At high latitudes, about 10% of the population suffers from depression in winter. Although it has become a serious public health issue, its underlying mechanism remains unknown. Interestingly, animals also show depression-like behavior in winter, and small teleosts have emerged as powerful models for the study of complex brain disorders. Here, we show that medaka exhibit decreased sociability and increased anxiety-like behavior under winter-like conditions. Using metabolomic and transcriptomic analyses, we found changes in multiple signaling pathways involved in depression, including the NRF2 antioxidant pathway. Chemical genomics and targeted mutation of the NRF2 gene revealed that seasonal changes in the NRF2 pathway regulate winter depression-like behavior. This study provides insights into the understanding and treatment of seasonally regulated affective disorders.

Seasonal changes in the environment lead to depression-like behaviors in humans and animals. The underlying mechanisms, however, are unknown. We observed decreased sociability and increased anxiety-like behavior in medaka fish exposed to winter-like conditions. Whole brain metabolomic analysis revealed seasonal changes in 68 metabolites, including neurotransmitters and antioxidants associated with depression. Transcriptome analysis identified 3,306 differentially expressed transcripts, including inflammatory markers, melanopsins, and circadian clock genes. Further analyses revealed seasonal changes in multiple signaling pathways implicated in depression, including the nuclear factor erythroid-derived 2-like 2 (NRF2) antioxidant pathway. A broad-spectrum chemical screen revealed that celastrol (a traditional Chinese medicine) uniquely reversed winter behavior. NRF2 is a celastrol target expressed in the habenula (HB), known to play a critical role in the pathophysiology of depression. Another NRF2 chemical activator phenocopied these effects, and an NRF2 mutant showed decreased sociability. Our study provides important insights into winter depression and offers potential therapeutic targets involving NRF2.

In temperate zones, organisms are exposed to dynamic annual changes in the environment, including day length, temperature, and rainfall. To better adapt to these seasonal changes, animals change their physiology and behavior, such as their ability to reproduce, hibernate, and molt (1). Several environmental and nutritional factors, however, influence seasonality. For example, goats are generally considered seasonal breeders and mate during the fall. This seasonality is obvious at high latitudes (>35°) but less marked at low latitudes. Indeed, when seasonally breeding goats are transferred from high latitudes to the tropics, they are no longer seasonal and start breeding all year round (2). In contrast, rats are believed to be nonseasonal animals. However, Fischer 344 rats exhibit clear seasonality when their nutrition is restricted (3). Thus, differences between seasonal and nonseasonal animals appears to be a measure of the strength of their seasonal responses. Since seasonal changes in physiology and behavior are fundamental to an organism, it is plausible that all living species have the potential for seasonal adaptation. Although less obvious, humans are sometimes considered seasonal as seasonal changes in birth rates and the occurrence of infectious, heart, and cerebrovascular diseases have been reported (4, 5).

Seasonal affective disorder (SAD) is a subtype of depression characterized by recurrent episodes that manifest annually, generally in winter (also known as winter depression) (6). Typical symptoms of SAD include low mood, lethargy, sleep problems, disrupted circadian rhythms, social withdrawal, decreased libido, and changes in appetite and body weight (5). Interestingly, seasonal changes in the environment can lead to similar depression- and anxiety-like behaviors in animals (7). We recently reported robust seasonal changes in the behavior of medaka fish (Oryzias latipes) (8). Under winter conditions, medaka are infertile, relatively immobile, exhibit lower light sensitivity, as well as changes in color perception. Notably, seasonal changes in color perception have also been reported in humans (9). Furthermore, patients with SAD show lower light sensitivity in winter compared to healthy subjects as measured by the electroretinogram, and this decreased retinal sensitivity, along with depressed mood, are normalized in summer or following bright light therapy (10). Most behavioral abnormalities are not caused by single gene mutations and are likely the result of dysregulation in multiple brain structures and neural pathways. Therefore, both forward and reverse genetic approaches are quite difficult. We aim to overcome these challenges and address the underlying mechanisms of seasonally regulated behaviors using more global approaches, including metabolomic and transcriptomic analyses of the whole brain of medaka fish. Furthermore, we used a chemical genomics approach in vivo. Understanding the molecular basis of these behaviors has important implications for both the basic pathology of SAD and the possible therapeutic intervention for depressive behaviors.

