Anxiety disorder is a prevalent psychological condition among individuals who seek clinical treatment. It refers to the response of the mind and body to stress, danger, or unfamiliar situations [1]. Anxiety disorder is often considered to be a protracted response to imprecise future threats and broad attentional focus. The primary symptoms of anxiety disorder are excessive or repetitive worries and intrusive thoughts preceding significant events, which may induce uneasiness, discomfort, or terror [2]. Anxiety disorders, as defined by the American Psychiatric Association’s diagnostic criteria, comprise specific phobias, panic disorder with or without agoraphobia (PDA), social anxiety disorder (SAD), and generalized anxiety disorder [3]. Specific phobia has a 12-month incidence rate of 10.3%, making it the most common anxiety disorder [4]. PDA is the second most common type, with an incidence rate of 6.0%, followed by SAD and generalized anxiety disorder (GDA), with incidence rates of 2.7% and 2.2%, respectively [5]. Anxiety disorders typically have a slow onset, and symptom severity fluctuates between generalized anxiety disorder and PDA [6, 7]. Women are 1.5–2 times more likely than men to develop anxiety disorders, which often co-occur with depression, obsessive-compulsive disorder, post-traumatic stress disorder, and other mental disorders [8]. More than 70% of individuals who attempt suicide have an anxiety disorder. Identifying individuals with anxiety disorders is challenging due to the lack of specific biological markers. The diagnosis and treatment of anxiety disorders and their comorbidities impose a significant economic burden on patients, families, and society. Therefore, defining the pathogenesis of anxiety disorders and finding new strategies for their treatment is both necessary and a high-priority.

The balance between neuronal excitation and inhibition (E/I) is crucial for maintaining normal brain function. E/I imbalance leads to various mental disorders. For instance, in anxiety and depressive disorders, a significant imbalance of the excitatory glutamatergic and inhibitory gamma-aminobutyric acid-ergic (GABAergic) systems occurs in the medial prefrontal cortex and hippocampus [9, 10, 11, 12]. The specific mechanisms underlying E/I imbalance-induced anxiety and depression, however, remain unclear. GABAergic dysfunction is known to induce hippocampal neuronal signal-transduction inhibition, which, in turn, has been linked to the development of neurological diseases and neuropsychiatric disorders, especially in cases of anxiety disorders or depression [13]. These inhibitory neurons provide a necessary safeguard for excitatory neural transmission via synaptic inhibition caused by hyperpolarization [14]. Therefore, the reduction of inhibitory GABAergic signaling and the elevation of glutamatergic neurotransmission are strongly associated with the occurrence of anxiety disorders.

Melatonin receptors are a type of conserved G-protein-coupled receptor comprising melatonin receptor 1 (encoded by the MTNR1A gene), melatonin receptor 2 (encoded by the MTNR1B gene), and in rare cases, melatonin receptor 3 in mammals. In the suprachiasmatic nucleus, melatonin primarily regulates neuronal firing and clock-gene expression through MTNR1A and MTNR1B, thereby coordinating the circadian rhythms of the human body [15]. MTNR1A and MTNR1B have different modes of coupling with G proteins upon activation. MTNR1A primarily couples to the G${}_i$ and insensitive G${}_{q/11}$ families to inhibit forskolin-stimulated cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signaling pathways, as well as the phosphorylation of nuclear cAMP response element binding (CREB) [16]. It also enhances potassium conductance by increasing Kir inward rectifying channels [17]. MTNR1B mainly couples to G${}_{i/o}$ type G proteins, which inhibit the production of cAMP stimulated by forskolin and formation of cyclic guanosine monophosphate (cGMP), activates the protein kinase C (PKC) signaling pathway in the suprachiasmatic nucleus, and reduces dopamine release from the retina [18, 19]. MTNR1B expression occurs mainly in the brain and pancreas. Some studies suggest that MTNR1A and MTNR1B exhibit receptor heterodimerization, which may regulate neurological and psychiatric disorders [20, 21]. Despite their important regulatory roles in various physiological and pathological processes, assessing the biological functions of MTNR1A and MTNR1B remains challenging due to their extremely low expression levels, particularly MTNR1B [22]. Genome-wide association studies (GWAS) have shown that variations in the human MTNR1B gene sequence are closely related to the development of diabetes [23]. Melatonin receptors have become new pharmaceutical targets for treating mental disorders. Comai et al. [24] demonstrated that the MTNR1B agonist UCM765 reduced anxiety-like behavior in mice and increased nonrapid eye movement (non-REM) sleep. Conversely, mice with systemic knockout of Mtnr1b receptors showed disrupted sleep patterns and increased anxiety-like behavior [25, 26]. Clinically, agomelatine acts as a new type of anti-depressant/anti-anxiety medication by stimulating MTNR1A/MTNR1B receptors [27]. Furthermore, analysis of the human protein atlas (HPA) database (https://www.proteinatlas.org/ENSG00000134640-MTNR1B/single+cell+type) by us revealed that MTNR1B is primarily expressed in astrocytes in the brain. Astrocytes are the most abundant glial cell type in the central nervous system and release a range of neurotransmitters (such as glutamate, gliotransmitters), thereby affecting synaptic excitability and playing an important role in emotional regulation [28, 29, 30]. Therefore, in the present study we further investigated whether astrocyte-specific ablation of the Mtnr1b gene in mice is associated with similar behavioral abnormalities. The underlying biological mechanism was also explored, with the aim of providing new strategies for treating anxiety disorders.

