Evidence for co-existence of CCK-8 and GnRH in neurons of the mesencephalic tegmentum in the chameleon M. Bennis a, *, J. Repe ´rant b , G. Tramu c a Unite ´ de Neurosciences du Comportement, De ´ partement de Biologie, Faculte ´ des Sciences Semlalia, BP. 15, dBd Prince My Abdallah, 40 000 Marrakech, Morocco b INSERM, U-106, Laboratoire de Neuromorphologie, De ´ veloppement et Evolution, Ho ˆ pital de la Salpe ´ trie ` re, Paris, France c Laboratoire de Neurocytochimie Fonctionnelle, Universite ´ de Bordeaux I, Talence, France Received 21 October 1997; received in revised form 4 December 1997; accepted 10 December 1997 Abstract A double-label immunofluorescence technique was used to demonstrate the co-localization of cholecystokinin-8 (CCK-8) and gonadotrophin-releasing hormone (GnRH) in individual neurons and processes of the chameleon brain. Co-localization was limited to a small population of cells in the dorsomedial tegmentum; in other regions of the brain, neurons were observed to be either CCK-8-immunopositive or GnRH-immunopositive but never both. However, double-labeled fibers and terminals were found to be distributed at a low density throughout the thalamus, the medial hypothalamus, the tegmentum and the spinal cord. These data provide the first indication for the co-localization of CCK-8 and GnRH, whose functional significance remains to be established.  1998 Elsevier Science Ireland Ltd. Keywords: Cholecystokinin-8; Gonadotrophin-releasing hormone; Co-localization; Dorsomedial tegmentum Peptides are widely distributed in the central nervous system of vertebrates (reviews in [6]). Several authors have described the co-localization of two or more peptides within individual neurons in many brain structures of a variety of vertebrate species [4,16,18,19]. This co-localiza- tion has led to the suggestion that the neuropeptides may play different roles in the nervous system, and may in some systems be involved in conventional neurotransmission as well as neuromodulation. In the chameleon, a gonadotrophin-releasing hormone (GnRH)-like neuropeptide is present in restricted cell groups of the central nervous system [1], one of these being located in the dorsomedial tegmentum. A cholecys- tokinin-8 (CCK-8)-like peptide is also widely distributed in the brain of this species, one group of immunoreactive neu- rons also lying in the dorsomedial tegmentum [2]. The aim of the present study is to provide evidence for a co-localization of CCK-8- and GnRH-like peptides both in neurons of the dorsomedial tegmentum and in brain stem fibers in the chameleon. Experiments were performed on six chameleons (Chame- leo chameleon) taken in the wild in southern Morocco. Under Nembutal anesthesia (75 mg/kg, intraperitoneal injection), each animal was perfused through the heart with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4. The brain was removed and stored in fresh fixative for 3 h and then placed in 30% sucrose in PB overnight at 4°C. Brains were sectioned on the frontal plane at 15–20 mm using a freezing microtome, collected and air dried onto gelatin coated-slides. Slides were washed in cold PB and then placed overnight at room temperature in a mixture of rabbit polyclonal anti-CCK-8 (1/1000) and guinea-pig anti- GnRH (1/5000) (generously provided by le Laboratoire de Neurocytochimie fonctionnelle, Bordeaux I, France) in PB containing 0.3% Triton X-100. Slides were then rinsed with PB and placed in the secondary antisera mixture containing rhodamine isothiocyanate (RITC)-conjugate swine anti-rab- bit IgG (1/100), fluorescein isothiocyanate (FITC)-conju- Neuroscience Letters 240 (1998) 155–158 0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(97)00939-7 * Corresponding author. Tel.: +212 4 434649; fax: +212 4 437412/ 436769.