Rasd2 Mediates Acute Fasting-Induced Antidepressant-Like Effects via Dopamine D2 Receptor Activation in Ovariectomized Mice

Abstract Background Previous studies have shown that estrogen and acute fasting for 9 hours have antidepressant-like effects by reducing immobility time in the forced swimming test. Estrogen and acute fasting share a common regulatory gene, Rasd2. RASD2 regulates dopamine D2 receptor (DRD2) transmission, but the role of Rasd2 in the DRD2-mediated antidepressant-like effect of acute fasting has not been examined. Methods In this study, open field test, forced swimming test, tail suspension test and sucrose preference test were used for behavioral assessments. RNA-seq, western blot, enzyme-linked immunosorbent assay, and co-immunoprecipitation were used to explore the role of Rasd2 in a depression model induced by ovariectomy and the antidepressant-like effects of 9-hour fasting. Results The RNA seq results showed that acute fasting induced a significant change in Rasd2 gene expression. Depression-like behaviors induced by ovariectomy were associated with decreased RASD2 and DRD2 protein levels in the hippocampus, and Rasd2 overexpression in the hippocampus alleviated depression-like behaviors and increased DRD2 expression. Nine-hour fasting had antidepressant-like effects in ovariectomized mice by upregulating the protein levels of RASD2, DRD2, CREB-BDNF, Akt, and estrogen receptor beta, and these effects can be blocked by DRD2 antagonists. Conclusions Our results suggest that Rasd2 and DRD2 play pivotal roles in depression-like behavior induced by ovariectomy. Rasd2 regulates DRD2-mediated antidepressant-like effects of acute fasting in ovariectomized mice. Rasd2 can therefore be postulated to be a potential therapeutic target for depression and perhaps also a potential predictive marker for depression.


Significance Statement
Previous studies have shown that estrogen and acute fasting for 9 hours have antidepressant-like effects by reducing immobility time in the forced swimming test. Estrogen and acute fasting share a common regulatory gene, Rasd2. RASD2 regulates dopamine D2 receptor (DRD2) transmission, but the role of Rasd2 in the DRD2-mediated antidepressant-like effect of acute fasting has not been examined. In this study, behavioral assessments of antidepressant action were detected by open field test, forced swimming test, tail suspension test, and sucrose preference test. RNA-seq, western blot, enzyme-linked immunosorbent assay, and co-immunoprecipitation were used to explore the role of Rasd2 in a depression model induced by ovariectomy and the antidepressant-like effects of 9-hour fasting. The present study suggests that Rasd2 and DRD2 play pivotal roles in depression-like behavior induced by ovariectomy. Rasd2 regulates DRD2-mediated antidepressant-like effects of acute fasting in ovariectomized mice. Rasd2 can therefore be postulated to be a potential therapeutic target for depression and perhaps also a potential predictive marker for depression.

