Effect of agomelatine on memory deficits and hippocampal gene expression induced by chronic social defeat stress in mice

Chronic stress is known to induce not only anxiety and depressive-like phenotypes in mice but also cognitive impairments, for which the action of classical antidepressant compounds remains unsatisfactory. In this context, we investigated the effects of chronic social defeat stress (CSDS) on anxiety-, social- and cognitive-related behaviors, as well as hippocampal Bdnf, synaptic plasticity markers (PSD-95, Synaptophysin, Spinophilin, Synapsin I and MAP-2), and epigenetic modifying enzymes (MYST2, HDAC2, HDAC6, MLL3, KDM5B, DNMT3B, GADD45B) gene expression in C57BL/6J mice. CSDS for 10 days provoked long-lasting anxious-like phenotype in the open field and episodic memory deficits in the novel object recognition test. While total Bdnf mRNA level was unchanged, Bdnf exon IV, MAP-2, HDAC2, HDAC6 and MLL3 gene expression was significantly decreased in the CSDS mouse hippocampus. In CSDS mice treated 3 weeks with 50 mg/kg/d agomelatine, an antidepressant with melatonergic receptor agonist and 5-HT2C receptor antagonist properties, the anxious-like phenotype was not reversed, but the treatment successfully prevented the cognitive impairments and hippocampal gene expression modifications. Altogether, these data evidenced that, in mice, agomelatine was effective in alleviating stress-induced altered cognitive functions, possibly through a mechanism involving BDNF signaling, synaptic plasticity and epigenetic remodeling.

Chronic social defeat stress (CSDS). Chronic social defeat stress was performed as previously described 9,10 . Male CD-1 retired breeder mice were screened for aggressive behavioral response measured by the latency to initial aggression that should be less than 60 s in at least two consecutive 180 s-screening sessions 31 . 24 h before the first defeat, mice were singly housed in one side of a standard cage separated by a clear perforated Plexiglas divider, which allowed sensory but not physical contact. An experimental C57BL/6J intruder mouse was exposed to a CD-1 aggressor for 10 min, during which the intruder mouse was attacked and displayed subordinate posturing. To avoid physical injury, mice were briefly isolated in case of over-aggressive behavior from the CD-1. After a 10-min defeat session, the experimental mouse was placed for the rest of the day on the opposite side of the divider. This procedure was repeated for 10 consecutive days (D1-D10), using a different aggressor CD-1 mouse every day, between 10:00 and 11:00 am. Control mice were housed by pair in a similar cage, separated by a clear Plexiglas divider, and never exposed to CD-1 mice. Immediately after the last defeat session, stressed and control mice were singly housed in a new standard cage.
In order to assess the effect of chronic agomelatine treatment on stress-induced behavioral deficits, susceptible mice were divided into two homogenous groups: "stressed-AGO" that included stressed mice treated for 3 weeks with agomelatine (50 mg/kg/day, i.p.) and "stressed-HEC" that included stressed mice treated with vehicle (HEC) in the same conditions. Control mice treated for 3 weeks with HEC were called "control-HEC". Mice were weighted once a week in order to monitor body weight gain. Neither stress nor treatments significantly modified body weight (See Table S1), although there is a trend for a higher increase in weight after social stress compared to controls which actually fits with previous data on the leptin resistance-induced by social stress 32 . Behavioral testing. On D11, defeated and control animals were tested in the open field and social avoidance tests, in order to select the susceptible, which displayed anxiety-and social avoidance phenotypes, and the resilient mice 10 , and agomelatine or HEC chronic treatments were initiated at the same day. Similar behavioral tests were replicated on D29 to assess the effect of treatments. In addition, on D30 and D31, a novel object recognition test was carried out to evaluate the effect of stress and treatments on episodic memory (see Fig. 1). 24  Social avoidance test. Social avoidance was assessed three hours after the open field test, as previously described 9,10 . The test consisted in two consecutive 150 s-sessions, under low light conditions (5 lux). Mice were subjected to the test in the same open field boxes they were previously habituated during the open field test. In the first session, experimental mice were allowed to explore the open field containing an empty circular wire cage (18 × 9 cm) located at one end of the field. In the second session, conditions were identical except that the circular cage contained a CD-1 aggressive mouse (defined as "target"). A virtual interaction zone (area projecting 8 cm around wire cage) was delimited, and the time spent in this zone was scored during both sessions using a video tracking system (Viewpoint). Social interaction behavior was estimated as interaction ratio: (time spent in the SCientifiC RepoRts | 7:45907 | DOI: 10.1038/srep45907 interaction zone in the presence of target/time spent in the interaction zone in the absence of target) * 100. Mice were classified either as susceptible with ratio below 100 or as resilient with ratio above 100 on a social avoidance test performed at D11, as described in the literature 10 . Within the CSDS-subjected group, only susceptible mice were included for the subsequent experiments. No such sorting was performed within the control group.

