Effects of pre-gestational exposure to the stressors and perinatal mirtazapine administration on the excitability of hippocampal glutamate and brainstem monoaminergic neurons, hippocampal neuroplasticity, and anxiety-like behavior in rats

Effects of pre-gestational exposure to the stressors and perinatal mirtazapine administration on the excitability of hippocampal glutamate and brainstem monoaminergic neurons, hippocampal neuroplasticity, and anxiety-like behavior in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of pre-gestational exposure to the stressors and perinatal mirtazapine administration on the excitability of hippocampal glutamate and brainstem monoaminergic neurons, hippocampal neuroplasticity, and anxiety-like behavior in rats Eliyahu Dremencov, Ruslan Paliokha, Mireia Viñas-Noguera, Stanislavá Bukatova, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5448456/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Aug, 2025 Read the published version in Molecular Psychiatry → Version 1 posted 10 You are reading this latest preprint version Abstract When accompanied by excessive exposure to the stressors, pregnancy may result in prenatal depression, that has in turn negative influence on the offspring’s brain. Mirtazapine, among other antidepressants, is commonly used to treat prenatal depression. Even though mirtazapine is generally considered safe for pregnant women, its effect on the offspring brain have not been sufficiently investigated. The present study aimed to examine the effects of chronic unpredictable stress (CUS) in pregestational rats, prenatal mirtazapine treatment, and their combination, on offspring behavior and brain function. We assessed offspring anxiety levels during the elevated plus maze (EPM) test, the expression of pro-neuroplastic proteins in the offspring brain, the excitability of brainstem monoamine and hippocampal glutamate neurons, and the expression and activity of ryanodine receptors (RyR2). Prenatal mirtazapine had an anxiolytic effect on the offspring of the stressed dams. This effect was associated with an increased excitability of serotonin (5-HT) neurons and elevated expression of the brain-derived neurotrophic factor (BDNF). Regarding the offspring glutamate and dopamine neurons, the combination of maternal stress and mirtazapine inhibited their burst firing, potentially due to decreased expression of the glutamate receptors. Even though calcium signaling is important for the burst firing of the neurons, the effects of maternal stress and mirtazapine on the burst activity of the offspring glutamate and dopamine might not be mediated via mechanism(s) involving the RyR2. Summarizing, mirtazapine may diminish the negative influence of maternal stress and depression on the offspring brain, via mechanism(s) putatively involving 5-HT neurotransmission and BDNF. Biological sciences/Neuroscience Health sciences/Diseases/Psychiatric disorders/Depression prenatal depression prenatal antidepressant treatment anxiety hippocampus monoamines brain-derived neurotrophic factor (BDNF) glutamate receptor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Pregnancy is linked with robust endocrine and neurochemical changes in female organism, rarely observed in other non-pathological conditions. The concentrations of the steroid hormones estradiol and cortisol are gradually increasing during pregnancy, reaching the maximum toward its end. The levels of the brain derived neurotrophic factor (BDNF) are, vice versa , progressively decreasing during pregnancy. BDNF is known to play a key role in the pathophysiology of depression and related mood and anxiety disorders. Likely, due to the decreased BDNF, depressed mood and increased anxiety, not necessarily reaching the level of clinical depression, are frequently experienced during the pregnancy and postpartum 1 . Similarly to pregnancy, stress is generally associated with increased corticosteroids and decreased BDNF. It is thus possible that stress experienced during or before pregnancy may, in certain conditions, lead to neuroendocrine changes extreme enough to induce clinical depression. Indeed, prenatal, and postpartum depression are common complications of pregnancy 2 , and excessive exposure to stressors 3 , 4 and/or strong decline in BDNF 1 are known risk factors for these disorders. Depression is known to induce long-lasting changes in the brain, such as decreased neuroplasticity. When depression occurs during pregnancy, it affects not only the maternal, but also embryonal CNS. Increased rates of the attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (AUD), depression, and schizophrenia were indeed observed in children of depressed mothers 5 , 6 . Pharmacotherapy is a primary treatment strategy in prenatal depression. Literally, all antidepressants act on monoamines, and monoamines play a role in neurogenesis. Since antidepressants pass through the placenta, the putative effects of these drugs on the offspring brain development must be considered. In our previous studies 7 – 10 , we examined the effects of pre-gestational chronic unpredictable stress (CUS) and prenatal treatment with catecholamine releaser bupropion and with α 2 -adrenergic and 5-HT 2A/2C/3 serotonergic antagonist mirtazapine on the offspring behavioral and neurophysiological characteristics. We found that the maternal CUS resulted in hyperactivity-like behavior and decreased spatial memory in female adolescent offspring 7 , 8 . Forced swim test uncovered decreased immobility time in adolescent females and increased swimming in adolescents of both sexes 8 . Elevated plus maze (EPM) test detected increased time spent in closed arms in adolescent males, decreased intersection time in adult males, and decreased number of entries to the open arms in adults of both sexes 8 , 10 . Prenatal mirtazapine potentiated hyperactivity- and antidepressant-like effects of the maternal CUS 8 . With respect to the anxiogenic effect of the maternal CUS, both prenatal mirtazapine and bupropion diminished it 10 . Proteins like BDNF, postsynaptic density protein 95 (PSD95), glial fibrillary acidic protein, (GFAP), and glutamate (GLUR) and ryanodine (RyR2) receptors were used in neurobehavioral studies where offspring rats were exposed to CUS and/or treatment with mirtazapine. They are key markers of brain functioning and plasticity. BDNF is crucial for neuronal survival, growth, and synaptic plasticity, often reduced by stress but enhanced by antidepressants. PSD95 is essential for synaptic stability and signaling, reflecting synaptic integrity. GFAP is a marker of astrocyte activation, indicating neuroinflammatory responses to stress. GLUR are critical for excitatory neurotransmission, which is often disrupted by stress. Together, these proteins provide insight into the molecular changes associated with stress, neuroplasticity, and antidepressant effects in brain development 11 . It was indeed reported in our previous study that the maternal CUS enhanced the excitability of 5-HT neurons in offspring, and prenatal bupropion potentiated the stimulatory effect of the CUS on 5-HT neuronal firing activity in offspring 10 . The present study aims to test the hypothesis that the offspring behavioral changes induced by prenatal mirtazapine, administered by its own or in combination with the CUS, are mediated via the altered hippocampus Cornu Ammonis-1/3 (CA1/3) glutamate, dorsal raphe nucleus (DRN) 5-HT, and ventral tegmental area (VTA) dopamine neuronal firing activity. The present study aimed to investigate the effects of the maternal pregestational CUS, prenatal mirtazapine treatment, and their combination, on the anxious behavior of the adult male and female offspring, expression of the pro-neuroplastic proteins in their brains, excitability of brainstem monoamine and hippocampal glutamate neurons, as well as expression and activity of RyR2 channels. Methods Animals Female nulliparous Wistar rats, weighing 200–220 g, were obtained from the Department of Toxicology and Laboratory Animal Breeding, Institute of Experimental Pharmacology and Toxicology, Centre of Experimental Medicine of the Slovak Academy of Sciences, Dobra Voda, Slovakia. Pre-gestational stress Female nulliparous Wistar rats were allowed to acclimatize for at least one week, and then randomly divided into the CUS or non-CUS groups, as previously described 7 – 10 . For the detailed protocol of the CUS administration, see Supplementary Materials. Mating, perinatal antidepressant treatment, and subsequent manipulations One week after the end of the CUS procedure, females were mated with males. Mirtazapine (10 mg/kg/day, per oral) was administered from day 10 of gestation until delivery. For the detailed protocol of mirtazapine administration, see Supplementary Materials. The offspring were weaned on postpartum day 21 and housed in litter groups of four animals per cage of the same sex. All experiments were carried out on male and female offspring of antidepressant or vehicle treated CUS or non-CUS dams who had reached the age of 48–56 days. As in our previous studies 7 – 10 , electrophysiological and proteomic experiments were carried out on different animals, at least 24 hours after the performance of behavioral experiments. EPM test The anxiety behavior of the adult offspring of the stressed and non-stressed dams treated with mirtazapine or vehicle during the gestation was measured using EMP. For the detailed protocol, see the Supplementary Materials. Assessment of the expression of pro-neuroplasticity proteins Brain samples were extracted and whole hippocampi were excised for quantitative protein Western blot analysis. For the detailed protocol of the Western blot analysis of NDNF, PSD95, GFAP, and GLUR levels, see the Supplementary Materials. Quantification of the optical densities of synaptophysin Sections of the dorsal hippocampus were analyzed for optical densities of synaptophysin. For the detailed protocol, see Supplementary Materials. Electrophysiology in vivo The spontaneous firing activity of the hippocampal glutamate, DRN 5-HT, and VTA dopamine neurons were performed using extracellular single-unit in vivo electrophysiology. For the detailed protocol, see Supplementary Materials. Expression and activity of RyR2 The brain endoplasmic reticulum (ER) microsomes enriched in RyR2 channels were isolated from rat subcortical structures located beneath the cerebral cortex, following isolation protocol described by Bilmen and Michelangeli 12 . For details, see see Supplementary Materials. Data analysis Action potentials (spikes) were detected using the spike sorting algorithm, with version 6.02 of Spike2 software (Cambridge Electronic Design, Cambridge, UK). The neuronal firing rate and burst activity characteristics were calculated using the