Different postnatal development profiles of neurons containing distinct GABAA receptor beta subunit mRNAs (beta 1, beta 2, and beta 3) in the rat forebrain.

The expression of three beta subunit (beta 1, beta 2, and beta 3) mRNAs for gamma-aminobutyric acidA receptor in the postnatal rat forebrain was examined by in situ hybridization histochemistry with probes synthesized for the respective subunit mRNAs. The developmental expression of these subunit mRNAs conformed to one of three patterns. Pattern I was high expression of the mRNA at birth and a constant or increasing expression thereafter. In contrast, pattern II was no or very low expression of the mRNA at birth, with expression quickly increasing to reach the adult level in the early postnatal period. Pattern III was the transient expression of the subunit mRNA or else a marked decrease of its expression after a peak in the early postnatal period. On the basis of this classification, the expression of beta 3 subunit mRNA followed pattern I in most regions of the forebrain, such as the isocortex, the olfactory bulb and some of its related areas, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the caudate-putamen, the nucleus accumbens, the globus pallidus, the ventral pallidum, and the hypothalamus. In some areas, such as the magnocellular preoptic nucleus, the thalamus, and the subthalamic nucleus, pattern III was seen for this subunit. However, none of the regions of the brain showed pattern II expression of beta 3 subunit mRNA. In contrast, the expression of beta 1 and beta 2 subunit mRNAs followed pattern II in most regions of the forebrain. These included the expression of beta 1 subunit mRNA in the isocortex, the olfactory bulb, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the thalamus, and the hypothalamus, and the expression of beta 2 subunit mRNA in the isocortex, the olfactory bulb and some of its related areas, the amygdala, the nucleus of the diagonal band, the caudate-putamen, the thalamus, and the hypothalamus. Pattern I was not found for beta 1 subunit mRNA, although it was seen in some areas for beta 2 subunit mRNA, such as the ventral pallidum, the globus pallidus, and the magnocellular preoptic nucleus. On the other hand, pattern III was followed by beta 1 subunit mRNA in the anterior olfactory nucleus, the olfactory tubercle, and the piriform cortex, and the same pattern for the beta 2 subunit was also found in the olfactory tubercle, the hippocampal formation, the septum, the bed nucleus of the stria terminalis, and the nucleus accumbens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential expression of GABAA/benzodiazepine receptor beta 1, beta 2, and beta 3 subunit mRNAs in the developing mouse cerebellum.

Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian cerebellum. Cerebellar granule, Purkinje, and deep nuclear neurons are known to receive GABAergic afferents. Since GABA exerts its inhibitory effects via GABA receptors, it is of interest to determine the temporal relationship between the formation of GABAergic synapses and the expression of genes coding for the GABA receptor. In a previous study, we have examined the developmental expression of binding sites for [3H]muscimol, which binds with high affinity to the beta subunits of the GABAA/benzodiazepine (GABAA/BZ) receptor. In the present study, [35S]cRNA probes were used to examine the appearance and distribution of GABAA/BZ beta 1, beta 2, and beta 3 subunit mRNAs in the developing C57BL/6 mouse cerebellum by in situ hybridization. In the adult cerebellum, the distribution of the three subunit mRNAs was clearly different, despite considerable overlap, and their temporal expression differed throughout postnatal development. The beta 1 hybridization signal appeared within the cerebellar cortex during the second postnatal week as a discrete band at the interface of the molecular and granule cell layers. Grains were distributed diffusely over small densely staining cells surrounding the Purkinje cells; relatively few grains were visible over Purkinje cell bodies themselves. This distribution may reflect an association with Bergmann glia or basket cells. The beta 2 and beta 3 hybridization signals were present considerably earlier than that of the beta 1 mRNA. The beta 2 signal was present at birth in the molecular/Purkinje cell layer; as development progressed, the signal became increasingly intense over both granule and Purkinje cells. At birth, the beta 3 subunit mRNA was present in the external germinal and molecular layers, later becoming largely localized within the granule cell layer. Dense beta 2 and beta 3 cRNA probe labeling was present over the adult granule cell layer. Moderate levels of beta 2 signal were seen over Purkinje cell bodies; considerably less labeling was observed with the beta 3 probe. The adult distribution of beta 2 and beta 3 cRNA probes showed good spatial correspondence with the known GABAA receptor beta subunit markers, [3H]-muscimol and the mAb 62-3G1 antibody, each being present within the granule cell layer. Our results indicate that the temporal expression of GABAA/BZ receptor beta subunit messages within a given cell type may be independently regulated, and that acquisition of the beta 2 and beta 3 mRNAs occurs before these cells become integrated into mature synaptic circuits.     

