Fluvoxamine reverses estrogen-dependent decline in voluntary activities and decreased amygdala levels of serotonin in ovariectomized rats

Studies suggest that increased expression of brain-derived neurotrophic factor (BDNF) could mediate the antidepressant effects of drugs. We analyzed the effects of fluvoxamine on locomotor activities, serotonin levels in the amygdala, and hippocampal expression of BDNF mRNA in ovariectomized (OVX) rats. Female Wistar rats (8 weeks, 180–200 g) were divided into four groups: sham; OVX; OVX with estrogen; and OVX with fluvoxamine. Six weeks after ovariectomy, rats were assessed according to spontaneous locomotor activity, forced-swimming test (FST), and microdialyses experiments. Body and uterine weight of OVX rats 6 weeks after surgery were significantly increased and decreased, respectively, compared with those of the sham group, but these changes were returned to sham-group levels upon chronic administration of estrogen and fluvoxamine. More potent decreases in voluntary activities were observed in OVX rats compared with rats in the sham group, but were increased markedly upon administration of estrogen and fluvoxamine. In the FST, immobility time and beat counts were increased and decreased significantly by ovariectomy compared with those of the sham group, respectively, but estrogen and fluvoxamine treatment reversed these changes significantly. More potent decreases in serotonin release in the amygdala were observed in OVX rats compared with those of sham rats, but were reversed upon estrogen Journal of Brain Science, June 30, 2016,Vol.46 7 replacement. Similar recovery was observed in OVX rats upon fluvoxamine treatment. These data suggest that, in OVX rats, chronic administration of fluvoxamine can recover estrogen-dependent changes in behaviors, decreased serotonin release in the amygdala, and reduced expression of BDNF mRNA. Key wards: Amygdala, Serotonin, Estrogen, Fluvoxamine, OVX; Abbreviations BDNF, brain-derived neurotrophic factor; ER, endoplasmic reticulum; OVX, ovariectmized; RT-PCR, reverse transcription-polymerase chain reaction; SSRI, selective serotonin reuptake inhibitor; XBP-1, X-box binding protein-1; mRNA, messenger ribonucleic acid Introduction The female hormone estrogen has an important role in bone physiology (reviewed in [15, 16]). Moreover, there is a close relationship between estrogen and memory, learning, and emotion in the brain (reviewed in [16]). Also, it has been reported that replenishment with estrogen can improve recognition, learning and memory (reviewed in [3, 5, 14]). We reported that, in female rats 6 weeks after their ovaries had been removed, depression-like symptoms (as manifested by decreases in spontaneous locomotor activities and serotonin levels in the amygdala) could be observed [9]. Depression is a complex disorder brought about by genetic and environmental conditions. Depression involves brain abnormalities as well as dysfunction of the endocrine system, inflammation, altered glucose metabolism and, in some cases, coronary artery disease [8, 18]. Selective serotonin reuptake inhibitors (SSRIs) are first-line treatment for depression and depression-like symptoms. However, resistance to the effects of antidepressants has been documented, so development of new agents with new mechanisms of action is needed [13]. Journal of Brain Science, June 30, 2016,Vol.46 8 Studies have suggested that brain-derived neurotrophic factor (BDNF) signaling is necessary and sufficient for the action of antidepressant drugs [20]. BDNF signaling is associated with cyclic adenosine monophosphate responsive element binding protein, which induces neurogenesis [20]. Moreover, high levels of BDNF have been observed in post mortem hippocampal samples from individuals suffering from depression [20]. Such findings suggest a correlation between decreased expression of BDNF and the onset of depression. Furthermore, a recent report demonstrated that the SSRI fluvoxamine reversed the reduced expression of BDNF messenger ribonucleic acid (mRNA) by chronic infusion of dexamethasone in mice exhibiting depression-like behaviors [17]. Therefore, the therapeutic effects of antidepressants could be mediated by increased expression of BDNF. In the present study, we analyzed the chronic (6-week) effects of the antidepressant fluvoxamine on locomotor activities, serotonin levels in the amygdala, and expression of BDNF mRNA in the hippocampus in ovariectomized (OVX) rats. Materials and Methods Ethical approval of the study protocol The study protocol was approved by the Ethics Committee of Yokohama College of Pharmacy (Kanagawa, Japan). All experiments using animals were carried out based on Guidelines for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) as approved by the Japanese Pharmacological Society. Animals, ovariectomy, and drug administration Animal care and ovariectomy were as described previously with minor modification [9]. Briefly, 36 female Wistar rats (8 weeks, 180–200 g) were divided into