Mesolimbic neuropeptide W coordinatesstress responses under novel environments

Item Type Article

Authors Motoike, Toshiyuki; Long, Jeffrey M.; Tanaka, Hirokazu; Sinton,Christopher M.; Skach, Amber; Williams, S. Clay; Hammer,Robert E.; Sakurai, Takeshi; Yanagisawa, Masashi

Citation Mesolimbic neuropeptide W coordinates stress responses undernovel environments 2016, 113 (21):6023 Proceedings of theNational Academy of Sciences

DOI 10.1073/pnas.1518658113

Publisher NATL ACAD SCIENCES

Journal Proceedings of the National Academy of Sciences

Rights Copyright © 2016 The Authors. Published by National Academy ofSciences.

Download date 04/02/2021 02:39:40

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Mesolimbic neuropeptide W coordinates stressresponses under novel environmentsT. MOTOIKEa,d, J. M. LONGb,c, H. TANAKAa,d1, C. M. SINTONe, A. G. SKACHa, S. C. WILLIAMSa, R. E. HAMMERb, T.SAKURAId,f, AND M. YANAGISAWAa,d,g*

a Howard Hughes Medical Institute and Department of Molecular Genetics, b Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX75390-8584, USA; c Laboratory of Behavioral Neuroscience, National Institute on Aging, National Institutes of Health, 251 Bayview Blvd. Baltimore, MD21224, USA; d Exploratory Research for Advanced Technology, Yanagisawa Orphan Receptor Project, Japan Science and Technology Agency, Tokyo 135-0064,Japan; eArizona Respiratory Center, Department of Medicine, University of Arizona, Tucson, AZ 85724-5030, USA; f Department of Molecular Neuroscienceand Integrative Physiology, Faculty of Medicine, Institute of Medical, Pharmacological, and Health Sciences, Kanazawa University, Kanazawa 920-8640,Japan; g International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba 305-8575, Japan

Submitted to Proceedings of the National Academy of Sciences of the United States of America

Neuropeptide B (NPB) and neuropeptide W (NPW) are endogenousneuropeptide ligands for the G protein-coupled receptors NPBWR1and NPBWR2. Here we report that the majority of NPW neuronsin the mesolimbic region possess tyrosine hydroxylase (TH) im-munoreactivity, indicating that a small subset of dopaminergicneurons co-express NPW. These NPW-containing neurons denselyand exclusively innervate two limbic system nuclei in adult mousebrain: the lateral bed nucleus of the stria terminalis (BSTL) and thelateral part of the central amygdala nucleus (CeAL). In the CeAL ofwild-type mice, restraint stress resulted in an inhibition of cellularactivity, but this stress-induced inhibition was attenuated in theCeAL neurons of NPW-/- mice. Moreover, the response of NPW-/-

mice to either formalin-induced pain stimuli or a live rat (i.e., apotential predator) was abnormal only when they were placed in anovel environment: they failed to show the normal species-specificself-protective and aversive reactions. In contrast, the behaviorof NPW-/- mice in a habituated environment was indistinguish-able from that of wild-type mice. These results indicate that theNPW/NPBWR1 system could play a critical role in the gating ofstressful stimuli during exposure to novel environments.

Amygdala | Fear | Pain | Dopaminergic | Mouse

INTRODUCTION

Limbic structures, including the amygdala and bed nucleus ofthe stria terminalis (BST), where different modalities of envi-ronmental stimuli can converge and interact, are critical for theresponse to stress (1). The basolateral amygdala (BLA) receivessensory information from the thalamus and cortex, and transfersthat information to the central amygdala (CeA), an output centerfor the amygdaloid complex. Subsequently, the CeA relays thisinformation through axonal projections to nuclei in diverse areas,such as the hypothalamus, brain stem, and pons (2, 3). Thesecircuits are essential for mediating fear and anxiety, especially forfear conditioning using auditory or visual conditioned stimuli(4,5). Indeed, in a recent report, optogenetic manipulation of theBLA-CeA pathway was found to modulate the expression ofanxiety in the mouse (6).

