Effects of gamma-aminobutyric acid receptor agonists and antagonist on LHRH-synthesizing neurons as detected by immunocytochemistry and in situ hybridization.

Abstract

Gamma-aminobutyric acid (GABA) is thought to play an important role in the regulation of luteinizing hormone-releasing hormone (LHRH) release but its role in the regulation of LHRH gene expression and LHRH synthesis is not known. We hypothesized that since GABA appears to have primarily inhibitory effects on LHRH cells (at the level of the cell body), GABA may act to decrease LHRH gene expression and peptide synthesis. This hypothesis was tested by examining the effect of GABA receptor activation and GABA receptor blockade on LHRH mRNA and peptide levels employing in situ hybridization and immunocytochemistry. Cells in the preoptic area (POA) of ovariectomized (ovx) rats were selectively exposed in vivo to specific GABA-ergic receptor agonists or an antagonist for up to 24 h. THIP, a specific GABAA receptor agonist, did not have a significant effect on either the intensity of LHRH immunoreactivity, or the number of LHRH-ir cells, observed as compared to controls. Baclofen, a GABAB receptor agonist appeared to decrease the number of cells with the greatest intensity of LHRH immunoreactivity, compared to controls. In situ hybridization, with either a tritiated RNA probe or a 32P-labelled oligonucleotide, complementary to LHRH mRNA, revealed that THIP either had no effect on the labelling intensity (32P-labelled oligonucleotide) or (contrary to our hypothesis) a slight excitatory effect on the level of LHRH mRNA detected per cell (tritiated RNA probe). Bicuculline (a specific GABAA receptor antagonist) decreased both the number of labelled cells observed per section through the POA, and the intensity of labelling observed in sections hybridized with the 32P-labelled oligonucleotide. These results suggest that in the POA GABAA receptors do not exert an inhibitory effect on LHRH gene expression, but rather could affect LH perhaps by electrically inhibiting LHRH neurons. In contrast, baclofen appeared to exert an inhibitory effect on LHRH gene expression, since the number of grains per labelled cell in the POA of baclofen-treated rats was lower than the grains per labelled cell of control rats. Also, similar to the results obtained with immunocytochemistry, in situ hybridization following baclofen treatment suggested that activation of GABAB receptors is able to reduce the number of neurons with the highest levels of LHRH mRNA. Thus, in the POA, GABA acting through GABAB receptors could be effective through changes in mRNA or peptide synthesis.