burstiDAtor software ( www.github.com/nno/burstidator ), in accordance with our previous publications 10 , 13 – 15 . Statistical assessments were carried out using SigmaPlot 12.5 software (Systat Software Inc, Chicago, IL, USA). A three-way analysis of variance (ANOVA), with the factors of sex, maternal CUS, and perinatal bupropion, followed by a Bonferroni post-hoc test, was used to evaluate the impacts of maternal CUS and antidepressant treatment and the sex of the offspring, on the offspring behaviour, expression of pro-neuroplastic proteins in the offspring brain, synaptophysin optical density, firing activity characteristics of the hippocampal glutamate and brainstem monoamine neurons, and opening of the RyR2. A probability of p ≤ 0.05 was considered significant. Results Effects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the offspring behavior during the EPM test There was a significant effect sex (F 1,76 =11.54, p = 0.001) and maternal CUS (F 1,76 =5.87, p = 0.02, three-way ANOVA) on the number of entries to the closed arms of the EPM apparatus in the offspring (Fig. 1 A). There was no effect of maternal mirtazapine and no interactions between the factors of the comparison. Bonferroni post-hoc test did not detect any significant difference between any specific groups of animals as well. Nevertheless, the number of entries to the closed arms tended to be higher in females compared to males and in offspring of non-CUS dams in comparison to the offspring of the CUS dams. With respect to the number of entries to open arms, there was no significant effect of any of the factors of the comparison (Fig. 1 B). There was, however, significant sex × maternal CUS × maternal mirtazapine interaction (F 1,78 =6.73, p = 0.01, three-way ANOVA). Bonferroni post-hoc test did not detect any significant difference between any specific groups of animals as well. Nevertheless, maternal mirtazapine tended to decrease the number of entries to the open arms in male offspring of non-stressed dams and female offspring of the stressed dams and to increase this factor in the female offspring of the stressed dams. There were no effects of sex or maternal stress and mirtazapine and no interactions between these factors on the time spent in the closed arms of the EPM apparatus (Fig. 1 C). With respect to the time spent in the open arms (Fig. 1 D), there was no significant effect of any of the factors of the comparison. There was, however, significant offspring sex × maternal CUS × maternal mirtazapine interaction (F 1,78 =4.33, p = 0.04, three-way ANOVA). Bonferroni post-hoc test did not detect any significant difference between any specific groups of animals as well. Nevertheless, maternal mirtazapine tended to increase this variable r in the male offspring of the stressed dams and female offspring of non-stressed dams. Effects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the expression of pro-neuroplastic proteins With respect to the BDNF protein levels (Fig. 2 A), there was a significant effect of the offspring sex (F 1,45 =15.50, p < 0.001), offspring sex × maternal mirtazapine (F1,45 = 11.74, p = 0.001), maternal stress × maternal mirtazapine (F 1,45 =34.89, p < 0.001), and offspring sex × maternal stress × maternal mirtazapine interactions (F 1,45 =10.76, p = 0.002, three-way ANOVA). Bonferroni post-hoc test detected suppressing effect of the maternal CUS on BDNF level in male offspring (p < 0.001, regardless maternal mirtazapine treatment), significant difference between male and female offspring of the dams exposed to both CUS and mirtazapine (p < 0.001, with the value higher in females), and a robust increasing effect of the maternal CUS and in female offspring of mirtazapine-treated dams (p < 0.001). Regarding the PSD95 (Fig. 2 B), there was a significant effect of the maternal stress (F 1,49 =6.05, p = 0.02) and significant interaction between the offspring sex, maternal stress, and maternal mirtazapine treatment (F 1,49 =11.49, p = 0.002, three-way ANOVA). Bonferroni post-hoc test also unveiled a significant increasing effect of maternal stress in male offspring of the vehicle-, but not mirtazapine-treated dams (p = 0.02). With respect to the GFAP (Fig. 2 C), there was a significant effect of the offspring sex (F 1,46 =13.28, p < 0.001) and significant offspring sex × maternal stress × maternal mirtazapine interaction (F 1,46 =18.75, p < 0.001, three-way ANOVA). Bonferroni post-hoc test detected sex differences in the offspring of non-stressed dams treated with mirtazapine and stressed dams treated with vehicle (p < 0.002, and the value in males higher than in females in both cases), as well as an increasing effect of the maternal stress and female offspring of mirtazapine-treated dams (p < 0.002). Regarding the GLUR (Fig. 2 D), there was a significant offspring sex × maternal stress × maternal mirtazapine interaction (F 1,49 =11.49, p < 0.002, three-way ANOVA). Bonferroni post-hoc test unveiled suppressing effect of the maternal CUS in male offspring of mirtazapine-treated dams (p < 0.001). Effects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the synaptophysin optical density in the hippocampus Figure 3 illustrates the synaptophysin optical density in the hippocampal Cornu Ammonis areas CA3 (A) and CA4 (B) and in the dentate gyrus (C). In the CA3, there was significant effect of the offspring sex (F 1,58 =12.03, p = 0.001) and significant interactions between offspring sex and maternal CUS (F 1,58 =4.16, p = 0.05), and maternal stress and CUS (F 1,58 =4.61, p = 0.04). Bonferroni post-hoc test reported sex differences in the offspring of vehicle- (p = 0.01) and mirtazapine-treated (p = 0.02) rats, regardless of maternal CUS exposure (values in females lower compared to t males in both cases). With respect to the CA4, there was significant effect of the offspring sex (F 1,59 =30.06, p < 0.001) and significant offspring sex × maternal CUS interaction (F 1,59 =9.61, p = 0.003). Bonferroni post-hoc test detected effect of the maternal CUS on CA4 synaptophysin in females (p < 0.001), but not in males. There was a statistically significant difference between the sex in the offspring of the stressed (p < 0.001), but not in non-stressed dams. CA4 synaptophysin levels were higher in females compared to males. In the dentate gyrus, neither the difference between the sexes nor the effect of the maternal CUS or mirtazapine, or any interaction between the factors of comparison, were observed. Effects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of glutamate neurons in the hippocampus of male and female offspring Representative recording from a hippocampal glutamate neuron is shown on Fig. 4 A. With respect to the mean spontaneous firing rate of the neurons (Fig. 4 B), there was a significant interaction between the offspring sex and maternal CUS (F 1,490 =12.90, p < 0.001, three-way ANOVA). Bonferroni post-hoc test detected a sex difference in the offspring of the stressed dams (p < 0.003, with the value higher in males). Regarding the average number of the spontaneously active neurons per electrode descend (Fig. 4 C), three-way ANOVA revealed significant offspring sex × maternal stress interaction (F 1,150 =8.47, p < 0.004, three-way ANOVA). Bonferroni post-hoc test detected sex differences in the offspring of the stressed dams (p = 0.002, with the value in males higher than in females) and emphasized the increasing effect of the maternal CUS in male (p = 0.004, regardless maternal mirtazapine treatment), but not in female offspring. With respect to the bursts’ frequency (Fig. 4 D), there was a significant effect of the maternal stress (F 1,150 =6.83, p = 0.009) and significant interaction between offspring sex and maternal stress (F 1,150 =6.78, p = 0.01, three-way ANOVA). Bonferroni post-hoc test showed significant sex differences in the offspring of the stressed dams (p < 0.03, with the value in males higher than in females) and suppressing effect of the maternal stress in female (p < 0.001), but not male offspring. Other characteristics of the burst firing, namely, percent of the spikes occurring in the bursts (Fig. 4 E) and the mean number of spikes in bursts (Fig. 4 F), were not affected by the maternal stress or mirtazapine treatment, neither they had sex differences. Effects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of 5-HT neurons in the DRN of male and female offspring Representative recording from a DRN 5-HT neuron is shown on Fig. 5 A. With respect to the mean spontaneous firing rate of the neurons (Fig. 5 B), three-way ANOVA revealed the significant effect of the offspring sex (F 1,400 =10.99, p < 0.001). Even though Bonferroni post-hoc test did not reveal any between-group differences, the values in females tended to be lower comparing to the males. When two-way ANOVA (using the maternal stress and maternal mirtazapine treatment as factors of comparison) was applied on the male offspring only, it revealed significant effect of the maternal stress (F 1,263 =5.22, p = 0.02). Maternal stress increased the firing rate of 5-HT neurons in the male offspring of the vehicle-, but not mirtazapine-treated rats (p = 0.04, Bonferroni post-hoc test). Regarding the average number of the density of the spontaneously active neurons (Fig. 5 C), only a sex difference (F 1,93 =4.84, p < 0.04, three-way ANOVA) was unveiled. The values in females tended to be lower compared to the males. With respect to the parameters of the burst firing, such as bursts’ frequency (Fig. 5 D), percent of the spikes occurring in the bursts (Fig. 5 E) and the mean number of spikes in bursts (Fig. 5 F), were not affected by the offspring sex, maternal stress nor mirtazapine treatment. Effects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of dopamine neurons in the VTA of male and female offspring Representative recording from a DRN 5-HT neuron is shown on Fig. 6 A. Offspring sex, maternal stress or mirtazapine treatment did not affect the firing rate (Fig. 6 B) and the average number of the spontaneously active neurons per electrode track (Fig. 6 C). With respect to the bursts’ frequency (Fig. 6 D), significant effects of the offspring sex (F 1,346 =7.48, p = 0.007) and maternal stress (F 1,346 =6.83, p = 0.009) and significant interaction between maternal sex and maternal mirtazapine treatment (F 1,346 =10.59, p = 0.001, three-way ANOVA) were observed. Bonferroni pos-hoc test revealed the suppressing effects of the maternal CUS in female offspring (p = 0.001) and maternal mirtazapine in female offspring of stressed dams (p = 0.001). Regarding the percentage of the spikes occurring in the bursts (Fig. 6 E), there were significant effects of the offspring sex (F 1,346 =36.49, p < 0.001), maternal stress (F 1,346 =19.19, p < 0.001) and mirtazapine treatment (F 1,346 =6.99, p < 0.009), as well as significant offspring sex × maternal mirtazapine (F 1,346 =8.20, p = 0.004), maternal stress × maternal mirtazapine (F 1,346 =6.28, p < 0.02), and offspring sex × maternal stress × maternal mirtazapine (F 1,346 =6.16, p = 0.01) interactions. Bonferroni post-hoc test detected a sex difference in the offspring of the mirtazapine-treated non-stressed (p = 0.007) and stressed dams (p < 0.001, the value in females lower compared to the males in both cases) and suppressing effect of maternal stress in female offspring of mirtazapine-treated dams (p < 0.001). With respect to the mean number of spikes in burst (Fig. 6 F), there was a significant effect of the offspring sex (F 1,346 =7.49, p < 0.007) and significant offspring sex × maternal stress × maternal mirtazapine interaction (F 1,346 =5.34, p < 0.02). Bonferroni post-hoc test revealed a sex difference in the offspring of the stressed dams, regardless of mirtazapine treatment (p = 0.004), with the value in females lower than in males. Effects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the expression and activity of RyR2 channels Expression of RyR2 protein and opening of RyR2 channels were not affected by the offspring sex or by maternal stress or mirtazapine (see Supplementary Materials, Fig S1 ). Discussion We found that pregestational CUS, combined with prenatal mirtazapine, tended to have an anxiolytic effect on male and an anxiogenic effect on female offspring, measured by the EPM test. Maternal CUS increased PSD95 expression in male offspring, increased synaptophysin in females, and decreased BDNF in both sexes. In females, pregestational CUS-induced decrease in BDNF expression was reversed by perinatal mirtazapine. Combination of pre-gestational CUS and perinatal mirtazapine increased the GFAP expression in the females and decreased GLUR expression in the males. Pre-gestational CUS decreased the excitability of the hippocampal glutamate and VTA dopamine neurons; mirtazapine enhanced this effect. With respect to 5-HT neurons, maternal CUS increased their firing activity. Similarly to our previous studies 7 , 8 , 10 , we found that maternal pre-gestational CUS and perinatal antidepressant treatment by their own did not alter offspring behavior during the EPM test. Pre-gestational CUS, combined with prenatal mirtazapine, tended to increase these characteristics in males, suggesting an anxiolytic effect, and to decrease it in females, suggesting an anxiogenic effect. It was found that pregestational CUS decreased BDNF expression in the offspring brain. In females, but not in males, perinatal mirtazapine reversed the suppressing effect of the pre-gestational CUS on BDNF expression in the offspring brain. With respect to PSD95, maternal CUS increased its expression in the brain of the male, but not female offspring. Perinatal mirtazapine also tended to increase PSD95 in male offspring brain. Thus, statistically significant difference between the offspring of the stressed and non-stressed dams was observed only in animals prenatally exposed to the vehicle, but not to mirtazapine. Combination of pre-gestational CUS and perinatal mirtazapine, but none of these factors by their own, increased the GFAP expression in the female and decreased GLUR expression in the male offspring brain. CUS may diminish BDNF expression in female offspring but not in male offspring due to sex-specific differences in stress response and brain plasticity. These differences are largely driven by hormonal influences, particularly the interaction of sex hormones like estrogen and testosterone with neurotrophic factors such as BDNF 16 . In females, estrogen plays a critical role in regulating BDNF expression, especially in brain areas like the hippocampus, which is sensitive to stress 17 . Chronic stress can disrupt the estrogen-BDNF pathway, leading to reduced BDNF levels. Males, on the other hand, have different hormonal responses and may be more resilient to the BDNF-lowering effects of stress due to testosterone or differential activation of stress-related brain circuits 18 . Additionally, stress may trigger different neuroinflammatory responses or epigenetic changes in males and females, contributing to the sex-specific regulation of BDNF expression 19 . The interaction of mirtazapine with chronic stress produces sex-specific neurobiological outcomes-enhancing GFAP and astrocyte activation in females, potentially exacerbating anxiety-related behaviors, while reducing glutamate receptor levels and improving stress resilience in males, leading to anxiolytic effects. These sex differences highlight the complex interplay between stress, neuroinflammation, and neurotransmission in shaping behavioral responses. It was found that the maternal CUS increased synaptophysin optical density in the offspring hippocampus, especially, in its CA3 area. This effect was observed in female, but not in male offspring. Prenatal mirtazapine did not affect offspring hippocampal synaptophysin; neither did pregnancy with the effect of pregestational CUS. The increase in synaptophysin optical density in female but not male offspring may be due to sex-specific hormonal regulation, differential stress responses, and possible compensatory plasticity mechanisms in the female brain, which may help offset the adverse effects of maternal chronic stress. Chronic stress during pregnancy can induce epigenetic changes that influence brain development in offspring. These changes might alter the expression of synaptic proteins like synaptophysin in a sex-dependent manner 20 . In females, maternal stress might induce compensatory mechanisms, such as increased synaptic plasticity, as a way to cope with the altered brain environment, leading to higher synaptophysin levels 21 . In addition, estrogen may interact with synaptic proteins like synaptophysin more prominently in female offspring. This hormone may enhance synaptic resilience to stress, potentially increasing synaptophysin expression in females but not in males, who rely more on testosterone, which might have different effects on synaptic function 22 . We found that maternal CUS decreased the density of the spontaneously active hippocampal neurons, their mean firing rate, and the burst mode of their firing. This effect was sex-specific and observed in female, but not in male rats. It is known that the burst firing of glutamate neurons enhances the nerve terminal neurotransmitter release, in comparison with the same amount of action potentials fired in a single-spike mode 23 . Pregestational stress is therefore likely to decrease hippocampal glutamate transmission in female offspring. It is thus possible that the decrease in hippocampal glutamate transmission, observed in females, but not in males, is responsible, at least in part, for the anxiogenic effect of pregestational maternal stress, that was also observed in female, but not in male offspring. The link between reduced hippocampal glutamate transmission and anxiety has been shown in previous studies. Widman and coauthors demonstrated that the low novelty response (LR) rats, a genetic breed characterized by high anxiety, had decreased hippocampal long-term potentiation (LTP) and reduced density of the dendritic spines on hippocampal pyramidal neurons 24 . On the other hand, high levels of anxiety in rats experienced social defeat stress 25 and in mice lacking serotonin-1A (5-HT 1A ) receptor 26 were associated with increased LTP and excitatory post-synaptic currents (EPSC), respectively. In our previous study, an anxiolytic effect of a selective delta-opioid receptor (DOR) agonist SNC80 was accompanied by an elevated firing rate and increased burst activity of hippocampal neurons 27 . It was found that the mean firing rate of the spontaneously active 5-HT neurons, as well as their density, were higher in the males comparing to the females. Similar sex-related differences in the firing activity of 5-HT neurons were observed in the previous studies from our 10 and other laboratories 28 . Similarly to our previous study 10 , we found that pregestational CUS increased the basal firing rate of 5-HT neurons in male, but not in female offspring. Since the firing activity of the DRN 5-HT neurons determines limbic 5-HT neurotransmission 29 , pregestational CUS might increase 5-HT tone in the male offspring. As it was already suggested 10 , maternal CUS-induced increase in limbic 5-HT transmission in the male offspring might be a compensatory mechanism designated to prevent the negative effect of the maternal stress on the offering brain. Perinatal mirtazapine tended to increase the firing activity of 5-HT neurons in male rats as well. Thus, statistically significant difference between the offspring of the stressed and non-stressed dams was observed only in animals prenatally exposed to the vehicle, but not to mirtazapine. This observation is consistent with the stimulatory effect of the sustained mirtazapine on 5-HT neurons reported in previous studies 30 , 31 . It is well established that 5-HT neurotransmission is fundamental in anxiety 32 . Maternal and CUS and mirtazapine-induced stimulation of the offspring 5-HT neurons might be therefore consistent with the anxiolytic effects of these maternal factors on the offspring behavior during the EPM test. The presence of this compensatory mechanism in male, but not in female rats might explain the fact that the anxiolytic effect of the maternal stress and mirtazapine was observed in male, but not in female offspring. We found that pregestational CUS led to the decreased burst firing of dopamine neurons in female, but not in male offsprings’ VTA. Perinatal mirtazapine robustly potentiated the effect of pregestational CUS on the burst firing of dopamine neurons in female offspring’ VTA. Burst firing of dopamine neurons is associated with higher efficiency of neurotransmitter release from the nerve terminals 33 . Maternal CUS and mirtazapine may thus suppress dopamine neurotransmission in female offspring. Maternal CUS and mirtazapine-induced suppression of dopamine neurotransmission might be thus involved in the anxiogenic effect of these factors, that was also observed in female, but not male offspring. The involvement of the mesolimbic dopamine transmission in anxiety was indeed reported in previous studies. It was stated that postpartum anxiety and depressive-like behavior in rats were associated with attenuated population activity in the VTA 34 . On the other hand, increased anxiety in a rat model of neuropathic pain was associated with elevated accumulation of dopamine levels 35 . Finally, we had recently described that the anxiolytic effect of DOR agonist SNC80 was accompanied by elevated firing rate and increased burst activity of the VTA dopamine neurons 27 . Ca 2+ signaling