The lateral BST (BSTL) and CeA (CeAL) are two majorcomponents of the central extended amygdala, a continuum of te-lencephalic structures of the forebrain (7, 8). Both the BSTL andCeAL contain numerous GABAergic as well as peptidergic neu-rons, such as those producing enkephalin, corticotropin releasingfactor (CRF) and somatostatin (9-13), and these GABAergicneurons form dense local inhibitory circuits within these nuclei.The GABAergic microcircuit in the CeAL is thought to playpivotal roles in mediating fear and anxiety, as recently dissected atthe cellular level by studies that combined optogenetics and elec-trophysiology (14-17). A subpopulation of GABAergic neurons inthe CeAL, which are inhibited by a conditioned stimulus (CS), aredifferentiated by the expression of protein kinase C-delta (PKC-

delta) (14). These PKC-delta positive GABAergic neurons wereshown to provide feed-forward disinhibition on output neuronsin the medial CeA (CeAM), resulting in a conditioned freezingresponse following exposure to a CS stimulus (14, 15). However,PKC-delta negative GABAergic neurons, which co-express so-matostatin and make reciprocal inhibitory synapses with PKC-delta positive GABAergic neurons, do not send any significantintra-amygdaloid inhibitory projections to the CeAM. Instead,they send long-range projections to innervate the periaqueductalgray (PAG) and the paraventricular nucleus of the thalamus(PVT), i.e., extra-amygdala areas implicated in defensive behav-ior, and thus contribute to fear conditioning independent of theCeAM (16, 17).

The G protein-coupled receptors (GPCRs) NPBWR1 andNPBWR2 (originally termed GPR7 and GPR8) were identifiedas closely-related orphan GPCRs by a degenerative PCR screenusing primers based on sequences from opioid receptors (18).Whereas humans and other primates express both NPBWR1 andNPBWR2, no functional NPBWR2 ortholog has been demon-strated in rodents (19). A role for NPBWR1 has been character-ized by the phenotypical analysis of NPBWR1 null mice, whichdemonstrate late onset obesity with a hyperphagic phenotype(20). These mice also show abnormal social interaction, and anaberrant autonomic response to physical stress (21). Recently,a single nucleotide polymorphism of the human NPBWR1 gene

Significance

The study proposes a physiological role of Neuropeptide W(NPW) in modulating mouse behaviors under stress. Here wefound that NPW-producing neurons (which are a small subsetof mesolimbic dopaminergic neurons) exclusively innervatesthe extended central amygdala, where the peptide plays anessential role in stress-induced inhibition of the amygdalaneurons. The response of NPW-null mice to either formalin-induced pain stimuli or a live rat (a potential predator) wasabnormal when they were placed in a novel environment:they failed to show the normal self-protective and aversivereactions. In contrast, the behavior of NPW-null mice in a ha-bituated environment was normal. These results demonstratea critical role of NPW in the gating of stressful stimuli duringexposure to novel environments.

Reserved for Publication Footnotes

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Submission PDFFig. 1. Neuroanatomy of NPW neurons and their projection sites. (A-F)Immunostaining of NPW in the BSTL (A-C) and the CeAL (D-F). The arrowsindicate NPW-immunoreactivity in the BSTL (A) and CeAL (D). No immunore-activity was observed in NPW -/- mice (B and E). (C and F) High magnificationviews of A and D, respectively. +/+, wild-type mice; -/-, NPW -/- mice. (G-J)Distribution of NPW and tyrosine hydroxylase (TH) in the midbrain. NPWmRNA in the PAG/VTA (G) and the DRD (I), and immunostaining of thesame regions with anti-TH antibody (H and J). ac, anterior commissure; CPu,caudate putamen; opt, optic tract; BLA, basolateral amygdaloid nucleus;PAG, periaqueductal gray; VTA, ventral tegmental area; IP, interpeduncularnucleus; DRD, dorsal part of dorsal raphe nucleus. Scale bars: 500 µm.

