Introduction

Social isolation is widely used as stress among juvenile and is also considered a risk factor for developing psychiatric disorders [1,2,3,4]. Considering the recent stay-at-home orders in response to the COVID-19 pandemic globally, understanding the consequence of social isolation is critical for informing some interventions that may alleviate the development of mental disorders after the isolation period. People suffering long-lasting periods of isolation usually report feelings of loneliness, anger, post-traumatic stress, anxiety and depression, and boredom [5, 6]; increased expression of inflammatory markers and high blood pressure [7]; and increased risk of hypofunction and early mortality [8, 9]. Exploring how these results may differentially influence behavior in older adults is particularly important, as this population frequently experiences social isolation during their late-life [7, 10].

Some studies in adult animals have also demonstrated that social isolation during adolescence increased sensitivity to stress [11,12,13], decreased social behaviors [14, 15], increased anxiety-like behavior [11, 12, 14, 16, 17] and depressive-like behavior [18], and reduced hippocampal cell proliferation and impaired spatial memory [2]. It is worth mentioning that the effects of stress are often relied on the age of animals that undergo the stress experience [19]. For example, in measuring of anhedonia, some investigators found that sucrose consumption or preference was increased in animals isolated during adolescence [20, 21] while it decreased after isolation during adulthood [22]. Previous researches about social isolation have mainly focused on animals that were isolated during post-weaning. However, whether aged animals exposed to social isolation stress show similar effects on their behaviors and the further neurobiology mechanism being involved is unknown right now.

The hippocampus is a part of the brain with great importance for stress response given that it is rich in stress hormone targets [23]. Early studies in rodents have found that stress may lead to structural and functional alterations in corresponding brain regions by reducing brain weight [24, 25], mediating hippocampal atrophy, and affecting neurogenesis [26]. Homocysteine (Hcy), a sulfur-containing amino acid, is an important product of methionine metabolism [27, 28]. The main source of Hcy is methionine in food intake, and Hcy is catabolized mainly in the liver in the presence of B vitamins as coenzymes [29], which means that deficiency of these vitamins such as B12, folate (B9), and pyridoxine (B6) leads to hyperhomocysteinemia (Hhcy) [30, 31]. Meanwhile, some evidence indicated that there is intimate relationship between Hcy and stress. According to previous study, chronic stress can increase Hcy levels by affecting the expression of Hcy metabolic enzymes in the liver [32]. But whether Hcy is involved in depression induced by stress during late-life period is unknown.

Brain-derived neurotrophic factor (BDNF) plays important roles in the maintenance of cognitive function [33, 34]. Several studies have found that various stress patterns, such as PTSD, prenatal stress, and negative early-life events, affect BDNF expression. All of these processes are closely associated with methylation [35, 36]. However, whether BDNF is regulated during chronic social isolation stress needs further study.

Therefore, in this study, we wanted to evaluate the effects of social isolation on depressive-like behaviors in aged mice. Moreover, considering the intimate relationship between stress and Hcy [32], we also investigated whether this amino acid was involved in our hypothesis.

Materials and Methods

Animals

Male C57BL/6J mice obtained at 18 months of age were used and randomly assigned to different groups (the number of mice used in each group was described in the legend of figures). Mice were exposed to a 12-h/12-h light/dark cycle in standard laboratory cages temperature at 21–25°C and had free access to pure water and food except during the stress intervention and behavioral experiments. All of the animal experiments were approved by the Chinese Council on Animal Care Guidelines and experimental operations were approved by Southern Medical University ethics committees (the number of the approval document is D2017221). We made all efforts to minimize animal suffering.

Social Isolation Protocol

Male C57BL/6J mice aged at 18 months were housed individually. While isolated mice could hear and smell other mice within the housing facility, there was no physical interaction with other mice. Additionally, the number of investigators handling the isolated mice during weekly cage changes was kept to a minimum. Mice were isolated for 2 months before the initiation of any experimental procedure.

Open Field Test

The open field test was performed as previous study described [37,38,39]. It is performed in a rectangular chamber (40×40×30 cm) that was made of gray polyvinyl chloride. At the beginning of the test, mice were gently placed on the center of the chamber. Then mice can freely move for 5 min, which could be monitored by an automated video tracking system. The total distance of experimental mice can be recorded and analyzed automatically by using EthoVision 11.0 software.

