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.

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.

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.

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.

How Stress Shapes Neuroimmune Function: Implications for the Neurobiology of Psychiatric Disorders.

Chronic stress causes physiological and hormonal adaptations that lead to neurobiological consequences and behavioral and cognitive impairments. In particular, chronic stress has been shown to drive reduced neurogenesis and altered synaptic plasticity in brain regions that regulate mood and motivation. The neurobiological and behavioral effects of stress resemble the pathophysiology and symptoms observed in psychiatric disorders, suggesting that there are similar underlying mechanisms. Accumulating evidence indicates that neuroimmune systems, particularly microglia, have a critical role in regulating the neurobiology of stress. Preclinical models indicate that chronic stress provokes changes in microglia phenotype and increases inflammatory cytokine signaling, which affects neuronal function and leads to synaptic plasticity deficits and impaired neurogenesis. More recent work has shown that microglia can also phagocytose neuronal elements and contribute to structural remodeling of neurons in response to chronic stress. In this review we highlight work by the Duman research group (as well as others) that has revealed how chronic stress shapes neuroimmune function and, in turn, how inflammatory mediators and microglia contribute to the neurobiological effects of chronic stress. We also provide considerations to engage the therapeutic potential of neuroimmune systems, with the goal of improving treatment for psychiatric disorders.

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.

GABA interneurons are the cellular trigger for ketamine's rapid antidepressant actions.

A single subanesthetic dose of ketamine, an NMDA receptor (NMDAR) antagonist, produces rapid and sustained antidepressant actions in depressed patients, addressing a major unmet need for the treatment of mood disorders. Ketamine produces a rapid increase in extracellular glutamate and synaptic formation in the prefrontal cortex, but the initial cellular trigger that initiates this increase and ketamine's behavioral actions has not been identified. To address this question, we used a combination of viral shRNA and conditional mutation to produce cell-specific knockdown or deletion of a key NMDAR subunit, GluN2B, implicated in the actions of ketamine. The results demonstrated that the antidepressant actions of ketamine were blocked by GluN2B-NMDAR knockdown on GABA (Gad1) interneurons, as well as subtypes expressing somatostatin (Sst) or parvalbumin (Pvalb), but not glutamate principle neurons in the medial prefrontal cortex (mPFC). Further analysis of GABA subtypes showed that cell-specific knockdown or deletion of GluN2B in Sst interneurons blocked or occluded the antidepressant actions of ketamine and revealed sex-specific differences that are associated with excitatory postsynaptic currents on mPFC principle neurons. These findings demonstrate that GluN2B-NMDARs on GABA interneurons are the initial cellular trigger for the rapid antidepressant actions of ketamine and show sex-specific adaptive mechanisms to GluN2B modulation.

Prefrontal cortex interneurons display dynamic sex-specific stress-induced transcriptomes.

γ-aminobutyric acid (GABA) inhibitory interneurons play a key role in efferent and afferent control of principle neuron activity in the prefrontal cortex (PFC), thereby regulating signal integrity of cognitive and behavioral processes. Recent evidence suggests that specific subtypes of interneurons in the PFC mediate stress-induced depressive-like behaviors. Abnormalities of GABA interneurons, particularly the somatostatin (human, SST; mouse, Sst) subtype, have been reported in postmortem brains of depressed subjects and include sex differences that could explain the increased incidence of depression in women. Here, we analyze the transcriptional profiles and the effects of chronic stress in males vs. females on GABA interneuron subtypes in the PFC. Using Sst- and Parvalbumin-fluorescence tagged reporter mice and fluorescence-activated cell sorting (FACS) combined with RNA sequencing, we identify distinct transcriptome profiles for these interneuron subtypes in the medial PFC. Based on evidence that SST interneurons are altered in depression, we then determined the effects of chronic stress on this interneuron subtype. Chronic stress causes significant dysregulation of several key pathways, including sex-specific differences in the Sst interneuron profiles. The transcriptional pathways altered by chronic stress in males overlap with enriched pathways in non-stressed females. These changes occurred predominantly in decreased expression of elongation initiation factor 2 (EIF2) signaling, suggesting that dysfunction of the translational machinery of SST interneurons could be critical to the development of depressive-like behaviors in males. In addition, SST interneurons from females exposed to chronic stress show dysregulation of different, growth factor signaling pathways.

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.

Role of Neuronal VEGF Signaling in the Prefrontal Cortex in the Rapid Antidepressant Effects of Ketamine.

