Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior

Journal Pre-proof Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior

Shunji Yamada, Mohammad Shyful Islam, Nienke van Kooten, Sonny Bovee, Yoon-Mi Oh, Atsushi Tsujimura, Yoshihisa Watanabe, Masaki Tanaka PII:

S0014-4886(20)30047-9

DOI:

https://doi.org/10.1016/j.expneurol.2020.113216

Reference:

YEXNR 113216

To appear in:

Experimental Neurology

Received date:

28 October 2019

Revised date:

7 January 2020

Accepted date:

30 January 2020

Please cite this article as: S. Yamada, M.S. Islam, N. van Kooten, et al., Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior, Experimental Neurology (2019), https://doi.org/10.1016/j.expneurol.2020.113216

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© 2019 Published by Elsevier.

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Neuropeptide Y neurons in the nucleus accumbens modulate anxiety-like behavior

Shunji Yamada a, Mohammad Shyful Islama , Nienke van Kootena, Sonny Bovee a, Yoon-Mi Oha, Atsushi Tsujimura b , Yoshihisa Watanabe b , Masaki Tanaka a,*

a

Department of Anatomy and Neurobiology, Graduate School of Medical Science, Kyoto

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Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan

Department of Basic Geriatrics, Graduate School of Medical Science, Kyoto Prefectural

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University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566,

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Japan

*Corresponding author:

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E-mail: [email protected] (MT)

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Abstract Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that is widely expressed in the central nervous system, including the cerebral cortex, nucleus accumbens (NAc) and hypothalamus. We previously analyzed the behavior of transgenic mice exclusively expressing an unedited RNA isoform of the 5-HT2C receptor. These mice showed decreased NPY gene expression in the NAc and exhibited behavioral despair, suggesting that NAc NPY neurons may be involved in mood

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disorder; however, their role in this behavior remained unknown. Therefore, in the present study, we investigated the functional role of NAc NPY neurons in anxiety-like behavior by examining

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the impact of specific ablation or activation of NAc NPY neurons using NPY-Cre mice and Cre-dependent adeno-associated virus. Diphtheria toxin-mediated ablation of NAc NPY neurons

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significantly increased anxiety-like behavior in the open field and elevated plus maze tests,

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compared with before toxin treatment. Moreover, chemogenetic activation of NAc NPY neurons reduced anxiety-like behavior in both behavioral tests compared with control mice. These

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results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.

Neuropeptide Y

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Keywords

Nucleus accumbens

Anxiety-like behaviors

Diphtheria toxin receptor Designer receptors exclusively activated by designer drugs

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1. Introduction Neuropeptide Y (NPY) is a highly conserved 36-amino acid peptide that is expressed widely in the central nervous system. Several lines of evidence demonstrate that NPY exerts an anxiolytic-like effect (Heilig, 2004; Kask et al., 2002). For example, intracerebroventricular (ICV) administration of NPY in rats induces strong preference for the open arms in the elevated plus maze test (EPM), which is one of the most widely used behavioral paradigms for assessing

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anxiety-like behavior (Broqua et al., 1995). Center distance is significantly reduced in NPY knockout (KO) mice relative to wild-type (WT) mice in the open field test (OFT), which is

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another common test for anxiety-like behavior in rodents (Bannon et al., 2000). ICV administration of NPY increases food intake and produces anxiolytic-like behavior in rats, but

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microinjection of NPY into the amygdala has only an anxiolytic effect (Heilig et al., 1993),

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suggesting that distinct mechanisms underlie NPY-mediated regulation of food intake and anxiety-like behavior. The role of NPY neurons in the hypothalamic arcuate nucleus in the

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regulation of food intake has been extensively studied (Gehlert, 1999; Inui, 1999; Kalra et al.,

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1999). On the other hand, it is difficult to elucidate where NPY neurons related to anxiety located in by the pharmacological experiments, even if NPY neurons locate in the

2002).