Sucrose Preference Test

Sucrose preference test was performed as previous study described [38, 40]. In brief, mice were habituated to 1% sucrose for 2 days with two 50-ml bottles (A and B). After that, bottle A contained 1% sucrose solution, and bottle B contained water (s/w), and the sucrose preference was measured weekly for 1 h following a 24-h period of water and food deprivation. The bottle positions were switched weekly to avoid a side bias. The fluid that was consumed from each bottle was measured every time. The sucrose preference = VolA/ (VolA + VolB).

Coat score assay was performed as previous study described [40]. The total coat score was measured weekly as the sum of the score of seven different body parts: head, dorsal coat, neck, tail, ventral coat, hindpaws, and forepaws. A score of 1 was given for a well-groomed coat while a score of 0 was given for an unkempt coat.

Forced Swimming Test

The forced swimming test (FST) apparatus was a clear glass cylinder (45-cm height and 19-cm diameter) filled with water (22–25°C) to 23 cm. In the 6-min test, the duration of immobility was measured during the final 4 min using Ethovision XT software [38, 40].

Tail Suspension Test

Tail suspension test (TST) was conducted using tail suspension cubicles (PHM-300, MED-Associates). Mice were individually suspended by the tail to a metal hanger with adhesive tape affixed 1.0–1.5 cm from the tip of the tail for 6 min. The sum of the immobility time for the total 6 min were recorded using Med Associates Tail Suspension software version 2 (MED-Associates).

T-maze Test

The T-maze test was conducted in a T-shaped elevated maze (30×10 cm start arm and two 30×10 cm goal arms, with stripes or circles on the walls of the goal arms). During training, both of the two arms were open, and mice were placed in the start arm facing away from the choice point, and allowed to freely explore the maze for 20min. Once the mice entered the arm, we closed the chosen door. The mice were returned to a new cage 1min later. The retention test was performed 5min after training. During this test, both goal arms were open, and mice were placed at the end of the start arm facing away from the choice point and allowed to freely explore for 5 min. The percent of time spent in each arm and the total exploration (in meters) were measured using the EthoVision tracking software (Noldus). New arm preference was calculated by dividing the percentage of time spent in the new arm (that was closed during training) by the percentage of time spent in both goal arms (new and old). Data were analyzed using a two-tailed Student’s t-test.

Novel Object Recognition Test

The test was carried out in a square open field box (33 cm × 33 cm × 20 cm). The test was divided into two steps: adaption and testing. Mice were placed in the empty arena to adapt for 5 min on the first day. On the second day, mice were placed in the same cognitive box containing two identical white cube blocks and allowed to explore for a total of 20 s, up to 10 min. On the third day, a novel object replaced one familiar object, and the times of exploring (including limb contact, whisker exploration, etc.) the new (Tnew) and the old (Told) objects within 10 min were measured separately. Cognitive index was calculated as follows: Cognitive index = (Tnew / (Tnew + Told).

Contextual Fear Conditioning Test

The contextual fear conditioning test was carried out similar to studies previously described [41, 42]. Firstly, mice were trained in a conditioning chamber A. They were exposed to four tone-foot-shock pairing paradigms (foot shock, 1 s, 0.4 mA; tone, 30 s, 80 dB; 80-s interval). Then, their contextual fear learning was evaluated by placing mice returned to the chamber A 24 h later and monitored the freezing time automatically with Med Associates Video-Tracking and Scoring software.

Plasma Hcy Analysis

High-performance liquid chromatography (HPLC) with fluorimetric detection and isocratic elution was used to measure plasma Hcy level. The HPLC containing a WATERS LC2695 instrument, WATERS 2475 fluorescence detector, and a Symmetry Shield RP18 column of C18 model (3.9 mmi.d.×150 mm, 5-μm microparticles) was used for chromatographic separation. The final conditions of excitation light of 390 nm and emission light of 470 nm were used to detect the compounds in the liquid column. The known concentrations of Hcy and N-acetyl-l-cysteine were also used as calibration curves to calculate the Hcy content.

Homocysteine and Diet Intervention

To study the effects of Hcy, mice were fed a diet containing 1% methionine to elevate the Hcy level in vivo [43]. The homocysteine intervention group was given compound vitamin B intragastrically at 9 AM every day during the stress process; the total volume of gavage was 1.5 mL, and the content of each component was as follows: vitamin B 6 24 mg/kg body weight per day; vitamin B 12 20 μg/kg body weight per day; folic acid 10 mg/kg body weight per day [44].

