MINIREVIEW Neuropeptide S as a novel arousal promoting peptide transmitter Rainer K. Reinscheid and Yan-Ling Xu Department of Pharmacology, University of California, Irvine, CA, USA Introduction The importance of neuropeptides for the regulation of sleep-wake cycles has only become visible in the recent past. For many years, sleep neurobiology focused on the major small molecule transmitters in the brain, however, this work has produced a complex picture of how sleep and wakefulness might be modulated at the neurochemical level. Basically, aminergic transmitters such as noradrenaline, histamine, acetylcholine, dop- amine and serotonin are responsible for particular sta- ges of wakefulness or its maintenance [1,2]. Also, the excitatory transmitter, glutamate, is involved in arousal and therefore stabilizes an awake state. On the other hand, the major inhibitory transmitter in the brain, GABA, is necessary to reduce cortical activity and plays an important role in sleep onset and mainten- ance. Acetylcholine (ACh) appears to serve a dual role: ACh release coincides with elevated arousal as well as the onset of paradoxical sleep, also known as rapid eye Keywords anxiety; brainstem; locus coeruleus; neuropeptide; sleep ⁄ wakefulness Correspondence R. K. Reinscheid, Department of Pharmacology, University of California Irvine, 360 Med Surge II, Irvine, CA 92697-4625, USA Fax: +1 949 824 4855 Tel: +1 949 824 9228 E-mail: rreinsch@uci.edu (Received 21 June 2005, accepted 18 August 2005) doi:10.1111/j.1742-4658.2005.04982.x Behavioral arousal requires integration of multiple neurotransmitter and neuromodulatory systems. Identifying these systems is the key to not only a better understanding of the neurobiology of sleep ⁄ wakefulness but may also lead to the discovery of potential therapeutic targets for various sleep disorders. We review here a novel arousal promoting neuropeptide system, neuropeptide S (NPS) and its receptor. Pharmacologically, NPS activates NPS receptors at low nanomolar concentration to increase concentrations of intracellular Ca 2+ . Anatomically, both NPS precursor and receptor mRNAs are found predominately in the central nervous system. NPS pre- cursor mRNA is expressed only in several discrete regions located mainly in the brainstem. In particular, it is highly expressed in a previously undes- cribed group of neurons localized between locus coeruleus and Barring- ton’s nucleus. NPS receptor mRNA is widely distributed in many brain areas with high expression levels in cortex, hypothalamus, amygdala and multiple midline thalamic nuclei. Functionally, central administration of NPS increases locomotor activity in both naı ¨ ve and habituated mice. It also significantly increases wakefulness and decreases paradoxical (rapid eye movement) sleep and slow wave sleep in rats. In addition, NPS sup- presses anxiety-like behaviors in mice exposed to different behavioral para- digms measuring responses to novelty or stress. These studies indicate that the NPS system is a newly discovered transmitter system that regulates vigilance and emotional states. NPS appears to possess a unique pharmaco- logical profile in producing both anxiolytic-like and hypervigilant effects. Abbreviations ACh, acetylcholine; CRF, corticotropin-releasing factor; GPCR, G-protein coupled receptor; NPS, neuropeptide S; NPSR, neuropeptide S receptor; TH, tyrosine hydroxylase. FEBS Journal 272 (2005) 5689–5693 ª 2005 FEBS 5689 movement sleep [3]. In addition to these major neuro- chemical systems, subtle roles for prostaglandins and adenosine have been described in the modulation of sleep and wakefulness [4]. Despite this detailed descrip- tion of the neurobiological basis of sleep-wakefulness regulation, many aspects are still incompletely under- stood. For example, the neuronal mechanisms orches- trating the transition between sleep and wakefulness, and vice versa, or disorders such as narcolepsy were not explained by these neurotransmitter systems. Also, the function of sleep for metabolic homeostasis, immune function or complex brain processes such as learning and memory are under intense investigation [5]. The work on orphan G-protein coupled receptors (GPCRs) during the last decade has greatly, and unex- pectedly, advanced our knowledge about neurobio- logical mechanisms underlying sleep-wakefulness modulation. The first step was marked by the discov- ery that the neuropeptide hypocretin ⁄ orexin could potently induce wakefulness, and its absence or a null- mutation in one of its receptors was associated with narcolepsy [6–8]. Another important, and even less expected, finding was the discovery that another pep- tide, termed prokineticin 2 was signaling the circadian clock rhythm from the suprachiasmatic nucleus in order to control circadian behavior [9]. Both of these peptides were initially discovered as ligands of orphan GPCRs. The neuropeptide cortistatin, which activates somatostatin receptors, was found to suppress cortical activity and antagonize ACh-induced cortical excita- tion, indicating that it might be involved in cortical synchronization [10]. The newest example of yet another novel ligand of an orphan GPCR involved in sleep-wakefulness regula- tion is Neuropeptide S (NPS) [11]. This paper will summarize our current knowledge about the pharma- cology, distribution and behavioral effects of NPS and will outline some strategies for future research. Structure, biosynthesis, distribution and pharmacology of NPS Bioinformatic analysis showed that the primary struc- ture of NPS is highly conserved among vertebrates. At the time of writing this