Neuron, Vol. 38, 701–713, June 5, 2003, Copyright 2003 by Cell Press Hypothalamic Orexin Neurons Regulate Arousal According to Energy Balance in Mice Akihiro Yamanaka,1,2,5,7 Carsten T. Beuckmann,6,7 The molecular and physiological basis of this evolution- Jon T. Willie,6,7 Junko Hara,1,2,5 Natsuko Tsujino,1,2,5 arily conserved phenomenon remains poorly under- Michihiro Mieda,6 Makoto Tominaga,1,3 stood. Ken-ichi Yagami,1,4 Fumihiro Sugiyama,1,4 Orexins, also called hypocretins, are a pair of neuro- Katsutoshi Goto,1,2 Masashi Yanagisawa,1,5,6,* peptides expressed by a specific population of neurons and Takeshi Sakurai1,2,5,* in the lateral hypothalamic area (LHA), a region of the 1Institute of Basic Medical Science brain implicated in feeding, arousal, and motivated be- 2 Department of Pharmacology havior (de Lecea et al., 1998; Sakurai et al., 1998; Chemelli 3 Department of Molecular Neurobiology et al., 1999; Willie et al., 2001). Orexin-A (hypocretin-1) 4 Laboratory Animal Research Center and orexin-B (hypocretin-2) are derived from a common University of Tsukuba precursor peptide, the product of the prepro-orexin Ibaraki 305-8575 gene (Sakurai et al., 1998, 1999). The actions of orexins Japan are mediated by two G protein-coupled receptors 5 ERATO Yanagisawa Orphan Receptor Project termed orexin receptor type 1 and orexin receptor type 2 Japan Science and Technology Corporation (Sakurai et al., 1998). Orexin-containing neurons project Tokyo 135-0064 from the LHA to numerous brain regions, with the limbic Japan system, hypothalamus, and monoaminergic and cholin- 6 Howard Hughes Medical Institute ergic nuclei of brainstem receiving particularly strong Department of Molecular Genetics innervations (Peyron et al., 1998; Date et al., 1999; University of Texas Southwestern Medical Center Nambu et al., 1999). Thus, the orexinergic system is Dallas, Texas 75390 anatomically well placed to influence the arousal, moti- vational, metabolic, autonomic, and motor processes necessary to elicit homeostatically appropriate be- Summary haviors. When orexins are administered centrally to rodents, Mammals respond to reduced food availability by be- they are reported to elevate sympathetic tone, plasma coming more wakeful and active, yet the central path- corticosterone levels (Hagan et al., 1999), metabolic rate ways regulating arousal and instinctual motor programs (Lubkin and Stricker-Krongrad, 1998), food intake (Sa- (such as food seeking) according to homeostatic need kurai et al., 1998), locomotor activity (Nakamura et al., are not well understood. We demonstrate that hypo- 2000), and wakefulness (Hagan et al., 1999). While these thalamic orexin neurons monitor indicators of energy effects highlight the complexity of the orexin system balance and mediate adaptive augmentation of arousal and the responses it regulates, most of these effects in response to fasting. Activity of isolated orexin neu- resemble those observed in fasted animals (Itoh et al., 1990; Challet et al., 1997; Dewasmes et al., 1989). In- rons is inhibited by glucose and leptin and stimulated deed, dopamine antagonists attenuate increases in lo- by ghrelin. Orexin expression of normal and ob/ob comotor activity induced by either fasting or orexin ad- mice correlates negatively with changes in blood glu- ministration in rodents (Nakamura et al., 2000; Itoh et cose, leptin, and food intake. Transgenic mice, in which al., 1990). Importantly, pharmacological antagonism of orexin neurons are ablated, fail to respond to fasting orexin receptor type 1 is associated with reduced food with increased wakefulness and activity. These find- intake and weight reduction in rodents (Smart et al., ings indicate that orexin neurons provide a crucial link 2002). These observations are all consistent with the between energy balance and arousal. hypothesis that orexin neurons provide a link between metabolic status and motivated behavior. Introduction The importance of orexin in promoting arousal is high- lighted by the discovery that mice lacking either the Motivated behaviors such as food seeking are critically orexin gene (prepro-orexin knockout mice) or orexin dependent upon arousal pathways. Periods of foraging neurons (orexin/ataxin-3 transgenic mice), as well as and rest are both essential, but these mutually exclusive mice and dogs with null mutations in the orexin receptor behaviors require appropriate coordination and cycling type 2 gene, all have phenotypes remarkably similar to at environmentally advantageous times and in response the human sleep disorder narcolepsy (Lin et al., 1999; to homeostatic needs. When faced with negative energy Chemelli et al., 1999; Hara et al., 2001; Willie et al., balance due to reduced food availability, mammals re- 2003 [this issue of Neuron]). Narcolepsy is a neurological spond with phases of increased wakefulness and loco- disease characterized by excessive sleepiness and motor activity that support food seeking (Borbely, 1977; pathologic intrusions of rapid eye movement (REM) Danguir and Nicolaidis, 1979; Dewasmes et al., 1989; sleep-related phenomena into wakefulness. Consistent Itoh et al., 1990; Challet et al., 1997; Williams et al., 2002). with these findings, recent reports suggest that human narcolepsy is accompanied by a specific destruction of *Correspondence: [email protected] (T.S.), masashi. orexin neurons in the hypothalamus (Peyron et al., 2000; [email protected] (M.Y.) Thannickal et al., 2000). Human narcolepsy also is asso- 7These authors contributed equally to this work. ciated with metabolic abnormalities, including increased Neuron 702 frequency of non-insulin-dependent diabetes mellitus dissection of LHA regions followed by enzymatic dis- and increased body mass index (Honda et al., 1986; persal of tissues. EGFP-expressing orexin neurons were Schuld et al., 2000; Nishino et al., 2001). Complex disrup- identified by fluorescence microscopy and immediately tions of energy homeostasis in prepro-orexin knockout subjected to whole-cell patch-clamp recordings in phys- and orexin/ataxin-3 transgenic mice are indicated by iological extracellular solution. Additionally, we per- hypophagia, obesity, and inactivity relative to control formed whole-cell patch-clamp studies with hypothala- littermates (Willie et al., 2001; Hara et al., 2001). Orexin mic slice preparations from orexin/EGFP transgenic neurons appear to be sensitive to nutritional status as mice. prepro-orexin mRNA is upregulated in rats during fasting In current-clamp mode, dispersed orexin neurons (Sakurai et al., 1998). Since insulin-induced hypoglyce- showed a resting membrane potential of Ϫ47.4 Ϯ 0.8 mia also increases orexin mRNA expression as well as mV and spontaneous firing at 15.0 Ϯ 2.6 Hz (n ϭ 20). expression by orexin neurons of c-Fos, a marker of neu- Membrane capacitance was 6.3 Ϯ 0.5 pF. Isolated orexin ronal activation, changes in circulating glucose concen- neurons exhibited shallower resting potentials and tration might directly or indirectly mediate some of these higher spontaneous firing rates as compared with those effects (Griffond et al., 1999; Moriguchi et al., 1999). observed using hypothalamic slice preparations from In order to identify orexin-containing neurons for di- orexin/EGFP mice (Ϫ61.4 Ϯ 4.9 mV and 5.5 Ϯ 3.9 Hz, rect electrophysiological recording, we generated trans- n ϭ 24). This may suggest that orexin neurons in slice genic mice expressing GFP exclusively in these neurons. preparations have robust innervation from inhibitory We demonstrate that isolated orexin neurons are able neurons within the slice. In the present study, we used to monitor humoral and neural indicators of energy bal- only isolated neurons showing resting potentials be- ance in mice. To probe the physiological relevance of low Ϫ45 mV for further analysis. Typical action potentials this interaction, we show that the presence of orexin of isolated neurons (Figure 2A) were followed by a nar- neurons is required for mice to respond appropriately row shoulder and deep, brief after-hyperpolarizations in to fasting by increasing wakefulness and locomotor ac- all orexin neurons examined, regardless of donor age, tivity. gender, or transgenic line (data not shown). Results Extracellular Factors that Influence the Activity of Orexin Neurons Specific Expression of a GFP Transgene To characterize which extracellular factors directly mod- in Orexin-Containing Neurons ulate the activity of orexin neurons, we first collected Because orexin neurons of the LHA are diffusely distrib- data from dissociated single neurons in order to exclude uted and lack distinct morphological features, it is chal- confounding synaptic effects. We applied various neu- lenging to directly examine their electrophysiological rotransmitters and neuromodulators in superfused solu- activity and regulation. To facilitate identification of ore- tion during continuous recording (Table 1). Like most xin neurons, we generated transgenic mouse lines in CNS neurons, all orexin neurons examined (10/10) were which the enhanced green fluorescence protein (EGFP) robustly depolarized (with associated increase in action is expressed under control of the human prepro-orexin potential frequency) by a relatively low dose of gluta- mate (10 ␮M) and hyperpolarized (with associated inhibi- promoter (orexin/EGFP transgenic mice) (Figure 1A). Un- tion of action potentials) by a relatively low dose of GABA der fluorescence microscopy, we observed green fluo- (10 ␮M) (Table 1). This suggests that these