Ketogenic diet alters microglial morphology and changes the hippocampal lipidomic profile distinctively in stress susceptible versus resistant male mice upon repeated social defeat.

Psychological stress confers an increased risk for several diseases including psychiatric conditions. The susceptibility to psychological stress is modulated by various factors, many of them being modifiable lifestyle choices. The ketogenic diet (KD) has emerged as a dietary regime that offers positive outcomes on mood and health status. Psychological stress and elevated inflammation are common features of neuropsychiatric disorders such as certain types of major depressive disorder. KD has been attributed anti-inflammatory properties that could underlie its beneficial consequences on the brain and behavior. Microglia are the main drivers of inflammation in the central nervous system. They are known to respond to both dietary changes and psychological stress, notably by modifying their production of cytokines and relationships among the brain parenchyma. To assess the interactions between KD and the stress response, including effects on microglia, we examined adult male mice on control diet (CD) versus KD that underwent 10 days of repeated social defeat (RSD) or remained non-stressed (controls; CTRLs). Through a social interaction test, stressed mice were classified as susceptible (SUS) or resistant (RES) to RSD. The mouse population fed a KD tended to have a higher proportion of individuals classified as RES following RSD. Microglial morphology and ultrastructure were then analyzed in the ventral hippocampus CA1, a brain region known to present structural alterations as a response to psychological stress. Distinct changes in microglial soma and arborization linked to the KD, SUS and RES phenotypes were revealed. Ultrastructural analysis by electron microscopy showed a clear reduction of cellular stress markers in microglia from KD fed animals. Furthermore, ultrastructural analysis showed that microglial contacts with synaptic elements were reduced in the SUS compared to the RES and CTRL groups. Hippocampal lipidomic analyses lastly identified a distinct lipid profile in SUS animals compared to CTRLs. These key differences, combined with the distinct microglial responses to diet and stress, indicate that unique metabolic changes may underlie the stress susceptibility phenotypes. Altogether, our results reveal novel mechanisms by which a KD might improve the resistance to psychological stress.

Ketogenic diet changes microglial morphology and the hippocampal lipidomic profile differently in stress susceptible versus resistant male mice upon repeated social defeat.

Psychological stress confers an increased risk for several diseases including psychiatric conditions. The susceptibility to psychological stress is modulated by various factors, many of them being modifiable lifestyle choices. The ketogenic diet (KD) has emerged as a dietary regime that offers positive outcomes on mood and health status. Psychological stress and elevated inflammation are common features of neuropsychiatric disorders such as certain types of major depressive disorder. KD has been attributed anti-inflammatory properties that could underlie its beneficial consequences on the brain and behavior. Microglia are the main drivers of inflammation in the central nervous system. They are known to respond to both dietary changes and psychological stress, notably by modifying their production of cytokines and relationships among the brain parenchyma. To assess the interactions between KD and the stress response, including effects on microglia, we examined adult male mice on control diet (CD) versus KD that underwent 10 days of repeated social defeat (RSD) or remained non-stressed (controls; CTRLs). Through a social interaction test, stressed mice were classified as susceptible (SUS) or resistant (RES) to RSD. The mouse population fed a KD tended to have a higher proportion of individuals classified as RES following RSD. Microglial morphology and ultrastructure were then analyzed in the ventral hippocampus CA1, a brain region known to present structural alterations as a response to psychological stress. Distinct changes in microglial soma and arborization linked to the KD, SUS and RES phenotypes were revealed. Ultrastructural analysis by electron microscopy showed a clear reduction of cellular stress markers in microglia from KD fed animals. Furthermore, ultrastructural analysis showed that microglial contacts with synaptic elements were reduced in the SUS compared to the RES and CTRL groups. Hippocampal lipidomic analyses lastly identified a distinct lipid profile in SUS animals compared to CTRLs. These key differences, combined with the distinct microglial responses to diet and stress, indicate that unique metabolic changes may underlie the stress susceptibility phenotypes. Altogether, our results reveal novel mechanisms by which a KD might improve the resistance to psychological stress.

