Chronic psychosocial stress induces microglial activation and inflammatory responses that lead to neuronal dysfunction and depressive-like behavior

Chronic psychosocial stress induces microglial activation and inflammatory responses that lead to neuronal dysfunction and depressive-like behavior | 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 Chronic psychosocial stress induces microglial activation and inflammatory responses that lead to neuronal dysfunction and depressive-like behavior Stella Tsirka, Alexandros Kokkosis, Miguel Madeira, Kimonas Valais, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1991809/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Repeated stress can lead to the development of anxiety and is considered a risk factor for major depressive disorder (MDD). Clinical studies and animal models of repeated and chronic stress have reported that symptom severity is correlated with microglial activation and upregulation of neuroinflammatory cytokine signaling in brain areas implicated in mood regulation. Despite mounting evidence implicating impairments of neuroplasticity and synaptic signaling deficits into the pathophysiology of stress-related mental disorders, whether microglial activation modulates neuronal homeostasis in response to chronic stress has been debated. Here, using the repeated social defeat stress (RSDS) mouse model we demonstrate that microglial activation and related inflammatory responses are regulating neuronal plasticity associated with depressive-like behavior. Specifically, we show that chronic stress induces a swift activation and proliferation of microglia as well as macrophage infiltration in the mPFC, which are spatially related to neuronal activation. Moreover, we report a remarkable association of microglial spectrum of reactivity and concomitant inflammatory responses with susceptibility or resilience to chronic stress. In addition, we find that exposure to chronic stress exacerbates phagocytosis of synaptic elements and significant neuronal plasticity deficits associated with depressive-like behavior. Importantly, by utilizing two different CSF1R inhibitors (the brain penetrant PLX5622 and the non-penetrant PLX73086) we determine the contributions of microglial and infiltrating macrophages in the depression pathophenotype. Our findings highlight a crucial role for microglia (and secondarily macrophages) in catalyzing the pathological manifestations of depression in response to chronic stress by promoting neuroinflammation and neuronal deficits in mPFC. Biological sciences/Neuroscience/Molecular neuroscience Biological sciences/Physiology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Microglia, the innate immune cells of the central nervous system (CNS), have been shown to be active participants in the normal physiological, homeostatic functions of the CNS, as well as to serve as modulators of pathologic events that take place in the brain and spinal cord, including excitotoxic and neurodegenerative processes, stroke and autoimmune diseases 1 . Additionally, microglia share common features with bone marrow-derived monocytes, yet they possess a unique gene expression profile 2 . The modulatory properties of microglia are regulated by toll-like (TLRs), complement, purinergic and scavenger receptors, leading to secretion of cytokines, chemokines, and other signaling molecules 1 , 3 , 4 . A large body of evidence suggests that neuroimmunologic processes affect neuronal homeostasis contributing to the pathophysiology of mental disorders, as well 5 – 7 . More specifically, elevated levels of proinflammatory cytokines, such as IL-6, TNF-a and C-reactive protein (CRP), have been measured both peripherally and centrally in patients with Major Depression Disorder (MDD); and autoimmune/ inflammatory diseases are considered to be co-morbid with MDD 5,7−9 . To this end, experimental adaptations in animals using either lipopolysaccharide (LPS) or cytokine administration (IL-1b or IL-6) resulted in loss of interest and reduction in reward sensitivity 10 , 11 Neuroimaging studies of MDD patients have also revealed that symptom severity is correlated with elevated levels of translocator protein (TSPO; neuroinflammation marker) in brain regions specifically implicated in mood regulation, including the medial prefrontal cortex (mPFC), the anterior cingulate gyrus and infralimbic cortices 12 – 14 . The neuroimmunological responses to chronic stress observed in humans are also evident in rodent models of psychosocial stress. In models of chronic social defeat (CSD) and repeated social defeat stress (RSDS) we and others have shown that stress may result in increased local generation of reactive oxygen species (ROS) 15 , 16 . In addition, elimination of microglia (in the context of CSD) using inhibitors of Colony Stimulating Factor Receptor 1 (CSF1R) mitigated the high levels of ROS and protected from the development of depressive-like behavior in mice 16 . Similar results have been shown for chronic unpredictable mild stress and the use of the microglial inhibitor minocycline 17 . Although a strong relationship between the presence of activated microglia and MDD-like symptoms has been suggested, a requisite role for microglia for the development of depressive behavior has not been established. In the present study we use the RSDS paradigm to study the cellular and molecular responses of microglia in the mPFC, and examine their role in the modulation of synaptic changes and neuroplasticity, as well as the related behavioral manifestations of stress-induced depressive-like behavior in mice. We demonstrate that microglia are swiftly activated in response to chronic stress, spatially relating to increased neuronal activation in threat-appraisal areas. Moreover, we reveal an important association of microglial and infiltrating-macrophage reactivity and respective inflammatory responses, with susceptibility or resilience towards chronic stress. We also report that microglia are negatively regulating neuronal plasticity and modulate levels of synaptic elements in response to chronic stress, leading to depressive-like behavior. Importantly, we assess the contribution of peripheral macrophages to the depressive-like traits upon chronic stress. Finally, we find that depression-related phenotypic changes are suppressed with microglial ablation but are rapidly re-established after microglial repopulation, suggesting that microglia are critical dynamic mediators for the establishment of depressive-like behavior in mice. Results Chronic stress induces microglial recruitment and increased microglial proliferation in the mPFC, leading to depressive-like behavior We utilized the repeated social defeat paradigm (RSDS; 10 days) to induce depressive-like behavior in adult male mice, as previously described 15 , 18 (Fig. 1 a). Briefly, twenty-four hours after RSDS (D10) behavioral tests were performed (BH; D11-D14) and the socially defeated (SD) mice were divided (see Methods) in two groups: SD-Sus (~ 80%; susceptible) and SD-Res (~ 20%; resilient). Both groups exhibited significant anxiety-like behavior, however the SD-Sus demonstrated characteristic depressive-like phenotypic features: social avoidance, despair-like behavior, reduction of reward under stress, anhedonia, and elevated plasma corticosterone levels (~ 130 ng/ml; data not shown), as previously demonstrated 15 (Fig. 1 b, Supplementary Fig. 1a-d ). The naïve mice (Con) were only exposed to wt (C57BL/6J) mice for 1 minute. To determine the effects of chronic stress on microglial density and proliferation in the mPFC, 5-Bromo-2′-deoxyuridine (BrdU) was administered ad libitum throughout RSDS, and experimental groups were euthanized on D15. Histological analysis of the mPFC revealed a significant increase of microglial cell density (microglial/macrophage Iba1 + marker and CX3CR1 -GFP + mice) and microglial proliferation capacity (%BrdU + of CX3CR1 + ), compared to Con and SD-Res (Fig. 1 c-g). Importantly, microglial recruitment was also observed at an earlier time point (D6; CX3CR1 + ) after a short-term RSDS paradigm adaptation (miniSD for 3 days/BH on D4-D6; see Methods for more details 15 ) and was spatially related to a significant increase of neuronal activity (NeuN + cFos + immunoreactivity) in mPFC ( Supplementary Fig. 2a-d ). Of interest, the microglia in the mPFC of SD-Sus mice adopted a more reactive morphology which was characterized by shorter and fewer process intersections, increased cell body surface area and reduced process complexity index, as determined by Sholl and branched structure analyses of Iba1 + hyperstack traces (Fig. 1 h-k; Methods). These morphological attributes have been previously identified in neuroinflammatory 19 and neurodegenerative models 20 , and strongly associate with a pro-inflammatory activation state 21 , 22 . Interestingly, the SD-Res microglia also exhibited reactive morphology which was different from that of Con and SD-Sus, displaying fewer intersections closer to the cell body (but longer and numerous processes distally from it), but intermediary changes in cell body and branch complexity, suggesting a distinct microglial activation state. Chronic stress susceptibility/resilience is associated with the inflammatory response profile and polarization of microglial/macrophages in the mPFC. Psychosocial stress can promptly activate the release of glucocorticoids 5 , 15 , 22 (we have previously shown elevated plasma corticosterone levels in SD-Sus mice 15 ), through which they can signal the recruitment of peripheral monocytes into the brain parenchyma. We performed flow cytometry in CX3CR1 -GFP + mice and observed significant monocytic population trafficking [CD11b + CD45 high (macrophages)] into the mPFC of SD-Sus compared to the Con and SD-Res groups on D15 post-RSDS (Fig. 2 a-b). These findings were further supported by immunoblot analysis of key adhesion molecules responsible for monocytic infiltration (VCAM-1 and ICAM-1) which were significantly increased in the SD-Sus group (Fig. 2 c-d). Further characterization of the microglia/macrophage populations revealed a significant increase of the pro-inflammatory marker CD86 and a concomitant decrease of the anti-inflammatory marker CD206 in the SD-Sus mice compared to the Con and SD-Res groups (Fig. 2 e-j), suggesting a pro-inflammatory inclination of the SD-Sus phenotype. Accordingly, a significant upregulation of CD206 + expression was noted in the SD-Res microglia/macrophages compared to Con and SD-Sus, suggesting an anti-inflammatory phenotype (Fig. 2 e-j). This was further corroborated by RT-qPCR and immunoblot assays of mPFC after RSDS. Specifically, the mRNA levels of TNF-α, CXCL10 and IL-1β and protein levels of CD86, iNOS (all pro-inflammatory markers) were significantly upregulated in the SD-Sus, while the IL-10 mRNA and CD206 protein levels (both anti-inflammatory markers) were significantly decreased compared to the Con and SD-Res (Fig. 2 k-n and Supplementary Fig. 3e ). Respectively, the SD-Res mice exhibited an increase of the mRNA levels of IL-10 and the protein levels of CD206 and Arg1 (anti-inflammatory markers) (Fig. 2 k-n and Supplementary Fig. 3e ). Consistent with the proinflammatory responses observed in the SD-Sus mice, total and microglial-specific translocator protein (TSPO; microgliosis and astrogliosis marker indicative of neuroinflammation) levels were markedly elevated in mPFC compared to Con and SD-Res groups, possibly through a TLR4-dependent pathway ( Supplementary Fig. 3a-d ), depicting significant inflammatory responses in the area of mPFC in the SD-Sus mice. Microglial phagocytosis of neuronal synaptic elements is exacerbated in mPFC post-chronic stress. Delving into the microglial reactivity after chronic stress, we sought to assess their phagocytic activity in mPFC using the lysosomal marker CD68, and whether this could be correlated with the expression of the proinflammatory marker iNOS during susceptibility (Fig. 2 o). The SD-Sus microglia exhibited a dramatic increase in phagocytosis which was noticeably colocalized with the iNOS pro-inflammatory marker (iNOS + CD68 + Iba1 + ), compared to Con and SD-Res groups (Fig. 2 o-r). In stark contrast the microglia from the SD-Res group, even though had significantly increased CD68 levels compared to Con (Fig. 2 p), did not display a pro-inflammatory phenotype (Fig. 2 q,r), indicating a distinct reactivity phenotype. It is known that microglia are heavily involved in synaptic reorganization and circuitry refinement through synaptic pruning in the adult brain 23 . Excessive pruning, however, may lead to loss or weakening of synaptic connections in the neuronal circuits 24 . Therefore, we next asked whether the elevated phagocytic capacity of microglia in the SD-Sus mice would result in increased engulfment of synaptic components in the mPFC neurons. We imaged CX3CR1 GFP/+ mice along with lysosomal marker CD68 and either PSD95 (postsynaptic marker) or Synapsin-1 (Syn-1; presynaptic marker) (Fig. 3 ). The microglial phagocytosis of PSD95 (CX3CR1 + CD68 + PSD95 + ; Fig. 3 a,d) and of Syn-1 punctae (CX3CR1 + CD68 + Syn-1+ + Fig. 3 b,f), were significantly increased in the SD-Sus mice compared to Con and SD-Res. This resulted in concomitant reductions in the PSD95 and Syn-1 immunoreactivity (Fig. 3 a-c,e) and protein expression levels (Fig. 6 e) in the mPFC of SD-Sus mice. Evident of the microglial reactivity in the depressive-like mice was also the significant increase of microglial phagocytosis of myelin observed in the SD-Sus mice (CX3CR1 + CD68 + CNP + ), leading to significant myelin deficits in the mPFC area (Fig. 4 a-c), as we have previously demonstrated 15 . The interaction of cAMP response element-binding protein (CREB) with brain-derived neurotrophic factor (BDNF) is an essential element in signal transduction pathways involved in mental disorders and critical for cellular resilience and neuroplasticity 25 , 26 . Due to the significant microglial-mediated synaptic element reductions observed in the mPFC area, we sought to assess the potential effects of chronic stress on neuronal plasticity using the markers p-CREB(Ser133) and BDNF in the mPFC of the SD groups. The immunoreactivity of neuronal p-CREB in SD-Sus mice was significantly decreased compared to Con and was in stark contrast to the significant upregulation evident in the SD-Res mice (Fig. 4 d-e). Analogous bimodal responses were noted in the BDNF protein levels of the SD groups (Fig. 6 f), suggesting that the chronic-stress susceptibility/resilience of the SD mice is interlinked with neuroplasticity, synaptic connectivity, and cellular resilience of the neuronal circuitry in the mPFC. Chronic stress induces long-lasting monocytic infiltration into the mPFC, mediating depressive-like behavior in mice. Due to the persisting inflammatory responses in mPFC, we next used the CX3CR1- Cre ERT 2 - eYFP:: Rosa26- tdTom mouse line (Fig. 5 a; see Methods for more details) to trace the monocytic infiltration on D25 post-RSDS. For the induction of Cre-recombination, TMX was administered i.p. in CX3CR1 CreER/+ ::R26 tdTOM/+ mice for 5 days 27 , starting at P30. The RSDS paradigm was performed 1 month after the induction of Cre, and behavioral tests were performed 2 weeks after the end of the paradigm (Fig. 5 b; BH, D22-D25), as we have previously shown 15 . The first days after Cre-recombination, all microglia and peripheral macrophages (CX3CR1 + ) became YFP + tdTom + cells (data not shown). Contrary to the self-renewing microglia 28 , the bone marrow–derived monocytic/macrophage populations, have an estimated half-life of 3–4 weeks, allowing for fate mapping of the recruited monocytes (YFP + tdTom − ) 28 . We euthanized the CX3CR1 CreER/+ ::R26 tdTOM/+ mice on D25, revealing a significant increase of monocytic populations in the mPFC of SD-Sus mice compared to the Con and SD-Res groups (Fig. 5 c-e). In addition, we visualized the expression of ICAM-1 (a key monocyte-adhesion molecule): ICAM-1 immunoreactivity was significantly upregulated on the endothelial cells in SD-Sus mice, with a large portion of it colocalizing with the microglia/macrophage populations (CX3CR1-eYFP + ICAM-1 + ) (Fig. 5 f-g). Microglial and monocytic elimination during chronic stress exerts significant anti-depressant action. To determine whether the long-lasting monocytic recruitment (Fig. 2 a-b, Fig. 5 c,e) and concomitant inflammatory responses (Fig. 2 e-j) are responsible for the emergence of depressive-like behavior after chronic stress, we dissected and investigated separately the pathophenotypic contribution of the microglia and monocytes. We utilized PLX73086 (see Methods for details), a CSF1R inhibitor which depletes peripheral macrophage populations (dose 200 mg/kg) but does not affect the CNS resident microglia because of its low blood-brain barrier (BBB) penetration ( Supplementary Fig. 4a ) 29 . Experimental groups starting at D0 had ad libitum access either to Control-chow or the drug-containing chow (PLX73086) throughout the RSDS until they were euthanized on D15 (Fig. 5 h). As summarized in Fig. 5 i the PLX73086 treatment exerted moderate but significant anti-depressant effects, as measured by a battery of behavioral tests (SI, EPM, FST and SPT) (Fig. 5 h-m). Notably, the percentage of the SD-Sus mice dropped from 80–50%, based on the social interaction ratio, suggesting that the recruited monocytes play a significant role in the development of depression (Fig. 5 i, j). We next determined the contribution of microglia in the development of the depressive-like behavior in chronically stressed mice. PLX5622 (see Methods for more details) is a CSF1R inhibitor which can enter the CNS and ablate 90–95% of microglial/macrophage populations after 7 days of treatment (1,200 mg/kg dose) 29 , 30 . PLX5622-chow treatment or Control-chow was introduced on D0 of the RSDS paradigm and was administered until mice were euthanized for further analysis (D15) (Fig. 6 a). In the experimental groups treated with PLX5622-chow significant microglial ablation (~ 95%) was observed compared to the Control-chow on D15 after the RSDS paradigm ( Supplementary Fig. 4b-c ). Remarkably, microglial elimination during chronic stress (PLX56-Res group) was able to protect 100% of the SD mice (in comparison to the 20% of the naturally resilient SD-Res group in animals receiving Control-show) from the emergence of depressive-like behavior (social avoidance, anhedonia, anxiety, reward under stress and despair), suggestive of an essential role that microglia have in the progression of the disorder (Fig. 6 b,d and Supplementary Fig. 5a-h ). PLX56-treated naïve mice (no social defeat; PLX56-Con) were also included in the study and did not demonstrate any significant behavioral alterations compared to the Con animals, as previously described 24 , 30 . To exclude the implication of any PLX56-related side effects upon the sensory/recognition input of the treated mice, we also performed olfaction (buried food test) and memory recognition tests (novel object recognition), but no significant deficits were noted in the performance of mice 24 (Data not shown). In response to microglial elimination, the previously observed increase in the neuroinflammatory marker TSPO was rescued in the PLX56-Res group compared to the SD-Sus non-treated group (Fig. 6 c), suggesting that TSPO could be used as a potential marker for monitoring neuroinflammatory responses in MDD and other mental disorders 14 . Of interest, the TSPO protein levels in the non-stressed PLX56-Con were significantly downregulated compared to the rest of the groups (and especially the PLX56-Res), indicating that astrocytes and endothelial cells (which also express TSPO) contribute to the inflammatory burden during chronic stress. Based on the reduction of neuroinflammation and the prominent anti-depressant state after microglial elimination, we sought to examine if this affected synaptic plasticity and connectivity elements dysregulated during chronic stress (Fig. 6 e-f). As demonstrated by immunoblot analyses, microglial elimination in the socially defeated PLX56-Res mice significantly rescued the decreased levels previously observed for the pre- and post-synaptic elements Synapsin-I and PSD95 in the mPFC of SD-Sus animals (Fig. 6 e). Notably, the non-defeated PLX56-Con group showed a considerable increase of the synaptic elements, indicative of the importance of microglia as sculptors of neuronal connectivity and circuitry in an activity-dependent manner 23 . Considering that the neurotrophin BDNF and serotonin receptor 5-HTR1A 2 6 , 31 are components of two major regulatory signaling systems involved in depressive disorders, we measured their protein levels in the mPFC of non-treated and treated SD groups. Treatment of the SD mice with PLX5622 was able to substantially protect the PLX56-Res from deficits in BDNF and 5-HTR1A protein levels (SD-Sus), as depicted by the comparable protein levels with the Con and SD-Res not-treated groups (Fig. 6 f). Microglia are essential mediators for the sensitization to chronic stress and required for the establishment of depressive-like phenotypes in mice. To further examine whether microglia play a critical modulatory role in the development of stress-induced behaviors, we took advantage of the quick repopulation properties of the microglia, once the mice treated with PLX5622-chow are reverted to normal chow 32 , 33 . Microglia were eliminated by PLX5622-treatment during the RSDS paradigm (Con-chow group was also included), and behavioral tests were performed between D11-D14 (BH1). Starting on D15, the PLX5622 chow was withdrawn, and normal/Con-chow was introduced for all groups, to allow for microglial repopulation (Fig. 6 g). The repopulation depends solely on local clonal expansion of the surviving microglial cells 32 , 34 , which can rapidly replenish their numbers within 5 to 7 days after removal of the CSF1R inhibitor 32 , 33 . As shown here, microglia repopulated the area during the 10 days of PLX5622 withdrawal, surpassing the microglial population of the non-treated PLX56-Con (Fig. 6 g-h, Supplementary Fig. 4d ). At the end of the repopulation phase, mice underwent a second round of behavioral tests (D22-D25;BH2) (Fig. 6 g). Microglial repopulation was sufficient to trigger depressive-like behaviors in the previously categorized PLX56-Res, inducing social avoidance, anxiogenesis, despair-like behavior and anhedonia, comparable with those observed in the non-treated SD-Sus mice (Fig. 6 i-j; Supplementary Fig. 6a-d ). This can also be depicted by the significant deterioration of the individual performances after paired analyses (before and after microglial repopulation) of the previously categorized PLX56-Res mice ( right panels in Fig. 6 i; Supplementary Fig. 6a-d ). It is important to note that a portion of the PLX56-Res mice also include the naturally occurring SD-Res mice (~ 20%), which we extensively characterized in previous sections. This becomes also evident by the paired analyses in the behavioral tasks, since approximately 20% of the PLX56-Res mice (D11-14), remained resilient (SD-Res) on the second round of behavioral tests (D22-25) ( right panels in Fig. 6 i; Supplementary Fig. 6a-d ). To examine whether the striking attenuation of depressive-like behavior upon microglial elimination was attributed to the onset of PLX5622 treatment at the beginning of chronic stress sensitization of SD mice (PLX5622-treatment starts together with RSDS paradigm), we initiated PLX treatment after the establishment of depressive-like behavior (post-RSDS; Supplementary Fig. 7a ). PLX5622-chow was introduced on D15 (after the end of RSDS and the first round of behavioral testing; RSDS phase) until D25 (Treatment phase; Supplementary Fig. 7a ). Α second round of behavioral tests was performed between D22-25 (ΒΗ2) for behavioral assessment of the experimental groups before and after PLX5622-treatment. It is important to note that both SD-Sus and SD-Res groups were included in the PLX5622 treatment, following their behavioral categorization. Remarkably, the PLX5622 treatment post-RSDS was able to result in significant antidepressant action, rescuing key depression-related indices (social avoidance, anxiogenesis, reward under stress and despair-like behavior) in all the SD-Sus treated mice; Supplementary Fig. 7b-f ). This is also illustrated by the significant individual scores of the SD groups in response to the PLX5622 treatment ( right panels in Supplementary Fig. 7c-f ). Discussion A large body of evidence from clinical research and animal models indicates that the immune system and associated inflammatory responses have a bidirectional regulatory impact on stress- and mood-related neuronal circuits, modulating susceptibility and resilience to a variety of psychosocial stressors 5 , 35 , 36 . Here, we employed the RSDS protocol, a mouse model of stress-induced depression-like behaviors 15 , 18 , to elucidate the cellular dynamics and inflammatory responses of microglia. In line with our previous studies 15 , 18 , the majority (~ 80%) of SD mice displayed long-lasting depressive-like behavioral features (SD-Sus), while a smaller cohort (~ 20%) exhibited resilience to stress (SD-Res), reflecting the heterogeneous responses to chronic stress in humans 37 . The microglial responses and neuroimmune interplay mechanisms in mPFC were investigated both in the SD-Res mice, which have been relatively uncharacterized, as well as the SD-Sus animals. We detected a rapid (D6) and long-lasting (D15) increase of microglial density and proliferation after RSDS in the mPFC of the SD-Sus mice. Our findings are in line with previous studies reporting increased microglial immunoreactivity 38 , 39 in mPFC, but also contrast reports showing microglial recruitment and proliferation only after acute stress 40 . Possible reasons for these discrepancies in the literature might be attributed to the differences in the stress paradigms (the nature, length, and intensity of stressor). We also demonstrated that the microglial recruitment in the mPFC of the SD-Sus mice was spatially related to a significant increase of neuronal activity (cFOS). The cFOS upregulation in response to chronic stress has been previously attributed to neuronal sensitization 41 . Microglia are known to respond rapidly to psychosocial stressors through ATP/purinergic signaling in an neuronal activity-dependent manner 42 . Notably, even though the SD-Res mice had comparable stress/anxiety levels with the SD-Sus (evident via EPM test), they exhibited only a minor increase of cFOS reactivity, suggesting a compensatory neuronal circuitry mechanism 43 . The microglial density increases displayed after RSDS could be attributed to local microglial proliferation, but also to monocytic recruitment (the markers Iba1 and CX3CR1 label both microglia and macrophages). However there has been ambiguity in the literature regarding the latter, with some studies suggesting significant peripheral macrophage recruitment in the mPFC after chronic stress 17 , 44 and some others reporting no macrophage involvement 40 . These deviations could be attributed to the use of different mouse models of stress, or variability in the duration of the stress paradigms. Here, we demonstrated a lasting peripheral monocytic recruitment (D15 and D25 post-RSDS) in the SD-Sus mice, which was facilitated by a significant upregulation of the adhesion molecules VCAM-1 and ICAM-1 in the mPFC. These results corroborate with previously described mechanisms implicating microglia, endothelial cells, and macrophage interactions contributing to changes in mood behavior 45 , 46 . Further characterization of the microglia/macrophage inflammatory responses revealed a significant increase of several pro-inflammatory markers (CD86, iNOS, TNF-α, CXCL10 and IL-1β) and a reactive hypertrophic morphology characterized by shorter and fewer processes and increased cell body area 47 , followed by a concomitant decrease of anti-inflammatory markers (CD206, Arg1 and IL-10) in the SD-Sus mice compared to the Con and SD-Res groups. The morphology of the SD-Res microglia was also distinct with long distally hyper-ramified morphology, previously described for microglia responding to increased glutamatergic neurotransmission 48 . These findings indicate that susceptibility or resilience is associated with the inflammatory response profile and polarization spectrum (pro-inflammatory/anti-inflammatory) of microglial/macrophages in the mPFC. Consistent with the inflammatory responses observed in the SD-Sus mice, total and microglial-specific TSPO levels were elevated in the mPFC compared to Con and SD-Res groups. In agreement with our findings, recent studies in MDD and schizophrenia patients have reported a significant upregulation of TSPO levels in the cingulate cortex and PFC 12 , 49 , further supporting a role for microglial activation and associated neuroinflammatory responses in the mental disorder pathophysiology. In accordance with previous studies reporting increased phagocytic activity of microglia in the mPFC 38 , 40 , we observed a dramatic increase in microglial phagocytosis which was colocalized with the iNOS proinflammatory marker, which were previously shown to involve a TLR4-dependent pathway 38 . Interestingly, the microglia from SD-Res group, even though had significantly increased CD68 levels compared to Con, they did not display a pro-inflammatory phenotype, consistent with a distinct activation status compared to the proinflammatory SD-Sus microglia. As microglia are crucial regulators of neuronal connectivity and of circuitry refinement through synaptic pruning 8 , we observed a significant increase in microglial phagocytosis of pre- and post-synaptic elements in the mPFC of SD-Sus mice and significant reductions of PSD95 and Syn-1 levels, which was followed by substantial synaptic plasticity deficits as indicated by the levels of BDNF, p-CREB and serotonin receptor 5-HTR1A, all known regulatory signaling components involved in depressive disorders 26 , 31 . Microglia and infiltrating macrophages have been previously reported to be in close interaction and act in synergism 1 inducing stress and depressive disorders 17 , 36 . In this study we investigated their individual pathophenotypic contribution to depression through treatment with two different CSF1R inhibitors: PLX73086 (which depletes peripheral macrophage populationsby about 80% 50 ) and PLX5622 (which depletes both microglia and macrophage populations). We demonstrated that peripheral macrophage ablation (PLX73086) exerted a moderate but significant anti-depressant action in the SD-treated groups (50% of protection), suggesting that the recruited monocytes contribute to the development of depression. The effect that microglial/macrophage elimination (PLX5622) had during chronic stress was remarkable, since it prevented depressive-like behaviors in the total SD population (100% protection). The lack of depressive symptoms was accompanied by reduction of the levels of TSPO in the PLX56-Res group, potentially relating the TSPO marker with the behavioral performance in depressive disorders 12 . Our findings are consistent with previous studies using PLX5622 to ablate microglia two weeks prior to the initiation of chronic stress using different paradigm formats 16 , 51 , 52 . Our study is