in the brain plays a crucial role in neuronal viability, synaptic plasticity, and higher cognitive processes 36 – 39 , suggesting that Ca 2+ mishandling contributes to various neuropsychiatric and neurodegenerative disorders 40 – 46 . RyR2, a key intracellular Ca 2+ channel, is significantly implicated in neuronal Ca²⁺ signaling and several studies suggest that phosphorylation-induced increase in RyR activity may contribute to AD pathogenesis 43 , 46 – 49 . Implication of RyR2 channels in pathogenesis of neuropsychiatric disorders has started to emerge only recently 50 , 51 . However, the primary efforts have focused on identifying mutations in the RYR genes that encode three distinct isoforms, rather than directly investigating potential RyR dysfunction and post-translational modifications. We found that neither amount of the RyR2 channel (a dominant isoform in the brain 52 , 53 ), its resting activity, or phosphorylation state was altered. Thus, we can exclude the potential contribution of this Ca 2+ channel in changes in neuronal excitability of the offspring caused by the pre-gestational maternal CUS, prenatal mirtazapine, and their combination. In conclusion, maternal mirtazapine did not alter anxious behavior of the offspring by its own. Prenatal mirtazapine, however, had an anxiolytic effect in male offspring in the stressed dams. Prenatal mirtazapine also tended to stimulate 5-HT neurons in male offspring; this stimulatory effect on 5-HT neurons might underline the anxiolytic effect of prenatal mirtazapine. Stimulatory effect of prenatal mirtazapine on BDNF expression in the offspring of the stressed dams might be linked with increased 5-HT neurotransmission and with decreased anxiety as well. Maternal stress and mirtazapine had a suppressing effect on the burst firing of the offspring VTA dopamine and hippocampal glutamate neurons; it might be linked with the decreased expression of the GLUR. The effects of maternal stress and mirtazapine on the burst firing of the central neurons in offspring are not mediated via mechanism(s) involving the RyR2 channel. Declarations Acknowledgement The authors thank Katya Sketa for the technical assistance. Funding This work of was supported by the grants APVV-19-0435, APVV-20-0202, APVV-APVV-22-0061, VEGA-2/0057/22, VEGA-2/0133/23, and VEGA-2/0045/24. Author contribution RP, MVN, SB, DG, JG, MG, LL, ED and MD formulated the working hypothesis and planned the experiments, RP, MVN, SB, DG, JG, MG, RD, and HO performed the experiments, RP, MVN, SB, DG, JG, MG, RD, TK, and ED analysed the results, RP, MVN, ED, and MD wrote the manuscript, all authors proof-read the manuscript and approved it for publication. Data availability Data will be made available on request. Compliance with ethical standards Conflict of interests The authors declare no conflict of interest. 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1","display":"","copyAsset":false,"role":"figure","size":632856,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of entries to the closed (A) and open (B) arms and time spent in the closed (C) and open (D) arms of the elevated plus maze (EPM) apparatus for male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/059b2f4ffcca8727dd4b5173.png"},{"id":73411797,"identity":"40445682-1666-4b6f-b35e-a1b469c359a1","added_by":"auto","created_at":"2025-01-09 16:15:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4700378,"visible":true,"origin":"","legend":"\u003cp\u003eProtein levels of the brain-derived neurotrophic factor (BDNF; A), postsynaptic density protein 95 (PSD95; B), glial fibrillary acidic protein (GFAP; C) and glutamate receptor (GLUR; D) in the hippocampus of male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation. **p\u0026lt;0.01 and ***p\u0026lt;0.001, between-group comparison, Bonferroni post-hoc tests.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/886b624c0840123e23a1d03f.png"},{"id":73411799,"identity":"89f8a92a-9a18-456b-8c7d-d7b2f441e243","added_by":"auto","created_at":"2025-01-09 16:15:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":528397,"visible":true,"origin":"","legend":"\u003cp\u003eSynaptophysin optical density in the cornu Ammonis 3 (CA3; A), cornu Ammonis 4 (CA4; B), and dentate gyrus of male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation. **p\u0026lt;0.01 and ***p\u0026lt;0.001, between-group comparison, Bonferroni post-hoc tests.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/7d0cbffc3884df77ca8b2631.png"},{"id":73411800,"identity":"6470e026-1d15-4bc7-88d7-51ffbaaf5e68","added_by":"auto","created_at":"2025-01-09 16:15:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4127060,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative recording from a hippocampal glutamate neuron (A; male offspring of the vehicle-treated not stressed dam) and mean spontaneous firing rate (B), number of spontaneously active neurons per electrode descend (C), burst frequency (D), percent of spikes occurring in the bursts (E), and number of spikes per burst (F) of the hippocampal glutamate neurons of male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001, between-group comparison, Bonferroni post-hoc tests.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/3abca0a15e9e6488d92cff55.png"},{"id":73411805,"identity":"ea4aced4-0ef2-43d9-9da4-a2755f192f80","added_by":"auto","created_at":"2025-01-09 16:15:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4099664,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative recording from a dorsal raphe nucleus (DRN) serotonin (5-HT) neuron (A; male offspring of the vehicle-treated not stressed dam) and mean spontaneous firing rate (B), number of spontaneously active neurons per electrode descend (C), burst frequency (D), percent of spikes occurring in the bursts (E), and number of spikes per burst (F) of the DRN 5-HT neurons of male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001, between-group comparison, Bonferroni post-hoc tests.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/53420a92e43e2dbaa336a03e.png"},{"id":73411802,"identity":"7774e806-92b8-4ded-91f8-7d92e5398b77","added_by":"auto","created_at":"2025-01-09 16:15:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4737116,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative recording from a ventral tegmental area (VTA) dopamine neuron (A; male offspring of the vehicle-treated not stressed dam) and mean spontaneous firing rate (B), number of spontaneously active neurons per electrode descend (C), burst frequency (D), percent of spikes occurring in the bursts (E), and number of spikes per burst (F) of the VTA dopamine neurons of male and female (fem) offspring of the dams experienced (CUS) or not experienced (non-CUS) pregestational chronic unpredictable stress and treated with mirtazapine or vehicle during the gestation. *p\u0026lt;0.05, **p\u0026lt;0.01 and ***p\u0026lt;0.001, between-group comparison, Bonferroni post-hoc tests.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/15be32199c9e145543cbb402.png"},{"id":89889314,"identity":"d489544e-8283-4851-a8a1-8937d10dad19","added_by":"auto","created_at":"2025-08-26 07:11:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17775808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/e6cd4fd9-b79d-4294-876f-b082cd4d2ab8.pdf"},{"id":73411796,"identity":"d04f6e85-5ca5-4ba3-8936-709274f0a20d","added_by":"auto","created_at":"2025-01-09 16:15:56","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1805957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/40bf2b1a03fe14671af31cda.png"},{"id":73411803,"identity":"dfed2d26-0ff2-469f-94b1-f274f2b6ee2c","added_by":"auto","created_at":"2025-01-09 16:15:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3471409,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementtoPaliokhaetal.docx","url":"https://assets-eu.researchsquare.com/files/rs-5448456/v1/4fed1dc68c1f004c4e1cf03e.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Effects of pre-gestational exposure to the stressors and perinatal mirtazapine administration on the excitability of hippocampal glutamate and brainstem monoaminergic neurons, hippocampal neuroplasticity, and anxiety-like behavior in rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePregnancy is linked with robust endocrine and neurochemical changes in female organism, rarely observed in other non-pathological conditions. The concentrations of the steroid hormones estradiol and cortisol are gradually increasing during pregnancy, reaching the maximum toward its end. The levels of the brain derived neurotrophic factor (BDNF) are, \u003cem\u003evice versa\u003c/em\u003e, progressively decreasing during pregnancy. BDNF is known to play a key role in the pathophysiology of depression and related mood and anxiety disorders. Likely, due to the decreased BDNF, depressed mood and increased anxiety, not necessarily reaching the level of clinical depression, are frequently experienced during the pregnancy and postpartum\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSimilarly to pregnancy, stress is generally associated with increased corticosteroids and decreased BDNF. It is thus possible that stress experienced during or before pregnancy may, in certain conditions, lead to neuroendocrine changes extreme enough to induce clinical depression. Indeed, prenatal, and postpartum depression are common complications of pregnancy\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, and excessive exposure to stressors\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and/or strong decline in BDNF\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e are known risk factors for these disorders.