Immunocytochemical detection of corticotropin-releasing factor: Multiple cross-reactions of a widely used carboxy-terminally directed corticotropin-releasing factor antiserum (code rC70) in rat hypothalamus

Abstract

Forty-one-residue corticotropin-releasing factor is a physiologically significant mediator of the hypothalamic control of corticotropin secretion by the anterior pituitary gland. This releasing hormone is produced by parvicellular neurons in the hypothalamic paraventricular nucleus that project to the external zone of the median eminence. Recent immunocytochemical evidence based on work with a rabbit antiserum against rat corticotropin-releasing factor (code rC70) suggests that about half of the parvicellular corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus synthesize vasopressin, another potent corticotropin secretagogue, while the rest of the cells do not. If this is indeed the case, the neurohumoral control of corticotropin release may be mediated via distinct hypothalamic effector pathways utilizing releasing hormone cocktails of varying composition. In the present study we have examined the specificity of various antisera against rat corticotropin-releasing factor in immunocytochemical staining. Male Wistar rats pretreated with colchicine were used throughout. The brain was fixed by perfusion with a Zamboni type fixative solution. Vibratome sections of the hypothalamus were immunostained with three different primary antisera (codes rC70, rCRF-3, oCRF-N) using the peroxidase-antiperoxidase or avidin-biotin complex methods. All three antisera stained cell groups previously described to be immunopositive for corticotropin-releasing factor. Most notably, however, rC70 labelled a significant number of additional cells, most readily identified in the arcuate and suprachiasmatic nuclei, as well as in the dorsolateral hypothalamic area caudal to the paraventricular nucleus. Liquid-phase preabsorption of rC70 with N- and C-terminal fragments of corticotropin-releasing factor revealed that the immunostaining in these latter areas could be blocked by the C-terminal hexapeptide of rat corticotropin-releasing factor, whereas in other hypothalamic regions the hexapeptide caused no noticeable change in the staining pattern. Moreover, the staining with rC70 in the arcuate nucleus and the dorsolateral hypothalamic area could be blocked by α-melanocyte stimulating hormone and that in the suprachiasmatic nucleus by peptide histidine/isoleucineamide. This may be readily explained by sequence homologies between the aforementioned peptides and the C-terminal hexapeptide of rat corticotropin-releasing factor. Thus, we communicate yet another case where an apparently highly specific antiserum may contain antibodies that recognize small portions (two amino acid residues) of the original antigen even when these are presented within relatively small and otherwise structurally unrelated neuropeptide transmitters.