Individual differences in the chronic stress-induced depression-like behavior are mediated by the expression of Kcnj2 gene in the lateral septum

Individual differences in the chronic stress-induced depression-like behavior are mediated by the expression of Kcnj2 gene in the lateral septum | 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 Individual differences in the chronic stress-induced depression-like behavior are mediated by the expression of Kcnj2 gene in the lateral septum Masayoshi Okada, Marcel AG van der Heyden This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8030199/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract It has been demonstrated that chronic unpredictable mild stress (CUMS) induces depression-like behaviors in mice. Notably, there are differences among individuals in the extent to which these behaviors are exhibited, similar to humans. In this study, we focused on individual differences and examined which brain areas have differences in neuronal excitability, and which genes are differentially expressed between stress-susceptible mice with depression-like behaviors and stress-resilient mice without depression-like behaviors (Stress-S and -R). After 8 weeks of CUMS, the number of c-Fos positive cells was reduced in Stress-R mice compared to Stress-S mice only in the cingulate cortex and lateral septum. RNA-seq analysis revealed an upregulation of Kcnj2 mRNA, the gene for the inward-rectifying potassium channel (Kir2.1), in the lateral septum of Stress-R mice, without affecting the expression of other potassium channels. The expression of genes related to adult neurogenesis, neuroinflammation, hypothalamic-pituitary-adrenal axis, BDNF, and monoamine hypotheses were similar between Stress-S and -R mice. Intra-septal injections of Kir2.1 activators, flecainide, and GPV0057, showed antidepressant effects in the tail-suspension test. These findings suggest that the Stress-R behavior is the result of the upregulation of Kir2.1 and decreased neuronal excitability in the lateral septum. They also suggest the uniqueness of this model and the potential to reveal new mechanisms. Biological sciences/Genetics Biological sciences/Neuroscience Chronic stress depression Kir2.1 lateral septum c-Fos RNA-Seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Major depression is a common mood disorder largely triggered by chronic stress [ 1 ]. Notably, from experience, we know that not everyone who experiences stress develops depression, and that people differ in their susceptibility to stress. The mechanism that distinguishes stress resilience from susceptibility is unclear. Patients with depression are mostly treated with antidepressants, such as selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and noradrenergic and specific serotonergic antidepressant (NaSSA), which enhance the transmission of monoamines. However, according to the STAR*D study, conducted by the American National Institute of Mental Health, current drugs are ineffective in 30% of patients with major depression [ 2 ]. This suggests the presence of mechanisms of onset that are different from monoamine transmission. It is noteworthy that neurostimulation therapies, such as electric convulsive therapy, deep brain stimulation, and transcranial magnetic stimulation, are effective in some drug-resistant depression [ 3 , 4 ]. It is assumed that the neuronal excitabilities of some brain regions are dysregulated in patients: these neurostimulation therapies compensate for the dysregulated neuronal excitability. Since major depressive episodes are caused by stressful life events [ 1 ], stress may lead to dysregulated neural excitability in a specific area, leading to depression. This dysregulation may result from changes in the expression of genes that control neuronal excitability. Intrinsic neuronal excitability is mostly controlled by K⁺ channels, which control the resting membrane potential and the frequency and shape of the action potential. There are 77 subtypes of K⁺ channels, which are classified into four types according to their number of transmembrane domains and opening features: two-pore domain, voltage-gated, Ca²⁺-activated, and inwardly rectifying potassium channels [ 5 ]. Those channels are widely expressed in the central nervous system, but their roles in stress resilience and depression remain largely unknown. Recent studies have suggested the involvement of K⁺ channels in mood disorders. For instance, single nucleotide polymorphisms (SNPs) associated with some K⁺ channel loci, and both up-and down-regulations of K⁺ channel mRNAs from brain samples of depression [ 6 ]. A mutation in a K⁺ channel (kcnh7) resulted in familial bipolar disorder [ 7 ]. These findings suggest that dysregulation in K⁺ channels result in pathogenesis and that modulation of the channel involved could potentially have therapeutic effects. Indeed, some modulators for K⁺ channels (Kcnq and Kcnk2) successfully showed an antidepressive effect in mice [ 8 , 9 ]. We also demonstrated that ostruthin, a two-pore domain K⁺ channel (TREK-1, Kcnk2) activator, exhibits antidepressant and anxiolytic properties [ 10 ], and an inwardly rectifying potassium channel (Kir1.1, Kcnj1) blocking peptide, tertiapin-RQ, exhibited antidepressive and anxiogenic effects [ 11 ]. Interestingly, these two antidepressant compounds suppressed the neural activities in the lateral septum, suggesting an involvement of this region in the antidepressant effects. Based on these findings, we hypothesized that chronic stress alters K⁺ channel expression in specific brain areas, resulting in abnormal excitability and, thereby, depression-like behavior. Since individual differences have been noted in the depression-like behavior among the mice under chronic stress [ 12 ], we focused on individual differences in stress-induced depression-like behavior and analyzed the brain regions whose neuronal excitability differed between stress-susceptible (S) and -resilient (R) mice. We then identified a K⁺ channel gene, Kcnj2, whose mRNA expression was upregulated in the Stress-R mice, and the antidepressant effects of the channel activators. RESULTS Individual differences in depression-like behavior and classification to Stress-S and -R mice Chronic unpredictable mild stress (CUMS) is considered as mimicking human depression in some aspects [ 13 ], and individual differences in the depression-like behavior induced by CUMS was shown to be useful for the examination of the mechanisms of the depression like behavior [ 12 ]. As we hypothesized that chronic stress causes a difference in the neuronal excitability and, consequently, a difference in behavior, we subjected male ICR mice to CUMS for 8 weeks and then analyzed the depression-like behavior in forced swim test (FST) and tail suspension test (TST) (Fig. 1 ). CUMS treatment significantly increased the immobile time in FST (Fig. 2 A) and insignificantly increased that in the TST (Fig. 2 B), compared with those of control (unstressed) mice. These results suggest that the CUMS-treatment had the expected effect, i.e., depressive effect, on the mice’s behavior. Among CUMS-treated mice, some showed a depressed phenotype, in which the immobile time were more than 100 seconds, but others showed a resilient phenotype, in which the immobile time were shorter than the average time of control mice. These findings suggest the presence of individual differences in this model. The individual differences in chronic stress-sensitive, depression-like behavior in CUMS-treated mice were further analyzed with novelty-suppressed feeding (NSF) and sucrose preference test (SPT). Table 1 shows the raw data of a round of four tests and the median values of each test. The values exhibited more depression-like behavior than the median are shaded in gray. Then we classified the stressed mice into three groups: those with three grays were classified as Stress-S mice, and those with one gray were classified as Stress-R mice. Mice with two grays were grouped into the middle category and were not subjected to further analysis. Table 1 An example of classification of Stress-S and -R mice. This table shows the raw data of four behavioral tests of each mouse in a CUMS round. The slots showing results as more depressed than the median for each mouse's behavioral test are shaded gray. The mice were classified based on the number of gray slots. The classification was repeated on a total of 36 mice, which were divided into three groups: 11 Stress-S mice, 13 Stress-R mice, and 12 Middle mice. Middle mice were not analyzed. Mouse # FST TST NSF SPT Classfication 1 210 153 56 58 Stress-S 2 105 53 68 63 Stress-R 3 120 30 80 68 Middle 4 132 28 69 90 Middle 5 110 99 98 65 Stress-S 6 113 127 99 90 Middle 7 182 70 13 23 Stress-S 8 207 60 58 70 Stress-R 9 97 10 123 48 Middle 10 37 1 41 63 Stress-R 11 94 68 28 90 Stress-R 12 155 99 108 89 Stress-S Median 116.5 64 68.5 67 Middle Reduced neuronal activities in the lateral septum and cingulate cortex of Stress-R mice To determine which brain area is involved in the individual differences in susceptibility to chronic stress, brain slices from control, Stress-S, and -R mice (n = 10, 7, 9) were immunostained with an anti-c-Fos antibody, which is a marker of neuronal excitability. The control mice were not stressed but experienced FST and TST, which were the first and acute stressful experience for them, on the previous and last day. The mouse brains were fixed two hours after TST. We examined anti-c-Fos immunoreactivity in the brain areas that are known to be involved in the depression-like behavior and stress, i.e., hippocampal dentate gyrus (DG), dorsal raphe nucleus (DRN), locus coeruleus (LC), lateral habenular nucleus (lHB), hypothalamic paraventricular nucleus (PVN), thalamic paraventricular nucleus (PVT), cingulate cortex (CG), and lateral septum (LS). Because the ventral part of LS is more involved in the regulation of emotional behaviors than the dorsal part, we only analyzed the ventral part. There were no significant differences in the c-Fos-immunoreactive cell number among Control, Stress-S, and Stress-R groups in the DG, DRN, LC, lHB, or PVN. Significant changes were found in the CG and LS (Fig. 3 ). In these regions, the number of c-Fos-positive cells was similar between the Control and Stress-S mice. However, the number of positive cells in Stress-R mice was significantly reduced, suggesting that the observed individual differences in this model resulted from decreased neuronal excitability in these regions. c-Fos-positive cells were significantly increased in the Control mice compared to Stress-S and -R mice in the PVT, but