To further examine the role of NRF2 in winter depression-like behavior, we generated a genome-edited NRF2 mutant using the CRISPR/Cas9 system. We crossed an injected G0 founder with a wild-type fish to obtain F1 heterozygotes, and then F1 heterozygotes were crossed to generate F2 mutant and wild-type siblings. NRF2 mutant medaka had 4-bp deletion in exon 2 of the NRF2 gene, and this deletion caused a frame shift, introducing a premature stop codon upstream of the DNA binding and dimerization domains ( SI Appendix, Fig. S8 ). We did not observe any developmental, morphological, or behavioral abnormalities in the mutant fish under normal housing conditions. We then examined sociability under summer-like conditions using the three-chamber sociability test. Mutant medaka were less social and spent less time in the preferential area ( Fig. 5E ). Although we attempted to examine the effect of celastrol on mutant fish under winter-like conditions, many NRF2 mutant fish died, suggesting that they are highly sensitive to environmental changes.

Drug screening treating winter behavior. (A) Of 112 existing drugs, 21 potential hits (blue and red dots) were detected in the three-chamber sociability test (first screen, P < 0.05, and n = 8). Celastrol (red dot) was the only drug that passed the first, second, and third screenings. The y axis shows the log2 ratio between the time spent in the preferential area and the avoidant area. (B) Celastrol-treated medaka spent more time in the preferential area, while dimethyl sulfoxide (DMSO)-treated medaka did not under winter-like conditions (two-way ANOVA and Sidak’s multiple comparison test. **P < 0.01, N.S. is not significant, mean ± SEM, and n = 8–15). (C) Structurally distinct NRF2 activator, DMF, rescued winter behavior by increasing sociability (two-way ANOVA and Sidak’s multiple comparison test. **P < 0.01, N.S. is not significant, mean ± SEM, and n = 8–14). (D) Expression of NRF2 in the HB examined by in situ hybridization. The black arrow indicates the hybridization signal. (Scale bar, 50 μm.) HB: habenula; E: epiphysis; DM: nucleus dorsomedialis thalami, VM: nucleus ventromedialis thalami; tro: tractus opticus. (E) Genome-edited NRF2 mutant spent less time in the preferential area and more time in the avoidant area under summer-like conditions (*P < 0.05, mean ± SEM, and n = 26 to 27).

In addition to circadian clock genes, we discovered up-regulation of several inflammatory markers, including cytokines (e.g., fish specific interleukin-6 [IL6: M17], IL10, tumor necrosis factor ligand superfamily member 13B (also known as B-cell activating factor [BAFF]), and a cytokine receptor (interleukin-1 receptor type 2 [IL1R2]) under SC conditions ( Fig. 3 F and G ). The inflammatory response and oxidative stimuli have been shown to change spine density in several brain regions, including the hippocampus ("neuroplasticity hypothesis") ( 15 , 16 ). We, therefore, analyzed spine morphology in the medaka brain using DiOlistic labeling with the lipophilic dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) ( SI Appendix, SI Text ). We examined the pars lateralis of the dorsal telencephalic area (Dl), which is considered analogous to the mammalian hippocampus ( 19 ). Although the percentage of each spine type (i.e., mushroom, thin, and stubby) did not show statistical differences, an overall decrease in spine density in the medaka brain was observed in the Dl region in SC compared with LW conditions ( Fig. 3 H and I ).

