Microglial Cell Population Research Articles

TMIC-71. ELUCIDATING IMMUNE MICROENVIRONMENTAL DISTINCTIONS BETWEEN IDH-MUTANT ASTROCYTOMA AND OLIGODENDROGLIOMA USING SINGLE-CELL TRANSCRIPTIONAL PROFILING

Abstract Despite exhibiting longer overall survival that IDH-wt counterparts, IDH-mutant gliomas remain uniformly deadly and impact patients in the prime of adult life. The immune microenvironment of IDH-mutant gliomas remains incompletely characterized, particularly with regard to distinctions between IDH-mutant astrocytoma and IDH-mutant, 1p/19q codeleted oligodendroglioma. To address this knowledge gap, we subjected dissociated tumor suspensions derived from 7 astrocytoma and 7 oligodendroglioma clinical samples to flow cytometry-based sorting for the pan-immune marker, CD45, and the microglial marker, P2RY12. This process yielded both microglial and non-microglial immune cell populations, which were then analyzed by scRNA sequencing. This workflow revealed a single large cluster of microglia, characterized by expression of Galectin-1 and lysosomal Lipase A, which was present at a significantly higher level in oligodendroglioma. While the biological significance of this cluster is unclear, gene set enrichment analysis exploring phenotypic differences between microglia in astrocytoma and oligodendroglioma revealed significant increases in expression of gene sets related to antigen presentation in astrocytoma. Parallel analysis of the non-microglial immune fraction showed an increase in the expression of pro-inflammatory gene sets, such as genes related to cytokine secretion, within the CD8 T-cell compartment of astrocytomas. We also observed upregulation of the expression of inflammatory gene sets within the dendritic cells of astrocytomas. Taken together, these changes suggest that the immune compartment of astrocytoma is more active and inflamed, and targeting the immune system in these tumors may represent a potential therapeutic avenue.

Brain volume and microglial density changes are correlated in a juvenile mouse model of cranial radiation and CSF1R inhibitor treatment.

Microglia have been shown to proliferate and become activated following cranial radiotherapy (CRT), resulting in a chronic inflammatory response. We investigated the role of microglia in contributing to widespread volume losses observed in the brain following CRT in juvenile mice. To manipulate microglia, we used low-dose treatment with a highly selective CSF1R inhibitor called PLX5622 (PLX). We hypothesized that alteration of the post-CRT microglia population would lead to changes in brain development outcomes, as evaluated by structural MRI. Wild-type C57BL/6J mice were provided with daily intraperitoneal injections of PLX (25 mg/kg) or vehicle from postnatal day (P)14 to P19. Mice also received whole-brain irradiation (7 Gy) or sham irradiation (0 Gy) at 16 days of age. In one cohort of mice, immunohistochemical assessment in tissue sections was conducted to assess the impact of the selected PLX and CRT doses as well as their combination. In a separate cohort, mice were imaged using MRI at P14 (pretreatment), P19, P23, P42 and P63 in order to assess induced volume changes, which were measured based on structures from a predefined atlas. We observed that PLX and radiation treatments led to sex-specific changes in the microglial cell population. Across treatment groups, MRI-detected anatomical volumes at P19 and P63 were associated with microglia and proliferating microglia densities, respectively. Overall, our study demonstrates that low-dose PLX treatment produces a sex-dependent response in juvenile mice, that manipulation of microglia alters CRT-induced volume changes and that microglia density and MRI-derived volume changes are correlated in this model.

High-throughput ultrastructural analysis of macular telangiectasia type 2.

Macular Telangiectasia type 2 (MacTel), is an uncommon form of late-onset, slowly-progressive macular degeneration. Associated with regional Müller glial cell loss in the retina and the amino acid serine synthesized by Müller cells, the disease is functionally confined to a central retinal region - the MacTel zone. We have used high-throughput multi-resolution electron microscopy techniques, optimized for disease analysis, to study the retinas from two women, mother and daughter, aged 79 and 48 years respectively, suffering from MacTel. In both eyes, the principal observations made were changes specific to mitochondrial structure both outside and within the MacTel zone in all retinal cell types, with the exception of those in the retinal pigment epithelium (RPE). The lesion areas, which are a hallmark of MacTel, extend from Bruch's membrane and the choriocapillaris, through all depths of the retina, and include cells from the RPE, retinal vascular elements, and extensive hypertrophic basement membrane material. Where the Müller glial cells are lost, we have identified a significant population of microglial cells, exclusively within the Henle fiber layer, which appear to ensheathe the Henle fibers, similar to that seen normally by Müller cells. Since Müller cells synthesize retinal serine, whereas retinal neurons do not, we propose that serine deficiency, required for normal mitochondrial function, may relate to mitochondrial changes that underlie the development of MacTel. With mitochondrial changes occurring retina-wide, the question remains as to why the Müller cells are uniquely susceptible within the MacTel zone.

Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer’s disease cortex

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer’s disease (AD). New MethodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Image processing and supervised machine learning for retinal microglia characterization in senescence.

The process of senescence impairs the function of cells and can ultimately be a key factor in the development of disease. With an aging population, senescence-related diseases are increasing in prevalence. Therefore, understanding the mechanisms of cellular senescence within the central nervous system (CNS), including the retina, may yield new therapeutic pathways to slow or even prevent the development of neuro- and retinal degenerative diseases. One method of probing the changing functions of senescent retinal cells is to observe retinal microglial cells. Their morphological structure may change in response to their surrounding cellular environment. In this chapter, we show how microglial cells in the retina, which are implicated in aging and diseases of the CNS, can be identified, quantified, and classified into five distinct morphotypes using image processing and supervised machine learning algorithms. The process involves dissecting, staining, and mounting mouse retinas, before image capture via fluorescence microscopy. The resulting images can then be classified by morphotype using a support vector machine (SVM) we have recently described showing high accuracy. This SVM model uses shape metrics found to correspond with qualitative descriptions of the shape of each morphotype taken from existing literature. We encourage more objective and widespread use of methods of quantification such as this. We believe automatic delineation of the population of microglial cells in the retina, could potentially lead to their use as retinal imaging biomarkers for disease prediction in the future.

A Distinct Microglial Cell Population Expressing Both CD86 and CD206 Constitutes a Dominant Type and Executes Phagocytosis in Two Mouse Models of Retinal Degeneration.

Microglial cells are the key regulators of inflammation during retinal degeneration (RD) and are conventionally classified as M1 or M2. However, whether the M1/M2 classification exactly reflects the functional classification of microglial cells in the retina remains debatable. We examined the spatiotemporal changes of microglial cells in the blue-LED and NaIO3-induced RD mice models using M1/M2 markers and functional genes. TUNEL assay was performed to detect photoreceptor cell death, and microglial cells were labeled with anti-IBA1, P2RY12, CD86, and CD206 antibodies. FACS was used to isolate microglial cells with anti-CD206 and CD86 antibodies, and qRT-PCR was performed to evaluate Il-10, Il-6, Trem-2, Apoe, and Lyz2 expression. TUNEL-positive cells were detected in the outer nuclear layer (ONL) from 24 h to 72 h post-RD induction. At 24 h, P2RY12 was decreased and CD86 was increased, and CD86/CD206 double-labeled cells occupied the dominant population at 72 h. And CD86/CD206 double-labeled cells showed a significant increase in Apoe, Trem2, and Lyz2 levels but not in those of Il-6 and Il-10. Our results demonstrate that microglial cells in active RD cannot be classified as M1 or M2, and the majority of microglia express both CD86 and CD206, which are involved in phagocytosis rather than inflammation.

Sigma-1 receptor expression in a subpopulation of lumbar spinal cord microglia in response to peripheral nerve injury

