Astrocyte-Ablation of Mtnr1b Increases Anxiety-Like Behavior in Adult Male Mice

¹ Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, National Clinical Research Center for Child Health and Disorders, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, Children's Hospital of Chongqing Medical University, 400015 Chongqing, China

² The Second Affiliated Hospital and Yuying Children's Hospital, Key Laboratory of Alzheimer's Disease of Zhejiang Province, Zhejiang Provincial Clinical Research Center for Mental Disorders, School of Mental Health and Wenzhou Kangning Hospital, Institute of Aging, Wenzhou Medical University, 325035 Wenzhou, Zhejiang, China

³ Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), 325001 Wenzhou, Zhejiang, China

^*Correspondence: zwh@hospital.cqmu.edu.cn (Weihui Zhou); weihong@wmu.edu.cn (Weihong Song)

J. Integr. Neurosci. 2023, 22(6), 154; https://doi.org/10.31083/j.jin2206154

Submitted: 14 August 2023 | Revised: 9 September 2023 | Accepted: 15 September 2023 | Published: 30 October 2023

This is an open access article under the CC BY 4.0 license.

Abstract

Background: Astrocytes are essential for synaptic transmission, and their dysfunction can result in neuropsychiatric disorders such as anxiety and depression. Many studies have shown that global knockout of Melatonin receptor 2 (Mtnr1b) is associated with the development of various mental disorders. Aim: This study aimed to investigate the effects of astrocyte ablation of Mtnr1b on cognitive function and anxiety-like behavior in mice, as well as the potential biological mechanisms. Methods: A conditional Cre-loxP system allowing deletion of Mtnr1b from astrocytes was developed to investigate the specific role Mtnr1b. Control and Mtnr1b cKO ${}^{\text{Gfap}}$ mice were selected for cognitive function behavioral testing (Morris water maze test, novel object recognition test) and emotion-related behavioral testing (open field, elevated plus maze). After testing, brain tissue was collected and examined by immunofluorescence for the expression of neuronal nuclei (NeuN), glutamate decarboxylase 67 (GAD67), and vesicular glutamate transporter 1 (vGluT1). RNA-seq was performed on hippocampal tissue from control and Mtnr1b cKO ${}^{\text{Gfap}}$ mice to identify differentially expressed genes. Additional confirmation of differential gene expression was performed using real-time quantitative polymerase chain reaction (qRT-PCR). Results: Mtnr1b cKO ${}^{\text{Gfap}}$ mice were not significantly different from control mice in the Morris water maze and novel object recognition tests. Results from the open field and elevated plus maze tests showed that Mtnr1b cKO ${}^{\text{Gfap}}$ mice exhibited significantly more anxiety-like behavior than did controls. Immunofluorescence revealed that the number of mature neurons did not differ significantly between Mtnr1b cKO ${}^{\text{Gfap}}$ mice and controls. The expression of GAD67 in the hippocampal CA1 and CA3 areas of Mtnr1b cKO ${}^{\text{Gfap}}$ mice was significantly lower than in the control group, but no significant difference was detected for vGluT1 expression. RNA-seq and qRT-PCR results showed that Mtnr1b knockout in astrocytes led to a decrease in the levels of gamma-aminobutyric acid sub-type A (GABA ${}_{\text{A}}$ ) receptors and Kir2.2. Conclusions: The astrocyte-specific knockout in Mtnr1b cKO ${}^{\text{Gfap}}$ mice results in anxiety-like behavior, which is caused by down-regulation of gamma-aminobutyric acid-ergic (GABAergic) synaptic function.

Keywords

astrocyte

anxiety

GABA

melatonin receptor 2

Funding

Key Laboratory of Alzheimer’s Disease of Zhejiang Province

Zhejiang Provincial Clinical Research Center for Mental Disorders (WS)

Figures

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Fig. 1.

Astrocyte-ablation of Mtnr1b induces anxiety-like behavior in adult male mice. (A) Representative tracks of open field exploration of Mtnr1b cKO ${}^{\text{Gfap}}$ and control mice. (B) Mtnr1b cKO ${}^{\text{Gfap}}$ mice exhibited a significant reduction in exploration time in the central area of the open field than did control mice (n = 6, Mtnr1b cKO ${}^{\text{Gfap}}$ and control; p = 0.0220). (C) In the open field test, Mtnr1b cKO ${}^{\text{Gfap}}$ mice traveled a significantly shorter distance than did controls (n = 6, Mtnr1b cKO ${}^{\text{Gfap}}$ and control; p = 0.0456). (D) Representative tracks of elevated plus maze exploration of Mtnr1b cKO ${}^{\text{Gfap}}$ and control mice. (E,F) Mtnr1b cKO ${}^{\text{Gfap}}$ mice spent less time (p = 0.0474) in the open arms and entered the open arms of the elevated plus maze less frequently than did control mice (p = 0.0082) (n = 6, Mtnr1b cKO ${}^{\text{Gfap}}$ and control). Data are presented as the mean $\pm{}$ SEM. * p $<$ 0.05, ** p $<$ 0.01. SEM, standard error of the mean; Mtnr1b, melatonin receptor 2.

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J. Integr. Neurosci. Print ISSN 0219-6352 Electronic ISSN 1757-448X