Stress-induced dysfunction of neurovascular astrocytes contributes to sex-specific behavioral deficits.

Astrocytes form an integral component of the neurovascular unit, ensheathing brain blood vessels with projections high in aquaporin-4 (AQP4) expression. These AQP4-rich projections facilitate interaction between the vascular endothelium, astrocytes, and neurons, and help stabilize vascular morphology. Studies using preclinical models of psychological stress and post-mortem tissue from patients with major depressive disorder (MDD) have reported reductions in AQP4, loss of astrocytic structures, and vascular impairment in the prefrontal cortex (PFC). Though compelling, the role of AQP4 in mediating stress-induced alterations in blood vessel function and behavior remains unclear. Here, we address this, alongside potential sex differences in chronic unpredictable stress (CUS) effects on astrocyte phenotype, blood-brain barrier integrity, and behavior. CUS led to pronounced shifts in stress-coping behavior and working memory deficits in male -but not female- mice. Following behavioral testing, astrocytes from the frontal cortex were isolated for gene expression analyses. We found that CUS increased various transcripts associated with blood vessel maintenance in astrocytes from males, but either had no effect on-or decreased-these genes in females. Furthermore, CUS caused a reduction in vascular-localized AQP4 and elevated extravasation of a small molecule fluorescent reporter (Dextran) in the PFC in males but not females. Studies showed that knockdown of AQP4 in the PFC in males is sufficient to disrupt astrocyte phenotype and increase behavioral susceptibility to a sub-chronic stressor. Collectively, these findings provide initial evidence that sex-specific alterations in astrocyte phenotype and neurovascular integrity in the PFC contribute to behavioral and cognitive consequences following chronic stress.

Repeated Activation of Pyramidal Neurons in the Prefrontal Cortex Alters Microglial Phenotype in Male Mice.

Aberrant neuronal activity in the cortex alters microglia phenotype and function in several contexts, including chronic psychologic stress and neurodegenerative disease. Recent findings even suggest that heightened levels of neuronal activity spur microglia to phagocytose synapses, with potential impacts on cognition and behavior. Thus, the present studies were designed to determine if activation of neurons alone-independent of disease or dysfunction-is sufficient to alter microglial phenotype in the medial prefrontal cortex (mPFC), a brain region critical in emotion regulation and cognition. In these studies, we used both an adeno-associated virus-mediated and Cre-dependent chemogenetic [designer receptors exclusively activated by designer drugs (DREADD)] approach to repeatedly activate excitatory pyramidal neurons (CaMKIIa+) neurons in the mPFC. Various molecular, cytometric, and behavioral endpoints were examined. Recurrent DREADD-induced neuronal activation led to pronounced changes in microglial density, clustering, and morphology in the mPFC and increased microglia-specific transcripts implicated in synaptic pruning (e.g., Csf1r, Cd11b). Further analyses revealed that the magnitude of DREADD-induced neuronal activation was significantly correlated with measures of microglial morphology in the mPFC. These alterations in microglial phenotype coincided with an increase in microglial lysosome volume in the mPFC and selective deficits in working memory function. Altogether, these findings indicate that repeated neuronal activation alone is sufficient to drive changes in microglia phenotype and function in the mPFC. Future studies using optogenetic and chemogenetic approaches to manipulate neural circuits need to consider microglial and other nonneuronal contributions to physiologic and behavioral outcomes. SIGNIFICANCE STATEMENT: Microglia are highly attuned to fluctuations in neuronal activity. Here we show that repeated activation of pyramidal neurons in the prefrontal cortex induces broad changes in microglia phenotype; this includes upregulation of pathways associated with microglial proliferation, microglia-neuron interactions, and lysosome induction. Our findings suggest that studies using chemogenetic or optogenetic approaches to manipulate neural circuits should be mindful of indirect effects on nonneuronal cells and their potential contribution to measured outcomes.

Molecular neurobiology of loss: a role for basolateral amygdala extracellular matrix.