Postnatal ontogenesis of neurons containing GABAA alpha 1 subunit mRNA in the rat forebrain.

The expression of GABAA receptor alpha 1 subunit mRNA in the postnatal rat forebrain was examined by in situ hybridization histochemistry. In most regions, including the isocortex, olfactory bulb, amygdala, septum, nucleus of the diagonal band, bed nucleus of the stria terminalis, basal ganglia, thalamus, and hypothalamus, the expression of alpha 1 subunit mRNA was low at birth but showed a dramatic increase during the early postnatal period. Adult levels of expression were reached at around the second or third week of life in these regions. However, in the caudate-putamen, and the nucleus accumbens, the expression of this subunit was only transient.     

Postnatal development of GABAA receptor beta1, beta2/3, and gamma2 immunoreactivity in the rat retina.

GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the mammalian central nervous system and plays an important role in neuronal physiology during ontogenesis. The distribution of the beta1-, beta2/3-, and gamma2-subunit of the GABAA receptor in the rat retina was studied during postnatal development using immunohistochemical methods. All subunits were found at birth. However, each subunit showed a unique staining pattern with a different local distribution. The immunoreactivity pattern changed during the time course of postnatal development for each of the proteins investigated. A clustered distribution at presumptive synaptic sites as indicated by a punctate staining pattern of the inner plexiform layer was detected as early as the second day of postnatal development. However, diffuse staining of presumptive extrasynaptic sites was found throughout development. The typical adult layering of immunoreactivity into distinctive bands appeared later in development, characteristically in the second postnatal week. The results of the present study suggest that GABAA receptor expression precedes the formation of functional synapses and changes along with cellular differentiation of the rat retina. Developmentally regulated changes in GABAA receptor composition and distribution indicate possible functions for this receptor during retinal ontogeny.     

Postnatal development of the alpha1 containing GABAA receptor subunit in rat hippocampus

Here we have studied the developmental expression of alpha1 subunit of the GABAA receptor in comparison with the expression of alpha2 subunit and several GABAergic markers (parvalbumin (PV), calretinin (CR), somatostatin (SOM), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP)). The alpha1 expression (mRNA and protein) was low at birth and increased progressively until the adulthood. This expression pattern was similar to that observed for PV, opposite to that of CR (high at birth and decreased continuously until the adulthood) and differed from that observed for the alpha2 and neuropeptides (SOM, NPY and VIP) (in all cases, a clear peak in expression was observed at P10). We further investigated the expression of alpha1, PV and CR by immunohistochemistry. As expected, the alpha1 and the PV expression were low at birth and increased progressively until the adulthood. Both alpha1 and PV were co-expressed by the same interneuronal population, however, the maturation of the alpha1 subunit preceded to that of PV. Finally, we observed a gradient of maturation between the different fields of the hippocampus proper (CA2-3 preceded to CA1 and DG). This gradient could be related to the high expression of CR positive cells and fibers during the first 10 postnatal days, located principally in the stratum lacunosum moleculare of the CA2-3 layers.     

Transient expression of GABAA receptor subunit mRNAs in the cellular processes of cultured cortical neurons and glia.

In this study, we have studied by in situ hybridisation histochemistry the expression and intracellular distribution of the GABAA receptor subunit mRNAs in cultured neurons obtained from postnatal day 1-3 rats in order to determine how neurotransmitter receptor expression may be regulated during development of the nervous system. In postnatal cortical cells, we found that GABAA receptor subunit mRNAs coding for alpha2, alpha5, beta2, beta3 and gamma2 subunits were transiently expressed in the cellular processes and growth cones after 1-3 days in culture. These observations indicate that GABAA receptor subunit mRNAs are transported (or trafficked) into the cellular processes of early postnatal cortical cells. These selective localisations were rarely observed after 5 days in culture and only in cells which had not made cell-to-cell contact. The localisation of subunit mRNAs in the processes was more effectively maintained up to 5 days or even longer if cell-to-cell contact was avoided by culturing the cells at low density or by inhibiting neurite sprouting pharmacologically with the GABA receptor channel antagonist TBPS. Finally, immunocytochemistry revealed the expression of GABAA receptors in the growth cones of pyramidal neurons in culture. Thus, the expression of mRNA correlates to the expression of protein. These results suggest that the selective trafficking of GABAA receptor subunit mRNAs during synaptogenesis may be regulated by synapse formation and/or glial-neural communication.     

