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.

Adolescent high fat diet alters the transcriptional response of microglia in the prefrontal cortex in response to stressors in both male and female mice.

Both obesity and high fat diets (HFD) have been associated with an increase in inflammatory gene expression within the brain. Microglia play an important role in early cortical development and may be responsive to HFD, particularly during sensitive windows, such as adolescence. We hypothesized that HFD during adolescence would increase proinflammatory gene expression in microglia at baseline and potentiate the microglial stress response. Two stressors were examined, a physiological stressor [lipopolysaccharide (LPS), IP] and a psychological stressor [15 min restraint (RST)]. From 3 to 7 weeks of age, male and female mice were fed standard control diet (SC, 20% energy from fat) or HFD (60% energy from fat). On P49, 1 h before sacrifice, mice were randomly assigned to either stressor exposure or control conditions. Microglia from the frontal cortex were enriched using a Percoll density gradient and isolated via fluorescence-activated cell sorting (FACS), followed by RNA expression analysis of 30 genes (27 target genes, three housekeeping genes) using Fluidigm, a medium throughput qPCR platform. We found that adolescent HFD induced sex-specific transcriptional response in cortical microglia, both at baseline and in response to a stressor. Contrary to our hypothesis, adolescent HFD did not potentiate the transcriptional response to stressors in males, but rather in some cases, resulted in a blunted or absent response to the stressor. This was most apparent in males treated with LPS. However, in females, potentiation of the LPS response was observed for select proinflammatory genes, including Tnfa and Socs3. Further, HFD increased the expression of Itgam, Ikbkb, and Apoe in cortical microglia of both sexes, while adrenergic receptor expression (Adrb1 and Adra2a) was changed in response to stressor exposure with no effect of diet. These data identify classes of genes that are uniquely affected by adolescent exposure to HFD and different stressor modalities in males and females.

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.

Influence of Nutritional Status and Leptin Action on Agrp and Pomc Co-Expression in Hypothalamic Melanocortin System Neurons.

OBJECTIVES: The control of energy balance relies on the counterbalancing release of neuropeptides encoded by the pro-opiomelanocortin (Pomc) and agouti-related protein (Agrp) genes, expressed by 2 distinct neuronal populations of the arcuate (ARC) nucleus of the hypothalamus. Although largely segregated, single-cell resolution techniques demonstrate some degree of co-expression. We studied whether challenges to the control of energy balance influence the degree of Agrp and Pomc co-expression in ARC melanocortin neurons. METHODS: We used fluorescence-activated cell sorting followed by quantitative polymerase chain reaction and fluorescent in situ hybridization to measure Pomc and Agrp gene co-expression in POMC or AGRP neurons in response to (1) acute or chronic calorie restriction, or (2) obesity due to loss of leptin receptor expression or chronic high-fat diet feeding in male mice. RESULTS: Melanocortin ARC neurons of fed mice exhibited low, yet detectable, levels of Pomc and Agrp gene co-expression. Calorie restriction significantly increased and decreased total Agrp and Pomc expression, respectively, and reduced the expression of Pomc relative to Agrp in AGRP neurons. Leptin-deficient db/db mice showed increased total Agrp levels and decreased Pomc expression, as well as significantly increased Agrp expression relative to Pomc in POMC neurons. Expression or co-expression levels did not differ between diet-induced obese mice and lean controls. CONCLUSIONS: Changes in Agrp and Pomc co-expression within POMC and AGRP neurons following chronic calorie restriction or in db/db mice suggest an additional mechanism to further suppress the melanocortin signaling during conditions of severely reduced leptin action.

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.

Integrating neuroimmune systems in the neurobiology of depression.

Data from clinical and preclinical studies indicate that immune dysregulation, specifically of inflammatory processes, is associated with symptoms of major depressive disorder (MDD). In particular, increased levels of circulating pro-inflammatory cytokines and concomitant activation of brain-resident microglia can lead to depressive behavioural symptoms. Repeated exposure to psychological stress has a profound impact on peripheral immune responses and perturbs the function of brain microglia, which may contribute to neurobiological changes underlying MDD. Here, we review these findings and discuss ongoing studies examining neuroimmune mechanisms that influence neuronal activity as well as synaptic plasticity. Interventions targeting immune-related cellular and molecular pathways may benefit subsets of MDD patients with immune dysregulation.