INTRODUCTION
Depression is a mental disease characterized by low mood, psychomotor retardation, and cognitive impairment, which severely reduce quality of life (Nestler et al., 2002). Currently, depression is one of the leading causes of disability and a major contributor to the overall global burden of disease (Lancet, 2022). It is worth noting that a meta-analysis has shown that the heritability for major depression is approximately 37% (Flint and Kendler, 2014), and the prevalence of depression in women is almost twice that of men worldwide (Martin et al., 2013). In particular, women are at high risk of depression during hormonal transition phases (peripartum, perimenopause, etc.) (Freeman et al., 2014;Borgsted et al., 2022). However, preclinical study of depression in females remains understudied (Jiang et al., 2022;Lima et al., 2022).
Calorie restriction has been shown to extend the life span of several species over the past few decades (Fontana and Partridge, 2015) and has positive effects on neurological diseases, including Alzheimer's disease and Parkinson's disease (Zhang et al., 2021b;Ezzati and Pak, 2023;Govic et al., 2022). In our previous studies, mice treated with 9-hour fasting significantly shortened the immobility time of the forced swimming test (FST), whereas mice fasted for 3 hours and 18 hours had no significant changes (Li et al., 2014). Further studies revealed that acute fasting produces antidepressant-like effects through the activation of the cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling pathway in the prefrontal cortex (PFC) and hippocampus (HP) (Lutter et al., 2008;Li et al., 2014;Cui et al., 2018;. Additionally, caloric restriction upregulates estrogen receptor expression but has no effects on androgen receptor (Słuczanowska-Głąbowska et al., 2015). Fasting produces estrogenic effects in ovariectomized mice (Bigsby et al., 1997), and estrogen enhances the antidepressant-like effects of acute fasting via the activation of the CREB-BDNF signaling pathway in the PFC and HP . Therefore, fasting might be used as an adjunct to estrogen replacement therapy for depression.
RNA-seq data suggest that estrogen and acute fasting exert antidepressant-like effects through a common gene, Rasd2 . Whether Rasd2 participates in the antidepressant-like effects of fasting has not been directly examined yet, to our knowledge. RASD2 is a GTP binding protein that is highly enriched in the striatum and found at lower levels of expression in the HP, cerebral cortex, olfactory bulb, etc. (Vargiu et al., 2004). Rasd2 negatively regulates G protein-coupled receptor-mediated cAMP production, and the targeted deletion of Rasd2 in mice can significantly activate the cAMP/protein kinase A signaling pathway in the striatum (Vargiu et al., 2004;Errico et al., 2008;Ghiglieri et al., 2015). In addition, recent research indicates that Rasd2 regulates the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin signaling pathway and consequently has a role in several neurological and psychiatric diseases, such as schizophrenia and Huntington's disease (Emamian et al., 2004;Subramaniam et al., 2011;Lee et al., 2015). However, the role of Rasd2 in depression remains unclear.
Rasd2 function is closely tied to dopamine function. Depleting the striatum of dopaminergic input decreases Rasd2 mRNA expression in the striatum (Harrison and LaHoste, 2006). In addition, activation of dopamine D2 receptors (DRD2) produces exaggerated stereotypy in Rasd2 knockout mice (Quintero et al., 2008). Sciamanna et al. (2015) found that Rasd2 deficiency produces aberrant DRD2-dependent activity through an abnormal Ca 2+ -dependent modulation of PI3K/Akt signaling. Rasd2 mRNA has been located in dopamine D1 receptor-medium spiny neurons, DRD2-medium spiny neurons, and cholinergic interneurons . Rasd2 regulates dopamine-dependent neurotransmission by affecting the survival of nigrostriatal dopaminergic neurons Pinna et al., 2016). These findings suggest that Rasd2 effects on other aspects of dopamine signaling may be involved in depression. In addition to DRD2 signaling pathways, dopamine supersensitivity in response to antidepressant treatment is mediated by the activation of the CREB-BDNF signaling pathways in the nucleus accumbens (Guillin et al., 2001;Gershon et al., 2007).
In this study, we used RNA-seq, behavioral tests, western blot (WB), enzyme-linked immunosorbent assay, and co-immunoprecipitation (Co-IP) to comprehensively investigate the role of transcription factor RASD2 in 9-hour fasting on the improvement of depression-like behavior induced by ovariectomy and whether this effect is regulated by DRD2. Considering the significant effects of fasting and estrogen on the BDNF-CREB signaling pathway in the HP and PFC of mice and the fact that RASD2 is enriched in the striatum while interacting with DRD2, in this study, we aimed to investigate the molecular mechanisms involved in the HP, PFC, and striatum.

Animals
Female ICR mice (6-10 weeks, 25 ± 2 g) were purchased from Jilin University (Changchun, China). The mice were kept in plastic cages (25.5 × 15 × 14 cm) under standard laboratory conditions: room temperature 23°C ± 1°C, a 12-hour-light/-dark cycle (7:00 am-7:00 pm light period). Food and water were available ad libitum. Before experiments, mice were randomly assigned to each group. Five mice were housed in 1 cage before surgery and were housed in a single cage after surgery to prevent the mice from biting each other. Transparent cages were used to allow the mice to see each other, and toys were placed in the cages throughout the single-cage rearing period. All experiments were conducted according to the standards set forth in the Laboratory Animal-Guideline for ethical review of animal welfare (GB/T 35892-2018) and under protocols approved by the Institutional Animal Care and Use Committee of Jilin University.

