Repeated Social Defeat Stress Research Articles

Ramelteon protects against social defeat stress-associated abnormal behaviors

Psychological stress affects the neuroendocrine regulation, which modulates mental status and behaviors. Melatonin, a hormone synthesized primarily by the pineal gland, regulates many brain functions, including circadian rhythms, pain, sleep, and mood. Selective pharmacological melatonin agonist ramelteon has been clinically used to treat mood and sleep disorders. Posttraumatic stress disorder (PTSD) is a psychiatric condition associated with severe trauma; it is generally triggered by traumatic events, which lead to severe anxiety and uncontrollable trauma recall. We recently reported that repeated social defeat stress (RSDS) may induce robust anxiety-like behaviors and social avoidance in mice. In the present study, we investigated whether melatonin receptor activation by melatonin and ramelteon regulates RSDS-induced behavioral changes. Melatonin treatment improved social avoidance and anxiety-like behaviors in RSDS mice. Moreover, treatment of the non-selective MT1/MT2 receptor agonist, ramelteon, markedly ameliorated RSDS-induced social avoidance and anxiety-like behaviors. Moreover, activating melatonin receptors also balanced the expression of monoamine oxidases, glucocorticoid receptors, and endogenous antioxidants in the hippocampus. Taken together, our findings indicate that the activation of both melatonin and ramelteon regulates RSDS-induced anxiety-like behaviors and PTSD symptoms. The current study also showed that the regulatory effects of neuroendocrine mechanisms and cognitive behaviors on melatonin receptor activation in repeated social defeat stress.

Autonomic Regulation of T-Lymphocyte Inflammation after Psychological Trauma

Post-traumatic stress disorder (PTSD) is a complex psychiatric condition that is accompanied by systemic inflammation, increased sympathetic activity, and an elevated risk of cardiovascular disease development. These physiological alterations hint at disturbances in the immune system, yet the precise signaling mechanisms linking psychological trauma to immune responses remain poorly understood. In a preclinical murine model of PTSD, termed repeated social defeat stress (RSDS), we previously demonstrated that psychological trauma induces systemic inflammation, notably the production of interleukin 17A (IL-17A) and 22 (IL-22) from T-lymphocytes. Targeted denervation of the spleen successfully abolished the T-lymphocyte inflammatory response following psychological trauma, suggesting a neural origin for the signal. Furthermore, we showed that T-lymphocytes lacking tyrosine hydroxylase (i.e., devoid of the ability to produce catecholamines) were unable to produce IL-17A and IL-22 after RSDS, further suggesting an autonomic role in the expression of these proinflammatory cytokines. Therefore, we postulated that psychological trauma elicits T-lymphocyte inflammation via sympathetic modulation. To investigate this hypothesis, we employed chemogenetic technology known as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to inhibit sympathetic signaling from the rostral ventrolateral medulla (RVLM), a central region of sympathetic signaling, during RSDS. RVLM DREADD-injected RSDS mice showed blunted blood pressure (-1.17 fold, p<0.0001) and heart rate (-1.07 fold, p=0.0026) compared to wild-type RSDS mice, confirming the effcacy of the sympathetic blockade. Moreover, after RSDS with sympathetic blockade, mice possessed completely ablated systemic circulating protein levels of IL-17A and IL-22 (-5.8 fold and -4.7 fold, respectively) compared to control GFP injected RSDS mice. To investigate the role of sympathetic regulation further, we next examined mice lacking beta 1 and 2-adrenergic receptors, given the previously described abundance of these receptors on T-lymphocytes. Confirming the results of the sympathetic blockade, mice lacking beta 1 and 2 adrenergic receptors had significantly diminished systemic circulating protein levels of IL-17A and IL-22 (-7.5 fold, p=0.0011; -31.5 fold, respectively) compared to wild-type mice after RSDS. Moreover, systemic infusion of the beta 1 and 2-adrenergic receptor antagonist propranolol also showed attenuated circulating levels of IL-17A and IL-22 (-2.57 fold and -14.59 fold, respectively) in wild-type mice after RSDS. Taken together, these data support a role for sympathetic modulation of T-lymphocyte inflammation after psychological trauma, which provides new insights into how mental health leads to somatic pathophysiology. R01HL158521. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Elucidation of the Molecular Signal Linking Psychological Trauma to T-lymphocyte Inflammation

