Characterisation of GABAA receptor gamma subunit expression by magnocellular neurones in rat hypothalamus

Gamma-aminobutyric acid (GABA) is known to inhibit the electrical and secretory activity of oxytocin and vasopressin neurones located in the supraoptic and paraventricular nuclei following osmotic, cardiovascular or suckling stimuli. To understand fully the nature of GABA actions on these magnocellular neurones it is important to define the heteropentameric GABAA receptor proteins they express. In the present study, single and dual labelling in situ hybridisation and immunocytochemical experiments were undertaken to define the GABAA receptor gamma subunits expressed by these cells. In situ hybridisation with 35S-labelled antisense oligonucleotides showed that all magnocellular neurones in the supraoptic and paraventricular nuclei of the female rat expressed mRNA encoding the gamma 2 subunit of the GABAA receptor but not the gamma 1 or gamma 3 subunits. Immunocytochemical experiments using a specific polyclonal rabbit antibody directed against the gamma 2 subunit of the GABAA receptor showed that all hypothalamic magnocellular neurones were strongly immunoreactive for gamma 2 subunit protein. Dual in situ hybridisation experiments using the gamma 2 subunit 35 S-labelled oligonucleotide with alkaline phosphatase-labelled antisense oligonucleotides specific for either oxytocin or vasopressin revealed that essentially all oxytocin and vasopressin neurones in both the supraoptic and paraventricular nuclei expressed the gamma 2 subunit of the GABAA receptor. Similarly, sequential double immunoperoxidase staining revealed that all oxytocin and vasopressin neurones in both magnocellular nuclei of the hypothalamus were immunoreactive for the gamma 2 subunit. This study shows that only the gamma 2 subunit of the GABAA receptor gamma subunit family is expressed by hypothalamic oxytocin and vasopressin neurones. In conjunction with our previous results, these findings indicate that individual magnocellular neurones express a complement of alpha 1, alpha 2, beta 2, beta 3 and gamma 2 subunits of the GABAA receptor. The observation of strong gamma 2 subunit expression by neurones known to also express alpha 1 and alpha 2 subunit proteins suggests that these magnocellular cells may express GABAA receptors with both benzodiazepine type-1 and type-2 pharmacology.