Inhibition of rostral basal forebrain neurons promotes wakefulness and induces FOS in orexin neurons Shinsuke Satoh, 1 Hitoshi Matsumura, 2 Tomoko Nakajima, 2 Ken-ichi Nakahama, 3 Tuyoshi Kanbayashi, 4 Seiji Nishino, 5 Hiroshi Yoneda 2 and Yasufumi Shigeyoshi 1 1 Second Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589–8511, Japan 2 Department of Neuropsychiatry, Osaka Medical College, Takatsuki, Osaka, Japan 3 Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Bunkyo, Tokyo, Japan 4 Department of Neuropsychiatry, Akita University School of Medicine, Hondo, Akita, Japan 5 Stanford University Center for Narcolepsy, Palo Alto, CA, USA Keywords: microdialysis, muscimol, orexin rat, sleep-wake activity Abstract The present study examined whether the activities of the rostral basal forebrain neurons alter the activities of the orexin (also known as hypocretin) neurons in the tuberal part of the hypothalamus in rats. We performed microdialysis perfusion of the ventromedial portion of the rostral basal forebrain with the GABA A receptor agonist muscimol to inhibit focally the neuronal activities in the rostral basal forebrain. Then, we monitored sleep/wake behaviour and investigated the pattern of activities of orexin neurons by examining the expression of FOS as an indicator of cellular activation. Bilateral perfusion with muscimol (5, 15, and 50 mM) produced a dose- dependent decrease in the amount of sleep. This perfusion with muscimol at 50 mM produced FOS-like immunoreactivity in 37% of the orexin neurons located in the tuberal part of the hypothalamus, whereas the FOS-like immunoreactivity was sparse in orexin neurons of the sleeping control rats (P ¼ 0.001 by Mann–Whitney U-test). Unilateral perfusion with muscimol (50 mM) also suppressed sleep. In this case, FOS-like immunoreactivity was seen in 40% of the orexin neurons on the side ipsilateral to the perfusion site but only in 10% of orexin neurons on the contralateral side (P ¼ 0.018 by Wilcoxon signed rank test). These functional data suggested that a sleep- generating element in the ventromedial part of the rostral basal forebrain provides an inhibitory influence on the activities of the orexin neurons in the tuberal part of the hypothalamus. Introduction Orexin (also known as hypocretin) is a neuropeptide that is synthesized by the neurons exclusively located in the perifornical/lateral hypo- thalamic areas (de Lecea et al., 1998; Sakurai et al., 1998). Orexin plays a crucial role in the regulation of feeding, sleep/wake states and muscle tone (Siegel, 1999; Kilduff & Peyron, 2000; Willie et al., 2001). Dysfunction of the orexin system has been found in animal and human narcolepsy, a sleep disorder involving a difficulty in maintaining wakefulness or sleep for an extended period and with muscle atonia triggered by emotional excitation (Lin et al., 1999; Chemelli et al., 1999; Nishino et al., 2000; Peyron et al., 2000; Thannickal et al., 2000). The factors regulating the activities of orexin-containing neurons have been examined from the aspect of feeding and metabolism. The fasting and insulin-induced hypoglycemia increased the expression of prepro-orexin mRNA or FOS in orexin neurons (Sakurai et al., 1998; Griffond et al., 1999). On the other hand, a circadian variation in FOS in orexin neurons has been demonstrated (Estabrook et al., 2001; Martı ´nez et al., 2002), which raises the possibility that the activities of the neuronal elements regulating sleep/wake states might also influ- ence the activities of the orexin-containing neurons. However, this possibility has not been examined in detail. The ventromedial portion of the rostral basal forebrain is thought to contain the neuronal elements regulating the sleep/wake states. Wake- fulness is promoted by the application of analogues of norepinephrine or amphetamine into this region (Berridge & Foote, 1996, 1999). In contrast, the application of prostaglandins, interleukin 1b, tumour necrosis factor or adenosine and its derivatives promotes sleep (Matsumura et al., 1994; Satoh et al., 1996, 1998, 1999; Portas et al., 1997; Terao et al., 1998a,b; Yoshida et al., 2000). The diagonal band nucleus, septum nucleus, accumbens nucleus, and pre-optic nucleus are supposedly the target structures on which these chemicals act. The present study investigated whether the activities of the rostral basal forebrain neurons alter the activities of the orexin neurons. Firstly, we performed microdialysis-perfusion of the ventromedial portion of the rostral basal forebrain of rats with muscimol to make a focal inactivation in this brain region, and then examined its influence on their behavioural state. Muscimol is a typical agonist for GABA A receptors and exerts an inhibitory action on neuronal excitation. Secondly, we performed double immunohistochemistry to detect FOS and orexin to examine how this focal inactivation of the rostral basal forebrain neurons influences the activities of orexin neurons. FOS is a nuclear protein and the product of the immediate-early gene c-fos, which is rapidly and transiently expressed in response to a variety of stimulations. FOS has been used to determine the potential European Journal of Neuroscience, Vol. 17, pp. 1635–1645, 2003 ß Federation of European Neuroscience Societies doi:10.1046/j.1460-9568.2003.02577.x Correspondence: Dr S. Satoh, as above. E-mail: satohs@med.kindai.ac.jp Received 3 September 2002, revised 1 February 2003, accepted 4 February 2003