review, genomic DNA sequences corresponding to parts of the NPS precursor were available from the following species: human, chimpanzee, macaque, bovine, dog, elephant, mouse, rat, rabbit, guinea pig, chicken, frog (Xenopus tropicalis) and opossum. However, the gene appears to be absent from the currently available fish genomes (zebrafish and fugu), indicating that the NPS precursor gene occurred late during vertebrate evolution. The amino- terminal residue of NPS in all species is always serine (single amino acid code ‘S’) and therefore we termed this molecule neuropeptide S, or NPS. The NPS pre- cursor protein contains the typical structural features of a neuropeptide precursor. A hydrophobic signal peptide immediately follows the initiator methionine. The immature peptide is preceded by a pair of basic amino acids (Lys, Arg) that might serve as processing sites for proteolytic cleavage (Fig. 1). The NPS recep- tor is a typical GPCR containing seven membrane- spanning domains. It shares moderate homology with other members of the GPCR supergene family, especially neuropeptide receptors. The highest degree of similarity is found with vasopressin or oxytocin receptors. Using in situ hybridization we studied the distribu- tion of NPS precursor and receptor mRNA in rat brain (Fig. 2). These experiments showed that the NPS receptor (NPSR) mRNA is widely expressed throughout the nervous system, with highest levels found in cortex, thalamus, hypothalamus, and amy- gdala. Low levels of NPSR mRNA were detected in brainstem. In contrast, the NPS precursor mRNA was mainly expressed in brainstem nuclei such as the locus coeruleus area, the principle 5 sensory nucleus and the lateral parabrachial nucleus of the brain- stem. A small number of scattered NPS-positive cells were found in other brain areas, such as amygdala and hypothalamus. The NPS-producing neurons in the locus coeruleus area were found to define a novel nucleus that lies between the noradrenergic locus coeruleus proper and Barrington’s nucleus. Double in situ hybridization revealed that NPS precursor mRNA is neither colocal- ized with tyrosine hydroxylase (TH; a marker of noradrenergic neurons) nor with corticotropin-releas- ing factor (CRF; a marker for neurons of Barrington’s nucleus). This unique anatomical pattern of NPS expressing neurons defines a previously unrecognized population of cells in the brainstem. It is also evident from our in situ hybridization data that there are still other cells in this area that express none of these neuro- chemical markers (TH, CRF or NPS) and thus might contain other known or novel transmitters. Fig. 1. Primary structure of the human NPS precursor. The hydro- phobic signal peptide is shown by broken underlining. Endopro- tease cleavage at a pair of basic amino acids (KR; double underlined) is presumed to release the mature NPS peptide (single underlined). NPS produces arousal and anxiolysis R. K. Reinscheid and Y L. Xu 5690 FEBS Journal 272 (2005) 5689–5693 ª 2005 FEBS Cells stably expressing NPSR were used to charac- terize the in vitro pharmacology of NPS. Nanomolar concentrations of NPS produce a transient increase in intracellular free Ca 2+ , indicating that NPS might be an excitatory transmitter in vivo by elevating intracellu- lar Ca 2+ . A radiolabled analog of NPS ( 125 I-labeled Tyr 10 -NPS) shows displaceable binding with high affin- ity (K d ¼ 0.3 nm) [11]. High affinity receptor binding and high potency to evoke intracellular second messen- ger responses are important pharmacological para- meters to classify NPS as a typical neuropeptide transmitter which is active at low concentrations. NPS promotes arousal and reduces anxiety-like behavior in rats and mice The first step in studying the physiological functions of NPS in the nervous system was a detailed analysis of locomotor behavior produced by central administra- tion of NPS in mice. Mice that were naı ¨ ve to the test chamber showed a profound increase in locomotion, measured as the total distance traveled over one hour. It is well known that animals naturally show increased exploratory activity when they are exposed to a novel environment and therefore the NPS-induced locomo- tion seen in these naı ¨ ve mice could have two possible reasons: (a) NPS might enhance the exploratory com- ponent by increasing the response to novelty, or (b) the stimulatory effect might be independent of novelty and thus genuine arousal. To distinguish between these two possibilities we injected mice that had been habitu- ated to the test chamber for one hour before adminis- tration of the drug. In habituated mice, injection of saline (control) did not produce any increase in loco- motion because they had already explored the test chamber extensively before. However, low concentra- tions (0.1 or 1 nmole) of NPS were able to reinstate exploration in habituated mice that lasted for almost one hour. In both naı ¨ ve and habituated mice NPS sig- nificantly reduced inactivity, i.e., time the animals rest. These experiments show that NPS produces profound arousal that is independent of novelty [11]. Because arousal is an important component of wakefulness, we also analyzed the effect of NPS on sleep patterns in rats during their normal period of inactivity, i.e., during the light phase. Low doses of NPS significantly increased wakefulness and conversely suppressed all stages of sleep during the first hour post administration. These studies indicate that NPS might be involved in the induction or maintenance of wake- fulness. The arousal-promoting effect of NPS might be partially mediated by NPSRs expressed in