Ketamine rapidly reverses stress-induced impairments in GABAergic transmission in the prefrontal cortex in male rodents.

Dysfunction of medial prefrontal cortex (mPFC) in association with imbalance of inhibitory and excitatory neurotransmission has been implicated in depression. However, the precise cellular mechanisms underlying this imbalance, particularly for GABAergic transmission in the mPFC, and the link with the rapid acting antidepressant ketamine remains poorly understood. Here we determined the influence of chronic unpredictable stress (CUS), an ethologically validated model of depression, on synaptic markers of GABA neurotransmission, and the influence of a single dose of ketamine on CUS-induced synaptic deficits in mPFC of male rodents. The results demonstrate that CUS decreases GABAergic proteins and the frequency of inhibitory post synaptic currents (IPSCs) of layer V mPFC pyramidal neurons, concomitant with depression-like behaviors. In contrast, a single dose of ketamine can reverse CUS-induced deficits of GABA markers, in conjunction with reversal of CUS-induced depressive-like behaviors. These findings provide further evidence of impairments of GABAergic synapses as key determinants of depressive behavior and highlight ketamine-induced synaptic responses that restore GABA inhibitory, as well as glutamate neurotransmission.

Glucocorticoid receptor antagonism prevents microglia-mediated neuronal remodeling and behavioral despair following chronic unpredictable stress.

Synaptic deficits and neuronal dystrophy in the prefrontal cortex (PFC) are linked to behavioral and cognitive symptoms in depressed individuals. Preclinical studies indicate that chronic stress causes synaptic deficits on pyramidal neurons in the PFC that contribute to behavioral and cognitive impairments. Our recent work shows that chronic stress provokes microglia-mediated neuronal remodeling via neuronal colony stimulating factor (CSF)-1 signaling, leading to synaptic deficits and depressive-like behaviors. Other reports indicate that elevated corticosterone causes pyramidal neuron atrophy and microglia activation in the medial PFC, implicating glucocorticoid signaling in microglia-mediated neuronal remodeling following chronic stress. In this study, male mice were exposed to chronic unpredictable stress (CUS) and received daily administration of glucocorticoid receptor antagonist RU486 (25 mg/kg, i.p.). As expected, CUS exposure caused adrenal hypertrophy and elevated plasma corticosterone levels. Glucocorticoid receptor blockade prevented behavioral despair and cognitive impairments following CUS. Moreover, RU486 administration diminished CUS-induced CSF1 signaling in the PFC and reduced markers of phagocytosis on purified microglia. Confocal imaging in Thy1-GFP(M) mice showed that CUS increased microglia-mediated neuronal remodeling, and RU486 administration attenuated microglial engulfment of neuronal elements and prevented dendritic spine density deficits on pyramidal neurons following CUS. These results demonstrate that chronic stress-induced glucocorticoid signaling promotes CSF1 signaling and microglia-mediated neuronal remodeling in the medial PFC, which contributes to development of behavioral despair and cognitive impairments. This study presents primary evidence that neuroendocrine responses engage neuron-microglia interactions in the PFC; further implicating microglia in stress-induced neuronal remodeling, PFC dysfunction, and associated behavioral consequences.