Computed tomography angiographic study of surgical anatomy of thyroid arteries: Clinical implications in neck dissection.

BACKGROUND: The course and variations of thyroid arteries must be understood by surgeons to prevent bleeding during operative procedures of the thyroid gland. There is limited scientific literature regarding the radiological anatomy of thyroid arteries in this geographical area, the Garhwal region of Sub-Himalayan belt, which is considered to be the endemic belt of goiter. Computed tomography angiography provides a three-dimensional orientation of the vascular and surgical anatomy of the entire cervical region. AIM: To estimate the proportion of variation in origin of thyroid arteries using Computed Tomography Angiography. METHODS: Using Computed Tomography Angiography, the presence and origin of the superior thyroid artery, inferior thyroid artery, and thyroid ima artery were observed and assessed. RESULTS: Out of total 210 subjects, superior thyroid artery was seen to be emerging from external carotid artery in 77.1% cases. The artery was found to be originating at the level of bifurcation of common carotid artery in 14.3% cases, whereas in 8.6% cases, it emerged as a direct branch of the common carotid artery. Similarly, the inferior thyroid artery was observed to be emerging from thyrocervical trunk, subclavian artery and vertebral artery in 95.7% cases, 3.3% and 1% cases, respectively. Thyroid ima artery was also reported in a subject, arising from the brachiocephalic trunk. CONCLUSION: To avoid vascular injuries, excessive and uncontrollable bleeding, intra-operative difficulties, and post-operative issues, it is imperative for surgeons to be aware of the course and variations of thyroid arteries.

Amyloid ß Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's Disease.

Several microglia-expressed genes have emerged as top risk variants for Alzheimer's disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (Aß), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent upon age and disease progression and is more prominent in the hippocampus in mice and humans. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD-laden microglia exhibited a deficit in Aß phagocytosis. Unbiased lipidomic analysis identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation. We demonstrate that DGAT2, a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting DGAT2 improved microglial uptake of Aß. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

Surgical Anatomy of Vertebral Artery in Relation to Atlantoaxial Instrumentation: A Cadaveric Study.

BACKGROUND: With the advent of pedicle screws and advanced instrumentation techniques, internal fixation and stabilization of upper cervical vertebrae are possible in fractures of an axis. However, the proximity of vertebral arteries (VAs) poses a unique challenge to surgeons during these procedures and can result in profound physical impairment to patients. Cadaveric studies contributing to fine anatomical details necessitate conducting such studies. METHODS: After receiving due ethical permission, this descriptive cross-sectional study was carried out on 10 cadavers in the department of Anatomy, All India Institute of Medical Science (AIIMS) Rishikesh. Twenty VAs were dissected along their course, and measurements of parameters related to the axis and atlas vertebra were noted. RESULTS: The length of the pre-osseous segment related to the axis (VAX-1) on the right and left sides were from 3.8 to 14.5 mm (7.48±3.88 mm) and 4.46 to 10.5mm (6.94±2.01mm) respectively. The length of the osseous segment related to the axis (VAX-2) on the right side and left sides were from 6.82 to 31 mm (17.9±7.84mm) and 7.35 to 20 mm (15.6±4.53). The osseous segment of the VA related to the axis (VAX-2) shows genu (bend), which extends to a variable distance towards the midline. The mean distance of VA genu from the midline of the axis vertebral body on the right and left sides was 15.6mm and 17.5 mm, respectively. The percentage of superior articular facet (SAF) surface area of the axis occupied by the VA was 25-50% in nine and 50-75% in 11 cadavers, reflecting incomplete occupancy. CONCLUSION: The study suggests that for instrumentation of the axis vertebra in the midline, the minimum distance between the genu of both sides of VA segments, related to an osseous segment of the axis (VAX-2) and medial extent of the VA groove of the atlas, should be considered as a safe zone to minimize inadvertent VA injury. During atlantoaxial fixation through a posterior approach in interarticular, pars, and pedicle screws, the surgical anatomy of the VA in relation to the osseous segment of the VA within the transverse process of the axis should be kept in mind to avoid inadvertent VA injury.