there were no differences between Stress-S and -R mice, excluding the involvement in the individual differences (Fig. 4 ). Since PVT is known to quickly respond to acute stress, this increase in the c-Fos expression may be attributable to the fact that the FST and TST were the first stress experience to the control mice whereas Stress-S and R had experienced it for weeks already. RNA-seq analysis revealed increased expression of an inwardly rectifying K⁺ channel gene, kcnj2, in the lateral septum Intrinsic neuronal activity is mainly controlled by K⁺ channels, and some K⁺ channel modulators have antidepressant effects, through modulation of neuronal activities. Especially, in our previous studies, two K⁺ channel modulator compounds, ostruthin and tertiapin-RQ, were found to have antidepressant effects and decreased the neuronal excitabilities in the LS [ 10 , 11 , 14 ]. Therefore, we compared the expression of mRNAs in the LS of Stress-S and -R mice for various K⁺ channels with RNA-Seq analysis (n = 4 and 4). We found an increase in expression in the Kcnj2 gene, which encodes an inwardly rectifying K⁺ channel (Kir2.1), in the LS of Stress-R mice compared to Stress-S mice (Fig. 5 A and C). The expressions of other channels, Kcnj9 and Kcnh4, were also changed, but the difference was insignificant (Fig. 5 D) and the expression level was very low (Fig. 5 E). The expression levels of the majority of K⁺ channel genes, including Kcnk2, Kcnq2, and Kcnq3, which were reported to be involved in antidepressant effects [ 10 , 11 , 15 , 16 ] remained at the same level (Fig. 5 F, G, and H). The expression of Kcnh7, which was reported to be the causative gene for familial bipolar disease [ 7 ], remained the same (Fig. 5 I). RNA-Seq revealed little differences in the expression of genes associated with known hypotheses regarding depression We also analyzed the other genes that are present in the known hypotheses for depression, i.e., hypothalamus-pituitary-adrenal (HPA)-axis, neuroinflammation, ketamine NMDA receptor, adult neurogenesis, and Bdnf and its receptor Ntrk2. There were no significant differences between Stress-S and -R mice (Fig. 6 A and C). The most well-known hypothesis for depression is the monoamine hypothesis: depression is caused by a deficiency in the brain's monoamine neurotransmitters, serotonin, norepinephrine, and dopamine, and antidepressant medications are believed to enhance these neurotransmissions, thereby alleviating symptoms. The expression levels of monoamine-degrading enzymes, Comt, Maoa and Maob, remained the same (Fig. 6 B and D). The expression levels of monoamine synthesizing enzymes, Th (tyrosine hydroxylase) and Tph (tryptophan hydroxylase), exhibited no significant increase and remained at low levels. The expression of Slc6a4 (serotonin transporter) was below the cut-off level. The expression level of most receptors for 5-HT, norepinephrine, and dopamine remained constant. But those of Drd2 and Drd1 (dopamine D₂ and D₁ receptors) were increased significantly and insignificantly (p = 0.06), respectively, in Stress-R mice. A similar increase in 5-HT1b (5-HT receptor 1b) was also observed in the Stress-R mice. These increases may be implicated in Stress-R behavior, at least to some extent. Involvement of inwardly rectifying K⁺ channel in the antidepressive behavior To confirm the involvement of the Kir2.1 channel in individual differences in depression-like behavior, we injected 1 µL of Kir2.1 channel activators, flecainide (2 mM) and GPV0057 (5 mM), and blockers, ML133 (2 mM) and PA-6 (10 mM), into the septum of mice. The administration of these agents are supposed to mimic the upregulation and downregulation of the Kir2.1 channel expression, respectively. Both administrations of flecainide showed significant antidepressant effects on TST compared with PBS injection (n = 15 and 15, Fig. 7 A). Similarly, the administration tended to decrease the immobile time in FST (Fig. 7 B). Consistently, the administration of GPV0057 decreased the immobile time in TST and tended to in FST (n = 15 and 15, Fig. 7 C and D). The administration of a Kir2.1 channel blocker ML133, showed only an insignificant increase the immobile times of TST and FST (n = 14 and 16, Fig. 7 E and F). Similarly, PA-6 did not show depressant effects in both tests (n = 14 and 16, Fig. 7 G and H). Administrations of fivefold doses of the compounds above via intracerebroventricular injection elicited similar results. The administrations of the channel blockers exhibited only an insignificant effect on depression-like behavior, likely attributed to the low baseline expression of the channel gene, which consequently restricts efficacy of channel blockade. Therefore, these findings suggest a crucial role for upregulation of functional Kir2.1 in the Stress-R mice, rather than the downregulation in the Stress-S mice. DISCUSSION In this study, genetically homogeneous closed colony-bred mice showed individual differences in depression-like behavior in response to chronic stress, as humans do. We then examined mRNA expression in the ventral lateral septum according to the difference in the neuronal excitability between Stress-S and -R mice. RNA-seq analysis revealed that expression of the Kir2.1 channel gene (Kcnj2) was increased in Stress-R mice, potentially suppressing neural excitability. Indeed, the overexpression of Kir2.1 channels is a well-established method of artificially suppressing neuronal excitability [ 17 , 18 ]. In support, the administration of Kir2.1 channel activators exhibited antidepressant effects in TST. These results suggest that the upregulation of Kcnj2 gene led to Stress-R behavior through the suppression of neural activity in the lateral septum. This hypothesis that the Kcnj2 gene plays a role in depression-like behavior is supported by previous human studies. A meta-analysis of gene expression profiles revealed decreased Kcnj2 expression in the postmortem brains of individuals who died by suicide [ 19 ]. A heterozygous missense mutation (Thr192Ile) in the Kcnj2 gene has been associated with major depression, as well as Andersen-Tawil syndrome [ 20 ]. Reportedly, the expression of Kir2.1 was increased in the hippocampal granule neurons, which became epileptic by the injection of kainic acid, to compensate for the higher excitability [ 21 ]. It was also reported that neurons can alter the expression of various ion channels to maintain homeostatic stability in neural activity [ 22 ]. When excessive stress information is input in the form of neural excitability, differences in the ability to alter K⁺ channel expression as a compensatory mechanism may have led to individual differences in depressive-like behavior as shown here. The expression of mRNA for dopamine D₂ receptors increased, and the expression of mRNA for D₁ receptors tended to increase as well. Similar increases in D 2 receptor mRNA expression were observed in Stress-R mice in the mesolimbic region after five weeks of chronic stress, but not after two weeks [ 23 ]. Dopamine receptor partial agonists show therapeutic effect on major depression [ 24 ], these increases might be involved in the Stress-R behavior, improving the depressive symptoms like anhedonia and low motivation. The expression of serotonin 5-HT 1b receptor was also increased in the Stress-R mice. The relationship between 5-HT 1b receptor expression and depressive-like behavior is not clear [ 25 ]. Both agonists and antagonists have demonstrated antidepressant effects in animal studies. Furthermore, postmortem brain studies in humans revealed decreased mRNA expression of this receptor in the hippocampus and frontopolar cortex compared with control subjects. Low 5-HT 1B receptor binding in limbic regions has been found in patients with depression. The decreased expression observed in Stress-S mice may lead to depression-like behavior. A recent study has shown a rapid antidepressant effect of a selective 5-HT 1b receptor agonist, CP-94253, in FST [ 26 ]. The increase in expression might also contribute to the Stress-R phenotype through a similar mechanism. The involvement of the lateral septum in depression-like behavior has been documented for over two decades; however, the specific roles of the lateral septum in depressive-like behavior remain a subject of controversy [ 27 ]. A recent elegant study examining individual differences in reward-seeking behavior following social defeat stress in female mice suggests that the antidepressant effects are attributable to the inhibition of the neurons [ 28 ]. Given that the role of the lateral septum is to mediate adverse information [ 29 ], its suppression is predicted to impede the transmission of stress information, thereby inducing stress-R behavior. Wirtshafter and Wilson [ 30 ] theorized that the lateral septum functions as a central nexus, thereby serving as a focal point for the aggregation of information that governs mood, anxiety, and motivation. In fact, the neurons in non-treated mice that were only anesthetized were c-Fos-negative, but became positive after the acute stress of TST [ 10 ]. Notably, there were little differences in the expression of genes associated with other well-known depression hypotheses, i.e., adult neurogenesis, BDNF, HPA-axis, neuroinflammation, and ketamine NMDA receptor. Previous studies reported that chronic stress induced changes in expression of these mRNAs or proteins in other areas, e.g., reduction in BDNF mRNA in the hippocampus [ 31 ]. These findings suggest that the possible mechanism of the onset of Stress-S and -R behaviors in this model is different from previous ones. The etiology of major depression is understood to be genetically heterogeneous [ 32 ], suggesting the presence of novel mechanisms of onset. Consequently, the lack of difference in the expression of depression-associated genes suggests the uniqueness of this mouse model. This may be useful for the identification of novel genes and the elucidation of previously unrecognized mechanisms underlying the onset of depression. In this study, we used male mice of a closed colony strain (Slc:ICR). The genetic information of those mice was supposed to be highly similar. The observed individual differences in behavior may be attributable to epigenetic modifications of the genome. For instance, epigenetic differences in the genomic DNA methylation may lead to individual differences in the Kir2.1 channel expression, which can result in alterations in excitability and behavior. Indeed, individual differences in DNA methylation were shown between human monozygotic twins [ 33 ]. Alternatively, individual differences in the acetylation/deacetylation of histone