Figure 1. Study design.
Mice were submitted to CSDS for 10 days and then treated 21 days with either agomelatine or its vehicle HEC i.p. Anxiety-and depressive-like phenotypes were assessed at day 11 and day 29 using the social avoidance and open field test, and memory was tested using the novel object recognition test at days 30   , mice subjected to CSDS can be split into two groups: the susceptible mice, which display a significant reduction in the interaction ratio compared to control mice, and the resilient mice, which do not behave differently from non-stressed group and display a significant higher interaction ratio than the susceptible mice. (B) On Day 29, no significant difference was found between control-, stressed mice treated with HEC and stressed mice treated with agomelatine. Each bar is the mean ± S.E.M. of n = 19 (control mice), 11 (stressed HEC mice) and 11 (stressed AGO mice). AGO, agomelatine; HEC, hydroxyethylcellulose. During the acquisition phase, two identical objects were placed in the arena, on the right and left corners, at 7 cm-distance from the walls. Mice were introduced in the arena and left free to explore for 5 min, and returned to their home cage. After a 1h-intertrial interval (ITI1), one of the objects was replaced by a different one, and the mice were reintroduced in the arena for another 5 min-session (recognition phase 1, D30). The procedure was replicated 24 h (ITI24) after the first recognition phase, with a different new object (recognition phase 2, D31). In both testing phases, the time spent by the mice exploring the objects (touching with their nose or forelimbs, or sniffing at a distance less than 1 cm) was manually scored from recorded video using XNote StopWatch software. The recognition index was calculated as follow: (time spent exploring the novel object -time spent exploring the familiar object)/(time exploring both novel and identical objects). Mice that explored both objects less than 5 s during recognition phase 1 or 2 were excluded. Statistical analysis. Data (displayed as mean ± S.E.M.) were analyzed using Prism 5 Software (GraphPad, San Diego, USA). To compare "control-HEC", "stressed-HEC" and "stressed-AGO" groups, a one-way analysis of variance (ANOVA) was used, followed by Newman Keuls multiple comparisons test. When variances were significantly different, a Kruskal-Wallis test was used, followed by a Dunn's multiple comparisons tests. For comparing "control" and "susceptible" groups, an unpaired Student's t-test was used, with Welch's correction if needed.

Results
Effect of CSDS and chronic agomelatine treatment on depression-related behavior in the social avoidance test. The interaction ratio was used to discriminate susceptible and resilient mice to chronic stress among a batch of mice all subjected to CSDS. As shown in Fig. 2A and S1, social avoidance test led to the segregation of susceptible mice (n = 24), and resilient mice (n = 17). Social avoidance test was then replicated on D29, after a 3-week treatment with either agomelatine or HEC in CSDS-exposed mice, or HEC in control mice ( Fig. 2B and S1). Although a Kruskal-Wallis test failed to reveal any overall difference between the three groups of mice (p = 0.16), it can be noted that stress still tended to reduce interaction ratio, but there was no effect of the agomelatine treatment. The open field test was repeated on D29, to evaluate the effect of treatments on stress-induced deficits (Fig. 3B). After a 3 week chronic vehicle treatment, one way ANOVA followed by Newman-Keuls multiple comparisons test showed that stressed-HEC mice still showed reduced locomotion [F(2,41) = 5.737, p < 0.001] and entry number in the center of the open field (F(2,41) = 4.461, p < 0.01). Although there was a slightly significant overall difference in the mean time spent at the center between the three groups (p = 0.0479), a Newman Keuls multiple comparisons test failed to reach statistical significance. Chronic agomelatine treatment was not able to prevent stress-induced hypolocomotion and decreased exploration in the center of the open field [F(2,41) = 5.529, p < 0.001; F(2,41) = 3.966, p < 0.01, respectively].

Effect of CSDS and chronic agomelatine treatment on anxiety-related behavior in
Effect of CSDS and chronic agomelatine treatment on episodic memory in the novel object recognition test. Novel object recognition test performed on D30 and D31 was used to determine the effect of CSDS and that of chronic agomelatine on mouse episodic memory. ITI 1 h and 24 h allowed evaluating respectively the short-and long-term memory. In the recognition phase 1 with ITI1, a Kruskal-Wallis test revealed no overall difference between the different groups (p = 0.3377) (Fig. 4A). However, in the recognition phase 2 with ITI24, a one-way ANOVA followed by Newman-Keuls multiple comparisons test showed that stressed-HEC displayed a significantly decreased in the recognition index compared to control-HEC mice [F(2,30) = 3.209, p < 0.05] (Fig. 4B). In contrast, a one-way ANOVA showed that stressed-AGO mice did not significantly differ from control-HEC [F(2,30) = 0.8906, p > 0.05] and that the recognition ratio was significantly increased in comparison to that of stressed-HEC mice [F(2,30) = 3.679, p < 0.05].  Table 2). In contrast, a one-way ANOVA followed by Newman-Keuls multiple comparisons test showed that MAP-2 hippocampal mRNA level was decreased by CSDS in HEC-treated mice [F(2,37) = 3.538, p < 0.05], and that this effect was prevented by chronic agomelatine administration [F(2,37) = 3.555, p < 0.05].
Effect of CSDS and chronic agomelatine treatment on epigenetic modifying enzymes mRNA expression. A one-way ANOVA showed that gene expression of histone acetyltransferase MYST2 was not modified in any of the groups [F(2,39) = 0.3391, p = 0.7145] ( Table 3). In contrast, CSDS decreased the expression of histone deacetylases HDAC2 [F(2,37) = 4.534, p < 0.05, one-way ANOVA followed by Newman-Keuls multiple comparisons test] and HDAC6 mRNA (p < 0.05, Kruskal-Wallis test followed by Dunn's multiple comparisons test). Interestingly, this effect was prevented by chronic agomelatine treatment [HDAC2: F(2,37) = 3.249, p < 0.05 and HDAC6: p < 0.05, respectively]. At histone methylation level, a one-way ANOVA followed by Newman-Keuls multiple comparisons test indicated that CSDS reduced the expression of the histone methyltransferase The effects of CSDS and agomelatine treatment on hippocampal Bdnf gene expression were determined after 3 weeks of agomelatine (50 mg/kg/day, i.p.) treatment following 10 days of CSDS. CSDS and chronic agomelatine treatment did not induce any change in total Bdnf and Bdnf exon VI mRNA expression. However, Bdnf exon IV gene expression was significantly reduced in stressed-HEC mice, but the level of Bdnf exon IV mRNA was not different between control-HEC and stressed-AGO mice. *p < 0.05, one-way ANOVA followed by Newman-Keuls multiple comparisons test. Each result is expressed as the mean ± S.E.M. of n = 21 (control mice), 12 (stressed HEC mice) and 12 (stressed AGO mice). AGO, agomelatine; HEC, hydroxyethylcellulose.    (Table 3).