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that not only exhibits significant behavioral pathology, but also increases the likelihood of comorbid inflammatory conditions such as autoimmune and cardiovascular diseases. While PTSD patients experience heightened systemic inflammation, the exact link between psychological trauma and immune dysregulation remains to be elucidated. Our group has recapitulated the psychological trauma-induced inflammatory phenotype in a pre-clinical murine model of PTSD known as repeated social defeat stress (RSDS). Importantly, a key site of this inflammation appears to originate from T-lymphocytes in the spleen, and targeted splenic nerve denervation was able to completely prevent splenic T-lymphocyte inflammation after RSDS (p<0.0001). This critical observation suggests the T-lymphocyte response to psychological trauma is neural derived as opposed to circulating, therefore, we began systematic investigation into possible neural-derived signals that may lead to enhanced T-lymphocyte inflammation. To investigate whether the signal involves the T-cell receptor (TCR), we utilized OT-I and OT-II mice that possess non-mouse antigen-restricted TCRs. RSDS induced T-lymphocyte inflammation in these mice similar to wild-type, therefore, suggesting the signal is not antigenic in nature (p>0.05). Next, we examined the role of norepinephrine and adrenergic signaling using both pharmacological blockade and genetic knockouts of both alpha and beta adrenergic receptors, but this too was insuffcient to reverse the RSDS-induced T-lymphocyte inflammation (p>0.05). Following this, we examined the role of damage associated molecular patterns (DAMPs) by pharmacological blockade of the toll-like receptor 4 (TLR4), yet again, this intervention did not attenuate T-lymphocyte inflammation after RSDS (p>0.05). Last, our data suggests interleukin 6 (IL-6) is upregulated early after psychological trauma; therefore, we examined the role for cytokine-induced T-lymphocyte inflammation. Using anti-IL-6 antibodies during RSDS, T-lymphocyte inflammation appeared blunted compared to wild-type animals (p<0.05). Moreover, T-lymphocytes treated with only IL-6 expressed similar inflammation gene profiles as those from RSDS animals (p<0.05). Taken together, our data suggest IL-6 may be the T-lymphocyte inflammation-inducing signal, but additional studies are warranted to understand the exact source of IL-6 and mechanisms underlying IL-6’s ability to drive T-lymphocyte inflammation. R01HL158521. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Role of GABA in the Dorsal Striatum-Raphe Nucleus Circuit Regulating Stress Vulnerability in Male Mice with High Levels of Shati/Nat8l.

Depression is a frequent and serious illness, and stress is considered the main risk factor for its onset. First-line antidepressants increase serotonin (5-hydroxytryptamine; 5-HT) levels in the brain. We previously reported that an N-acetyltransferase, Shati/Nat8l, is upregulated in the dorsal striatum (dSTR) of stress-susceptible mice exposed to repeated social defeat stress (RSDS) and that dSTR Shati/Nat8l overexpression in mice (dSTR-Shati OE) induces stress vulnerability and local reduction in 5-HT content. Male mice were used in this study, and we found that dSTR 5-HT content decreased in stress-susceptible but not in resilient mice. Moreover, vulnerability to stress in dSTR-Shati OE mice was suppressed by the activation of serotonergic neurons projecting from the dorsal raphe nucleus (dRN) to the dSTR, followed by upregulation of 5-HT content in the dSTR using designer receptors exclusively activated by designer drugs (DREADD). We evaluated the role of GABA in modulating the serotonergic system in the dRN. Stress-susceptible after RSDS and dSTR-Shati OE mice exhibited an increase in dRN GABA content. Furthermore, dRN GABA content was correlated with stress sensitivity. We found that the blockade of GABA signaling in the dRN suppressed stress susceptibility in dSTR-Shati OE mice. In conclusion, we propose that dSTR 5-HT and dRN GABA, controlled by striatal Shati/Nat8l via the dSTR-dRN neuronal circuitry, critically regulate stress sensitivity. Our study provides insights into the neural processes that underlie stress and suggests that dSTR Shati/Nat8l could be a novel therapeutic target for drugs against depression, allowing direct control of the dRN serotonergic system.