Experimental Design
The experimental design and timeline are shown in Figure 1. To investigate the effect of acute fasting on gene expression in mouse brain, mice were killed after 9-hour fasting or normal diet, and brain tissues (PFC) were dissected and processed for RNAseq ( Figure 1A). To investigate the effect of ovarian removal on depression-like behavior and related changes in protein expression, behavioral tests (FST, n = 13 each group; open field test [OFT; n = 13 each group], tail suspension test [TST; n = 6-7 each group], and sucrose preference test [SPT; n = 8 each group]) and serum (n = 8 each group) and brain tissue (PFC and HP, n = 3-6 each group) extraction were performed 7 days after ovariectomy ( Figure 1B).

Surgery
All animals were adapted to the laboratory environment for 3 days before undergoing ovariectomy. The surgical procedure for ovariectomy followed the same procedure described in our previous report (Liu et al., 2012). Briefly, mice were anesthetized with pentobarbital sodium (65 mg/kg, i.p., Dingguo Changsheng Biotechnology, Beijing, China), and the mice were kept in a lateral position. Hair was removed 1 cm horizontally from both sides of the spine, and the skin was disinfected with betadine. A small incision was made parallel to the spine at the intersection of the upper thigh and the lateral spine of the mice, and then the ovaries were removed bilaterally. A week was allowed for recovery before further testing. Sham-operated animals only had incisions without removing the ovaries.
To overexpress Rasd2 in the HP, a lentiviral expression vector was synthesized by Obio Technology (Shanghai, P.R. China). Viral titers were 4.77*108 particles/mL for pLenti-Ubc-EGFP-2A-3FLAG-Rasd2 and 1.55*109 particles/mL for pLenti-Ubc-EGFP-3FLAGcontrol. After anesthesia with pentobarbital sodium, mice were placed on the stereotactic frame and the scalp and connective tissue were cut to fully expose the skull. After holes were drilled at the appropriate locations, the virus was microinjected bilaterally into the HP (-1.8 mm anterior-posterior, ±1.6 mm medial-lateral, and -1.5 mm dorsal-ventral from bregma; Figure 1C) at a speed of 0.2 μL/min.

RNA Isolation, Sequencing, and Bioinformatic Analysis
The animals were decapitated, and the PFC was quickly removed, placed on ice, labeled, and stored in a refrigerator at -80°C for later processing and analysis. Tissue was processed following the instructions of the Trizol kit to extract total RNA and then using RNase-free DNase I to remove genomic DNA. RNA purity and concentration were determined using a Nano Photometer spectrophotometer (IMPLEN, Westlake Village, CA, USA) and a Qubit 2.0 kit. High-quality RNA samples were transported on dry ice to Sangon Biotech (Shanghai, China) for sequencing and testing. The sequencing of the established library was performed with the Illumina HiSeq XTen platform (Illumina, San Diego, CA, USA), and paired-end reads at 150 bp were obtained. The Bioconductor software package was used to correct for multiple testing (false discovery rate cutoff <0.1) and to identify differentially expressed transcripts based on counts per million values. P < .05 was considered statistically significant.

Open Field Test
Mice were placed in the center of an acrylic apparatus (48.8-cm diameter, 16 cm high) (Liu et al., 2012). The floor of the apparatus was divided into 16 equal squares. The test lasted 6 minutes and was recorded with a video camera (DCR-SX83E, Sony, Shanghai, China). Horizontal locomotor activity (the number of grid lines crossing traversed by all 4 paws of the mouse) and vertical locomotor activity (number of times the mouse stood with both forepaws off the ground) were counted by an observer blind to the treatment conditions.