Psychological loss is a common experience that erodes well-being and negatively impacts quality of life. The molecular underpinnings of loss are poorly understood. Here, we investigate the mechanisms of loss using an environmental enrichment removal (ER) paradigm in male rats. The basolateral amygdala (BLA) was identified as a region of interest, demonstrating differential Fos responsivity to ER and having an established role in stress processing and adaptation. A comprehensive multi-omics investigation of the BLA, spanning multiple cohorts, platforms, and analyses, revealed alterations in microglia and the extracellular matrix (ECM). Follow-up studies indicated that ER decreased microglia size, complexity, and phagocytosis, suggesting reduced immune surveillance. Loss also substantially increased ECM coverage, specifically targeting perineuronal nets surrounding parvalbumin interneurons, suggesting decreased plasticity and increased inhibition within the BLA following loss. Behavioral analyses suggest that these molecular effects are linked to impaired BLA salience evaluation, leading to a mismatch between stimulus and reaction intensity. These loss-like behaviors could be rescued by depleting BLA ECM during the removal period, helping us understand the mechanisms underlying loss and revealing novel molecular targets to ameliorate its impact.

Depletion of microglial BDNF increases susceptibility to the behavioral and synaptic effects of chronic unpredictable stress.

In the medial prefrontal cortex (PFC), chronic stress reduces synaptic expression of glutamate receptors, leading to decreased excitatory signaling from layer V pyramidal neurons and working memory deficits. One key element driving these changes is a reduction in brain-derived neurotrophic factor (BDNF) signaling. BDNF is a potent mediator of synaptic growth and deficient BDNF signaling has been linked to stress susceptibility. Prior studies indicated that neurons are the primary source of BDNF, but more recent work suggests that microglia are also an important source of BDNF. Adding to this, our work showed that 14 days of chronic unpredictable stress (CUS) reduced Bdnf transcript in PFC microglia, evincing its relevance in the effects of stress. To explore this further, we utilized transgenic mice with microglia-specific depletion of BDNF (Cx3cr1Cre/+:Bdnffl/fl) and genotype controls (Cx3cr1Cre/+:Bdnf+/+). In the following experiments, mice were exposed to a shortened CUS paradigm (7 days) to determine if microglial Bdnf depletion promotes stress susceptibility. Analyses of PFC microglia revealed that Cx3cr1Cre/+:Bdnffl/fl mice had shifts in phenotypic markers and gene expression. In a separate cohort, synaptoneurosomes were collected from the PFC and western blotting was performed for synaptic markers. These experiments showed that Cx3cr1Cre/+:Bdnffl/fl mice had baseline deficits in GluN2B, and that 7 days of CUS additionally reduced GluN2A levels in Cx3cr1Cre/+:Bdnffl/fl mice, but not genotype controls. Behavioral and cognitive testing showed that this coincided with exacerbated stress effects on temporal object recognition in Cx3cr1Cre/+:Bdnffl/fl mice. These results indicate that microglial BDNF promotes glutamate receptor expression in the PFC. As such, mice with deficient microglial BDNF had increased susceptibility to the behavioral and cognitive consequences of stress.

Microglial P2Y12 mediates chronic stress-induced synapse loss in the prefrontal cortex and associated behavioral consequences.

Chronic unpredictable stress (CUS) drives microglia-mediated neuronal remodeling and synapse loss in the prefrontal cortex (PFC), contributing to deficits in cognition and behavior. However, it remains unclear what mechanisms guide microglia-neuron interactions in stress. Evidence indicates that neuronal activity-dependent purinergic signaling directs microglial processes and synaptic engagement via P2Y12, a purinergic receptor exclusively expressed by microglia in the brain. Stress alters excitatory neurotransmission in the PFC, thus we aimed to determine if P2Y12 signaling promotes functional changes in microglia in chronic stress. Here we used genetic ablation of P2Y12 (P2ry12-/-) or pharmacological blockade (clopidogrel, ticagrelor) to examine the role of purinergic signaling in stress-induced microglia-neuron interaction. Multiple behavioral, physiological, and cytometric endpoints were analyzed. Deletion of P2Y12 led to a number of fundamental alterations in the PFC, including the heightened microglial number and increased dendritic spine density. Flow cytometry revealed that microglia in P2ry12-/- mice had shifts in surface levels of CX3CR1, CSF1R, and CD11b, suggesting changes in synaptic engagement and phagocytosis in the PFC. In line with this, pharmacological blockade of P2Y12 prevented CUS-induced increases in the proportion of microglia with neuronal inclusions, limited dendritic spine loss in the PFC, and attenuated alterations in stress coping behavior and working memory function. Overall, these findings indicate that microglial P2Y12 is a critical mediator of stress-induced synapse loss in the PFC and subsequent behavioral deficits.

Acyloxyacyl hydrolase regulates microglia-mediated pelvic pain.