distinct, however, as we investigated the effects of microglia/macrophage ablation on behavioral and neuronal homeostasis both during the induction of chronic stress (PLX5622 during RSDS), and after the induction of chronic stress and the establishment of a neuroinflammatory environment in the mPFC area (PLX5622 post-RSDS). We find that microglial/macrophage ablation during chronic stress was able to prevent essential neuronal plasticity deficits (BDNF and 5-HTR1A) and the loss of synaptic elements (Syn-1 and PSD95). A key finding of our study was the re-emergence of a depressive-like phenotype after microglial repopulation. Considering that microglial repopulation occurs entirely from surviving residual microglia 53 , 54 , it would be important to examine in the future whether chronic stress induces specific depressive-like behavior epigenetic changes to microglia, which can be passed on after their clonal expansion, and if neuronal sensitization of the mPFC is contributing to the microglial “re-education” into a pro-inflammatory state when they reappear. With these in mind, it may be interesting to explore potential approaches in which microglia are epigenetically “re-educated’’ towards a neuroprotective phenotype (similar to the anti-inflammatory microglial phenotype of the SD-Res) or utilize an anti-inflammatory treatment in conjunction with first line anti-depressant treatments. Limitations of our study are the fact that the RSDS model uses most commonly male animals, and that it is debated whether avoidance in the social interaction test represents a 'depressive-like' behavior or impaired learning 55 . Collectively, the present study: i) reveals an important association of microglial/macrophage polarization spectrum (pro-inflammatory/anti-inflammatory) and their respective inflammatory responses with susceptibility or resilience to chronic stress, ii) demonstrates that proinflammatory microglial activation leads to deficits in neuroplasticity and synaptic-connectivity, iii) signifies the contribution of monocytic recruitment to the neuroinflammatory burden and associated behavioral deficits (possibly by microglial-mediated signaling, as previously suggested 24 ) and iv) indicates that microglia are not only essential mediators for the sensitization of SD groups to chronic stress but also necessary for the establishment of depressive-like phenotype. These findings are further solidifying the neuroinflammatory hypothesis of depression and support a critical role for microglia and macrophages in the depression pathophysiology. Materials And Methods Animals All animal procedures were approved by the Institutional Animal Care and Use Committee (covered by Animal welfare assurance No A3011-0), at Stony Brook University, and conducted in accordance with the guidelines of the National Institutes of Health “Guide for the Care and Use of Laboratory Animals”. Experiments were performed using adult (2–3 months old) male mice [C57BL/6J (wt), CX3CR1 -GFP (Jackson Labs, 005582 model B6.129P-Cx3cr1tm1Litt/J), CX3CR1 -Cre ERT 2 -eYFP (Jackson Labs, 021160 model B6.129P2 (Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ), td-Tomato (Jackson Labs, 007905 model number B6;129S6-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze/J)], were used in the study. All the mouse lines were backcrossed to a C57BL/6J background, bred in-house, and genotyped by PCR. CD-1 retired-breeder male mice (Charles River Laboratories, CD-1 IGS mice, strain code:022) were used as aggressors. The CD-1 male mice exert aggression only towards male mice, therefore female mice were excluded in the study. For induction of recombination in the CX3CR1- Cre ERT 2 - eYFP:: Rosa26- tdTom mice, tamoxifen (Sigma CAS # 10540-29‐1) dissolved in ethanol : sunflower seed oil was injected intraperitoneally (i.p.) starting at P30, at a daily dose of 75 µg/g body weight (from a 10 mg/ml stock), for 5 consecutive days, as in 15 , 27 . All animals were housed in 12-hr light/dark cycle. Food and water were provided ad libitum by the experimenters. Repeated social defeat stress paradigm (RSDS) Adult male mice were subjected to repeated bouts of physical aggression (‘‘defeats’’) from aggressive CD-1 mice for 10 consecutive days, as previously described 15 , 18 . The CD-1 aggressors were screened during a 3-day screening period for adequate aggressive behavior and then housed on one side of the divided mouse cage (known as the home cage), at least overnight prior to the start of defeat sessions. All defeat experiments were performed within that compartment, while the intruders were rotated across defeat days, so that the experimental animals would not habituate to a single aggressor. On day 0 (D0) of RSDS the ‘socially defeated-to-be’ (SD) mice were placed into the aggressor’s space for 10 min (physical stress), and after the end of the encounter they were returned to their side overnight. The mice could see and smell the aggressor through the clear perforated divider (sensory stress). The naïve mice (Con) were exposed to wt (C57BL/6J) mice, instead, for 1 minute. About 80% of the mice that were subjected to social defeat stress displayed depressive-like behavior (SD-Sus for Susceptible), whereas the rest 20% were the nonresponding mice (SD-Res for Resilient) did not, thus modeling the heterogeneity in individual responses to stress in humans 56 . At the end of RSDS paradigms all animals were singly housed. Socially defeated mice were categorized based on the SI output, and further behavioral analyses for each group followed this categorization. Both SD-Sus and SD-Res exhibited anxiety-like behavior, however the SD-Sus additionally demonstrated social avoidance, reduction of reward under stress, anhedonia, despair-like behavior, and elevated plasma corticosterone levels (~ 130 ng/ml) 15,56 . In addition to this paradigm, a short-term RSDS paradigm adaptation (miniSD; 3 days) followed by 3 days of behavioral tests (BH; D4-D6) was also performed in the results shown in Supplementary Fig. 2 , which resulted in ~ 50% of the SD mice becoming SD-Sus, as we have previously demonstrated 15 . Although we find that the model produces very reproducible / reliable results, it does have the limitation that in the current format the model uses male mice only for the experiments. Behavioral analyses For endpoints D15 and D25, mice were behaviorally tested for 4 days (1 experiment/day). Behavioral testing and recordings were performed during the light phase. Both naïve and SD mouse groups were tested for behavioral alterations using the i) social interaction test (SI; measures social avoidance), ii) elevated plus-maze test (EPM; measures stress/anxiety), iii) forced swimming test (FST; measures despair-like behavior), iv) sucrose preference test (SPT; assesses anhedonia-lack of feeling pleasure), and v) novelty suppressed feeding test (NSF; assesses reward under stress). All experiments (except for SPT) were performed in a light-controlled and sound-isolated behavioral analysis room. The mice were acclimatized to the experimental room for 1 hour before the start of each experiment. The SI and EPM were performed under red light conditions; the remaining behavioral tests were performed with lights on. The software Noldus Ethovision XT16 was used for the automated tracking and scoring during the behavioral tests. White noise generator (70–75 dB) was used to mask intermittent disturbing sounds (from surrounding areas) that would potentially startle the animals. Social Interaction (SI) One day after the final social defeat stress interaction, a SI test was performed to determine whether the animals display social avoidance, as previously established 15 , 18 . Mice were placed into the social interaction open-field arena [(42 cm (w) × 42 cm (d) × 42 cm (h)], and the time spent in the interaction zone (< 8 cm from the wire-mesh enclosure) was monitored for the two 2.5-min phases (without or with a novel CD-1 aggressor present in the enclosure), separated by a duration of 30 s. The socially defeated mice with SI ratio [(Time in interaction zone with aggressor) / (Time in interaction zone without aggressor)] > 1 were classified as SD-Res, and mice with a ratio below 1 (social avoidance) were classified as SD-Sus 15 . The naïve mice were categorized as Control (C). Elevated plus-maze test (EPM) The apparatus used for this test was cross-shaped with two open arms (30 cm x 5 cm) and two closed arms (30 cm x 5 cm x15 cm) that extended from a central platform (5 cm x 5 cm), as previously described 15 , 57 . The entire maze was elevated 40 cm above the floor. The enclosed arms offered safety when the mouse was stressed; on the other hand, the open arms offered the motive of exploration. Each mouse was placed in the central square of the apparatus, facing an enclosed arm. The mice were let to roam freely for 10 min totally; an arm entry was defined when all four paws entered an arm. The total mobility, time spent in open arms and time closed arms were recorded, as an index of stress/anxiety 57 . Forced swimming test (FSM) Each mouse was placed in a transparent tank [inescapable Plexiglas cylindrical tank (height: 30 cm, diameter: 22.5 cm)] that was filled with water (to a depth of 15 cm and maintained at 25°C) and their escape related mobility behavior was measured. Once the mouse was in the water, it was left to swim for 6 min in total, assessing the behavior only during the last 4 min, as previously performed 15 . The mice that acquired an immobile posture, characterized by motionless floating in the water, were termed immobile (immobility time), making only the necessary movements to keep the head above the water. Before returning the animals to their home cages, they were dried gently using paper towels to prevent hypothermia. Sucrose preference test (SPT) The SPT was performed as previously 15 . The experimental groups were habituated for 72 h to 2% sucrose, in which the bottles were alternated to avoid bias for a specific cage side. The day before the testing the mice underwent an 18 h water deprivation period. The day of the testing bottles filled with sucrose 2% or water were weighed, and consumption was determined for a 24 h period. Bottles were weighed again at the end of the 24 h period. Sucrose preference was expressed as (Δweightsucrose) / Δweightsucrose + Δweightwater) x100. Sucrose preference score less than 70% was displaying anhedonia. Novelty suppressed feeding test (NSF) The NSF test measures the time that mice take to approach and eat food in a novel environment following an extended period of food deprivation. Mice were food-deprived for 24 h and moved in freshly prepared home-cages (to avoid any pellets remaining on the bedding). The next day, each mouse was placed at the corner of an open-field arena [(42 cm (w) × 42 cm (d) × 42 cm (h)] with a pellet of chow already positioned at the center of it. The time until the first bite of the chow pellet was recorded, with maximum trial time the 10 min. After the trial, the mice were returned to their home cage which contained a pre-weighed food pellet, and food consumption was measured for a period of 5 min, as described 15 . BrdU labeling For the labeling of proliferating cells, 5-Bromo-2′-deoxyuridine (BrdU;Sigma-B5022) was dissolved in drinking water (1 mg/ml), and all mouse groups were given access to the water ad libitum throughout the 10 days of the RSDS paradigm 15 . Microglial depletion with PLX5622 Experimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX5622 (PLX56, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 1200 mg/kg of food weight (Research Diets, Inc.), as previously 58 . Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Seven days of PLX administration can achieve > 90% microglial ablation 58 , 59 . Exact treatment duration varied depending on the experimental design and is defined on each section. Peripheral macrophage depletion with PLX73086 Experimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX73086 (PLX73, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 200 mg/kg of food weight (Research Diets, Inc.), as previously 59 . Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Exact treatment duration varied depending on the experimental design and is defined on each section. Immunoblot analysis Mouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain ~ 500-µm thick brain coronal sections. Medial PFC was dissected out (with the help of a dissecting microscope) from + 1.2 mm to + 2.5 mm anterior to bregma slices. Protein lysate was prepared using RIPA buffer (120–150 µl) with protease and phosphatase inhibitors (200 mM PMSF, 100 mM sodium orthovanadate, and protease inhibitor cocktail; Santa Cruz, sc-24948A). Lysates were shaken for 15 min at 4°C, cleared by centrifugation at 15,000 g for 10 min at 4°C, and protein concentration was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific; 23225). Samples (15–20 µg) were boiled for 5 min with 5X SDS-PAGE sample loading buffer (Thermo Fisher Scientific; 39000), separated by SDS-PAGE, and transferred to PVDF membranes (ThermoFisher Scientific; 88518) at 30 V for 16–18 h at 4°C. Membranes were blocked with 5% w/v nonfat dry milk (Cell Signal; 9999S) and incubated with primary antibodies ( Supplementary Table 2a ) for 16–18 h at 4°C. Washes with TBST (Cell Signal; 9997S) were followed by incubation with HRP-coupled secondary antibodies ( Supplementary Table 2b ). Signal was visualized by Sapphire Biomolecular Imager (Azure Biosystems) using a chemiluminescent substrate mixture (Supersignal West Pico Plus; ThermoFisher Scientific, 34580 or Immobilon Western; Millipore, WBKLS0500). Optical densities were collected with Fiji ImageJ software 60 . Where indicated, protein levels were expressed as fold change (versus Control) of the arbitrary units (A.U.) following normalization to the corresponding loading controls (β-Actin, GAPDH). Samples were normalized to the total protein for each sample, as determined by the BCA assay. For a detailed antibody list used see Supplementary Table 2a . Immunohistochemistry Deeply anesthetized mice (isoflurane) were transcardially perfused with cold 1X PBS (15 ml) followed by cold PFA fixative solution (4% paraformaldehyde in 1X PBS; 20ml) and brains were dissected and post-fixed for an additional 24 hours. Then brains were cryoprotected in 30% sucrose for 24 hours and sectioned with a sliding microtome. Free-floating brain sections (30–40 µm thick) containing the mPFC, were blocked with 10% goat serum (ThermoFisher Scientific; 16210072) in 0.3% TritonX-100 in 1X PBS for 1–2 hour at room temperature. Tissue sections were incubated with primary antibodies overnight at 4°C at the indicated concentrations ( Supplementary Table 2a ). The following day, sections were washed in PBS-Triton and incubated with the appropriate cross-absorbed secondary antibodies ( Table II-3) . After 4 additional washes, nuclei were stained with DAPI (Sigma; D9542) and sections were mounted using MOWIOL mounting media. For BrdU IHC, sections were pretreated in 2M HCl for 40 minutes at 37°C, followed by 2-3x 5-minute washes in 0.1 M Boric acid (pH 8.5). For a detailed antibody list used see Supplementary Table 2c . Microscopy and histological quantification The confocal laser-scanning microscopes Leica TCS SP8X and Leica TCS-SP5 were used for imaging of FITC, EGFP, EYFP, Alexa-488, Alexa-555, Alexa-594, Alexa-647 and CY3 fluorophores. Optical sections (z = 1.0µm; total stack of 10–16µm for 1 cell layer) of confocal epifluorescence images were sequentially acquired using 10x, 20x, 40x (N.A. 1.30), 63x (N.A. 1.20) and 100x (N.A. 1.40) objectives, with LAS AF software. Fiji ImageJ software 60 was used for image reconstruction, cell