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Psychological stress is the major risk factor for major depressive disorder. Sustained stress causes changes in behaviour, brain connectivity and in its cells and organelles. Resilience to stress is understood as the ability to recover from stress in a positive way or the resistance to the negative effects of psychological stress. Microglia, the resident immune cells of the brain, are known players of stress susceptibility, but less is known about their role in stress resilience and the cellular changes involved. Ultrastructural analysis has been a useful tool in the study of microglia and their function across contexts of health and disease. Despite increased access to electron microscopy, the interpretation of electron micrographs remains much less accessible. In this review, we will first present microglia and the concepts of psychological stress susceptibility and resilience. Afterwards, we will describe ultrastructural analysis, notably of microglia, as a readout to study the mechanisms underlying psychological stress resilience. Lastly, we will cover nutritional ketosis as a therapeutic intervention that was shown to be effective in promoting psychological stress resilience as well as modifying microglial function and ultrastructure.

Abstract Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage triggers a variety of secondary damage mechanisms at the injury site which significantly contribute to a larger lesion size and increased functional damage. Inflammatory mechanisms which directly involve both microglia (MG) and monocyte-derived macrophages (MDM) play important roles in the post-injury processes, including inflammation and debris clearing. In the current study, we investigated changes in the structure and function of MG/MDM in the injured spinal cord of adult female mice, 7 days after a thoracic contusion SCI. With the use of chip mapping scanning electron microscopy, which allows to image large samples at the nanoscale, we performed an ultrastructural comparison of MG/MDM located near the lesion vs adjacent regions to provide novel insights into the mechanisms at play post-injury. We found that MG/MDM located near the lesion had more mitochondria overall, including mitochondria with and without morphological alterations, and had a higher proportion of altered mitochondria. MG/MDM near the lesion also showed an increased number of phagosomes, including phagosomes containing myelin and partiallydigested materials. MG/MDM near the injury interacted differently with the spinal cord parenchyma, as shown by their reduced number of direct contacts with synaptic elements, axon terminals and dendritic spines. In this study, we characterized the ultrastructural changes of MG/MDM in response to spinal cord tissue damage in mice, uncovering changes in phagocytic activity, mitochondrial ultrastructure, and inter-cellular interactions within the spinal cord parenchyma.

Abstract A diverse heterogeneity of microglial cells was previously described in Alzheimer’s disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aβ) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aβ plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed.

Abstract Background Microglia participate in the immune response upon central nervous system (CNS) infections. However, the role of these cells during herpes simplex encephalitis (HSE) has not been fully characterized. We sought to identify different microglia/microglia-like cells and describe the potential mechanisms and signaling pathways involved during HSE. Methods The transcriptional response of CD11b+ immune cells, including microglia/microglia-like cells, was investigated using single-cell RNA sequencing (scRNA-seq) on cells isolated from the ventral posterolateral nucleus (VPL)-enriched thalamic regions of C57BL/6 N mice intranasally infected with herpes simplex virus-1 (HSV-1) (6 × 105 PFUs/20 µl). We further performed scanning electronic microscopy (SEM) analysis in VPL regions on day 6 post-infection (p.i.) to provide insight into microglial functions. Results We describe a novel microglia-like transcriptional response associated with a rare cell population (7% of all analyzed cells), named “in transition” microglia/microglia-like cells in HSE. This new microglia-like transcriptional signature, found in the highly infected thalamic regions, was enriched in specific genes (Retnlg, Cxcr2, Il1f9) usually associated with neutrophils. Pathway analysis of this cell-type transcriptome showed increased NLRP3-inflammasome-mediated interleukin IL-1β production, promoting a pro-inflammatory response. These cells' increased expression of viral transcripts suggests that the distinct “in transition” transcriptome corresponds to the intrinsic antiviral immune signaling of HSV-1-infected microglia/microglia-like cells in the thalamus. In accordance with this phenotype, we observed several TMEM119+/IBA-I+ microglia/microglia-like cells immunostained for HSV-1 in highly infected regions. Conclusions A new microglia/microglia-like state may potentially shed light on how microglia could react to HSV-1 infection. Our observations suggest that infected microglia/microglia-like cells contribute to an exacerbated CNS inflammation. Further characterization of this transitory state of the microglia/microglia-like cell transcriptome may allow the development of novel immunomodulatory approaches to improve HSE outcomes by regulating the microglial immune response.