four groups of nine. Rats were allowed to acclimatize for ≥1 week to their surroundings before the start of experimentation (12-h light–dark cycle; Journal of Brain Science, June 30, 2016,Vol.46 9 lights on at 7 am and off at 7 pm). After 1 week, OVX and sham operations were undertaken as described previously (Fukushima et al., 2000). One week after ovariectomy, fluvoxamine (50 mg/kg body weight; Wako Pure Chemical Industries, Osaka, Japan) and β-estradiol (50 μg/kg body weight; Sigma–Aldrich, Saint Louis, MO, USA) were administered (p.o. and i.p., respectively) five times per week for 6 weeks. Voluntary momentum, forced-swim test (FST), and microdialyses Six weeks after ovariectomy, rats were assessed according to spontaneous locomotor activity, FST, and microdialyses experiments, as described previously [9, 17]. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) RT-PCR was carried out as described previously [17]. Statistical analyses Statistical analyses were undertaken as described previously [17]. Results Body weight and uterine weight of OVX rats 6 weeks after surgery were increased and decreased significantly, respectively, compared with that of the sham group (Fig. 1a and b; sham and OVX groups). The increased body weight and decreased uterine weight of OVX rats returned to sham-group levels upon chronic administration of estrogen (Fig. 1a and b; sham and estrogen groups). A tendency for recovery was observed upon chronic administration of fluvoxamine in terms of increases and decreases of body weight and uterine weight, but the differences were not significant (Fig 1a and b; sham and fluvoxamine groups). Figure 2a shows spontaneous locomotor activity for 12 h in the dark phase (7 pm to 7 am) for the four groups 6 weeks after ovariectomy. More potent decreases in voluntary activities were observed in OVX rats compared with rats in the sham group (Fig. 2b). These Journal of Brain Science, June 30, 2016,Vol.46 10 data were similar to those of our previous report [9]. These decreases in locomotor activities in OVX rats in the dark phase were increased markedly upon administration of estrogen and fluvoxamine (Fig. 2b). Next, we analyzed the immobility time and beat counts on FST of the four rat groups (Fig. 3). Immobility time and beat counts were increased and decreased significantly by ovariectomy compared with those of the sham group, respectively (Fig. 3a and b; sham and OVX groups). These data were consistent with our previous report [9]. Treatment with estrogen and fluvoxamine reversed these changes significantly to levels seen in control rats (Figs. 3a and b). Next, we analyzed serotonin levels in the amygdala under identical conditions (Fig. 4). More potent decreases in serotonin release in the amygdala were observed in OVX rats compared with those of sham rats (Fig. 4a). These observations were consistent with our previous report [9]. These decreases in serotonin levels in the amygdala were reversed upon estrogen replacement (Fig. 4a; OVX and estrogen), suggesting that decreases in serotonin level by ovariectomy were dependent upon the disappearance of estrogen. A similar recovery was observed in OVX rats upon fluvoxamine treatment (Fig. 4a; OVX and fluvoxamine). However, this recovery in serotonin release was not detected in the case of dopamine release in the amygdala (Fig. 4b). This result was expected because SSRIs have a mechanism of action specific for serotonin. Next, we analyzed hippocampal BDNF expression of sham rats and OVX rats using RT-PCR (Fig. 5). No significant changes were observed in samples from any group obtained from the cerebral cortex or brainstem (Fig. 5; middle and lower panels). More potent decreases in hippocampal BDNF expression were observed in the OVX group compared with that in sham Journal of Brain Science, June 30, 2016,Vol.46 11 groups (Fig. 5; upper panel). This elevation of BDNF expression was recovered markedly upon estrogen treatment (Fig. 5; upper panel). Hippocampal BDNF expression in OVX rats was recovered significantly upon fluvoxamine treatment (Fig. 5; upper panel). Hippocampal expression of the estrogen receptor-1 gene was not changed in all groups (data not shown). Discussion The present study showed that the body weight and uterine weight of OVX rats 6 weeks after ovariectomy were increased and decreased significantly compared with that of the sham group, respectively (Fig. 1), and that this phenomenon was consistent with previous reports [1,4,9]. These results suggest that we carried out ovariectomy successfully. Such increases in body weight and decreases in uterine weight in OVX rats were returned to sham-group levels upon chronic administration of estrogen (Fig. 1), suggesting that the increased weight of OVX rats was significantly dependent upon disappearance of estrogen. Moreover, this tendency for recovery in increases and decreases in body weight and uterine weight was also observed upon chronic administration of fluvoxamine except that the differences were not significant (Fig.