counting, integrated density analysis and colocalization analysis. For mPFC analysis, 6 fields (located between + 1.2mm to + 2.5mm anterior to bregma), containing the cingulate cortex (Cg1), prelimbic (PL), infralimbic (IL) and medial orbital cortex (MO) were taken from each animal, for quantitative analysis. Typically, 3–4 brain slices were analyzed per animal, and at least 3 different animals for each experimental condition were evaluated. Cell counts were presented as the number of marker + cells/section (section corresponding to 0.2 mm 2 ). Each section quantification depicts the sum of the marker + cells from all the analyzed fields. For the integrated density analysis, the IsoData thresholding was applied to each image, and the integrated density [is defined as the product of pixel intensity (255 = the maximum pixel intensity for an 8-bit image) x area] was measured. For the colocalization analysis, the IsoData thresholding was applied to each image, images were merged, compartmentalized, and the integrated density was measured for the colocalized signal. Each section with integrated density quantification is depicted by the mean of the integrated density from all the analyzed fields. All experimental group comparisons were conducted on sections stained and imaged with identical exposure and acquisition settings. All analyses were performed on raw images, prior to any image processing by Adobe Illustrator CS6 for representation purposes. The cell counting was performed in a blinded manner by 4 experimenters collectively, and tissue sections were matched across samples. Microglial morphological analysis To quantitatively examine microglial morphology, unprocessed 30µm z-stack confocal images (100x objective; 20 individual images, step size: 1µm) of cells for Iba1 activity in the mPFC area were imported into Neurolucida software, as previously described 15 . Cell bodies and processes were manually traced (6 cells/mouse), and a 3D rendering was created. The 3D reconstructions were imported into Neurolucida Explorer for branched structure analysis and Sholl analysis (5µm starting radius, 50µm ending radius, and 5µm step size). Number of process intersections, cell surface area, and cell complexity were measured. Microglial complexity = [Sum of the terminal orders + Number of terminals] * [Total dendritic length / Number of primary dendrites]. Terminal orders: Number of "sister" branches encountered as proceeding from the terminal to the cell body (calculated for each terminal); Terminal: Refers to process endings. Flow cytometry Flow cytometry was performed as previously, with minor modifications 61 . Mice were deeply anesthetized (isoflurane) and transcardially perfused with 20 ml of ice cold 1X HBSS (pH 7.4). The mPFC of CX3CR1 -GFP mice was collected in ice-cold Hibernate-A Medium (ThermoFisher Scientific; A1247501) isolated and enzymatically digested in a pre-warmed (10 min) 1.54 mg/ml papain solution (Worthington; LS003127), containing 1.1 mM EDTA, 0.067 mM β-mercaptoethanol and 5.5 mM cysteine-HCl, for 20 minutes at 37°C with manual inversion every 5 minutes. Further processing was performed at 4°C. Tissue was triturated with a p100 tip, followed by syringe G20 and then G25 trituration (5 times each). Tissue debris was removed by passing the cell suspension through a 40-µm cell strainer and centrifuged for 5 minutes at 2000 g at 4°C to remove papain solution. Myelin was removed using a discontinuous Percoll gradient. The cell pellet was resuspended in 30% Percoll (Sigma; P1644) in 1X HBSS, layered over a 37% and a 70% Percoll cushion, and centrifuged at 800 g for 40 min (800 x g) at 10°C. The supernatant containing myelin was removed, and cells were collected at the 37–70% Percoll interface. The remaining solution was diluted with 1X HBSS, and samples were centrifuged for 10 minutes at 1000 g, 4°C to pellet the cells. The pellet was then resuspended in flow cytometry (FC) buffer [0.5% bovine serum albumin (BSA) in 1X PBS]; cells were counted using trypan blue exclusion. Non-specific binding was blocked using anti- CD16/32 (1:50 in FC buffer; Tonbo biosciences, TB-70-0161-U500) for 30 minutes on ice and then stained with CD11b-APC, CD45-PerCP/Cy5.5, CD86-Pacific Blue, CD206-PE, antibodies (1:100 in FC buffer, Biolegend) in various combinations for 30 minutes on ice in dark conditions. The staining solution was removed by centrifugation for 5 minutes at 1000 g, and cells were washed twice with FC buffer before resuspending for flow cytometric analysis on a BD LSR Fortessa. The FC analysis was performed in the endpoint population of 15,000 CX3CR1 -GFP + cells/mouse. Data were further analyzed on the FlowJo software. RNA isolation and quantitative PCR Mouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain ~ 500-µm thick brain coronal sections. RNA from the mPFC (+ 1.2 mm to + 2.5 mm anterior to bregma) tissue or cells was isolated using Qiazol (Qiagen; 79306). The total RNA was separated from the aqueous phase using a RNeasy MicroKit (Qiagen; 74004). cDNA was reverse transcribed using random primers and Superscript IV (Invitrogen; 18090200). Quantitative PCR was performed from cDNA using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific; F548L) as per the manufacturer’s protocol on an Applied Biosystems 7900HT real time PCR system (Ct values above 35 cycles were not considered for the analysis). Fold changes were calculated using the ∆∆Ct method. For the primer sequences, see ( Supplementary Table 2d) . All gene expression levels were normalized to Gapdh mRNA levels. Statistical analysis No statistical methods were used to predetermine sample size a priori , but the sample sizes used were similar to those reported in previous studies 15 . Furthermore, representative data from each experiment were examined by Shapiro-Wilk’s test (p > 0.05) 62 and a visual inspection of their histograms to confirm normal data distribution 63 , 64 . The majority of the experimental analyses was performed by parametric ordinary one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison post-hoc tests. For the glial morphological analyses two-way ANOVA was performed, followed by Tukey’s multiple comparison post-hoc tests. For the PLX5622 repopulation behavioral tests a paired t-test was performed. The GraphPad Prism 8 and Excel (Microsoft) were used for all statistical analyses. The data were reported as mean ± S.D. with symbols indicating the following P value ranges: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and **** P ≤ 0.0001). Declarations Acknowledgements We are grateful to Drs. Ramin Parsey, Christine DeLorenzo, Joel Levine and Holly Colognato for their valuable insight and discussions. This work was supported by the National Institute of Mental Health, Grant/Award Numbers: R01MH123093-01, R01MH123093-01S1 (MMM), the American Heart Association (19PRE34370044; AGK) and Scholars in BioMedical Sciences Program (T32GM127253). Author Contributions A.G.K. and S.E.T. conceptualized the project. A.G.K. designed, performed experiments, analyzed data. 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Microglial recruitment of IL-1beta-producing monocytes to brain endothelium causes stress-induced anxiety. Mol Psychiatry 23, 1421–1431, doi: 10.1038/mp.2017.64 (2018). Weber, M. D., McKim, D. B., Niraula, A., Witcher, K. G., Yin, W., Sobol, C. G., Wang, Y., Sawicki, C. M., Sheridan, J. F. & Godbout, J. P. The Influence of Microglial Elimination and Repopulation on Stress Sensitization Induced by Repeated Social Defeat. Biological Psychiatry, doi: https://doi.org/10.1016/j.biopsych.2018.10.009 (2018). Tay, T. L., Mai, D., Dautzenberg, J., Fernández-Klett, F., Lin, G. et al. A new fate mapping system reveals context-dependent random or clonal expansion of microglia. Nature Neuroscience 20, 793–803, doi: 10.1038/nn.4547 (2017). Huang, Y., Xu, Z., Xiong, S., Sun, F., Qin, G., Hu, G., Wang, J., Zhao, L., Liang, Y.-X., Wu, T., Lu, Z., Humayun, M. S., So, K.-F., Pan, Y., Li, N., Yuan, T.-F., Rao, Y. & Peng, B. Repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion. Nature Neuroscience 21, 530–540, doi: 10.1038/s41593-018-0090-8 (2018). Ayash, S., Schmitt, U., Lyons, D. M. & Muller, M. B. Stress inoculation in mice induces global resilience. Transl Psychiatry 10, 200, doi: 10.1038/s41398-020-00889-0 (2020). Krishnan, V., Han, M. H., Graham, D. L., Berton, O., Renthal, W. et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131, 391–404, doi: 10.1016/j.cell.2007.09.018 (2007). Ferlemi, A. V., Avgoustatos, D., Kokkosis, A. G., Protonotarios, V., Constantinou, C. & Margarity, M. Lead-induced effects on learning/memory and fear/anxiety are correlated with disturbances in specific cholinesterase isoform activity and redox imbalance in adult brain. Physiol Behav 131, 115–122, doi: 10.1016/j.physbeh.2014.04.033 (2014). Nissen, J. C., Thompson, K. K., West, B. L. & Tsirka, S. E. Csf1R inhibition attenuates experimental autoimmune encephalomyelitis and promotes recovery. Experimental neurology 307, 24–36, doi: 10.1016/j.expneurol.2018.05.021 (2018). Bellver-Landete, V., Bretheau, F., Mailhot, B., Vallières, N., Lessard, M., Janelle, M.-E., Vernoux, N., Tremblay, M.-È., Fuehrmann, T., Shoichet, M. S. & Lacroix, S. Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury. Nature Communications 10, 518, doi: 10.1038/s41467-019-08446-0 (2019). Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. & Cardona, A. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676–682, doi: 10.1038/nmeth.2019 (2012). Thompson, K. K., Nissen, J. C., Pretory, A. & Tsirka, S. E. Tuftsin Combines With Remyelinating Therapy and Improves Outcomes in Models of CNS Demyelinating Disease. Front Immunol 9, 2784, doi: 10.3389/fimmu.2018.02784 (2018). Shapiro, S. S. & Wilk, M. B. An analysis of variance test for normality (complete samples). Biometrika 52, 591–611 (1965). The SAGE Dictionary of Statistics. The SAGE Dictionary of Statistics. SAGE Publications, Ltd. (SAGE Publications, Ltd). E, D. P. D. a. L. Measuring Skewness: A Forgotten Statistic? Journal of Statistics Education 19, 1–18 (2011). Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files SupplementaryFigureswithLegendsKokkosisetal.2022.pdf SupplementaryTable1.pdf SupplementaryTable2.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-1991809","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":145840466,"identity":"d5c48d4a-f65f-4831-aa9f-a5809c04e2ac","order_by":0,"name":"Stella 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6","display":"","copyAsset":false,"role":"figure","size":2463306,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"MainFigure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1991809/v1/7ff973ef789fc10bd121bc6e.jpg"},{"id":29431058,"identity":"49e589c2-2028-4769-b3d5-a06462dec95a","added_by":"auto","created_at":"2022-11-23 11:42:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1636243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1991809/v1/bdb38167-3808-4d99-858d-024901739c2d.pdf"},{"id":28291286,"identity":"3b1b98a6-3925-40f8-8339-fd341d6b0a4f","added_by":"auto","created_at":"2022-10-26 17:31:21","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":423476866,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureswithLegendsKokkosisetal.2022.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1991809/v1/cd068a4867e2d4da6a6d1594.pdf"},{"id":28291400,"identity":"633580be-51b8-4e1a-83bc-ff44b1c0c627","added_by":"auto","created_at":"2022-10-26 17:35:54","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":750136,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1991809/v1/606d50309c81c6106235c765.pdf"},{"id":28291773,"identity":"38d1a8dc-74a0-49f6-9bc7-1ca64728b295","added_by":"auto","created_at":"2022-10-26 17:45:54","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":632658,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1991809/v1/7d3fa2e015154fb69e31e5a9.pdf"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Chronic psychosocial stress induces microglial activation and inflammatory responses that lead to neuronal dysfunction and depressive-like behavior","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroglia, the innate immune cells of the central nervous system (CNS), have been shown to be active participants in the normal physiological, homeostatic functions of the CNS, as well as to serve as modulators of pathologic events that take place in the brain and spinal cord, including excitotoxic and neurodegenerative processes, stroke and autoimmune diseases\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Additionally, microglia share common features with bone marrow-derived monocytes, yet they possess a unique gene expression profile\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The modulatory properties of microglia are regulated by toll-like (TLRs), complement, purinergic and scavenger receptors, leading to secretion of cytokines, chemokines, and other signaling molecules\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA large body of evidence suggests that neuroimmunologic processes affect neuronal homeostasis contributing to the pathophysiology of mental disorders, as well\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. More specifically, elevated levels of proinflammatory cytokines, such as IL-6, TNF-a and C-reactive protein (CRP), have been measured both peripherally and centrally in patients with Major Depression Disorder (MDD); and autoimmune/ inflammatory diseases are considered to be co-morbid with MDD\u003csup\u003e5,7\u0026minus;9\u003c/sup\u003e. To this end, experimental adaptations in animals using either lipopolysaccharide (LPS) or cytokine administration (IL-1b or IL-6) resulted in loss of interest and reduction in reward sensitivity\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Neuroimaging studies of MDD patients have also revealed that symptom severity is correlated with elevated levels of translocator protein (TSPO; neuroinflammation marker) in brain regions specifically implicated in mood regulation, including the medial prefrontal cortex (mPFC), the anterior cingulate gyrus and infralimbic cortices\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe neuroimmunological responses to chronic stress observed in humans are also evident in rodent models of psychosocial stress. In models of chronic social defeat (CSD) and repeated social defeat stress (RSDS) we and others have shown that stress may result in increased local generation of reactive oxygen species (ROS)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In addition, elimination of microglia (in the context of CSD) using inhibitors of Colony Stimulating Factor Receptor 1 (CSF1R) mitigated the high levels of ROS and protected from the development of depressive-like behavior in mice\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Similar results have been shown for chronic unpredictable mild stress and the use of the microglial inhibitor minocycline\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Although a strong relationship between the presence of activated microglia and MDD-like symptoms has been suggested, a requisite role for microglia for the development of depressive behavior has not been established.