Repeated social defeat stress leads to immunometabolic shifts in innate immune cells of the spleen

Psychosocial stress has been shown to prime peripheral innate immune cells, which take on hyper-inflammatory phenotypes and are implicated in depressive-like behavior in mouse models. However, the impact of stress on cellular metabolic states that are thought to fuel inflammatory phenotypes in immune cells are unknown. Using single cell RNA-sequencing, we investigated mRNA enrichment of immunometabolic pathways in innate immune cells of the spleen in mice subjected to repeated social defeat stress (RSDS) or no stress (NS). RSDS mice displayed a significant increase in the number of splenic macrophages and granulocytes (p < 0.05) compared to NS littermates. RSDS-upregulated genes in macrophages, monocytes, and granulocytes significantly enriched immunometabolic pathways thought to play a role in myeloid-driven inflammation (glycolysis, HIF-1 signaling, MTORC1 signaling) as well as pathways related to oxidative phosphorylation (OXPHOS) and oxidative stress (p < 0.05 and FDR<0.1). These results suggest that the metabolic enhancement reflected by upregulation of glycolytic and OXPHOS pathways may be important for cellular proliferation of splenic macrophages and granulocytes following repeated stress exposure. A better understanding of these intracellular metabolic mechanisms may ultimately help develop novel strategies to reverse the impact of stress and associated peripheral immune changes on the brain and behavior.

Chronic psychosocial stress triggers microglial-/macrophage-induced inflammatory responses leading to neuronal dysfunction and depressive-related behavior.

Chronic environmental stress and traumatic social experiences induce maladaptive behavioral changes and is a risk factor for major depressive disorder (MDD) and various anxiety-related psychiatric disorders. Clinical studies and animal models of chronic stress have reported that symptom severity is correlated with innate immune responses and upregulation of neuroinflammatory cytokine signaling in brain areas implicated in mood regulation (mPFC; medial Prefrontal Cortex). Despite increasing evidence implicating impairments of neuroplasticity and synaptic signaling deficits into the pathophysiology of stress-related mental disorders, how microglia may modulate neuronal homeostasis in response to chronic stress has not been defined. Here, using the repeated social defeat stress (RSDS) mouse model we demonstrate that microglial-induced inflammatory responses are regulating neuronal plasticity associated with psychosocial stress. Specifically, we show that chronic stress induces a rapid activation and proliferation of microglia as well as macrophage infiltration in the mPFC, and these processes are spatially related to neuronal activation. Moreover, we report a significant association of microglial inflammatory responses with susceptibility or resilience to chronic stress. In addition, we find that exposure to chronic stress exacerbates phagocytosis of synaptic elements and deficits in neuronal plasticity. Importantly, by utilizing two different CSF1R inhibitors (the brain penetrant PLX5622 and the non-penetrant PLX73086) we highlight a crucial role for microglia (and secondarily macrophages) in catalyzing the pathological manifestations linked to psychosocial stress in the mPFC and the resulting behavioral deficits usually associated with depression.

Neuropeptide Y potentiates T-Lymphocyte inflammation after psychological trauma

Post-traumatic stress disorder (PTSD) is a psychiatric disorder characterized by severe behavioral alterations, but also demonstrates elevated levels of systemic inflammation, sympathoexcitation, and a potentiated risk for autoimmune and cardiovascular diseases. These physiological changes suggest perturbations to the immune system, however, the signaling mechanisms linking psychological trauma to the immune system are not well understood. Utilizing a pre-clinical mouse model of PTSD known as repeated social defeat stress (RSDS), we previously demonstrated that psychological trauma causes robust T-lymphocyte-driven inflammation in the spleen as evidenced by increased pro-inflammatory cytokine production (primarily interleukin 17A, IL-17A), inflammatory polarization, and enhanced mitochondrial reactive oxygen species. Moreover, targeted denervation of the spleen was able to completely ablate this T-lymphocyte inflammatory response after psychological trauma, suggesting the signal was autonomic and neural in nature. Given that the splenic nerve possesses only sympathetic neurons (no direct parasympathetic innervation), we hypothesized that psychological trauma mediates splenic T-lymphocyte inflammation via enhanced release of sympathetic neurotransmitters. To test this, we first exposed naïve primary T-lymphocytes to various sympathetic catecholamines, including dopamine, norepinephrine, and epinephrine. Contrary to our original hypothesis, we observed no significant changes in IL-17A expression or mitochondria reactive oxygen species. Next, we assessed the physiological response of naïve T-lymphocytes exposed to neuropeptide Y (NPY) given its role in both sympathoexcitation as well as in PTSD. Interestingly, naïve T-lymphocytes treated with NPY also demonstrated no effect, but when NPY was given in combination of norepinephrine, we observed both an increase in IL-17A gene expression (~2 fold) and intracellular redox (~2 fold). To further investigate this effect, we examined NPY levels in our RSDS mouse model over-time. Circulating NPY levels spiked immediately after only one day of RSDS (~4.5 fold, p=0.0317) but steadily declined over the remaining 10 day psychological trauma induction. Intriguingly, NPY levels were strongly correlated with both circulating IL-17A and T-lymphocyte mitochondrial reactive oxygen species levels after RSDS, supporting our in vitro observations. Overall, these data suggest NPY may be playing a critical role in shaping the inflammatory milieu after psychological trauma, which may enhance the likelihood of inflammatory comorbidities. NIH R01HL158521 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Repeated Social Defeat Stress Induces an Inflammatory Gut Milieu by Altering the Mucosal Barrier Integrity and Gut Microbiota Homeostasis

