Effects of serotonin on perifornical-lateral hypothalamic area neurons in rat Sunil Kumar, 1,2,5 Ronald Szymusiak, 1,2,3 Tariq Bashir, 1,2 Seema Rai, 1 Dennis McGinty 1,4 and Md. Noor Alam 1,4 1 Research Service (151A3), Veteran Affairs Greater Los Angeles Healthcare System, 16111 Plummer Street, Sepulveda, California 91343, USA 2 Department of Medicine and 3 Department of Neurobiology, School of Medicine, University of California, Los Angeles, California, USA 4 Department of Psychology, University of California, Los Angeles, California, USA 5 Department of Zoology, Patna University, Patna, India Keywords: hypocretin, melanin-concentrating hormone, orexin, serotonin, sleep Abstract The hypocretin (HCRT) system of the perifornical-lateral hypothalamic area (PF-LHA) has been implicated in the facilitation of behavioral arousal. HCRT neurons receive serotonergic afferents from the dorsal raphe nucleus. Although in-vitro pharmacological studies suggest that serotonin (5-HT) inhibits HCRT neurons, the in-vivo effects of 5-HT on HCRT neurons in the PF-LHA and associated behavioral changes have not been described. We examined the effects of 5-HT delivered locally into the PF-LHA using reverse microdialysis on its neuronal activity and the consequent sleep–wake changes in rats. First, we quantified Fos expression (Fos-IR) in HCRT and other PF-LHA neurons following unilateral 5-HT perfusion in awake rats. Second, we determined the transient effects of 5-HT perfusion on the extracellular activity of the PF-LHA neurons recorded via microwires placed adjacent to the microdialysis probe. Third, we examined the effects of 5-HT perfusion into the PF-LHA on the sleep–wake profiles of the rats during the lights-off period. Unilateral perfusion of 5-HT into the PF-LHA in awake rats dose-dependently decreased the number of HCRT neurons exhibiting Fos-IR. 5-HT also inhibited the discharge activity of four of five responsive wake-related, putative HCRT neurons. However, unilateral perfusion of 5-HT into the PF-LHA did not produce significant behavioral changes during the 2-h recording period. These results confirm the in-vitro findings that 5-HT exerts inhibitory influences on HCRT neurons but further suggest that the inactivation of a limited number of HCRT neurons by unilateral 5-HT microdialysis may not be sufficient to induce behavioral changes. Introduction The perifornical-lateral hypothalamic area (PF-LHA) has been impli- cated in the regulation of behavioral arousal (McGinty & Szymusiak, 2003; Gerashchenko & Shiromani, 2004; Jones, 2005). Electrical stimulation of the PF-LHA evokes locomotor activity and electroen- cephalogram (EEG) activation, and most of the neurons within the PF-LHA exhibit wake-related discharge activity (Krolicki et al., 1985; Sinnamon et al., 1999; Alam et al., 2002; Koyama et al., 2003). The PF-LHA contains several cell types including those expressing hypocretin (HCRT) and melanin-concentrating hormone (MCH) peptides (Broberger et al., 1998; Peyron et al., 1998; Abrahamson & Moore, 2001). Evidence suggests that the HCRT system contributes to the facilitation and ⁄ or maintenance of arousal (Kilduff & Peyron, 2000; Taheri et al., 2002; Scammell, 2003; Siegel, 2004). For example, HCRT neurons exhibit wake-associated discharge and c-Fos expres- sion (Fos-IR), and are quiescent during non-rapid eye movement (REM) ⁄ REM sleep (Estabrooke et al., 2001; Espana et al., 2003; Lee et al., 2005; Mileykovskiy et al., 2005). Local applications of HCRT into various brain sites, e.g. basal forebrain, preoptic area and locus coeruleus, promote waking (Bourgin et al., 2000; Methippara et al., 2000; Thakkar et al., 2001). Human narcoleptics exhibit HCRT cell loss (Peyron et al., 2000; Thannickal et al., 2000). Some of the symptoms of narcolepsy, including excessive sleepiness and cataplexy, are exhibited by mice lacking the HCRT gene or HCRT neurons and rats with a targeted destruction of HCRT-receptor-expressing neurons in the PF-LHA (Chemelli et al., 1999; Gerashchenko et al., 2001; Hara et al., 2001). In contrast to HCRT neurons, MCH neurons may facilitate sleep. MCH neurons exhibit REM-associated Fos-IR and the intracerebroventricular administration of MCH increases both non- REM and REM sleep (Verret et al., 2003; Modirrousta et al., 2005). The serotonergic system in the dorsal raphe nucleus has been implicated in sleep–wake regulation and projects to the HCRT field (Peyron et al., 1998; Portas et al., 2000; Datta et al., 2003; Adrien, 2004; Wang et al., 2005). The discharge of serotonin (5-HT) neurons is also tightly coupled with behavioral arousal, exhibiting the highest discharge in waking, a lower discharge in non-REM and a near quiescence in REM sleep (McGinty & Harper, 1976; Guzman-Marin et al., 2000). 5-HT 1A receptor immunoreactivity has been reported on HCRT neurons (Collin et al., 2002; Muraki et al., 2004). Although in-vitro pharmacological studies suggest that 5-HT inhibits HCRT neurons (Li et al., 2002; Muraki et al., 2004), the regulatory effects of Correspondence: Dr Md. Noor Alam, 1 Research Service (151A3), as above. E-mail: noor@ucla.edu Received 30 August 2006, revised 26 October 2006, accepted 31 October 2006 European Journal of Neuroscience, Vol. 25, pp. 201–212, 2007 doi:10.1111/j.1460-9568.2006.05268.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd