DOI: https://doi.org/10.21203/rs.3.rs-2118589/v1
Social isolation is an unpleasant experience associated with an increased risk of mental disorders. Exploring whether these experiences affect behaviors in aged adults is particularly important, as the elderly is very likely to suffer periods of social isolation during their late-life. In this study, we analyzed the depressive-like behaviors, plasma concentrations of homocysteine (Hcy), and brain-derived neurotropic factor (BDNF) levels in aged mice undergoing social isolation. Results showed that depressive-like behavioral performance and decreased BDNF level were correlated with hyperhomocysteinemia (HHcy) levels that were detected in 2-month isolated mice. Elevated Hcy induced by high methionine diet 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 indicated that Hcy played a critical role in social isolation-induced depressive-like behaviors and BDNF reduction, suggesting the possibility of Hcy as a potential therapeutic target and vitamin B intake as a potential value in the prevention of stress-induced depression.
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 [1–2], increased expression of inflammatory markers and high blood pressure [3], increased risk of hypofunction and early mortality [4–5]. 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 [3, 6].
Social isolation is widely used as stress among juvenile and is also considered as a risk factor for developing psychiatric disorders [7–10]. Some studies have demonstrated that social isolation during adolescent increased sensitivity to stress [11–13], decreased social behaviors [14–15], increased anxiety-like behaviors [11–12, 14, 16–17] and depressive behaviors [18], reduced hippocampal cell proliferation and impaired spatial memory [8] in adult animals. 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 adolescents [20–21] while decreased after isolation during adulthood [22]. Previous researches about social isolation have mainly focused on animals that isolated during post-weaning. However, whether aged animals experienced with social isolation stress produce some effects on their behaviors and the further neurobiology involved have no idea 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 to 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 that induced by stress during late-life period have no idea.
Therefore, in this study, we want 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.
Male C57BL/6J mice obtained at 18 months of age were used and randomly assigned to different groups. 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. We made all efforts to minimize animal suffering.
The open field test was performed as previous study described [33–35]. 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 a 5min, 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 was performed as previous study described [34, 36]. 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 [36]. 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.
The FST apparatus was a clear glass cylinder (45 cm height and 19 cm diameter) filled with water (22°C–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 [34, 36].
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).
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, 5µm 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.
To study the effects of Hcy on the cognitive function of rats, rats were fed a diet containing 1% methionine to simulate the HHcy status in vivo [37]. 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: VB6 24 mg/kg bw·d; VB12 20 µg/kg bw·d; folic acid 10 mg/kg bw·d [38].
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 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 ELISA kit (Promega) with accord to the manufacturer’s instructions.
All of the data are listed as mean ± standard error of the mean (SEM). 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 (PLSD) 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.
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 decreased sucrose preference (Fig. 1C) and coat deterioration (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 total duration of immobility in forced swimming test (FST) (Fig. 1F) and tail suspension test (TST) (Fig. 1G). 2-month social isolation had no effect on locomotor activity in aged mice (Fig. 1B). These results proved the fact that two-month social isolation induced depressive-like behaviors in aged mice.
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 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 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.
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-month of methionine diet significantly elevated 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 that similar to those caused by social isolation, with a reduced sucrose preference and coat score in the sucrose preference test (Fig. 3D, E), increased immobility time in 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).
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 to reduce Hcy. Plasma Hcy was measured to confirm the effect of the Hcy intervention (Fig. 4A). We found that VBco gavage significantly reversed 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 sucrose preference to the levels comparable to control mice, whereas a 4-week treatment with control diet was ineffective (Fig. 4D, E). Moreover, application of vitamin B complex decreased the duration of immobility in FST (Fig. 4G) and TST (Fig. 4H). Administration of VBco diet had no effect on locomotor activity (Fig. 4C) or body weight (Fig. 4F).
The hippocampal protein levels of BDNF, a gene closely related to depression [39–41], were measured at different time points of social isolation. We found that the level of BDNF 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 reduced the hippocampal BDNF levels that similar to social isolation (Fig. 5C). In contrast, VBco gavage significantly elevated BDNF levels, compared with the socially isolated mice (Fig. 5D). Taken together, the hippocampal BDNF may be negatively regulated by Hcy during social isolation in aged mice.
The major findings of the present study were as follows. First, two-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 [42]. 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. Rodents, like humans, are social creatures that thrive in group housing conditions. 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 two weeks of late-life isolation in aged rodents [43–44]. Here, we found that two-month late-life isolation induced depressive-like behaviors, enriching 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 [45], 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 [38]. Zhou, Cong et al. found that 6 weeks of chronic stress resulted in decreased autonomic activity and increased Hcy in rats; however, the administration of folic acid resulted in decreased Hcy and remission of symptoms, which the authors linked to decreased IL-6 release [46]. 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 [47]. 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 [48]. In addition, Hcy is an independent risk factor for Alzheimer’s disease [49], and administration of B-complex vitamins may alleviate cognitive symptoms in AD patients [50]. Moreover, Hcy level negatively correlates with scores on the Cambridge Cognition Scale in the elderly [51]. Taken together, Hcy may be an important mediator of stress-induced depressive-like behaviors.
Neurotrophic factors play an important role in the production and maintenance of depression in mammals [52]. 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 [53–54]. Our study in mice showed that both stress and high levels of Hcy caused a decrease in BDNF level in the hippocampus induced depressive-like behaviors. 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 [55], chronic social defeat stress [56], and chronic mild stress [57], 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 in rats [58], and knockdown of BDNF in mice inhibits neuronal plasticity and induces the onset of psychiatric symptoms such as anxiety in mice [59]. Moreover, some people found that interference with BDNF at the dentate gyrus triggers depressive-like behavior and affects neuronal regeneration in rats [58]. 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 [60] and that administration of methyl donors reverses the psychiatric symptoms caused by Hcy during stress [46]. 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 that 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.
Ethics Approval
Not applicable.
Author contributions
W. D. Wei designed the experiments; W. D. Wei, Y. X. Lan, K. Lu, Y. Wang and W. Y. Chen performed the experiments; W. D. Wei and K. Lu analyzed the data; W. D. Wei and K. Lu wrote the manuscript; and all authors commented on this manuscript.
Availability of Data and Materials
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Consent for Publication
This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All authors read and approved the final manuscript. The authors consent for publication in Molecular Neurobiology.
Funding
This study was supported by President Foundation of the Third Affiliated Hospital of Southern Medical University (YQ2021013).
Research Involving Human Participants and/or Animals
All procedures were performed in accordance with the guidelines in the National Institutes of Health Guide for Care and Use of Laboratory Animals, and the study design was approved by the appropriate ethics review board.
Competing Interests
The authors declare that they have no competing interests.
Code Availability
Not applicable.