BackgroundPosttraumatic stress disorder (PTSD) is a mental health condition triggered by exposure to traumatic events in an individual’s life. Patients with PTSD are also at a higher risk for comorbidities. However, it is not well understood how PTSD affects human health and/or promotes the risk for comorbidities. Nevertheless, patients with PTSD harbor a proinflammatory milieu and dysbiotic gut microbiota. Gut barrier integrity helps to maintain normal gut homeostasis and its dysregulation promotes gut dysbiosis and inflammation. MethodsWe used a mouse model of repeated social defeat stress (RSDS), a preclinical model of PTSD. Behavioral studies, metagenomics analysis of the microbiome, gut permeability assay (on mouse colon, using an Ussing chamber), immunoblotting, and immunohistochemical analyses were performed. Polarized intestinal epithelial cells and 3-dimensional crypt cultures were used for mechanistic analysis. ResultsThe RSDS mice harbor a heightened proinflammatory gut environment and microbiota dysbiosis. The RSDS mice further showed significant dysregulation of gut barrier functions, including transepithelial electrical resistance, mucin homeostasis, and antimicrobial responses. RSDS mice also showed a specific increase in intestinal expression of claudin-2, a tight junction protein, and epinephrine, a stress-induced neurotransmitter. Treating intestinal epithelial cells or 3-dimensional cultured crypts with norepinephrine or intestinal luminal contents (fecal contents) upregulated claudin-2 expression and inhibited transepithelial electrical resistance. ConclusionsTraumatic stress induces dysregulation of gut barrier functions, which may underlie the observed gut microbiota changes and proinflammatory gut milieu, all of which may have an interdependent effect on the health and increased risk of comorbidities in patients with PTSD.

Transthyretin Is Commonly Upregulated in the Hippocampus of Two Stress-Induced Depression Mouse Models.

Chronic stress can affect gene expression in the hippocampus, which alters neural and cerebrovascular functions, thereby contributing to the development of mental disorders such as depression. Although several differentially expressed genes in the depressed brain have been reported, gene expression changes in the stressed brain remain underexplored. Therefore, this study examines hippocampal gene expression in two mouse models of depression induced by forced swim stress (FSS) and repeated social defeat stress (R-SDS). Transthyretin (Ttr) was commonly upregulated in the hippocampus of both mouse models, as determined by microarray, RT-qPCR, and Western blot analyses. Evaluation of the effects of overexpressed Ttr in the hippocampus using adeno-associated virus-mediated gene transfer revealed that TTR overexpression induced depression-like behavior and upregulation of Lcn2 and several proinflammatory genes (Icam1 and Vcam1) in the hippocampus. Upregulation of these inflammation-related genes was confirmed in the hippocampus obtained from mice vulnerable to R-SDS. These results suggest that chronic stress upregulates Ttr expression in the hippocampus and that Ttr upregulation may be involved in the induction of depression-like behavior.

S100a9 Protects Against the Effects of Repeated Social Defeat Stress

BackgroundPosttraumatic stress disorder, a consequence of psychological trauma, is associated with increased inflammation and an elevated risk of developing comorbid inflammatory diseases. However, the mechanistic link between this mental health disorder and inflammation remains elusive. We previously found that S100a8 and S100a9 messenger RNA, genes that encode the protein calprotectin, were significantly upregulated in T lymphocytes and positively correlated with inflammatory gene expression and the mitochondrial redox environment in these cells. Therefore, we hypothesized that genetic deletion of calprotectin would attenuate the inflammatory and redox phenotype displayed after psychological trauma. MethodsWe used a preclinical mouse model of posttraumatic stress disorder known as repeated social defeat stress (RSDS) combined with pharmacological and genetic manipulation of S100a9 (which functionally eliminates calprotectin). A total of 186 animals (93 control, 93 RSDS) were used in these studies. ResultsUnexpectedly, we observed worsening of behavioral pathology, inflammation, and the mitochondrial redox environment in mice after RSDS compared with wild-type animals. Furthermore, loss of calprotectin significantly enhanced the metabolic demand on T lymphocytes, suggesting that this protein may play an undescribed role in mitochondrial regulation. This was further supported by single-cell RNA sequencing analysis demonstrating that RSDS and loss of S100a9 primarily altered genes associated with mitochondrial function and oxidative phosphorylation. ConclusionsThese data demonstrate that the loss of calprotectin potentiates the RSDS-induced phenotype, which suggests that its observed upregulation after psychological trauma may provide previously unexplored protective functions.