In the present study, adult male mice with deletion of Mtnr1b in astrocytes were found to exhibit anxiety-like behaviors. This was accompanied by a reduction in inhibitory neurons, but no significant changes in excitatory neurons in the hippocampal CA1 and CA3 region. It was also found that deletion of Mtnr1b in astrocytes led to transcriptional changes in the hippocampus and downregulation of the Gaba2 gene in Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice. This blocked inhibitory synaptic neurotransmission, leading to anxiety-like behaviors. The present findings revealed a critical role for astrocytic Mtnr1b in the hippocampus in anxiety-like behaviors. Furthermore, this regulatory effect on anxiety is achieved through down-regulation of GABAergic synaptic function.

In the present study, Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice were generated using the Cre-loxP system. The effects of astrocyte-specific deletion of Mtnr1b knockout on mouse emotions were assessed by conducting open field and elevated plus maze tests. In addition, Morris water maze and novel object recognition tests were performed to evaluate the impact of Mtnr1b knockout on learning and memory. Astrocyte-specific deletion of Mtnr1b was found to result in anxiety-like behaviors in mice, without affecting their learning and memory abilities. Evaluation of excitatory and inhibitory neurons showed significantly lower GAD67 levels in the hippocampal CA1 and CA3 regions of Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice than in controls, whereas the number of excitatory neurons did not differ significantly. Transcriptome sequencing of mouse hippocampal tissue further revealed enriched pathways related to potassium channel activity and GABA receptor gated chloride channel activity. qRT-PCR confirmed the consistency of the related gene expression with RNA-seq results. These findings suggest the anxiety-like behaviors observed in Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice are associated with a decrease in hippocampal GABA levels.

Astrocytes are the most abundant glial cells in brain tissue and possess the ability to release neuroactive factors, clear excessive neurotransmitters between synapses, and maintain ion homeostasis to influence communication between synapses [41, 42]. Although astrocytes do not transmit electrical signals, they modulate neuronal activity by releasing chemical signals and exchanging ions [43, 44]. Disordered astrocyte function contributes to a range of neurological and psychiatric disorders. Yamashita et al. [31] eported that activation of astrocytes in the anterior cingulate cortex through channel rhodopsin leads to reduced non-REM sleep during the light period in mice. Specific knockout of connexin 43 in the astrocytes of mice promotes sleep during the dark period by silencing wake-promoting neurons. Li et al. [32] reported that specific knockout of astrocytic Liver X receptor $\beta$ (Lxr$\beta$) leads to an E/I imbalance, resulting in anxiety-like behavior in mice. Rodolphi et al. [45] demonstrated that ceftriaxone upregulates expression of the astrocytic glutamate transporter-1 (GLT-1), thereby counteracting the excessive glutamate induced by nandrolone decanoate, and reducing aggressive behavior in mice. In addition, the present study indicated that deletion of Mtnr1b in astrocytes induced anxiety-like behavior in male mice, thus providing additional evidence for the involvement of aberrant astrocyte function in behavioral disturbances.

