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The purpose of the present study was to localize luteinizing hormone-releasing hormone (LHRH) mRNA within the male rat forebrain using an in situ hybridization approach. The expression of LHRH mRNA was compared in castrate and intact males to approach questions on the chronic influences of circulating testicular steroids on the gene expression of the peptide. Frozen 10 micron sections fixed in paraformaldehyde were obtained from the forebrain region of intact and 2 week post-castrate adult male rats. LHRH mRNA was autoradiographically detected using an oligomer (59mer) complementary to the mRNA coding for amino acids -5 to 15 of the human LHRH preprohormone. Individual brain sections were incubated in prehybridization buffer for 2 h to reduce nonspecific binding. Following this, 20 microliter of hybridization buffer containing 65,000-120,000 cpm of the 59mer were applied to sections and hybridized at 37 degrees C for 3 days. The sections were then rinsed over a 48 h period, dehydrated, dipped in Kodak NTB2 liquid emulsion and exposed for 22 days. Autoradiograms were developed and counterstained with fast green and cresyl violet. As reported in the female, LHRH message-containing cells were localized in ventral septal regions, the diagonal bands of Broca, preoptic area and anterior hypothalamus. On occasion, LHRH gene expressing cells were found to appear in loose clusters. Labeled cells were never found in control sections treated with hybridization buffer lacking the 59mer. The total number of LHRH mRNA-containing cells localized in intact rats did not differ significantly from the castrate group.(ABSTRACT TRUNCATED AT 250 WORDS)

was linear over the range of specific staining. When the optimum conditions were exceeded, marked overestimations of hormone levels occurred due to the detection of nonstained tissues (24). However, automated equipment has become available in which the microscopic image ofthe specimen is viewed by a modified television camera and the detected image is converted to output signals, which are rapidly evaluated and quantified with logic circuitry (8). A major limitation 12 GROSS, ROTHFELD in the use of such quantitative image analysis systems for the evaluation of histological preparations has been the requirement for high contrast between the specific features to be detected and the surrounding background (8). The unlabeled antibody immunocytochemical technique overcomes this lim-Literature Cited

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