Cadaveric Study on the Morphology and Morphometry of Heart Papillary Muscles.

Introduction A normal atrioventricular valve complex of the heart consists of the atrioventricular (A-V) ring, cusps, chordae tendineae, and papillary muscles. The right ventricle contains three while the left ventricle contains only two papillary muscles, which are named according to their location. A thorough understanding of the normal anatomy as well as possible variations can help surgeons in various corrective surgeries involving papillary muscles. Material & methods The study included 50 formalin-preserved hearts procured from human cadavers of unknown age and cause of death. The number of papillary muscles along with their shape, size, and pattern were noted separately for each ventricle. Data were analyzed using SPSS Version 21.0 (IBM Corp., Armonk, NY). Results The left and right ventricles contained two and three papillary muscles, respectively, in all the hearts. In the right ventricles, conical shape and the single base and divided apex (SBDA) pattern were found to be most prevalent. Anterior papillary muscles exhibited the mean length of 12.71±3.81 and 16.41±4.33 in the right and left ventricles, respectively. Similarly, posterior papillary muscles exhibited a mean length of 12.40±3.03 and 14.64±3.92 in the right and left ventricles, respectively. Both differences were found to be statistically significant Conclusion For the appropriate functioning of valves, both anatomical and mechanical coherence of the papillary muscles is required. A very keen understanding of this valvular complex is thus essential for anatomists, physiologists, and cardiologists to deal with normal as well as pathological valvular conditions.

A Comparative Morphometric and Histological Study of Human Fetus and Fetal Pancreas in Hyperglycemic and Normoglycemic Mothers.

BACKGROUND: A significant percentage of pregnancies with gestational diabetes mellitus (GDM) has been found to result in the delivery of macrosomic babies. The current study intends to highlight the correlation between maternal diabetes and fetal parameters as well as the histogenesis of the fetal pancreas in humans. MATERIALS AND METHODS: The study included thirty aborted fetuses, categorized into seven groups according to their gestational age. Morphometric analysis of fetal parameters and fetal pancreas was done, and the values were compared within different gestational age groups. Pancreatic tissue was processed, stained with Hematoxylin & Eosin, and examined. A comparison was then made between fetuses with and without gestational diabetes.  Results: All the fetal biometrics as well as pancreatic parameters showed greater numeric values in mothers with GDM as compared to the controls of the same gestational age groups. However, the difference was not statistically significant. Histogenesis in such fetuses revealed GDM-related hyperplasia of islets of Langerhans. CONCLUSION: A timely diagnosis of GDM is thus of paramount significance due to its potential implications so that appropriate interventions can be done on time, to improve the overall outcome.

Capillary-associated microglia regulate vascular structure and function through PANX1-P2RY12 coupling in mice.

Microglia are brain-resident immune cells with a repertoire of functions in the brain. However, the extent of their interactions with the vasculature and potential regulation of vascular physiology has been insufficiently explored. Here, we document interactions between ramified CX3CR1 + myeloid cell somata and brain capillaries. We confirm that these cells are bona fide microglia by molecular, morphological and ultrastructural approaches. Then, we give a detailed spatio-temporal characterization of these capillary-associated microglia (CAMs) comparing them with parenchymal microglia (PCMs) in their morphological activities including during microglial depletion and repopulation. Molecularly, we identify P2RY12 receptors as a regulator of CAM interactions under the control of released purines from pannexin 1 (PANX1) channels. Furthermore, microglial elimination triggered capillary dilation, blood flow increase, and impaired vasodilation that were recapitulated in P2RY12-/- and PANX1-/- mice suggesting purines released through PANX1 channels play important roles in activating microglial P2RY12 receptors to regulate neurovascular structure and function.

Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice.

Chronic psychological stress is one of the most important triggers and environmental risk factors for neuropsychiatric disorders. Chronic stress can influence all organs via the secretion of stress hormones, including glucocorticoids by the adrenal glands, which coordinate the stress response across the body. In the brain, glucocorticoid receptors (GR) are expressed by various cell types including microglia, which are its resident immune cells regulating stress-induced inflammatory processes. To study the roles of microglial GR under normal homeostatic conditions and following chronic stress, we generated a mouse model in which the GR gene is depleted in microglia specifically at adulthood to prevent developmental confounds. We first confirmed that microglia were depleted in GR in our model in males and females among the cingulate cortex and the hippocampus, both stress-sensitive brain regions. Then, cohorts of microglial-GR depleted and wild-type (WT) adult female mice were housed for 3 weeks in a standard or stressful condition, using a chronic unpredictable mild stress (CUMS) paradigm. CUMS induced stress-related behavior in both microglial-GR depleted and WT animals as demonstrated by a decrease of both saccharine preference and progressive ratio breakpoint. Nevertheless, the hippocampal microglial and neural mechanisms underlying the adaptation to stress occurred differently between the two genotypes. Upon CUMS exposure, microglial morphology was altered in the WT controls, without any apparent effect in microglial-GR depleted mice. Furthermore, in the standard environment condition, GR depleted-microglia showed increased expression of pro-inflammatory genes, and genes involved in microglial homeostatic functions (such as Trem2, Cx3cr1 and Mertk). On the contrary, in CUMS condition, GR depleted-microglia showed reduced expression levels of pro-inflammatory genes and increased neuroprotective as well as anti-inflammatory genes compared to WT-microglia. Moreover, in microglial-GR depleted mice, but not in WT mice, CUMS led to a significant reduction of CA1 long-term potentiation and paired-pulse ratio. Lastly, differences in adult hippocampal neurogenesis were observed between the genotypes during normal homeostatic conditions, with microglial-GR deficiency increasing the formation of newborn neurons in the dentate gyrus subgranular zone independently from stress exposure. Together, these findings indicate that, although the deletion of microglial GR did not prevent the animal's ability to respond to stress, it contributed to modulating hippocampal functions in both standard and stressful conditions, notably by shaping the microglial response to chronic stress.

Microglia contribute to social behavioral adaptation to chronic stress.

Microglial activation has been regarded mainly as an exacerbator of stress response, a common symptom in psychiatric disorders. This study aimed to determine whether microglia contribute to adaptive response of the brain and behavior toward stress using a mild and adaptive stress model - chronic restraint stress (CRS) - with wild type (WT) and CX3CR1-GFP (CX3CR1[G]) mice and human schizophrenia patients' data. Our results revealed that CRS did not exacerbate anxiety and depressive-like behaviors, but instead strengthened social dominance and short-term spatial learning in WT mice. Compared to WT and CX3CR1(+/G) heterozygous mice, CX3CR1(G/G) homozygotes were subordinate in social interaction before and after CRS. Microglia in WT mice underwent a series of region-specific changes involving their phagocytosis of presynaptic vesicular glutamate transporter 2 protein, contacts with synaptic elements, CD206+ microglial proportion, and gene expressions such as Cx3cr1. By contrast, CX3CR1-deficient microglia showed decreased CD206+ while increased MHCII+ subpopulations and hypo-ramification in the hippocampus, as well as sensitized polarization and morphological change in response to CRS. Furthermore, CD206+ microglial abundancy was positively correlated with social dominancy and microglial ramification in CX3CR1-GFP mice. Moreover, CX3CR1 mRNA level was reduced in CRS-treated mouse brains and showed a smaller interactome with other brain genes in the dorsal-lateral prefrontal cortices of patients with schizophrenia. Our findings overall highlight microglia and its receptor CX3CR1 as key contributors in regulation of social behavioral adaptation to chronic stress.