Fig. 2. Projection of NPW/TH neurons in the central amygdala. (A and B)Immunostaining of NPW and TH in the CeAL from a preproenkephalin1-EGFP transgenic mouse at low magnification: NPW and TH were stained withAlexa 594 (red). GFP-expressing neurons are enkephalinergic neurons. (C-E) Immunostaining of NPW (green) and TH (red) in the CeAL from a wildtype mouse at high magnification: yellow arrows indicate the co-localizationof NPW and TH in nerve terminals in the CeAL. ic, intercalated cells; BLA,basolateral amygdaloid nucleus. Scale bars: A and B, 100 µm; C-E, 50 µm

has been shown to influence the evaluation of facial expressions(22). We and other groups have isolated neuropeptide B (NPB)and neuropeptide W (NPW) as cognate endogenous ligands for

Fig. 3. NPW neurons co-express TH. (A-C) Co-localization of NPW (green)and TH (red) in cell bodies of the PAG. Immunostaining of midbrain sectionswas performed on colchicine-treated brains: yellow arrows indicate the co-localization of NPW and TH in the PAG. (D-F) Co-localization of NPW mRNA(maroon) and TH (red) in the PAG. After the colorimetric in situ hybridizationof NPW , immunostaining of TH was performed. Scale bars: A-C, 50 µm; D-F,100 µm.

Fig. 4. NPW-dependent suppression of zif268 expression in the CeAL understress. (A) Immunostaining of the CeAL with the anti-zi268 antibody. (upperleft) wild-type, no stress; (upper right) wild-type, after 30-min restraint stress;(lower left) NPW -/-, no stress; (lower right) NPW -/-, after 30-min restraintstress. Red dotted lines demarcate the CeAL and the black dotted linedelineates the intermediate capsule between the CeAL and BLA. opt, optictract; ic, intercalated cells; st, stria terminalis; BLA, basolateral amygdaloidnucleus; M, medial part of the central amygdaloid nucleus. (B) Quantificationof zif268 positive cells (mean ± SEM) in the CeAL. White bars, wild-typemice; gray bars, NPW -/- mice. ** P < 0.05, comparing wild-type mice withand without restraint stress (t-test). *** P < 0.05, comparing wild-type andNPW -/- mice, both after 30-min restraint stress (t-test). (C) In situ hybridizationof NPBWR1 (left) and immunohistochemistry using anti-PKC-delta antiserum(right) on adjacent sections from the CeAL of a wild-type mouse. Redlines demarcate the lateral boundary of the CeAL. opt, optic tract; st, striaterminalis. Scale bars: A, 100 µm; C, 50 µm.

NPBWR1 and NPBWR2 (23-26). NPB and NPW are encoded bytwo separate genes and together with NPBWR1 (and NPBWR2in primates) constitute a neuropeptide receptor system (27). NPBconsists of 29 amino acids with a unique brominated N-terminaltryptophan moiety (23, 25), whereas NPW, as the mature peptide,exists in two forms: NPW [1-23] and NPW [1-30]. Both NPW23

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Fig. 5. Abnormal response of NPW -/- mice to noxious stimuli in novelenvironment.(A) The latency in seconds to withdraw the tail in response toa noxious thermal stimulus. White bars, wild-type mice; gray bars, NPW -/-

mice. (B and C) Time course of paw licking after the subcutaneous injectionof 20 µl of 5% formalin into the plantar surface of the right hind paw ofeach test mouse. Each point represents the amount of time (sec) the mousespent licking the injected paw during a 5-min observation period. (B) Afterthe formalin injection, each test mouse was placed into the home cage. (C)After the formalin injection, each test mouse was placed into a novel cage.** P < 0.05, comparing wild-type (blue) and NPW -/- (red) mice (t-test). Valuesare displayed as mean ± SEM.

and NPW30 bind and activate NPBWR1 and NPBWR2 withsimilar nanomolar affinities (24, 27).

In a previous report, we described the distribution ofNPBWR1, NPB and NPW mRNA in mouse brain by in situhybridization (23). In contrast to relatively widespread distri-butions of NPBWR1 and NPB mRNA, the neurons expressingNPW mRNA are primarily localized in specific midbrain regions,including the periaqueductal gray (PAG), the ventral tegmentalarea (VTA), the Edinger Westphal nucleus, and the dorsal part ofthe dorsal raphe nucleus (23, 28). This characteristic expressionpattern implies a potential role for NPW in the regulation of lim-bic function. In this regard, we also observed a diffuse expressionof NPW mRNA in the CA3 region of adult mouse hippocampus(29). Here we investigated the potential physiological functionsof NPW using histochemical studies and phenotypical analysesof NPW null mice, with a particular focus on their behavioralcharacteristics under environmental stress.