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anxiety-related brain regions, such as amygdala and dorsal periaqueductal gray (Kask et al.,

The nucleus accumbens (NAc), which has a central role in reward systems (Sesack and Grace, 2010), is an area of the brain rich in NPY-expressing cells (Chronwall et al., 1985; de Quidt and Emson, 1986; Morris, 1989). We previously demonstrated that NPY-containing neurons are distributed in both the core and shell of the NAc (Aoki et al., 2016). In addition, we analyzed the behavior of transgenic mice expressing an unedited RNA isoform of the 5-HT2C receptor, and found that these mice showed a reduction in NPY gene expression in the NAc and exhibited behavioral despair (Aoki et al., 2016). Likewise, ICV administration of cholecystockinin-4 causes a decrease in NPY-immunoreactive fibers and cell bodies in the NAc

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and induces anxiety-like behaviors (Desai et al., 2014). These observations suggest that NAc NPY neurons may be linked to mood disorders. In the present study, to directly investigate functional role of NAc NPY neurons, we constructed adeno-associated virus (AAV) encoding Cre-dependent diphtheria toxin receptor (DTR) and performed region-specific ablation of NAc NPY neurons using NPY-Cre mice and diphtheria toxin (DT)-mediated cell ablation/dysfunction technique. Moreover, using designer receptors

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exclusively activated by designer drugs (DREADD) technology, we examine the impact of the

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activation of NAc NPY neurons on anxiety-like behavior.

2. Materials & Methods Animals

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2.1.

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To produce NPY-Cre mice, B6.FVB(Cg)-Tg(NPY-cre)RH26Gsat/Mmucd (037423-UCD, NPY-Cre) sperm was purchased from Mutant Mouse Resource & Research Centers (CA, USA).

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We entrusted the generation of NPY-Cre mice using the sperm to the RIKEN BioResource

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Research Center (Ibaraki, Japan). The mice were kept under a 12-h light/dark cycle (lights on at 08:00). Standard food pellets and water were provided ad libitum. Behavioral testing was

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performed between 10:00 and 13:00. All animal procedures, including the production and maintenance protocols, and behavioral studies, were reviewed and approved by the Animal Care and Use Committee of the Kyoto Prefectural University of Medicine (M30-187).

2.2.

Adeno-associated virus preparation

The human HB-EGF (hDTR) fragment was inserted into the NheI and Xbal sites in the pAAV-EF1a-double-floxed-mCherry-WPRE-HGHpA vector (to obtain pAAV-EF1a-floxed-hDTR-mCherry). To substitute the mCherry for EGFP, the EGFP fragment was cut from pEGFP-N1 by SmaI and BsrGI digestion and inserted into the BsaBI and BsrGI sites in pAAV-EF1a-floxed-hDTR-mCherry (to obtain pAAV-EF1a-floxed-hDTR-EGFP).

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pAAV-EF1a-DIO-hM3D(Gq)-mCherry (Addgene plasmid #50460; http://n2t.net/addgene:50460; RRID: Addgene_50460) and pAAV-EF1a-double floxed-mCherry-WPRE-HGHpA (Addgene plasmid # 20299 ; http://n2t.net/addgene:20299 ; RRID:Addgene_20299) were a gift from Bryan Roth and Karl Deisseroth, respectively. For recombinant AAV (rAAV) production, the vectors were cotransfected into HEK293 cells with pAAV-DJ and pHelper (Cell Biolabs, San Diego, CA, USA), according to the manufacturer’s

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instructions. rAAV particles were purified by discontinuous step gradient using iodixanol (Opti-Prep; Nycomed Pharma, Oslo, Norway), as described previously (Aoki et al., 2016;

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Shirahase et al., 2018). Iodixanol fractions were concentrated and dialyzed by centrifugation through a Biomax 100K filter (Millipore, Billerica, MA, USA) with phosphate-buffered saline

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(PBS) containing 5% sorbitol. rAAV titers were determined by quantitative PCR using the

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AAVpro titration kit (Takara Bio, Shiga, Japan), and aliquoted rAAVs were stored at −80 ºC. To eliminate NAc NPY neurons, we used the DT-mediated cell ablation technique. Briefly,

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AAV-EF1a-floxed-hDTR-EGFP was injected into the NAc in NPY-Cre mice to induce

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hDTR-EGFP expression in the NPY neurons in this nucleus. These mice were subsequently

neurons.