Abstract Background Zika virus (ZIKV) has been associated with several neurological complications in adult patients. Methods We used a mouse model deficient in TRIF and IPS-1 adaptor proteins, which are involved in type I interferon production, to study the role of microglia during brain infection by ZIKV. Young adult mice were infected intravenously with the contemporary ZIKV strain PRVABC59 (1 × 105 PFUs/100 µL). Results Infected mice did not present overt clinical signs of the disease nor body weight loss compared with noninfected animals. However, mice exhibited a viremia and a brain viral load that were maximal (1.3 × 105 genome copies/mL and 9.8 × 107 genome copies/g of brain) on days 3 and 7 post-infection (p.i.), respectively. Immunohistochemistry analysis showed that ZIKV antigens were distributed in several regions of the brain, especially the dorsal hippocampus. The number of Iba1+/TMEM119+ microglia remained similar in infected versus noninfected mice, but their cell body and arborization areas significantly increased in the stratum radiatum and stratum lacunosum-moleculare layers of the dorsal hippocampus cornu ammoni (CA)1, indicating a reactive state. Ultrastructural analyses also revealed that microglia displayed increased phagocytic activities and extracellular digestion of degraded elements during infection. Mice pharmacologically depleted in microglia with PLX5622 presented a higher brain viral load compared to untreated group (2.8 × 1010 versus 8.5 × 108 genome copies/g of brain on day 10 p.i.) as well as an increased number of ZIKV antigens labeled with immunogold in the cytoplasm and endoplasmic reticulum of neurons and astrocytes indicating an enhanced viral replication. Furthermore, endosomes of astrocytes contained nanogold particles together with digested materials, suggesting a compensatory phagocytic activity upon microglial depletion. Conclusions These results indicate that microglia are involved in the control of ZIKV replication and/or its elimination in the brain. After depletion of microglia, the removal of ZIKV-infected cells by phagocytosis could be partly compensated by astrocytes.

Abstract Background Maternal nutrition is critical for proper fetal development. While increased nutrient intake is essential during pregnancy, an excessive consumption of certain nutrients, like fat, can lead to long-lasting detrimental consequences on the offspring. Animal work investigating the consequences of maternal high-fat diet (mHFD) revealed in the offspring a maternal immune activation (MIA) phenotype associated with increased inflammatory signals. This inflammation was proposed as one of the mechanisms causing neuronal circuit dysfunction, notably in the hippocampus, by altering the brain-resident macrophages—microglia. However, the understanding of mechanisms linking inflammation and microglial activities to pathological brain development remains limited. We hypothesized that mHFD-induced inflammation could prime microglia by altering their specific gene expression signature, population density, and/or functions. Methods We used an integrative approach combining molecular (i.e., multiplex-ELISA, rt-qPCR) and cellular (i.e., histochemistry, electron microscopy) techniques to investigate the effects of mHFD (saturated and unsaturated fats) vs control diet on inflammatory priming, as well as microglial transcriptomic signature, density, distribution, morphology, and ultrastructure in mice. These analyses were performed on the mothers and/or their adolescent offspring at postnatal day 30. Results Our study revealed that mHFD results in MIA defined by increased circulating levels of interleukin (IL)-6 in the mothers. This phenotype was associated with an exacerbated inflammatory response to peripheral lipopolysaccharide in mHFD-exposed offspring of both sexes. Microglial morphology was also altered, and there were increased microglial interactions with astrocytes in the hippocampus CA1 of mHFD-exposed male offspring, as well as decreased microglia-associated extracellular space pockets in the same region of mHFD-exposed offspring of the two sexes. A decreased mRNA expression of the inflammatory-regulating cytokine Tgfb1 and microglial receptors Tmem119, Trem2, and Cx3cr1 was additionally measured in the hippocampus of mHFD-exposed offspring, especially in males. Conclusions Here, we described how dietary habits during pregnancy and nurturing, particularly the consumption of an enriched fat diet, can influence peripheral immune priming in the offspring. We also found that microglia are affected in terms of gene expression signature, morphology, and interactions with the hippocampal parenchyma, in a partially sexually dimorphic manner, which may contribute to the adverse neurodevelopmental outcomes on the offspring.