proteins may also be involved. METHODS Experimental design and typical schedule of CUMS Figure 1 describes the experimental design of this study. Male ICR mice (5-week-old) were purchased from Shimidzu Laboratory Supplies (Kyoto, Japan). They had free access to food and water and were kept in a 12-h light/dark cycle (08:00–20:00). The animals were maintained in groups in plastic cages except for the last week of CUMS, in which they were individually housed in small cages. Experiments were conducted in a quiet and air-conditioned room (23 ± 2℃). Animal experiments were carried out following the guidelines of the Physiological Society of Japan and The ARRIVE guidelines 2.0. All experiments were approved by the Committee on Animal Experiments at Kurashiki University of Science and the Arts. After one week of adaptation, mice were subjected to CUMS for 8 weeks and underwent behavioral tests according to the previous methods [ 34 ]. The stressors included: swimming in a 2 L beaker of water at a temperature of 27℃ for 10 min, confinement in a breathable tube for 1 hour, food and water deprivation for overnight (14 hours), exposure to wet bedding (250 mL water and soggy sawdust) for 3 hours, shaking of the cage (reciprocal movement of 3–4 cm at 120 rpm) for 1 hour, tilting of the cage at a 45° angle for 1 hour, exposure to an inverted light and dark cycle (from 8:00–20:00 to 20:00–8:00) for 48 hours, and exposure to strobe light (120 flashes/min) for 3 hours. Control mice were kept in enriched cages with cardboard tubes and weighed daily. A typical CUMS schedule is shown in Fig. 1 , and the combinations of stressors were changed weekly. Tests for depression-like behavior and classification of the CUMS-treated mice into Stress-S and -R mice Depression-like behaviors were examined with four behavioral tests: tail-suspension test (TST), forced swim test (FST), sucrose preference test (SPT), and novelty-suppressed feeding (NSF). Each behavioral test was performed according to a method reported previously [ 10 , 11 , 14 ] Novelty-suppressed feeding (NSF) On the two days before the last, food and water were deprived at 20:00. On the next morning, the test mouse was placed in a corner of the arena and allowed to explore the open field (90 × 90 × 45 cm), in which food pellets were placed on filter paper (10 cm in diameter) at the center of the arena until the mouse approached and took the first bite of the chow or certain cut-off time (5 min) was reached. Sucrose preference test (SPT) Mice were initially acclimated to sucrose solution, i.e., free choice of drinking either 1% (w/v) sucrose solution or tap water three times in the week before the last and in the last week. After the NSF test, consumption of sucrose solution and water for 24 h was then estimated by measuring the difference in weight of the respective bottles before and after. The percentage of sucrose preference was calculated according to the following formula: sucrose preference (%) = sucrose intake / (sucrose intake + water intake) * 100. Forced swim test (FST) After the NSF, mice were put back in the original cage and had free access to food and water/sucrose water for 3–4 hours. Then a mouse was placed into a 2 L beaker containing 12 cm of water (27℃). The mouse was allowed to swim for 6 min. The last 4 min of data were used for analysis. Immobility is defined as the absence of activity, such as escape-oriented behaviors. The slow movement of the forelimbs only for breathing was considered immobility. Tail suspension test (TST) On the last day, the mouse was hung on a hook using adhesive tape placed 1 cm from the extremity of its tail 50 cm above the table. The amount of time spent immobile was recorded for 5 min. Classification to Stress-S and -R mice Three rounds of CUMS treatment and behavioral testing were conducted. Subsequently, brains were used for c-Fos immunostaining (n = 24) and RNA-seq (n = 12). Table 1 shows the raw data of a round and the median values of each test of a round. Values that exhibited more depression-like behavior than the median are shaded in gray. Then, mice with three grays were classified as Stress-S, whereas those with zero or one gray were classified as Stress-R mice. Mice with two grays were not analyzed further. Anti-c-Fos immunostaining Two hours after being subjected to the TST, the mouse was anesthetized with isoflurane (4%, 400 mL/min) using a vaporizer (NARCOBIT-E, KN-1071-I, Natsume, Tokyo, Japan). After confirming that the mouse was deeply anesthetized, the brain was fixed by transcardiac perfusion with 4% paraformaldehyde in PBS. The brains were sliced coronally (75 µm in thickness) using a microslicer (PRO7, Dosaka, Kyoto, Japan). The free-floating slices were reacted with an anti-c-Fos antibody (1: 200, rabbit mAB (9F6), Cell Signaling Technology, MA) diluted in PBS containing 0.3% BSA and 0.3% Triton-X-100, at 4˚C for 48 h. The immunoreaction was visualized using a secondary antibody (Vectestain Universal Elite ABC Kit, peroxidase, Vector Labs, Burlingame, CA) and 3,3’-Diaminobenzidine (DAB; Tokyo Chemical Industry, Tokyo, Japan) at room temperature in accordance with the manufacturer’s instructions. For statistical analysis, the number of c-Fos immunoreactive cells was quantified using ImageJ software. Briefly, fields (0.16 mm²) of c-Fos immunoreactivity were acquired using a microscope with a CCD camera. DAB-positive nuclei were considered as anti-c-Fos-immunoreactive neurons. RNA-Seq analysis Two hours after the behavioral tests, mice were anesthetized with isoflurane (4%, 400 mL/min) and decapitated with scissors. The brains were removed, frozen with dry ice powder, and stored at -80°C. Semi-melted brains were mounted in a brain slicer (Muromachi Kikai, Kyoto, Japan) and sliced to a thickness of 2 mm. The lateral septum was cut out with a scalpel and immersed in TRIzol (Thermo Fisher Scientific, Waltham, MA). Total RNA was purified using the SV Total RNA Isolation System (Promega, Fitchburg, WI) according to the manufacturer's instructions. The RNA samples were quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the quality was confirmed using a Tapestation (Agilent Technologies, Santa Clara, CA). Sequencing libraries were prepared from 200 ng of total RNA using an MGIEasy RRNA Depletion Kit and an MGIEasy RNA Directional Library Prep Set (MGI Tech Shenzhen, China), following the manufacturers' instructions. The libraries were sequenced on the DNBSEQ-G400 FAST sequencer with a paired-end 150-nt strategy. All sequencing reads were trimmed for low-quality bases and adapters using Trimmomatic (v. 0.38). We estimated raw reads (counts) for each gene in each sample using RSEM version 1.3.0 and Bowtie 2. We used the edgeR program to detect differentially expressed genes. The gene counts were normalized to reads per million (RPM), and p-values were estimated using likelihood analysis. Genes with p < 0.05 were considered differently expressed genes. Genes, of which the read number/sample is < 1, were considered negative. Behavioral effects of the intra-septal injection of the activators and blockers for Kir2.1 channel The effect of the activators and blockers for Kir2.1 channel, which mimic the effect of upregulation and downregulation of the Kcnj2 gene, on the depression-like behaviors was examined with TST and FST after the injection of these drugs into the septum in accordance with our previous study [ 11 , 14 ]. The Kir2.1 channel activators (Flecainide [ 35 ], GPV0057 [ 36 ]) and blockers (ML133 [ 37 ], PA-6 [ 38 ]) were first dissolved in dimethyl sulfoxide (DMSO) at higher concentration and were diluted with phosphate-buffered saline (PBS) by vigorous mixing for several minutes. An equal volume of DMSO was added to PBS as a control. The concentrations of intraseptal injections were 2 mM (Flecainide), 5 mM (GPV0057), 2 mM (ML133), and 10 mM (PA-6). Male ICR mice (5-week-old) were adapted, only being weighed, for a week, and then subjected to the behavioral tests, without being chronically stressed. After confirming that the mouse was deeply anesthetized (isoflurane 2–4%, 400 mL/min), we injected drugs into the septum (1 µL/mouse) over 60 seconds (coordinates: 0 mm caudal to bregma, 0 mm lateral to the midline, and 3.5 mm below the skull), using a microsyringe (MS-N05(5 µL) 28G needle, ITO Corp., Fuji, Shizuoka, Japan). The injection needle was kept for 30 seconds after the injection. The dosage was set such that, even if each compound was distributed throughout the whole brain (0.5 mL), concentration would exceed the EC 50 and IC 50 values. Then the mouse was returned to its home cage. Each animal was subjected to TST and FST 30 and 50 min after injection, respectively. Statistical analysis Data are given as the mean ± SEM. The normality of data distribution was confirmed with the Shapiro-Wilk test. Statistical significance between two groups was determined using Student’s t-test. Those obtained from three or more groups were analyzed statistically by one-way analysis of variance (ANOVA) followed by Bonferroni test. A p-value of < 0.05 was considered significant. The number of asterisks indicates the p values: *, p < 0.05; **, p < 0.01; ***, p < 0.005. Declarations Funding MO was supported by KAKENHI (22K11818), Kobayashi Foundation, Ryobi Teien Memorial Foundation, and Wesco Scientific Promotion Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Ethics declarations Competing interests The authors have declared that no competing interests exist. Ethical approval The animal ethics were approved by the Committee on Animal Experiments at Kurashiki University of Science and the Arts, in accordance with the guidelines of the Physiological Society of Japan and The ARRIVE 2.0 ( https://arriveguidelines.org ). Consent for publication All authors consented to the submission and publication of this study. Data availability All data used in this study were available in the original research. Data generated in this study were included in the main text and supplementary files. The datasets generated and/or analyzed during the current study are available on the website ( https://osf.io/kmvsg/overview ) can be obtained by contacting the corresponding author. Author Contribution MO carried out experimental design, investigation, analysis, and writing. MH did initial characterization of the Kir2.1 modulating compounds and writing. Acknowledgement The authors thank R. Kawakami, T. Ono, and N. Sato, who have done the pilot experiments of this study, and Dr. G. Ecker (Vienna University) for synthesizing GPV0057. Data Availability All data used in this study were available in the original research. Data generated in this study were included in the main text and supplementary files. The datasets generated and/or analyzed during the current study are available on the website (https://osf.io/kmvsg/overview) can be obtained by contacting the corresponding author. References Hammen, C. Stress and depression. Annu. Rev. Clin. Psychol. 