\u003c/p\u003e \u003cp\u003eIn the present study we use the RSDS paradigm to study the cellular and molecular responses of microglia in the mPFC, and examine their role in the modulation of synaptic changes and neuroplasticity, as well as the related behavioral manifestations of stress-induced depressive-like behavior in mice. We demonstrate that microglia are swiftly activated in response to chronic stress, spatially relating to increased neuronal activation in threat-appraisal areas. Moreover, we reveal an important association of microglial and infiltrating-macrophage reactivity and respective inflammatory responses, with susceptibility or resilience towards chronic stress. We also report that microglia are negatively regulating neuronal plasticity and modulate levels of synaptic elements in response to chronic stress, leading to depressive-like behavior. Importantly, we assess the contribution of peripheral macrophages to the depressive-like traits upon chronic stress. Finally, we find that depression-related phenotypic changes are suppressed with microglial ablation but are rapidly re-established after microglial repopulation, suggesting that microglia are critical dynamic mediators for the establishment of depressive-like behavior in mice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eChronic stress induces microglial recruitment and increased microglial proliferation in the mPFC, leading to depressive-like behavior\u003c/p\u003e \u003cp\u003eWe utilized the repeated social defeat paradigm (RSDS; 10 days) to induce depressive-like behavior in adult male mice, as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Briefly, twenty-four hours after RSDS (D10) behavioral tests were performed (BH; D11-D14) and the socially defeated (SD) mice were divided (see Methods) in two groups: SD-Sus (~\u0026thinsp;80%; susceptible) and SD-Res (~\u0026thinsp;20%; resilient). Both groups exhibited significant anxiety-like behavior, however the SD-Sus demonstrated characteristic depressive-like phenotypic features: social avoidance, despair-like behavior, reduction of reward under stress, anhedonia, and elevated plasma corticosterone levels (~\u0026thinsp;130 ng/ml; data not shown), as previously demonstrated\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, \u003cb\u003eSupplementary Fig.\u0026nbsp;1a-d\u003c/b\u003e). The na\u0026iuml;ve mice (Con) were only exposed to wt (C57BL/6J) mice for 1 minute.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine the effects of chronic stress on microglial density and proliferation in the mPFC, 5-Bromo-2\u0026prime;-deoxyuridine (BrdU) was administered \u003cem\u003ead libitum\u003c/em\u003e throughout RSDS, and experimental groups were euthanized on D15. Histological analysis of the mPFC revealed a significant increase of microglial cell density (microglial/macrophage Iba1\u003csup\u003e+\u003c/sup\u003e marker and \u003cem\u003eCX3CR1\u003c/em\u003e-GFP\u003csup\u003e+\u003c/sup\u003e mice) and microglial proliferation capacity (%BrdU\u003csup\u003e+\u003c/sup\u003e of CX3CR1\u003csup\u003e+\u003c/sup\u003e), compared to Con and SD-Res (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-g). Importantly, microglial recruitment was also observed at an earlier time point (D6; CX3CR1\u003csup\u003e+\u003c/sup\u003e) after a short-term RSDS paradigm adaptation (miniSD for 3 days/BH on D4-D6; see Methods for more details\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e) and was spatially related to a significant increase of neuronal activity (NeuN\u003csup\u003e+\u003c/sup\u003ecFos\u003csup\u003e+\u003c/sup\u003e immunoreactivity) in mPFC (\u003cb\u003eSupplementary Fig.\u0026nbsp;2a-d\u003c/b\u003e). Of interest, the microglia in the mPFC of SD-Sus mice adopted a more reactive morphology which was characterized by shorter and fewer process intersections, increased cell body surface area and reduced process complexity index, as determined by Sholl and branched structure analyses of Iba1\u003csup\u003e+\u003c/sup\u003e hyperstack traces (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh-k; Methods). These morphological attributes have been previously identified in neuroinflammatory\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and neurodegenerative models\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, and strongly associate with a pro-inflammatory activation state\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Interestingly, the SD-Res microglia also exhibited reactive morphology which was different from that of Con and SD-Sus, displaying fewer intersections closer to the cell body (but longer and numerous processes distally from it), but intermediary changes in cell body and branch complexity, suggesting a distinct microglial activation state.\u003c/p\u003e \u003cp\u003eChronic stress susceptibility/resilience is associated with the inflammatory response profile and polarization of microglial/macrophages in the mPFC.\u003c/p\u003e \u003cp\u003ePsychosocial stress can promptly activate the release of glucocorticoids\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e (we have previously shown elevated plasma corticosterone levels in SD-Sus mice\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e), through which they can signal the recruitment of peripheral monocytes into the brain parenchyma. We performed flow cytometry in \u003cem\u003eCX3CR1\u003c/em\u003e-GFP\u003csup\u003e+\u003c/sup\u003e mice and observed significant monocytic population trafficking [CD11b\u003csup\u003e+\u003c/sup\u003eCD45\u003csup\u003ehigh\u003c/sup\u003e (macrophages)] into the mPFC of SD-Sus compared to the Con and SD-Res groups on D15 post-RSDS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b). These findings were further supported by immunoblot analysis of key adhesion molecules responsible for monocytic infiltration (VCAM-1 and ICAM-1) which were significantly increased in the SD-Sus group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec-d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther characterization of the microglia/macrophage populations revealed a significant increase of the pro-inflammatory marker CD86 and a concomitant decrease of the anti-inflammatory marker CD206 in the SD-Sus mice compared to the Con and SD-Res groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-j), suggesting a pro-inflammatory inclination of the SD-Sus phenotype. Accordingly, a significant upregulation of CD206\u003csup\u003e+\u003c/sup\u003e expression was noted in the SD-Res microglia/macrophages compared to Con and SD-Sus, suggesting an anti-inflammatory phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-j). This was further corroborated by RT-qPCR and immunoblot assays of mPFC after RSDS. Specifically, the mRNA levels of TNF-α, CXCL10 and IL-1β and protein levels of CD86, iNOS (all pro-inflammatory markers) were significantly upregulated in the SD-Sus, while the IL-10 mRNA and CD206 protein levels (both anti-inflammatory markers) were significantly decreased compared to the Con and SD-Res (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek-n and \u003cb\u003eSupplementary Fig.\u0026nbsp;3e\u003c/b\u003e). Respectively, the SD-Res mice exhibited an increase of the mRNA levels of IL-10 and the protein levels of CD206 and Arg1 (anti-inflammatory markers) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek-n and \u003cb\u003eSupplementary Fig.\u0026nbsp;3e\u003c/b\u003e). Consistent with the proinflammatory responses observed in the SD-Sus mice, total and microglial-specific translocator protein (TSPO; microgliosis and astrogliosis marker indicative of neuroinflammation) levels were markedly elevated in mPFC compared to Con and SD-Res groups, possibly through a TLR4-dependent pathway (\u003cb\u003eSupplementary Fig.\u0026nbsp;3a-d\u003c/b\u003e), depicting significant inflammatory responses in the area of mPFC in the SD-Sus mice.\u003c/p\u003e \u003cp\u003eMicroglial phagocytosis of neuronal synaptic elements is exacerbated in mPFC post-chronic stress.\u003c/p\u003e \u003cp\u003eDelving into the microglial reactivity after chronic stress, we sought to assess their phagocytic activity in mPFC using the lysosomal marker CD68, and whether this could be correlated with the expression of the proinflammatory marker iNOS during susceptibility (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo). The SD-Sus microglia exhibited a dramatic increase in phagocytosis which was noticeably colocalized with the iNOS pro-inflammatory marker (iNOS\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003eIba1\u003csup\u003e+\u003c/sup\u003e), compared to Con and SD-Res groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eo-r). In stark contrast the microglia from the SD-Res group, even though had significantly increased CD68 levels compared to Con (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep), did not display a pro-inflammatory phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eq,r), indicating a distinct reactivity phenotype.\u003c/p\u003e \u003cp\u003eIt is known that microglia are heavily involved in synaptic reorganization and circuitry refinement through synaptic pruning in the adult brain\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Excessive pruning, however, may lead to loss or weakening of synaptic connections in the neuronal circuits\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Therefore, we next asked whether the elevated phagocytic capacity of microglia in the SD-Sus mice would result in increased engulfment of synaptic components in the mPFC neurons. We imaged CX3CR1\u003csup\u003eGFP/+\u003c/sup\u003e mice along with lysosomal marker CD68 and either PSD95 (postsynaptic marker) or Synapsin-1 (Syn-1; presynaptic marker) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The microglial phagocytosis of PSD95 (CX3CR1\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003ePSD95\u003csup\u003e+\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,d) and of Syn-1 punctae (CX3CR1\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003eSyn-1+\u003csup\u003e+\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,f), were significantly increased in the SD-Sus mice compared to Con and SD-Res. This resulted in concomitant reductions in the PSD95 and Syn-1 immunoreactivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c,e) and protein expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee) in the mPFC of SD-Sus mice. Evident of the microglial reactivity in the depressive-like mice was also the significant increase of microglial phagocytosis of myelin observed in the SD-Sus mice (CX3CR1\u003csup\u003e+\u003c/sup\u003eCD68\u003csup\u003e+\u003c/sup\u003eCNP\u003csup\u003e+\u003c/sup\u003e), leading to significant myelin deficits in the mPFC area (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-c), as we have previously demonstrated\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe interaction of cAMP response element-binding protein (CREB) with brain-derived neurotrophic factor (BDNF) is an essential element in signal transduction pathways involved in mental disorders and critical for cellular resilience and neuroplasticity\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Due to the significant microglial-mediated synaptic element reductions observed in the mPFC area, we sought to assess the potential effects of chronic stress on neuronal plasticity using the markers p-CREB(Ser133) and BDNF in the mPFC of the SD groups. The immunoreactivity of neuronal p-CREB in SD-Sus mice was significantly decreased compared to Con and was in stark contrast to the significant upregulation evident in the SD-Res mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-e). Analogous bimodal responses were noted in the BDNF protein levels of the SD groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ef), suggesting that the chronic-stress susceptibility/resilience of the SD mice is interlinked with neuroplasticity, synaptic connectivity, and cellular resilience of the neuronal circuitry in the mPFC.\u003c/p\u003e \u003cp\u003eChronic stress induces long-lasting monocytic infiltration into the mPFC, mediating depressive-like behavior in mice.\u003c/p\u003e \u003cp\u003eDue to the persisting inflammatory responses in mPFC, we next used the \u003cem\u003eCX3CR1-\u003c/em\u003eCre\u003csup\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003cem\u003e-\u003c/em\u003eeYFP::\u003cem\u003eRosa26-\u003c/em\u003etdTom mouse line (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea; see Methods for more details) to trace the monocytic infiltration on D25 post-RSDS. For the induction of Cre-recombination, TMX was administered i.p. in CX3CR1\u003csup\u003eCreER/+\u003c/sup\u003e::R26\u003csup\u003etdTOM/+\u003c/sup\u003e mice for 5 days\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, starting at P30. The RSDS paradigm was performed 1 month after the induction of Cre, and behavioral tests were performed 2 weeks after the end of the paradigm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb; BH, D22-D25), as we have previously shown\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The first days after Cre-recombination, all microglia and peripheral macrophages (CX3CR1\u003csup\u003e+\u003c/sup\u003e) became YFP\u003csup\u003e+\u003c/sup\u003etdTom\u003csup\u003e+\u003c/sup\u003e cells (data not shown). Contrary to the self-renewing microglia\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, the bone marrow\u0026ndash;derived monocytic/macrophage populations, have an estimated half-life of 3\u0026ndash;4 weeks, allowing for fate mapping of the recruited monocytes (YFP\u003csup\u003e+\u003c/sup\u003etdTom\u003csup\u003e\u0026minus;\u003c/sup\u003e)\u003csup\u003e28\u003c/sup\u003e. We euthanized the CX3CR1\u003csup\u003eCreER/+\u003c/sup\u003e::R26\u003csup\u003etdTOM/+\u003c/sup\u003e mice on D25, revealing a significant increase of monocytic populations in the mPFC of SD-Sus mice compared to the Con and SD-Res groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec-e). In addition, we visualized the expression of ICAM-1 (a key monocyte-adhesion molecule): ICAM-1 immunoreactivity was significantly upregulated on the endothelial cells in SD-Sus mice, with a large portion of it colocalizing with the microglia/macrophage populations (CX3CR1-eYFP\u003csup\u003e+\u003c/sup\u003eICAM-1\u003csup\u003e+\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ef-g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicroglial and monocytic elimination during chronic stress exerts significant anti-depressant action.\u003c/p\u003e \u003cp\u003eTo determine whether the long-lasting monocytic recruitment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,e) and concomitant inflammatory responses (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-j) are responsible for the emergence of depressive-like behavior after chronic stress, we dissected and investigated separately the pathophenotypic contribution of the microglia and monocytes. We utilized PLX73086 (see Methods for details), a CSF1R inhibitor which depletes peripheral macrophage populations (dose 200 mg/kg) but does not affect the CNS resident microglia because of its low blood-brain barrier (BBB) penetration (\u003cb\u003eSupplementary Fig.\u0026nbsp;4a\u003c/b\u003e)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Experimental groups starting at D0 had \u003cem\u003ead libitum\u003c/em\u003e access either to Control-chow or the drug-containing chow (PLX73086) throughout the RSDS until they were euthanized on D15 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eh). As summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei the PLX73086 treatment exerted moderate but significant anti-depressant effects, as measured by a battery of behavioral tests (SI, EPM, FST and SPT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eh-m). Notably, the percentage of the SD-Sus mice dropped from 80\u0026ndash;50%, based on the social interaction ratio, suggesting that the recruited monocytes play a significant role in the development of depression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, j).\u003c/p\u003e \u003cp\u003eWe next determined the contribution of microglia in the development of the depressive-like behavior in chronically stressed mice. PLX5622 (see Methods for more details) is a CSF1R inhibitor which can enter the CNS and ablate 90\u0026ndash;95% of microglial/macrophage populations after 7 days of treatment (1,200 mg/kg dose)\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. PLX5622-chow treatment or Control-chow was introduced on D0 of the RSDS paradigm and was administered until mice were euthanized for further analysis (D15) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). In the experimental groups treated with PLX5622-chow significant microglial ablation (~\u0026thinsp;95%) was observed compared to the Control-chow on D15 after the RSDS paradigm (\u003cb\u003eSupplementary Fig.\u0026nbsp;4b-c\u003c/b\u003e). Remarkably, microglial elimination during chronic stress (PLX56-Res group) was able to protect 100% of the SD mice (in comparison to the 20% of the naturally resilient SD-Res group in animals receiving Control-show) from the emergence of depressive-like behavior (social avoidance, anhedonia, anxiety, reward under stress and despair), suggestive of an essential role that microglia have in the progression of the disorder (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eb,d \u003cb\u003eand Supplementary Fig.\u0026nbsp;5a-h\u003c/b\u003e). PLX56-treated na\u0026iuml;ve mice (no social defeat; PLX56-Con) were also included in the study and did not demonstrate any significant behavioral alterations compared to the Con animals, as previously described\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To exclude the implication of any PLX56-related side effects upon the sensory/recognition input of the treated mice, we also performed olfaction (buried food test) and memory recognition tests (novel object recognition), but no significant deficits were noted in the performance of mice\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e (Data not shown).\u003c/p\u003e \u003cp\u003eIn response to microglial elimination, the previously observed increase in the neuroinflammatory marker TSPO was rescued in the PLX56-Res group compared to the SD-Sus non-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ec), suggesting that TSPO could be used as a potential marker for monitoring neuroinflammatory responses in MDD and other mental disorders\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Of interest, the TSPO protein levels in the non-stressed PLX56-Con were significantly downregulated compared to the rest of the groups (and especially the PLX56-Res), indicating that astrocytes and endothelial cells (which also express TSPO) contribute to the inflammatory burden during chronic stress.\u003c/p\u003e \u003cp\u003eBased on the reduction of neuroinflammation and the prominent anti-depressant state after microglial elimination, we sought to examine if this affected synaptic plasticity and connectivity elements dysregulated during chronic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee-f). As demonstrated by immunoblot analyses, microglial elimination in the socially defeated PLX56-Res mice significantly rescued the decreased levels previously observed for the pre- and post-synaptic elements Synapsin-I and PSD95 in the mPFC of SD-Sus animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Notably, the non-defeated PLX56-Con group showed a considerable increase of the synaptic elements, indicative of the importance of microglia as sculptors of neuronal connectivity and circuitry in an activity-dependent manner\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Considering that the neurotrophin BDNF and serotonin receptor 5-HTR1A\u003csup\u003e2\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e are components of two major regulatory signaling systems involved in depressive disorders, we measured their protein levels in the mPFC of non-treated and treated SD groups. Treatment of the SD mice with PLX5622 was able to substantially protect the PLX56-Res from deficits in BDNF and 5-HTR1A protein levels (SD-Sus), as depicted by the comparable protein levels with the Con and SD-Res not-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003eMicroglia are essential mediators for the sensitization to chronic stress and required for the establishment of depressive-like phenotypes in mice.\u003c/p\u003e \u003cp\u003eTo further examine whether microglia play a critical modulatory role in the development of stress-induced behaviors, we took advantage of the quick repopulation properties of the microglia, once the mice treated with PLX5622-chow are reverted to normal chow\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Microglia were eliminated by PLX5622-treatment during the RSDS paradigm (Con-chow group was also included), and behavioral tests were performed between D11-D14 (BH1). Starting on D15, the PLX5622 chow was withdrawn, and normal/Con-chow was introduced for all groups, to allow for microglial repopulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The repopulation depends solely on local clonal expansion of the surviving microglial cells\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, which can rapidly replenish their numbers within 5 to 7 days after removal of the CSF1R inhibitor\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. As shown here, microglia repopulated the area during the 10 days of PLX5622 withdrawal, surpassing the microglial population of the non-treated PLX56-Con (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eg-h, \u003cb\u003eSupplementary Fig.\u0026nbsp;4d\u003c/b\u003e). At the end of the repopulation phase, mice underwent a second round of behavioral tests (D22-D25;BH2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eMicroglial repopulation was sufficient to trigger depressive-like behaviors in the previously categorized PLX56-Res, inducing social avoidance, anxiogenesis, despair-like behavior and anhedonia, comparable with those observed in the non-treated SD-Sus mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ei-j; \u003cb\u003eSupplementary Fig.\u0026nbsp;6a-d\u003c/b\u003e). This can also be depicted by the significant deterioration of the individual performances after paired analyses (before and after microglial repopulation) of the previously categorized PLX56-Res mice (\u003cb\u003eright panels in\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ei; \u003cb\u003eSupplementary Fig.\u0026nbsp;6a-d\u003c/b\u003e). It is important to note that a portion of the PLX56-Res mice also include the naturally occurring SD-Res mice (~\u0026thinsp;20%), which we extensively characterized in previous sections. This becomes also evident by the paired analyses in the behavioral tasks, since approximately 20% of the PLX56-Res mice (D11-14), remained resilient (SD-Res) on the second round of behavioral tests (D22-25) (\u003cb\u003eright panels in\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ei; \u003cb\u003eSupplementary Fig.\u0026nbsp;6a-d\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eTo examine whether the striking attenuation of depressive-like behavior upon microglial elimination was attributed to the onset of PLX5622 treatment at the beginning of chronic stress sensitization of SD mice (PLX5622-treatment starts together with RSDS paradigm), we initiated PLX treatment after the establishment of depressive-like behavior (post-RSDS; \u003cb\u003eSupplementary Fig.\u0026nbsp;7a\u003c/b\u003e). PLX5622-chow was introduced on D15 (after the end of RSDS and the first round of behavioral testing; RSDS phase) until D25 (Treatment phase; \u003cb\u003eSupplementary Fig.\u0026nbsp;7a\u003c/b\u003e). Α second round of behavioral tests was performed between D22-25 (ΒΗ2) for behavioral assessment of the experimental groups before and after PLX5622-treatment. It is important to note that both SD-Sus and SD-Res groups were included in the PLX5622 treatment, following their behavioral categorization. Remarkably, the PLX5622 treatment post-RSDS was able to result in significant antidepressant action, rescuing key depression-related indices (social avoidance, anxiogenesis, reward under stress and despair-like behavior) in all the SD-Sus treated mice; \u003cb\u003eSupplementary Fig.\u0026nbsp;7b-f\u003c/b\u003e). This is also illustrated by the significant individual scores of the SD groups in response to the PLX5622 treatment (\u003cb\u003eright panels in Supplementary Fig.\u0026nbsp;7c-f\u003c/b\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eA large body of evidence from clinical research and animal models indicates that the immune system and associated inflammatory responses have a bidirectional regulatory impact on stress- and mood-related neuronal circuits, modulating susceptibility and resilience to a variety of psychosocial stressors\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Here, we employed the RSDS protocol, a mouse model of stress-induced depression-like behaviors\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, to elucidate the cellular dynamics and inflammatory responses of microglia. In line with our previous studies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the majority (~\u0026thinsp;80%) of SD mice displayed long-lasting depressive-like behavioral features (SD-Sus), while a smaller cohort (~\u0026thinsp;20%) exhibited resilience to stress (SD-Res), reflecting the heterogeneous responses to chronic stress in humans\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The microglial responses and neuroimmune interplay mechanisms in mPFC were investigated both in the SD-Res mice, which have been relatively uncharacterized, as well as the SD-Sus animals.\u003c/p\u003e \u003cp\u003eWe detected a rapid (D6) and long-lasting (D15) increase of microglial density and proliferation after RSDS in the mPFC of the SD-Sus mice. Our findings are in line with previous studies reporting increased microglial immunoreactivity\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e in mPFC, but also contrast reports showing microglial recruitment and proliferation only after acute stress\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Possible reasons for these discrepancies in the literature might be attributed to the differences in the stress paradigms (the nature, length, and intensity of stressor).\u003c/p\u003e \u003cp\u003eWe also demonstrated that the microglial recruitment in the mPFC of the SD-Sus mice was spatially related to a significant increase of neuronal activity (cFOS). The cFOS upregulation in response to chronic stress has been previously attributed to neuronal sensitization\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Microglia are known to respond rapidly to psychosocial stressors through ATP/purinergic signaling in an neuronal activity-dependent manner\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Notably, even though the SD-Res mice had comparable stress/anxiety levels with the SD-Sus (evident via EPM test), they exhibited only a minor increase of cFOS reactivity, suggesting a compensatory neuronal circuitry mechanism\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe microglial density increases displayed after RSDS could be attributed to local microglial proliferation, but also to monocytic recruitment (the markers Iba1 and CX3CR1 label both microglia and macrophages). However there has been ambiguity in the literature regarding the latter, with some studies suggesting significant peripheral macrophage recruitment in the mPFC after chronic stress\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e and some others reporting no macrophage involvement\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. These deviations could be attributed to the use of different mouse models of stress, or variability in the duration of the stress paradigms. Here, we demonstrated a lasting peripheral monocytic recruitment (D15 and D25 post-RSDS) in the SD-Sus mice, which was facilitated by a significant upregulation of the adhesion molecules VCAM-1 and ICAM-1 in the mPFC. These results corroborate with previously described mechanisms implicating microglia, endothelial cells, and macrophage interactions contributing to changes in mood behavior\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFurther characterization of the microglia/macrophage inflammatory responses revealed a significant increase of several pro-inflammatory markers (CD86, iNOS, TNF-α, CXCL10 and IL-1β) and a reactive hypertrophic morphology characterized by shorter and fewer processes and increased cell body area\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, followed by a concomitant decrease of anti-inflammatory markers (CD206, Arg1 and IL-10) in the SD-Sus mice compared to the Con and SD-Res groups. The morphology of the SD-Res microglia was also distinct with long distally hyper-ramified morphology, previously described for microglia responding to increased glutamatergic neurotransmission\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These findings indicate that susceptibility or resilience is associated with the inflammatory response profile and polarization spectrum (pro-inflammatory/anti-inflammatory) of microglial/macrophages in the mPFC.\u003c/p\u003e \u003cp\u003eConsistent with the inflammatory responses observed in the SD-Sus mice, total and microglial-specific TSPO levels were elevated in the mPFC compared to Con and SD-Res groups. In agreement with our findings, recent studies in MDD and schizophrenia patients have reported a significant upregulation of TSPO levels in the cingulate cortex and PFC\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, further supporting a role for microglial activation and associated neuroinflammatory responses in the mental disorder pathophysiology.\u003c/p\u003e \u003cp\u003eIn accordance with previous studies reporting increased phagocytic activity of microglia in the mPFC\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, we observed a dramatic increase in microglial phagocytosis which was colocalized with the iNOS proinflammatory marker, which were previously shown to involve a TLR4-dependent pathway\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Interestingly, the microglia from SD-Res group, even though had significantly increased CD68 levels compared to Con, they did not display a pro-inflammatory phenotype, consistent with a distinct activation status compared to the proinflammatory SD-Sus microglia. As microglia are crucial regulators of neuronal connectivity and of circuitry refinement through synaptic pruning\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, we observed a significant increase in microglial phagocytosis of pre- and post-synaptic elements in the mPFC of SD-Sus mice and significant reductions of PSD95 and Syn-1 levels, which was followed by substantial synaptic plasticity deficits as indicated by the levels of BDNF, p-CREB and serotonin receptor 5-HTR1A, all known regulatory signaling components involved in depressive disorders\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicroglia and infiltrating macrophages have been previously reported to be in close interaction and act in synergism\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e inducing stress and depressive disorders\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In this study we investigated their individual pathophenotypic contribution to depression through treatment with two different CSF1R inhibitors: PLX73086 (which depletes peripheral macrophage populationsby about 80%\u003csup\u003e50\u003c/sup\u003e) and PLX5622 (which depletes both microglia and macrophage populations). We demonstrated that peripheral macrophage ablation (PLX73086) exerted a moderate but significant anti-depressant action in the SD-treated groups (50% of protection), suggesting that the recruited monocytes contribute to the development of depression. The effect that microglial/macrophage elimination (PLX5622) had during chronic stress was remarkable, since it prevented depressive-like behaviors in the total SD population (100% protection). The lack of depressive symptoms was accompanied by reduction of the levels of TSPO in the PLX56-Res group, potentially relating the TSPO marker with the behavioral performance in depressive disorders\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Our findings are consistent with previous studies using PLX5622 to ablate microglia two weeks prior to the initiation of chronic stress using different paradigm formats\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Our study is distinct, however, as we investigated the effects of microglia/macrophage ablation on behavioral and neuronal homeostasis both during the induction of chronic stress (PLX5622 during RSDS), and after the induction of chronic stress and the establishment of a neuroinflammatory environment in the mPFC area (PLX5622 post-RSDS). We find that microglial/macrophage ablation during chronic stress was able to prevent essential neuronal plasticity deficits (BDNF and 5-HTR1A) and the loss of synaptic elements (Syn-1 and PSD95).