Enzyme-Linked Immunosorbent Assay Detection

After deeply anesthetized, the hippocampal tissues of experimental mice were harvested and dissected, which were then disrupted by sonication in NP-40 lysis buffer containing with 50 mM Tris (pH=7.4), 150 mM NaCl, 1% NP-40, 0.1% Triton X-100, and 0.1% SDS supplemented with protease inhibitors. After being centrifuged at 20,000×g for 15 min, the cleared supernatant was collected and treated with 1 N HCl for 15 min at room temperature, followed by neutralization with 1 N NaOH. BDNF levels were measured using the Emax Immuno-Assay System Enzyme-Linked Immunosorbent Assay kit (Promega) with accord to the manufacturer’s instructions [45].

Statistical analyses

All of the data are listed as mean ± standard error of the mean. For comparisons between two independent groups, we used two-tailed Student’s t-test; for comparisons of more than two groups, we used one-way ANOVA followed by Fisher’s protected least significant difference post hoc analysis. All of the analyses were performed by the GraphPad Prism 8.0 statistical package, and p < 0.05 was considered to be significantly different.

Results

Social Isolation Stress Induced Depressive-Like Behaviors in Aged Mice

To substantiate the effects of social isolation on depressive-like behaviors for aged mice, 18-month mice were socially isolated or were reared in normal conditions. Sucrose preference tests were performed weekly to assess anhedonia, a core symptom of depression (Fig. 1A). We found that 7-week isolation led to a decrease in sucrose preference (Fig. 1C) and deterioration in coat score (Fig. 1D), which were more serious after 8-week isolation (Fig. 1C, D). However, this isolation paradigm had no effect on body weight between the two groups (Fig. 1E). Furthermore, 2-month isolation dramatically increased the immobility time in the FST (Fig. 1F) and TST (Fig. 1G). Two-month social isolation had no effect on locomotor activity in aged mice (Fig. 1B). We also observed impaired cognitive ability in isolated mice, with lower alternative rate in the T-maze test (Fig. 1H) and discrimination ratio in the novel object recognition (NOR) test (Fig. 1I) and less freezing time in the contextual fear conditioning test (Fig. 1J). These results proved the fact that two-month social isolation induced depressive-like behaviors in aged mice.

Fig. 1
figure 1

Two-month social isolation induced depressive-like behaviors in aged mice. A Schematic of the experiments tests. ISO, isolation. B Two-month social isolation did not affect locomotor activity (n=12/group, two-tailed Student’s t-test, P=0.673). Sucrose preference test (C), coat score assay (D), and body weight (E) after 2-month social isolation in aged mice (n=12 mice/group; repeated measures two-way ANOVA; for C, F(1, 22)=7.568, P=0.012; for D, F(1, 22)=9.638, P=0.006; for E, F(1, 22)=3.177, P=0.386). Increased the immobility time of isolated mice in FST (F) and TST (G) after 2-month social isolation in aged mice (n = 12 mice/group; two-tailed Student’s t-test, for F, P =0.003; for G, P=0.012). H–J Two-month social isolation impaired cognitive ability in aged mice (n=12/group, two-tailed Student’s t-test, for H, P=0.023; for I, P=0.006; for J, P=0.018). Data show mean ± s.e.m. *P<0.05, **P< 0.01, ***P< 0.001

Full size image

A Direct Correlation Between Hcy Levels and the Social Isolation–Induced Depressive-Related Performance

To determine whether Hcy was involved in social isolation–induced depressive-like behaviors, we measured the level of Hcy between control mice and socially isolated mice (Fig. 2A). We found the level of Hcy in the plasma was increased gradually during social isolation time (Fig. 2B). Most importantly, we found that the sucrose preference (Fig. 2C), the coat score of the sucrose preference tests (Fig. 2D), the immobility time of the FST (Fig. 2E), and the TST (Fig. 2F) showed significant correlation with plasma Hcy levels, respectively. These findings suggest that Hcy is closely related to the depressive-like behaviors in aged mice during social isolation. Thus, it is particularly important to investigate whether Hcy is involved in the development of depressive-like behaviors.

