Neuroendocrine cells express high levels of neuropeptide genes and thus are useful models to examine regulation of gene expression in the CNS. We have found that a wide variety of postnatal neuroendocrine cells survive and express specific neuropeptide genes in organotypic slice- explant cultures. We have characterized luteinizing hormone-releasing hormone neurons (LHRH) and oxytocin (OT) neurons in these cultures maintained in serum-free media, in the presence or absence of tetrodoxin (TTX). Using defined media and TTX, we examined the effects of estradiol (E2) treatment on peptide mRNA levels in LHRH neurons and OT neurons using semiquantitative in situ hybridization histochemistry. Results indicate that E2 acts differentially on LHRH gene expression, depending on the anatomical location of the LHRH cell examined. For most LHRH cells, the effect of E2 was blocked by TTX, indicating that the action of E2 on LHRH gene expression was via an interneuron. However, in one anatomical region, the effect of E2 was maintained in the presence of TTX, suggesting that a small subpopulation of LHRH cells can respond to E2 directly. We are currently investigating this phenomenon in vivo and in vitro by analysis of neuronal phenotypes expressing E2-receptor mRNA. In addition, using these cultures in defined media +/- TTX, we are now examining the signal transduction pathways active in these peptidergic neurons. Currently, we are studying the effects of forskolin and/or phorbol 12-myristate, 13-acetate treatment on both LHRH and OT mRNA maintained in organotypic cultures. Analysis of these explants are currently being done. Finally, by retrograde labeling cells which project to circumventricular organs in vivo and then generating cultures, we are able to identify a subpopulation of neuroendocrine cells in situ. Using this technique, we are patch-clamping, recording and marking neuroendocrine cells whose efferent projections are known. After characterizing the electrophysiological properties of these cells we will identify their neuropeptide phenotype.
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