thalamic Fig. 2. Schematic drawings of NPS receptor mRNA expression in the rat brain. Representative regions with high levels of NPS receptor mRNA signals (small circles) are depicted in the drawings. Numbers at the bottom left of each drawing correspond to the anteroposterior distance of the plate relative to bregma according to the rat brain atlas of Paxinos & Watson [20]. Strong NPS receptor mRNA expression is found in the anterior olfactory nucleus, endopiriform nucleus, piriform cortex, motor cortex, retrosplenial cortex and subiculum. Multiple tha- lamic nuclei including the midline nuclei of the thalamus (indicated by an arrow) show significant levels of NPSR expression. Substantial expression of NPSR mRNA is also observed in the hypothalamus and the amygdala complex. DEn, dorsal endopiriform nucleus; AON, anter- ior olfactory nucleus; En, endopiriform nucleus (dorsal and ventral); M2, motor cortex 2; Hyp, hypothalamus; Amg, amygdala; RSA, retrosple- nial agranular cortex; S, subiculum; Prc, precommissural nucleus, Pvp, paraventricular thalamic nucleus; PH, posterior hypothalamus. R. K. Reinscheid and Y L. Xu NPS produces arousal and anxiolysis FEBS Journal 272 (2005) 5689–5693 ª 2005 FEBS 5691 midline nuclei, as this brain structure is known to act as a relay between arousal centers of the brainstem and the cortex [12]. High levels of NPS receptor expression were also found in the amygdala. The amygdala is a brain struc- ture that is closely involved in the processing of emo- tional behavior and memories [13]. The well-established role of the amygdala in modulation of fear and anxiety led us to hypothesize that the NPS system might also be involved in emotional behavior. Therefore, the effect of NPS administration was tested in mice using four different paradigms which are able to measure fearful responses and have been validated using anxiolytic drugs such as diazepam. We found that centrally administered NPS could produce an anxiolytic-like pro- file that was independent of the motor-activating effects of the peptide [11]. NPS increased the time the animals spent exploring the less protected or brighter areas of the different test chambers (open field, light-dark box, elevated plus maze) similar to classical anxiolytic drugs. In order to control for possible confounding effects of the NPS-induced hyperlocomotion, we used the marble burying test. This is a behavioral paradigm in which anxiolytic drugs have been shown to selectively reduce a natural defensive behavior [14]. NPS adminis- tration reduced the time mice engaged in burying the unfamiliar objects placed in their cages [11]. In summary, the behavioral studies showed that NPS can produce arousal independent of novelty while also alleviating anxiety responses triggered by stressful or unfamiliar environments. Comparison with other neuro- transmitters involved in arousal and anxiety The present examples demonstrate that NPS can potently modulate arousal and stress. This pharmaco- logical spectrum of NPS is quite unique as compared to other transmitters or drugs that influence sleep and ⁄ or emotional behavior. For example, stimulants such as amphetamine or cocaine promote arousal and suppress sleep but appear to have anxiogenic-like effects in tests of emotional behavior [15,16]. Hypocre- tin ⁄ orexin is able to suppress sleep and induce pro- found wakefulness, but the peptide shows no effects on anxiety-like behavior [17]. The antinarcolepsy drug modafinil (ProvigilÒ), whose mechanism of action is still unknown, induces long-lasting wakefulness but does not modulate anxiety [18]. Typical anxiolytic drugs such as benzodiazepines (diazepam, ValiumÒ) do not affect locomotion at anxiolytic doses but tend to inhibit motor activity at higher doses [19]. These examples show that NPS produces a unique spectrum of behavioral effects. Future research will have to dem- onstrate how release of endogenous NPS is involved in modulating sleep-wake states and emotional behavior. NPS agonists could have unique applications in the treatment of hypersomnia and anxiety disorders while NPS antagonists might be novel therapeutic tools to treat insomnia. Synthetic NPS agonists and antago- nists will also be crucial to discover and study further physiological functions of NPS and validate its poten- tial as a drug target. NPS and its receptor are a very recent example for the impact of orphan receptor research on neuro- science and our understanding of brain functions. The identification of NPS as a modulator of arousal and anxiety represents a first step to elucidate its complete spectrum of physiological functions and sheds new light on the neurochemistry and biological basis of sleep-wakefulness regulation and fear. Acknowledgements R.K.R. and Y.L.X. were supported in part by grants from the National Institutes of Mental Health (NIMH). 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K. Reinscheid and Y L. Xu NPS produces arousal and anxiolysis FEBS Journal 272 (2005) 5689–5693 ª 2005 FEBS 5693 . para- digms measuring responses to novelty or stress. These studies indicate that the NPS system is a newly discovered transmitter system that regulates vigilance and emotional states. NPS appears. sleep onset and mainten- ance. Acetylcholine (ACh) appears to serve a dual role: ACh release coincides with elevated arousal as well as the onset of paradoxical sleep, also known as rapid eye Keywords anxiety;. increases wakefulness and decreases paradoxical (rapid eye movement) sleep and slow wave sleep in rats. In addition, NPS sup- presses anxiety-like behaviors in mice exposed to different behavioral