Behavioral effects and inflammatory markers in the brain and periphery after repeated social defeat stress burdened by Opisthorchis felineus infection in mice

The combination of 4-week repeated social defeat stress (RSDS) and Opisthorchis felineus infection was modeled in C57BL/6 mice. Various parameters were compared between three experimental groups of male mice (SS: mice subjected to RSDS, OF: mice infected with O. felineus, and OF + SS: mice subjected to both adverse factors) and behavior-tested and intact (INT) controls. The combination caused liver hypertrophy and increased the blood level of proinflammatory cytokine interleukin 6 and proteolytic activity of cathepsin B in the hippocampus. Meanwhile, hypertrophy of the spleen and of adrenal glands was noticeable. Anxious behavior in the elevated plus-maze test was predominantly due to the infection, with synergistic effects of an interaction of the two adverse factors on multiple parameters in OF + SS mice. Depression-like behavior in the forced swimming test was caused only by RSDS and was equally pronounced in SS mice and OF + SS mice. Helminths attenuated the activities of cathepsin B in the liver and hypothalamus (which were high in SS mice) and increased cathepsin L activity in the liver. The highest blood level of corticosterone was seen in SS mice but was decreased to control levels by the trematode infection. OF mice had the lowest level of corticosterone, comparable to that in INT mice. Thus, the first data were obtained on the ability of O. felineus helminths—even at the immature stage—to modulate the effects of RSDS, thereby affecting functional connections of the host, namely “helminths → liver↔brain axis.”

T-lymphocyte tyrosine hydroxylase regulates TH17 T-lymphocytes during repeated social defeat stress

Posttraumatic stress disorder (PTSD) is a debilitating psychiatric disorder which results in deleterious changes to psychological and physical health. Patients with PTSD are especially susceptible to life-threatening co-morbid inflammation-driven pathologies, such as autoimmunity, while also demonstrating increased T-helper 17 (TH17) lymphocyte-driven inflammation. While the exact mechanism of this increased inflammation is unknown, overactivity of the sympathetic nervous system is a hallmark of PTSD. Neurotransmitters of the sympathetic nervous system (i.e., catecholamines) can alter T-lymphocyte function, which we have previously demonstrated to be partially mitochondrial redox-mediated. Furthermore, we have previously elucidated that T-lymphocytes generate their own catecholamines, and strong associations exist between tyrosine hydroxylase (TH; the rate-limiting enzyme in the synthesis of catecholamines) and pro-inflammatory interleukin 17A (IL-17A) expression within purified T-lymphocytes in a rodent model of psychological trauma. Therefore, we hypothesized that T-lymphocyte-generated catecholamines drive TH17 T-lymphocyte polarization through a mitochondrial superoxide-dependent mechanism during psychological trauma. To test this, T-lymphocyte-specific TH knockout mice (THT-KO) were subjected to psychological trauma utilizing repeated social defeat stress (RSDS). RSDS characteristically increased tumor necrosis factor-α (TNFα), IL-6, IL-17A, and IL-22, however, IL-17A and IL-22 (TH17 produced cytokines) were selectively attenuated in circulation and in T-lymphocytes of THT-KO animals. When activated ex vivo, secretion of IL-17A and IL-22 by THT-KO T-lymphocytes was also found to be reduced, but could be partially rescued with supplementation of norepinephrine specifically. Interestingly, THT-KO T-lymphocytes were still able to polarize to TH17 under exogenous polarizing conditions. Last, contrary to our hypothesis, we found RSDS-exposed THT-KO T-lymphocytes still displayed elevated mitochondrial superoxide, suggesting increased mitochondrial superoxide is upstream of T-lymphocyte TH induction, activity, and TH17 regulation. Overall, these data demonstrate TH in T-lymphocytes plays a critical role in RSDS-induced TH17 T-lymphocytes and offer a previously undescribed regulator of inflammation in RSDS.

Single-Shot 10K Proteome Approach: Over 10,000 Protein Identifications by Data-Independent Acquisition-Based Single-Shot Proteomics with Ion Mobility Spectrometry.