RESULTS

NPW neurons in midbrain are dopaminergic and project ex-clusively to the central extended amygdala. A systematic im-munohistochemical survey of the entire central nervous system(CNS) with verified anti-NPW antisera showed localization ofimmunoreactivity exclusively to the BSTL and CeAL (Fig. 1 Aand D). A high magnification view (Fig. 1 C and F) revealedthat specific immunoreactivity surrounded neuronal cell bodiesin these two regions, indicating that axonally transported NPWwas accumulating at the axonal terminals. Consistent with thisobservation, abundant NPBWR1 mRNA expression has beenpreviously reported in both the BSTL and CeAL (23). No im-munoreactive staining was observed in these regions in brainslices from NPW-/- mice, indicating the fidelity and specificity ofthe antiserum (Fig. 1 B and E) (see Supplementary Informationfor the generation of NPW-/- mice). In summary, these resultsindicate that NPBWR1-expressing neurons in the mouse BSTLand CeAL are extensively innervated by NPW neurons.

The dorsocaudal part of the ventral tegmental area (VTA),collectively defined as the A10dc, encompassing the dorsal raphenucleus and the periaqueductal gray (PAG), contains many NPWneurons (Fig. 1 G and I) as well as dopaminergic neurons (30)

Fig. 6. Abnormal predator-fear response of NPW -/- mice in novel envi-ronment. (A) c-fos immunoreactivity in the paraventricular nucleus (PVN)induced by the stress of a 60-min exposure to a rat. Upper panels: wild-typemice; lower panels: NPW -/- mice. (B) Changes in plasma corticosterone levelafter a 30-min exposure to a rat. ** P < 0.05, comparing mice of the samegenotype with and without rat stress (t-test). (C) Thigmotaxis: the proportionof time spent in the center area of the open field. After a 1-h exposure to arat in the rat cage, each mouse was tested in the open field and its activitywas recorded for 15 min. ** P < 0.05, comparing wild-type mice with andwithout exposure to rat stress (t-test). *** P < 0.05, comparing wild-type andNPW -/- mice, both after rat stress (t-test). (D) The proportion of time spentin the lighted compartment of the light-dark box. After a 1-h exposure toa rat in the rat cage, each mouse was tested in the light-dark box and itsactivity was recorded for 12 min. ** P < 0.05, comparing mice of the samegenotype with and without exposure to rat stress (t-test). (E and F) The timethat each test mouse spent vigilantly staring at the rat was measured duringa 1-h observation period. (E) The test mouse was placed into a large cagewhere a rat had been singly housed for several days. ** P < 0.05, comparingwild-type and NPW -/- mice (t-test). (F) A rat was placed into a large cagewhere the test mouse had been singly housed for 2-3 days. White bars, wild-type mice; gray bars, NPW -/- mice. Values are displayed as mean ± SEM. n.s.,not significant.

(Fig. 1 H and J). Immunohistochemistry using NPW antiserum(Fig. 1D and Fig. 2A) and a tyrosine hydroxylase (TH) antibody(Fig. 2B) revealed dense localization of NPW and TH in the me-dial region of the CeAL. Double immunohistochemistry showedsignificant co-localization of NPW and TH in axon terminals inthe CeAL (Fig. 2 C-E). After colchicine treatment, the majorityof the NPW-containing cell bodies in the PAG region were foundto co-express TH (Fig. 3 A-C). Essentially the same results werealso obtained by combining NPW in situ hybridization and TH im-munohistochemistry (Fig. 3 D-F). These results demonstrate thatthe majority of NPW neurons in the midbrain are dopaminergic,and that a subset of A10dc dopaminergic neurons express NPW.