\u003c/p\u003e \u003cp\u003eDepression is known to induce long-lasting changes in the brain, such as decreased neuroplasticity. When depression occurs during pregnancy, it affects not only the maternal, but also embryonal CNS. Increased rates of the attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (AUD), depression, and schizophrenia were indeed observed in children of depressed mothers\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePharmacotherapy is a primary treatment strategy in prenatal depression. Literally, all antidepressants act on monoamines, and monoamines play a role in neurogenesis. Since antidepressants pass through the placenta, the putative effects of these drugs on the offspring brain development must be considered. In our previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we examined the effects of pre-gestational chronic unpredictable stress (CUS) and prenatal treatment with catecholamine releaser bupropion and with α\u003csub\u003e2\u003c/sub\u003e-adrenergic and 5-HT\u003csub\u003e2A/2C/3\u003c/sub\u003e serotonergic antagonist mirtazapine on the offspring behavioral and neurophysiological characteristics. We found that the maternal CUS resulted in hyperactivity-like behavior and decreased spatial memory in female adolescent offspring\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Forced swim test uncovered decreased immobility time in adolescent females and increased swimming in adolescents of both sexes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Elevated plus maze (EPM) test detected increased time spent in closed arms in adolescent males, decreased intersection time in adult males, and decreased number of entries to the open arms in adults of both sexes\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Prenatal mirtazapine potentiated hyperactivity- and antidepressant-like effects of the maternal CUS\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. With respect to the anxiogenic effect of the maternal CUS, both prenatal mirtazapine and bupropion diminished it\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eProteins like BDNF, postsynaptic density protein 95 (PSD95), glial fibrillary acidic protein, (GFAP), and glutamate (GLUR) and ryanodine (RyR2) receptors were used in neurobehavioral studies where offspring rats were exposed to CUS and/or treatment with mirtazapine. They are key markers of brain functioning and plasticity. BDNF is crucial for neuronal survival, growth, and synaptic plasticity, often reduced by stress but enhanced by antidepressants. PSD95 is essential for synaptic stability and signaling, reflecting synaptic integrity. GFAP is a marker of astrocyte activation, indicating neuroinflammatory responses to stress. GLUR are critical for excitatory neurotransmission, which is often disrupted by stress. Together, these proteins provide insight into the molecular changes associated with stress, neuroplasticity, and antidepressant effects in brain development\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt was indeed reported in our previous study that the maternal CUS enhanced the excitability of 5-HT neurons in offspring, and prenatal bupropion potentiated the stimulatory effect of the CUS on 5-HT neuronal firing activity in offspring\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The present study aims to test the hypothesis that the offspring behavioral changes induced by prenatal mirtazapine, administered by its own or in combination with the CUS, are mediated via the altered hippocampus Cornu Ammonis-1/3 (CA1/3) glutamate, dorsal raphe nucleus (DRN) 5-HT, and ventral tegmental area (VTA) dopamine neuronal firing activity.\u003c/p\u003e \u003cp\u003eThe present study aimed to investigate the effects of the maternal pregestational CUS, prenatal mirtazapine treatment, and their combination, on the anxious behavior of the adult male and female offspring, expression of the pro-neuroplastic proteins in their brains, excitability of brainstem monoamine and hippocampal glutamate neurons, as well as expression and activity of RyR2 channels.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFemale nulliparous Wistar rats, weighing 200\u0026ndash;220 g, were obtained from the Department of Toxicology and Laboratory Animal Breeding, Institute of Experimental Pharmacology and Toxicology, Centre of Experimental Medicine of the Slovak Academy of Sciences, Dobra Voda, Slovakia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePre-gestational stress\u003c/h3\u003e\n\u003cp\u003eFemale nulliparous Wistar rats were allowed to acclimatize for at least one week, and then randomly divided into the CUS or non-CUS groups, as previously described\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. For the detailed protocol of the CUS administration, see Supplementary Materials.\u003c/p\u003e\n\u003ch3\u003eMating, perinatal antidepressant treatment, and subsequent manipulations\u003c/h3\u003e\n\u003cp\u003eOne week after the end of the CUS procedure, females were mated with males. Mirtazapine (10 mg/kg/day, per oral) was administered from day 10 of gestation until delivery. For the detailed protocol of mirtazapine administration, see Supplementary Materials. The offspring were weaned on postpartum day 21 and housed in litter groups of four animals per cage of the same sex. All experiments were carried out on male and female offspring of antidepressant or vehicle treated CUS or non-CUS dams who had reached the age of 48\u0026ndash;56 days. As in our previous studies\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, electrophysiological and proteomic experiments were carried out on different animals, at least 24 hours after the performance of behavioral experiments.\u003c/p\u003e\n\u003ch3\u003eEPM test\u003c/h3\u003e\n\u003cp\u003eThe anxiety behavior of the adult offspring of the stressed and non-stressed dams treated with mirtazapine or vehicle during the gestation was measured using EMP. For the detailed protocol, see the Supplementary Materials.\u003c/p\u003e\n\u003ch3\u003eAssessment of the expression of pro-neuroplasticity proteins\u003c/h3\u003e\n\u003cp\u003eBrain samples were extracted and whole hippocampi were excised for quantitative protein Western blot analysis. For the detailed protocol of the Western blot analysis of NDNF, PSD95, GFAP, and GLUR levels, see the Supplementary Materials.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of the optical densities of synaptophysin\u003c/h2\u003e \u003cp\u003eSections of the dorsal hippocampus were analyzed for optical densities of synaptophysin. For the detailed protocol, see Supplementary Materials.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eElectrophysiology in vivo\u003c/h3\u003e\n\u003cp\u003eThe spontaneous firing activity of the hippocampal glutamate, DRN 5-HT, and VTA dopamine neurons were performed using extracellular single-unit \u003cem\u003ein vivo\u003c/em\u003e electrophysiology. For the detailed protocol, see Supplementary Materials.\u003c/p\u003e\n\u003ch3\u003eExpression and activity of RyR2\u003c/h3\u003e\n\u003cp\u003eThe brain endoplasmic reticulum (ER) microsomes enriched in RyR2 channels were isolated from rat subcortical structures located beneath the cerebral cortex, following isolation protocol described by Bilmen and Michelangeli\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. For details, see see Supplementary Materials.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eAction potentials (spikes) were detected using the spike sorting algorithm, with version 6.02 of Spike2 software (Cambridge Electronic Design, Cambridge, UK). The neuronal firing rate and burst activity characteristics were calculated using the burstiDAtor software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.github.com/nno/burstidator\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.github.com/nno/burstidator\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), in accordance with our previous publications\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Statistical assessments were carried out using SigmaPlot 12.5 software (Systat Software Inc, Chicago, IL, USA). A three-way analysis of variance (ANOVA), with the factors of sex, maternal CUS, and perinatal bupropion, followed by a Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test, was used to evaluate the impacts of maternal CUS and antidepressant treatment and the sex of the offspring, on the offspring behaviour, expression of pro-neuroplastic proteins in the offspring brain, synaptophysin optical density, firing activity characteristics of the hippocampal glutamate and brainstem monoamine neurons, and opening of the RyR2. A probability of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the offspring behavior during the EPM test\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThere was a significant effect sex (F\u003csub\u003e1,76\u003c/sub\u003e=11.54, p\u0026thinsp;=\u0026thinsp;0.001) and maternal CUS (F\u003csub\u003e1,76\u003c/sub\u003e=5.87, p\u0026thinsp;=\u0026thinsp;0.02, three-way ANOVA) on the number of entries to the closed arms of the EPM apparatus in the offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). There was no effect of maternal mirtazapine and no interactions between the factors of the comparison. Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test did not detect any significant difference between any specific groups of animals as well. Nevertheless, the number of entries to the closed arms tended to be higher in females compared to males and in offspring of non-CUS dams in comparison to the offspring of the CUS dams.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith respect to the number of entries to open arms, there was no significant effect of any of the factors of the comparison (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). There was, however, significant sex \u0026times; maternal CUS \u0026times; maternal mirtazapine interaction (F\u003csub\u003e1,78\u003c/sub\u003e=6.73, p\u0026thinsp;=\u0026thinsp;0.01, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test did not detect any significant difference between any specific groups of animals as well. Nevertheless, maternal mirtazapine tended to decrease the number of entries to the open arms in male offspring of non-stressed dams and female offspring of the stressed dams and to increase this factor in the female offspring of the stressed dams.\u003c/p\u003e \u003cp\u003eThere were no effects of sex or maternal stress and mirtazapine and no interactions between these factors on the time spent in the closed arms of the EPM apparatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eWith respect to the time spent in the open arms (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), there was no significant effect of any of the factors of the comparison. There was, however, significant offspring sex \u0026times; maternal CUS \u0026times; maternal mirtazapine interaction (F\u003csub\u003e1,78\u003c/sub\u003e=4.33, p\u0026thinsp;=\u0026thinsp;0.04, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test did not detect any significant difference between any specific groups of animals as well. Nevertheless, maternal mirtazapine tended to increase this variable r in the male offspring of the stressed dams and female offspring of non-stressed dams.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the expression of pro-neuroplastic proteins\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWith respect to the BDNF protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), there was a significant effect of the offspring sex (F\u003csub\u003e1,45\u003c/sub\u003e=15.