Forced Swimming Test
Each mouse was individually placed in a cylindrical container (11 cm diameter × 25 cm high), filled with water (12 cm depth), with the water temperature maintained at 25°C ± 1°C (Liu et al., 2012). The test lasted 6 minutes and was recorded with a video camera (DCR-SX83E, Sony). The first 2 minutes of the test were considered adaptation time, and behavior was recorded for the only final 4 minutes of the test. Duration of immobility, swimming, and climbing as well as defecation (number of fecal boli) were determined by an observer blinded to the experimental conditions. Specific discrimination of behavior in the FST (immobility, swimming, and climbing) was according to the criteria previously reported (Cryan et al., 2002). Immobility was defined as having no additional movement other than that necessary to keep the head above the water. Swimming was defined as swimming with the body parallel to the wall. Climbing was characterized by pawing movements oriented at the side of the chamber with the animal oriented perpendicularly to the wall (Cryan et al., 2002).

Tail Suspension Test
The TST was referred to in previously published articles (Kim et al., 2021;Zhang et al., 2021a). Tape was attached 2 cm from the mouse tail-tip, and the mouse was held in an inverted state with the head approximately 20 cm above the ground with tape. The behavior of the mice within 5 minutes was recorded by a video camera (DCR-SX83E, Sony). The cumulative immobility time (the body of mice was vertically inverted and immobile) during the last 4 minutes was recorded by an observer blind to the treatment conditions.

Sucrose Preference Test
Mice were trained to acclimate to 1% sucrose solution (two 1% sucrose water bottles per cage) 2 days before the formal test. Mice were water deprived for 12 hours, then 2 weighed water bottles (one 1% sucrose solution and one pure water) were placed in each cage. After 1 hour, all bottles were weighed to calculate sucrose solution and water consumption (Zhang et al., 2021a). Sucrose preference = sucrose solution consumption / (sucrose solution consumption + pure water consumption) * 100%.

Enzyme-Linked Immunosorbent Assay
Mice were anesthetized and their whiskers were clipped. Blood was collected by retro-orbital bleeding and placed at room temperature for 1 hour, followed by centrifugation at 2000 rpm for 10 minutes. The plasma supernatant was collected and stored at -80°C until use. Assay was performed by recommended protocol of kit (Feiya Biotechnology Co., Ltd, Jiangsu, China). To the wells were added standard or samples and added sample diluent. We then added horseradish peroxidase (HRP)-conjugate reagent to each well and incubated for 60 minutes at 37°C. After washing, chromogen A and B were added to each well and incubated for 15 minutes at 37°C. Finally, stop solution was added to each well. We read optical density at 450 nm by using a microtiter plate reader (Variosbon Flsh, Thermo Scientific, Waltham, MA, USA) within 15 minutes.

Co-immunoprecipitation
Co-IP was performed by the recommended protocol of kit manufacturer (Abs955, Absin, Shanghai, China). Firstly, RIPA buffer (R0020, Solarbio) with 1% PMSF solution was added to the collected tissue, and the tissue was homogenized by homogenizer. Then, the samples were centrifuged at 12 000 rpm for 20 minutes at 4°C, and the supernatant was removed for use. Primary antibody (RASD2, RHES-101AP, Fabgennix, Frisco, TX, USA) was added to the samples, while homologous antibodies (Rabbit IgG, abs20035, Absin) from nonspecific immunization were used as controls and the samples were incubated overnight at 4°C. Protein A and G were added to the samples and gently mixed overnight at 4°C then centrifuged at 12 000 rpm for 1 minute to retain the precipitate. Precipitate was washed by wash buffer 3 times. 1*SDS sample buffer was added to resuspend the precipitate, and the sample was held at 95°C-100°C for 5 minutes. All samples were subsequently analyzed by WB.