Discussion
The effects of chronic social defeat stress and chronic agomelatine treatment on mouse behavior and plasticity gene expression reported here showed that agomelatine failed to reverse stress-induced alterations on anxietyand social-like behaviors, but was able to prevent CSDS deleterious effects on cognitive performances and hippocampal gene expression. Krishnan et al. 10 initially demonstrated that a population of C57BL/6J subjected to the CSDS paradigm could be separated into susceptible mice, with behavioral and molecular alterations, and resilient mice that were less prone to develop such deficits. Those two groups could be separated using social avoidance test, based on the natural propensity of mice to interact with unfamiliar mouse 34 . Here, the social avoidance test, performed 24 h after the end of the last defeat session, revealed that 59% of total mice subjected to the stress protocol were susceptible and 41% were resilient, as expected from data in the literature 10 . We assessed the effect of chronic agomelatine treatment on stress-induced behavior and molecular modifications on susceptible mice only. To this aim, social avoidance test was replicated on D29, after a 3-week treatment with either agomelatine or its vehicle in CSDS-exposed mice, or vehicle in control mice. However, after this 3-week treatment delay, no statistical difference in the social avoidance test could be found between the 3 groups of mice. The loss of the stress effect, which was clearly present on D11, could result from a marked decrease in the interaction ratio mean of control group on D29 (−42%). According to previous work 9 , mice habituation or disinterest to arena exploration is unlikely to explain this behavior. Animal isolation 11 and daily repeated vehicle injections 35 which are known to be meaningful stressors may have contributed to this confounding effect. However, the fact that the interaction ratio of CSDS-exposed mice treated with agomelatine did not differ from that of vehicle-treated mice suggest that this antidepressant was ineffective to reverse a depression-related behavior in the present social avoidance test. Those results did not meet previous results since agomelatine after chronic administration at the same dose ranges has been shown to display significant efficacy in various preclinical depression-like models related to stress including the transgenic GR-i mouse 36,37 , the corticosterone-treated mouse 38 , the chronic mild stress in rats 39 and the prenatally stressed rat 29,40 , but for most of them, the stress procedure was different and based on much milder stressors than those applied here. As expected from CSDS experiments, susceptible mice displayed a strong decreased in the exploration of the center of an open field on D11, indicating a clear-cut anxious-like phenotype 41,42 . On D29, the effect of social defeat stress on anxiety-related behavior was still present in stressed-HEC mice, but again, agomelatine was not able to reverse this behavior. These data are not in line with previous results since agomelatine at antidepressant doses was shown to possess anxiolytic properties in naïve rats 43,44 and also in various rodent depression-like models 37,38,40 . In particular, in the open field, a chronic agomelatine treatment was shown to reverse the anxiety-like phenotype induced by chronic corticosterone consumption in drinking water 38 . It was also demonstrated that agomelatine administered either acutely or sub-chronically was able to reduce anxiety-like behaviors of rats after a social defeat, an effect that needs the integrity of the suprachiasmathic nucleus 45 . Again, and as already suggested in the social avoidance test, CSDS is known to be very stressful 3,46 and to induce stronger anxiety-related deficits than other types of stress such as chronic mild stress 11 . Agomelatine, which positive effects  Table 3. Long-lasting effect of chronic social defeat stress and chronic agomelatine on histone acetylation/ methylation and DNA methylation modifying enzymes mRNA expression in the hippocampus. The effects of CSDS and agomelatine treatment on hippocampal gene expression of enzymes known to modify histone acetylation and methylation as well as DNA methylation were tested after 3 weeks of agomelatine (50 mg/kg/ day, i.p.) treatment following 10 days of CSDS. No significant differences were found between the three groups for MYST2, KDM5B, DNMT3B and GADD45B mRNA expression. However, concerning HDAC2, HDAC6 and MLL3, stressed mice chronically treated with the vehicle showed reduced gene expression, and this effect was reversed by chronic agomelatine treatment. *p < 0.05 "Control HEC" vs "Stressed HEC", # p < 0.05 "Stressed HEC" vs "Stressed AGO", one-way ANOVA followed by Newman-Keuls multiple comparisons test (HDAC2 and MLL3) or Kruskal-Wallis test followed by Dunn's multiple comparisons test (HDAC6). Each result is expressed as the mean ± S.E.M. of n = 19 (control mice), 10 (stressed HEC mice) and 10 (stressed AGO mice). AGO, agomelatine; HEC, hydroxyethylcellulose.