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), offspring sex \u0026times; maternal mirtazapine (F1,45\u0026thinsp;=\u0026thinsp;11.74, p\u0026thinsp;=\u0026thinsp;0.001), maternal stress \u0026times; maternal mirtazapine (F\u003csub\u003e1,45\u003c/sub\u003e=34.89, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and offspring sex \u0026times; maternal stress \u0026times; maternal mirtazapine interactions (F\u003csub\u003e1,45\u003c/sub\u003e=10.76, p\u0026thinsp;=\u0026thinsp;0.002, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test detected suppressing effect of the maternal CUS on BDNF level in male offspring (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, regardless maternal mirtazapine treatment), significant difference between male and female offspring of the dams exposed to both CUS and mirtazapine (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, with the value higher in females), and a robust increasing effect of the maternal CUS and in female offspring of mirtazapine-treated dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the PSD95 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), there was a significant effect of the maternal stress (F\u003csub\u003e1,49\u003c/sub\u003e=6.05, p\u0026thinsp;=\u0026thinsp;0.02) and significant interaction between the offspring sex, maternal stress, and maternal mirtazapine treatment (F\u003csub\u003e1,49\u003c/sub\u003e=11.49, p\u0026thinsp;=\u0026thinsp;0.002, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test also unveiled a significant increasing effect of maternal stress in male offspring of the vehicle-, but not mirtazapine-treated dams (p\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e \u003cp\u003eWith respect to the GFAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), there was a significant effect of the offspring sex (F\u003csub\u003e1,46\u003c/sub\u003e=13.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and significant offspring sex \u0026times; maternal stress \u0026times; maternal mirtazapine interaction (F\u003csub\u003e1,46\u003c/sub\u003e=18.75, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test detected sex differences in the offspring of non-stressed dams treated with mirtazapine and stressed dams treated with vehicle (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002, and the value in males higher than in females in both cases), as well as an increasing effect of the maternal stress and female offspring of mirtazapine-treated dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.002).\u003c/p\u003e \u003cp\u003eRegarding the GLUR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), there was a significant offspring sex \u0026times; maternal stress \u0026times; maternal mirtazapine interaction (F\u003csub\u003e1,49\u003c/sub\u003e=11.49, p\u0026thinsp;\u0026lt;\u0026thinsp;0.002, three-way ANOVA). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test unveiled suppressing effect of the maternal CUS in male offspring of mirtazapine-treated dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational maternal CUS, prenatal mirtazapine, and their combination on the synaptophysin optical density in the hippocampus\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the synaptophysin optical density in the hippocampal \u003cem\u003eCornu Ammonis\u003c/em\u003e areas CA3 (A) and CA4 (B) and in the dentate gyrus (C). In the CA3, there was significant effect of the offspring sex (F\u003csub\u003e1,58\u003c/sub\u003e=12.03, p\u0026thinsp;=\u0026thinsp;0.001) and significant interactions between offspring sex and maternal CUS (F\u003csub\u003e1,58\u003c/sub\u003e=4.16, p\u0026thinsp;=\u0026thinsp;0.05), and maternal stress and CUS (F\u003csub\u003e1,58\u003c/sub\u003e=4.61, p\u0026thinsp;=\u0026thinsp;0.04). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test reported sex differences in the offspring of vehicle- (p\u0026thinsp;=\u0026thinsp;0.01) and mirtazapine-treated (p\u0026thinsp;=\u0026thinsp;0.02) rats, regardless of maternal CUS exposure (values in females lower compared to t males in both cases).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith respect to the CA4, there was significant effect of the offspring sex (F\u003csub\u003e1,59\u003c/sub\u003e=30.06, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and significant offspring sex \u0026times; maternal CUS interaction (F\u003csub\u003e1,59\u003c/sub\u003e=9.61, p\u0026thinsp;=\u0026thinsp;0.003). Bonferroni \u003cem\u003epost-hoc\u003c/em\u003e test detected effect of the maternal CUS on CA4 synaptophysin in females (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not in males. There was a statistically significant difference between the sex in the offspring of the stressed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not in non-stressed dams. CA4 synaptophysin levels were higher in females compared to males.\u003c/p\u003e \u003cp\u003eIn the dentate gyrus, neither the difference between the sexes nor the effect of the maternal CUS or mirtazapine, or any interaction between the factors of comparison, were observed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of glutamate neurons in the hippocampus of male and female offspring\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRepresentative recording from a hippocampal glutamate neuron is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. With respect to the mean spontaneous firing rate of the neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), there was a significant interaction between the offspring sex and maternal CUS (F\u003csub\u003e1,490\u003c/sub\u003e=12.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, three-way ANOVA). Bonferroni post-hoc test detected a sex difference in the offspring of the stressed dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.003, with the value higher in males).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the average number of the spontaneously active neurons per electrode descend (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), three-way ANOVA revealed significant offspring sex \u0026times; maternal stress interaction (F\u003csub\u003e1,150\u003c/sub\u003e=8.47, p\u0026thinsp;\u0026lt;\u0026thinsp;0.004, three-way ANOVA). Bonferroni post-hoc test detected sex differences in the offspring of the stressed dams (p\u0026thinsp;=\u0026thinsp;0.002, with the value in males higher than in females) and emphasized the increasing effect of the maternal CUS in male (p\u0026thinsp;=\u0026thinsp;0.004, regardless maternal mirtazapine treatment), but not in female offspring.\u003c/p\u003e \u003cp\u003eWith respect to the bursts\u0026rsquo; frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), there was a significant effect of the maternal stress (F\u003csub\u003e1,150\u003c/sub\u003e=6.83, p\u0026thinsp;=\u0026thinsp;0.009) and significant interaction between offspring sex and maternal stress (F\u003csub\u003e1,150\u003c/sub\u003e=6.78, p\u0026thinsp;=\u0026thinsp;0.01, three-way ANOVA). Bonferroni post-hoc test showed significant sex differences in the offspring of the stressed dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.03, with the value in males higher than in females) and suppressing effect of the maternal stress in female (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not male offspring.\u003c/p\u003e \u003cp\u003eOther characteristics of the burst firing, namely, percent of the spikes occurring in the bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and the mean number of spikes in bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF), were not affected by the maternal stress or mirtazapine treatment, neither they had sex differences.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of 5-HT neurons in the DRN of male and female offspring\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRepresentative recording from a DRN 5-HT neuron is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA. With respect to the mean spontaneous firing rate of the neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), three-way ANOVA revealed the significant effect of the offspring sex (F\u003csub\u003e1,400\u003c/sub\u003e=10.99, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Even though Bonferroni post-hoc test did not reveal any between-group differences, the values in females tended to be lower comparing to the males. When two-way ANOVA (using the maternal stress and maternal mirtazapine treatment as factors of comparison) was applied on the male offspring only, it revealed significant effect of the maternal stress (F\u003csub\u003e1,263\u003c/sub\u003e=5.22, p\u0026thinsp;=\u0026thinsp;0.02). Maternal stress increased the firing rate of 5-HT neurons in the male offspring of the vehicle-, but not mirtazapine-treated rats (p\u0026thinsp;=\u0026thinsp;0.04, Bonferroni post-hoc test).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the average number of the density of the spontaneously active neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), only a sex difference (F\u003csub\u003e1,93\u003c/sub\u003e=4.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.04, three-way ANOVA) was unveiled. The values in females tended to be lower compared to the males.\u003c/p\u003e \u003cp\u003eWith respect to the parameters of the burst firing, such as bursts\u0026rsquo; frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), percent of the spikes occurring in the bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and the mean number of spikes in bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), were not affected by the offspring sex, maternal stress nor mirtazapine treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the firing activity of dopamine neurons in the VTA of male and female offspring\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRepresentative recording from a DRN 5-HT neuron is shown on Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. Offspring sex, maternal stress or mirtazapine treatment did not affect the firing rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) and the average number of the spontaneously active neurons per electrode track (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWith respect to the bursts\u0026rsquo; frequency (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), significant effects of the offspring sex (F\u003csub\u003e1,346\u003c/sub\u003e=7.48, p\u0026thinsp;=\u0026thinsp;0.007) and maternal stress (F\u003csub\u003e1,346\u003c/sub\u003e=6.83, p\u0026thinsp;=\u0026thinsp;0.009) and significant interaction between maternal sex and maternal mirtazapine treatment (F\u003csub\u003e1,346\u003c/sub\u003e=10.59, p\u0026thinsp;=\u0026thinsp;0.001, three-way ANOVA) were observed. Bonferroni pos-hoc test revealed the suppressing effects of the maternal CUS in female offspring (p\u0026thinsp;=\u0026thinsp;0.001) and maternal mirtazapine in female offspring of stressed dams (p\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eRegarding the percentage of the spikes occurring in the bursts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), there were significant effects of the offspring sex (F\u003csub\u003e1,346\u003c/sub\u003e=36.49, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), maternal stress (F\u003csub\u003e1,346\u003c/sub\u003e=19.