\u003c/p\u003e \u003cp\u003eA key finding of our study was the re-emergence of a depressive-like phenotype after microglial repopulation. Considering that microglial repopulation occurs entirely from surviving residual microglia\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, it would be important to examine in the future whether chronic stress induces specific depressive-like behavior epigenetic changes to microglia, which can be passed on after their clonal expansion, and if neuronal sensitization of the mPFC is contributing to the microglial \u0026ldquo;re-education\u0026rdquo; into a pro-inflammatory state when they reappear. With these in mind, it may be interesting to explore potential approaches in which microglia are epigenetically \u0026ldquo;re-educated\u0026rsquo;\u0026rsquo; towards a neuroprotective phenotype (similar to the anti-inflammatory microglial phenotype of the SD-Res) or utilize an anti-inflammatory treatment in conjunction with first line anti-depressant treatments.\u003c/p\u003e \u003cp\u003eLimitations of our study are the fact that the RSDS model uses most commonly male animals, and that it is debated whether avoidance in the social interaction test represents a 'depressive-like' behavior or impaired learning\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCollectively, the present study: i) reveals an important association of microglial/macrophage polarization spectrum (pro-inflammatory/anti-inflammatory) and their respective inflammatory responses with susceptibility or resilience to chronic stress, ii) demonstrates that proinflammatory microglial activation leads to deficits in neuroplasticity and synaptic-connectivity, iii) signifies the contribution of monocytic recruitment to the neuroinflammatory burden and associated behavioral deficits (possibly by microglial-mediated signaling, as previously suggested\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e) and iv) indicates that microglia are not only essential mediators for the sensitization of SD groups to chronic stress but also necessary for the establishment of depressive-like phenotype. These findings are further solidifying the neuroinflammatory hypothesis of depression and support a critical role for microglia and macrophages in the depression pathophysiology.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cp\u003eAnimals\u003c/p\u003e \u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee (covered by Animal welfare assurance No A3011-0), at Stony Brook University, and conducted in accordance with the guidelines of the National Institutes of Health \u0026ldquo;Guide for the Care and Use of Laboratory Animals\u0026rdquo;. Experiments were performed using adult (2\u0026ndash;3 months old) male mice [C57BL/6J (wt), \u003cem\u003eCX3CR1\u003c/em\u003e-GFP (Jackson Labs, 005582 model B6.129P-Cx3cr1tm1Litt/J), \u003cem\u003eCX3CR1\u003c/em\u003e-Cre\u003csup\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e-eYFP (Jackson Labs, 021160 model B6.129P2 (Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ), td-Tomato (Jackson Labs, 007905 model number B6;129S6-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze/J)], were used in the study. All the mouse lines were backcrossed to a C57BL/6J background, bred in-house, and genotyped by PCR. CD-1 retired-breeder male mice (Charles River Laboratories, CD-1 IGS mice, strain code:022) were used as aggressors. The CD-1 male mice exert aggression only towards male mice, therefore female mice were excluded in the study. For induction of recombination in the \u003cem\u003eCX3CR1-\u003c/em\u003eCre\u003csup\u003eERT\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003cem\u003e-\u003c/em\u003eeYFP::\u003cem\u003eRosa26-\u003c/em\u003etdTom mice, tamoxifen (Sigma CAS # 10540-29‐1) dissolved in ethanol : sunflower seed oil was injected intraperitoneally (i.p.) starting at P30, at a daily dose of 75 \u0026micro;g/g body weight (from a 10 mg/ml stock), for 5 consecutive days, as in\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. All animals were housed in 12-hr light/dark cycle. Food and water were provided \u003cem\u003ead libitum\u003c/em\u003e by the experimenters.\u003c/p\u003e \u003cp\u003eRepeated social defeat stress paradigm (RSDS)\u003c/p\u003e \u003cp\u003eAdult male mice were subjected to repeated bouts of physical aggression (\u0026lsquo;\u0026lsquo;defeats\u0026rsquo;\u0026rsquo;) from aggressive CD-1 mice for 10 consecutive days, as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. The CD-1 aggressors were screened during a 3-day screening period for adequate aggressive behavior and then housed on one side of the divided mouse cage (known as the home cage), at least overnight prior to the start of defeat sessions. All defeat experiments were performed within that compartment, while the intruders were rotated across defeat days, so that the experimental animals would not habituate to a single aggressor. On day 0 (D0) of RSDS the \u0026lsquo;socially defeated-to-be\u0026rsquo; (SD) mice were placed into the aggressor\u0026rsquo;s space for 10 min (physical stress), and after the end of the encounter they were returned to their side overnight. The mice could see and smell the aggressor through the clear perforated divider (sensory stress). The na\u0026iuml;ve mice (Con) were exposed to wt (C57BL/6J) mice, instead, for 1 minute. About 80% of the mice that were subjected to social defeat stress displayed depressive-like behavior (SD-Sus for Susceptible), whereas the rest 20% were the nonresponding mice (SD-Res for Resilient) did not, thus modeling the heterogeneity in individual responses to stress in humans\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. At the end of RSDS paradigms all animals were singly housed. Socially defeated mice were categorized based on the SI output, and further behavioral analyses for each group followed this categorization. Both SD-Sus and SD-Res exhibited anxiety-like behavior, however the SD-Sus additionally demonstrated social avoidance, reduction of reward under stress, anhedonia, despair-like behavior, and elevated plasma corticosterone levels (~\u0026thinsp;130 ng/ml)\u003csup\u003e15,56\u003c/sup\u003e. In addition to this paradigm, a short-term RSDS paradigm adaptation (miniSD; 3 days) followed by 3 days of behavioral tests (BH; D4-D6) was also performed in the results shown in \u003cb\u003eSupplementary Fig.\u0026nbsp;2\u003c/b\u003e, which resulted in ~\u0026thinsp;50% of the SD mice becoming SD-Sus, as we have previously demonstrated\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Although we find that the model produces very reproducible / reliable results, it does have the limitation that in the current format the model uses male mice only for the experiments.\u003c/p\u003e \u003cp\u003eBehavioral analyses\u003c/p\u003e \u003cp\u003eFor endpoints D15 and D25, mice were behaviorally tested for 4 days (1 experiment/day). Behavioral testing and recordings were performed during the light phase. Both na\u0026iuml;ve and SD mouse groups were tested for behavioral alterations using the i) social interaction test (SI; measures social avoidance), ii) elevated plus-maze test (EPM; measures stress/anxiety), iii) forced swimming test (FST; measures despair-like behavior), iv) sucrose preference test (SPT; assesses anhedonia-lack of feeling pleasure), and v) novelty suppressed feeding test (NSF; assesses reward under stress). All experiments (except for SPT) were performed in a light-controlled and sound-isolated behavioral analysis room. The mice were acclimatized to the experimental room for 1 hour before the start of each experiment. The SI and EPM were performed under red light conditions; the remaining behavioral tests were performed with lights on. The software Noldus Ethovision XT16 was used for the automated tracking and scoring during the behavioral tests. White noise generator (70\u0026ndash;75 dB) was used to mask intermittent disturbing sounds (from surrounding areas) that would potentially startle the animals.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSocial Interaction (SI)\u003c/b\u003e One day after the final social defeat stress interaction, a SI test was performed to determine whether the animals display social avoidance, as previously established\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Mice were placed into the social interaction open-field arena [(42 cm (w) \u0026times; 42 cm (d) \u0026times; 42 cm (h)], and the time spent in the interaction zone (\u0026lt;\u0026thinsp;8 cm from the wire-mesh enclosure) was monitored for the two 2.5-min phases (without or with a novel CD-1 aggressor present in the enclosure), separated by a duration of 30 s. The socially defeated mice with SI ratio [(Time in interaction zone with aggressor) / (Time in interaction zone without aggressor)]\u0026thinsp;\u0026gt;\u0026thinsp;1 were classified as SD-Res, and mice with a ratio below 1 (social avoidance) were classified as SD-Sus\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The na\u0026iuml;ve mice were categorized as Control (C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eElevated plus-maze test (EPM)\u003c/b\u003e The apparatus used for this test was cross-shaped with two open arms (30 cm x 5 cm) and two closed arms (30 cm x 5 cm x15 cm) that extended from a central platform (5 cm x 5 cm), as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. The entire maze was elevated 40 cm above the floor. The enclosed arms offered safety when the mouse was stressed; on the other hand, the open arms offered the motive of exploration. Each mouse was placed in the central square of the apparatus, facing an enclosed arm. The mice were let to roam freely for 10 min totally; an arm entry was defined when all four paws entered an arm. The total mobility, time spent in open arms and time closed arms were recorded, as an index of stress/anxiety\u003csup\u003e57\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eForced swimming test (FSM)\u003c/b\u003e Each mouse was placed in a transparent tank [inescapable Plexiglas cylindrical tank (height: 30 cm, diameter: 22.5 cm)] that was filled with water (to a depth of 15 cm and maintained at 25\u0026deg;C) and their escape related mobility behavior was measured. Once the mouse was in the water, it was left to swim for 6 min in total, assessing the behavior only during the last 4 min, as previously performed\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The mice that acquired an immobile posture, characterized by motionless floating in the water, were termed immobile (immobility time), making only the necessary movements to keep the head above the water. Before returning the animals to their home cages, they were dried gently using paper towels to prevent hypothermia.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSucrose preference test (SPT)\u003c/b\u003e The SPT was performed as previously\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The experimental groups were habituated for 72 h to 2% sucrose, in which the bottles were alternated to avoid bias for a specific cage side. The day before the testing the mice underwent an 18 h water deprivation period. The day of the testing bottles filled with sucrose 2% or water were weighed, and consumption was determined for a 24 h period. Bottles were weighed again at the end of the 24 h period. Sucrose preference was expressed as (Δweightsucrose) / Δweightsucrose\u0026thinsp;+\u0026thinsp;Δweightwater) x100. Sucrose preference score less than 70% was displaying anhedonia.\u003c/p\u003e \u003cp\u003e \u003cb\u003eNovelty suppressed feeding test (NSF)\u003c/b\u003e The NSF test measures the time that mice take to approach and eat food in a novel environment following an extended period of food deprivation. Mice were food-deprived for 24 h and moved in freshly prepared home-cages (to avoid any pellets remaining on the bedding). The next day, each mouse was placed at the corner of an open-field arena [(42 cm (w) \u0026times; 42 cm (d) \u0026times; 42 cm (h)] with a pellet of chow already positioned at the center of it. The time until the first bite of the chow pellet was recorded, with maximum trial time the 10 min. After the trial, the mice were returned to their home cage which contained a pre-weighed food pellet, and food consumption was measured for a period of 5 min, as described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBrdU labeling\u003c/p\u003e \u003cp\u003eFor the labeling of proliferating cells, 5-Bromo-2\u0026prime;-deoxyuridine (BrdU;Sigma-B5022) was dissolved in drinking water (1 mg/ml), and all mouse groups were given access to the water \u003cem\u003ead libitum\u003c/em\u003e throughout the 10 days of the RSDS paradigm\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMicroglial depletion with PLX5622\u003c/p\u003e \u003cp\u003eExperimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX5622 (PLX56, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 1200 mg/kg of food weight (Research Diets, Inc.), as previously \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Seven days of PLX administration can achieve\u0026thinsp;\u0026gt;\u0026thinsp;90% microglial ablation \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Exact treatment duration varied depending on the experimental design and is defined on each section.\u003c/p\u003e \u003cp\u003ePeripheral macrophage depletion with PLX73086\u003c/p\u003e \u003cp\u003eExperimental groups were fed with colony stimulating factor-1 receptor (CSF1R) inhibitor, PLX73086 (PLX73, provided by Plexxikon Inc.), formulated in AIN-76A standard chow at 200 mg/kg of food weight (Research Diets, Inc.), as previously\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Control animals were fed AIN-76A standard chow (Research Diets, Inc.). Exact treatment duration varied depending on the experimental design and is defined on each section.