Sigma-1 Receptor has been shown to localize to sites of peripheral nerve injury and back pain. Radioligand probes have been developed to localize Sigma-1 Receptor and thus image pain source. However, in non-pain conditions, Sigma-1 Receptor expression has also been demonstrated in the central nervous system and dorsal root ganglion. This work aimed to study Sigma-1 Receptor expression in a microglial cell population in the lumbar spine following peripheral nerve injury. A publicly available transcriptomic dataset of 102,691 L4/5 mouse microglial cells from a sciatic-sural nerve spared nerve injury model and 93,027 age and sex matched cells from a sham model was used. At each of three time points—postoperative day 3, postoperative day 14, and postoperative month 5—gene expression data was recorded for both spared nerve injury and Sham cell groups. For all cells, 27,998 genes were sequenced. All cells were clustered into 12 distinct subclusters and gene set enrichment pathway analysis was performed. For both the spared nerve injury and Sham groups, Sigma-1 Receptor expression significantly decreased at each time point following surgery. At the 5-month postoperative time point, only one of twelve subclusters showed significantly increased Sigma-1 Receptor expression in spared nerve injury cells as compared to Sham cells (p = 0.0064). Pathway analysis of this cluster showed a significantly increased expression of the inflammatory response pathway in the spared nerve injury cells relative to Sham cells at the 5-month time point (p = 6.74e−05). A distinct subcluster of L4/5 microglia was identified which overexpress Sigma-1 Receptor following peripheral nerve injury consistent with neuropathic pain inflammatory response functioning. This indicates that upregulated Sigma-1 Receptor in the central nervous system characterizes post-acute peripheral nerve injury and may be further developed for clinical use in the differentiation between low back pain secondary to peripheral nerve injury and low back pain not associated with peripheral nerve injury in cases where the pain cannot be localized.

Seizure-induced increase in microglial cell population in the developing zebrafish brain

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, in particular following early-life seizures, remains poorly understood due in part to the lack of suitable experimental models allowing the in vivo imaging of microglial activity. Given the advantage of zebrafish larvae for minimally-invasive imaging approaches, we sought for the first time to describe the microglial responses after acute seizures in two different zebrafish larval models: a chemically-induced epileptic model by the systemic injection of kainate at 3 days post-fertilization, and the didys552 genetic epilepsy model, which carries a mutation in scn1lab that leads to spontaneous epileptiform discharges. Kainate-treated larvae exhibited transient brain damage as shown by increased numbers of apoptotic nuclei as early as one day post-injection, which was followed by an increase in the number of microglia in the brain. A similar microglial phenotype was also observed in didys552-/- mutants, suggesting that microglia numbers change in response to seizure-like activity in the brain. Interestingly, kainate-treated larvae also displayed a decreased seizure threshold towards subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity in comparison with vehicle-injected larvae. These results are comparable to kainate-induced rodent seizure models and suggest the suitability of these zebrafish seizure models for future studies, in particular to elucidate the links between epileptogenesis and microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility.

Roles and regulation of microglia activity in multiple sclerosis: insights from animal models.

As resident macrophages of the CNS, microglia are critical immune effectors of inflammatory lesions and associated neural dysfunctions. In multiple sclerosis (MS) and its animal models, chronic microglial inflammatory activity damages myelin and disrupts axonal and synaptic activity. In contrast to these detrimental effects, the potent phagocytic and tissue-remodelling capabilities of microglia support critical endogenous repair mechanisms. Although these opposing capabilities have long been appreciated, a precise understanding of their underlying molecular effectors is only beginning to emerge. Here, we review recent advances in our understanding of the roles of microglia in animal models of MS and demyelinating lesions and the mechanisms that underlie their damaging and repairing activities. We also discuss how the structured organization and regulation of the genome enables complex transcriptional heterogeneity within the microglial cell population at demyelinating lesions.

Morphological comparison of the microglial cell population between organotypic retinal cultures and axotomised retinas in vivo

AbstractPurpose: Retinal organotypic cultures (ROCs) have been used since the early 1930s as in vivo surrogates to study retinal ganglion cell (RGC) loss and neuroprotection. In this work we purposed to analyse and compare the morphological changes of microglial cells (MCs) along time in ROCs and axotomised retinas.Methods: C57/Bl6 mice were used. For the in vitro model, retinas were dissected and cultured in supplemented medium. In the in vivo model, the left optic nerve (ONC) was crushed. In both models, retinas were analysed at 24‐h intervals for up to 7 days. MCs were then immunodetected with anti‐Iba1 antibody and then photographed in the ganglion cell layer (GCL) of the retina using a confocal microscope. Finally, their morphology was quantitatively analysed using the free Image J Fiji software.Results: After ONC, the number of MC processes decreased significantly from day 1 to 7 compared to intact retinas. Similarly, we observed a significant decrease in the number of processes of in vitro MCs, which was significantly greater than after axotomy in vivo The total length of the processes decreased in both models up to 7 days. When studying the size of the MCs, it was observed that from 1 to 7 days MCs were bigger in vitro than in vitro and in both models than in intact retinas.Conclusions: The morphology of MCs differs in vivo and in vitro, suggesting a differential activation.