1 , 293–319 (2005). Dibernardo, A. et al. Humanistic outcomes in treatment resistant depression: a secondary analysis of the STAR*D study. BMC Psychiatry . 18 , 352 (2018). Ressler, K. J. & Mayberg, H. S. Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic NIH Public Access Author Manuscript. Nat. Neurosci. 10 , 1116–1124 (2007). Mayberg, H. S. et al. 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Antidepressive effect of an inward rectifier K + channel blocker peptide, tertiapin-RQ. PLoS One . 15 , 1–22 (2020). Ebner, K. & Singewald, N. Individual differences in stress susceptibility and stress inhibitory mechanisms. Curr. Opin. Behav. Sci. 14 , 54–64 (2017). Antoniuk, S., Bijata, M., Ponimaskin, E. & Wlodarczyk, J. Chronic unpredictable mild stress for modeling depression in rodents: Meta-analysis of model reliability. Neurosci. Biobehav Rev. 99 , 101–116 (2019). Okada, M. & Tran, T. T. T. Effect of chronic administration of ostruthin on depression-like behavior in chronically stressed mice. IBRO Neurosci. Rep. 16 , 622–628 (2024). Heurteaux, C. et al. Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype. Nat. Neurosci. 9, 1134–1141 (2006). (2006). Tan, A. et al. Effects of the KCNQ channel opener ezogabine on functional connectivity of the ventral striatum and clinical symptoms in patients with major depressive disorder. Mol. Psychiatry . 25 , 1323–1333 (2020). Burrone, J., O’Byrne, M. & Murthy, V. N. Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons. Nature 420 , 414–418 (2002). Okada, M. & Matsuda, H. Chronic lentiviral expression of inwardly rectifying K + channels (Kir2.1) reduces neuronal activity and downregulates voltage-gated potassium currents in hippocampus. Neuroscience 156 , 289–297 (2008). Piras, I. S. et al. A review and meta-analysis of gene expression profiles in suicide. Eur. Neuropsychopharmacol. 56 , 39–49 (2022). Chan, H. F. et al. A novel neuropsychiatric phenotype of KCNJ2 mutation in one Taiwanese family with Andersen-Tawil syndrome. J. Hum. Genet. 55 , 186–188 (2010). Young, C. C. et al. Upregulation of inward rectifier K+ (Kir2) channels in dentate gyrus granule cells in temporal lobe epilepsy. J Physiol 587 , (2009). Marder, E. & Prinz, A. A. 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Regulation of affect by the lateral septum: Implications for neuropsychiatry. Brain Res. Rev. 46, 71–117 at (2004). https://doi.org/10.1016/j.brainresrev.2004.04.009 Li, L. et al. Social trauma engages lateral septum circuitry to occlude social reward. Nature 613 , 696–703 (2023). Mongeau, R., Miller, G. A., Chiang, E. & Anderson, D. J. Neural correlates of competing fear behaviors evoked by an innately aversive stimulus. J. Neurosci. 23 , 3855–3868 (2003). Wirtshafter, H. S. & Wilson, M. A. Lateral septum as a nexus for mood, motivation, and movement. Neurosci. Biobehav. Rev. 126, 544–559 at (2021). https://doi.org/10.1016/j.neubiorev.2021.03.029 Smith, M. A., Makino, S., Kvetnansky, R. & Post, R. M. Stress and glucocorticoids affect the expression of brain-derived neurotrophic factor and neurotrophin-3 mRNAs in the hippocampus. J. Neurosci. 15 , 1678–1777 (1995). Nguyen, T. D. et al. Genetic Contribution to the Heterogeneity of Major Depressive Disorder: Evidence From a Sibling-Based Design Using Swedish National Registers. Am. J. Psychiatry . 180 , 714–722 (2023). Watanabe, M., Honda, C. & Iwatani, Y. Within-pair differences of DNA methylation levels between monozygotic twins are different between male and female pairs. BMC Med. Genomics . 9 , 55 (2016). Wen, G. et al. Regulation of tau protein on the antidepressant effects of ketamine in the chronic unpredictable mild stress model. Front. Psychiatry . 10 , 447263 (2019). Caballero, R. et al. Flecainide increases Kir2.1 currents by interacting with cysteine 311, decreasing the polyamine-induced rectification. Proc. Natl. Acad. Sci. U. S. A. 107, 15631–15636 (2010). Li, E. et al. Development of new Kir2.1 channel openers from propafenone analogues. Br. J. Pharmacol. 182 , 633–650 (2025). Wang, H. R. et al. Selective inhibition of the K(ir)2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR, and pharmacological characterization of ML133. ACS Chem. Biol. 6 , 845–856 (2011). Takanari, H. et al. Efficient and specific cardiac IK₁ inhibition by a new pentamidine analogue. Cardiovasc. Res. 99 , 203–214 (2013). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8030199","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":554520915,"identity":"59a28a2c-5896-444c-a7ff-28568a6a3d45","order_by":0,"name":"Masayoshi Okada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIie3QMQrCMBiG4a8EOqV0VQQ9gVAJCILYq1gKdRUcFSwI6aarg97BIwiBdCm6FhxdOzi5WMQoCE5RN5E8gZAhL+QPYBg/yQYougBR68n7IIm+T8T9RPQXn5oJD07FZO+7CQijZQ9ussVoqEnamRTVlTwESwESOjxEJeuDLXVJPohr1D70PTWLcGL1uBxgVJ/MLvS68x8JLadovE8iWXP41tqoJKS2gPc2yWTUWc9DNYs1a615SltZEOtnSTnLi3PPdxdCVIpyXK+nQjLdj72w4vuunmRx9lnxghy/TgzDMP7ZDblKROfCYC0bAAAAAElFTkSuQmCC","orcid":"","institution":"Kurashiki University of Science and the Arts","correspondingAuthor":true,"prefix":"","firstName":"Masayoshi","middleName":"","lastName":"Okada","suffix":""},{"id":554520917,"identity":"7862170c-3d2a-4dac-86af-f9d64283fa5a","order_by":1,"name":"Marcel AG van der Heyden","email":"","orcid":"","institution":"University Medical Center Utrecht","correspondingAuthor":false,"prefix":"","firstName":"Marcel","middleName":"AG van der","lastName":"Heyden","suffix":""}],"badges":[],"createdAt":"2025-11-04 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18:43:43","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95247,"visible":true,"origin":"","legend":"","description":"","filename":"2db37bcfa6f149d9b884e7d857068d151structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/6b4b5e2503a3135da3638ed4.xml"},{"id":97672744,"identity":"27a35fd3-192e-4e93-a466-86a81f43b1dc","added_by":"auto","created_at":"2025-12-08 09:38:44","extension":"html","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104565,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/d2abb5bd60e97321e59996b3.html"},{"id":97556048,"identity":"087e338c-89e3-431c-be6e-fd06acb22dce","added_by":"auto","created_at":"2025-12-05 18:43:42","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252820,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design.\u003c/p\u003e\n\u003cp\u003eAfter a one-week adaptation period, male ICR mice were subjected to CUMS for eight weeks, while control mice were only weighed. Then, at the end of the CUMS, the stressed mice were classified into Stress-S and Stress-R groups according to the results of four behavioral tests, i.e., NSF, FST, SPT, and TST. The mice's brains were subsequently analyzed using anti-c-Fos immunostaining and RNA sequencing. A typical CUMS schedule is shown below. Mice were subjected to stress daily in the morning and afternoon in a pseudo-randomized order. The order was changed weekly.\u003c/p\u003e","description":"","filename":"FigureMS151.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/1ca2969deef191e64d1d55d0.jpg"},{"id":97556049,"identity":"dc509cba-f197-4c44-a0ff-10e3c4297b54","added_by":"auto","created_at":"2025-12-05 18:43:42","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":111303,"visible":true,"origin":"","legend":"\u003cp\u003eDepression-like behavior induced by the CUMS.\u003c/p\u003e\n\u003cp\u003eCUMS treatment significantly increased and tended to increase the immobile time in FST (A) and TST (B), respectively.\u003c/p\u003e","description":"","filename":"FigureMS152.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/585b7568b3db10aa444e4e04.jpg"},{"id":97556050,"identity":"aae7dfb2-53d8-4c0c-af7a-7a464547c1e9","added_by":"auto","created_at":"2025-12-05 18:43:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":532540,"visible":true,"origin":"","legend":"\u003cp\u003eSignificant change in c-Fos-positive cells in the CG and LS, but not in other regions.\u003c/p\u003e\n\u003cp\u003e(A an B) The numbers of c-Fos positive cells were decreased in the CG and LS of Stress-R mice, compared with those of Control and Stress-S mice. Bar 100 μm. (C) Those numbers did not change in the Hippocampal DG, DRN, LC, lHb, and PVN.\u003c/p\u003e","description":"","filename":"FigureMS153.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/1d3d28ea50db6222fb6358c1.jpg"},{"id":97556053,"identity":"293391c4-b537-44cc-a61a-ecf664e7e1f7","added_by":"auto","created_at":"2025-12-05 18:43:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":211702,"visible":true,"origin":"","legend":"\u003cp\u003eIncrease in the c-Fos positive cell in the PVT of Control mice.\u003c/p\u003e\n\u003cp\u003eThe number of c-Fos-immunoreactive cells in the PVT was increased in control mice experiencing acute stress of TST and FST. Bar 100 μm.\u003c/p\u003e","description":"","filename":"FigureMS154.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/d1b6f318922311d9c7807dae.jpg"},{"id":97672675,"identity":"88958c35-66e5-4692-b5c3-3bac4886d491","added_by":"auto","created_at":"2025-12-08 09:38:33","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1051055,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-Seq analysis of mRNAs for K⁺channels.\u003c/p\u003e\n\u003cp\u003e(A) No substantial changes were observed in the expression of nearly all mRNA encoding K⁺ channels between Stress-S and -R mice (n = 4). However, a significant increase was noted in Kcnj2, which encodes the Kir2.1 channel, in the LS of Stress-R mice. Slots of significant and large difference were shaded in gray (p \u0026lt; 0.05 and ㇑Log₂FC ㇑\u0026gt; 0.75, respectively). (B) Volcano-plot of the mRNAs for K⁺ channels. (C, D, and E) Expressions of Kcnj2, Kcnj9, and Kcnh4. The mRNA expression levels of the latter two weren't statistically significant different. (F, G, H, and I) The mRNA expressions of Kcnk2, Kcnq2, Kcnq3, and Kcnh7, which were reported to be involved in depression and bipolar disorder, did not show significant changes.\u003c/p\u003e","description":"","filename":"FigureMS155.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/0c40fcb906ed3ef4e4dbff76.jpg"},{"id":97556056,"identity":"01f3e9c6-79d1-4fb3-814e-50c59c6668a4","added_by":"auto","created_at":"2025-12-05 18:43:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":723678,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-Seq analysis of mRNAs related to other known hypotheses for depression.