Stress-induced, NPW-dependent modulation of neuronal ac-tivity in the CeAL. Using the immediate early gene zif268 as amarker for neuronal activity (31), we noted in wild-type micethat the number of zif268-positive cells decreases in the CeALafter a 30-min exposure to restraint stress. In contrast, this acutestress-induced decline in the number of zif268-positive CeAL

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cells does not occur in NPW-/- mice (Fig. 4 A and B). Thissuggested that, in the absence of NPW innervation, a subset ofCeAL neurons might be disinhibited during exposure to stressfulsituations. Two populations of CeAL neurons were identifiedwith opposite responses to a CS and shown to make reciprocalinhibitory connections (15). These two populations of neuronsare differentiated by the expression of protein kinase C-delta(PKC-delta). We examined whether such CS-inhibited PKC-deltapositive neurons in the CeAL express NPBWR1. Using NPBWR1in situ hybridization and PKC-delta immunohistochemistry, wenoted a clear difference in the localization pattern of these twomolecules in the CeAL, i.e., NPBWR1 mRNA is expressed pref-erentially in the medial region of the CeAL, whereas the majorityof PKC-delta expressing neurons are located in the lateral regionof the CeAL (Fig. 4C). From these results, we hypothesized thatNPBWR1 expressing neurons do not innervate and disinhibit theoutput neurons in the CeAM. Rather, these NPBWR1 neuronsmay modulate CeAL neurons, such as the PKC-delta neurons.

NPW-/- mice showed an abnormal response to noxious stimuliwhen placed in a novel environment. In a previous study, wefound that intracerebroventricular (icv) administration of NPBin the rat induced analgesia in the formalin test (23). Also, asnoted above, NPW mRNA is highly expressed in the PAG, an areaimplicated in the descending analgesic system (23). We thereforesubmitted NPW-/- mice to the tail-flick test to investigate whetherthey exhibited any deficiency in their response to acute pain.Wild-type and NPW-/- mice were not different in their responsein this test (Fig. 5A). We then examined whether NPW-/- micedemonstrated an altered pain response to formalin injected in ahindpaw. Wild-type and NPW-/- mice exhibited essentially identi-cal licking behaviors to the formalin injection site after they werereturned to the home cage, confirming that NPW-/- mice havea normal response to pain under baseline conditions (Fig. 5B).In contrast, however, we observed a marked reduction in lickingbehavior in NPW-/- mice when they were returned to a novelcage after the injection (Fig. 5C). Wild-type mice exhibited thesame licking behavior when returned to either cage. A novel cageenvironment is known both to be acutely stressful and to stimulateexploratory behavior in mice (32). We thus hypothesized thatNPW-/- mice exhibited reduced pain-induced behavior becauseof the additional stress and distraction of a novel environmentfollowing the formalin injection. Exposure to a new testing cageor laboratory environment has been shown to induce analgesiain rats and mice (33, 34). Here, however, we did not observeany analgesic effect of the novel cage environment in wild-typemice. This absence of an analgesic effect in wild-type mice may bedue to the balance between the salience of the pain experiencedhere versus the novelty of the environment: i.e., the pain causedby 20 µl of 5% formalin in wild-type mice would surpass thenovelty effect caused by a different cage. This will be examined infuture studies, in which a more balanced condition using a milderpain stimulus combined with a more significant environmentalchange may evoke an analgesic effect in wild-type mice. Mostimportantly, however, even with such a relatively minor changeto environmental conditions in the present study, NPW-/- miceexhibited a significant reduction in licking after the formalininjection. In view of this result, we thus also considered whetherexposure to environmental stimuli might be necessary as an ad-junct to reveal further behavioral abnormalities in NPW-/- mice.

NPW-/- mice showed abnormal fear-oriented behaviors whenplaced in a novel environment. As a way of imposing a compellingstress, we introduced an awake rat, which is a potential predatorfor a mouse. In order to verify the efficacy of this stressor, weexamined two established stress indicators, c-fos expression in theparaventricular nucleus (PVN) of the hypothalamus and plasmacorticosterone levels. As shown in Fig. 6A, a 60-min exposure

to a rat induced significant c-fos expression in the PVN regionin both wild-type and NPW-/- mice. Plasma corticosterone levelswere also increased to a similar extent in response to exposure toa rat in both genotypes (Fig. 6B). These results showed that theparadigm of rat exposure was effective at inducing the intrinsicstress response independently of NPW. Importantly, these re-sults also demonstrated that NPW-/- mice had normal awarenessof a potentially life-threatening change in their environment.Furthermore, these data showed that NPW-/- mice had intactpathways for the acute neuroendocrine stress response and pro-duced normal and robust activation of the hypothalamo-pituitary-adrenocortical (HPA) axis in response to the predator stress.

