Microglia are the immune cells of the brain and become activated during any type of brain injury. In the middle cerebral artery occlusion (MCAo) model, a mouse model for ischemic stroke, we have previously shown that microglia and invaded monocytes upregulate the expression of the muscarinic acetylcholine receptor 3 (M3R) in the ischemic lesion. Here we tested whether this upregulation has an impact on the pathogenesis of MCAo. We depleted the m3R receptor in microglia, but not in circulating monocytes by giving tamoxifen to CX3CR1-CreERT+/+M3Rflox/flox (M3RKOmi) animals 3 weeks prior to MCAo. We found that M3RKOmi male mice had bigger lesions, more pronounced motor deficits after one week and cognitive deficits after about one month compared to control males. The density of Iba1+ cells was lower in the lesions of M3RKO male mice in the early, but not in the late disease phase. In females, these differences were not significant. By giving tamoxifen 1 week prior to MCAo, we depleted m3R in microglia and in circulating monocytes (M3RKOmi/mo). Male M3RKOmi/mo did not differ in lesion size, but had a lower survival rate, showed motor deficits and a reduced accumulation of Iba1+ positive cells into the lesion site. In conclusion, our data suggest that the upregulation of m3R in microglia and monocytes in stroke has a beneficial effect on the clinical outcome in male mice.

Despite the observed severe effects of microgravity on mammalian cells, many astronauts have completed long term stays in space without suffering from severe health problems. This raises questions about the cellular capacity for adaptation to a new gravitational environment. The International Space Station (ISS) experiment TRIPLE LUX A, performed in the BIOLAB laboratory of the ISS COLUMBUS module, allowed for the first time the direct measurement of a cellular function in real time and on orbit. We measured the oxidative burst reaction in mammalian macrophages (NR8383 rat alveolar macrophages) exposed to a centrifuge regime of internal 0 g and 1 g controls and step-wise increase or decrease of the gravitational force in four independent experiments. Surprisingly, we found that these macrophages adapted to microgravity in an ultra-fast manner within seconds, after an immediate inhibitory effect on the oxidative burst reaction. For the first time, we provided direct evidence of cellular sensitivity to gravity, through real-time on orbit measurements and by using an experimental system, in which all factors except gravity were constant. The surprisingly ultra-fast adaptation to microgravity indicates that mammalian macrophages are equipped with a highly efficient adaptation potential to a low gravity environment. This opens new avenues for the exploration of adaptation of mammalian cells to gravitational changes.

Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma.

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy.

As the immunocompetent cells of the central nervous system, microglia accumulate at amyloid beta plaques in Alzheimer's disease (AD) and acquire a morphological phenotype of activated microglia. Recent functional studies, however, indicate that in mouse models of amyloidosis and AD, these cells are rather dysfunctional indicated by a reduced phagocytic activity. Here, we report that this reduction in phagocytic activity is associated with perturbed purinergic receptor signaling, since phagocytosis could be stimulated by P2Y6 receptor activation in control, but not in 5xFAD transgenic animals, an animal model of amyloid deposition. Impaired phagocytosis is not innate, and develops only at later stages of amyloidosis. Furthermore, we show that membrane currents induced by uridine diphosphate, a ligand activating P2Y6 receptors, are altered in response rate and amplitude in microglia in close vicinity to plaques, but not in plaque-free areas of 5xFAD animals. These changes were accompanied by changes in membrane properties and potassium channel activity of plaque-associated microglia in early and late stages of amyloidosis. As a conclusion, the physiological properties of plaque-associated microglia are altered with a strong impact on purinergic signaling.

Microglia are the immune effector cells of the central nervous system (CNS) and react to pathologic events with a complex process including the release of nitric oxide (NO). NO is a free radical, which is toxic for all cells at high concentrations. To target an exaggerated NO release, we tested a library of 16 544 chemical compounds for their effect on lipopolysaccharide (LPS)-induced NO release in cell line and primary neonatal microglia. We identified a compound (C1) which significantly reduced NO release in a dose-dependent manner, with a low IC50 (252 nM) and no toxic side effects in vitro or in vivo. Target finding strategies such as in silico modelling and mass spectroscopy hint towards a direct interaction between C1 and the nitric oxide synthase making C1 a great candidate for specific intra-cellular interaction with the NO producing machinery.