Constitutively active microglial populations limit anorexia induced by the food contaminant deoxynivalenol

Microglia are involved in neuroinflammatory processes during diverse pathophysiological conditions. To date, the possible contribution of these cells to deoxynivalenol (DON)-induced brain inflammation and anorexia has not yet been evaluated. DON, one of the most abundant trichothecenes found in cereals, has been implicated in mycotoxicosis in both humans and farm animals. DON-induced toxicity is characterized by reduced food intake, weight gain, and immunological effects. We previously showed that exposure to DON induces an inflammatory response within the hypothalamus and dorsal vagal complex (DVC) which contributes to DON-induced anorexia. Here, in response to anorectic DON doses, we reported microglial activation within two circumventricular organs (CVOs), the area postrema (AP) and median eminence (ME) located in the DVC and the hypothalamus, respectively. Interestingly, this microglial activation was observed while DON-induced anorexia was ongoing (i.e., 3 and 6 h after DON administration). Next, we took advantage of pharmacological microglia deletion using PLX3397, a colony-stimulating factor 1 receptor (CSF1R)-inhibitor. Surprisingly, microglia-depleted mice exhibited an increased sensitivity to DON since non-anorectic DON doses reduced food intake in PLX3397-treated mice. Moreover, low DON doses induced c-Fos expression within feeding behavior-associated structures in PLX3397-treated mice but not in control mice. In parallel, we have highlighted heterogeneity in the phenotype of microglial cells present in and around the AP and ME of control animals. In these areas, microglial subpopulations expressed IBA1, TMEM119, CD11b and CD68 to varying degrees. In addition, a CD68 positive subpopulation showed, under resting conditions, a noticeable phagocytotic/endocytotic activity. We observed that DON strongly reduced CD68 in the hypothalamus and DVC. Finally, inactivation of constitutively active microglia by intraperitoneal administration of minocycline resulted in anorexia with a DON dose ineffective in control mice. Taken together, these results strongly suggest that various populations of microglial cells residing in and around the CVOs are maintained in a functionally active state even under physiological conditions. We propose that these microglial cell populations are attempting to protect the brain parenchyma from hazardous molecules coming from the blood. This study could contribute to a better understanding of how microglia respond to environmental contaminants.

Microglial cells: Sensors for neuronal activity and microbiota-derived molecules.

Microglial cells play pleiotropic homeostatic activities in the brain, during development and in adulthood. Microglia regulate synaptic activity and maturation, and continuously patrol brain parenchyma monitoring for and reacting to eventual alterations or damages. In the last two decades microglia were given a central role as an indicator to monitor the inflammatory state of brain parenchyma. However, the recent introduction of single cell scRNA analyses in several studies on the functional role of microglia, revealed a not-negligible spatio-temporal heterogeneity of microglial cell populations in the brain, both during healthy and in pathological conditions. Furthermore, the recent advances in the knowledge of the mechanisms involved in the modulation of cerebral activity induced by gut microbe-derived molecules open new perspectives for deciphering the role of microglial cells as possible mediators of these interactions. The aim of this review is to summarize the most recent studies correlating gut-derived molecules and vagal stimulation, as well as dysbiotic events, to alteration of brain functioning, and the contribution of microglial cells.

Reactive morphology of dividing microglia following kainic acid administration.

The microglial response to a pathological microenvironment is hallmarked by a change in cellular morphology. Following a pathological stimulus, microglia become reactive and simultaneously divide to create daughter cells. Although a wide array of microglial morphologies has been observed, the exact functions of these distinct morphologies are unknown, as are the morphology and reactivity status of dividing microglia. In this study, we used kainic acid to trigger microglial activation and cell division. Following a cortical kainic acid injection, microglial morphology and proliferation were examined at 3 days post-injection using immunohistochemistry for ionized calcium binding adapter molecule 1 (Iba1) to stain for microglia, and KI67 as a marker of cell division. Individual microglial cells were isolated from photomicrographs and skeletal and fractal analyses were used to examine cell size and spatial complexity. We examined the morphology of microglia in both wildtype and microglia-specific tumor necrosis factor (TNF)-α knockout mice. Data were analyzed using generalized linear mixed models or a two-way ANOVA. We found that dividing microglia had a more reactive morphology (larger cell body area, longer cell perimeter, and less ramification) compared to microglia that were not dividing, regardless of microglial release of TNF-α. However, we also observed dividing microglia with a complex, more ramified morphology. Changes in microglial morphology and division were greatest near the kainic acid injection site. This study uses robust and quantitative techniques to better understand microglial cell division, morphology, and population dynamics, which are essential for the development of novel therapeutics that target microglia.