Fig. 2
figure 2

A direct correlation between Hcy levels and depressive-related behavioral test. A Schematic of the experiments tests. B Plasma Hcy levels of control and isolation group at different time points (n=8–12 per group; one-way ANOVA; F(4, 39)=37.087, P=0.006). C Correlation between Hcy levels and the sucrose preference of the sucrose preference test (n=12; r2=0.6564, P=0.0014). D Correlation between Hcy levels and the coat score of the sucrose preference test (n=12; r2=0.5886, P=0.0036). E, F Correlation between Hcy levels and the immobility time in the FST (E, n=8; r2=0.4554, P=0.0161) and the TST (F, n=12; r2=0.5263, P=0.0076). Data show mean ± s.e.m. *P<0.05

Full size image

Diet-Induced Increased Hcy Mimicked the Depressive-Like Behaviors in the Same Manner as Social Isolation

To confirm whether Hcy is involved in social isolation–induced depressive-like behaviors, we employed the high methionine diet which was used to elevate Hcy (Fig. 3A). Two months of methionine diet significantly increased the Hcy level in control mice, thereby mimicking the effect of social isolation stress on Hcy (Fig. 3B). Behavior tests showed that high methionine diet led to depressive-like behaviors similar to those caused by social isolation, with a decreased sucrose preference and coat score in the sucrose preference test (Fig. 3D, E), increased the immobility time in the FST (Fig. 3G) and TST (Fig. 3H). Administration of high methionine diet had no effect on locomotor activity (Fig. 3C) or body weight (Fig. 3F). Also, administration of high methionine diet impaired the cognitive performance (Fig. 3I–K).

Fig. 3
figure 3

Diet-induced HHcy mimicked the depressive-like behaviors in the same manner as social isolation. A Schematic of the experiments tests. B Two-month of methionine diet significantly elevated Hcy level (n=8 per group; two-tailed Student’s t-test, P=0.008). C Two-month of methionine diet had no effect on locomotor activity (n=12 per group; two-tailed Student’s t-test, P=0.374). Sucrose preference test (D), coat score assay (E), and body weight (F) after 2-month administration of methionine diet in aged mice (n=12 mice/group; repeated measures two-way ANOVA; for D, F(1, 22)=7.124, P=0.042; for E, F(1, 22)=8.158, P=0.011; for F, F(1, 22)=3.074, P=0.414). Increased the immobility time of isolated mice in FST (G) and TST (H) after 2-month administration of methionine diet in aged mice (n = 12 mice/group; two-tailed Student’s t-test, for G, P =0.012; for H, P=0.017). I–K Two-month of methionine diet impaired cognitive ability in aged mice (n=12/group, two-tailed Student’s t-test, for I, P=0.036; for J, P=0.013; for K, P=0.032). Data show mean ± s.e.m. *P<0.05, **P< 0.01

Full size image

Hcy Reduction Alleviated the Depressive-Like Behaviors in Socially Isolated Aged Mice

To further confirm whether Hcy is involved in social isolation–induced depressive-like behaviors, socially isolated aged mice were gavaged with a vitamin B complex (VBco) to reduce Hcy. Plasma Hcy was measured to confirm the effect of the Hcy intervention (Fig. 4A). We found that VBco gavage significantly reversed the elevated plasma Hcy levels in socially isolated mice (Fig. 4B). Meanwhile, treatment with vitamin B complex markedly improved the physical state of the coat and increased the sucrose preference to the levels comparable to control mice, whereas treatment with control diet was ineffective (Fig. 4D, E). Moreover, application of vitamin B complex decreased the immobility time in the FST (Fig. 4G) and TST (Fig. 4H). Administration of VBco diet had no effect on locomotor activity (Fig. 4C) or body weight (Fig. 4F). Also, administration of VBco complex diet reversed the impaired cognitive performance in isolated aged mice (Fig. 4I–K).

Fig. 4
figure 4

Hcy reduction alleviated the depressive-like behaviors in socially isolated aged mice. A Schematic of the experiments tests. B Administration of vitamin B complex diet normalized Hcy level in socially isolated aged mice (n=8 per group; two-tailed Student’s t-test, P=0.028). C Administration of vitamin B complex diet had no effect on locomotor activity (n=12 per group; two-tailed Student’s t-test, P=0.864). Sucrose preference test (D), coat score assay (E), and body weight (F) after administration of vitamin B complex diet in aged mice (n=12 mice/group; repeated measures two-way ANOVA; for D, F(1, 22)=6.742, P=0.045; for E, F(1, 22)=7.108, P=0.037; for F, F(1, 22)=3.368, P=0.573). Increased the immobility time in FST (G) and TST (H) was reversed by administration of vitamin B complex diet in isolated aged mice (n = 12 mice/group; two-tailed Student’s t-test, for G, P =0.024; for H, P=0.034). I–K Administration of vitamin B complex diet reversed the impaired cognitive ability in isolated aged mice (n=12/group, two-tailed Student’s t-test, for I, P=0.022; for J, P=0.016; for K, P=0.039). Data show mean ± s.e.m. *P<0.05