Statistical Analysis
All data values are expressed as mean ± SEM and were analyzed by GraphPad Prism Software (version 8.0.1). Student's t test was used to compare means between 2 groups (sham vs ovariectomy; control vs Rasd2 overexpression). Two-way ANOVA was used to compare the effects of factorial designs (factor 1: fasting; factor 2: sulpiride). When a significant difference was obtained in an ANOVA, post hoc comparisons were performed between means using Tukey's honestly significant difference test (Tukey's HSD). P < .05 was considered statistically significant. The Shapiro-Wilk test was used to evaluate the normality of the data by SPSS (version 23). Effect size was assessed calculating η 2 or Cohen's d as needed by SPSS. Following Cohen (1988), we interpreted estimated η 2 and d values as follows: η 2 = 0.01 small, 0.06 medium, 0.14 large; d = 0.2 small, 0.5 medium, 0.8 large.

Effect of Acute Fasting on Brain Gene Expression Changes
Gene expression significantly changed in the PFC as a result of 9-hour fasting. Bioinformatic analysis of the pattern of significantly altered genes is shown in Figure 2 for biological processes (A), cellular components (B), molecular functions (C), and overall functions (D). Figure 2A identified the first 20 biological processes related to differential expressed genes. Among them, dopaminergic synaptic transmission, as well as several biological functions involving dopaminergic neurotransmission, were altered. Figure 2B shows the top 20 cellular components showing altered gene expression. Figure 2C shows the top 20 molecular functions related to differentially expressed genes. Among them, neuropeptide hormone activity, dopamine binding, and syntaxin binding are obviously related to central nervous system functions. Figure 2D shows the top 20 overall results from the analysis of GO enrichment in which the neuronal cell body, neuropeptide signaling pathway, myelin sheath, synaptic transmission (dopaminergic), and neuropeptide hormone activity are related to central nervous system function. The above data suggest that the changes in differentially expressed genes induced by 9-hour fasting may be involved in nerve cell growth and development, hormone regulation, and signal transmission as well as other processes. Further gene function analysis was carried out from the biological process of synaptic transmission (dopaminergic), and genes with significant differences were screened out as shown in Figure 2E: Adenosine A2a receptor (Adora2a), Drd2, Drd1, Tyrosine hydroxylase (TH), and Rasd2. In the enrichment analysis of the KEGG pathway in the PFC after fasting ( Figure 2F-H), the PI3K-Akt pathway has the largest number of differentially expressed genes (16 differentially expressed genes), although the analysis again identified several gene sets related to dopaminergic function (e.g., cocaine addiction, Parkinson's disease, and dopaminergic synapse), shown in Figure 2H. Ribosomes, Parkinson's disease, and herpes simplex infection ranked next in this analysis with 11 differentially expressed genes, followed by the cAMP signaling pathway with 10 differentially expressed genes.