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and mirtazapine treatment (F\u003csub\u003e1,346\u003c/sub\u003e=6.99, p\u0026thinsp;\u0026lt;\u0026thinsp;0.009), as well as significant offspring sex \u0026times; maternal mirtazapine (F\u003csub\u003e1,346\u003c/sub\u003e=8.20, p\u0026thinsp;=\u0026thinsp;0.004), maternal stress \u0026times; maternal mirtazapine (F\u003csub\u003e1,346\u003c/sub\u003e=6.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.02), and offspring sex \u0026times; maternal stress \u0026times; maternal mirtazapine (F\u003csub\u003e1,346\u003c/sub\u003e=6.16, p\u0026thinsp;=\u0026thinsp;0.01) interactions. Bonferroni post-hoc test detected a sex difference in the offspring of the mirtazapine-treated non-stressed (p\u0026thinsp;=\u0026thinsp;0.007) and stressed dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, the value in females lower compared to the males in both cases) and suppressing effect of maternal stress in female offspring of mirtazapine-treated dams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eWith respect to the mean number of spikes in burst (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), there was a significant effect of the offspring sex (F\u003csub\u003e1,346\u003c/sub\u003e=7.49, p\u0026thinsp;\u0026lt;\u0026thinsp;0.007) and significant offspring sex \u0026times; maternal stress \u0026times; maternal mirtazapine interaction (F\u003csub\u003e1,346\u003c/sub\u003e=5.34, p\u0026thinsp;\u0026lt;\u0026thinsp;0.02). Bonferroni post-hoc test revealed a sex difference in the offspring of the stressed dams, regardless of mirtazapine treatment (p\u0026thinsp;=\u0026thinsp;0.004), with the value in females lower than in males.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of the pre-gestational CUS, perinatal mirtazapine, and their combination on the expression and activity of RyR2 channels\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExpression of RyR2 protein and opening of RyR2 channels were not affected by the offspring sex or by maternal stress or mirtazapine (see Supplementary Materials, Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe found that pregestational CUS, combined with prenatal mirtazapine, tended to have an anxiolytic effect on male and an anxiogenic effect on female offspring, measured by the EPM test. Maternal CUS increased PSD95 expression in male offspring, increased synaptophysin in females, and decreased BDNF in both sexes. In females, pregestational CUS-induced decrease in BDNF expression was reversed by perinatal mirtazapine. Combination of pre-gestational CUS and perinatal mirtazapine increased the GFAP expression in the females and decreased GLUR expression in the males. Pre-gestational CUS decreased the excitability of the hippocampal glutamate and VTA dopamine neurons; mirtazapine enhanced this effect. With respect to 5-HT neurons, maternal CUS increased their firing activity.\u003c/p\u003e \u003cp\u003eSimilarly to our previous studies\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we found that maternal pre-gestational CUS and perinatal antidepressant treatment by their own did not alter offspring behavior during the EPM test. Pre-gestational CUS, combined with prenatal mirtazapine, tended to increase these characteristics in males, suggesting an anxiolytic effect, and to decrease it in females, suggesting an anxiogenic effect.\u003c/p\u003e \u003cp\u003eIt was found that pregestational CUS decreased BDNF expression in the offspring brain. In females, but not in males, perinatal mirtazapine reversed the suppressing effect of the pre-gestational CUS on BDNF expression in the offspring brain. With respect to PSD95, maternal CUS increased its expression in the brain of the male, but not female offspring. Perinatal mirtazapine also tended to increase PSD95 in male offspring brain. Thus, statistically significant difference between the offspring of the stressed and non-stressed dams was observed only in animals prenatally exposed to the vehicle, but not to mirtazapine. Combination of pre-gestational CUS and perinatal mirtazapine, but none of these factors by their own, increased the GFAP expression in the female and decreased GLUR expression in the male offspring brain.\u003c/p\u003e \u003cp\u003eCUS may diminish BDNF expression in female offspring but not in male offspring due to sex-specific differences in stress response and brain plasticity. These differences are largely driven by hormonal influences, particularly the interaction of sex hormones like estrogen and testosterone with neurotrophic factors such as BDNF\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In females, estrogen plays a critical role in regulating BDNF expression, especially in brain areas like the hippocampus, which is sensitive to stress\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Chronic stress can disrupt the estrogen-BDNF pathway, leading to reduced BDNF levels. Males, on the other hand, have different hormonal responses and may be more resilient to the BDNF-lowering effects of stress due to testosterone or differential activation of stress-related brain circuits\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Additionally, stress may trigger different neuroinflammatory responses or epigenetic changes in males and females, contributing to the sex-specific regulation of BDNF expression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe interaction of mirtazapine with chronic stress produces sex-specific neurobiological outcomes-enhancing GFAP and astrocyte activation in females, potentially exacerbating anxiety-related behaviors, while reducing glutamate receptor levels and improving stress resilience in males, leading to anxiolytic effects. These sex differences highlight the complex interplay between stress, neuroinflammation, and neurotransmission in shaping behavioral responses.\u003c/p\u003e \u003cp\u003eIt was found that the maternal CUS increased synaptophysin optical density in the offspring hippocampus, especially, in its CA3 area. This effect was observed in female, but not in male offspring. Prenatal mirtazapine did not affect offspring hippocampal synaptophysin; neither did pregnancy with the effect of pregestational CUS.\u003c/p\u003e \u003cp\u003eThe increase in synaptophysin optical density in female but not male offspring may be due to sex-specific hormonal regulation, differential stress responses, and possible compensatory plasticity mechanisms in the female brain, which may help offset the adverse effects of maternal chronic stress.\u003c/p\u003e \u003cp\u003eChronic stress during pregnancy can induce epigenetic changes that influence brain development in offspring. These changes might alter the expression of synaptic proteins like synaptophysin in a sex-dependent manner\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In females, maternal stress might induce compensatory mechanisms, such as increased synaptic plasticity, as a way to cope with the altered brain environment, leading to higher synaptophysin levels\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. In addition, estrogen may interact with synaptic proteins like synaptophysin more prominently in female offspring. This hormone may enhance synaptic resilience to stress, potentially increasing synaptophysin expression in females but not in males, who rely more on testosterone, which might have different effects on synaptic function\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe found that maternal CUS decreased the density of the spontaneously active hippocampal neurons, their mean firing rate, and the burst mode of their firing. This effect was sex-specific and observed in female, but not in male rats. It is known that the burst firing of glutamate neurons enhances the nerve terminal neurotransmitter release, in comparison with the same amount of action potentials fired in a single-spike mode\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Pregestational stress is therefore likely to decrease hippocampal glutamate transmission in female offspring. It is thus possible that the decrease in hippocampal glutamate transmission, observed in females, but not in males, is responsible, at least in part, for the anxiogenic effect of pregestational maternal stress, that was also observed in female, but not in male offspring.\u003c/p\u003e \u003cp\u003eThe link between reduced hippocampal glutamate transmission and anxiety has been shown in previous studies. Widman and coauthors demonstrated that the low novelty response (LR) rats, a genetic breed characterized by high anxiety, had decreased hippocampal long-term potentiation (LTP) and reduced density of the dendritic spines on hippocampal pyramidal neurons\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. On the other hand, high levels of anxiety in rats experienced social defeat stress\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and in mice lacking serotonin-1A (5-HT\u003csub\u003e1A\u003c/sub\u003e) receptor\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e were associated with increased LTP and excitatory post-synaptic currents (EPSC), respectively. In our previous study, an anxiolytic effect of a selective delta-opioid receptor (DOR) agonist SNC80 was accompanied by an elevated firing rate and increased burst activity of hippocampal neurons\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt was found that the mean firing rate of the spontaneously active 5-HT neurons, as well as their density, were higher in the males comparing to the females. Similar sex-related differences in the firing activity of 5-HT neurons were observed in the previous studies from our \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and other laboratories\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSimilarly to our previous study\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, we found that pregestational CUS increased the basal firing rate of 5-HT neurons in male, but not in female offspring. Since the firing activity of the DRN 5-HT neurons determines limbic 5-HT neurotransmission\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, pregestational CUS might increase 5-HT tone in the male offspring. As it was already suggested\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, maternal CUS-induced increase in limbic 5-HT transmission in the male offspring might be a compensatory mechanism designated to prevent the negative effect of the maternal stress on the offering brain.