A Comparative Biology of Microglia Across Species.

Microglia are unique brain-resident, myeloid cells. They have received growing interest for their implication in an increasing number of neurodevelopmental, acute injury, and neurodegenerative disorders of the central nervous system (CNS). Fate-mapping studies establish microglial ontogeny from the periphery during development, while recent transcriptomic studies highlight microglial identity as distinct from other CNS cells and peripheral myeloid cells. This evidence for a unique microglial ontogeny and identity raises questions regarding their identity and functions across species. This review will examine the available evidence for microglia in invertebrate and vertebrate species to clarify similarities and differences in microglial identity, ontogeny, and physiology across species. This discussion highlights conserved and divergent microglial properties through evolution. Finally, we suggest several interesting research directions from an evolutionary perspective to adequately understand the significance of microglia emergence. A proper appreciation of microglia from this perspective could inform the development of specific therapies geared at targeting microglia in various pathologies.

Sex Differences of Microglia and Synapses in the Hippocampal Dentate Gyrus of Adult Mouse Offspring Exposed to Maternal Immune Activation.

Schizophrenia is a psychiatric disorder affecting ∼1% of humans worldwide. It is earlier and more frequently diagnosed in men than woman, and men display more pronounced negative symptoms together with greater gray matter reductions. Our previous findings utilizing a maternal immune activation (mIA) mouse model of schizophrenia revealed exacerbated anxiety-like behavior and sensorimotor gating deficits in adult male offspring that were associated with increased microglial reactivity and inflammation in the hippocampal dentate gyrus (DG). However, both male and female adult offspring displayed stereotypy and impairment of sociability. We hypothesized that mIA may lead to sex-specific alterations in microglial pruning activity, resulting in abnormal synaptic connectivity in the DG. Using the same mIA model, we show in the current study sex-specific differences in microglia and synapses within the DG of adult offspring. Specifically, microglial levels of cluster of differentiation (CD)68 and CD11b were increased in mIA-exposed females. Sex-specific differences in excitatory and inhibitory synapse densities were also observed following mIA. Additionally, inhibitory synaptic tone was increased in DG granule cells of both males and females, while changes in excitatory synaptic transmission occurred only in females with mIA. These findings suggest that phagocytic and complement pathways may together contribute to a sexual dimorphism in synaptic pruning and neuronal dysfunction in mIA, and may propose sex-specific therapeutic targets to prevent schizophrenia-like behaviors.

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice.

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain.

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

Ultrastructural evidence of microglial heterogeneity in Alzheimer's disease amyloid pathology.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by the deposition of extracellular fibrillar amyloid ß (fΑß) and the intracellular accumulation of neurofibrillary tangles. As AD progresses, Aß drives a robust and prolonged inflammatory response via its recognition by microglia, the brain's immune cells. Microglial reactivity to fAß plaques may impair their normal surveillance duties, facilitating synaptic loss and neuronal death, as well as cognitive decline in AD. METHODS: In the current study, we performed correlative light, transmission, and scanning electron microscopy to provide insights into microglial structural and functional heterogeneity. We analyzed microglial cell bodies and processes in areas containing fAß plaques and neuronal dystrophy, dystrophy only, or appearing healthy, among the hippocampus CA1 of 14-month-old APPSwe-PS1Δe9 mice versus wild-type littermates. RESULTS: Our quantitative analysis revealed that microglial cell bodies in the AD model mice were larger and displayed ultrastructural signs of cellular stress, especially nearby plaques. Microglial cell bodies and processes were overall less phagocytic in AD model mice. However, they contained increased fibrillar materials and non-empty inclusions proximal to plaques. Microglial cell bodies and processes in AD model mice also displayed reduced association with extracellular space pockets that contained debris. In addition, microglial processes in healthy subregions of AD model mice encircled synaptic elements more often compared with plaque-associated processes. These observations in mice were qualitatively replicated in post-mortem hippocampal samples from two patients with AD (Braak stage 5). CONCLUSION: Together, our findings identify at the ultrastructural level distinct microglial transformations common to mouse and human in association with amyloid pathology.