SCientifiC RepoRts | 7:45907 | DOI: 10.1038/srep45907 have been observed in mild stress paradigms, was not able to reverse the behavioral deficits induced by CSDS, in contrast to antidepressants of different families such as imipramine or fluoxetine 9,47,48 . However, and although it had no significant efficacy on anxiety-and depressive-like behaviors in the present study, agomelatine showed clear cut effects on CSDS-induced memory impairments measured with the NOR test, a well-validated paradigm for evaluating episodic memory in rodents 49 . When testing the mice on D30, no deficit in the short-term memory of CSDS-subjected animals was observed in ITI1 conditions, but the memory performances of stressed-HEC mice were significantly altered in the ITI24 ones. These data corroborated previous studies that also described impaired cognition in social-defeated mice using the NOR test 16,41 with mnesic alterations in that case recorded at both short and long term, but measured 5 days only after the last defeat session. Interestingly, we showed that long term memory was still affected 3 weeks after CSDS. In addition, chronic agomelatine treatment reversed CSDS-induced cognitive dysfunction in this behavior, as previously demonstrated in various memory-like models. Indeed, agomelatine was shown to produce memory facilitating effects in the NOR task in naïve rats 25,26 or to improve mnesic alteration in mice exposed to unpredictable mild stress 27 . Agomelatine was also effective in enhancing animal performances in other cognition tests such as the radial-arm water maze 28 and the Morris water maze 27 and it improves social memory in prenatal stressed rats 29 . The positive profile of agomelatine on memory processes makes it stands out from classical antidepressant drugs, for which the effect on memory is still matter of debate (for exhaustive review see ref. 5).
The involvement of the neurotrophic factor BDNF in mnesic processes has been clearly demonstrated 50,51 . Lesion studies and inactivation models have shown that in the NOR test object recognition memory was hippocampus-dependent 52,53 when using long ITI (24 h), as we did presently, and not a short one, further supporting a delay-dependent role for the hippocampus in object recognition memory. Therefore, we quantified mRNA levels of Bdnf gene in this structure, and also two of its exons which have been shown to be reduced in CSDS, the Bdnf exons IV and VI 47 . In our conditions, CSDS failed to induce any modification in total Bdnf gene expression. This does not meet previous results that demonstrated a clear downregulation of total Bdnf gene in the hippocampus of social defeated mice, at both short and long term 47,[54][55][56][57] , although one study reported no change in the same conditions 58 . While Bdnf exon VI mRNA gene expression was also unchanged, Bdnf exon IV mRNA was significantly downregulated in CSDS stressed mice. Interestingly, this effect was abolished after a 3-week treatment with agomelatine. These data could be related to those published using imipramine, where a 10 day social defeat stress paradigm downregulated hippocampal Bdnf exon IV and a 4-week imipramine treatment restored basal mRNA level 47 .
Previous studies had also demonstrated the enhancing effect of chronic agomelatine (40-50 mg/kg i.p.) on hippocampal Bdnf gene expression both in naïve rodents 26,[59][60][61] and in depressive like-models 27,37 . The particular regulation of the Bdnf IV transcript had already been proposed to be crucial in the mechanism of action of antidepressant drugs 37 . Interestingly, this transcript is known to selectively stimulate proximal dendrites growth in primary cultures of rat hippocampal neurons 62 . Therefore, it can be proposed that in CSDS, the decrease in Bdnf exon IV gene expression might led to a reduced synaptic plasticity leading to the cognitive impairments observed in the NOR test.
To further evaluate this hypothesis, we then quantified hippocampal mRNA levels of synaptic plasticity markers, especially those of genes that have already been associated with cognitive alterations [63][64][65] . While PSD-95, Synaptophysin, Spinophilin and Synapsin I mRNA levels remained unchanged whatever the conditions, CSDS decreased MAP-2 gene expression and this effect was prevented by agomelatine treatment. MAP-2 is a protein that regulates microtubules and dendritic remodeling 26 . Long-term reduced hippocampal expression of the latter gene has been previously described in mice subjected to stress protocols 66 , but to our knowledge, this is the first study revealing social stress-induced MAP-2 gene expression modifications. Ladurelle et al. 26 have shown that a 22-day agomelatine treatment was able to strongly increase the expression of MAP-2 protein in the hippocampus of naïve rats. These last data supported our present results that showed that chronic agomelatine administration also prevented the alterations on MAP-2 gene expression induced by CSDS, although in our conditions, changes in MAP-2 gene expression induced by CSDS are rather mild, and should be supported by further morphological studies, to address its functional relevance. However, those changes suggested that social stress-induced downregulation of MAP-2 along with Bdnf exon IV might has provoked dendritic or synaptic alterations, leading to cognitive impairments and that chronic agomelatine could exert a protective effect on mice mnemonic functions by preventing Bdnf IV and MAP-2 gene expression deficits.
