1 subunit. In thalamus, a prominent increase in alpha3 and alpha4 subunit immunoreactivity was evident, but without change in regional distribution. In the ventrobasal complex, which contains primarily postsynaptic alpha1- and extrasynaptic alpha4-GABA(A) receptors, the loss of alpha1 subunit was accompanied by disruption of gamma2 subunit and gephyrin clustering, in spite of the increased alpha4 subunit expression. However, in the reticular nucleus, which lacks alpha1-GABA(A) receptors in wild-type mice, postsynaptic alpha3/gamma2/gephyrin clusters were unaffected. These results demonstrate that adaptive responses in the brain of alpha1(0/0) mice involve reorganization of GABAergic circuits and not merely replacement of the missing alpha1 subunit by another receptor subtype. In addition, clustering of gephyrin at synaptic sites in cerebellum and thalamus appears to be dependent on expression of a GABA(A) receptor subtype localized postsynaptically.     

Long-range interactions in neuronal gene expression: evidence from gene targeting in the GABA(A) receptor beta2-alpha6-alpha1-gamma2 subunit gene cluster

Clustering of GABA(A) receptor alpha1, alpha6, beta2, and gamma2 subunit genes on mouse chromosome 11/human chromosome 5 may have functional significance for coordinating expression patterns, but until now there has been no evidence for cross-talk between the genes. However, altering the structure of the alpha6 gene, specifically expressed in the cerebellum, with neomycin gene insertions in two different experiments unexpectedly reduced the expression of the widespread alpha1 and beta2 genes in the forebrain. There were corresponding reductions in the levels of alpha1 and beta2 subunit proteins and in autoradiographic ligand binding densities to GABA(A) receptors in the forebrain of alpha6-/- mice. The gamma2 mRNA level was not changed, nor were beta3 and delta mRNAs. The data suggest that elements in the neo gene may have an influence over long distances in the GABA(A) subunit gene complex on as yet undefined structures coordinating the expression of the alpha1 and beta2 genes.     

Five mutations in the GABA A alpha6 gene 5' flanking region are associated with a reduced basal and ethanol-induced alpha6 upregulation in mutated Sardinian alcohol non-preferring rats

The presence of four nucleotide changes and a three base-pair deletion in the GABA A alpha6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q alpha6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the alpha6 gene was investigated by measuring cerebellar alpha6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased alpha6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of alpha6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the alpha6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5' flanking alpha6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced alpha6 gene expression.     

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.     

Subunit composition of rat ventral spinal cord GABA(A) receptors, assessed by single cell RT-multiplex PCR

We analyzed the expression of native GABA(A) receptors in choline acetyltransferase and glutamic acid decarboxilase positive cells, from lamina IX of the lumbar region of rat spinal cord. More than one isoform of each subunit was detected within a single cell. The alpha3, alpha5, alpha1, beta3 and gamma2 subunit was the most frequent combination in both cell populations. However, the total number of subunit expressed by each cell type was different, being the ChAT positive cells the simplest. Interestingly, the ChAT and GAD positive cells also displayed a different pattern of distribution of both spliced isoforms of the gamma2 subunit. These results indicate that several GABA(A) receptors, with different molecular composition, are expressed in a single cell and that different cell types can express different GABA(A) receptors.     

Delayed upregulation of GABA(A) alpha1 receptor subunit mRNA in somatosensory cortex of mice following learning-dependent plasticity of cortical representations.

Experience-dependent modifications of cortical representational maps are accompanied by changes in several components of GABAergic inhibitory neurotransmission system. We examined with in situ hybridization to 35S-labeled oligoprobe changes of expression of GABA(A) receptor alpha1 subunit mRNA in the barrel cortex of mice after sensory conditioning training. One day and 5 days after the end of short lasting (3 daily sessions) training an increased expression of GABA(A) alpha1 mRNA was observed at the cortical site where the plastic changes were previously found. Learning associated activation of the cerebral cortex increases expression of GABA(A) receptor mRNA after a short post-training delays.     

