Prolactin (PRL) is an anterior pituitary hormone and acts through receptors present in the hypothalamus as well as in other regions of the brain. The aim of this experiment was to investigate whether these prolactin receptor (PRLR)-expressing neuronal populations co-express the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). We examined this using three complimentary approaches; (1) dual label in situ hybridisation for glutamic acid decarboxylase (Gad) (a marker of GABAergic neurones) and PRLR mRNA, (2) immunohistochemistry for phosphorylated STAT5 (pSTAT5), a marker of PRLR activation, in reporter mice that express tdTomato fluorescent protein in GABAergic neurones (under Vgat promoter control) and (3) immunohistochemistry for pSTAT5 and green fluorescent protein (GFP) in conditional knock-out mice that are lacking PRLRs in GABAergic neurones (Vgat-cre, PRLRlox/lox). These mice express cre-recombinase specifically in GABAergic neurones which catalyses inversion of the floxed fragment of the PRLR gene and thus removes functional PRLRs selectively from this cell type. Cre-mediated recombination can be detected by activation of expression of GFP from the modified locus.
Taken together, the results from these three approaches have identified PRL-sensitive GABAergic neurones in the AVPV/PeN, mPOA, PVN, DMN, MePD and the Arc. PRL-induced labeling for pSTAT5 was present in both Vgat-cre, PRLRlox/lox mice and control mice (PRLRlox/lox) but the levels were significantly reduced in the mPOA and MePD of Vgat-cre, PRLRlox/lox mice.
To investigate whether tuberoinfundibular dopaminergic (TIDA) neurones, that are known to be PRL-responsive, were also GABAergic, we evaluated the phenotype of GABAergic neurones activated by PRL in the reporter mouse. Triple label fluorescence immunohistochemistry for pSTAT5, tdTomato and tyrosine hydroxylase showed that recombination occurred in the arcuate nucleus but the majority of PRL-sensitive GABAergic neurones were not TIDA neurones.
These results show that PRLRs are co-localised with GABAergic neurones throughout the hypothalamus but the level of co-expression varies across different nuclei.