Intracerebral hemorrhage induces monocyte-related gene expression within six hours: Global transcriptional profiling in swine ICH.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Our prior research identified a significant association with monocyte level and ICH mortality. To advance our understanding, we sought to identify gene expression after ICH using a swine model to test the hypothesis that ICH would induce peripheral blood mononuclear cell (PBMC) gene expression. In 10 pigs with ICH, two PBMC samples were drawn from each with the first immediately prior to ICH induction and the second six hours later. RNA-seq was performed with subsequent bioinformatics analysis using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Ingenuity® Pathway Analysis (IPA). There were 182 significantly upregulated and 153 significantly down-regulated differentially expressed genes (DEGs) after ICH. Consistent with findings in humans, significant GO and KEGG pathways were primarily related to inflammation and the immune response. Five genes, all upregulated post-ICH and known to be associated with monocyte activation, were repeatedly DEGs in the significant KEGG pathways: CD14, TLR4, CXCL8, IL-18, and CXCL2. In IPA, the majority of upregulated disease/function categories were related to inflammation and immune cell activation. TNF and LPS were the most significantly activated upstream regulators, and ERK was the most highly connected node in the top network. ICH induced changes in PBMC gene expression within 6 h of onset related to inflammation, the immune response, and, more specifically, monocyte activation. Further research is needed to determine if these changes affect outcomes and may represent new therapeutic targets.

Correction of MFG-E8 Resolves Inflammation and Promotes Cutaneous Wound Healing in Diabetes.

Milk fat globule epidermal growth factor-factor 8 (MFG-E8) is a peripheral glycoprotein that acts as a bridging molecule between the macrophage and apoptotic cells, thus executing a pivotal role in the scavenging of apoptotic cells from affected tissue. We have previously reported that apoptotic cell clearance activity or efferocytosis is compromised in diabetic wound macrophages. In this work, we test the hypothesis that MFG-E8 helps resolve inflammation, supports angiogenesis, and accelerates wound closure. MFG-E8(-/-) mice displayed impaired efferocytosis associated with exaggerated inflammatory response, poor angiogenesis, and wound closure. Wound macrophage-derived MFG-E8 was recognized as a critical driver of wound angiogenesis. Transplantation of MFG-E8(-/-) bone marrow to MFG-E8(+/+) mice resulted in impaired wound closure and compromised wound vascularization. In contrast, MFG-E8(-/-) mice that received wild-type bone marrow showed improved wound closure and improved wound vascularization. Hyperglycemia and exposure to advanced glycated end products inactivated MFG-E8, recognizing a key mechanism that complicates diabetic wound healing. Diabetic db/db mice suffered from impaired efferocytosis accompanied with persistent inflammation and slow wound closure. Topical recombinant MFG-E8 induced resolution of wound inflammation, improvements in angiogenesis, and acceleration of closure, upholding the potential of MFG-E8-directed therapeutics in diabetic wound care.

Optogenetic stimulation of infralimbic PFC reproduces ketamine's rapid and sustained antidepressant actions.

Ketamine produces rapid and sustained antidepressant actions in depressed patients, but the precise cellular mechanisms underlying these effects have not been identified. Here we determined if modulation of neuronal activity in the infralimbic prefrontal cortex (IL-PFC) underlies the antidepressant and anxiolytic actions of ketamine. We found that neuronal inactivation of the IL-PFC completely blocked the antidepressant and anxiolytic effects of systemic ketamine in rodent models and that ketamine microinfusion into IL-PFC reproduced these behavioral actions of systemic ketamine. We also found that optogenetic stimulation of the IL-PFC produced rapid and long-lasting antidepressant and anxiolytic effects and that these effects are associated with increased number and function of spine synapses of layer V pyramidal neurons. The results demonstrate that ketamine infusions or optogenetic stimulation of IL-PFC are sufficient to produce long-lasting antidepressant behavioral and synaptic responses similar to the effects of systemic ketamine administration.

Neuroinflammatory Dynamics Underlie Memory Impairments after Repeated Social Defeat.