Suicide is a complex public health challenge associated worldwide with one death every 40 s. Research advances in the neuropathology of suicidal behaviors (SB) have defined discrete brain changes which may hold the key to suicide prevention. Physiological differences in microglia, the resident immune cells of the brain, are present in post-mortem tissue samples of individuals who died by suicide. Furthermore, microglia are mechanistically implicated in the outcomes of important risk factors for SB, including early-life adversity, stressful life events, and psychiatric disorders. SB risk factors result in inflammatory and oxidative stress activities which could converge to microglial synaptic remodeling affecting susceptibility or resistance to SB. To push further this perspective, in this Review we summarize current areas of opportunity that could untangle the functional participation of microglia in the context of suicide. Our discussion centers around microglial state diversity in respect to morphology, gene and protein expression, as well as function, depending on various factors, namely brain region, age, and sex.

The immune system is essential for maintaining homeostasis, as well as promoting growth and healing throughout the brain and body. Considering that immune cells respond rapidly to changes in their microenvironment, they are very difficult to study without affecting their structure and function. The advancement of non-invasive imaging methods greatly contributed to elucidating the physiological roles performed by immune cells in the brain across stages of the lifespan and contexts of health and disease. For instance, techniques like two-photon in vivo microscopy were pivotal for studying microglial functional dynamics in the healthy brain. Through these observations, their interactions with neurons, astrocytes, blood vessels and synapses were uncovered. High-resolution electron microscopy with immunostaining and 3D-reconstruction, as well as super-resolution fluorescence microscopy, provided complementary insights by revealing microglial interventions at synapses (phagocytosis, trogocytosis, synaptic stripping, etc.). In addition, serial block-face scanning electron microscopy has provided the first 3D reconstruction of a microglial cell at nanoscale resolution. This review will discuss the technical toolbox that currently allows to study microglia and other immune cells in the brain, as well as introduce emerging methods that were developed and could be used to increase the spatial and temporal resolution of neuroimmune imaging. A special attention will also be placed on positron emission tomography and the development of selective functional radiotracers for microglia and peripheral macrophages, considering their strong potential for research translation between animals and humans, notably when paired with other imaging modalities such as magnetic resonance imaging.

The first electron microscope was constructed in 1931. Several decades later, techniques were developed to allow the first ultrastructural analysis of microglia by transmission electron microscopy (EM). In the 50 years that followed, important roles of microglia have been identified, specifically due to the ultrastructural resolution currently available only with EM. In particular, the addition of electron-dense staining using immunohistochemical EM methods has allowed the identification of microglial cell bodies, as well as processes, which are difficult to recognize in EM, and to uncover their complex interactions with neurons and synapses. The ability to recognize neuronal, astrocytic, and oligodendrocytic compartments in the neuropil without any staining is another invaluable advantage of EM over light microscopy for studying intimate cell–cell contacts. The technique has been essential in defining microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic stripping and pruning via phagocytosis of extraneous synapses. Recent technological advances in EM including serial block-face imaging and focused-ion beam scanning EM have also facilitated automated acquisition of large tissue volumes required to reconstruct neuronal circuits in 3D at nanometer-resolution. These cutting-edge techniques which are now becoming increasingly available will further revolutionize the study of microglia across stages of the lifespan, brain regions, and contexts of health and disease. In this mini-review, we will focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that were provided by EM.

Traumatic spinal cord injury can cause immediate physical damage to the spinal cord and result in severe neurological deficits. The primary, mechanical tissue damage triggers a variety of secondary damage mechanisms at the injury site which significantly contribute to a larger lesion size and increased functional damage. Inflammatory mechanisms which directly involve both microglia (MG) and monocyte-derived macrophages (MDM) play important roles in the post-injury processes, including inflammation and debris clearing. In the current study, we investigated changes in the structure and function of MG/MDM in the injured spinal cord of adult female mice, 7 days after a thoracic contusion SCI. With the use of chip mapping scanning electron microscopy, which allows to image large samples at the nanoscale, we performed an ultrastructural comparison of MG/MDM located near the lesion vs adjacent regions to provide novel insights into the mechanisms at play post-injury. We found that MG/MDM located near the lesion had more mitochondria overall, including mitochondria with and without morphological alterations, and had a higher proportion of altered mitochondria. MG/MDM near the lesion also showed an increased number of phagosomes, including phagosomes containing myelin and partiallydigested materials. MG/MDM near the injury interacted differently with the spinal cord parenchyma, as shown by their reduced number of direct contacts with synaptic elements, axon terminals and dendritic spines. In this study, we characterized the ultrastructural changes of MG/MDM in response to spinal cord tissue damage in mice, uncovering changes in phagocytic activity, mitochondrial ultrastructure, and inter-cellular interactions within the spinal cord parenchyma. [ABSTRACT FROM AUTHOR]