Chronic pelvic pain conditions such as interstitial cystitis/bladder pain syndrome (IC/BPS) remain clinical and mechanistic enigmas. Microglia are resident immune cells of the central nervous system (CNS) that respond to changes in the gut microbiome, and studies have linked microglial activation to acute and chronic pain in a variety of models, including pelvic pain. We have previously reported that mice deficient for the lipase acyloxyacyl hydrolase (AOAH) develop pelvic allodynia and exhibit symptoms, comorbidities, and gut dysbiosis mimicking IC/BPS. Here, we assessed the role of AOAH in microglial activation and pelvic pain. RNAseq analyses using the ARCHS4 database and confocal microscopy revealed that AOAH is highly expressed in wild type microglia but at low levels in astrocytes, suggesting a functional role for AOAH in microglia. Pharmacologic ablation of CNS microglia with PLX5622 resulted in decreased pelvic allodynia in AOAH-deficient mice and resurgence of pelvic pain upon drug washout. Skeletal analyses revealed that AOAH-deficient mice have an activated microglia morphology in the medial prefrontal cortex and paraventricular nucleus, brain regions associated with pain modulation. Because microglia express Toll-like receptors and respond to microbial components, we also examine the potential role of dysbiosis in microglial activation. Consistent with our hypothesis of microglia activation by leakage of gut microbes, we observed increased serum endotoxins in AOAH-deficient mice and increased activation of cultured BV2 microglial cells by stool of AOAH-deficient mice. Together, these findings demonstrate a role for AOAH in microglial modulation of pelvic pain and thus identify a novel therapeutic target for IC/BPS.

The semantics of microglia activation: neuroinflammation, homeostasis, and stress.

Microglia are emerging as critical regulators of neuronal function and behavior in nearly every area of neuroscience. Initial reports focused on classical immune functions of microglia in pathological contexts, however, immunological concepts from these studies have been applied to describe neuro-immune interactions in the absence of disease, injury, or infection. Indeed, terms such as 'microglia activation' or 'neuroinflammation' are used ubiquitously to describe changes in neuro-immune function in disparate contexts; particularly in stress research, where these terms prompt undue comparisons to pathological conditions. This creates a barrier for investigators new to neuro-immunology and ultimately hinders our understanding of stress effects on microglia. As more studies seek to understand the role of microglia in neurobiology and behavior, it is increasingly important to develop standard methods to study and define microglial phenotype and function. In this review, we summarize primary research on the role of microglia in pathological and physiological contexts. Further, we propose a framework to better describe changes in microglia1 phenotype and function in chronic stress. This approach will enable more precise characterization of microglia in different contexts, which should facilitate development of microglia-directed therapeutics in psychiatric and neurological disease.

Synaptic and behavioral effects of chronic stress are linked to dynamic and sex-specific changes in microglia function and astrocyte dystrophy.

Emerging evidence indicates that males and females display different neurobiological responses to chronic stress which contribute to varied behavioral adaptations. In particular, pyramidal neurons undergo dendritic atrophy and synapse loss in the prefrontal cortex (PFC) of male, but not female, mice. Our recent work shows that chronic stress also provokes microglia-mediated neuronal remodeling, which contributes to synaptic deficits in the PFC and associated behavioral consequences in males. Separate studies indicate that chronic stress promotes astrocyte dystrophy in the PFC which is associated with behavioral despair. Notably, these prior reports focused primarily on stress effects in males. In the present studies, male and female mice were exposed to 14 or 28 days of chronic unpredictable stress (CUS) to assess molecular and cellular adaptations of microglia, astrocytes, and neurons in the medial PFC. Consistent with our recent work, male, but not female, mice displayed behavioral and cognitive deficits with corresponding perturbations of neuroimmune factors in the PFC after 14 days of CUS. Fluorescence-activated cell sorting and gene expression analyses revealed that CUS increased expression of select markers of phagocytosis in male PFC microglia. Confocal imaging in Thy1-GFP(M) mice showed that CUS reduced dendritic spine density, decreased GFAP immunolabeling, and increased microglia-mediated neuronal remodeling only in male mice. After 28 days of CUS, both male and female mice displayed behavioral and cognitive impairments. Interestingly, there were limited stress effects on neuroimmune factors and measures of microglial phagocytosis in the PFC of both sexes. Despite limited changes in neuroimmune function, reduced GFAP immunolabeling and dendritic spine deficits persisted in male mice. Further, GFAP immunolabeling and dendritic spine density remained unaltered in the PFC of females. These findings indicate that chronic stress causes sex-specific and temporally dynamic changes in microglial function which are associated with different neurobiological and behavioral adaptations. In all, these results suggest that microglia-mediated neuronal remodeling, astrocyte dystrophy, and synapse loss contribute to stress-induced PFC dysfunction and associated behavioral consequences in male mice.