We then evaluated the locomotor activity of wild-type andNPW-/- mice in the open field test after exposure to a rat. Duringa 15-min session in the open field, both wild-type and NPW-/-

mice, without exposure to a rat, showed similar locomotor activityin terms of total distance traveled and center time (Fig. 6C).After a 1-h exposure to a rat, wild-type mice showed a significantdecrease in center time (Fig. 6C). In contrast, NPW-/- mice didnot show this decrease in time in the central area of the openfield after rat exposure. Thigmotaxis, or the aversion to openspaces, is a characteristic rodent behavior which is considered acorrelate of anxiety (35). This finding, that NPW-/- mice showedless thigmotaxis than wild-type mice, suggested that NPW-/- micewere apparently less anxious after exposure to a rat.

We next assessed this apparent hypoanxiety observed inNPW-/- mice after rat exposure by using the light-dark box test,in which a less anxious mouse tends to spend more time in thebrightly lit compartment. Under control conditions and after a 1-hexposure to a rat, both wild-type and NPW-/- mice showed similarlocomotor activity in terms of total distance traversed. The timespent in the lighted compartment was significantly decreased inwild-type mice after exposure to a rat, as expected. In contrast,NPW-/- mice exhibited a significantly weakened aversion towardsthe lit, open compartment (Fig. 6D). These results support theopen-field thigmotaxis data, and indicate less anxiety in NPW-/-

mice after a 1-h exposure to a rat in a novel environment. How-ever, the fact that NPW-/- mice were exhibiting less overt anxietyin these tests following exposure to the rat did not necessarily con-firm a hypoanxious state. It could, for example, reflect increaseddistraction after rat exposure - as we had postulated as a feasibleexplanation for the results following the formalin injection.

During the time of exposure to the rat, we frequently ob-served unusual behaviors in NPW-/- mice. For a naïve wild-typemouse, a rat in the same cage is potentially threatening and thesituation is thus highly stressful for the mouse. Hence, wild-typemice typically demonstrated species-specific, innate fear-orientedbehaviors towards the rat, such as marked avoidance, maintainingmaximal distance and full vigilance, and orienting themselvestowards the rat. In contrast, NPW-/- mice often appeared indif-ferent towards the rat, i.e., they fell asleep, approached the ratfrequently and closely, and in some cases they ate and drankin the presence of the rat. These behaviors were quantified bymeasuring the time that each mouse spent vigilantly directingits gaze towards the rat during the 1-h exposure. As displayedin Fig. 6E, NPW-/- mice vigilantly stared at the rat significantlyless than wild-type mice. Importantly, this difference in behaviorwas not observed in an alternative paradigm, in which the rat wasplaced as an intruder into a cage in which a mouse had been singlyhoused and well acclimated. NPW-/- mice, like wild-type mice,were highly vigilant towards the intruder rat under these circum-stances (Fig. 6F). These data, taken in conjunction with resultsfrom the formalin test, indicate that NPW-/- mice behave inap-propriately and do not express normal self-preservation behaviorswhen they encounter a life-threatening event concurrently with

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a novel environment. In other words, a novel environment maybe overly distracting for these mice and this has a major impacton their ongoing behavior. These results support the hypothesisthat NPW-/- mice may also have been distracted and thus didnot initiate typical self-protective behaviors in the open field andlight-dark box tests after exposure to a rat.

DISCUSSIONContrary to our prediction and to the previous published dataobtained from icv injection experiments (36), NPW-/- mice werenot different in bodyweight or metabolic state from wild-typemice (Fig. S2). We thus hypothesize that it is NPB, and notNPW, that regulates feeding behavior and energy homeostasis inadult mice by acting at NPBWR1. Consistent with this hypothesis,NPB-/- mice exhibit an obese phenotype (29).

