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.

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.

On-chip estimation of hematocrit level for diagnosing anemic conditions by Impedimetric techniques.

An on-chip device has been fabricated on Si platform for precise estimation of the hematocrit (Hct) level of human blood with rapid turn out. Impedance/capacitance spectroscopy and current-voltage (I-V) measurements have been employed to observe the variation of electrical parameters of erythrocyte suspensions with varying Hct level. Experimentally obtained values of capacitance, impedance and conductance with Hct level suggests a linear variation. Current-time measurement has also been performed to ensure the susceptibility of red blood cells under a fixed external electric field for certain duration of time. The online real time sample analyzes have also been performed by the device connected with an embedded electronic circuit interfaced with a laptop through appropriate software. This has enabled the development of a novel Si-chip based compact point-of-care (POC) diagnosis system for Hct level estimation by measuring the dielectric/capacitive variation of an erythrocyte cell suspension. The relevant performance parameters of such a compact system including range, resolution, limit of detection and throughput have also been evaluated.

Dark microglia: A new phenotype predominantly associated with pathological states.

The past decade has witnessed a revolution in our understanding of microglia. These immune cells were shown to actively remodel neuronal circuits, leading to propose new pathogenic mechanisms. To study microglial implication in the loss of synapses, the best pathological correlate of cognitive decline across chronic stress, aging, and diseases, we recently conducted ultrastructural analyses. Our work uncovered the existence of a new microglial phenotype that is rarely present under steady state conditions, in hippocampus, cerebral cortex, amygdala, and hypothalamus, but becomes abundant during chronic stress, aging, fractalkine signaling deficiency (CX3 CR1 knockout mice), and Alzheimer's disease pathology (APP-PS1 mice). Even though these cells display ultrastructural features of microglia, they are strikingly distinct from the other phenotypes described so far at the ultrastructural level. They exhibit several signs of oxidative stress, including a condensed, electron-dense cytoplasm and nucleoplasm making them as "dark" as mitochondria, accompanied by a pronounced remodeling of their nuclear chromatin. Dark microglia appear to be much more active than the normal microglia, reaching for synaptic clefts, while extensively encircling axon terminals and dendritic spines with their highly ramified and thin processes. They stain for the myeloid cell markers IBA1 and GFP (in CX3 CR1-GFP mice), and strongly express CD11b and microglia-specific 4D4 in their processes encircling synaptic elements, and TREM2 when they associate with amyloid plaques. Overall, these findings suggest that dark microglia, a new phenotype that we identified based on their unique properties, could play a significant role in the pathological remodeling of neuronal circuits, especially at synapses.

Histone deacetylase inhibition facilitates massed pattern-induced synaptic plasticity and memory.

Massed training is less effective for long-term memory formation than the spaced training. The role of acetylation in synaptic plasticity and memory is now well established. However, the role of this important protein modification in synaptic plasticity induced by massed pattern of stimulation or memory induced by massed training is not well understood. Here we show that increasing the level of acetylation enhances long-term potentiation induced by massed pattern of high frequency stimulation. Furthermore, enhancing acetylation level facilitates long-term memory by massed training. Thus, increasing acetylation level facilitates synaptic plasticity and memory by massed patterns.

Identification of Biomarkers for Arsenicosis Employing Multiple Kernel Learning Embedded Multiobjective Swarm Intelligence.

Arsenic is a carcinogen, and long-term exposure to it may result in the development of multi-organ disease. Understanding the underlying intricate molecular network of toxicity and carcinogenicity is crucial for identifying a small set of differentially expressed biomarker genes to predict the risk of the exposed population. In this paper, a multiple kernel learning (MKL) embedded multi-objective swarm intelligence technique has been proposed to identify the candidate biomarker genes from the transcriptomic profile of arsenicosis samples. To achieve the optimal classification accuracy along with the minimum number of genes, a multi-objective random spatial local best particle swarm optimization (MO-RSplbestPSO) has been utilized. The proposed MO-RSplbestPSO also guides the multiple kernel learning mechanism which provides data specific classification. The proposed computational framework has been applied to the developed whole genome DNA microarray prepared using blood samples collected from a specific arsenic exposed area of the Indian state of West Bengal. A set of twelve biomarker genes, with four novel genes, are successfully identified for the classification of exposure to arsenic and its subcategories, which can be used as future prognostic biomarkers for screening of arsenic exposed populations. Also, the biological significance of each gene is detailed to delineate the complex molecular networking and mode of toxicity.