• Ketogenic diet tends to promote resistance to psychological stress. • Hippocampal microglia show morphological adaptations to stress and diet. • Microglia of stress-susceptible mice make less synaptic contacts. • Microglia of ketogenic diet-fed mice show less signs of cellular stress. • Lipids are differentially regulated in the hippocampi of susceptible mice. 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. [ABSTRACT FROM AUTHOR]

A diverse heterogeneity of microglial cells was previously described in Alzheimer's disease (AD) pathology, including dark microglia, a state characterized by ultrastructural markers of cellular stress. To provide novel insights into the roles of dark microglia during aging in the context of AD pathology, we performed a quantitative density and ultrastructural analysis of these cells using high-throughput scanning electron microscopy in the ventral hippocampus CA1 stratum lacunosum-moleculare of 20-month-old APP-PS1 vs C57BL/6J male mice. The density of dark microglia was significantly higher in APP-PS1 vs C57BL/6J mice, with these cells accounting for nearly half of all microglia observed near amyloid-beta (Aβ) plaques. This dark microglial state interacted more with dystrophic neurites compared to other APP-PS1 microglia and possessed glycogen granules, associated with a metabolic shift toward glycolysis, which provides the first ultrastructural evidence of their presence in microglia. Dark microglia were further observed in aging human post-mortem brain samples showing similar ultrastructural features as in mouse. Overall, our results provide a quantitative ultrastructural characterization of a microglial state associated with cellular stress (i.e., dark microglia) that is primarily restricted near Aβ plaques and dystrophic neurites. The presence of this microglial state in the aging human post-mortem brain is further revealed. [ABSTRACT FROM AUTHOR]

The hippocampus is a plastic brain area that shows functional segregation along its longitudinal axis, reflected by a higher level of long-term potentiation (LTP) in the CA1 region of the dorsal hippocampus (DH) compared to the ventral hippocampus (VH), but the mechanisms underlying this difference remain elusive. Numerous studies have highlighted the importance of microglia–neuronal communication in modulating synaptic transmission and hippocampal plasticity, although its role in physiological contexts is still largely unknown. We characterized in depth the features of microglia in the two hippocampal poles and investigated their contribution to CA1 plasticity under physiological conditions. We unveiled the influence of microglia in differentially modulating the amplitude of LTP in the DH and VH, showing that minocycline or PLX5622 treatment reduced LTP amplitude in the DH, while increasing it in the VH. This was recapitulated in Cx3cr1 knockout mice, indicating that microglia have a key role in setting the conditions for plasticity processes in a region-specific manner, and that the CX3CL1–CX3CR1 pathway is a key element in determining the basal level of CA1 LTP in the two regions. The observed LTP differences at the two poles were associated with transcriptional changes in the expression of genes encoding for Il-1, Tnf-α, Il-6, and Bdnf, essential players of neuronal plasticity. Furthermore, microglia in the CA1 SR region showed an increase in soma and a more extensive arborization, an increased prevalence of immature lysosomes accompanied by an elevation in mRNA expression of phagocytic markers Mertk and Cd68 and a surge in the expression of microglial outward K+ currents in the VH compared to DH, suggesting a distinct basal phenotypic state of microglia across the two hippocampal poles. Overall, we characterized the molecular, morphological, ultrastructural, and functional profile of microglia at the two poles, suggesting that modifications in hippocampal subregions related to different microglial statuses can contribute to dissect the phenotypical aspects of many diseases in which microglia are known to be involved. [ABSTRACT FROM AUTHOR]

Suicide is a complex public health challenge associated worldwide with one death every 40 s. Research advances in the neuropathology of suicidal behaviors (SB) have defined discrete brain changes which may hold the key to suicide prevention. Physiological differences in microglia, the resident immune cells of the brain, are present in post-mortem tissue samples of individuals who died by suicide. Furthermore, microglia are mechanistically implicated in the outcomes of important risk factors for SB, including early-life adversity, stressful life events, and psychiatric disorders. SB risk factors result in inflammatory and oxidative stress activities which could converge to microglial synaptic remodeling affecting susceptibility or resistance to SB. To push further this perspective, in this Review we summarize current areas of opportunity that could untangle the functional participation of microglia in the context of suicide. Our discussion centers around microglial state diversity in respect to morphology, gene and protein expression, as well as function, depending on various factors, namely brain region, age, and sex. [ABSTRACT FROM AUTHOR]