OBJECTIVE: The N-methyl-d-aspartate receptor antagonist ketamine produces rapid and sustained antidepressant actions even in patients with treatment-resistant depression. Vascular endothelial growth factor (VEGF) has been implicated in the effects of conventional monoamine-based antidepressants, but the role of VEGF in the rapid antidepressant actions of ketamine remains unclear. The authors examined whether neuronal VEGF signaling in the medial prefrontal cortex (mPFC) mediates the rapid antidepressant actions of ketamine. METHODS: The authors used a combination of approaches, including conditional, neuron-specific knockout of VEGF or its receptor, Flk-1; antibody neutralization; viral-mediated knockdown of Flk-1; and pharmacological inhibitors. Further in vitro and in vivo experiments were performed to examine whether neuronal VEGF signaling was required for the neurotrophic and synaptogenic actions of ketamine that underlie its behavioral actions. RESULTS: The behavioral actions of systemic ketamine are blocked by forebrain excitatory neuron-specific deletion of either VEGF or Flk-1 or by intra-mPFC infusion of a VEGF neutralizing antibody. Moreover, intra-mPFC infusions of VEGF are sufficient to produce rapid ketamine-like behavioral actions, and these effects are blocked by neuron-specific Flk-1 deletion. The results also show that local knockdown of Flk-1 in mPFC excitatory neurons in adulthood blocks the behavioral effects of systemic ketamine. Moreover, inhibition of neuronal VEGF signaling blocks the neurotrophic and synaptogenic effects of ketamine. CONCLUSIONS: Together, these findings indicate that neuronal VEGF-Flk-1 signaling in the mPFC plays an essential role in the antidepressant actions of ketamine.

Stress-Induced Neuronal Colony Stimulating Factor 1 Provokes Microglia-Mediated Neuronal Remodeling and Depressive-like Behavior.

BACKGROUND: Chronic stress exposure causes neuronal atrophy and synaptic deficits in the medial prefrontal cortex (PFC), contributing to development of anxiety- and depressive-like behaviors. Concomitantly, microglia in the PFC undergo morphological and functional changes following stress exposure, suggesting that microglia contribute to synaptic deficits underlying behavioral consequences. METHODS: Male and female mice were exposed to chronic unpredictable stress (CUS) to examine the role of neuron-microglia interactions in the medial PFC during development of anxiety- and depressive-like behaviors. Thy1-GFP-M mice were used to assess microglia-mediated neuronal remodeling and dendritic spine density in the medial PFC. Viral-mediated knockdown of neuronal colony stimulating factor 1 (CSF1) was used to modulate microglia function and behavioral consequences after CUS. RESULTS: CUS promoted anxiety- and depressive-like behaviors that were associated with increased messenger RNA levels of CSF1 in the PFC. Increased CSF1 messenger RNA levels were also detected in the postmortem dorsolateral PFC of individuals with depression. Moreover, microglia isolated from the frontal cortex of mice exposed to CUS show elevated CSF1 receptor expression and increased phagocytosis of neuronal elements. Notably, functional alterations in microglia were more pronounced in male mice compared with female mice. These functional changes in microglia corresponded with reduced dendritic spine density on pyramidal neurons in layer 1 of the medial PFC. Viral-mediated knockdown of neuronal CSF1 in the medial PFC attenuated microglia-mediated neuronal remodeling and prevented behavioral deficits caused by CUS. CONCLUSIONS: These findings revealed that stress-induced elevations in neuronal CSF1 provokes microglia-mediated neuronal remodeling in the medial PFC, contributing to synaptic deficits and development of anxiety- and depressive-like behavior.

Persistent Increase in Microglial RAGE Contributes to Chronic Stress-Induced Priming of Depressive-like Behavior.

BACKGROUND: Chronic stress-induced inflammatory responses occur in part via danger-associated molecular pattern (DAMP) molecules, such as high mobility group box 1 protein (HMGB1), but the receptor(s) underlying DAMP signaling have not been identified. METHODS: Microglia morphology and DAMP signaling in enriched rat hippocampal microglia were examined during the development and expression of chronic unpredictable stress (CUS)-induced behavioral deficits, including long-term, persistent changes after CUS. RESULTS: The results show that CUS promotes significant morphological changes and causes robust upregulation of HMGB1 messenger RNA in enriched hippocampal microglia, an effect that persists for up to 6 weeks after CUS exposure. This coincides with robust and persistent upregulation of receptor for advanced glycation end products (RAGE) messenger RNA, but not toll-like receptor 4 in hippocampal microglia. CUS also increased surface expression of RAGE protein on hippocampal microglia as determined by flow cytometry and returned to basal levels 5 weeks after CUS. Importantly, exposure to short-term stress was sufficient to increase RAGE surface expression as well as anhedonic behavior, reflecting a primed state that results from a persistent increase in RAGE messenger RNA expression. Further evidence for DAMP signaling in behavioral responses is provided by evidence that HMGB1 infusion into the hippocampus was sufficient to cause anhedonic behavior and by evidence that RAGE knockout mice were resilient to stress-induced anhedonia. CONCLUSIONS: Together, the results provide evidence of persistent microglial HMGB1-RAGE expression that increases vulnerability to depressive-like behaviors long after chronic stress exposure.