The evolution of mass spectrometry (MS) and analytical techniques has led to the demand for proteome analysis with high proteome coverage in single-shot measurements. Focus has been placed on data-independent acquisition (DIA)-MS and ion mobility spectrometry as techniques for deep proteome analysis. We aimed to expand the proteome coverage by single-shot measurements using optimizing high-field asymmetric waveform ion mobility spectrometry parameters in DIA-MS. With our established proteome analysis system, more than 10,000 protein groups were identified from HEK293 cell digests within 120 min of MS measurement time. Additionally, we applied our approach to the analysis of host proteins in mouse feces and detected as many as 892 host protein groups (771 upregulated/121 downregulated proteins) in a mouse model of repeated social defeat stress (R-SDS) used in studying depression. Interestingly, 285 proteins elevated by R-SDS were related to mental disorders. The fecal host protein profiling by deep proteome analysis may help us understand mental illness pathologies noninvasively. Thus, our approach will be helpful for an in-depth comparison of protein expression levels for biological and medical research because it enables the analysis of highly proteome coverage in a single-shot measurement.

Immune Phenotypes of Oligodendroglial‐Lineage Cells in MDD and in Response to Chronic Stress‐Induced Microglial Inflammation

BackgroundMajor Depressive Disorder (MDD) is a complex and heterogenous psychiatric disorder affecting more than 300 million people worldwide. Though the mechanisms underlying MDD are not fully understood, a subset of depressive patients can manifest chronic neuroinflammatory responses. In addition, brain‐imaging studies in MDD patients have provided evidence of white matter reductions and such changes are hypothesized to lead to disruption of Oligodendroglial‐Lineage cells (OLN) homeostatic mechanisms, resulting in destabilization of emotional/cognitive circuitry. The present goal of the study is to investigate whether these histopathological alterations are linked with chronic inflammation through single‐nucleus transcriptomics of MDD patients and in a depression mouse model.MethodsBioinformatics: Raw counts were downloaded from GEO (GSE144136), and expression objects created using Seurat. OLN were subsetted, clustered (using UMAP), and annotated based on their expression of early and late OLN markers. Pseudotime trajectory analysis was conducted using Moncle3. In vivo: The Repeated Social Defeat Stress (RSDS) paradigm (10 days) was used to induce depressive‐like behavior in 8–12‐week‐old male CX3CR1‐GFP+ and CSPG4‐EGFP+ mice. Behavioral tasks (BH) were performed to stratify the defeated mice to susceptible (S; depressive‐like) and resilient (R; non‐depressive) to stress groups. The study focuses on the MDD‐affected prefrontal cortex (mPFC) area (Fig).ResultsSeparate clustering of MDD and Control samples yielded four similar clusters labeled based on their gene expression as: OPCs, Committed OPCs, Immature Oligodendrocytes (OLs), and Mature OLs. One MDD‐specific cluster was also obtained and labeled as Immune OLs (ImOL), given its expression of immune genes, such as: ARGHAP24, ADAM28, LPAR6, C3, CD74, and P2RY12. Further analysis of the ImOL cluster revealedexpression of markers associated with later phases of OLN progeny. Finally, a predictive model of pseudotime trajectory revealed alterations in the progression of OLN progeny in MDD samples. We then sought to identify this novel subset of ImOL in the RSDS model of depression. MHCII, C3 and P2RY12 were examined in conjunction with OLN markers (CSPG4, O4, CNP), with significant changes observed in S groups. Interestingly, microglial phagocytosis of myelin elements (CX3CR1‐GFP, MHCII, CNP), was significantly increased in S groups post RSDS, rendering the ImOL populations as potential recruiters of innate immunity.ConclusionsTogether, our computational data in MDD patients and depression mouse model, show that OLN can swiftly respond to chronic stress, and adopt an immune‐like phenotype, potentially contributing to the white matter disturbances observed in MDD.

Abstract 210: Susceptibility To Social Stress Modulates Hematopoiesis And Impairs Atherosclerosis Resolution