2.3.

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administered DT. The binding of DT to DTR would then result in the cell death of these NPY

Stereotaxic surgery

Mice, 10–14 weeks of age, were anesthetized with 20 mg/ml chloral hydrate (Nacalai Tesque, Kyoto, Japan) and placed on a stereotaxic apparatus (Narishige, Tokyo, Japan). AAV-DIO-mCherry (0.8 × 107 viral genome particles/mL), AAV-DIO-hM3Dq-mCherry (1.4 × 1011 viral genome particles/mL) or AAV-EF1a-floxed-hDTR-EGFP (3.3 × 1011 viral genome particles/mL) together with Fast Green dye (Nacalai Tesque) was bilaterally microinjected (0.5 l/site) into the NAc (AP, +1.9 mm from the bregma; ML, ±0.8 mm from the midline; DV, 4.3 mm below the skull surface) using a 30-gauge Hamilton syringe needle (Hamilton, Reno, NV,

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USA) at a rate of 0.1 l/min. These injected mice are hereafter referred to as mCherry, NPY-hM3Dq and NPY-DTR mice, respectively. The needle was kept in place for 5 min after each injection, and then, the needle was slowly removed. mCherry, hM3Gq-mCherry and DTR-EGFP expression was allowed to develop for more than 2 weeks.

2.4.

Behavioral tests

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To examine anxiety-like behavior, we performed two behavioral tests—the open field test (OFT) and the elevated plus maze test (EPM). Each test was performed once per day. In the

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NAc NPY neuron lesion experiment, NPY-DTR and mCherry mice were given the two behavioral tests before DT injection. After these tests, the mice were administered 50 ng/g

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(body weight) DT (Bioacademia, Osaka, Japan) by intraperitoneal injection on two consecutive

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days. After 3 weeks, the DT-injected mice were tested again. For NAc NPY neuron-specific activation, 0.1 mg/ml clozapine-N-oxide (CNO; Cayman,

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Michigan, USA) was administered intraperitoneally at 5 mg/kg (body weight) to

Open field test

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2.4.1.

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hM3Dq-mCherry and mCherry mice 30–60 min before behavioral testing.

The OFT was performed as described previously (Shirahase et al., 2018). Each mouse was allowed to move freely in the open field box (60 × 60 cm) for 30 min. The percentage of time spent and the percentage of distance traveled in the center area of the open field (30 × 30 cm), and the total distance traveled in the open field were measured using SMART v3.0 software (Panlab SL, Barcelona, Spain).

2.4.2.

Elevated plus maze test

The EPM test was performed as described previously (Aoki et al., 2016). The EPM consisted of two open arms (30 × 5 cm) and two enclosed arms of the same size, with 13-cm-high

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transparent walls. The arms and central square were constructed from gray plastic sheets elevated to a height of 50 cm above the floor. To minimize the likelihood of animals falling from the apparatus, 5-mm-high Plexiglas aides were used for the open arms. Arms of the same type were arranged opposite each other. The device was set up under low illumination (center square, 200 lux). Each mouse was placed in a closed arm of the maze. Mouse behavior was recorded during a 10-min test period. The number of entries into and the time spent in the open

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arms were recorded. For data analysis, the following two measures were used: percentage of entries into the open arms and duration of stay in the open arms. Data acquisition and analysis

Immunohistochemistry (IHC)

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2.5.

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were performed using SMART v3.0 software.