We have also demonstrated that the majority of NPW neu-rons in the A10dc region co-express dopamine, and that the BSTLand CeAL are the exclusive target regions of NPW neurons inmice. Eliava et al. (37) reported that dopaminergic axons, but notnoradrenergic or serotonergic axons, form a dense plexus in theCeAL. A dense dopaminergic innervation of the CeAL as well asthe dorsolateral subdivision of the BST has also been described(38). By combining retrograde dye and TH immunostaining,dopaminergic neurons of the A10dc group have been shown tocontain approximately half of the total number of retrograde-dye/TH double-positive neurons projecting to the CeA and BSTL(30). Thus, mesolimbic dopaminergic neurons extensively inner-vate cell bodies in the CeAL and BSTL, and these dopaminergicneurons are likely to exert a crucial role in these nuclei (39).Together with our findings that the majority of NPW neuronslocalized in the A10dc region co-express dopamine and sendaxons exclusively to the CeAL and BSTL, we now hypothesizethat NPW also plays an essential role in modulating limbic systemfunction.

In our previous study, we observed a diffuse expression ofNPW mRNA in the CA3 region of adult mouse hippocampus(29). Consistent with this, a diffuse expression of NPBWR1mRNA was detectable in the CA1 region, which is the primarytarget of the CA3 pyramidal neurons (23). However, we wereunable to detect any distinct expression of NPW mRNA in theadult hippocampus using colorimetric ISH (40). Furthermore, wedetected NPW immunoreactivity only in the BSTL and CeAL,but not in the target regions of CA3 pyramidal neurons, such asthe lateral septal nucleus, CA1 and CA3 regions. We assume,therefore, that a subpopulation of CA3 neurons might expressNPW mRNA but, if any, at a very low level and that any involve-ment of the hippocampal NPW/NPBWR1 system in the behaviorsexamined here would be small.

Burst activity in VTA dopaminergic neurons is elicited bysudden auditory or visual orienting stimuli in the awake cat(41). Restraint stress also increases dopaminergic burst firing inputative VTA neurons in awake rats (42). In these studies, thedischarge of dopaminergic neurons in the A10dc region was notspecifically investigated, but it is likely that novel sensory stimulisuch as those used in these studies would evoke the same patternof excitation as that noted for dopaminergic neurons throughoutthe VTA. NPW-containing dopaminergic neurons in the A10dcregion may therefore also be activated by these stimuli in the sametime frame and transduce signals to the CeAL and BSTL. In ratbrain, particularly high levels of dopamine D2 receptor (D2R)ligand binding was observed in the CeAL (43), and D2R mRNA-positive neurons have been localized to the medial region of theCeAL (37). We have observed the localization of NPW- and TH-immunoreactive fibers, as well as NPBWR1 expression, in themedial region of the CeAL (Fig. 1D and Fig. 2 A and B) (21). Al-though the cellular co-localization of NPBWR1 and D2R remainsunconfirmed, considering that both NPBWR1 and D2R are Gi-

coupled inhibitory receptors, any activity of NPBWR1+/D2R+neurons in the CeAL would be under potent inhibitory regula-tion by NPW/dopamine neurons. Our histological analysis hasshown that the localization patterns of NPBWR1 neurons andPKC-delta positive neurons in the CeAL are distinct. This resultsuggests that NPBWR1 neurons are not those that are inhibitedby a CS and do not disinhibit the output neurons in the CeAM (14,15). Instead, NPBWR1 neurons may co-express somatostatin andsend inhibitory axons to PKC-delta positive neurons in the CeALand/or send long-range projections to extra-amygdala areas. Thisinterpretation is supported by our previous finding that thoseCeAL neurons that were hyperpolarized by bath application ofNPB or NPW in whole cell recordings, send short axons within theCeAL or relatively long axons outside the CeAL. This was shownby neurobiotin injection after recordings were completed (21).Importantly, therefore, a role of NPBWR1 neurons in conveyingfear-mediating signals would likely involve such projections.