Developmental profile of polyadenylated and non-polyadenylated GABAA receptor subunit mRNAs.

The ratio of mRNA not selected for polyadenylation (non-poly(A)+ selected) to mRNA selected for polyadenylation (poly(A)+) for the beta 1, alpha 1 and gamma 2 subunits of the GABAA receptor complex was examined in rats as a function of age. RNA was extracted from whole brain of rats that were either 0, 1, 3, 5 or over 60 days of postnatal age. Poly(A)+ mRNA was purified by oligo(dT)-cellulose chromatography. Non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor beta 1, alpha 1 and gamma 2 subunits were examined by Northern blot analysis using cDNA probes specific for these subunits. Levels of GABAA receptor beta 1 subunit mRNA were also examined by solution hybridization analysis with a beta 1 riboprobe. Analysis of Northern blots revealed that levels of poly(A)+ beta 1 subunit mRNA were highest at 0 days of age, but decreased and reached adult levels by 5 days of postnatal age. However, levels of the beta 1 subunit message extracted from non-poly(A)+ selected mRNA were not significantly different at any of the ages examined, suggesting the existence of a population of beta 1 subunit mRNA that is not polyadenylated. The age-related discrepancy between beta 1 subunit levels measured in non-poly(A)+ selected mRNA and poly(A)+ mRNA was also observed using solution hybridization analysis. In contrast, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the alpha 1 subunit of the GABAA complex increased from 0 days of age to adulthood. Similarly, levels of both non-poly(A)+ selected mRNA and poly(A)+ mRNA for the GABAA receptor gamma 2 subunit increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-expression of glycine receptor beta subunit and GABAA receptor gamma subunit mRNA in the rat dorsal root ganglion cells.

We examined the expression of the beta subunit mRNA of the glycine receptor and the gamma subunit mRNA of the GABAA receptor in the rat dorsal root ganglion (DRG) using in situ hybridization histochemistry with oligonucleotide probes. About 44% and 37% of the all DRG neurons were labeled by the probes for glycine receptor beta subunit and GABAA receptor gamma subunit mRNAs. Labeled neurons were mostly large cells that simultaneously expressed both glycine receptor beta subunit and GABAA receptor gamma subunit mRNA as demonstrated using consecutive sections. Thus, we suggest the possibility that both GABA and glycine presynaptically regulate the activity of neurons involved in low-threshold mechanoreception at axo-axonic synapses in the spinal cord.     

GABAA receptor beta3 subunit gene-deficient heterozygous mice show parent-of-origin and gender-related differences in beta3 subunit levels, EEG, and behavior

The homozygous knockout mouse for the beta3 subunit of the GABAA receptor has been proposed as a model for the neurodevelopmental disorder, Angelman syndrome, based on phenotypic similarities of craniofacial abnormalities, cognitive defects, hyperactivity, motor incoordination, disturbed rest-activity cycles, and epilepsy. Since most children with Angelman syndrome are autosomal heterozygotes of maternal origin, apparently through genomic imprinting, we used gabrb3-deficient heterozygote mice of defined parental origin to investigate whether this phenotype is also maternally imprinted in mouse. Whole brain extracts showed greatly reduced beta3 subunit levels in male mice of maternal origin but not in male mice of paternal origin. Females of both parental origin showed greatly reduced beta3 subunit levels. Heterozygotes did not exhibit hyperactive circling behavior, convulsions, or electrographically recorded seizures. EEGs showed qualitative differences among heterozygotes, with male mice of maternal origin demonstrating more abnormalities including increased theta activity. Ethosuximide inhibited theta bursts, suggesting an alteration in the thalamocortical relay. Carbamazepine induced EEG slowing in males and EEG acceleration in females, with a larger effect in paternal-origin heterozygotes. Evidence thus suggests both parent-of-origin and gender-related components in developmental regulation of beta3 expression, in particular, that the maternally-derived male heterozygote may carry a developmental modification resulting in less beta3 protein, which may reflect partial genomic imprinting of the gabrb3 gene in mice.     

