Francisco Araujo

GABAA receptor subunit expression changes in the rat cerebellum and cerebral cortex during aging

Significant aging-related decreased expression of various GABAAR subunit mRNAs (alpha 1, gamma 2, beta 2, beta 3 and sigma) was found in both cerebellum and cerebral cortex using quantitative dot blot and in situ hybridization techniques. Contrary to the other subunits, the alpha 6 mRNA expression was significantly increased in the aged cerebellum. Parallel age-related changes in protein expression for gamma 2 and beta 2/3 (decrease) and alpha 6 (increase) were revealed in cerebellum by quantitative immunocytochemistry. However, no significant changes in alpha 1 protein expression nor in the number or affinity of [3H]zolpidem binding sites were detected in cerebellum even though alpha 1 mRNA expression was significantly decreased in the aged rat. Age-related increased expression of alpha 6 mRNA and protein in the cerebellum was accompanied by no significant changes in the number of diazepam-insensitive [3H]Ro15-4513 binding sites. In the cerebral cortex, no changes in the protein expression of the main GABAA receptor subunits (alpha 1, gamma 2 and beta 2/3) were observed which contrasted with the age-related decreased expression of the corresponding mRNAs. No significant changes in the number or affinity of [3H]zolpidem binding sites were observed in the cerebral cortex. Thus, age-related changes in the mRNA expression of a particular subunit does not necessarily lead to similar changes in protein or assembly into mature GABAA receptors. The results reveal the existence of complex regulatory mechanisms of GABAA receptor expression, at the transcriptional, translational and post-translational and/or assembly levels, which vary with the subunit and brain area.

Molecular and Pharmacological Characterization of Native Cortical γ-Aminobutyric AcidA Receptors Containing Both α1 and α3 Subunits

We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical α1-α3 γ-aminobutyric acidA (GABAA) receptors using subunit-specific antibodies. The co-existence of α1 and α3 subunits in native GABAA receptors was demonstrated by immunoblot analysis of the anti-α1- or anti-α3-immunopurified receptors and by immunoprecipitation experiments of the [3H]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the α1-α3 GABAA receptors represented 54.7 ± 5.0 and 23.6 ± 3.3% of the α3 and α1 populations, respectively. Therefore, α1 and α3 subunits are associated in the same native GABAA receptor complex, but, on the other hand, these α1-α3 GABAA receptors from the cortex constitute a large proportion of the total α3 population and a relatively minor component of the α1 population. The pharmacological analysis of the α1- or α3-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and Cl 218,872. These results indicate the existence of native GABAA receptors possessing both α1 and α3 subunits, with α1 and α3 subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-α1-anti-α3 double-immunopurified receptors demonstrated the presence of stoichiometric amounts of α1 and α3 subunits, associated with β2/3, and γ2 subunits. The pharmacological analysis of α1-α3 GABAA receptors demonstrated that, despite the fact that each α subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [3H]Ro15-4513-photolabeled peptides was 70:30. Therefore, the α1 subunit is pharmacologically predominant over the α3 subunit. These results indicate the existence of active and nonactive α subunits in the native α1-α3 GABAA receptors from rat cortex.

Absence of association between δ and γ2 subunits in native GABAA receptors from rat brain

We investigated the possible association between δ and γ2 subunits in native GABAA receptors, from different rat brain regions, using subunit-specific anti-δ and anti-γ2 antibodies. Previous reports have provided somewhat controversial results, indicating both the presence and the absence of association between these two subunits in native receptors. Our results indicate the absence of co-localization between δ and γ2 subunits. In immunoprecipitation experiments, anti-δ antibody consistently immunoprecipitated []muscimol binding activity (GABA binding sites) from all brain areas tested (10–20% of the total binding). However, under the same conditions, no significant []flumazenil or []ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a]-[1,4]benzodiazepine-3-carboxylate (Ro15-4513) binding (benzodiazepine binding sites) activity was detected in the immunopellets. These results indicate the absence of association between δ and γ2 subunits. This question was directly addressed by immunopurification and Western blot experiments. As expected, no γ2 subunits were detected in anti-δ immunoaffinity purified receptors. Conversely, no δ subunits were identified in anti-γ2 immunopurified receptors. Thus, these results demonstrate the absence of association between δ and γ2 subunits in native GABAA receptors. Finally, our results also indicate the relevance of the solubilization conditions on the apparent association between different subunits of the native GABAA receptor complex.