Disruption of circadian rhythms and elevation of inflammatory markers in winter-like conditions. (A) Two sets of microarray experiments identified 5,309 differential probes between SC and LW conditions. Further analysis using a fold-change (FC) cutoff of 1.5 identified 3,601 probes representing 3,306 transcripts. (B) Clustered organization of 3,601 probes between SC and LW conditions. Data were normalized over the entire data set. The color scale at the Left represents the normalized signal intensity. (C) Temporal expression profiles of circadian clock genes under SC (blue) and LW (red) conditions verified by qPCR. The bars at the bottom of each graph represent the lighting conditions (two-way ANOVA, P < 0.05, mean ± SEM, and n = 4 to 5). Note that for PER2 and CRY2, the y axis values are different for SC and LW conditions. (D and E) Seasonal changes in OPN4B and OPN4XB expressions validated by qPCR (D) and in situ hybridization (E). The black arrow indicates the hybridization signal. (Scale bar, 40 μm) (two-way ANOVA, P < 0.05, mean ± SEM, and n = 4 to 5). BO: bulbus olfactorius; Dl: pars lateralis of the dorsal telencephalic area; Dm: pars medialis of the dorsal telencephalic area; E: epiphysis; PO: nucleus preopticus; PS: nucleus pretectalis superficialis; SCN: suprachiasmatic nucleus; tro: tractus opticus; VM: nucleus ventromedialis thalami. (F and G) Seasonal changes in the cytokines and cytokine receptor measured by microarray (F) and qPCR (G) (Welch’s t test, *P < 0.05, **P < 0.01, mean ± SEM, and n = 5). (H) Representative images of DiI-labeled dendritic spines within the DI. (Scale bar, 5 μm.) (I) Spine morphology and density within the DI, analogous to the mammalian hippocampus, in SC and LW conditions (Welch’s t test, **P < 0.01, N.S. is not significant, mean ± SEM, and n = 34 and 37 dendritic segments analyzed for SC and LW conditions, respectively).

We next performed microarray analysis to examine the seasonal transcriptional landscape of the medaka brain. To minimize the number of false positives, we performed two sets of experiments: SC to LW and LW to SC. In the SC-to-LW experiment, adult male medaka maintained in SC conditions either remained in SC or were transferred into LW conditions. By contrast, in the LW-to-SC experiment, adult male medaka kept under LW conditions either remained in LW or were transferred into SC conditions. In both experiments, 2 wk after transfer, whole brains were collected in the middle of the light phase. Microarray analysis identified 5,309 probes in common between the two experiments (Moderated t test, P < 0.05, false discovery rate [FDR] < 0.05, SC to LW: n = 6; LW to SC: n = 5). Of these, 3,601 probes were identified by a 1.5-fold cutoff that represents 3,306 transcripts ( Fig. 3 A and B and SI Appendix, Table S3 ). Disrupted circadian rhythms, such as abnormal phase and diminished amplitude, are often observed in SAD ("circadian rhythm hypothesis") ( 17 , 18 ). The microarray analyses identified several differentially expressed genes, including those encoding melanopsin photopigments OPN4B and OPN4XB and circadian clock genes PER2, PER3, and CRY2 ( SI Appendix, Table S3 ). These results were confirmed by qPCR (two-way ANOVA, P < 0.05) and/or in situ hybridization ( Fig. 3 C–E ) ( SI Appendix, SI Text ). In addition to the clock genes above, we performed qPCR analysis on CLOCKB, BMAL1, and NPAS2 and discovered that they were also differentially expressed (two-way ANOVA and P < 0.05) ( Fig. 3C ). Interestingly, expression of these circadian clock genes was no longer rhythmic, or their amplitude was significantly reduced in winter- compared to summer-like conditions ( Fig. 3C and SI Appendix, Table S4 ).

Medaka are less social and show more anxiety-like behaviors under winter-like conditions. (A) Schematic of the novel tank test (Left). The time spent in bottom area was shorter in medaka kept under LW conditions than those kept under SC conditions (Welch’s t test, **P < 0.01, mean ± SEM, and n = 18–20) (Right). (B) Schematic of the tank for the three-chamber sociability test (Left). Medaka kept under LW conditions spent more time in the preferential area, whereas medaka kept under SC conditions failed to show any interest in other fish (two-way ANOVA and Sidak’s multiple comparison test. **P < 0.01, N.S. is not significant, mean ± SEM, and n = 15) (Right). (C) Schematic of the mirror biting test (Left). The numbers of crossings into the mirror contact zone (outlined) were significantly decreased under SC than LW conditions. (Welch’s t test, **P < 0.01, mean ± SEM, and n = 17–20) (Right). (D) Schematic of the tank for the light–dark test (Left). Medaka in LW conditions spent more time in the light area than medaka kept under SC conditions. Also, medaka kept under LW conditions showed significantly more transitions between light and dark areas. (Welch’s t test, **P < 0.01, mean ± SEM, and n = 15) (Right).