\u003c/p\u003e \u003cp\u003eImmunoblot analysis\u003c/p\u003e \u003cp\u003eMouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain\u0026thinsp;~\u0026thinsp;500-\u0026micro;m thick brain coronal sections. Medial PFC was dissected out (with the help of a dissecting microscope) from +\u0026thinsp;1.2 mm to +\u0026thinsp;2.5 mm anterior to bregma slices. Protein lysate was prepared using RIPA buffer (120\u0026ndash;150 \u0026micro;l) with protease and phosphatase inhibitors (200 mM PMSF, 100 mM sodium orthovanadate, and protease inhibitor cocktail; Santa Cruz, sc-24948A). Lysates were shaken for 15 min at 4\u0026deg;C, cleared by centrifugation at 15,000 g for 10 min at 4\u0026deg;C, and protein concentration was determined using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific; 23225). Samples (15\u0026ndash;20 \u0026micro;g) were boiled for 5 min with 5X SDS-PAGE sample loading buffer (Thermo Fisher Scientific; 39000), separated by SDS-PAGE, and transferred to PVDF membranes (ThermoFisher Scientific; 88518) at 30 V for 16\u0026ndash;18 h at 4\u0026deg;C. Membranes were blocked with 5% w/v nonfat dry milk (Cell Signal; 9999S) and incubated with primary antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;2a\u003c/b\u003e) for 16\u0026ndash;18 h at 4\u0026deg;C. Washes with TBST (Cell Signal; 9997S) were followed by incubation with HRP-coupled secondary antibodies (\u003cb\u003eSupplementary Table\u0026nbsp;2b\u003c/b\u003e). Signal was visualized by Sapphire Biomolecular Imager (Azure Biosystems) using a chemiluminescent substrate mixture (Supersignal West Pico Plus; ThermoFisher Scientific, 34580 or Immobilon Western; Millipore, WBKLS0500). Optical densities were collected with Fiji ImageJ software\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Where indicated, protein levels were expressed as fold change (versus Control) of the arbitrary units (A.U.) following normalization to the corresponding loading controls (β-Actin, GAPDH). Samples were normalized to the total protein for each sample, as determined by the BCA assay. For a detailed antibody list used see \u003cb\u003eSupplementary Table\u0026nbsp;2a\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eImmunohistochemistry\u003c/p\u003e \u003cp\u003eDeeply anesthetized mice (isoflurane) were transcardially perfused with cold 1X PBS (15 ml) followed by cold PFA fixative solution (4% paraformaldehyde in 1X PBS; 20ml) and brains were dissected and post-fixed for an additional 24 hours. Then brains were cryoprotected in 30% sucrose for 24 hours and sectioned with a sliding microtome. Free-floating brain sections (30\u0026ndash;40 \u0026micro;m thick) containing the mPFC, were blocked with 10% goat serum (ThermoFisher Scientific; 16210072) in 0.3% TritonX-100 in 1X PBS for 1\u0026ndash;2 hour at room temperature. Tissue sections were incubated with primary antibodies overnight at 4\u0026deg;C at the indicated concentrations (\u003cb\u003eSupplementary Table\u0026nbsp;2a\u003c/b\u003e). The following day, sections were washed in PBS-Triton and incubated with the appropriate cross-absorbed secondary antibodies (\u003cb\u003eTable II-3)\u003c/b\u003e. After 4 additional washes, nuclei were stained with DAPI (Sigma; D9542) and sections were mounted using MOWIOL mounting media. For BrdU IHC, sections were pretreated in 2M HCl for 40 minutes at 37\u0026deg;C, followed by 2-3x 5-minute washes in 0.1 M Boric acid (pH 8.5). For a detailed antibody list used see \u003cb\u003eSupplementary Table\u0026nbsp;2c\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eMicroscopy and histological quantification\u003c/p\u003e \u003cp\u003eThe confocal laser-scanning microscopes Leica TCS SP8X and Leica TCS-SP5 were used for imaging of FITC, EGFP, EYFP, Alexa-488, Alexa-555, Alexa-594, Alexa-647 and CY3 fluorophores. Optical sections (z\u0026thinsp;=\u0026thinsp;1.0\u0026micro;m; total stack of 10\u0026ndash;16\u0026micro;m for 1 cell layer) of confocal epifluorescence images were sequentially acquired using 10x, 20x, 40x (N.A. 1.30), 63x (N.A. 1.20) and 100x (N.A. 1.40) objectives, with LAS AF software. Fiji ImageJ software\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e was used for image reconstruction, cell counting, integrated density analysis and colocalization analysis. For mPFC analysis, 6 fields (located between +\u0026thinsp;1.2mm to +\u0026thinsp;2.5mm anterior to bregma), containing the cingulate cortex (Cg1), prelimbic (PL), infralimbic (IL) and medial orbital cortex (MO) were taken from each animal, for quantitative analysis. Typically, 3\u0026ndash;4 brain slices were analyzed per animal, and at least 3 different animals for each experimental condition were evaluated. Cell counts were presented as the number of marker\u0026thinsp;+\u0026thinsp;cells/section (section corresponding to 0.2\u003csup\u003emm\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e). Each section quantification depicts the sum of the marker\u0026thinsp;+\u0026thinsp;cells from all the analyzed fields. For the integrated density analysis, the IsoData thresholding was applied to each image, and the integrated density [is defined as the product of pixel intensity (255\u0026thinsp;=\u0026thinsp;the maximum pixel intensity for an 8-bit image) x area] was measured. For the colocalization analysis, the IsoData thresholding was applied to each image, images were merged, compartmentalized, and the integrated density was measured for the colocalized signal. Each section with integrated density quantification is depicted by the mean of the integrated density from all the analyzed fields. All experimental group comparisons were conducted on sections stained and imaged with identical exposure and acquisition settings. All analyses were performed on raw images, prior to any image processing by Adobe Illustrator CS6 for representation purposes. The cell counting was performed in a blinded manner by 4 experimenters collectively, and tissue sections were matched across samples.\u003c/p\u003e \u003cp\u003eMicroglial morphological analysis\u003c/p\u003e \u003cp\u003eTo quantitatively examine microglial morphology, unprocessed 30\u0026micro;m z-stack confocal images (100x objective; 20 individual images, step size: 1\u0026micro;m) of cells for Iba1 activity in the mPFC area were imported into Neurolucida software, as previously described\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Cell bodies and processes were manually traced (6 cells/mouse), and a 3D rendering was created. The 3D reconstructions were imported into Neurolucida Explorer for branched structure analysis and Sholl analysis (5\u0026micro;m starting radius, 50\u0026micro;m ending radius, and 5\u0026micro;m step size). Number of process intersections, cell surface area, and cell complexity were measured. Microglial complexity = [Sum of the terminal orders\u0026thinsp;+\u0026thinsp;Number of terminals] * [Total dendritic length / Number of primary dendrites]. Terminal orders: Number of \"sister\" branches encountered as proceeding from the terminal to the cell body (calculated for each terminal); Terminal: Refers to process endings.\u003c/p\u003e \u003cp\u003eFlow cytometry\u003c/p\u003e \u003cp\u003eFlow cytometry was performed as previously, with minor modifications\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Mice were deeply anesthetized (isoflurane) and transcardially perfused with 20 ml of ice cold 1X HBSS (pH 7.4). The mPFC of \u003cem\u003eCX3CR1\u003c/em\u003e-GFP mice was collected in ice-cold Hibernate-A Medium (ThermoFisher Scientific; A1247501) isolated and enzymatically digested in a pre-warmed (10 min) 1.54 mg/ml papain solution (Worthington; LS003127), containing 1.1 mM EDTA, 0.067 mM β-mercaptoethanol and 5.5 mM cysteine-HCl, for 20 minutes at 37\u0026deg;C with manual inversion every 5 minutes. Further processing was performed at 4\u0026deg;C. Tissue was triturated with a p100 tip, followed by syringe G20 and then G25 trituration (5 times each). Tissue debris was removed by passing the cell suspension through a 40-\u0026micro;m cell strainer and centrifuged for 5 minutes at 2000 g at 4\u0026deg;C to remove papain solution. Myelin was removed using a discontinuous Percoll gradient. The cell pellet was resuspended in 30% Percoll (Sigma; P1644) in 1X HBSS, layered over a 37% and a 70% Percoll cushion, and centrifuged at 800 g for 40 min (800 x g) at 10\u0026deg;C. The supernatant containing myelin was removed, and cells were collected at the 37\u0026ndash;70% Percoll interface. The remaining solution was diluted with 1X HBSS, and samples were centrifuged for 10 minutes at 1000 g, 4\u0026deg;C to pellet the cells. The pellet was then resuspended in flow cytometry (FC) buffer [0.5% bovine serum albumin (BSA) in 1X PBS]; cells were counted using trypan blue exclusion. Non-specific binding was blocked using anti- CD16/32 (1:50 in FC buffer; Tonbo biosciences, TB-70-0161-U500) for 30 minutes on ice and then stained with CD11b-APC, CD45-PerCP/Cy5.5, CD86-Pacific Blue, CD206-PE, antibodies (1:100 in FC buffer, Biolegend) in various combinations for 30 minutes on ice in dark conditions. The staining solution was removed by centrifugation for 5 minutes at 1000 g, and cells were washed twice with FC buffer before resuspending for flow cytometric analysis on a BD LSR Fortessa. The FC analysis was performed in the endpoint population of 15,000 \u003cem\u003eCX3CR1\u003c/em\u003e-GFP\u003csup\u003e+\u003c/sup\u003e cells/mouse. Data were further analyzed on the FlowJo software.\u003c/p\u003e \u003cp\u003eRNA isolation and quantitative PCR\u003c/p\u003e \u003cp\u003eMouse subjects were euthanized with isoflurane overdose, and freshly extracted brains were micro-dissected using a McIlwain tissue chopper to obtain\u0026thinsp;~\u0026thinsp;500-\u0026micro;m thick brain coronal sections. RNA from the mPFC (+\u0026thinsp;1.2 mm to +\u0026thinsp;2.5 mm anterior to bregma) tissue or cells was isolated using Qiazol (Qiagen; 79306). The total RNA was separated from the aqueous phase using a RNeasy MicroKit (Qiagen; 74004). cDNA was reverse transcribed using random primers and Superscript IV (Invitrogen; 18090200). Quantitative PCR was performed from cDNA using Phusion Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific; F548L) as per the manufacturer\u0026rsquo;s protocol on an Applied Biosystems 7900HT real time PCR system (Ct values above 35 cycles were not considered for the analysis). Fold changes were calculated using the ∆∆Ct method. For the primer sequences, see (\u003cb\u003eSupplementary Table\u0026nbsp;2d)\u003c/b\u003e. All gene expression levels were normalized to \u003cem\u003eGapdh\u003c/em\u003e mRNA levels.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eNo statistical methods were used to predetermine sample size \u003cem\u003ea priori\u003c/em\u003e, but the sample sizes used were similar to those reported in previous studies\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Furthermore, representative data from each experiment were examined by Shapiro-Wilk\u0026rsquo;s test (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003csup\u003e62\u003c/sup\u003e and a visual inspection of their histograms to confirm normal data distribution\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. The majority of the experimental analyses was performed by parametric ordinary one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s multiple comparison post-hoc tests. For the glial morphological analyses two-way ANOVA was performed, followed by Tukey\u0026rsquo;s multiple comparison post-hoc tests. For the PLX5622 repopulation behavioral tests a paired t-test was performed. The GraphPad Prism 8 and Excel (Microsoft) were used for all statistical analyses. The data were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. with symbols indicating the following \u003cem\u003eP\u003c/em\u003e value ranges: *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001 and **** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001).\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eWe are grateful to Drs. Ramin Parsey, Christine DeLorenzo, Joel Levine and Holly Colognato for their valuable insight and discussions. This work was supported by the National Institute of Mental Health, Grant/Award Numbers: R01MH123093-01, R01MH123093-01S1 (MMM), the American Heart Association (19PRE34370044; AGK) and Scholars in BioMedical Sciences Program (T32GM127253).\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eA.G.K. and S.E.T. conceptualized the project. A.G.K. designed, performed experiments, analyzed data. M.M.M., K.V., and Z.H. performed image quantification and data analyses. A.G.K., M.M.M, and S.E.T drafted and edited the manuscript. S.E.T. provided technical expertise, funding, and supervised the project.\u003c/p\u003e\n\u003cp\u003eCompeting Interests statement\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, Q. \u0026amp; Barres, B. A. Microglia and macrophages in brain homeostasis and disease. 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Journal of Statistics Education 19, 1\u0026ndash;18 (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-1991809/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1991809/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRepeated stress can lead to the development of anxiety and is considered a risk factor for major depressive disorder (MDD). Clinical studies and animal models of repeated and chronic stress have reported that symptom severity is correlated with microglial activation and upregulation of neuroinflammatory cytokine signaling in brain areas implicated in mood regulation. Despite mounting evidence implicating impairments of neuroplasticity and synaptic signaling deficits into the pathophysiology of stress-related mental disorders, whether microglial activation modulates neuronal homeostasis in response to chronic stress has been debated. Here, using the repeated social defeat stress (RSDS) mouse model we demonstrate that microglial activation and related inflammatory responses are regulating neuronal plasticity associated with depressive-like behavior. Specifically, we show that chronic stress induces a swift activation and proliferation of microglia as well as macrophage infiltration in the mPFC, which are spatially related to neuronal activation. Moreover, we report a remarkable association of microglial spectrum of reactivity and concomitant inflammatory responses with susceptibility or resilience to chronic stress. In addition, we find that exposure to chronic stress exacerbates phagocytosis of synaptic elements and significant neuronal plasticity deficits associated with depressive-like behavior. Importantly, by utilizing two different CSF1R inhibitors (the brain penetrant PLX5622 and the non-penetrant PLX73086) we determine the contributions of microglial and infiltrating macrophages in the depression pathophenotype. Our findings highlight a crucial role for microglia (and secondarily macrophages) in catalyzing the pathological manifestations of depression in response to chronic stress by promoting neuroinflammation and neuronal deficits in mPFC.\u003c/p\u003e","manuscriptTitle":"Chronic psychosocial stress induces microglial activation and inflammatory responses that lead to neuronal dysfunction and depressive-like behavior","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-26 17:30:51","doi":"10.21203/rs.3.rs-1991809/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c8f7fb02-8ac1-44c2-9a50-85117a1e5345","owner":[],"postedDate":"October 26th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":16398311,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":16398312,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2022-11-23T11:42:22+00:00","versionOfRecord":[],"versionCreatedAt":"2022-10-26 17:30:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1991809","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1991809","identity":"rs-1991809","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}