\u003c/p\u003e\n\u003cp\u003e(A) Heatmaps of mRNAs related with HPA-axis, neuroinflammation, NMDA receptor, adult neurogenesis, BDNF, (B) Heatmaps of monoamine receptors (5-HT, epinephrine, dopamine), and monoamine metabolism. (C and D) Volcano-plots of those mRNAs.\u003c/p\u003e","description":"","filename":"FigureMS156.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/52e914d3fa6de1b240594ab5.jpg"},{"id":97671986,"identity":"381eb48e-37b4-49cc-befa-126433c07d40","added_by":"auto","created_at":"2025-12-08 09:33:39","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":285705,"visible":true,"origin":"","legend":"\u003cp\u003eThe antidepressive effect of Kir2.1 channel activators. (A and B) Intra-septal injection of flecainide significantly decreased immobility time in the TST and insignificantly in the FST. (C and D) Administration of GPV0057 significantly decreased immobility time in the TST and insignificantly in the FST. (E and F) The Kir2.1 channel blocker, ML133, insignificantly increased the immobile times in the TST and FST. (G and H) PA-6 did not show depressant effects.\u003c/p\u003e","description":"","filename":"FigureMS157.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/14bd6ad2fe0314f52a41bc22.jpg"},{"id":97893257,"identity":"cd64ff5c-2e2f-441f-a4c3-ae83ff478c10","added_by":"auto","created_at":"2025-12-10 15:29:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4129396,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8030199/v1/0436bc2e-b505-4cff-92b2-0dfdd9caea12.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Individual differences in the chronic stress-induced depression-like behavior are mediated by the expression of Kcnj2 gene in the lateral septum","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eMajor depression is a common mood disorder largely triggered by chronic stress [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Notably, from experience, we know that not everyone who experiences stress develops depression, and that people differ in their susceptibility to stress. The mechanism that distinguishes stress resilience from susceptibility is unclear. Patients with depression are mostly treated with antidepressants, such as selective serotonin reuptake inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRI), and noradrenergic and specific serotonergic antidepressant (NaSSA), which enhance the transmission of monoamines. However, according to the STAR*D study, conducted by the American National Institute of Mental Health, current drugs are ineffective in 30% of patients with major depression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This suggests the presence of mechanisms of onset that are different from monoamine transmission. It is noteworthy that neurostimulation therapies, such as electric convulsive therapy, deep brain stimulation, and transcranial magnetic stimulation, are effective in some drug-resistant depression [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It is assumed that the neuronal excitabilities of some brain regions are dysregulated in patients: these neurostimulation therapies compensate for the dysregulated neuronal excitability. Since major depressive episodes are caused by stressful life events [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], stress may lead to dysregulated neural excitability in a specific area, leading to depression. This dysregulation may result from changes in the expression of genes that control neuronal excitability.\u003c/p\u003e\u003cp\u003eIntrinsic neuronal excitability is mostly controlled by K⁺ channels, which control the resting membrane potential and the frequency and shape of the action potential. There are 77 subtypes of K⁺ channels, which are classified into four types according to their number of transmembrane domains and opening features: two-pore domain, voltage-gated, Ca\u0026sup2;⁺-activated, and inwardly rectifying potassium channels [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Those channels are widely expressed in the central nervous system, but their roles in stress resilience and depression remain largely unknown. Recent studies have suggested the involvement of K⁺ channels in mood disorders. For instance, single nucleotide polymorphisms (SNPs) associated with some K⁺ channel loci, and both up-and down-regulations of K⁺ channel mRNAs from brain samples of depression [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. A mutation in a K⁺ channel (kcnh7) resulted in familial bipolar disorder [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These findings suggest that dysregulation in K⁺ channels result in pathogenesis and that modulation of the channel involved could potentially have therapeutic effects. Indeed, some modulators for K⁺ channels (Kcnq and Kcnk2) successfully showed an antidepressive effect in mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We also demonstrated that ostruthin, a two-pore domain K⁺ channel (TREK-1, Kcnk2) activator, exhibits antidepressant and anxiolytic properties [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and an inwardly rectifying potassium channel (Kir1.1, Kcnj1) blocking peptide, tertiapin-RQ, exhibited antidepressive and anxiogenic effects [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Interestingly, these two antidepressant compounds suppressed the neural activities in the lateral septum, suggesting an involvement of this region in the antidepressant effects.\u003c/p\u003e\u003cp\u003eBased on these findings, we hypothesized that chronic stress alters K⁺ channel expression in specific brain areas, resulting in abnormal excitability and, thereby, depression-like behavior. Since individual differences have been noted in the depression-like behavior among the mice under chronic stress [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we focused on individual differences in stress-induced depression-like behavior and analyzed the brain regions whose neuronal excitability differed between stress-susceptible (S) and -resilient (R) mice. We then identified a K⁺ channel gene, Kcnj2, whose mRNA expression was upregulated in the Stress-R mice, and the antidepressant effects of the channel activators.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIndividual differences in depression-like behavior and classification to Stress-S and -R mice\u003c/h2\u003e\u003cp\u003eChronic unpredictable mild stress (CUMS) is considered as mimicking human depression in some aspects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and individual differences in the depression-like behavior induced by CUMS was shown to be useful for the examination of the mechanisms of the depression like behavior [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. As we hypothesized that chronic stress causes a difference in the neuronal excitability and, consequently, a difference in behavior, we subjected male ICR mice to CUMS for 8 weeks and then analyzed the depression-like behavior in forced swim test (FST) and tail suspension test (TST) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CUMS treatment significantly increased the immobile time in FST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and insignificantly increased that in the TST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), compared with those of control (unstressed) mice. These results suggest that the CUMS-treatment had the expected effect, i.e., depressive effect, on the mice\u0026rsquo;s behavior.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong CUMS-treated mice, some showed a depressed phenotype, in which the immobile time were more than 100 seconds, but others showed a resilient phenotype, in which the immobile time were shorter than the average time of control mice. These findings suggest the presence of individual differences in this model. The individual differences in chronic stress-sensitive, depression-like behavior in CUMS-treated mice were further analyzed with novelty-suppressed feeding (NSF) and sucrose preference test (SPT). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the raw data of a round of four tests and the median values of each test. The values exhibited more depression-like behavior than the median are shaded in gray. Then we classified the stressed mice into three groups: those with three grays were classified as Stress-S mice, and those with one gray were classified as Stress-R mice. Mice with two grays were grouped into the middle category and were not subjected to further analysis.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAn example of classification of Stress-S and -R mice. This table shows the raw data of four behavioral tests of each mouse in a CUMS round. The slots showing results as more depressed than the median for each mouse's behavioral test are shaded gray. The mice were classified based on the number of gray slots. The classification was repeated on a total of 36 mice, which were divided into three groups: 11 Stress-S mice, 13 Stress-R mice, and 12 Middle mice. Middle mice were not analyzed.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMouse #\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTST\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNSF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSPT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eClassfication\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e210\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e153\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-S\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e105\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-R\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMiddle\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e132\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMiddle\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e110\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-S\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e113\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e127\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMiddle\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e182\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-S\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e207\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-R\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e97\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e123\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMiddle\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-R\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e68\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-R\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e155\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e108\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eStress-S\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e116.