Medaka show striking differences in behavior depending on the season. Fish maintained in summer-like conditions are fertile, active, and swim throughout the tank, while those kept under winter-like conditions are infertile, relatively immobile, and remain at the bottom of the tank ( 8 ). For the emergence of these summer or winter phenotypes, both photoperiod and temperature are important in medaka fish. In the present study, we used adult male medaka maintained in conditions mimicking winter (short day and cool temperature [SC]: 10-h light/14-h dark; 10 °C) and divided them into two groups: one remained in SC, while the other group was transferred into summer-like conditions (long day and warm temperature [LW]: 14-h light/10-h dark; 26 °C). Two weeks after the transfer, behavioral tests were performed. We first examined behavior using a novel tank test. The time spent in the bottom area of the tank was significantly shorter in medaka kept under LW than those in SC conditions ( Fig. 1A ). Since medaka are highly social animals, we next performed a three-chamber sociability test ( Fig. 1B ). Medaka in LW conditions spent more time in the "preferential area" exhibiting interest in other individuals, whereas medaka kept under SC conditions swam randomly and failed to show a preference for other fish ( Fig. 1B ). Decreased sociability under SC conditions was confirmed in a mirror biting test. The number of approaches to the mirror was significantly decreased in SC compared with LW conditions ( Fig. 1C ). We further examined anxiety-like behavior using a light–dark tank test ( Fig. 1D ). Medaka kept under LW spent more time in the "light area" compared to medaka in SC conditions ( Fig. 1D ). The frequency of entering the light area was also significantly higher in medaka maintained in LW conditions ( Fig. 1D ). Similar results were observed in female medaka, demonstrating that these behaviors were not sex specific ( SI Appendix, Fig. S1 ). These results indicate that, regardless of sex, medaka are less social and show more anxiety-like behaviors in winter- compared to summer-like conditions.

Discussion

Seasonal changes in the environment may lead to depression- and anxiety-like behaviors in both humans and animals. However, the underlying mechanisms remain unknown. In the present study, we demonstrated that short days and cool temperatures mimicking winter conditions decreased sociability and increased anxiety-like behavior in medaka (Figs. 1 and 6A).

Fig. 6. Conservation of the HB pathway and possible mechanisms that cause winter behavior. (A) Hypothetical model of the underlying mechanisms regulating seasonal changes in behaviors. (B) Conservation of HB pathways in mammals and fish. Schematic of sagittal sections of rodent and fish brains. Red shows medial (mammal) and dorsal (fish) HB circuitries, while blue shows lateral (mammal) and ventral (fish) HB circuitries. Green shows feedback projections to the hippocampus and medial prefrontal cortex. Although the entopeduncular nucleus and ventral telencephalon send projections to fish HB, exact targets remain unclear (purple). mPFC: medial prefrontal cortex; VTA: ventral tegmental area; SNc: substantia nigra, pars compacta; RMTg: rostromedial tegmental nucleus; Dl: pars lateralis of the dorsal telencephalic area. Modified from Aizawa et al. (26) and Mizumori and Baker (28).

Bright light therapy is an effective treatment for patients with SAD, whereas decreased light exposure is often a risk factor (10). Melanopsin is involved in circadian photoentrainment, and its gene variants have been linked to SAD (29). Interestingly, we found differential expression of the melanopsin homologs OPN4B and OPN4XB (Fig. 3) between medaka kept under winter- and summer-like conditions. Candidate gene studies for SAD also reported SNPs in circadian clock genes, including PER2, PER3, BMAL1, CLOCK, NPAS2, and CRY2 (30⇓⇓⇓–34). We demonstrated a significant decrease in the amplitude of circadian clock gene rhythms in medaka kept under winter-like conditions compared to those in summer-like conditions (Figs. 3 and 6A). These results are consistent with the hypothesis that disruption of circadian rhythms is associated with SAD (18).