\u003c/p\u003e \u003cp\u003ePerinatal mirtazapine tended to increase the firing activity of 5-HT neurons in male rats as well. Thus, statistically significant difference between the offspring of the stressed and non-stressed dams was observed only in animals prenatally exposed to the vehicle, but not to mirtazapine. This observation is consistent with the stimulatory effect of the sustained mirtazapine on 5-HT neurons reported in previous studies\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIt is well established that 5-HT neurotransmission is fundamental in anxiety\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Maternal and CUS and mirtazapine-induced stimulation of the offspring 5-HT neurons might be therefore consistent with the anxiolytic effects of these maternal factors on the offspring behavior during the EPM test. The presence of this compensatory mechanism in male, but not in female rats might explain the fact that the anxiolytic effect of the maternal stress and mirtazapine was observed in male, but not in female offspring.\u003c/p\u003e \u003cp\u003eWe found that pregestational CUS led to the decreased burst firing of dopamine neurons in female, but not in male offsprings\u0026rsquo; VTA. Perinatal mirtazapine robustly potentiated the effect of pregestational CUS on the burst firing of dopamine neurons in female offspring\u0026rsquo; VTA. Burst firing of dopamine neurons is associated with higher efficiency of neurotransmitter release from the nerve terminals\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Maternal CUS and mirtazapine may thus suppress dopamine neurotransmission in female offspring. Maternal CUS and mirtazapine-induced suppression of dopamine neurotransmission might be thus involved in the anxiogenic effect of these factors, that was also observed in female, but not male offspring.\u003c/p\u003e \u003cp\u003eThe involvement of the mesolimbic dopamine transmission in anxiety was indeed reported in previous studies. It was stated that postpartum anxiety and depressive-like behavior in rats were associated with attenuated population activity in the VTA\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. On the other hand, increased anxiety in a rat model of neuropathic pain was associated with elevated accumulation of dopamine levels\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Finally, we had recently described that the anxiolytic effect of DOR agonist SNC80 was accompanied by elevated firing rate and increased burst activity of the VTA dopamine neurons\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCa\u003csup\u003e2+\u003c/sup\u003e signaling in the brain plays a crucial role in neuronal viability, synaptic plasticity, and higher cognitive processes\u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e, suggesting that Ca\u003csup\u003e2+\u003c/sup\u003e mishandling contributes to various neuropsychiatric and neurodegenerative disorders\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43 CR44 CR45\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. RyR2, a key intracellular Ca\u003csup\u003e2+\u003c/sup\u003e channel, is significantly implicated in neuronal Ca\u0026sup2;⁺ signaling and several studies suggest that phosphorylation-induced increase in RyR activity may contribute to AD pathogenesis\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eImplication of RyR2 channels in pathogenesis of neuropsychiatric disorders has started to emerge only recently\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. However, the primary efforts have focused on identifying mutations in the RYR genes that encode three distinct isoforms, rather than directly investigating potential RyR dysfunction and post-translational modifications. We found that neither amount of the RyR2 channel (a dominant isoform in the brain\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e), its resting activity, or phosphorylation state was altered. Thus, we can exclude the potential contribution of this Ca\u003csup\u003e2+\u003c/sup\u003e channel in changes in neuronal excitability of the offspring caused by the pre-gestational maternal CUS, prenatal mirtazapine, and their combination.\u003c/p\u003e \u003cp\u003eIn conclusion, maternal mirtazapine did not alter anxious behavior of the offspring by its own. Prenatal mirtazapine, however, had an anxiolytic effect in male offspring in the stressed dams. Prenatal mirtazapine also tended to stimulate 5-HT neurons in male offspring; this stimulatory effect on 5-HT neurons might underline the anxiolytic effect of prenatal mirtazapine. Stimulatory effect of prenatal mirtazapine on BDNF expression in the offspring of the stressed dams might be linked with increased 5-HT neurotransmission and with decreased anxiety as well. Maternal stress and mirtazapine had a suppressing effect on the burst firing of the offspring VTA dopamine and hippocampal glutamate neurons; it might be linked with the decreased expression of the GLUR. The effects of maternal stress and mirtazapine on the burst firing of the central neurons in offspring are not mediated via mechanism(s) involving the RyR2 channel.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Katya Sketa for the technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work of was supported by the grants APVV-19-0435, APVV-20-0202, APVV-APVV-22-0061, VEGA-2/0057/22, VEGA-2/0133/23, and VEGA-2/0045/24.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRP, MVN, SB, DG, JG, MG, LL, ED and MD formulated the working hypothesis and planned the experiments, RP, MVN, SB, DG, JG, MG, RD, and HO performed the experiments, RP, MVN, SB, DG, JG, MG, RD, TK, and ED analysed the results, RP, MVN, ED, and MD wrote the manuscript, all authors proof-read the manuscript and approved it for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of interests\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnimal experiments\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Animal Health and Animal Welfare Division of the State Veterinary and Food Administration of the Slovak Republic (Permit number Ro 3592/15-221) and confirmed to the Directive 2010/63/EU of the European Parliament and of the Council on the Protection of Animals Used for Scientific Purposes.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSingh S, Fereshetyan K, Shorter S, Paliokha R, Dremencov E, Yenkoyan K \u003cem\u003eet al.\u003c/em\u003e Brain-derived neurotrophic factor (BDNF) in perinatal depression: Side show or pivotal factor? 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Proceedings of the National Academy of Sciences of the United States of America 2011; 108: 3029\u0026ndash;3034.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"prenatal depression, prenatal antidepressant treatment, anxiety, hippocampus, monoamines, brain-derived neurotrophic factor (BDNF), glutamate receptor","lastPublishedDoi":"10.21203/rs.3.rs-5448456/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5448456/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWhen accompanied by excessive exposure to the stressors, pregnancy may result in prenatal depression, that has in turn negative influence on the offspring’s brain. Mirtazapine, among other antidepressants, is commonly used to treat prenatal depression. Even though mirtazapine is generally considered safe for pregnant women, its effect on the offspring brain have not been sufficiently investigated. The present study aimed to examine the effects of chronic unpredictable stress (CUS) in pregestational rats, prenatal mirtazapine treatment, and their combination, on offspring behavior and brain function. We assessed offspring anxiety levels during the elevated plus maze (EPM) test, the expression of pro-neuroplastic proteins in the offspring brain, the excitability of brainstem monoamine and hippocampal glutamate neurons, and the expression and activity of ryanodine receptors (RyR2). Prenatal mirtazapine had an anxiolytic effect on the offspring of the stressed dams. This effect was associated with an increased excitability of serotonin (5-HT) neurons and elevated expression of the brain-derived neurotrophic factor (BDNF). Regarding the offspring glutamate and dopamine neurons, the combination of maternal stress and mirtazapine inhibited their burst firing, potentially due to decreased expression of the glutamate receptors. Even though calcium signaling is important for the burst firing of the neurons, the effects of maternal stress and mirtazapine on the burst activity of the offspring glutamate and dopamine might not be mediated via mechanism(s) involving the RyR2. Summarizing, mirtazapine may diminish the negative influence of maternal stress and depression on the offspring brain, via mechanism(s) putatively involving 5-HT neurotransmission and BDNF.\u003c/p\u003e","manuscriptTitle":"Effects of pre-gestational exposure to the stressors and perinatal mirtazapine administration on the excitability of hippocampal glutamate and brainstem monoaminergic neurons, hippocampal neuroplasticity, and anxiety-like behavior in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-09 16:15:51","doi":"10.21203/rs.3.rs-5448456/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-01-31T14:25:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-01-25T17:29:07+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-01-21T21:42:24+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-01-09T06:10:26+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-01-08T18:14:03+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-01-07T11:08:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-19T12:31:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-18T11:50:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2024-11-15T12:01:08+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2024-11-14T11:18:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"40db5e5a-d636-4a50-9be9-07e8654632af","owner":[],"postedDate":"January 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":42490360,"name":"Biological sciences/Neuroscience"},{"id":42490361,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2025-08-26T07:11:10+00:00","versionOfRecord":{"articleIdentity":"rs-5448456","link":"https://doi.org/10.1038/s41380-025-03161-3","journal":{"identity":"molecular-psychiatry","isVorOnly":false,"title":"Molecular Psychiatry"},"publishedOn":"2025-08-25 04:00:00","publishedOnDateReadable":"August 25th, 2025"},"versionCreatedAt":"2025-01-09 16:15:51","video":"","vorDoi":"10.1038/s41380-025-03161-3","vorDoiUrl":"https://doi.org/10.1038/s41380-025-03161-3","workflowStages":[]},"version":"v1","identity":"rs-5448456","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5448456","identity":"rs-5448456","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}