MTNR1B is a G-protein-coupled receptor involved in various physiological processes, including sleep-wake rhythm, cell proliferation, and neurotransmitter release [46]. Studies have demonstrated that melatonin can reduce ischemia-related neuronal death in an Mtnr1b expression-dependent manner [47]. This indicates the importance of Mtnr1b in the development of neurological and psychiatric disorders [48, 49, 50]. In a previous study, Thomson et al. [25] reported that mice with a whole-body knockout of the Mtnr1b gene exhibited phenotypes associated with anxiety, reduced learning ability, and decreased attention. Liu et al. [51] reported similar results. In the present study, Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice were used as the research subjects, which has not previously been reported in the literature. The impact of astrocytic Mtnr1b gene knockout on mouse emotion was assessed using open field and elevated plus maze tests. This revealed that Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice showed marked anxiety-like behavior, which aligns with the findings of the Thomson and Liu studies [25, 51]. Despite small variations in mouse strains and Mtnr1b gene knockout ranges employed by different researchers, consistent anxiety results were observed across the different mouse strains.

In contrast to prior studies involving systemic knockout of the Mtnr1b gene in mice [25], the findings reported here demonstrate that targeted knockout of the Mtnr1b gene in astrocytes did not adversely affect learning and memory function in mice. We believe that previous studies used different mouse strain backgrounds (C57BL/6N and C3H/HeN), whereas the mouse background used here was C57BL/6J. C57BL/6J mice are known to have lower levels of melatonin production due to mutations in the melatonin synthesis enzyme, and thus have lower plasma melatonin levels [52]. Additionally, C57BL/6N mice produce melatonin in peripheral tissues, whereas C57BL/6J mice do not, which may affect their cognitive functions [53]. Another important factor is that previous studies used mice with a whole-body knockout of the Mtnr1b gene, whereas in the current study we used only mice with a specific knockout of the Mtnr1b gene in astrocytes. This may have affected the results of cognitive function tests. Finally, the specific experimental procedures were different. Thomson’s study used a continuous-performance task to evaluate cognitive function, whereas the tests used here were the Morris water maze and new object recognition. The continuous-performance test is used to evaluate the cognitive control ability and sustained attention of an animal, whereas the Morris water maze test is mainly used to evaluate spatial learning and memory abilities. The sensitivity of the continuous-performance task is higher than that of the Morris water maze task, which may also be a reason for the different cognitive function test results.

The influence of sex on Mtnr1b knockout mice also warrants thoughtful consideration. Melatonin levels have a significant impact on emotions, and it is important to highlight that in humans, females exhibit greater fluctuations in plasma melatonin levels than do males [54, 55]. This suggests that impaired melatonin function has different effects on individuals depending on their sex. Thomson et al. [25] reported that male Mtnr1b KO mice showed enhanced sociability, whereas female Mtnr1b KO mice exhibited an elevated anxiety response. Liu et al. [51] previously reported increased anxiety levels in Mtnr1b KO mice, without specifying the sex. Comai et al. [26] reported that male KO mice exhibited anxiety-like behavioral characteristics. Horst et al. [56] showed that ovarian hormones contribute to decreased anxiety levels in female mice exposed to stressful environments (such as elevated plus maze or open field test). In order to conduct a more comprehensive investigation into the effects of Mtnr1b deletion in astrocytes, male mice were therefore deliberately selected in the present work, thereby eliminating potential confounding factors arising from the influence of female hormones.

The excitability of central neurons is primarily influenced by glutamatergic neurons, and their level of excitation is continuously regulated by inhibitory synaptic inputs. The imbalance between excitatory and inhibitory synaptic transmission is a crucial process that underlies numerous neurological and psychiatric disorders, including epilepsy [57], schizophrenia [58], Alzheimer’s disease [59], anxiety [32], and depression [60]. An increasing number of reports have highlighted the significance of alterations in inhibitory neurons, particularly GABAergic synapses, that are vital in the development of numerous critical neuro-psychiatric disorders. Cheng et al. [61] reported that melatonin not only increases the amplitude of GABAergic miniature inhibitory postsynaptic currents (mIPSCs), but also enhances the frequency of mIPSCs in hippocampal neurons, indicating that melatonin enhances GABAergic signal transmission through both presynaptic and postsynaptic mechanisms. Astrocytes act as an important cell type in the regulation of neuronal activity [62]. It is therefore reasonable to hypothesize that blockade of Mtnr1b in astrocytes may also affect neurotransmission. GABA is synthesized by the enzyme glutamate decarboxylase (GAD) from glutamate. Multiple studies have implicated GAD67 protein as the molecule most strongly linked to various neuropsychiatric disorders [39, 63, 64]. Miyata et al. [65] demonstrated that global knockdown of GAD67 expression leads to anxiety-like behavior in mice. vGlut1 is a transport protein located in presynaptic neurons and is responsible for packaging glutamate into vesicles [66, 67]. It serves as a marker gene for glutamatergic neurons and has been implicated as a regulator of anxiety-like behavior [68]. The present study analyzed expression levels of the inhibitory neuron marker GAD67 and the excitatory neuron marker vGluT1 by immunofluorescence staining in various brain regions of both control and Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice. Additionally, transcriptome sequencing data revealed the presence of postsynaptic effects. GAD67 expression levels were significantly lower in the CA1 and CA3 regions of Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice than in their control counterparts, whereas vGluT1 levels remained unchanged. These results are in agreement with those of previous studies.