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After behavioral tests, DT-treated mCherry and NPY-DTR mice were anesthetized with pentobarbital (64.8 mg/ml Somnopentyl; Kyouritsu Seiyaku, Tokyo, Japan) and perfused with

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physiological saline followed by 4% paraformaldehyde (PFA) in 0.05 M phosphate buffer. The

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brain was immediately removed, postfixed with the same fixative overnight at 4 °C, and then kept in 30% sucrose in 0.05 M phosphate buffer (pH 7.5) at 4 °C. Every fourth coronal section

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(30 µm) containing the NAc (from 1.94 to 0.74 mm anterior to the bregma) was obtained using a cryostat (CM 3050 S; Leica, Wetzlar, Germany). Brains were processed for NPY IHC. Every fourth section through the NAc in the control (n = 5) and NPY-DTR (n = 5) mice was sequentially incubated with 0.3% H 2 O2 and 0.3% Triton X-100 in PBS for 30 min, and then in PBS containing 2% normal goat serum (NGS) and 0.1% Triton X-100 for 1 h at room temperature (RT). Sections were then incubated with rabbit antiserum against NPY (1:2,000; D7Y5A, Cell Signaling Technology, Danvers, USA) for 72 h at 4 °C. Immunoreactive neurons were visualized with a streptavidin-biotin kit (Nichirei, Tokyo, Japan) and 3,3′-diaminobenzidine (DAB), as described previously (Takanami et al., 2010; Yamada and Kawata, 2014).

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To confirm AAV-DTR-EGFP infection in NPY neurons in the NAc, some NPY-DTR mice were perfused before DT injection, and brain sections were obtained as described above. The brain sections were processed for NPY immunofluorescence. Every fourth section through the NAc in the NPY-DTR mice was incubated with 2% NGS in PBS for 1 h, and then with primary rabbit antiserum against NPY (1:2,000) for 72 h at 4 °C. After washing with PBS, the sections were incubated with Alexa Fluor 546-labeled anti-rabbit IgG (1:1,000; Thermo Fisher Scientific, MA,

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USA). AAV-DIO-mCherry and AAV-DIO-Gq-mCherry infection was verified by staining with rabbit anti-mCherry (1:2,000; ab167453, Abcam, Cambridge, UK). This revealed that infection

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by the AAVs encompassed the rostral (bregma +1.78) to caudal (bregma +0.86) regions of the NAc (supplemental Fig. 1).

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To confirm that CNO treatment induced activation of NAc NPY neurons, NPY-hM3Dq mice

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were perfused 90 min after intraperitoneal CNO injection, and brain sections were obtained as described above. The sections were processed for c-Fos & mCherry double fluorescence IHC.

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Every fourth section through the NAc in the NPY-hM3Dq mice was incubated with 2% NGS in

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PBS for 1 h, and then with primary rabbit antiserum against c-Fos (1:2,000; Ab-5, Calbiochem, Merck, Tokyo, Japan) and rat antiserum against mCherry (1:2,000; M11217, Thermo Fisher

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Scientific) for 24 h at RT. After washing with PBS, the sections were incubated with Alexa Fluor 488-labeled anti-rabbit IgG (1:1,000; Thermo Fisher Scientific) and Alexa Fluor 555-labeled anti-rat IgG (1:1,000; ab150154, Cambridge, UK).

2.6.

Statistical Analysis

Fluorescence images were obtained on a LSM510META confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). The sections reacted with DAB were observed under a light microscope (BX50; Olympus, Tokyo, Japan), and images were captured using a CCD camera (DP 21; Olympus). NPY-immunoreactive cells were counted in these images. All values are expressed as mean ± SEM. Significant differences between before and after DT treatment in the

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NPY-DTR and mCherry mice were evaluated by two-tailed paired t-test. Significant differences between mCherry and NPY-hM3Dq were evaluated by Student’s t-test.

3. Results 3.1. DT-mediated specific ablation of NAc NPY neurons We constructed and injected an AAV encoding a Cre-dependent DTR fused to an EGFP reporter

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into the NAc (Fig. 1A). Injection of the AAV into the NAc in the NPY-Cre mice induced specific expression of DTR-EGFP in NAc NPY neurons (Fig. 1B). Immunohistochemical

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analysis revealed that NPY-immunoreactive cells and fibers were densely localized in the NAc of DT-treated mCherry mice (Fig. 1C, left), while only a few NPY-immunoreactive cells were

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found in DT-treated NPY-DTR mice (Fig. 1C, right). The number of NPY-immunoreactive cells

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in the NAc was significantly lower in DT-treated NPY-DTR mice than in DT-treated mCherry mice (P < 0.01, Student’s t-test) (Fig. 1D). The densities of NPY-immunoreactive fibers in the

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paraventricular nucleus of the hypothalamus (PVN) and the numbers of NPY-immunoreactive

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cells in the basolateral amygdala (BLA) were comparable between DT-treated mCherry mice and DTR mice (Supplemental Fig. 2). These results indicate that ablation of NPY neurons was

mice.