Molecular characterization of Type I GABAA receptor complex from rat cerebral cortex and hippocampus

The molecular composition of the native gamma-aminobutyric acidA (GABAA) receptor complex is actually unknown. In the present communication we report a novel approach to characterize the minimal molecular conformation of the native GABAA receptor complex. This novel approach is based on the combination of subunit specific antibodies and specific 3H-labeled ligands in immunoprecipitation experiments. We have determined the presence of beta 2/3 and gamma 2 subunits in the Type I GABAA receptor complex, from rat cerebral cortex and hippocampus, by using two antibodies, the monoclonal 62-3G1 (specific for beta 2/3) and the polyclonal anti-gamma 2 (to the large intracellular loop of the gamma 2 short form) together with the Type I-specific ligand [3H]zolpidem. The association of gamma 2 and beta 2/3 subunits with the GABAA receptor complex was also tested using [3H]flumazenil. The results indicated that both gamma 2 and beta 2/3 were the most abundant subunits associated to either Type I or total benzodiazepine receptors from both cortex and hippocampus. Between 70-80% of Type I or total benzodiazepine binding activity was immunoprecipitated by either antibody. In addition, we have also investigated the coexistence of both subunits as part of the same population of Type I GABAA receptor complex by cross-immunoprecipitation experiments with 62-3G1 and anti-gamma 2. The results indicated that, in cerebral cortex, both gamma 2 and beta 2/3 subunits were part of the same population of Type I receptors. In hippocampus, an additional 20% of Type I receptors displayed either gamma 2 or beta 2/3 but not both subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related modifications on the GABAA receptor binding properties from Wistar rat prefrontal cortex

In the present communication we have investigated the pharmacological properties of the GABAA receptor from adult (3 months old) and aged (24 months old) Wistar rat prefrontal cortex. The prefrontal cortex is implicated in cognitive functions and stress and both processes seem to be altered during aging. These changes could be mediated by modifications in the GABAA receptor properties. Our results indicated the absence of generalized age-related modifications on the pharmacological properties of the GABAA receptor from prefrontal cortical membranes. Saturation experiments using the non-selective benzodiazepine [3H]flunitrazepam revealed that neither the Kd values or the Bmax were modified during aging. Moreover, Cl 218 872 displacement of [3H]flunitrazepam showed no age-related modifications on either the Kis or the relative proportion between the Type I and Type II benzodiazepine binding sites. Therefore, the benzodiazepine binding sites are well preserved in aged prefrontal cortex. On the other hand, saturation experiments using the GABA agonist [3H]muscimol demonstrated in the Bmax of the low affinity [3H]muscimol binding sites in aged rats (4.3 +/- 0.8 pmol/mg protein vs. 2.3 +/- 0.2 pmol/mg protein in adult and aged rats, respectively). However, no age-dependent modifications were observed in the allosteric interaction between GABA and benzodiazepine binding sites. These results demonstrate that the benzodiazepine binding sites and the GABA binding sites of the GABAA receptor complex from rat prefrontal cortical membranes are differentially affected by the aging process.

Distribution of the γ-aminobutyric acidA receptor complex alpha 5 subunit in chick brain. An immunocytochemical and autoradiographic study

Abstract This work reports the distribution of the γ-aminobutyric acidA (GABAA) receptor complex α5 subunit in the chick using an antibody raised against this subunit in the rat, an immunoprecipitation study and a comparative autoradiographic study using [3H]flunitrazepam in the presence of 1 μM zolpidem, which is considered to bind only to those areas presenting the α5 subunit. The specificity of the antibody for the chick GABAA receptor complex α5 subunit is supported by the similar bands obtained by Western blotting from rat and chick, the immunoprecipitation study and the general agreement in the distribution and pattern of labelling of this antibody in both species. The immunocytochemical and autoradiographic distributions in both the chick and rat are compared and some areas with disagreement between these distributions are discussed. The general conclusion is that the α5 subunit of the GABAA complex receptor seems to have been conserved along evolution.