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e68.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMiddle\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReduced neuronal activities in the lateral septum and cingulate cortex of Stress-R mice\u003c/h3\u003e\n\u003cp\u003eTo determine which brain area is involved in the individual differences in susceptibility to chronic stress, brain slices from control, Stress-S, and -R mice (n\u0026thinsp;=\u0026thinsp;10, 7, 9) were immunostained with an anti-c-Fos antibody, which is a marker of neuronal excitability. The control mice were not stressed but experienced FST and TST, which were the first and acute stressful experience for them, on the previous and last day. The mouse brains were fixed two hours after TST. We examined anti-c-Fos immunoreactivity in the brain areas that are known to be involved in the depression-like behavior and stress, i.e., hippocampal dentate gyrus (DG), dorsal raphe nucleus (DRN), locus coeruleus (LC), lateral habenular nucleus (lHB), hypothalamic paraventricular nucleus (PVN), thalamic paraventricular nucleus (PVT), cingulate cortex (CG), and lateral septum (LS). Because the ventral part of LS is more involved in the regulation of emotional behaviors than the dorsal part, we only analyzed the ventral part.\u003c/p\u003e\u003cp\u003eThere were no significant differences in the c-Fos-immunoreactive cell number among Control, Stress-S, and Stress-R groups in the DG, DRN, LC, lHB, or PVN. Significant changes were found in the CG and LS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In these regions, the number of c-Fos-positive cells was similar between the Control and Stress-S mice. However, the number of positive cells in Stress-R mice was significantly reduced, suggesting that the observed individual differences in this model resulted from decreased neuronal excitability in these regions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ec-Fos-positive cells were significantly increased in the Control mice compared to Stress-S and -R mice in the PVT, but there were no differences between Stress-S and -R mice, excluding the involvement in the individual differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Since PVT is known to quickly respond to acute stress, this increase in the c-Fos expression may be attributable to the fact that the FST and TST were the first stress experience to the control mice whereas Stress-S and R had experienced it for weeks already.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA-seq analysis revealed increased expression of an inwardly rectifying K⁺ channel gene, kcnj2, in the lateral septum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIntrinsic neuronal activity is mainly controlled by K⁺ channels, and some K⁺ channel modulators have antidepressant effects, through modulation of neuronal activities. Especially, in our previous studies, two K⁺ channel modulator compounds, ostruthin and tertiapin-RQ, were found to have antidepressant effects and decreased the neuronal excitabilities in the LS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Therefore, we compared the expression of mRNAs in the LS of Stress-S and -R mice for various K⁺ channels with RNA-Seq analysis (n\u0026thinsp;=\u0026thinsp;4 and 4). We found an increase in expression in the Kcnj2 gene, which encodes an inwardly rectifying K⁺ channel (Kir2.1), in the LS of Stress-R mice compared to Stress-S mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and C). The expressions of other channels, Kcnj9 and Kcnh4, were also changed, but the difference was insignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD) and the expression level was very low (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The expression levels of the majority of K⁺ channel genes, including Kcnk2, Kcnq2, and Kcnq3, which were reported to be involved in antidepressant effects [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] remained at the same level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G, and H). The expression of Kcnh7, which was reported to be the causative gene for familial bipolar disease [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], remained the same (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eRNA-Seq revealed little differences in the expression of genes associated with known hypotheses regarding depression\u003c/h3\u003e\n\u003cp\u003eWe also analyzed the other genes that are present in the known hypotheses for depression, i.e., hypothalamus-pituitary-adrenal (HPA)-axis, neuroinflammation, ketamine NMDA receptor, adult neurogenesis, and Bdnf and its receptor Ntrk2. There were no significant differences between Stress-S and -R mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe most well-known hypothesis for depression is the monoamine hypothesis: depression is caused by a deficiency in the brain's monoamine neurotransmitters, serotonin, norepinephrine, and dopamine, and antidepressant medications are believed to enhance these neurotransmissions, thereby alleviating symptoms. The expression levels of monoamine-degrading enzymes, Comt, Maoa and Maob, remained the same (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and D). The expression levels of monoamine synthesizing enzymes, Th (tyrosine hydroxylase) and Tph (tryptophan hydroxylase), exhibited no significant increase and remained at low levels. The expression of Slc6a4 (serotonin transporter) was below the cut-off level. The expression level of most receptors for 5-HT, norepinephrine, and dopamine remained constant. But those of Drd2 and Drd1 (dopamine D₂ and D₁ receptors) were increased significantly and insignificantly (p\u0026thinsp;=\u0026thinsp;0.06), respectively, in Stress-R mice. A similar increase in 5-HT1b (5-HT receptor 1b) was also observed in the Stress-R mice. These increases may be implicated in Stress-R behavior, at least to some extent.\u003c/p\u003e\n\u003ch3\u003eInvolvement of inwardly rectifying K⁺ channel in the antidepressive behavior\u003c/h3\u003e\n\u003cp\u003eTo confirm the involvement of the Kir2.1 channel in individual differences in depression-like behavior, we injected 1 \u0026micro;L of Kir2.1 channel activators, flecainide (2 mM) and GPV0057 (5 mM), and blockers, ML133 (2 mM) and PA-6 (10 mM), into the septum of mice. The administration of these agents are supposed to mimic the upregulation and downregulation of the Kir2.1 channel expression, respectively. Both administrations of flecainide showed significant antidepressant effects on TST compared with PBS injection (n\u0026thinsp;=\u0026thinsp;15 and 15, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Similarly, the administration tended to decrease the immobile time in FST (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Consistently, the administration of GPV0057 decreased the immobile time in TST and tended to in FST (n\u0026thinsp;=\u0026thinsp;15 and 15, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D). The administration of a Kir2.1 channel blocker ML133, showed only an insignificant increase the immobile times of TST and FST (n\u0026thinsp;=\u0026thinsp;14 and 16, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and F). Similarly, PA-6 did not show depressant effects in both tests (n\u0026thinsp;=\u0026thinsp;14 and 16, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and H). Administrations of fivefold doses of the compounds above via intracerebroventricular injection elicited similar results. The administrations of the channel blockers exhibited only an insignificant effect on depression-like behavior, likely attributed to the low baseline expression of the channel gene, which consequently restricts efficacy of channel blockade. Therefore, these findings suggest a crucial role for upregulation of functional Kir2.1 in the Stress-R mice, rather than the downregulation in the Stress-S mice.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, genetically homogeneous closed colony-bred mice showed individual differences in depression-like behavior in response to chronic stress, as humans do. We then examined mRNA expression in the ventral lateral septum according to the difference in the neuronal excitability between Stress-S and -R mice. RNA-seq analysis revealed that expression of the Kir2.1 channel gene (Kcnj2) was increased in Stress-R mice, potentially suppressing neural excitability. Indeed, the overexpression of Kir2.1 channels is a well-established method of artificially suppressing neuronal excitability [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In support, the administration of Kir2.1 channel activators exhibited antidepressant effects in TST. These results suggest that the upregulation of Kcnj2 gene led to Stress-R behavior through the suppression of neural activity in the lateral septum. This hypothesis that the Kcnj2 gene plays a role in depression-like behavior is supported by previous human studies. A meta-analysis of gene expression profiles revealed decreased Kcnj2 expression in the postmortem brains of individuals who died by suicide [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A heterozygous missense mutation (Thr192Ile) in the Kcnj2 gene has been associated with major depression, as well as Andersen-Tawil syndrome [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Reportedly, the expression of Kir2.1 was increased in the hippocampal granule neurons, which became epileptic by the injection of kainic acid, to compensate for the higher excitability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It was also reported that neurons can alter the expression of various ion channels to maintain homeostatic stability in neural activity [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. When excessive stress information is input in the form of neural excitability, differences in the ability to alter K⁺ channel expression as a compensatory mechanism may have led to individual differences in depressive-like behavior as shown here.