Diazepam limits microglia-mediated neuronal remodeling in the prefrontal cortex and associated behavioral consequences following chronic unpredictable stress.

Chronic stress induces neuronal atrophy and synaptic loss in the medial prefrontal cortex (PFC), and this leads to behavioral and cognitive impairments. Our recent findings indicate that microglia contribute to structural remodeling of neurons via increased colony-stimulating factor (CSF)-1 in the medial PFC. Other work shows that chronic stress induces aberrant neuronal activity in the medial PFC, and that neuronal hyperactivity increases CSF1 signaling and alters microglia function. Thus, the present studies were designed to examine the role of neuronal activity in stress-induced CSF1 signaling and microglia-mediated neuronal remodeling in the medial PFC. Additional analyses probed stress effects on the dorsal hippocampus (HPC), basolateral amygdala (BLA), and somatosensory cortex (SSCTX). Mice were exposed to chronic unpredictable stress (CUS) or handled intermittently as controls, and received daily injection of vehicle or diazepam (1 mg/kg). As anticipated, diazepam attenuated CUS-induced behavioral despair and cognitive impairments. Further studies showed that diazepam normalized Csf1 and C3 mRNA in the PFC, and prevented increases in Csf1r and Cd11b in frontal cortex microglia following CUS. Stress had no effect on neuroimmune gene expression in the HPC. Confocal imaging in Thy1-GFP(M) mice demonstrated that diazepam limited microglial engulfment of neuronal elements and blocked CUS-induced dendritic spine loss in the medial PFC. Altogether, these findings indicate that modulation of chronic stress-induced neuronal activity limits microglia-mediated neuronal remodeling in the medial PFC, and subsequent behavioral and cognitive consequences.

Effects of stress on the structure and function of the medial prefrontal cortex: Insights from animal models.

Stress alters both cognitive and emotional function, and increases risk for a variety of psychological disorders, such as depression and posttraumatic stress disorder. The prefrontal cortex is critical for executive function and emotion regulation, is a target for stress hormones, and is implicated in many stress-influenced psychological disorders. Therefore, understanding how stress-induced changes in the structure and function of the prefrontal cortex are related to stress-induced changes in behavior may elucidate some of the mechanisms contributing to stress-sensitive disorders. This review focuses on data from rodent models to describe the effects of chronic stress on behaviors mediated by the medial prefrontal cortex, the effects of chronic stress on the morphology and physiology of the medial prefrontal cortex, mechanisms that may mediate these effects, and evidence for sex differences in the effects of stress on the prefrontal cortex. Understanding how stress influences prefrontal cortex and behaviors mediated by it, as well as sex differences in this effect, will elucidate potential avenues for novel interventions for stress-sensitive disorders characterized by deficits in executive function and emotion regulation.

Gonadal hormones differentially regulate sex-specific stress effects on glia in the medial prefrontal cortex.

Women are more susceptible to various stress-linked psychopathologies, including depression. Dysfunction of the medial prefrontal cortex (mPFC) has been implicated in depression, and studies indicate sex differences in stress effects on mPFC structure and function. For example, chronic stress induces dendritic atrophy in the mPFC in male rats, yet dendritic growth in females. Recent findings suggest glial pathways toward depression. Glia are highly responsive to neuronal activity and function as critical regulators of synaptic plasticity. Preclinical models demonstrate stress-induced microglial activation in mPFC in males, yet deactivation in females. By contrast, stress reduces astrocyte complexity in mPFC in male rats, whereas the effects in females are unknown. Glia possess receptors for most gonadal hormones and gonadal hormones are known to modulate neuronal activity. Thus, gonadal hormones represent a potential mechanism underlying sex differences in glia, as well as divergent stress effects. Therefore, we examined the role of gonadal hormones in sex-specific stress effects on neuronal activity (ie FosB/ ΔFosB induction) and glia in the mPFC. The findings obtained indicate greater microglial activation in mPFC in females and a greater astrocyte area in males. Basal astrocyte morphology is modulated by androgens, whereas androgens or oestrogens dampen the microglial state in males. Astrocyte morphology is associated with neuronal activity in both sexes, regardless of hormonal condition. Chronic stress induced astrocytic atrophy in males, yet hypertrophy in females, with gonadal hormones partly regulating this difference. Stress effects on microglia are oestradiol-dependent in females. Taken together, these data suggest sex-specific, gonadal hormone-dependent stress effects on astrocytes and microglia in the mPFC.

