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

Previous double-label studies have demonstrated that progesterone receptors (PR) may coexist with nitric oxide synthase (NOS) and also with neuroactiv e peptides such as somatostatin (SOM), neurotensin (NT) and substance P (SP ) in neurons of the ventrolateral hypothalamic nucleus (VL) of the guinea p ig. In the present study, triple-label immunofluorescence histochemistry wa s used to determine whether neurons co-expressing both PR and NOS also cont ain one neuropeptide (SOM, NT or SP) and to quantify these colocalization r elationships. Ovariectomized guinea pigs were primed with estradiol to indu ce PR immunoreactivity and treated with colchicine to visualize immunoreact ive (IR) peptidergic neurons. Using three primary antibodies raised in diff erent species and labeled with three different markers, it is possible to r ecognize which type(s) of immunoreactivity each cell contains on the same s ections. 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 o f the VL. Combining our various quantitative observations, we found that SO M immunoreactivity appeared in 33% of the PR/NOS-IR neurons in the caudal a spect of the nucleus and that nearly all neurons containing SOM and NOS con tained PR. The occasional localization between PR, NOS and SP seen in the r ostral subdivision of the VL represented a very low proportion of the PR/NO S-IR neurons but 48% of NOS/SP-IR neurons. The relatively high proportion o f 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 funct ions. (C) 1999 Elsevier Science B.V. All rights reserved.