Background: Depressive disorders frequently involve prolonged stress and are associated with increased risk of cardiovascular disease (CVD). A major hurdle in gaining mechanistic insight is the paucity of animal models that accurately recapitulate the symptoms of depression disorders. The major question we investigated was whether psychological stress impairs the benefits of the common therapy used to reduce CV risk, namely, lipid lowering. Methods: We employed a standard protocol of repeated social defeat stress (RSDS) that elicits a depression-like phenotype in mice to test the effects on atherosclerosis resolution. LDLr-deficient mice were kept on an atherogenic diet for 24 wks. Then RSDS was applied for 10 days. Mice that developed a depressive-like phenotype, termed stress susceptible (SS), were identified with behavior testing. SS and control mice were then placed on cholesterol-reducing therapy. Results: SS mice vs. control mice had expansion of bone marrow (BM) myeloid progenitors (~80% increase for GMP cells; P&lt;0.001) and more circulating monocytes (~2X; P &lt;0.05), which exhibited higher recruitment (2.5X; P &lt;0.001) to atherosclerotic plaques. This resulted in increased (~50%; P &lt;0.05) plaque macrophage content after 3 wks of cholesterol lowering. To investigate whether the effects of RSDS on inflammation and atherosclerosis were mediated by re-programming of the BM progenitors of plaque macrophages, we performed BM transplantation from control or SS mice into LDLr-deficient mice. After the recovery period, the mice were fed an atherogenic diet for 24 wks. At that time, we found that compared to control BM recipients, SS BM recipients sustained the increase in myeloid progenitors (~16% increase, P&lt;0.05 for GMP population) and circulating inflammatory monocytes (50% increase; P &lt;0.001), as well as enhanced atherosclerotic plaque inflammation (23% more macrophage content; P&lt;0.001). Conclusions: Social stress impairs atherosclerosis inflammation resolution after cholesterol lowering, apparently by re-programming BM precursors of plaque macrophages. By applying molecular methods to this model, we hope to further understand the mechanistic basis for the increase in CVD risk in stressed people despite risk reducing therapies.

Abstract 126: Susceptibility To Social Stress Modulates Hematopoiesis And Impairs Atherosclerosis Resolution

Background: Depressive disorders frequently involve prolonged stress and are associated with increased risk of cardiovascular disease (CVD). A major hurdle in gaining mechanistic insight is the paucity of animal models that accurately recapitulate the symptoms of depression disorders. The major question we investigated was whether psychological stress impairs the benefits of the common therapy used to reduce CV risk, namely, lipid lowering. Methods: We employed a standard protocol of repeated social defeat stress (RSDS) that elicits a depression-like phenotype in mice to test the effects on atherosclerosis resolution. LDLr-deficient mice were kept on an atherogenic diet for 24 wks. Then RSDS was applied for 10 days. Mice that developed a depressive-like phenotype, termed stress susceptible (SS), were identified with behavior testing. SS and control mice were then placed on cholesterol-reducing therapy. Results: SS mice vs. control mice had expansion of bone marrow (BM) myeloid progenitors (~80% increase for GMP cells; P&lt;0.001) and more circulating monocytes (~2X; P &lt;0.05), which exhibited higher recruitment (2.5X; P &lt;0.001) to atherosclerotic plaques. This resulted in increased (~50%; P &lt;0.05) plaque macrophage content after 3 wks of cholesterol lowering. To investigate whether the effects of RSDS on inflammation and atherosclerosis were mediated by re-programming of the BM progenitors of plaque macrophages, we performed BM transplantation from control or SS mice into LDLr-deficient mice. After the recovery period, the mice were fed an atherogenic diet for 24 wks. At that time, we found that compared to control BM recipients, SS BM recipients sustained the increase in myeloid progenitors (~16% increase, P&lt;0.05 for GMP population) and circulating inflammatory monocytes (50% increase; P &lt;0.001), as well as enhanced atherosclerotic plaque inflammation (23% more macrophage content; P&lt;0.001). Conclusions: Social stress impairs atherosclerosis inflammation resolution after cholesterol lowering, apparently by re-programming BM precursors of plaque macrophages. By applying molecular methods to this model, we hope to further understand the mechanistic basis for the increase in CVD risk in stressed people despite risk reducing therapies.

Deficient Autophagy in Microglia Aggravates Repeated Social Defeat Stress-Induced Social Avoidance.

Major depressive disorder (MDD) is associated with repeated exposure to environmental stress. Autophagy is activated under various stress conditions that are associated with several diseases in the brain. This study was aimed at elucidating the autophagy signaling changes in the prefrontal cortex (PFC) under repeated social defeat (RSD) to investigate the involvement of microglial autophagy in RSD-induced behavioral changes. We found that RSD stress, an animal model of MDD, significantly induced initial autophagic signals followed by increased transcription of autophagy-related genes (Atg6, Atg7, and Atg12) in the PFC. Similarly, significantly increased transcripts of ATGs (Atg6, Atg7, Atg12, and Atg5) were confirmed in the postmortem PFC of patients with MDD. The protein levels of the prefrontal cortical LC3B were significantly increased, whereas p62 was significantly decreased in the resilient but not in susceptible mice and patients with MDD. This indicates that enhanced autophagic flux may alleviate stress-induced depression. Furthermore, we identified that FKBP5, an early-stage autophagy regulator, was significantly increased in the PFC of resilient mice at the transcript and protein levels. In addition, the resilient mice exhibited enhanced autophagic flux in the prefrontal cortical microglia, and the autophagic deficiency in microglia aggravated RSD-induced social avoidance, indicating that microglial autophagy involves stress-induced behavioral changes.