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NAc-specific. We confirmed the ablation of NAc NPY neurons in all DT-treated NPY-DTR

3.2. The impact of DT-mediated ablation of NAc NPY neurons on anxiety-like behaviors In the OFT, DT-mediated ablation of NAc NPY neurons significantly decreased the percentage of time spent in the central area (t7 = 12.18, P < 0.01, two-tailed paired t-test) and the percentage of distance traveled in the central area (t7 = 5.66, P < 0.01, two-tailed paired t-test) compared with before DT treatment (Fig. 2A). There was no significant difference in the total distance traveled in the open field between before and after DT treatment in NPY-DTR mice (t7 = 0.59, P = 0.955, two-tailed paired t-test; Fig. 2A). In the EPM, DT-mediated ablation of NAc

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NPY neurons significantly reduced the percentage of time spent in the open arms (t 6 = 2.75, P < 0.05, two-tailed paired t-test) and the number of entries into the open arms (t6 = 3.36, P < 0.05, two-tailed paired t-test; Fig. 2B) compared with before DT treatment. To confirm that the dose of DT and the second behavioral tests in the same individual do not influence anxiety-like behavior, we performed the same DT treatment in mCherry mice (Fig. 3). In the OFT, DT treatment in the mCherry mice did not affect the percentage of time spent or the

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distance traveled in the central area compared with before DT treatment (Fig. 3A). Likewise, there were no significant differences in the percentage of time spent in the open arms or the

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number of entries into the open arms in the EPM (Fig. 3B). This result suggests that influence of

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the DT treatment and second time in OFT and EPM test is small.

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3.3. The impact of NAc NPY neuronal activation on anxiety-like behaviors Because NAc NPY neurons are required for anxiolysis, we next examined whether activating

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these neurons is sufficient to induce anxiolytic-like behavior. We injected an AAV encoding a

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Cre-dependent excitatory designer receptor (hM3Dq) fused to mCherry or an AAV encoding Cre-dependent mCherry as a control. Chemogenetic activation of NAc NPY neurons was

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confirmed by c-Fos IHC. We found that c-Fos expression was induced in the majority of hM3Dq-mCherry-expressing neurons in the NAc, but not in mCherry-expressing neurons, in animals given CNO treatment (Fig. 4A). In the OFT, the percentage of time spent in the central area was significantly higher in CNO-treated hM3Dq-mCherry mice compared with CNO-treated mCherry mice (t15 = 2.75, P < 0.05, two-tailed unpaired Student t-test; Fig. 4B). No significant difference was observed between the two groups in the total distance traveled in the open field (t15 = 1.95, P = 0.68; Fig. 4B). In the EPM test, activation of hM3Dq with CNO significantly increased the percentage of time spent in the open arms (t15 = 2.71, P < 0.05, two-tailed unpaired Student’s t-test), without affecting the number of entries into the open arms (t15 = 1.18, P = 0.26; Fig. 4C).

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4. Discussion The NAc participates in various neurobehavioral functions, including wakefulness (Luo et al., 2018), motivation (Tsutsui-Kimura et al., 2017) and anxiety/depression (Feng et al., 2017). Although histological studies show that there are many NPY cell bodies and fibers in the NAc (Chronwall et al., 1985; de Quidt and Emson, 1986; Morris, 1989), their function remained

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unclear. Here, we show that mice with ablation of NPY neurons in the NAc exhibit an increase in anxiety-like behavior. We also found that DREADD-mediated activation of NAc NPY

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neurons enhanced the anxiolytic-like behavior.