DISCUSSION
In the present study, we found that 9-hour fasting altered differential gene expression in the PFC of ovariectomized mice. The results of GO enrichment analysis and KEGG pathway enrichment analysis of differentially expressed genes showed that fasting affected genes related with dopaminergic signaling, including Drd2, Drd1, TH, and Rasd2. A study also found that calorie restriction causes dopaminergic dysregulation in female mice (Carlin et al., 2016). In addition, calorie restriction delays the age-related or diabetes-related loss of DRD2 in rat brain (Roth et al., 1984; Rasd2 Mediates Acute Fasting-Induced Antidepressant-Like Effects | 225 Thanos et al., 2008;de Leeuw van Weenen et al., 2011). Our results are consistent with the above studies showing that the molecular mechanisms of fasting on depression may be closely linked to dopamine.
To further explore the molecular mechanisms underlying the effects of fasting in ovariectomized mice, Drd2 and Rasd2 were selected from the RNA-seq study for further study. Interestingly, in our studies, RASD2 protein was decreased in ovariectomized mice in the HP but not in the PFC. These results indicate that the depression model established by ovariectomy induces the downregulation of RASD2 in the HP (but not in the PFC), and the downregulation of RASD2 expression in the HP is one of the pathogeneses of depression. Although Rasd2 has been reported to be a common regulator of fasting and estrogen in the PFC , there may be other regulators involved in ovariectomized mice. It has been reported that short-term fasting increases autophagy in cortical neurons (Alirezaei et al., 2010), and overexpression of Rasd2 can also activate autophagy (Mealer et al., 2014). However, whether short-term fasting further increases autophagy through Rasd2 is still unknown, and this should be investigated in further studies. In addition, ovariectomy also reduced DRD2 expression in the HP.
Considering that there is a high density of DRD2 binding site in dorsal HP (Charuchinda et al., 1987;Edelmann and Lessmann, 2018), lentivirus vectors was microinjected into the dorsal HP to achieve Rasd2 overexpression in the HP of ovariectomized mice. Rasd2 overexpression in ovariectomized mice significantly decreased immobility in the FST and TST and increased swimming time and sucrose consumption, indicative of antidepressant effects. No effects were observed in the OFT, showing that the effects of Rasd2 overexpression were behaviorally specific and not just the result of elevated spontaneous activity. These results indicate that Rasd2 and DRD2 are fundamentally involved in ovariectomy-induced depression. Previous studies have shown that antidepressants work on catecholaminergic systems selectively increase climbing behavior, whereas antidepressants targeting serotonergic systems selectively increase swimming behavior (Cryan et al., 2005;Slattery and Cryan, 2012). In addition, emotional animals defecate more than non-emotional animals (Broadhurst, 1957;Craft et al., 2010). In our study, Rasd2 overexpression primarily increased swimming, with only modest effects on climbing and defecating behavior. Whether these effects involve dopamine or dopamine interactions with other monoaminergic systems is uncertain. In addition, Rasd2 overexpression in the HP of ovariectomized mice significantly increased DRD2 expression in the HP. Studies have found that Rasd2 affects DRD2-dependent activity  and also regulates striatal-dependent behaviors in a gender-specific manner . These results indicate that Rasd2 and DRD2 are likely to be involved in the molecular mechanisms underlying depressive-like symptoms induced by ovariectomy.
The role of RASD2 in DRD2-mediated antidepressant-like effects of 9-hour fasting based on ovariectomized mice was then examined. Immobility time in the FST was increased and sucrose consumption decreased, indicative of a depressive profile, and fasting significantly reversed the changes of depression-like behaviors. Moreover, DRD2 antagonists blocked the antidepressant-like effects of fasting. Antidepressant-like effects of fasting on climbing and swimming in the FST may involve dopamine or its interaction with other monoaminergic systems. This finding is consistent with previous studies showing that sulpiride antagonizes antidepressant effects on immobility (Borsini et al., 1988;Donato et al., 2013). Importantly, the present study suggests that DRD2 mediates the reduction in immobility time induced by fasting as well. Nonetheless, RASD2 and DRD2 appear to be intimately related in the effects of ovariectomy and fasting. Firstly, the antidepressant-like effect of 9-hour fasting involving DRD2 was shown to be closely related to RASD2 expression. Fasting increased the expression of RASD2 in the PFC, HP, and striatum, and DRD2 antagonists reversed this increase in RASD2 levels induced by fasting. Similarly, fasting increased DRD2 expression in the PFC and HP. These results suggest that the reduction in immobility caused by fasting is likely to be caused by regulating the expression of DRD2 and RASD2.