Repeated social defeat (RSD) is a murine stressor that recapitulates key physiological, immunological, and behavioral alterations observed in humans exposed to chronic psychosocial stress. Psychosocial stress promotes prolonged behavioral adaptations that are associated with neuroinflammatory signaling and impaired neuroplasticity. Here, we show that RSD promoted hippocampal neuroinflammatory activation that was characterized by proinflammatory gene expression and by microglia activation and monocyte trafficking that was particularly pronounced within the caudal extent of the hippocampus. Because the hippocampus is a key area involved in neuroplasticity, behavior, and cognition, we hypothesize that stress-induced neuroinflammation impairs hippocampal neurogenesis and promotes cognitive and affective behavioral deficits. We show here that RSD caused transient impairments in spatial memory recall that resolved within 28 d. In assessment of neurogenesis, the number of proliferating neural progenitor cells (NPCs) and the number of young, developing neurons were not affected initially after RSD. Nonetheless, the neuronal differentiation of NPCs that proliferated during RSD was significantly impaired when examined 10 and 28 d later. In addition, social avoidance, a measure of depressive-like behavior associated with caudal hippocampal circuitry, persisted 28 d after RSD. Treatment with minocycline during RSD prevented both microglia activation and monocyte recruitment. Inhibition of this neuroinflammatory activation in turn prevented impairments in spatial memory after RSD but did not prevent deficits in neurogenesis nor did it prevent the persistence of social avoidance behavior. These findings show that neuroinflammatory activation after psychosocial stress impairs spatial memory performance independent of deficits in neurogenesis and social avoidance. SIGNIFICANCE STATEMENT: Repeated exposure to stress alters the homeostatic environment of the brain, giving rise to various cognitive and mood disorders that impair everyday functioning and overall quality of life. The brain, previously thought of as an immune-privileged organ, is now known to communicate extensively with the peripheral immune system. This brain-body communication plays a significant role in various stress-induced inflammatory conditions, also characterized by psychological impairments. Findings from this study implicate neuroimmune activation rather than impaired neurogenesis in stress-induced cognitive deficits. This idea opens up possibilities for novel immune interventions in the treatment of cognitive and mood disturbances, while also adding to the complexity surrounding the functional implications of adult neurogenesis.

Knockdown of interleukin-1 receptor type-1 on endothelial cells attenuated stress-induced neuroinflammation and prevented anxiety-like behavior.

Interleukin-1ß (IL-1ß) is an inflammatory cytokine that plays a prominent role in stress-induced behavioral changes. In a model of repeated social defeat (RSD), elevated IL-1ß expression in the brain was associated with recruitment of primed macrophages that were necessary for development of anxiety-like behavior. Moreover, microglia activation and anxiety-like behavior associated with RSD did not occur in IL-1 receptor type-1 knock-out (IL-1R1(KO)) mice. Therefore, the objective of this study was to examine the role of IL-1 signaling in RSD-induced macrophage trafficking to the brain and anxiety-like behavior. Initial studies revealed that RSD did not increase circulating myeloid cells in IL-1R1(KO) mice, resulting in limited macrophage trafficking to the brain. In addition, IL-1R1(KO) bone marrow-chimera mice showed that IL-1R1 expression was essential for macrophage trafficking into the brain. To differentiate cellular mediators of stress-induced IL-1 signaling, endothelial-specific IL-1R1 knock-down (eIL-1R1kd) mice were used. Both wild-type (WT) and eIL-1R1kd mice had increased circulating monocytes, recruitment of macrophages to the brain, and altered microglia activation after RSD. Nonetheless, RSD-induced expression of IL-1ß, TNF-α, and IL-6 mRNA in brain CD11b(+) cells was attenuated in eIL-1R1kd mice compared with WT. Moreover, anxiety-like behavior did not develop in eIL-1R1kd mice. Collectively, these findings demonstrated that there was limited RSD-induced priming of myeloid cells in IL-1R1(KO) mice and disrupted propagation of neuroinflammatory signals in the brain of eIL-1R1kd mice. Furthermore, these data showed that transduction of IL-1 signaling by endothelial cells potentiates stress-induced neuroinflammation and promotes anxiety-like behavior.

Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants.

Stress-induced recruitment of bone marrow-derived monocytes to the brain promotes anxiety-like behavior.