Circuit and synaptic mechanisms of repeated stress: Perspectives from differing contexts, duration, and development.

The current review is meant to synthesize research presented as part of a symposium at the 2016 Neurobiology of Stress workshop in Irvine California. The focus of the symposium was "Stress and the Synapse: New Concepts and Methods" and featured the work of several junior investigators. The presentations focused on the impact of various forms of stress (altered maternal care, binge alcohol drinking, chronic social defeat, and chronic unpredictable stress) on synaptic function, neurodevelopment, and behavioral outcomes. One of the goals of the symposium was to highlight the mechanisms accounting for how the nervous system responds to stress and their impact on outcome measures with converging effects on the development of pathological behavior. Dr. Kevin Bath's presentation focused on the impact of disruptions in early maternal care and its impact on the timing of hippocampus maturation in mice, finding that this form of stress drove accelerated synaptic and behavioral maturation, and contributed to the later emergence of risk for cognitive and emotional disturbance. Dr. Scott Russo highlighted the impact of chronic social defeat stress in adolescent mice on the development and plasticity of reward circuity, with a focus on glutamatergic development in the nucleus accumbens and mesolimbic dopamine system, and the implications of these changes for disruptions in social and hedonic response, key processes disturbed in depressive pathology. Dr. Kristen Pleil described synaptic changes in the bed nuclei of the stria terminalis that underlie the behavioral consequences of allostatic load produced by repeated cycles of alcohol binge drinking and withdrawal. Dr. Eric Wohleb and Dr. Ron Duman provided new data associating decreased mammalian target of rapamycin (mTOR) signaling and neurobiological changes in the synapses in response to chronic unpredictable stress, and highlighted the potential for the novel antidepressant ketamine to rescue synaptic and behavioral effects. In aggregate, these presentations showcased how divergent perspectives provide new insights into the ways in which stress impacts circuit development and function, with implications for understanding emergence of affective pathology.

Dynamic cross-talk between microglia and peripheral monocytes underlies stress-induced neuroinflammation and behavioral consequences.

Psychological stress promotes the development and recurrence of anxiety and depressive behavioral symptoms. Basic and clinical research indicates that stress exposure can influence the neurobiology of mental health disorders through dysregulation of neuroimmune systems. Consistent with this idea several studies show that repeated stress exposure causes microglia activation and recruitment of peripheral monocytes to the brain contributing to development of anxiety- and depressive-like behavior. Further studies show that stress-induced re-distribution of peripheral monocytes leads to stress-sensitized neuroimmune responses and recurrent anxiety-like behavior. These stress-associated immune changes are important because brain resident and peripheral immune cells contribute to physiological processes that support neuroplasticity. Thus, perturbations in neuroimmune function can lead to impaired neuronal responses and synaptic plasticity deficits that underlie behavioral symptoms of mental health disorders. In this review we discuss recent advances in neuroimmune regulation of behavior and summarize studies showing that stress-induced microglia activation and monocyte trafficking in the brain contribute to the neurobiology of mental health disorders.

Molecular and Cellular Mechanisms of Rapid-Acting Antidepressants Ketamine and Scopolamine.

Major depressive disorder (MDD) is a prevalent neuropsychiatric disease that causes profound social and economic burdens. The impact of MDD is compounded by the limited therapeutic efficacy and delay of weeks to months of currently available medications. These issues highlight the need for more efficacious and faster-acting treatments to alleviate the burdens of MDD. Recent breakthroughs demonstrate that certain drugs, including ketamine and scopolamine, produce rapid and long-lasting antidepressant effects in MDD patients. Moreover, preclinical work has shown that the antidepressant actions of ketamine and scopolamine in rodent models are caused by an increase of extracellular glutamate, elevated BDNF, activation of the mammalian target of rapamycin complex 1 (mTORC1) cascade, and increased number and function of spine synapses in the prefrontal cortex (PFC). Here we review studies showing that both ketamine and scopolamine elicit rapid antidepressant effects through converging molecular and cellular mechanisms in the PFC. In addition, we discuss evidence that selective antagonists of NMDA and muscarinic acetylcholine (mACh) receptor subtypes (i.e., NR2B and M1-AChR) in the PFC produce comparable antidepressant responses. Furthermore, we discuss evidence that ketamine and scopolamine antagonize inhibitory interneurons in the PFC leading to disinhibition of pyramidal neurons and increased extracellular glutamate that promotes the rapid antidepressant responses to these agents. Collectively, these studies indicate that specific NMDA and mACh receptor subtypes on GABAergic interneurons are promising targets for novel rapid-acting antidepressant therapies.