\u003c/p\u003e\u003cp\u003eThe expression of mRNA for dopamine D₂ receptors increased, and the expression of mRNA for D₁ receptors tended to increase as well. Similar increases in D\u003csub\u003e2\u003c/sub\u003e receptor mRNA expression were observed in Stress-R mice in the mesolimbic region after five weeks of chronic stress, but not after two weeks [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Dopamine receptor partial agonists show therapeutic effect on major depression [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], these increases might be involved in the Stress-R behavior, improving the depressive symptoms like anhedonia and low motivation. The expression of serotonin 5-HT\u003csub\u003e1b\u003c/sub\u003e receptor was also increased in the Stress-R mice. The relationship between 5-HT\u003csub\u003e1b\u003c/sub\u003e receptor expression and depressive-like behavior is not clear [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Both agonists and antagonists have demonstrated antidepressant effects in animal studies. Furthermore, postmortem brain studies in humans revealed decreased mRNA expression of this receptor in the hippocampus and frontopolar cortex compared with control subjects. Low 5-HT\u003csub\u003e1B\u003c/sub\u003e receptor binding in limbic regions has been found in patients with depression. The decreased expression observed in Stress-S mice may lead to depression-like behavior. A recent study has shown a rapid antidepressant effect of a selective 5-HT\u003csub\u003e1b\u003c/sub\u003e receptor agonist, CP-94253, in FST [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The increase in expression might also contribute to the Stress-R phenotype through a similar mechanism.\u003c/p\u003e\u003cp\u003eThe involvement of the lateral septum in depression-like behavior has been documented for over two decades; however, the specific roles of the lateral septum in depressive-like behavior remain a subject of controversy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. A recent elegant study examining individual differences in reward-seeking behavior following social defeat stress in female mice suggests that the antidepressant effects are attributable to the inhibition of the neurons [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Given that the role of the lateral septum is to mediate adverse information [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], its suppression is predicted to impede the transmission of stress information, thereby inducing stress-R behavior. Wirtshafter and Wilson [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] theorized that the lateral septum functions as a central nexus, thereby serving as a focal point for the aggregation of information that governs mood, anxiety, and motivation. In fact, the neurons in non-treated mice that were only anesthetized were c-Fos-negative, but became positive after the acute stress of TST [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNotably, there were little differences in the expression of genes associated with other well-known depression hypotheses, i.e., adult neurogenesis, BDNF, HPA-axis, neuroinflammation, and ketamine NMDA receptor. Previous studies reported that chronic stress induced changes in expression of these mRNAs or proteins in other areas, e.g., reduction in BDNF mRNA in the hippocampus [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These findings suggest that the possible mechanism of the onset of Stress-S and -R behaviors in this model is different from previous ones. The etiology of major depression is understood to be genetically heterogeneous [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], suggesting the presence of novel mechanisms of onset. Consequently, the lack of difference in the expression of depression-associated genes suggests the uniqueness of this mouse model. This may be useful for the identification of novel genes and the elucidation of previously unrecognized mechanisms underlying the onset of depression.\u003c/p\u003e\u003cp\u003eIn this study, we used male mice of a closed colony strain (Slc:ICR). The genetic information of those mice was supposed to be highly similar. The observed individual differences in behavior may be attributable to epigenetic modifications of the genome. For instance, epigenetic differences in the genomic DNA methylation may lead to individual differences in the Kir2.1 channel expression, which can result in alterations in excitability and behavior. Indeed, individual differences in DNA methylation were shown between human monozygotic twins [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Alternatively, individual differences in the acetylation/deacetylation of histone proteins may also be involved.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003eExperimental design and typical schedule of CUMS\u003c/h2\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e describes the experimental design of this study. Male ICR mice (5-week-old) were purchased from Shimidzu Laboratory Supplies (Kyoto, Japan). They had free access to food and water and were kept in a 12-h light/dark cycle (08:00\u0026ndash;20:00). The animals were maintained in groups in plastic cages except for the last week of CUMS, in which they were individually housed in small cages. Experiments were conducted in a quiet and air-conditioned room (23\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃). Animal experiments were carried out following the guidelines of the Physiological Society of Japan and The ARRIVE guidelines 2.0. All experiments were approved by the Committee on Animal Experiments at Kurashiki University of Science and the Arts.\u003c/p\u003e\u003cp\u003eAfter one week of adaptation, mice were subjected to CUMS for 8 weeks and underwent behavioral tests according to the previous methods [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The stressors included: swimming in a 2 L beaker of water at a temperature of 27℃ for 10 min, confinement in a breathable tube for 1 hour, food and water deprivation for overnight (14 hours), exposure to wet bedding (250 mL water and soggy sawdust) for 3 hours, shaking of the cage (reciprocal movement of 3\u0026ndash;4 cm at 120 rpm) for 1 hour, tilting of the cage at a 45\u0026deg; angle for 1 hour, exposure to an inverted light and dark cycle (from 8:00\u0026ndash;20:00 to 20:00\u0026ndash;8:00) for 48 hours, and exposure to strobe light (120 flashes/min) for 3 hours. Control mice were kept in enriched cages with cardboard tubes and weighed daily. A typical CUMS schedule is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and the combinations of stressors were changed weekly.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eTests for depression-like behavior and classification of the CUMS-treated mice into Stress-S and -R mice\u003c/h3\u003e\n\u003cp\u003eDepression-like behaviors were examined with four behavioral tests: tail-suspension test (TST), forced swim test (FST), sucrose preference test (SPT), and novelty-suppressed feeding (NSF). Each behavioral test was performed according to a method reported previously [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eNovelty-suppressed feeding (NSF)\u003c/h2\u003e\u003cp\u003eOn the two days before the last, food and water were deprived at 20:00. On the next morning, the test mouse was placed in a corner of the arena and allowed to explore the open field (90 \u0026times; 90 \u0026times; 45 cm), in which food pellets were placed on filter paper (10 cm in diameter) at the center of the arena until the mouse approached and took the first bite of the chow or certain cut-off time (5 min) was reached.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eSucrose preference test (SPT)\u003c/h2\u003e\u003cp\u003eMice were initially acclimated to sucrose solution, i.e., free choice of drinking either 1% (w/v) sucrose solution or tap water three times in the week before the last and in the last week. After the NSF test, consumption of sucrose solution and water for 24 h was then estimated by measuring the difference in weight of the respective bottles before and after. The percentage of sucrose preference was calculated according to the following formula: sucrose preference (%)\u0026thinsp;=\u0026thinsp;sucrose intake / (sucrose intake\u0026thinsp;+\u0026thinsp;water intake) * 100.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eForced swim test (FST)\u003c/h2\u003e\u003cp\u003eAfter the NSF, mice were put back in the original cage and had free access to food and water/sucrose water for 3\u0026ndash;4 hours. Then a mouse was placed into a 2 L beaker containing 12 cm of water (27℃). The mouse was allowed to swim for 6 min. The last 4 min of data were used for analysis. Immobility is defined as the absence of activity, such as escape-oriented behaviors. The slow movement of the forelimbs only for breathing was considered immobility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eTail suspension test (TST)\u003c/h2\u003e\u003cp\u003eOn the last day, the mouse was hung on a hook using adhesive tape placed 1 cm from the extremity of its tail 50 cm above the table. The amount of time spent immobile was recorded for 5 min.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eClassification to Stress-S and -R mice\u003c/h2\u003e\u003cp\u003eThree rounds of CUMS treatment and behavioral testing were conducted. Subsequently, brains were used for c-Fos immunostaining (n\u0026thinsp;=\u0026thinsp;24) and RNA-seq (n\u0026thinsp;=\u0026thinsp;12). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the raw data of a round and the median values of each test of a round. Values that exhibited more depression-like behavior than the median are shaded in gray. Then, mice with three grays were classified as Stress-S, whereas those with zero or one gray were classified as Stress-R mice. Mice with two grays were not analyzed further.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAnti-c-Fos immunostaining\u003c/h2\u003e\u003cp\u003eTwo hours after being subjected to the TST, the mouse was anesthetized with isoflurane (4%, 400 mL/min) using a vaporizer (NARCOBIT-E, KN-1071-I, Natsume, Tokyo, Japan). After confirming that the mouse was deeply anesthetized, the brain was fixed by transcardiac perfusion with 4% paraformaldehyde in PBS. The brains were sliced coronally (75 \u0026micro;m in thickness) using a microslicer (PRO7, Dosaka, Kyoto, Japan). The free-floating slices were reacted with an anti-c-Fos antibody (1: 200, rabbit mAB (9F6), Cell Signaling Technology, MA) diluted in PBS containing 0.3% BSA and 0.3% Triton-X-100, at 4˚C for 48 h. The immunoreaction was visualized using a secondary antibody (Vectestain Universal Elite ABC Kit, peroxidase, Vector Labs, Burlingame, CA) and 3,3\u0026rsquo;-Diaminobenzidine (DAB; Tokyo Chemical Industry, Tokyo, Japan) at room temperature in accordance with the manufacturer\u0026rsquo;s instructions. For statistical analysis, the number of c-Fos immunoreactive cells was quantified using ImageJ software. Briefly, fields (0.16 mm\u0026sup2;) of c-Fos immunoreactivity were acquired using a microscope with a CCD camera. DAB-positive nuclei were considered as anti-c-Fos-immunoreactive neurons.