A monoclonal antibody directed against CLIP (ACTH 18-39). Anatomical distribution of immunoreactivity in the rat brain and hypophysis with quantification of the hypothalamic cell group

After the recent demonstration of the facilitatory effect exerted by corticotropin-like intermediate lobe peptide (CLIP or adrenocorticotropic hormone (ACTH) 18-39) on paradoxical sleep in the rat (Chastrette and Cespuglio, 1985), we undertook the production of monoclonal Antibodies against this peptide. Wistar rats were immunized against CLIP and their spleen cells fused with mouse myeloma cells. After recloning, 25 supernatants were found to give positive immunohistochemical reactions in the rat brain. In immunohistochemical tests performed by preabsorption, the 25 supernatants presented similar properties, i.e. recognized CLIP, ACTH (1-39) and ACTH (25-39), but not ACTH (1-24) and the C-terminal fragment (34-39). We assume that the epitope(s) recognized by the 25 supernatants is (are) located between the amino-acids Asn25 and Ala34 of the CLIP molecule. The immunoreactivity observed in the rat brain and hypophysis with this antibody was distributed with a pattern quite similar to that described for anti-ACTH Antibodies. A main group of immunoreactive cell bodies was located in the mediobasal hypothalamus and a small group in the nucleus of the solitary tract. Immunoreactive fibres were distributed from the olfactory nucleus to the spinal cord and formed particularly rich networks in the hypothalamus and preoptic area. Among Other locations, immunoreactive axons were also present in the brainstem centres involved in the control of the sleep-waking cycle, which is in accordance with the influence of CLIP on paradoxical sleep. Using Abercrombie's formula, the number of immunoreactive cells in the mediobasal hypothalamus was estimated at about 3000 neurons. We conclude that our monoclonal anti-CLIP antibody can be considered as a good marker of proopiomelanocortin neurons.