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.

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.

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.

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.

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.

lBest-HS algorithm based concurrent L1 adaptive control for non-Linear systems.

This paper proposes a new approach for designing stable hybrid L1 adaptive controller employing lbest topological model of harmony search (HS) algorithm. The proposed design approach guarantees desired stability and simultaneously provides satisfactory tracking performance for a class of non-linear systems. The design methodology for the controller utilizes the meta-heuristic global search feature of HS algorithm and the local search phenomenon of L1 adaptive control strategy in tandem. The paper also analytically describes the superiority of lbest topological model compared to the conventional HS algorithm in terms of convergence phenomenon, when hybridized with L1 adaptive control. The proposed hybrid control methodology has been implemented for benchmark simulation case studies and real-time experimentation to demonstrate its usefulness.

Calming Neurons with a Microglial Touch.

In vivo two-photon imaging of microglia in the intact brain has revealed that microglia constantly survey neuronal soma. Research over the past decade and a recent paper by Cserép et al. published in Science are now uncovering the nature, mechanisms, and consequences of these interactions in health and injury.

On-Device Reliability Assessment and Prediction of Missing Photoplethysmographic Data Using Deep Neural Networks.

Photoplethysmographic (PPG) measurements from ambulatory subjects may suffer from unreliability due to body movements and missing data segments due to loosening of sensor. This paper describes an on-device reliability assessment from PPG measurements using a stack denoising autoencoder (SDAE) and multilayer perceptron neural network (MLPNN). The missing segments were predicted by a personalized convolutional neural network (CNN) and long-short term memory (LSTM) model using a short history of the same channel data. Forty sets of volunteers' data, consisting of equal share of healthy and cardiovascular subjects were used for validation and testing. The PPG reliability assessment model (PRAM) achieved over 95% accuracy for correctly identifying acceptable PPG beats out of total 5000 using expert annotated data. Disagreement with experts' annotation was nearly 3.5%. The missing segment prediction model (MSPM) achieved a root mean square error (RMSE) of 0.22, and mean absolute error (MAE) of 0.11 for 40 missing beats prediction using only four beat history from the same channel PPG. The two models were integrated in a standalone device based on quad-core ARM Cortex-A53, 1.2 GHz, with 1 GB RAM, with 130 MB memory requirement and latency ∼0.35 s per beat prediction with a 30 s frame. The present method also provides improved performance with published works on PPG quality assessment and missing data prediction using two public datasets, CinC and MIMIC-II under PhysioNet.

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.

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.

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.

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.

Microglia under psychosocial stressors along the aging trajectory: Consequences on neuronal circuits, behavior, and brain diseases.

Mounting evidence indicates the importance of microglia for proper brain development and function, as well as in complex stress-related neuropsychiatric disorders and cognitive decline along the aging trajectory. Considering that microglia are resident immune cells of the brain, a homeostatic maintenance of their effector functions that impact neuronal circuitry, such as phagocytosis and secretion of inflammatory factors, is critical to prevent the onset and progression of these pathological conditions. However, the molecular mechanisms by which microglial functions can be properly regulated under healthy and pathological conditions are still largely unknown. We aim to summarize recent progress regarding the effects of psychosocial stress and oxidative stress on microglial phenotypes, leading to neuroinflammation and impaired microglia-synapse interactions, notably through our own studies of inbred mouse strains, and most importantly, to discuss about promising therapeutic strategies that take advantage of microglial functions to tackle such brain disorders in the context of adult psychosocial stress or aging-induced oxidative stress.

Dark microglia: Why are they dark?

Using transmission electron microscopy (TEM) we recently characterized a microglial phenotype that is induced by chronic stress, fractalkine receptor deficiency, aging, or Alzheimer disease pathology. These 'dark' microglia appear overly active compared with the normal microglia, reaching for synaptic clefts, and extensively engulfing pre-synaptic axon terminals and post-synaptic dendritic spines. From these findings we hypothesized that dark microglia could be specifically implicated in the pathological remodeling of neuronal circuits, which impairs learning, memory, and other essential cognitive functions. In the present addendum we further discuss about the possible causes of their dark appearance under TEM.