Forkhead Box 1(FoxO1) mediates psychological stress-induced neuroinflammation

ABSTRACT Objectives Neuroinflammation plays a key role in cerebrovascular disease (CVD). Neuropsychiatric disorders appear to share an epidemiological association with inflammation, but the mechanisms are unclear. Forkhead box 1 (FoxO1) regulates inflammatory signaling in diabetes and cardiovascular diseases, but its role in psychological stress-induced neuroinflammation remains unknown. Therefore, we investigated the potential involvement of FoxO1 in repeated social defeat stress (RSDS)-induced neuroinflammation. Methods 6-week-old male C57BL/6 J mice were randomly divided into RSDS or control groups. In the RSDS group, mice (18–22 g) were individually subjected to social defeat by an 8-week-old CD-1 mouse (28–32 g) for 10 min daily for 10 consecutive days. At 24 h after this 10-day process, corticosterone (CORT), epinephrine (EPI), hydrogen peroxide, and inflammatory factors (TNF-α, IL-6, IL-1β, and VCAM-1) from serum and brain tissues were assayed using ELISA, real-time PCR, and Western blot. Iba-1 was determined by immunofluorescence (IF), and FoxO1 siRNA was transfected into BV2 cells to further analyze the expression of inflammatory factors. Results RSDS significantly increased the levels of TNF-α, IL-6, IL-1β, and VCAM-1 in the serum; it also increased both mRNA and protein expression of these in the brain. FoxO1 was significantly increased after stress, while its knockdown significantly suppressed stress-induced inflammation. Immunofluorescence demonstrated the activation of microglia in the setting of RSDS. Conclusion RSDS induced a measurable inflammatory response in the blood and brain, and FoxO1 was demonstrated in vitro to aggravate stress-induced inflammation.

Induction of Repeated Social Defeat Stress in Rats.

Repeated social defeat stress (RSDS) is a model of chronic stress in rodents. There are several variants of social defeat procedures that exert robust effects in mice, but few published detailed protocols to produce a robust stress and altered immunological profile in rats. In this article, we describe the protocol for the induction of RSDS in adult male Sprague-Dawley rats. Using a resident-intruder paradigm, a physical component of stress is induced by direct attack from the resident aggressive retired breeder Long-Evans rats on the intruder experimental rats. A subsequent threat component is induced by the presence of the aggressor in the vicinity of the intruder, but with physical separation between them. The RSDS induced by this protocol produces robust immunological and behavioral changes in the experimental rats, as evidenced by development of anxiety-like behaviors in open field, social interaction, and elevated plus maze tests, as well as by changes in immune parameters (Munshi et al., 2020). This approach has been used as an ethologically relevant model of stressors that are potent enough to impact neural circuits that are similar to the neural circuits impacted in patients with depression and anxiety.

Inactivation of Zona Incerta Blocks Social Conditioned Place Aversion and Modulates Post-traumatic Stress Disorder-Like Behaviors in Mice.

Zona incerta (ZI), a largely inhibitory subthalamic region connected with many brain areas, has been suggested to serve as an integrative node for modulation of behaviors and physiological states, such as fear memory conditioning and aversion responses. It is, however, unclear whether ZI regulated the repeated social defeat stress (RSDS)-induced social conditioned place aversion (CPA) and post-traumatic stress disorder (PTSD)-like behaviors. In this study, the function of ZI was silenced via bilateral injection of tetanus toxin light chain (Tet-tox), a neurotoxin that completely blocks the evoked synaptic transmissions, expressing adeno-associated viruses (AAVs). We found ZI silencing: (1) significantly blocked the expression of RSDS-induced social CPA with no effect on the innate preference; (2) significantly enhanced the anxiety level in mice experienced RSDS with no effect on the locomotion activity; (3) altered the PTSD-associated behaviors, including the promotion of spatial cognitive impairment and the preventions of PPI deficit and social avoidance behavior. These effects were not observed on non-stressed mice. In summary, our results suggest the important role of ZI in modulating RSDS-induced social CPA and PTSD-like behaviors.