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.

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.

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.

miR-181a Mediates Inflammatory Gene Expression After Intracerebral Hemorrhage: An Integrated Analysis of miRNA-seq and mRNA-seq in a Swine ICH Model.

Intracerebral hemorrhage (ICH) is a severe neurological disorder with no proven treatment. Inflammation after ICH contributes to clinical outcomes, but the relevant molecular mechanisms remain poorly understood. In studies of peripheral leukocyte counts and mRNA-sequencing (mRNA-seq), our group previously reported that monocytes and Interleukin-8 (IL-8) were important contributors to post-ICH inflammation. microRNA (miRNA) are powerful regulators of gene expression and promising therapeutic targets. We now report findings from an integrated analysis of miRNA-seq and mRNA-seq in peripheral blood mononuclear cells (PBMCs) from a swine ICH model. In 10 pigs, one PBMC sample was collected immediately prior to ICH induction and a second 6 h later; miRNA-seq and mRNA-seq were completed for each sample. An aggregate score calculation determined which miRNA regulated the differentially expressed mRNA. Networks of molecular interactions were generated for the combined miRNA/target mRNA. A total of 227 miRNA were identified, and 46 were differentially expressed after ICH (FDR < 0.05). The anti-inflammatory miR-181a was decreased post-ICH, and it was the most highly connected miRNA in the miRNA/mRNA bioinformatic network analysis. miR-181a has interconnected pathophysiology with IL-8 and monocytes; in prior studies, we found that IL-8 and monocytes contributed to post-ICH inflammation and ICH clinical outcome, respectively. miR-181a was a significant mediator of post-ICH inflammation and is promising for further study, including as a potential therapeutic target. This investigation also demonstrated feasible methodology for miRNA-seq/mRNA-seq analysis in swine that is innovative, and with unique challenges, compared with transcriptomics research in more established species.

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.

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.

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.

Intracerebral Hemorrhage Induces Inflammatory Gene Expression in Peripheral Blood: Global Transcriptional Profiling in Intracerebral Hemorrhage Patients.

To perform global transcriptome profiling using RNA-seq in the peripheral blood of intracerebral hemorrhage (ICH) patients. In 11 patients with ICH, peripheral blood was collected within 24 h of symptom onset or last known well, and a second blood draw occurred 72 h (±6) after the first. RNA-seq identified differentially expressed genes (DEGs) between the first and second samples. Biological pathway enrichment analysis was performed with Ingenuity® Pathway Analysis (IPA). A total of 16,640 genes were identified and 218 were significant DEGs after ICH (false discovery rate <0.1). IPA identified 97 disease and functional categories that were significantly upregulated (z-score >2) post-ICH; 46 categories were specifically related to immune cell activation, 22 to general cellular activation processes, and 4 to other inflammation-related responses. In the canonical pathway and network analysis, inflammatory mediators of particular importance included interleukin-8, NF-κB, ERK1/2, and members of the integrin class. ICH induced peripheral blood gene expression at 72 to 96 h compared with 0 to 24 h from symptom onset. DEGs that were highly expressed included those related to inflammation and activation of the immune response. Further research is needed to determine whether these changes affect outcomes and may represent new therapeutic targets.

A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake.

The melanocortin system is a brain circuit that influences energy balance by regulating energy intake and expenditure. In addition, the brain-melanocortin system controls adipose tissue metabolism to optimize fuel mobilization and storage. Specifically, increased brain-melanocortin signaling or negative energy balance promotes lipid mobilization by increasing sympathetic nervous system input to adipose tissue. In contrast, calorie-independent mechanisms favoring energy storage are less understood. Here, we demonstrate that reduction of brain-melanocortin signaling actively promotes fat mass gain by activating the lipogenic program and adipocyte and endothelial cell proliferation in white fat depots independently of caloric intake via efferent nerve fibers conveyed by the common hepatic branch of the vagus nerve. Those vagally regulated obesogenic signals also contribute to the fat mass gain following chronic high-fat diet feeding. These data reveal a physiological mechanism whereby the brain controls energy stores that may contribute to increased susceptibility to obesity.

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.