Full size image

Downregulation of BDNF in the Hippocampus Was Involved in the Development of Depressive-Like Behaviors in Socially Isolated Mice

The hippocampal protein levels of BDNF, a gene closely related to depression [46,47,48], were measured at different time points of social isolation. We found that the level of BDNF was decreased with the duration of social isolation (Fig. 5A). Moreover, there was a significant negative correlation between plasma Hcy and BDNF levels in the hippocampus (Fig. 5B), which suggested that reduced BDNF levels during social isolation are closely associated with elevated Hcy. To further reveal whether BDNF is regulated by Hcy, we measured BDNF levels in the hippocampus after Hcy modulation. We found that high-methionine diet in control mice significantly decreased the hippocampal BDNF levels similar to social isolation (Fig. 5C). And supplementation of BDNF normalized the depressive-like behaviors in aged mice (Fig. 5D–F). In contrast, VBco gavage significantly increased the BDNF levels, compared with the socially isolated mice (Fig. 5G). And blockade of BDNF function with its receptor inhibitor K252a blocked the role of VBco gavage on depressive-like behaviors in isolated aged mice (Fig. 5H–K). Taken together, the hippocampal BDNF may be negatively regulated by Hcy during social isolation in aged mice.

Fig. 5
figure 5

Downregulation of BDNF in the hippocampus was involved in the development of cognitive decline in socially isolated mice. A Hippocampal BDNF levels of control and isolation group at different time points (n=8–12 per group; one-way ANOVA; F(4, 39)=28.073, P=0.031). B Correlation between Hcy levels and the hippocampal BDNF levels in isolated mice (n=12; r2=0.4723, P=0.0135). C Diet-induced HHcy mimicked the decrease of BDNF (n=8 per group; two-tailed Student’s t-test, P=0.038). D–G Microinjection of BDNF into the hippocampus normalized the depressive-like behaviors in aged mice that was treated with 2 months of methionine diet (n=12/group, two-tailed Student’s t-test, for D, P=0.682; for E, P=0.013; for F, P=0.031; for G, P=0.019). H Administration of vitamin B complex normalized the BDNF level in isolated aged mice (n=8 per group; two-tailed Student’s t-test, P=0.012). I–L Microinjection of K252a into the hippocampus blocked the role of administration of vitamin B complex diet in isolated aged mice on depressive-like behaviors (n=12/group, two-tailed Student’s t-test, for I, P=0.362; for J, P=0.022; for K, P=0.037; for L, P=0.028). Data show mean ± s.e.m. *P<0.05

Full size image

Discussion

The major findings of the present study were as follows. First, 2-month social isolation stress induced depressive-like behaviors in aged mice. Second, increased Hcy is closely related to the depressive-like behaviors in aged mice during social isolation stress. Third, diet-induced HHcy mimicked the depressive-like behaviors and BDNF downregulation in the same manner as social isolation, while administration of vitamin B complex supplements to reduce Hcy alleviated the depressive-like behaviors and BDNF reduction in socially isolated mice. Altogether, our results suggested Hcy is likely involved in social isolation stress–induced BDNF reduction and related depressive-like behaviors.

As a response to stimulation by internal and external environmental factors, stress involves systemic reactions in several organ systems, such as the nervous system, endocrine system, and immune system [49]. Rodent models of social isolation offer the ability to study the effects of isolation under highly controlled conditions, controlling for age, duration of isolation, housing conditions, and the developmental time point of isolation. The literature on the impact of social isolation on affective behavior in rodents to date has largely been limited to studying the effects of single-housing isolation-induced stress during critical developmental periods in adolescence and early adulthood. To date, limited studies have assessed the impact of social isolation in aged rodents, with only two studies, to our knowledge, having assessed social behavior following at least 2 weeks of late-life isolation in aged rodents [50, 51]. Here, we investigated the effects of social isolation on aged mice during their late-life and found that 2-month late-life isolation induced depressive-like behaviors. These results thus enriched the understanding of the impact of social isolation in aged mice.

