Báo cáo khoa học: Neuropeptide S as a novel arousal promoting peptide transmitter pdf

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

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 Ca2+ 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.

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 Barrcorticotropin-releas-ington’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).

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 Ca2+, indicating that NPS might be

an excitatory transmitter in vivo by elevating

intracellu-lar Ca2+ A radiolabled analog of NPS (125I-labeled

Tyr10-NPS) shows displaceable binding with high

affin-ity (Kd¼ 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.

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 (Provigil
), whose mechanism of action is

still unknown, induces long-lasting wakefulness but

does not modulate anxiety [18] Typical anxiolytic

drugs such as benzodiazepines (diazepam, Valium
)

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) R.K.R was also supported by a Young Investigator Award from the National Alliance for Research on Schizophrenia and Depression (NARSAD)

References

1 Siegel JM (2004) The neurotransmitters of sleep J Clin Psychiatry 65 (Suppl 16), 4–7

2 Jones BE (2003) Arousal systems Front Biosci 8, 438– 451

3 Jones BE (2004) Activity, modulation and role of basal forebrain cholinergic neurons innervating the cerebral cortex Prog Brain Res 145, 157–169

4 Gerashchenko D, Okano Y, Urade Y, Inoue S & Hayaishi O (2000) Strong rebound of wakefulness fol-lows prostaglandin D2- or adenosine A2a receptor agonist-induced sleep J Sleep Res 9, 81–87

5 Greene R & Siegel J (2004) Sleep: a functional enigma Neuromolecular Med 5, 59–68

6 Chemelli RM, Willie JT, Sinton CM, Elmquist JK, Scammell T, Lee C, Richardson JA, Williams SC, Xiong Y, Kisanuki Y et al (1999) Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation Cell 98, 437–451

7 Lin L, Faraco J, Li R, Kadotani H, Rogers W, Lin X, Qiu X, de Jong PJ, Nishino S & Mignot E (1999) The sleep disorder canine narcolepsy is caused by a mutation

in the hypocretin (orexin) receptor 2 gene Cell 98, 365– 376

8 de Lecea L & Sutcliffe JG (2005) The hypocretins and

sleep FEBS J 272, 5675–5688

9 Cheng MY, Bullock CM, Li C, Lee AG, Bermak JC,

Belluzzi J, Weaver DR, Leslie FM & Zhou QY (2002)

Prokineticin 2 transmits the behavioural circadian rhythm

of the suprachiasmatic nucleus Nature 417, 405–410

10 de Lecea L, Criado JR, Prospero-Garcia O, Gautvik

KM, Schweitzer P, Danielson PE, Dunlop CL, Siggins

GR, Henriksen SJ & Sutcliffe JG (1996) A cortical

neuro-peptide with neuronal depressant and sleep-modulating

properties Nature 381, 242–245

11 Xu YL, Reinscheid RK, Huitron-Resendiz S, Clark SD,

Wang Z, Lin SH, Brucher FA, Zeng J, Ly NK,

Henrik-sen SJ, de Lecea L & Civelli O (2004) Neuropeptide S:

a neuropeptide promoting arousal and anxiolytic-like

effects Neuron 43, 487–497

12 van der Werf YD, Witter MP & Groenewegen HJ

(2002) The intralaminar and midline nuclei of the

thala-mus Anatomical and functional evidence for

participa-tion in processes of arousal and awareness Brain Res

Rev 39, 107–140

13 Pare D, Quirk GJ & Ledoux JE (2004) New vistas on

amygdala networks in conditioned fear J Neurophysiol

92, 1–9

14 N’junge K & Handley SL (1991) Evaluation of

marble-burying behavior as a model of anxiety Pharmacol

Biochem Behav 38, 63–67

15 Hascoet M & Bourin M (1998) A new approach to the light⁄ dark test procedure in mice Pharmacol Biochem Behav 60, 645–653

16 Paine TA, Jackman SL & Olmstead MC (2002) Cocaine-induced anxiety: alleviation by diazepam, but not buspirone, dimenhydrinate or diphenhydramine Behav Pharmacol 13, 511–523

17 Hagan JJ, Leslie RA, Patel S, Evans ML, Wattam TA, Holmes S, Benham CD, Taylor SG, Routledge C, Hem-mati P et al (1999) Orexin A activates locus coeruleus cell firing and increases arousal in the rat Proc Natl Acad Sci USA 96, 10911–10916

18 Simon P, Panissaud C & Costentin J (1994) The stimu-lant effect of modafinil on wakefulness is not associated with an increase in anxiety in mice A comparison with dexamphetamine Psychopharmacology 114, 597–600

19 Chaouloff F, Durand M & Mormede P (1997) Anxiety-and activity-related effects of diazepam Anxiety-and chlordiazep-oxide in the rat light⁄ dark and dark ⁄ light tests Behav Brain Res 85, 27–35

20 Paxinos G & Watson C (1997) The Rat Brain in Stereo-taxic Coordinates Compact 3rd edn Academic Press, San Diego, CA