NPW-/- mice behaved inappropriately and did not expresstypical self-preserving behaviors when they abruptly encounteredlife-threatening events while concurrently placed in a novel en-vironment. Significantly, however, the stress-induced behaviorof NPW-/- mice under habituated conditions was identical tothat of wild-type mice. We hypothesize therefore that NPW-/-

mice are easily distracted and are unable to initiate normalspecies-specific self-protective reactions during and after expo-sure to novel environmental stimuli. At the cellular level, we havedemonstrated that restraint stress inhibited a subset of neuronsin the CeAL of wild-type mice. Similar results were previouslyreported using c-fos expression: several stressors, including re-straint stress, a loud noise or a novel environment, were found todecrease amphetamine-induced expression of c-fos in the CeALand BSTL (32, 44). Hence, inhibition of neuronal activity inthe CeAL and BSTL appears to be common to many, if notall, stressors. However, this inhibition was not fully exerted inCeAL neurons in NPW-/- mice, and this disinhibition of a groupof CeAL neurons may thus underlie the abnormal behavior ofNPW-/- mice observed here. Further studies to characterize theactivity of this specific subset of neurons in the CeAL of NPW-/-

mice in a novel environment may aid in the understanding of theneuronal mechanisms of the stress response, and by extension,the pathophysiology of those conditions in which impaired socialadaptation is symptomatic.

Our findings have demonstrated that behavioral adaptationin mice to a novel environment and the expression of an ap-propriate behavioral response are specifically dependent on con-current stimulus gating. Mesolimbic dopamine projections havelong been recognized as playing a role in orienting to, and ingating, significant stimuli (45). Our present results now indicatethat a subset of these dopaminergic projections, containing NPW,may play a critical role in the processing of, and adaptationto, novel environmental situations. Elsewhere, we have analyzedand described the behavioral phenotypes of mice with targeteddisruption of the NPBWR1 gene (21). NPBWR1 null mice alsoshowed a deficit in social interaction when they encountered anovel conspecific intruder and they exhibited abnormal auto-nomic and neuroendocrine responses to physical stress. Takentogether, these observations suggest an important role of theamygdala NPW/NPBWR1 system in the adaptation to stress innovel environments.

In conclusion, NPW-containing dopaminergic neurons in themesolimbic region may be critical for the transduction of stress-related information into the central extended amygdala whennovel environmental stimuli are encountered. We have demon-strated that NPW is essential for the appropriate expression ofnormal species-specific behaviors in response to life threatening,stressful stimuli under novel environments. Thus NPW-/- micebehaved abnormally in response to life-threatening events when

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the environment was novel and/or distracting. A possible inter-pretation is that these stimuli are not being gated appropriatelyin NPW-/- mice, and stimulus salience is either overwhelming orassigned an incorrect priority, so causing an inappropriate behav-ioral response. Determining the functional importance of thesefindings now requires additional studies, but our results havepotential implications for those psychiatric conditions, in whichstimulus gating, the orienting response and/or social adaptationare abnormal.

Materials and MethodsDetailed methods are provided in SI Materials and Methods. All animal proce-dures were approved by the Institutional Animal Care and Research AdvisoryCommittee (IACRAC) of the University of Texas Southwestern Medical Center,and were strictly in accordance with National Institutes of Health guidelines.Plasma corticosterone was estimated by the Endocrine Services Laboratory at

the Oregon National Primate Research Center (Beaverton, OR) as previouslydescribed (46).

CONFLICT OF INTERESTThe authors declare that no competing interests exist.

Acknowledgments .This work was supported in part by research funds from the Keck

Foundation; the Perot Family Foundation; the Exploratory Research forAdvanced Technology of Japan Science and Technology Agency; the WorldPremier International Research Center Initiative from MEXT, Japan; andthe Intramural Research Program of the NIH (J.M.L.). M.Y. is a formerInvestigator of the Howard Hughes Medical Institute during the periodwhen this research was performed. We thank Drs. Hiroshi Kuriyama, ShioriOgawa, Kenji Shibata, Norimasa Miyamoto and Yoji Kitamura for helpfuladvice and excellent technical assistance; Dr. David Hess for measuring serumcorticosterone; Claudia Erbel-Sieber, Sandi Jo Estill, and Carol Dudley fortechnical assistance in behavioral studies; Elizabeth Lummus for the injectionof ES cells for generating NPW -/- mice; and Shelley Dixon, Randal Floyd andMarcus Thornton for their technical supports.

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