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRNA-Seq analysis\u003c/h2\u003e\u003cp\u003eTwo hours after the behavioral tests, mice were anesthetized with isoflurane (4%, 400 mL/min) and decapitated with scissors. The brains were removed, frozen with dry ice powder, and stored at -80\u0026deg;C. Semi-melted brains were mounted in a brain slicer (Muromachi Kikai, Kyoto, Japan) and sliced to a thickness of 2 mm. The lateral septum was cut out with a scalpel and immersed in TRIzol (Thermo Fisher Scientific, Waltham, MA). Total RNA was purified using the SV Total RNA Isolation System (Promega, Fitchburg, WI) according to the manufacturer's instructions. The RNA samples were quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE), and the quality was confirmed using a Tapestation (Agilent Technologies, Santa Clara, CA). Sequencing libraries were prepared from 200 ng of total RNA using an MGIEasy RRNA Depletion Kit and an MGIEasy RNA Directional Library Prep Set (MGI Tech Shenzhen, China), following the manufacturers' instructions. The libraries were sequenced on the DNBSEQ-G400 FAST sequencer with a paired-end 150-nt strategy. All sequencing reads were trimmed for low-quality bases and adapters using Trimmomatic (v. 0.38). We estimated raw reads (counts) for each gene in each sample using RSEM version 1.3.0 and Bowtie 2. We used the edgeR program to detect differentially expressed genes. The gene counts were normalized to reads per million (RPM), and p-values were estimated using likelihood analysis. Genes with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered differently expressed genes. Genes, of which the read number/sample is \u0026lt;\u0026thinsp;1, were considered negative.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eBehavioral effects of the intra-septal injection of the activators and blockers for Kir2.1 channel\u003c/h2\u003e\u003cp\u003eThe effect of the activators and blockers for Kir2.1 channel, which mimic the effect of upregulation and downregulation of the Kcnj2 gene, on the depression-like behaviors was examined with TST and FST after the injection of these drugs into the septum in accordance with our previous study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The Kir2.1 channel activators (Flecainide [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], GPV0057 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]) and blockers (ML133 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], PA-6 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]) were first dissolved in dimethyl sulfoxide (DMSO) at higher concentration and were diluted with phosphate-buffered saline (PBS) by vigorous mixing for several minutes. An equal volume of DMSO was added to PBS as a control. The concentrations of intraseptal injections were 2 mM (Flecainide), 5 mM (GPV0057), 2 mM (ML133), and 10 mM (PA-6). Male ICR mice (5-week-old) were adapted, only being weighed, for a week, and then subjected to the behavioral tests, without being chronically stressed. After confirming that the mouse was deeply anesthetized (isoflurane 2\u0026ndash;4%, 400 mL/min), we injected drugs into the septum (1 \u0026micro;L/mouse) over 60 seconds (coordinates: 0 mm caudal to bregma, 0 mm lateral to the midline, and 3.5 mm below the skull), using a microsyringe (MS-N05(5 \u0026micro;L) 28G needle, ITO Corp., Fuji, Shizuoka, Japan). The injection needle was kept for 30 seconds after the injection. The dosage was set such that, even if each compound was distributed throughout the whole brain (0.5 mL), concentration would exceed the EC\u003csub\u003e50\u003c/sub\u003e and IC\u003csub\u003e50\u003c/sub\u003e values. Then the mouse was returned to its home cage. Each animal was subjected to TST and FST 30 and 50 min after injection, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are given as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The normality of data distribution was confirmed with the Shapiro-Wilk test. Statistical significance between two groups was determined using Student\u0026rsquo;s t-test. Those obtained from three or more groups were analyzed statistically by one-way analysis of variance (ANOVA) followed by Bonferroni test. A p-value of \u0026lt;\u0026thinsp;0.05 was considered significant. The number of asterisks indicates the p values: *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eMO was supported by KAKENHI (22K11818), Kobayashi Foundation, Ryobi Teien Memorial Foundation, and Wesco Scientific Promotion Foundation. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e\u003cp\u003eEthics declarations\u003c/p\u003e\u003cp\u003eCompeting interests\u003c/p\u003e\u003cp\u003eThe authors have declared that no competing interests exist.\u003c/p\u003e\u003cp\u003eEthical approval\u003c/p\u003e\u003cp\u003eThe animal ethics were approved by the Committee on Animal Experiments at Kurashiki University of Science and the Arts, in accordance with the guidelines of the Physiological Society of Japan and The ARRIVE 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eConsent for publication\u003c/p\u003e\u003cp\u003eAll authors consented to the submission and publication of this study.\u003c/p\u003e\u003cp\u003eData availability\u003c/p\u003e\u003cp\u003eAll data used in this study were available in the original research. Data generated in this study were included in the main text and supplementary files. The datasets generated and/or analyzed during the current study are available on the website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://osf.io/kmvsg/overview\u003c/span\u003e\u003cspan address=\"https://osf.io/kmvsg/overview\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) can be obtained by contacting the corresponding author.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMO carried out experimental design, investigation, analysis, and writing. MH did initial characterization of the Kir2.1 modulating compounds and writing.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank R. Kawakami, T. Ono, and N. Sato, who have done the pilot experiments of this study, and Dr. G. Ecker (Vienna University) for synthesizing GPV0057.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data used in this study were available in the original research. Data generated in this study were included in the main text and supplementary files. The datasets generated and/or analyzed during the current study are available on the website (https://osf.io/kmvsg/overview) can be obtained by contacting the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHammen, C. Stress and depression. \u003cem\u003eAnnu. Rev. Clin. Psychol.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 293\u0026ndash;319 (2005).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDibernardo, A. et al. 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Res.\u003c/em\u003e \u003cb\u003e99\u003c/b\u003e, 203\u0026ndash;214 (2013).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chronic stress, depression, Kir2.1, lateral septum, c-Fos, RNA-Seq","lastPublishedDoi":"10.21203/rs.3.rs-8030199/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8030199/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIt has been demonstrated that chronic unpredictable mild stress (CUMS) induces depression-like behaviors in mice. Notably, there are differences among individuals in the extent to which these behaviors are exhibited, similar to humans. In this study, we focused on individual differences and examined which brain areas have differences in neuronal excitability, and which genes are differentially expressed between stress-susceptible mice with depression-like behaviors and stress-resilient mice without depression-like behaviors (Stress-S and -R). After 8 weeks of CUMS, the number of c-Fos positive cells was reduced in Stress-R mice compared to Stress-S mice only in the cingulate cortex and lateral septum. RNA-seq analysis revealed an upregulation of Kcnj2 mRNA, the gene for the inward-rectifying potassium channel (Kir2.1), in the lateral septum of Stress-R mice, without affecting the expression of other potassium channels. The expression of genes related to adult neurogenesis, neuroinflammation, hypothalamic-pituitary-adrenal axis, BDNF, and monoamine hypotheses were similar between Stress-S and -R mice. Intra-septal injections of Kir2.1 activators, flecainide, and GPV0057, showed antidepressant effects in the tail-suspension test. These findings suggest that the Stress-R behavior is the result of the upregulation of Kir2.1 and decreased neuronal excitability in the lateral septum. They also suggest the uniqueness of this model and the potential to reveal new mechanisms.\u003c/p\u003e","manuscriptTitle":"Individual differences in the chronic stress-induced depression-like behavior are mediated by the expression of Kcnj2 gene in the lateral septum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-05 18:43:38","doi":"10.21203/rs.3.rs-8030199/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-02T04:51:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-25T01:38:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-21T07:28:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"310360992509628810810149590569434215501","date":"2026-02-15T06:05:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"68760566246597310115456842952840309409","date":"2026-02-13T05:37:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-04T17:10:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28430095832211048765496914751549930575","date":"2026-01-19T23:04:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-03T06:29:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-03T06:25:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-18T08:36:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-12T08:40:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-12T08:37:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"60aab994-dd95-498e-8056-2074f5ed9ae3","owner":[],"postedDate":"December 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":59007249,"name":"Biological sciences/Genetics"},{"id":59007250,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-05-18T06:38:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-05 18:43:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8030199","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8030199","identity":"rs-8030199","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}