Our study also highlights the role of Hcy, a small amino acid that was involved in vital processes such as energy metabolism and methylation [52], in the process of stress-induced cognitive dysfunction in mice. The main source of Hcy is methionine in food intake, and Hcy is catabolized mainly in the liver in the presence of B vitamins as coenzymes. Our study found that plasma Hcy levels increased with increasing duration of social isolation stress; this finding is similar to previous findings that chronic stress may lead to Hcy accumulation by inhibiting the transcription of cystathionine β-synthase, an Hcy isomerase, in the liver [32]. High-methionine diet and administration of VBco gavage induced the emergence of cognitive impairment and alleviated stress-induced cognitive impairment, respectively [44]. Zhou et al. found that 6 weeks of chronic stress resulted in decreased autonomic activity and increased Hcy; however, the administration of folic acid resulted in decreased Hcy and remission of symptoms, which the authors linked to decreased IL-6 release [53]. Another study suggested that elevated Hcy may be closely related to 5-HT metabolism in the relevant brain regions, and that methyl-donor deficiency may also lead to stress-like effects [54]. Results above suggest a potential role of Hcy in stress. Epidemiological studies have also found that Hcy is an important causative factor in neurodegenerative diseases [55]. In addition, Hcy is an independent risk factor for Alzheimer’s disease [56], and administration of B-complex vitamins may alleviate cognitive symptoms in AD patients [57]. Moreover, Hcy level negatively correlates with scores on the Cambridge Cognition Scale in the elderly [58]. Taken together, Hcy may be an important mediator of stress-induced depressive-like behaviors. However, in our study, 2-month social isolation stress induced depressive-like behaviors in middle-aged mice which were correlated with HHcy levels; and 2-month VBco gavage significantly reversed elevated plasma Hcy and depressive-like behaviors. So these results indicate a potential value of vitamin B intake in the prevention of stress-induced depressive-like behaviors. Nevertheless, we did not evaluate if the vitamin B intake could exhibit its therapeutic effect to alleviate the depressive-like behaviors induced by social isolation stress, which is the limitation and will be investigated in our future study.

Neurotrophic factors play an important role in the production and maintenance of depression in mammals [59]. As an important neurotrophic factor, BDNF plays an important role in maintaining normal brain function; it plays a key role in the formation of synapses, growth, and the differentiation and migration of neurons [60, 61]. Our study in mice showed that both stress and high levels of Hcy caused a decrease in BDNF level in the hippocampus which induced depressive-like behaviors. Meanwhile, intervention of Hcy by high-methionine diet or VBco gavage can decreased the hippocampal BDNF levels in normal mice or increased the BDNF levels in isolated mice, respectively. Correspondingly, supplementation of BDNF normalized the depressive-like behaviors in aged mice, while blockade of BDNF function with its receptor inhibitor K252a blocked the role of VBco gavage on depressive-like behaviors in isolated aged mice. These findings suggest that BDNF may be a downstream target molecule of Hcy in the process of stress-induced depressive-like behaviors. Inhibition of BDNF transcription results from chronic isolation stress [62], chronic social defeat stress [63], and chronic mild stress [64], suggesting that negative regulation of BDNF by stress is widespread in a variety of chronic stress processes. In addition, interference with BDNF at the dentate gyrus triggers depressive-like behavior and affects neuronal regeneration [65], and knockdown of BDNF in mice inhibits neuronal plasticity and induces the onset of psychiatric symptoms such as anxiety in mice [66]. Moreover, some people found that interference with BDNF at the dentate gyrus triggers depressive-like behavior and affects neuronal regeneration [65]. These findings indicate that BDNF is an important factor in the pathogenesis of mental disorders. Thus, it is particularly important to study how Hcy affects BDNF. Ma et al. found that the BDNF promoter region is regulated by methylation [67] and that administration of methyl donors reverses the psychiatric symptoms caused by Hcy during stress [53]. Therefore, the focus of our future study was to examine whether Hcy may affect BDNF transcription.

In summary, we found that social isolation stress can increase Hcy levels in aged mice, inhibit BDNF protein level, and lead to depressive-like behaviors in aged mice. These findings provide new insights into the mechanisms underlying depression caused by social isolation stress in aged mice. Finally, further interventional strategies for the occurrence and development of depression from a biological perspective should follow.