Projections from the raphe nuclei to the suprachiasmatic nucleus of the rat Anders Hay-Schmidt a, *, Niels Vrang b , Philip J. Larsen b , Jens D. Mikkelsen c a Department of Medical Anatomy, The Panum Institute, 18.2, University of Copenhagen, Blegdamsvej 3, Copenhagen DK-2200 N, Denmark b Rheoscience, Copenhagen, Denmark c Department of Molecular Anatomy and Physiology, Neurosearch, Copenhagen, Denmark Received 21 January 2003; received in revised form 26 April 2003; accepted 26 April 2003 Abstract The presence of serotonergic afferents in the hypothalamic suprachiasmatic nucleus (SCN) is well documented and several functional roles of serotonin (5-HT) in circadian function are well established. However, there is some controversy about the precise location of the serotonergic neurones from where this input arises. Discrete injection of the tracer Cholera toxin, subunit B, (ChB) was centred in the rat SCN, and a few retrograde labelled neurones were distributed in the dorsal and median raphe nuclei (MnR) and in the rostral part of the raphe magnus (RMg), but no neurones were found in the raphe pallidus or raphe obscurus. In addition, a group of neurones was consistently found in the medial part of the pontine supra lemniscal nucleus but not including the serotonergic B 9 region. A combination of retrograde tracing with Fluoro-Gold together with 5-HT-immunolabelling, showed few double-labelled neurones in the dorsal, MnR and B 9 . However, the majority of projecting neurones were not co-storing 5-HT immunoreactivity. Phaseolus vulgaris -leucoagglutinin (PHA-L) injections in the dorsal raphe resulted in faint labelling, whereas the MnR gave rise to several labelled fibres in the SCN. Individual delicate PHA-L nerve fibres were found in all compartments of the SCN both in terms of rostrocaudal, ventromedial and dorsomedial extent, without any sign of a topographical organisation of the MnR input to the SCN. PHA-L injections into RMg gave rise to labelling of a few processes within the SCN. In conclusion, the main serotonergic input to the rat SCN originates from a few neurones in the MnR. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Hypothalamus; Circadian rhythm; Serotonin; Nucleus; Neuronal connections; Phaseolus vulgaris -leucoagglutinin (PHA-L); Cholera toxin (ChB) 1. Introduction Brain serotonin originates from a relatively few serotonergic perikarya, located in the mesencephalic and metencephalic raphe system, which sends axons to almost all areas of the central nervous system, and influences several neuronal circuits including those implicated in circadian rhythmicity (for review, see Jacobs and Azmitia, 1992). The suprachiasmatic nucleus (SCN) houses the prin- cipal circadian pacemaker of the mammalian brain (Harrington et al., 1994; Hastings, 1991; Turek, 1985). Mammalian SCN neurones are sensitive to a number of chemical agents, which are contained in the afferents of the SCN (Turek, 1987; Wirz-Justice, 1987). The seroto- nergic projection is not essential for the circadian rhythm generation under constant conditions, but can Abbreviations: 5-HT, 5-hydroxy tryptamin, serotonin; AHA, anterior hypothalamic area, anterior; B 1 , serotonergic raphe pallidus; B 2 , serotonergic raphe obscurus; B 3 , serotonergic raphe magnus; B 4,6,7 , serotonergic dorsal raphe nucleus; B 8,5 , serotonergic median raphe nucleus; B 9 , serotonergic supralemniscal nucleus; BSA, bovine serum albumine; ChB, cholera toxin, subunit B; CT, circadian time; DRn, dorsal raphe nucleus; FG, Fluoro-Gold; LA, lateroanterior hypothalamic nucleus; KPBS, phosphate buffered saline with KCl; LD, light  /dark; LHA, lateral hypothalamus, anterior part; mlf, medial longitudinal fasciculus; MnR, median raphe nucleus; PHA-L, Phaseolus vulgaris leucoagglutinin; PBS, phosphate buffered saline; PVN, paraventricular hypothalamic nucleus; RMg, raphe magnus; ROb, raphe obscurus; RPa, raphe pallidus; SCN, suprachiasmatic nucleus; TX, triton X-100; VTg, ventral tegmental nucleus; xscp, decussatio pedunculus cerebellaris superiore. * Corresponding author. E-mail address: a.hay@mai.ku.dk (A. Hay-Schmidt). Journal of Chemical Neuroanatomy 25 (2003) 293  /310 www.elsevier.com/locate/jchemneu 0891-0618/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0891-0618(03)00042-5