A DUAL-IMMUNOCYTOCHEMICAL METHOD TO LOCALIZE C-FOS PROTEIN IN SPECIFIC NEURONS BASED ON THEIR CONTENT OF NEUROPEPTIDES AND CONNECTIVITY

Enhanced expression of the immediate early gene c-fos has been used as a marker of cellular activation in many different neuronal pathways. We wished to determine the neurochemical content and the connectivity of neurons, in which expression of c-fos is induced. For this purpose, a dual-immunocytochemical staining technique has been developed with avidin-biotin-peroxidase labelling using diaminobenzidine as the chrom ogen for c-fos protein located in the nucleus, and benzidine dihydroch loride (BDHC) in the presence of sodium nitroprusside to reveal cytopl asmic antigens (neuropeptide or retrograde tracer) in the same section . The blue granular BDHC reaction product in the cytoplasm combined wi th the homogeneous brown nuclear DAB staining for c-fos protein provid es excellent resolution of dual-labelled cells even in tissue sections of 40 mu m in thickness. The high sensitivity of the avidin-biotin-pe roxidase immunocytochemistry and the stability of the reaction product s provide an excellent tool for quantitative analysis of stimulated ce lls within a neurochemically defined cell group. The BDHC/DAB protocol was developed to identify activated cells in three experimental situa tions. Firstly, to investigate the phenotype of light-activated cells in the suprachiasmatic nucleus of the hypothalamus, c-fos protein DAB staining was carried out together with BDHC staining for peptide histi dine isoleucine (PHI) and vasoactive intestinal peptide (VIP). Secondl y, to identify activated neurons in female Syrian hamsters at the time of the proestrous luteinizing hormone surge, c-fos protein staining w ith DAB was carried out in combination with BDHC staining for gonadotr ophin-releasing hormone (GnRH). In both these studies, cells which co- localized the peptide and c-fos protein in the nucleus could be identi fied unequivocally. Thirdly, to analyse projections of c-fos-immunorea ctive neurons, the retrograde tracer, cholera toxin subunit B (ChB) wa s pressure-injected into the piriform cortex of rats, which were there after fully kindled in the contralateral amygdala. The tract tracer wa s stained with BDHC as the chromogen. Due to the advantages of the dua l-labelling methodology, the combination of retrograde tracing and c-f os protein histochemistry provides an excellent method for identifying projecting and activated neurons in the same section.