Quantitative studies of progesterone receptor and nitric oxide synthase colocalization with somatostatin, or neurotensin, or substance P in neurons of the guinea pig ventrolateral hypothalamic nucleus: an immunocytochemical triple-label analysis.

Abstract

Previous double-label studies have demonstrated that progesterone receptors (PR) may coexist with nitric oxide synthase (NOS) and also with neuroactive peptides such as somatostatin (SOM), neurotensin (NT) and substance P (SP) in neurons of the ventrolateral hypothalamic nucleus (VL) of the guinea pig. In the present study, triple-label immunofluorescence histochemistry was used to determine whether neurons co-expressing both PR and NOS also contain one neuropeptide (SOM, NT or SP) and to quantify these colocalization relationships. Ovariectomized guinea pigs were primed with estradiol to induce PR immunoreactivity and treated with colchicine to visualize immunoreactive (IR) peptidergic neurons. Using three primary antibodies raised in different species and labeled with three different markers, it is possible to recognize which type(s) of immunoreactivity each cell contains on the same sections. We observed that PR, NOS and SOM co-occurred extensively, whereas PR, NOS and NT or PR, NOS and SP were colocalized infrequently in neurons of the VL. Combining our various quantitative observations, we found that SOM immunoreactivity appeared in 33% of the PR/NOS-IR neurons in the caudal aspect of the nucleus and that nearly all neurons containing SOM and NOS contained PR. The occasional localization between PR, NOS and SP seen in the rostral subdivision of the VL represented a very low proportion of the PR/NOS-IR neurons but 48% of NOS/SP-IR neurons. The relatively high proportion of PR/NOS-IR cells expressing SOM in the VL suggests that these neurons are part of a neural circuitry involved in a variety of steroid-dependent functions.