Microglia in the developing prefrontal cortex of rats show dynamic changes following neonatal disconnection of the ventral hippocampus.

Impaired ventral hippocampal (VH)-prefrontal cortex (PFC) connectivity is implicated in many cognitive and behavioral disorders. Excitotoxic neonatal VH (nVH) lesion in rat pups has been shown to induce synaptic pruning in the PFC as well as behavioral changes of relevance to developmental neuropsychiatric disorders. In the current study, we hypothesized that microglia, immune cells required for proper brain development and plasticity, may play a role in the development of abnormal behaviors in the nVH-lesioned animals. Ibotenic acid-induced nVH lesion was induced in postnatal day (P)7 male rats. Developmental changes in microglial density, morphology, ultrastructure and gene expression were analyzed in the PFC at P20 and P60. Our results revealed increased microglial reactivity and phagocytic activity in the lesioned rats at P20. Increased mRNA levels of C3 and C1q, complement molecules involved in synaptic pruning, were concomitantly observed. Diminished, but maintained, microglial reactivity and reduced antioxidative defenses were identified in lesioned rats at P60. Behavioral deficits were significantly reduced in the post-pubertal rats by suppressing microglial reactivity by a one-week minocycline treatment immediately after the lesion, These results suggest that early-life disconnection of the VH has long-lasting consequences for microglial functions in the connected structures. Alterations in microglia may underlie synaptic reorganization and behavioral deficits observed following neonatal VH disconnection.

Reduced Microglial Activity and Enhanced Glutamate Transmission in the Basolateral Amygdala in Early CNS Autoimmunity.

Emotional dysfunction is common in multiple sclerosis (MS) patients and in mouse models of MS, including experimental autoimmune encephalomyelitis (EAE); however, the etiology of these behaviors is poorly understood. To identify CNS changes associated with these behaviors, we focused on the basolateral amygdala (BLA) because of its central role in the regulation of emotional behavior. Whole-cell recordings were performed in the principal neurons of the BLA in early EAE, before demyelination, T-cell invasion, and motor dysfunction. EAE female mice displayed increased frequency of mEPSCs, with no alteration in amplitude or evoked EPSC paired-pulse ratio compared with controls. We found an increase in the AMPA-NMDA ratio and dendritic spine density, indicating increased numbers of glutamatergic synapses. We saw similar electrophysiological changes in BLA principal neurons after microglia were either inactivated (minocycline) or depleted (Mac1-Saporin) in the BLA. Microglia regulate synapses through pruning, directed by complement protein 3 (C3) expression. C3 was downregulated in the BLA in EAE. Ultrastructural analysis of microglia revealed more complex ramifications and reduced extracellular digestion of cellular elements. We also observed reduced IBA-1 and CD68 staining and lack of proinflammatory cytokine expression in the amygdala. Thus, early EAE is a state of microglial "deactivation" associated with reduced synaptic pruning. This contrasts with the prototypic microglial activation commonly associated with inflammatory CNS disease. Additionally, these data support a role for the acquired immune system to influence both neuronal and microglial function in early CNS autoimmunity.SIGNIFICANCE STATEMENT Microglia help regulate synaptic homeostasis, but there has been little evidence for how this might be important in neuroinflammatory diseases. The data from this study reveal increased synaptic activity and spine density in early stages of experimental autoimmune encephalomyelitis (an animal model of multiple sclerosis) in the basolateral amygdala, a nucleus important in the types of behavioral changes we have previously described. These electrophysiological and morphological effects occurred without significant elevation of local inflammatory cytokines or local demyelination. Unexpectedly, in the context of inflammatory state, we found that microglia were "deactivated." This study provides strong evidence for a link between microglial activity and synaptic function; the conclusions contrast with the generally accepted view that microglia are activated in inflammatory disease.

