BASAL FOREBRAIN NEURONS MODULATE THE SYNTHESIS AND EXPRESSION OF NEUROPEPTIDES IN THE RAT SUPRACHIASMATIC NUCLEUS M. D. MADEIRA,* P. A. PEREIRA, S. M. SILVA, A. CADETE-LEITE AND M. M. PAULA-BARBOSA Department of Anatomy, Porto Medical School, Al. Prof. Herna ˆ ni Monteiro, 4200-319 Porto, Portugal Abstract—We tested the hypothesis that efferents from the nucleus basalis magnocellularis (NBM) play a direct role in the regulation of neuropeptide synthesis and expression by neurons of the rat suprachiasmatic nucleus (SCN). Adult male rats in which the NBM was destroyed with quinolinic acid, either unilaterally or bilaterally, were compared with rats injected with physiological saline and with control rats. The estimators used to assess the effects of cholinergic deaffer- entation on the neuroanatomy and neurochemistry of the SCN were the total number of SCN neurons, the total number and somatic size of SCN neurons producing vasopressin (VP) and vasoactive intestinal polypeptide (VIP), and the re- spective mRNA levels. Bilateral destruction of the NBM did not produce cell death in the SCN, but caused a marked reduction in the number and somatic size of SCN neurons expressing VP and VIP, and in the mRNA levels of these peptides. The decrease in the number of VP- and VIP- producing neurons provoked by unilateral lesions was less striking than that resulting from bilateral lesions. It was, how- ever, statistically significant in the ipsilateral hemisphere, but not in the contralateral hemisphere. The results show that the reduction of cholinergic inputs to the SCN impairs the synthesis, and thereby decreases the expression of neuropeptides by SCN neurons, and that the extent of the decline correlates with the amount of cholin- ergic afferents destroyed. This supports the notion that ace- tylcholine plays an important, and direct role in the regulation of the metabolic activity of SCN neurons. © 2004 IBRO. Pub- lished by Elsevier Ltd. All rights reserved. Key words: nucleus basalis magnocellularis, hypothalamus, acetylcholine, vasopressin, vasoactive intestinal poly- peptide, quinolinic acid. In mammals, the suprachiasmatic nucleus (SCN) functions as the dominant pacemaker of the circadian system. The generation of circadian rhythms is an intrinsic property of its neurons (reviewed in van Esseveldt et al., 2000), but environmental light is the critical cue responsible for stable entrainment of the circadian rhythms (reviewed in Ebling, 1996). Light information is transmitted directly to the ven- trolateral SCN via the retinohypothalamic tract (Moore et al., 1995), which uses glutamate as the primary neuroac- tive substance (reviewed in Ebling, 1996; see also Ding et al., 1994). In addition, the retinorecipient area of the SCN receives two other major neurochemical inputs that modify the SCN response to light and mediate nonphotic entrain- ment of circadian rhythms (for a review, see van Esseveldt et al., 2000). These include serotonin, which reaches the SCN mainly via projections from the median raphe nu- cleus, and neuropeptide Y that is delivered to the SCN through the geniculohypothalamic tract (reviewed in van Esseveldt et al., 2000; see also Moga and Moore, 1997). In addition to these major afferents, the SCN receives inputs from several other sources (see, for example, Moga and Moore, 1997) through moderately dense or even sub- stantial projections, whose neurotransmitters and involve- ment in the modulation of the circadian system are not known yet. In contrast with these projections are the cho- linergic afferents to the SCN. Actually, due to a series of investigations showing that carbachol, a nonselective cho- linergic agonist, caused phase shifts in circadian rhythms that mimic the effects of light on the mammalian circadian system (Zatz and Brownstein, 1979; Zatz and Herkenham, 1981; Earnest and Turek, 1985; Wee et al., 1992), acetyl- choline was one of the earliest proposed transmitters of the circadian system. However, only recently was the source of the cholinergic afferents to the SCN identified, and shown to be located in the basal forebrain complex and mesopontine tegmentum (Bina et al., 1993, 1997; Moga and Moore, 1997). In addition, from the few investigations that succeeded to identify cholinergic fibers within the SCN only one revealed the presence of dense choline acetyl- transferase (ChAT)-immunoreactive fibers in its ventrome- dial part (Ichikawa and Hirata, 1986), whereas the remain- ing showed that the SCN is sparsely innervated by cholin- ergic fibers (van den Pol and Tsujimoto, 1985; Bina et al., 1993; Kiss and Hala ´ sz, 1996). Despite these facts, several lines of research have provided convincing evidence that acetylcholine can act directly upon SCN neurons. First, SCN neurons possess cholinergic receptors (Block and Billiard, 1981; Miller and Billiard, 1986; Fuchs and Hop- pens, 1987; Pauly and Horseman, 1988; van der Zee et al., 1991; Okuda et al., 1993; Bina et al., 1998). Second, cholinergic fibers establish synaptic contacts with neurons located in the ventrolateral and dorsomedial parts of the SCN (Kiss and Hala ´sz, 1996). Finally, cholinergic agents cause phase-dependent phase shifts of the circadian sys- tem (Zatz and Brownstein, 1979; Zatz and Herkenham, *Corresponding author. Tel: +351-22-509-6808; fax: +351-22-550-5640. E-mail address: madeira@med.up.pt (M. D. Madeira). Abbreviations: CE, coefficient of error; ChAT, choline acetyltransferase; CV, coefficient of variation; DAB, diaminobenzidine; NBM, nucleus basalis magnocellularis; PB, phosphate buffer; PBS, phosphate-buffered saline; p75 NTR , p75 neurotrophin receptor; SCN, suprachiasmatic nucleus; VAChT, vesicular acetylcholine transporter; VIP, vasoactive intestinal polypeptide; V  N , mean somatic volume; VP, vasopressin. Neuroscience 125 (2004) 889 –901 0306-4522/04$30.00+0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.03.005 889