Morphological and functional characteristics of LPS-stimulated microglial cells under the action of orexin A

Interest to the orexin-containing neurons is caused by their recent discovery and perspectives of their usage for treatment of different diseases. The studies in this area were launched recently and are of special interest since the opportunity of modulating functional activity of the brain immune system is of pivotal significance for therapy of various central nervous system (CNS) disorders providing novel ways of search and promising data on therapeutic effects of orexins in inflammatory, autoimmune diseases as well as malignant tumors.
 Some data from literature show that orexins may exert therapeutic effects in different disorders caused by altered neuroimmune interactions. Participation of this neuromediator system is shown in pathogenesis of narcolepsia, obesity, multiple sclerosis, Alzheimer disease, intestinal disorders, septic shock and cancer, due to involvement of orexins in functional regulation of various components of immune syste, e.g., microglial cell populations. Despite only scarce data on these effects, some experimental results obtained over last years, add to our understanding of orexin effects upon functional activity of the brain immune system.
 A number of previous studies allowed to assess the orexin effects on morpho-functional features of microglial cells activated by lipopolysaccharide (LPS), thus presenting a prospective for development of novel approaches to therapy of infectious, inflammatory, neurodegenerative and autoimmune disorders affecting CNS. In the present study, we aimed for detecting the effects of neuromediator orexin A upon functional traits of of microglial cells activated by LPS (M1 phenotype) as evaluated by changes of their size and length of their processes, as well as density of cell distribution.
 We have studied the changes of microglia cell numbers following intraperitoneal LPS injection. It was shown that, the LPS causes higher activation degree of these cells, i.e., the contents of microglial cells becomes increased in somatosensory area of the brain cortex. A series of these studies allowed us to demonstrate that intracerebroventricular injection of orexin A in animals following LPS injection does not cause detectable changes of the processes initiated by LPS. The comparative analysis did not detect any changes in length of microglial processes localized in somatosensory or motor cortical areas, and corpus striatum. Other parameters of the microglial cell activation will be studied in future.

Pharmacological Depletion of Microglia Leads to a Dose-Dependent Reduction in Inflammation and Senescence in the Aged Murine Brain

Chronic macrophage activation was implicated as one of the main culprits for chronical, low-grade inflammation which significantly contributes to development of age-related diseases. Microglia as the brain macrophages have been recently implicated as key players in neuroinflammation and neurodegeneration in the aged brain. Microglial cell functions are indispensable in early development, however, activation or senescence of microglia in aging cells may be detrimental. Depletion of microglia using genetical or pharmacological approaches leads to opposite results regarding effects on brain cognition. In this study we pharmacologically depleted microglia using orally delivered low and high doses of the CSF1R inhibitor PLX5622 and assessed the expression levels of known inflammation markers (TNF-α, IL1-β, IL-6, IL-10), glia markers (Iba-1 and Gfap) and specific senescence marker p16Ink4a in the aged murine brain. Our results indicate that treatment with low and high doses of PLX5622 leads to a dose-dependent depletion of microglial cells with similar levels in young and aged mice. We also show that treatment with low and high PLX5622 differentially affected cytokine levels in young and old brains. By using low doses we could achieve reduction in inflammation circumventing the astrocyte activation. Removal of microglia cells led to decreased expression of the senescence marker p16Ink4a in the aged brain, indicating a relevant contribution of these cells to the expression of this marker and their senescent status in the healthy aging brain. Our results indicate that increased and detrimental brain inflammation in aged murine brain can be impaired by selectively reducing the microglial cell population.

Early-life thermal stress mediates long-term alterations in hypothalamic microglia.