Studies have found that Rasd2 deficiency leads to abnormal excitatory responses of cholinergic interneurons to activation of DRD2 receptors. Furthermore, PI3K inhibitors rescue the abnormal DRD2 response in Rasd2 knockout mice, and it has been found RASD2 acts as a bridge between PI3K and Akt signaling pathways (Subramaniam et al., 2011;Bang et al., 2012;Harrison et al., 2013;Lee et al., 2015). Fasting was shown to activate the PI3K-Akt pathway in the KEGG pathway analysis and lead to a decrease in Akt expression, and sulpiride treatment reverses the fasting-induced increases in Akt expression. These results indicate that changes in Akt expression may also participate in the reduction of immobility time mediated by DRD2.
The PI3K-Akt pathway is a DRD2-linked signaling pathways that has been linked to the pathogenesis of mood disorders and BDNF-mediated neuroprotection (Cao et al., 2019;Huang et al., 2021). Consistent with previous studies (Cui et al., 2018;Wang et al., 2019), in our studies, fasting increased CREB and BDNF expression in the PFC and HP of ovariectomized mice. Moreover, DRD2 antagonist antagonized fasting-induced increases in CREB and BDNF expression, indicating that the CREB-BDNF signaling pathway is likely to play a role in the antidepressant-like effects of fasting mediated by DRD2. Therefore, RASD2 may produce antidepressant-like effects by regulating the expression of Akt and further affecting the CREB-BDNF signaling pathway.
It has been reported that the mouse plasma levels of estrone, estradiol, and estriol were reduced 1 week after ovariectomy, and estradiol and estriol levels in plasma were similar between 1 week and 3 months post-ovariectomy and 17β-estradiol treatment . In the present study, ovariectomy induced an increase in immobility in the FST at 1 week after ovariectomy, similar to previous findings (Estrada-Camarena et al., 2011). In previous studies, caloric restriction produced estrogen-like effects (Bigsby et al., 1997) and increased estrogen levels; moreover, there was an additive antidepressant-like effect of fasting and estrogen in ovariectomized mice . Fasting increased ERβ expression in the HP and striatum, an effect antagonized by sulpiride. 17 β-Estradiol has no effect on immobility in ERβ knockout mice, but not ERα knockout mice in the FST Figure 6. Effect of sulpiride (SUL) on fasting-induced changes in protein expression. Figures represent the changes in the protein expression of RASD Family Member 2 (RASD2), brain-derived neurotrophic factor (BDNF), cAMP-response element binding protein (CREB), phospho-CREB (p-CREB), protein kinase B (Akt), dopamine D2 receptor (DRD2), estrogen receptor α (ERα), and estrogen receptor β (ERβ) in the hippocampus (HP) (A, a-h), prefrontal cortex (PFC) (B, i-p), and striatum (C, q-x) in ovariectomized mice. The data are expressed as mean ± SEM (n = 3-5). Two-way ANOVA with Tukey's honestly significant difference (HSD), *P < .05 vs ovariectomy (OV), **P < .01 vs vehicle (VEH):non-fasting; # P < .05, ## P < .01 vs VEH:fasting.
Rasd2 Mediates Acute Fasting-Induced Antidepressant-Like Effects | 227 (Rocha et al., 2005). These studies indicate that the increase in immobility time of ovariectomized mice is mainly related to ERβ. Moreover, the present study suggests that ERβ is involved in the effects mediated by DRD2 on the antidepressant-like effects of fasting, although the connection between RASD2, DRD2, and ERβ remains to be fully elucidated.

CONCLUSION
In summary, Rasd2 plays a role in depression-like behavior induced by ovariectomy, and this role is related to the regulation of DRD2. Nine-hour fasting has antidepressant-like effects in ovariectomized mice and upregulates the expression of RASD2, DRD2, CREB-BDNF, Akt, and ERβ ( Figure 7). Moreover, these effects are blocked by DRD2 antagonists. Rasd2 can therefore be postulated to be a potential therapeutic target for depression and perhaps also a potential predictive marker for depression. Finally, dopamine receptor-mediated gene regulation in antidepressant-like effects of acute fasting also provides new ideas for the treatment of depression. However, whether fasting has similar therapeutic effects on patients with depression and the conditions for implementing fasting (such as the specific time point and duration of fasting) need to be further explored in clinical studies.

Author Contributions
B.J.L. contributed conception and design of the study; Z.Q.C., C.H.Z., F.Y.Z., and J.J.P. performed the research; Z.Q.C. wrote the paper; and B.J.L. and R.J.C. provided the critical revisions. All authors read and approved the final manuscript.