Gephyrin-Independent Clustering of Postsynaptic GABAA Receptor Subtypes

Gephyrin has been shown to be essential for the synaptic localization of the inhibitory glycine receptor and major GABA(A) receptor (GABA(A)R) subtypes. However, in retina certain GABA(A)R subunits are found at synaptic sites in the absence of gephyrin. Here, we quantitatively analyzed GABA(A)R alpha1, alpha2, alpha3, alpha5, beta2/3, and gamma2 subunit immunoreactivities in spinal cord sections derived from wild-type and gephyrin-deficient (geph -/-) mice. The punctate staining of GABA(A)R alpha1 and alpha5 subunits was unaltered in geph -/- mice, whereas the numbers of alpha2-, alpha3-, beta2/3-, and gamma2-subunit-immunoreactive synaptic sites were significantly or even strikingly reduced in the mutant animals. Immunostaining with an antibody specific for the vesicular inhibitory amino acid transporter revealed that the number of inhibitory presynaptic terminals is unaltered upon gephyrin deficiency. These data show that in addition to gephyrin other clustering proteins must exist that mediate the synaptic localization of selected GABA(A)R subtypes.     

Congenital myasthenic syndrome caused by a mutation in the Ets-binding site of the promoter region of the acetylcholine receptor epsilon subunit gene.

Forty-two missense, truncation, or splice-site mutations of the acetylcholine receptor (AChR) subunit genes have been reported to date in patients with congenital myasthenic syndromes. Here we report a homozygous mutation, epsilon-155G --> A, in the promoter region of the AChR epsilon subunit gene that converts the Ets-binding site of the promoter region from CGGAA to CAGAA. The asymptomatic parents and brother are heterozygous and an affected sister is homozygous for epislon-155G --> A. The Ets-binding site mediates synapse specific expression of the AChR epsilon subunit gene. An identical G-to-A mutation in the mouse Ets-binding site was previously shown to decrease the binding affinity of the Ets-binding site for the GA binding protein, a transactivating factor for the Ets-binding site, and to reduce the synapse specific expression of the epsilon subunit. The decreased synaptic expression of the epsilon subunit readily accounts for the congenital myasthenic phenotype.     

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.     

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.     

Glycine and GABA(A) receptor subunits on Renshaw cells: relationship with presynaptic neurotransmitters and postsynaptic gephyrin clusters

Inhibitory synapses with large and gephyrin-rich postsynaptic receptor areas are likely indicative of higher synaptic strength. We investigated the presynaptic inhibitory neurotransmitter content (GABA, glycine, or both) and the presence and subunit composition of GABA(A) and glycine postsynaptic receptors in one example of gephyrin-rich synapses to determine neurochemical characteristics that could also contribute to enhance synaptic strength. Hence, we analyzed subunit receptor expression in gephyrin patches located on Renshaw cells, a type of spinal interneuron that receives powerful excitatory and inhibitory inputs and displays many large gephyrin patches on its surface. GABA(A) and glycine receptors were almost always colocalized inside Renshaw cell gephyrin clusters. According to the subunit-immunoreactivities detected, the composition of GABA(A) receptors was inferred to be either alpha(3)beta((2or3))gamma(2), alpha(5)beta((2or3))gamma(2), alpha(3)alpha(5)beta((2or3))gamma(2) or a combination of these. The types of neurotransmitters contained inside boutons presynaptic to Renshaw cell gephyrin patches were also investigated. The majority (60-75%) of terminals presynaptic to Renshaw cell gephyrin patches contained immunocytochemical markers for GABA as well as glycine, but a proportion contained markers only for glycine. Significantly, 40% of GABA(A) receptor clusters were opposed to presynaptic boutons that contained only glycinergic markers. We postulate that GABA and glycine corelease, and the presence of alpha3-containing GABA(A) receptors can enhance the postsynaptic current and contribute to strengthen inhibitory input on Renshaw cells. In addition, a certain degree of imprecision in the localization of postsynaptic GABA(A) receptors in regard to GABA release sites onto adult Renshaw cells was also found.