Stressful environmental events in early life have long-lasting consequences on later stress responses. We previously showed that heat conditioning of 3-day-old chicks during the critical period of heat-response development leads to heat vulnerability later in life. Here we assessed the role of early-life heat stress on the inflammatory response in the chick anterior hypothalamus (AH), focusing on hypothalamic microglia. We identified the microglial cell population in the chick AH using anti-KUL01 and anti-CD45 antibodies. Specific microglial features were also confirmed by expression of their signature genes. Under normal environmental conditions, hypothalamic microglia isolated from lipopolysaccharide (LPS)-injected chicks displayed a classical activated proinflammatory profile accompanied by a decreased homeostatic signature, demonstrating similarity of immune response with mammalian microglial cells. In accordance with our previous observations, conditioning of 3-day-old chicks under high ambient temperature decreased the number of newborn cells in the AH, among them microglial precursors. Although heat exposure did not affect microglial cell viability, it had a long-term impact on LPS-induced inflammatory response. Exposure to harsh heat led to heat vulnerability, and attenuated recruitment of peripheral monocytes and T cells into the AH following LPS challenge. Moreover, heat conditioning altered microglial reactivity, manifested as suppressed microglial activation in response to LPS. Innate immune memory developed by heat conditioning might underlie suppression of the microglial response to LPS challenge. We describe alterations in genome-wide CpG methylation profile of hypothalamic microglia, demonstrating probable epigenetic involvement in the reprogramming of microglial function, leading to heat-induced inflammatory cross-tolerance.

Astrocytic TSPO Upregulation Appears Before Microglial TSPO in Alzheimer's Disease.

Background:In vivo PET/SPECT imaging of neuroinflammation is primarily based on the estimation of the 18 kDa-translocator-protein (TSPO). However, TSPO is expressed by different cell types which complicates the interpretation.Objective:The present study evaluates the cellular origin of TSPO alterations in Alzheimer’s disease (AD).Methods:The TSPO cell origin was evaluated by combining radioactive imaging approaches using the TSPO radiotracer [125I]CLINDE and fluorescence-activated cell sorting, in a rat model of AD (TgF344-AD) and in AD subjects.Results:In the hippocampus of TgF344-AD rats, TSPO overexpression not only concerns glial cells but the increase is visible at 12 and 24 months in astrocytes and only at 24 months in microglia. In the temporal cortex of AD subjects, TSPO upregulation involved only glial cells. However, the mechanism of this upregulation appears different with an increase in the number of TSPO binding sites per cell without cell proliferation in the rat, and a microglial cell population expansion with a constant number of binding sites per cell in human AD.Conclusion:These data indicate an earlier astrocyte intervention than microglia and that TSPO in AD probably is an exclusive marker of glial activity without interference from other TSPO-expressing cells. This observation indicates that the interpretation of TSPO imaging depends on the stage of the pathology, and highlights the particular role of astrocytes.

GM-CSF induces noninflammatory proliferation of microglia and disturbs electrical neuronal network rhythms in situ

BackgroundThe granulocyte-macrophage colony-stimulating factor (GM-CSF) (or CSF-2) is involved in myeloid cell growth and differentiation, and, possibly, a major mediator of inflammation in body tissues. The role of GM-CSF in the activation of microglia (CNS resident macrophages) and the consequent impacts on neuronal survival, excitability, and synaptic transmission are widely unknown, however. Here, we focused on electrical neuronal network rhythms in the gamma frequency band (30–70 Hz). Gamma oscillations are fundamental to higher brain functions, such as perception, attention, and memory, and they are exquisitely sensitive to metabolic and oxidative stress.MethodsWe explored the effects of chronic GM-CSF exposure (72 h) on microglia in male rat organotypic hippocampal slice cultures (in situ), i.e., postnatal cortex tissue lacking leukocyte invasion (adaptive immunity). We applied extracellular electrophysiological recordings of local field potential, immunohistochemistry, design-based stereology, biochemical analysis, and pharmacological ablation of microglia.ResultsGM-CSF triggered substantial proliferation of microglia (microgliosis). By contrast, the release of proinflammatory cytokines (IL-6, TNF-α) and nitric oxide, the hippocampal cytoarchitecture as well as the morphology of parvalbumin-positive inhibitory interneurons were unaffected. Notably, GM-CSF induced concentration-dependent, long-lasting disturbances of gamma oscillations, such as slowing (beta frequency band) and neural burst firing (hyperexcitability), which were not mimicked by the T lymphocyte cytokine IL-17. These disturbances were attenuated by depletion of the microglial cell population with liposome-encapsulated clodronate. In contrast to priming with the cytokine IFN-γ (type II interferon), GM-CSF did not cause inflammatory neurodegeneration when paired with the TLR4 ligand LPS.ConclusionsGM-CSF has a unique role in the activation of microglia, including the potential to induce neuronal network dysfunction. These immunomodulatory properties might contribute to cognitive impairment and/or epileptic seizure development in disease featuring elevated GM-CSF levels, blood-brain barrier leakage, and/or T cell infiltration.