There are several lines of evidence indicating a close relationship between the dopaminergic

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system in the NAc and anxiety. Nicotine-induced reduction of time spent on the open arms in

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the EPM test is ameliorated by injection of dopamine D1 receptor (D1) or dopamine D2 receptor (D2) antagonists into the NAc (Zarrindast et al., 2012). Dopamine transporter

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knockdown in the NAc increases the time spent in the center area in the OFT and the time spent

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in the open arms in the EPM test (Bahi and Dreyer, 2019). Furthermore, chemogenetic inactivation of D2-expressing neurons in the NAc increases center preference in the OFT

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(Blomeley et al., 2018). These observations suggest that D1 and D2-expressing neurons in the NAc modulate anxiety-like behavior. Most D1 and D2-expressing neurons in the NAc are GABAergic medium spiny projection neurons (D1 MSNs and D2 MSNs, respectively), which comprise 95% of the total cell population in the ventral striatum (Klawonn and Malenka, 2018; Ribeiro et al., 2019). The remaining 5% of neurons are cholinergic or GABAergic aspiny interneurons (Ribeiro et al., 2019). One of these GABAergic aspiny interneurons in the neostriatum expresses somatostatin and NPY (Kawaguchi et al., 1995). Therefore, ablated/ activated-NPY neurons in the NAc in this study may be the interneurons. Because NAc interneurons strongly connect to D1 MSNs and D2 MSNs (Kawaguchi et al., 1995), NAc NPY neurons may directly regulate D1 MSNs and D2 MSNs to modulate anxiety.

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We previously demonstrated that overexpression of NPY in the NAc reduces behavioral despair in mice. Furthermore, NPY or NPY receptor type 1 (Y1-R) agonist injection into the NAc did not affect behavioral despair, suggesting that some NPY neurons in the NAc are projection neurons (Aoki et al., 2016). It has been reported that Y1-R agonist injection into the central amygdala has an anxiolytic effect in rats (Heilig et al., 1993; Kask et al., 2002). Moreover, NPY administration into the amygdala has an anxiolytic effect in the EPM test, and this effect is

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not observed in rats pre-injected with Y1-R antisense (Heilig, 1995). These results suggest that the anxiolytic effect of NPY is mediated by Y1-R in the amygdala. NAc NPY neurons may send

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some projections to the amygdala that exert an anxiolytic effect through the Y1-R. However, it must be noted that the ablation of NAc NPY neurons may have also affected other

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neurotransmitters. For example, NPY neurons in the arcuate nucleus of the hypothalamus

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(ARH) co-express agouti-related peptide (AgRP) and GABA. Thus, we cannot exclude a role of other neurotransmitters in the NAc NPY neurons in the modulation of anxiety-like behavior.

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Further study is needed to more fully characterize the NAc NPY neurons and to identify the

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neurons targeted by their projections that regulate anxiety. Comeras et al. suggested that activation of NPY neurons in the ARH increases food-seeking

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behavior and decreases anxiety and fear (Comeras et al., 2019). Our current results suggest that NAc NPY neurons are also involved in the modulation of anxiety-like behavior. However, deletion of NAc NPY neurons did not affect body weight in this study, suggesting that these neurons do not influence food-seeking or other hunger-related behaviors. Pharmacological studies show that manipulation of NPY or Y1-R agonist levels induces sedation, hypolocomotion and hyperlocomotion (Sorensen et al., 2004). However, ablation and activation of NAc NPY neurons did not affect total distance in the open field test in the present study, suggesting that NAc NPY neurons do not impact locomotion. Kask et al. postulated that NPY is continuously released, as a signal of safety in selected brain regions (Kask et al., 2002). Therefore, given that NPY neuron ablation in the NAc increased

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anxiety-like behavior, these neurons may be necessary to maintain baseline anxiolysis. As a corollary, dysfunction of NAc NPY neurons may be involved in the pathogenesis of anxiety disorder. Notably, we found that activation of NPY neurons in the NAc increased anxiolytic-like behavior. This suggests that NAc NPY neurons may activate to overcome anxiety; however, the signals that activate these neurons remain unknown. We previously demonstrated that administration of the selective 5-HT2CR agonist WAY-629 causes reduction of NAc NPY mRNA

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expression in wild-type mice (Aoki et al., 2016). Thus, 5-HT neurons may be a part of a circuit that modulates the activity of NAc NPY neurons, and thereby, anxiety-related behavior.