Alcohol-related expectancies are associated with the D2 dopamine receptor and GABAA receptor beta3 subunit genes

Molecular genetic research has identified promising markers of alcohol dependence, including alleles of the D2 dopamine receptor (DRD2) and the GABAA receptor beta3 subunit (GABRB3) genes. Whether such genetic risk manifests itself in stronger alcohol-related outcome expectancies, or in difficulty resisting alcohol, is unknown. In the present study, A1+ (A1A1 and A1A2 genotypes) and A1- (A2A2 genotype) alleles of the DRD2 and G1+ (G1G1 and G1 non-G1 genotypes) and G1- (non-G1 non-G1 genotype) alleles of the GABRB3 gene were determined in a group of 56 medically ill patients diagnosed with alcohol dependence. Mood-related alcohol expectancy (AE) and drinking refusal self-efficacy (DRSE) were assessed using the Drinking Expectancy Profile (Manual for the Drinking Expectancy Profile, Behaviour Research and Therapy Centre, Brisbane, 1996). Patients with the DRD2 A1+ allele, compared with those with the DRD2 A1- allele, reported significantly lower DRSE in situations of social pressure. Similarly, lower DRSE was reported under social pressure by patients with the GABRB3 G1+ allele when compared to those with the GABRB3 G1- alleles. Patients with the GABRB3 G1+ allele also revealed reduced DRSE in situations characterized by negative affect than those with the GABRB3 G1- alleles. Patients carrying the GABRB3 G1+ allele showed stronger AE relating to negative affective change (for example, increased depression) than their GABRB3 G1- counterparts. Biological influence in the development of some classes of cognitions is hypothesized. The clinical implications, particularly with regard to patient-treatment matching and the development of an integrated psychological and pharmacogenetic approach, are discussed.     

Ontogeny of GABAA/benzodiazepine receptor subunit mRNAs in the murine inferior olive: transient appearance of beta 3 subunit mRNA and [3H]muscimol binding sites.

The GABAA/benzodiazepine receptor consists of at least four subunits, alpha, beta, gamma and delta, each comprised of several variants. The developmental expression of the alpha 1, beta 1-3, gamma 2 and delta subunits was studied in the murine inferior olivary nucleus by in situ hybridization with antisense cRNA probes. The postnatal appearance and distribution of [3H]flunitrazepam and [3H]muscimol binding sites, alpha and beta subunit-specific ligands respectively, were also studied autoradiographically. The beta 3 subunit was transiently expressed in each of the subnuclei of the inferior olive: The signal was strong at birth, increased throughout postnatal week 1 and rapidly declined thereafter to low adult levels. A similar pattern of labeling was observed with [3H]muscimol. Detectable levels of alpha 1 subunit mRNA hybridization signal and [3H]flunitrazepam binding sites were also present in the inferior olive at birth, decreasing thereafter. Low to moderate levels of beta 1, beta 2, and gamma 2 subunit mRNAs were present in olivary neurons throughout postnatal development, while delta mRNAs were largely absent. It has been reported previously that, during the 2nd postnatal week, the ratio of climbing fiber terminals to Purkinje cells is reduced from 3:1, as observed in neonates, to the 1:1 relationship observed in the adult cerebellar cortex. Our results raise the possibility that the subunit composition of the GABAA/benzodiazepine receptor in inferior olivary neurons undergoes changes during development, and that this process may be related to the elimination of multiple climbing fiber innervation of cerebellar Purkinje cells.     

GABAA receptors containing alpha 1 and beta 2 subunits are mainly localized on neurons in the ventral pallidum.

The gamma-aminobutyric acid (GABA) projection from the nucleus accumbens to the ventral pallidum (VP) is important in the regulation of locomotion. Thus, stimulation and inhibition of GABAA receptors in the VP can alter locomotor activity. To determine whether the GABAA receptors are located presynaptically on accumbens efferents to the VP or postsynaptically on neurons intrinsic to the VP two experiments were performed. In the first, quinolinic acid lesions of the nucleus accumbens did not alter [3H]muscimol binding in the VP, while lesions in the VP significantly reduced (60-80%) binding as measured by light microscopic receptor autoradiography. In the second experiment, in situ hybridization with oligonucleotide probes for mRNAs of the alpha 1 and beta 2 subunits of the GABAA receptor was examined in the nucleus accumbens and VP. No mRNA for either subunit was observed in the nucleus accumbens, although many positively labeled neurons were present within the VP. By contrast, a moderate to high density of cells in both the nucleus accumbens and VP contained mRNA for glutamic acid decarboxylase. These data argue that the majority of GABAA receptors in the VP are not located presynaptically on axonal terminals originating from neurons in the nucleus accumbens.