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.

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.

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.

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 agedependent 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.

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.