In the present study, RNA-seq analysis of hippocampal tissue revealed that molecular functions were mainly enriched in pathways associated with potassium channel and GABA-receptor-gated chloride channel activities. This suggests that the astrocyte-specific knockout of Mtnr1b resulted in altered neuronal function. Melatonin enhances GABA${}_{\text{A}}$ receptor-mediated currents in cultured chick spinal cord and rat hippocampus neurons [61, 69]. Additionally, melatonin was found to enhance the amplitude of mIPSCs. These results suggested that melatonin is capable of increasing the activity of postsynaptic GABA${}_{\text{A}}$ receptors. Yu et al. [18] reported that melatonin can inhibit sympathetic neural regulation by enhancing GABA${}_{\text{A}}$ receptor activity in the hypothalamus. In the present study, a significant reduction of the Gabra2 gene in Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice was observed, suggesting that the astrocyte-specific knockout of Mtnr1b decreased the activity of the GABA${}_{\text{A}}$ receptor in the hippocampus. Taken together, these findings lead to the speculation that loss of the Mtnr1b gene in astrocytes results in downregulation of the GABAergic signaling pathway within the hippocampus of Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ mice.

Prior investigations have documented the activation of Kir3 channels via MTNR1A stimulation [70]. However, the present study used transcriptome sequencing and qRT-PCR analysis to show that deletion of the Mtnr1b gene in astrocytes elicits a significant decrease in the expression of Kcnj12, which is the gene responsible for encoding the Kir2.2 protein in hippocampal tissue [71]. Leonoudakis et al. [72] demonstrated that Kir2.2 is expressed not only in astrocytes but also in the granule cell layer. In the central nervous system, neuronal activity leads to K${}^{+}$ release into the extracellular space, and astrocytes modulate neuronal excitability by buffering extracellular K${}^{+}$ through Kir2.2. Previous studies have reported associations between intronic variants in the Kcne2 gene and psychiatric disorders [73]. Abbott et al. [74] reported that knockout of the Kcne2 gene increases neuronal excitability and enhances responsiveness to stress. Knockout of the Mtnr1b gene in astrocytes was also found to downregulate the expression of Kcne2 in the hippocampus. This evidence suggests that elimination of Mtnr1b cKO${}^{\text{𝐺𝑓𝑎𝑝}}$ in astrocytes affects the E/I balance through the regulation of genes involved in K${}^{+}$ channels.

In summary, astrocyte-specific elimination of the Mtnr1b gene in mice leads to anxiety-like behavior. This may be due to downregulation of the hippocampal GABAergic signaling pathway, resulting in an imbalance between excitatory and inhibitory synapses. The present results provide new insights into the potential of Mtnr1b as a novel therapeutic target for anxiety disorders.

The RNA-seq datasets analyzed in this study are available in the Genome sequence archive database (https://ngdc.cncb.ac.cn/search/?dbId=gsa&q=CRA012658, Accession: CRA012658). Described in Results and Methods section and available upon request.

ZM, WZhou and WS conceived and designed this research. ZM, SG, XD, QW, DH, XL, YJ, LJ, JZ, WZhu conducted the experiments. WZhou and WS contributed reagents, materials, and analytical tools. WS and WZhou supervised the project. ZM, QW, WZhou and WS wrote the manuscript and revised manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

All animal experiments were approved by the Ethics Committee of Chongqing Medical University and Animal Study Committee of the Children’s Hospital of Chongqing Medical University (no code–CHCMU-IACUC20221227006).

Not applicable.

The study was partially supported by Key Laboratory of Alzheimer’s Disease of Zhejiang Province and Zhejiang Provincial Clinical Research Center for Mental Disorders (WS).

The authors declare no conflict of interest.