Various environmental exposures during pregnancy, like maternal diet, can compromise, at critical periods of development, the neurovascular maturation of the offspring. Foetal exposure to maternal high-fat diet (mHFD), common to Western societies, has been shown to disturb neurovascular development in neonates and long-term permeability of the neurovasculature. Nevertheless, the effects of mHFD on the offspring's cerebrovascular health remains largely elusive. Here, we sought to address this knowledge gap by using a translational mouse model of mHFD exposure. Three-dimensional and ultrastructure analysis of the neurovascular unit (vasculature and parenchymal cells) in mHFD-exposed offspring revealed major alterations of the neurovascular organization and metabolism. These alterations were accompanied by changes in the expression of genes involved in metabolism and immunity, indicating that neurovascular changes may result from abnormal brain metabolism and immune regulation. In addition, mHFD-exposed offspring showed persisting behavioural alterations reminiscent of neurodevelopmental disorders, specifically an increase in stereotyped and repetitive behaviours into adulthood. In order to advance our understanding of the effects of maternal high-fat diet (mHFD) on the cerebrovascular health of offspring, Bordeleau et al. use a translational mouse model of mHFD exposure. They demonstrate that mHFD induces cerebrovascular and microglial changes in the offspring as well as behavioural alterations that are reminiscent of neurodevelopmental disorders associated with repetitive behaviours at adulthood. [ABSTRACT FROM AUTHOR]

The immune system is essential for maintaining homeostasis, as well as promoting growth and healing throughout the brain and body. Considering that immune cells respond rapidly to changes in their microenvironment, they are very difficult to study without affecting their structure and function. The advancement of non-invasive imaging methods greatly contributed to elucidating the physiological roles performed by immune cells in the brain across stages of the lifespan and contexts of health and disease. For instance, techniques like two-photon in vivo microscopy were pivotal for studying microglial functional dynamics in the healthy brain. Through these observations, their interactions with neurons, astrocytes, blood vessels and synapses were uncovered. High-resolution electron microscopy with immunostaining and 3D-reconstruction, as well as super-resolution fluorescence microscopy, provided complementary insights by revealing microglial interventions at synapses (phagocytosis, trogocytosis, synaptic stripping, etc.). In addition, serial block-face scanning electron microscopy has provided the first 3D reconstruction of a microglial cell at nanoscale resolution. This review will discuss the technical toolbox that currently allows to study microglia and other immune cells in the brain, as well as introduce emerging methods that were developed and could be used to increase the spatial and temporal resolution of neuroimmune imaging. A special attention will also be placed on positron emission tomography and the development of selective functional radiotracers for microglia and peripheral macrophages, considering their strong potential for research translation between animals and humans, notably when paired with other imaging modalities such as magnetic resonance imaging. [ABSTRACT FROM AUTHOR]

The first electron microscope was constructed in 1931. Several decades later, techniques were developed to allow the first ultrastructural analysis of microglia by transmission electron microscopy (EM). In the 50 years that followed, important roles of microglia have been identified, specifically due to the ultrastructural resolution currently available only with EM. In particular, the addition of electron-dense staining using immunohistochemical EM methods has allowed the identification of microglial cell bodies, as well as processes, which are difficult to recognize in EM, and to uncover their complex interactions with neurons and synapses. The ability to recognize neuronal, astrocytic, and oligodendrocytic compartments in the neuropil without any staining is another invaluable advantage of EM over light microscopy for studying intimate cell--cell contacts. The technique has been essential in defining microglial interactions with neurons and synapses, thus providing, among other discoveries, important insights into their roles in synaptic stripping and pruning via phagocytosis of extraneous synapses. Recent technological advances in EM including serial block-face imaging and focused-ion beam scanning EM have also facilitated automated acquisition of large tissue volumes required to reconstruct neuronal circuits in 3D at nanometer-resolution. These cutting-edge techniques which are now becoming increasingly available will further revolutionize the study of microglia across stages of the lifespan, brain regions, and contexts of health and disease. In this mini-review, we will focus on defining the distinctive ultrastructural features of microglia and the unique insights into their function that were provided by EM. [ABSTRACT FROM AUTHOR]

