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

https://doi.org/10.1016/j.expneurol.2020.113216Get rights and content

Highlights

  • We generated regional and temporal-specific NPY-lesioned mice.

  • Ablation of NPY neurons in the nucleus accumbens induced anxiety-like behavior.

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

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 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 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 significantly increased anxiety-like behavior in the open field and elevated plus maze tests, 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 results suggest that NPY neurons in the NAc are involved in the modulation of anxiety in mice.

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 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 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 microinjection of NPY into the amygdala has only an anxiolytic effect (Heilig et al., 1993), 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 regulation of food intake has been extensively studied (Gehlert, 1999; Inui, 1999; Kalra et al., 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 anxiety-related brain regions, such as amygdala and dorsal periaqueductal gray (Kask et al., 2002).

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 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 exclusively activated by designer drugs (DREADD) technology, we examine the impact of the activation of NAc NPY neurons on anxiety-like behavior.

Section snippets

Animals

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). We entrusted the generation of NPY-Cre mice using the sperm to the RIKEN BioResource 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 performed between 10:00 and 13:00. All animal procedures, including

DT-mediated specific ablation of NAc NPY neurons

We constructed and injected an AAV encoding a Cre-dependent hDTR fused to an EGFP reporter 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 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 found in DT-treated NPY-DTR mice (Fig. 1C, right). The

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

Declaration of Competing Interest

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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