Social stress is associated with altered immunity and higher incidence of anxiety-related disorders. Repeated social defeat (RSD) is a murine stressor that primes peripheral myeloid cells, activates microglia, and induces anxiety-like behavior. Here we show that RSD-induced anxiety-like behavior corresponded with an exposure-dependent increase in circulating monocytes (CD11b(+)/SSC(lo)/Ly6C(hi)) and brain macrophages (CD11b(+)/SSC(lo)/CD45(hi)). Moreover, RSD-induced anxiety-like behavior corresponded with brain region-dependent cytokine and chemokine responses involved with myeloid cell recruitment. Next, LysM-GFP(+) and GFP(+) bone marrow (BM)-chimeric mice were used to determine the neuroanatomical distribution of peripheral myeloid cells recruited to the brain during RSD. LysM-GFP(+) mice showed that RSD increased recruitment of GFP(+) macrophages to the brain and increased their presence within the perivascular space (PVS). In addition, RSD promoted recruitment of GFP(+) macrophages into the PVS and parenchyma of the prefrontal cortex, amygdala, and hippocampus of GFP(+) BM-chimeric mice. Furthermore, mice deficient in chemokine receptors associated with monocyte trafficking [chemokine receptor-2 knockout (CCR2(KO)) or fractalkine receptor knockout (CX3CR1(KO))] failed to recruit macrophages to the brain and did not develop anxiety-like behavior following RSD. Last, RSD-induced macrophage trafficking was prevented in BM-chimeric mice generated with CCR2(KO) or CX3CR1(KO) donor cells. These findings indicate that monocyte recruitment to the brain in response to social stress represents a novel cellular mechanism that contributes to the development of anxiety.

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.

ATG5 (autophagy related 5) in microglia controls hippocampal neurogenesis in Alzheimer disease.

Macroautophagy/autophagy is the intracellular degradation process of cytoplasmic content and damaged organelles. Autophagy is strongly associated with the progression of Alzheimer disease (AD). Microglia are brain-resident macrophages, and recent studies indicate that autophagy in microglia protects neurons from neurodegeneration. Postnatal neurogenesis, the generation of new neurons from adult neural stem cells (NSCs), is impaired in AD patients as well as in AD animal models. However, the extent to which microglial autophagy influences adult NSCs and neurogenesis in AD animal models has not been studied. Here, we showed that conditional knock out (cKO) of Atg5 (autophagy related 5) in microglia inhibited postnatal neurogenesis in the dentate gyrus (DG) of the hippocampus, but not in the subventricular zone (SVZ) of a 5×FAD mouse model. Interestingly, the protection of neurogenesis by Atg5 in microglia was only observed in female AD mice. To confirm the roles of autophagy in microglia for postnatal hippocampal neurogenesis, we generated additional cKO mice to delete autophagy essential genes Rb1cc1 or Atg14 in microglia. However, these rb1cc1 cKO and atg14 cKO mice did not exhibit neurogenesis defects in the context of a female AD mouse model. Last, we used the CSF1R antagonist to deplete ATG5-deficient microglia and this intervention restored neurogenesis in the hippocampus of 5×FAD mice. These results indicate that microglial ATG5 is essential to maintain postnatal hippocampal neurogenesis in a mouse model of AD. Our findings further support the notion that ATG5 in microglia supports NSC health and may prevent neurodegeneration.Abbreviations: 5×FAD: familial Alzheimer disease; Aß: ß-amyloid; AD: Alzheimer disease; AIF1: allograft inflammatory factor 1; ATG: autophagy related; BrdU: 5-bromo-2'-deoxyuridine; CA: Cornu Ammonis; cKO: conditional knock out; CSF1R: colony stimulating factor 1 receptor; Ctrl: control; DCX: doublecortin; DG: dentate gyrus; GFAP: glial fibrillary acidic protein; GZ: granular zone; H&E: hematoxylin and eosin; IF: immunofluorescence; LD: lipid droplet; LDAM: lipid droplets accumulated microglia; LPS: lipopolysaccharides; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; NSCs: neural stem cells; RB1CC1: RB1-inducible coiled-coil 1; SOX2: SRY (sex determining region Y)-box 2; SGZ: subgranular zone; SVZ: subventricular zone; WT: wild type.

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.

ß-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a ß-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1ß and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a ß-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of ß-adrenergic and IL-1 receptors.