Growing evidence points to a role for the immune system in the pathophysiology of depression, specifically, the inflammatory response and oxidative stress (14⇓–16). In human patients and animal models, depression is accompanied by a decrease in antioxidants (e.g., GSH) and amino acids (e.g., tryptophan and tyrosine) and an increase in inflammatory markers in the brain (14⇓–16). We discovered lower levels of GSH, tryptophan, and tyrosine in the brain of medaka kept under winter-like conditions (Fig. 2). We also demonstrated higher expression of inflammation markers, including cytokines (IL6, IL10, and BAFF) and the cytokine receptor (IL1R2) in the brain under winter-like conditions (Figs. 3 and 6A). It has been reported that the inflammatory cytokine IL-6 induces IL-10 production (35) and hence, BAFF production in the microglia (36). Several lines of evidence have indicated that inflammatory cytokines influence the monoamine serotonin as well as the excitatory amino acid glutamate (15). We also detected seasonal changes in serotonin and glutamate (Fig. 2). Although low serotonin levels are considered to be associated with depression, higher serotonin levels were observed in the medaka brain in winter-like compared to summer-like conditions. The ratio between serotonin metabolite 5-hydorxyindoleacetic acid (5-HIAA) and serotonin are typically used as a measure of synaptic transmission, but 5-HIAA was not detected in our analysis. In this case, serotonin turnover may be decreased, which suggests dysfunction of serotonergic neuronal activity. Due to its rapid antidepressant actions, the glutamatergic NMDA receptor antagonist ketamine has attracted attention in the mental health field (37). On the contrary, increased levels of glutamate and reduced taurine have been reported in the brains of patients with depression (38, 39). Consistent with this, we observed increased glutamate and reduced taurine levels in the brain of medaka kept under winter-like conditions (Fig. 2). In addition to the impact of cytokines on neurotransmitter systems, inflammation influences synaptic plasticity (16). Indeed, decreased spine density associated with depression was also observed in the medaka brain in the Dl region, which is considered to be homologous to the mammalian hippocampus (Figs. 3 and 6B).

Canonical pathway analysis identified winter-induced inactivation of multiple biological networks known to be associated with depression (Fig. 4A). Consistent with the antidepressive effects of endogenous estrogen in both men and women (40), transcriptome analysis revealed inactivation of estrogen receptor signaling under winter-like conditions. We also observed inactivation of activin signaling under winter-like conditions. An enriched environment activates the activin pathway and enhances cognitive performance and affective behaviors, while impairment of this pathway results in anxiety and depression (22). We also detected inactivation of the retinoic acid receptor (RAR) and glucocorticoid receptor signaling under winter-like conditions. These results are consistent with the notion that RAR signaling is involved in the modulation of the hypothalamic–pituitary–adrenal (HPA) axis (41). Dysregulation in the HPA axis has been demonstrated in a wide spectrum of psychiatric disorders (21). Although hyperactivity of the HPA axis is generally associated with depression, its hypoactivity may result in fatigue and tiredness (42). It is interesting that hyperactivity of the HPA axis is rarely observed in patients with SAD, unlike many other psychiatric conditions (43).

NRF2 is a transcriptional activator of antioxidant genes involved in anti-inflammatory processes in the brain. Reduced levels of GSH are associated with depression, and NRF2 induces GSH (23). Of note, we found inactivation of NRF2 signaling, reduction of GSH, and induction of inflammatory markers in medaka kept under winter-like conditions (Figs. 2–4). Orphan nuclear receptor NR4A1 (Nur77) is involved in the regulation of the inflammatory response (44). Therefore, inactivation of the Nur77 signaling pathway under winter-like conditions might be modulating the inflammatory response.