Whether the alterations in Bdnf and MAP-2 gene expression were supported by alterations in epigenetic regulations was assessed by studying hippocampal gene expression of various epigenetic modifying enzymes. RT-qPCR experiments revealed a downregulation of HDAC2, and HDAC6 gene expression in vehicle-treated CSDS mice. Interestingly, chronic agomelatine reversed these alterations. Since these epigenetic markers are a marker of active transcription 67 , it appears that they were probably not related to the downregulation of Bdnf exon IV and MAP-2 expression observed after CSDS. However, the effect of agomelatine on HDAC2 mRNA is consistent with the work of Boulle et al. 30 , wherein a 25-day agomelatine treatment (50 mg/kg/d i.p.) was able to prevent hippocampal HDAC2 downregulation observed in C57BL/6J mice exposed to chronic ultra mild stress, but had no effect by itself in non-stressed mice. Moreover, HDAC2 expression downregulation in the hippocampus was also observed just after a chronic restraint stress 68 , in a maternal separation model 69 and in a genetic model of depression based on impaired glucocorticoid receptor expression 70 , suggesting that HDAC2 alteration is a long lasting phenomenon. In CSDS, Covington et al. 71 also evidenced a reduced HDAC2 mRNA and protein levels in the nucleus accumbens 24 h and 2 weeks after the last defeat episode. HDAC6 mRNA level has already been shown to be decreased in the dorsal raphe of CSDS-exposed mice 72 , but to our knowledge, our study is the first which demonstrated such regulation in the hippocampus. In addition, we also described a stress-induced modification in the expression of MLL3. MLL3 is a histone H3K4-specific methyltransferase 73 , and hypermethylation at this site is known to have a positive impact on gene transcription 74,75 . Consequently, a reduced MLL3 mRNA SCientifiC RepoRts | 7:45907 | DOI: 10.1038/srep45907 level in this structure might lead to a general decrease in histone H3K4 methylation, therefore inhibiting gene transcription, which could explain CSDS-induced Bdnf exon IV and MAP-2 downregulation. Chronic agomelatine, normalizing MLL3 mRNA level, could prevent the deleterious stress effect on the latter gene expression. Nevertheless, no formal link can be found in the literature between MML3 and these two genes. Gupta et al. showed that in rats, memory formation was associated with increased global hippocampal H3K4 methylation 76 and data suggested that this hypermethylation was set at Bdnf exon I, but not exon IV, promoter. However, it is interesting to note that MLL3 has also been involved in genome-scale circadian transcription 77 further linking the antidepressant efficacy of agomelatine to its circadian effect.
In conclusion, we showed that agomelatine could reverse the cognitive and gene-related effects of social defeat stress in mice. In our conditions, CSDS induced long lasting deleterious effects on C57BL/6J social behavior, anxiety-and memory-related behaviors as well as hippocampal gene expression. While chronic agomelatine treatment was ineffective in social and anxiety-like behaviors, it significantly reversed the memory impairments observed in the NOR test. These data provide evidences that agomelatine could be a relevant therapy to alleviate depression-associated cognitive disorders 23 . CSDS-induced mnesic deficits might be related to Bdnf exon IV and MAP-2 gene expression alteration. Chronic agomelatine prevented these effects, possibly in part through epigenetic-related mechanisms. However, further studies are needed to evaluate the direct impact of CSDS and agomelatine on epigenetic marks and homeostasis at the Bdnf exon IV promoter and to clarify through which mechanisms this compounds could achieve its effects.