THE JOURNAL OF COMPARATWE NEUROLOGY 299:405-420 (1990) Hypothalamic Galanjn-Immunoreactive Neurons Projectingto the Posterior Lobe of the Rat Pituitary:ACombinedRetmgrade 'Ikacingandlinmunohktochemicd Study R. ARAI, B. ONTENIENTE, A. TREMBLEAU, M. LANDRY, ANDA. CALAS Departement de Cytologie, Institut des Neurosciences, CNRS UA 1199, Universitk Pierre et Marie Curie, F-75252 Paris Cedex 05 (R.A., A.T., M.L., A.C.), and INSERM U 161, F-75014 Paris Cedex (B.O.), France ABSTRACT To identify the galanin-immunoreactive neurons projecting to the posterior lobe of the pituitary in the rat hypothalamus, a retrograde tracer (complex of wheat germ agglutinin- enzymatically inactive horseradish peroxidase-colloidal gold) was injected into the posterior lobe of the pituitary. Sections of the hypothalamus were treated with a combination of silver enhancement of retrogradely transported tracer and immunohistochemistry of galanin. Of the total number of hypothalamic cells doubly labeled with retrograde tracing and galanin- immunostaining, 56-60% were found in the supraoptic nucleus, 18-23% in the retrochiasmatic nucleus, 8-10% in the lateral magnocellular portion of the paraventricular nucleus. The ratio of (number of doubly labeled cellsinumber of galanin-immunoreactive cells) in each of the above regions was similar to the ratio of (number of retrogradely labeled cellshumber of Nissl-stained cells) in the supraoptic nucleus. Of all retrogradely labeled cells in the hypothalamus, 511-5696 also contained galaninlike immunoreactivity. In conclusion: (1) galanin-immunoreactive fibers in the posterior lobe of the pituitary originate mainly in the supraoptic nucleus, retrochiasmatic nucleus, and lateral magnocellular portion of the paraventricular nucleus, (2) most of galanin-immunoreactive cells in these regions project to the posterior lobe of the pituitary, and (3) about half the neurons constituting the hypothalamo-neurohypophyseal system contain galaninlike immunoreactivity. Key words: hypothalamo-neurohypophyseal system, wheat germ agglutinin, colloidal gold, silver enhancement Galanin is a 29 amino acid peptide initially isolated from the pig intestine (Tatemoto et al., '83). Using radioimmu- noassay and immunohistochemistry with antiserum against pig galanin, galaninlike immunoreactivity has been found to be widely distributed in the rat central nervous system (Rokaeus et al., '84; Ch'ng et al., '85; Skofitsh and Jacobo- witz, '85, '86; Melander et al., '86). Galanin-immunoreactive fibers are concentrated in the posterior lobe of the rat pituitary (Ch'ng et al., '85; Skofitsch and Jacobowitz, '86). Hyperosmotic stimuli in- duced the secretion of galaninlike immunoreactivity from the rat posterior pituitary (Rokaeus et al., '88; Skofitsch et al., '891, and systemically administered galanin induced mild diuresis in the rat (Skofitsch et al., '89). In the rat hypothalamus, galanin-immunoreactive cells have been found in several nuclei including the supraoptic nucleus (SO), paraventricular nucleus (PV), and accessory neurosecretory nuclei (Skofitsch and Jacobowitz, '85; Me- lander et al., '86). Retrograde tracing studies showed that magnocellular neurons in these nuclei projected to the posterior pituitary (Sherlock et al., '75; Ono et al., '78). By means of hypothalamic lesions in the rat, Palkovits et al. ('87) suggested that galanin-immunoreactive fibers in the posterior pituitary might originate in the SO, PV, and accessory neurosecretory nuclei. However, the exact origin of galanin-immunoreactive fibers in the posterior pituitary is still uncertain. The aims of this study were: (1) to describe the location of the cell bodies of origin for the hypothalamo-neurohypophy- seal galanin-immunoreactive neuron system, and (2) to Accepted June 25,1990 Address reprint requests to R. Arai, who is now at the Laboratory of Clinical Science, NIMH, Building 10, Room 3D48, Bethesda, MD 20892. c 1990 WILEY-LISS, INC.