To further understand the underlying mechanism of winter depression-like behavior, we used a chemical genomics approach and discovered that the traditional Chinese medicine celastrol rescues winter behavior in medaka (Fig. 5). Celastrol is currently used for treatment of inflammatory diseases and cancer and has multiple molecular targets, including nuclear factor κB and heat shock protein 90 (45). Furthermore, increasing evidence suggests that celastrol exerts its anti-inflammatory effects by activating the NRF2 antioxidant pathway (24, 25). Importantly, the structurally distinct NRF2 activator, DMF, also rescued winter behavior, and a CRISPR/Cas9-generated NRF2 mutant showed decreased sociability under summer-like conditions (Fig. 5). Similarly, it has been reported that Nrf2-null mice show depressive-like behaviors, and that, in mouse models of depression, treatment with a NRF2 agonist shows antidepressive effects (46). Thus, NRF2 activators have been proposed as potential drug candidates for depression (23). It is particularly interesting to note that NRF2 expression is observed in the HB (Fig. 5). Although fish do not have a defined prefrontal cortex (PFC), the HB is evolutionarily highly conserved and connects the limbic forebrain and monoaminergic system (Fig. 6B). Recently, the LHb has emerged as a key brain region in the pathophysiology of depression and has been shown to mediate the antidepressive actions of ketamine in rat and mouse models of depression (37). Furthermore, deep brain stimulation has been used to regulate neural activity of the LHb in treatment-resistant depressed patients (47). It should be noted that recent studies in mice also demonstrated that the periHB nucleus, adjacent to the LHb, mediates the light-mediated mood alteration (48).

Treatment of multifactorial psychiatric disorders with just one drug, or monotherapy, is often insufficient, and comedication therapy is typically more effective. Therefore, there is an urgent need for new therapeutic targets and more effective therapies (49). Animal models play an essential role in the discovery of new drugs in virtually all fields of medicine. Small teleosts, such as zebrafish and medaka, are emerging models for the study of complex brain disorders and are becoming popular and more powerful models in pharmacogenetic studies. Despite the obvious differences between fish and humans, many features of their central nervous system are highly conserved, including neurotransmitters, their receptors and transporters, enzymes for their synthesis and degradation, as well as the neurons that underlie their function. Moreover, antipsychotics affect swimming patterns via conserved molecular targets and pathways that affect activity patterns in mammals (50, 51). Importantly, animal models of psychiatric disorders are required to satisfy three important criteria: i) face, ii) construct, and iii) predictive validities (52). i) Face validity refers to how similar the phenotypes are between an animal model and the human disorder. Although there are obvious behavioral differences between fish and humans, our previous and current studies of medaka kept under winter-like conditions demonstrated significant parallels with SAD symptoms, such as decreased locomotor and reproductive activity, lower light sensitivity, decreased sociability, and increased anxiety-like behavior (8). ii) Construct validity refers to how a mechanism is shared between an animal model and the human disorder. Our metabolomic, transcriptomic, and morphological analyses demonstrated seasonal changes at multiple levels, including metabolites (e.g., neurotransmitters, antioxidant, and amino acids), gene expression (e.g., circadian clock genes, cytokines, and cytokine receptor), and neuroplasticity (e.g., spine density). These phenotypes fit well with proposed hypotheses for depression including the circadian rhythm hypothesis, the inflammation hypothesis, and the neuroplasticity hypothesis (Fig. 6A). iii) Predictive validity refers to how medications similarly effect the behavior of an animal model and the disorder. For example, the antidepressant fluoxetine, a selective serotonin reuptake inhibitor, has been suggested to improve SAD. When we examined the effect of a 2-wk fluoxetine treatment of medaka kept under winter-like conditions, medaka moved to the top of the tank in the open field tank test, reflecting reduced anxiety-like behavior (SI Appendix, Fig. S9A). However, this treatment had no influence on behavior in the three-chamber sociability test (SI Appendix, Fig. S9B). This partial effect of fluoxetine might explain the view that there is not enough evidence to support the use of fluoxetine for the treatment of SAD (53). As mentioned above, SAD symptoms typically appear during fall to winter and disappear during spring to summer. A seasonal change in the environment from winter to spring is the ultimate treatment for SAD, and this appears to be true in medaka fish. Depression is considered an adaptation to a harsh environment, and to our knowledge, none of the currently available animal models for depression exhibit spontaneous recurrent mood episodes. Given the striking parallels between patients with SAD and medaka kept under winter-like conditions, we believe that our present findings provide insights into the understanding and treatment of seasonally regulated affective disorders. However, there exist clear differences between fish and mammals. For example, eyes are the only photoreceptive organ to reset the circadian clock in mammals, while light acts directly on peripheral clocks in fish (54). Since SAD-like symptoms have recently been reported in the diurnal primate, Rhesus macaque (55), future comparative studies on other organisms, such as macaques, are, indeed, important to better understand the evolution of vertebrate seasonal adaptation and seasonal changes in physiology and mood in humans.