Background: Maternal nutrition is critical for proper fetal development. While increased nutrient intake is essential during pregnancy, an excessive consumption of certain nutrients, like fat, can lead to long-lasting detrimental consequences on the offspring. Animal work investigating the consequences of maternal high-fat diet (mHFD) revealed in the offspring a maternal immune activation (MIA) phenotype associated with increased inflammatory signals. This inflammation was proposed as one of the mechanisms causing neuronal circuit dysfunction, notably in the hippocampus, by altering the brain-resident macrophages-microglia. However, the understanding of mechanisms linking inflammation and microglial activities to pathological brain development remains limited. We hypothesized that mHFD-induced inflammation could prime microglia by altering their specific gene expression signature, population density, and/or functions.Methods: We used an integrative approach combining molecular (i.e., multiplex-ELISA, rt-qPCR) and cellular (i.e., histochemistry, electron microscopy) techniques to investigate the effects of mHFD (saturated and unsaturated fats) vs control diet on inflammatory priming, as well as microglial transcriptomic signature, density, distribution, morphology, and ultrastructure in mice. These analyses were performed on the mothers and/or their adolescent offspring at postnatal day 30.Results: Our study revealed that mHFD results in MIA defined by increased circulating levels of interleukin (IL)-6 in the mothers. This phenotype was associated with an exacerbated inflammatory response to peripheral lipopolysaccharide in mHFD-exposed offspring of both sexes. Microglial morphology was also altered, and there were increased microglial interactions with astrocytes in the hippocampus CA1 of mHFD-exposed male offspring, as well as decreased microglia-associated extracellular space pockets in the same region of mHFD-exposed offspring of the two sexes. A decreased mRNA expression of the inflammatory-regulating cytokine Tgfb1 and microglial receptors Tmem119, Trem2, and Cx3cr1 was additionally measured in the hippocampus of mHFD-exposed offspring, especially in males.Conclusions: Here, we described how dietary habits during pregnancy and nurturing, particularly the consumption of an enriched fat diet, can influence peripheral immune priming in the offspring. We also found that microglia are affected in terms of gene expression signature, morphology, and interactions with the hippocampal parenchyma, in a partially sexually dimorphic manner, which may contribute to the adverse neurodevelopmental outcomes on the offspring. [ABSTRACT FROM AUTHOR]

• Dual-challenged males had schizophrenia-like behavioral alterations at adulthood. • Dual-challenged females instead displayed anxious-like behavior at adulthood. • Dual-challenged females reduced GABAergic transmission genes at adulthood. • Dual-challenged male microglia presented a blunted morphology at adulthood. • Dual-challenged female microglia instead adopted a hypertrophic morphology. A wide range of positive, negative, and cognitive symptoms compose the clinical presentation of schizophrenia. Schizophrenia is a multifactorial disorder in which genetic and environmental risk factors interact for a full emergence of the disorder. Infectious challenges during pregnancy are a well-known environmental risk factor for schizophrenia. Also, genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia. Translational animal models recapitulating these complex gene-environment associations have a great potential to untangle schizophrenia neurobiology and propose new therapeutic strategies. Given that genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia, we compared the outcomes of a well-characterized model of maternal immune activation induced using the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) in wild-type versus fractalkine receptor knockout mice. Possible behavioral and immune alterations were assessed in male and female offspring during adulthood. Considering the role of the hippocampus in schizophrenia, microglial analyses and bulk RNA sequencing were performed within this region to assess the neuroimmune dynamics at play. Males and females were examined separately. Offspring exposed to the dual challenge paradigm exhibited symptoms relevant to schizophrenia and unpredictably to mood disorders. Males displayed social and cognitive deficits related to schizophrenia, while females mainly presented anxiety-like behaviors related to mood disorders. Hippocampal microglia in females exposed to the dual challenge were hypertrophic, indicative of an increased surveillance, whereas those in males showed on the other end of the spectrum blunted morphologies with a reduced phagocytosis. Hippocampal bulk-RNA sequencing further revealed a downregulation in females of genes related to GABAergic transmission, which represents one of the main proposed causes of mood disorders. Building on previous results, we identified in the current study distinctive behavioral phenotypes in female mice exposed to a dual genetic and environmental challenge, thus proposing a new model of neurodevelopmentally-associated mood and affective symptoms. This paves the way to future sex-specific investigations into the susceptibility to developmental challenges using animal models based on genetic and immune vulnerability as presented here. [ABSTRACT FROM AUTHOR]