Chronic stress as a risk factor for Alzheimer's disease: Roles of microglia-mediated synaptic remodeling, inflammation, and oxidative stress.

Microglia are the predominant immune cells of the central nervous system (CNS) that exert key physiological roles required for maintaining CNS homeostasis, notably in response to chronic stress, as well as mediating synaptic plasticity, learning and memory. The repeated exposure to stress confers a higher risk of developing neurodegenerative diseases including sporadic Alzheimer's disease (AD). While microglia have been causally linked to amyloid beta (Aß) accumulation, tau pathology, neurodegeneration, and synaptic loss in AD, they were also attributed beneficial roles, notably in the phagocytic elimination of Aß. In this review, we discuss the interactions between chronic stress and AD pathology, overview the roles played by microglia in AD, especially focusing on chronic stress as an environmental risk factor modulating their function, and present recently-described microglial phenotypes associated with neuroprotection in AD. These microglial phenotypes observed under both chronic stress and AD pathology may provide novel opportunities for the development of better-targeted therapeutic interventions.

Delta Opioid Receptor Signaling Promotes Resilience to Stress Under the Repeated Social Defeat Paradigm in Mice.

The adaptation to chronic stress is highly variable across individuals. Resilience to stress is a complex process recruiting various brain regions and neurotransmitter systems. The aim of this study was to investigate the involvement of endogenous opioid enkephalin (ENK) signaling in the development of stress resilience in mice. The translational model of repeated social defeat (RSD) stress was selected to mimic the unpredictable disruptions of daily life and induce resilience or vulnerability to stress. As in humans, adult C57BL/6J mice demonstrated a great variability in their response to stress under this paradigm. A social interaction (SI) test was used to discriminate between the phenotypes of resilience or vulnerability to stress. After social defeat, the expression levels of ENK mRNA and their delta opioid receptors (DOPr) were quantified in the basolateral amygdala (BLA) and BLA-target areas by in situ hybridization. In this manner, ENK mRNA levels were found to decrease in the BLA and those of DOPr in the ventral hippocampus (HPC) CA1 of vulnerable mice only. Stimulating the DOPr pathway during social defeat by pharmacological treatment with the nonpeptide, selective DOPr agonist SNC80 further induced a resilient phenotype in a majority of stressed animals, with the proportion of resilient ones increasing from 33% to 58% of the total population. Ultrastructural analyses additionally revealed a reduction of oxidative stress markers in the pyramidal cells and interneurons of the ventral HPC CA1 upon SNC80 treatment, thus proposing a mechanism by which ENK-DOPr signaling may prevent the deleterious effects of chronic social stress.

Environmental stimuli shape microglial plasticity in glioma.

In glioma, microglia and infiltrating macrophages are exposed to factors that force them to produce cytokines and chemokines, which contribute to tumor growth and to maintaining a pro-tumorigenic, immunosuppressed microenvironment. We demonstrate that housing glioma-bearing mice in enriched environment (EE) reverts the immunosuppressive phenotype of infiltrating myeloid cells, by modulating inflammatory gene expression. Under these conditions, the branching and patrolling activity of myeloid cells is increased, and their phagocytic activity is promoted. Modulation of gene expression depends on interferon-(IFN)-γ produced by natural killer (NK) cells. This modulation disappears in mice depleted of NK cells or lacking IFN-γ, and was mimicked by exogenous interleukin-15 (IL-15). Further, we describe a key role for brain-derived neurotrophic factor (BDNF) that is produced in the brain of mice housed in EE, in mediating the expression of IL-15 in CD11b+ cells. These data define novel mechanisms linking environmental cues to the acquisition of a pro-inflammatory, anti-tumor microenvironment in mouse brain.