Sex Differences in Risk and Resilience: Stress Effects on the Neural Substrates of Emotion and Motivation.

Risk for stress-sensitive psychopathologies differs in men and women, yet little is known about sex-dependent effects of stress on cellular structure and function in corticolimbic regions implicated in these disorders. Determining how stress influences these regions in males and females will deepen our understanding of the mechanisms underlying sex-biased psychopathology. Here, we discuss sex differences in CRF regulation of arousal and cognition, glucocorticoid modulation of amygdalar physiology and alcohol consumption, the age-dependent impact of social stress on prefrontal pyramidal cell excitability, stress effects on the prefrontal parvalbumin system in relation to emotional behaviors, contributions of stress and gonadal hormones to stress effects on prefrontal glia, and alterations in corticolimbic structure and function after cessation of chronic stress. These studies demonstrate that, while sex differences in stress effects may be nuanced, nonuniform, and nonlinear, investigations of these differences are nonetheless critical for developing effective, sex-specific treatments for psychological disorders.

Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner.

Women are more susceptible to numerous stress-linked psychological disorders (e.g., depression) characterized by dysfunction of corticolimbic brain regions critical for emotion regulation and cognitive function. Although sparsely investigated, a number of studies indicate sex differences in stress effects on neuronal structure, function, and behaviors associated with these regions. We recently demonstrated a basal sex difference in- and differential effects of stress on- microglial activation in medial prefrontal cortex (mPFC). The resident immune cells of the brain, microglia are implicated in synaptic and dendritic plasticity, and cognitive-behavioral function. Here, we examined the effects of acute (3h/day, 1 day) and chronic (3h/day, 10 days) restraint stress on microglial density and morphology, as well as immune factor expression in orbitofrontal cortex (OFC), basolateral amygdala (BLA), and dorsal hippocampus (DHC) in male and female rats. Microglia were visualized, classified based on their morphology, and stereologically counted. Microglia-associated transcripts (CD40, iNOS, Arg1, CX3CL1, CX3CR1, CD200, and CD200R) were assessed in brain punches from each region. Expression of genes linked with cellular stress, neuroimmune state, and neuron-microglia communication varied between unstressed male and female rats in a region-specific manner. In OFC, chronic stress upregulated a wider variety of immune factors in females than in males. Acute stress increased microglia-associated transcripts in BLA in males, whereas chronic stress altered immune factor expression in BLA more broadly in females. In DHC, chronic stress increased immune factor expression in males but not females. Moreover, acute and chronic stress differentially affected microglial morphological activation state in male and female rats across all brain regions investigated. In males, chronic stress altered microglial activation in a pattern consistent with microglial involvement in stress-induced dendritic remodeling across OFC, BLA, and DHC. Together, these data suggest the potential for microglia-mediated sex differences in stress effects on neural structure, function, and behavior.

Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex.

Susceptibility to stress-linked psychological disorders, including post-traumatic stress disorder and depression, differs between men and women. Dysfunction of medial prefrontal cortex (mPFC) has been implicated in many of these disorders. Chronic stress affects mPFC in a sex-dependent manner, differentially remodeling dendritic morphology and disrupting prefrontally mediated behaviors in males and females. Chronic restraint stress induces microglial activation, reflected in altered microglial morphology and immune factor expression, in mPFC in male rats. Unstressed females exhibit increased microglial ramification in several brain regions compared to males, suggesting both heightened basal activation and a potential for sex-dependent effects of stress on microglial activation. Therefore, we assessed microglial density and ramification in the prelimbic region of mPFC, and immune-associated genes in dorsal mPFC in male and female rats following acute or chronic restraint stress. Control rats were left unstressed. On the final day of restraint, brains were collected for either qPCR or visualization of microglia using Iba-1 immunohistochemistry. Microglia in mPFC were classified as ramified, primed, reactive, or amoeboid, and counted stereologically. Expression of microglia-associated genes (MHCII, CD40, IL6, CX3CL1, and CX3CR1) was also assessed using qPCR. Unstressed females showed a greater proportion of primed to ramified microglia relative to males, alongside heightened CX3CL1-CX3CR1 expression. Acute and chronic restraint stress reduced the proportion of primed to ramified microglia and microglial CD40 expression in females, but did not significantly alter microglial activation in males. This sex difference in microglial activation could contribute to the differential effects of stress on mPFC structure and function in males versus females.