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Furthermore, the NAc appears to be strongly involved in reward seeking as well (Sesack and Grace, 2010). The satisfaction of hunger, thirst and sexual urge is a common reward system,

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and the suppression of anxiety and/or fear is a prerequisite for these behaviors (Comeras et al.,

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balance between reward and anxiety.

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2019). This suggests that NAc NPY neurons may form part of a neural network regulating the

Declaration of Competing Interest

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The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science Grants-in-Aid [grant no. 17H03553 to M.T. and grant no. 19K09032 to S.Y.]. Author Contributions S.Y., M.S., N.K., S.B., and Y.O. performed histological and behavioral experiments. A.T. generated the AAV. Y.W. supported behavioral experiments and provided the advice. S.Y. and M.T. wrote the paper. M.T. supervised the whole study.

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Figure legends Fig. 1. The effect of Cre-dependent AAV-HB-EGF (DTR)-EGFP injection into the NAc in NPY-Cre mice. (A) Schematic of pAAV-EF1-floxed-hDTR-EGFP. (B) Fluorescence image of EGFP (green, left), NPY (red, middle) and merged (right) in the NAc in the NPY-DTR mice

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before DT treatment. Scale bars, 20 m. (C) NPY immunostaining in the NAc in mCherry and NPY-DTR mice 3 weeks after intraperitoneal DT injection. NPY-immunoreactive neurons

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appear as brown punctae. Scale bars, 100 m. ac; anterior commissure. (D) Number of NPY-immunoreactive cells in the NAc in mCherry (mCh) and NPY-DTR (DTR) mice. Values

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are mean ± SEM. P-values were calculated by Student’s t-test.

Fig. 2. Analysis of anxiety-like behavior in NAc NPY-ablated mice. (A) NPY-DTR mice were

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subjected to the open field test (OFT) and the elevated plus maze test (EPM) before (Pre-DT) or

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3 weeks after intraperitoneal DT injection (Post-DT). Shown are the percentage of time spent in the central area, the percentage of distance traveled in the central area, and the total distance

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traveled in the open field during the 30-min OFT. (B) The percentage of time spent in and the number of entries into the open arms during the 10-min EPM. Sample numbers are indicated in parentheses. Values are mean ± SEM. P-values were calculated by paired t-test.

Fig. 3. Analysis of anxiety-like behavior in mCherry mice. mCherry mice were subjected to the OFT and the EPM before (Pre-DT) or 3 weeks after intraperitoneal DT injection (Post-DT). (A) The percentage of time spent in the central area, the percentage of distance traveled in the central area, and the total distance traveled in the open field during the 30-min OFT. (B) The percentage of time spent in and the number of entries into the open arms during the 10-min EPM. Sample numbers are indicated in parentheses. Values are mean ± SEM. P-values were

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calculated by paired t-test.

Fig. 4. Analysis of anxiety-like behavior in NAc NPY neuron-activated mice. (A) Fluorescence image of mCherry (red; NPY neurons) and c-Fos (green) in the NAc in the AAV-mCherry (left) and AAV-hM3Dq-mCherry (right)-injected mice 90 min after intraperitoneal CNO injection. Scale bars, 20 m. (B) The percentage of time spent in the central area and the total distance

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traveled in the open field during the 30-min OFT in the mCherry (mCh) and NPY-hM3Dq (hM3Dq) mice. (C) The percentage of time spent in and the number of entries into the open

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arms during the 10-min EPM in the mCherry and NPY-hM3Dq mice. Sample numbers are

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were calculated with Student’s t-test.

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indicated in parentheses. Values are mean ± SEM and raw values for individual mice. P-values

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Highlights:  We generated regional and temporal-specific NPY-lesioned mice  Ablation of NPY neurons in the nucleus accumbens induced anxiety-like behavior

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 Activation of NPY neurons in the nucleus accumbens induced anxiolytic-like behavior

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