Neuronal gamma oscillations and activity-dependent potassium transients remain regular after depletion of microglia in postnatal cortex tissue.

Microglial cells (resident macrophages) feature rapid activation in CNS disease and can acquire multiple phenotypes exerting neuroprotection or neurotoxicity. The functional impact of surveying ("resting") microglia on neural excitability and neurotransmission in physiology is widely unknown, however. We addressed this issue in male rat hippocampal slice cultures (in situ) by pharmacological microglial ablation within days and by characterizing neuronal gamma-band oscillations (30-70Hz) that are highly sensitive to neuromodulators and disturbances in ion and energy regulation. Gamma oscillations support action potential timing and synaptic plasticity, associate with higher brain functions like perception and memory, and require precise communication between excitatory pyramidal cells and inhibitory (GABAergic) interneurons. The slice cultures featured well-preserved hippocampal cytoarchitecture and parvalbumin-positive interneuron networks, microglia with ramified morphology, and low basal levels of IL-6, TNF-α, and nitric oxide (NO). Stimulation of slice cultures with the pro-inflammatory cytokine IFN-γ or bacterial LPS serving as positive controls for microglial reactivity induced MHC-II expression and increased cytokine and NO release. Chronic exposure of slice cultures to liposome-encapsulated clodronate reduced the microglial cell population by about 96%, whereas neuronal structures, astrocyte GFAP expression, and basal levels of cytokines and NO were unchanged. Notably, the properties of gamma oscillations reflecting frequency, number and synchronization of synapse activity were regular after microglial depletion. Also, electrical stimulus-induced transients of the extracellular potassium concentration ([K+ ]o ) reflecting cellular K+ efflux, clearance and buffering were unchanged. This suggests that nonreactive microglia are dispensable for neuronal homeostasis and neuromodulation underlying network signaling and rhythm generation in cortical tissue.

Interplay Between Human Gut Bacteria Escherichia coli and Lactobacillus mucosae in the Occurrence of Neuropsychiatric Disorders in Mice.

To understand the roles of human gut bacteria in the occurrence of neuropsychiatric disorders, we isolated inflammatory Escherichia coli K1 and anti-inflammatory Lactobacillus mucosae from healthy human feces and examined their effects on the occurrence of altered microbiota, cognitive decline, and depression in mice. Oral gavage of Escherichia coli K1 caused colitis, cognitive decline, and depression in mice in the elevated plus maze, tail suspension, and forced swimming tasks. However, NK41 treatment reduced K1-induced cognitive decline and anxiety/depression. Furthermore, NK41 treatment increased K1-suppressed brain-derived neurotrophic factor (BDNF) expression and BDNF+/NeuN+ cell population and suppressed K1-induced NF-κB activation and LPS+/Iba1+ and NF-κB+/Iba1+ (microglial) cell populations in the hippocampus. NK41 treatment also suppressed K1-induced TNF-α and LPS levels in the blood and TNF-α expression, myeloperoxidase activity, NF-κB+/CD11c+ and CD11b+/CD11c+ cell populations in the colon. Furthermore, NK41 treatment decreased K1-induced colonic MUC2 expression, gut Proteobacteria population, and fecal LPS levels and modified the bacterial abundance related to polysaccharide breaking and biosynthesis. In conclusion, the overgrowth of inflammatory bacteria such as Escherichia coli in the gastrointestinal tract can cause neuropsychiatric disorders with gut microbiota alteration and the superiority of anti-inflammatory bacteria such as Lactobacillus mucosae can alleviate neuropsychiatric disorders with the attenuation of altered microbiota.