High trait anxiety is a substantial risk factor for developing anxiety disorders and depression. While neuroinflammation has been identified to contribute to stress-induced anxiety, little is known about potential dysregulation in the neuroinflammatory system of genetically determined pathological anxiety or high trait anxiety individuals. We report microglial alterations in various brain regions in a mouse model of high trait anxiety (HAB). In particular, the dentate gyrus (DG) of the hippocampus of HABs exhibited enhanced density and average cell area of Iba1+, and density of phagocytic (CD68+/Iba1+) microglia compared to normal anxiety (NAB) controls. Minocycline was used to assess the capacity of a putative microglia 'inhibitor' in modulating hyperanxiety behavior of HABs. Chronic oral minocycline indeed reduced HAB hyperanxiety, which was associated with significant decreases in Iba1+ and CD68+Iba1+ cell densities in the DG. Addressing causality, it was demonstrated that longer (10 days), but not shorter (5 days), periods of minocycline microinfusions locally into the DG of HAB reduced Iba-1+ cell density and attenuated hyperanxiety-related behavior, indicating that neuroinflammation in the DG is at least partially involved in the maintenance of pathological anxiety. The present data reveal evidence of disturbances in the microglial system of individuals with high trait anxiety. Minocycline attenuated HAB hyperanxiety, likely by modulation of microglial activity within the DG. Thus, the present data suggest that drugs with microglia-targeted anti-inflammatory properties could be promising as novel alternative or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals genetically predisposed to hyperanxiety.

Neuroligin-4 (NLGN4) loss-of-function mutations are associated with monogenic heritable autism spectrum disorder (ASD) and cause alterations in both synaptic and behavioral phenotypes. Microglia, the resident CNS macrophages, are implicated in ASD development and progression. Here we studied the impact of NLGN4 loss in a mouse model, focusing on microglia phenotype and function in both male and female mice. NLGN4 depletion caused lower microglia density, less ramified morphology, reduced response to injury and purinergic signaling specifically in the hippocampal CA3 region predominantly in male mice. Proteomic analysis revealed disrupted energy metabolism in male microglia and provided further evidence for sexual dimorphism in the ASD associated microglial phenotype. In addition, we observed impaired gamma oscillations in a sex-dependent manner. Lastly, estradiol application in male NLGN4-/- mice restored the altered microglial phenotype and function. Together, these results indicate that loss of NLGN4 affects not only neuronal network activity, but also changes the microglia state in a sex-dependent manner that could be targeted by estradiol treatment.

Cerebral infection with the parasite Toxoplasma gondii is followed by activation of resident cells and recruitment of immune cells from the periphery to the CNS. In this study, we show that a subset of myeloid cells, namely Ly6C(high)CCR2(+) inflammatory monocytes that infiltrate the brain upon chronic T. gondii infection, plays a decisive role in host defense. Depletion of this monocyte subset resulted in elevated parasite load and decreased survival of infected mice, suggesting their crucial role. Notably, Ly6C(high)CCR2(+) monocytes governed parasite control due to production of proinflammatory mediators, such as IL-1α, IL-1β, IL-6, inducible NO synthase, TNF, and reactive oxygen intermediate. Interestingly, Ly6C(high)CCR2(+) monocytes were also able to produce the regulatory cytokine IL-10, revealing their dual feature. Moreover, we confirmed by adoptive transfer that the recruited monocytes further develop into two distinct subpopulations contributing to parasite control and profound host defense. The differentiated Ly6C(int)CCR2(+)F4/80(int) subset upregulated MHC I and MHC II molecules, suggesting dendritic cell properties such as interaction with T cells, whereas the Ly6C(neg)F4/80(high) cell subset displayed elevated phagocytic capacity while upregulating triggering receptor expressed on myeloid cells-2. Finally, we have shown that the recruitment of Ly6C(high) monocytes to the CNS is regulated by P-selectin glycoprotein ligand-1. These results indicate the critical importance of recruited Ly6C(high) monocytes upon cerebral toxoplasmosis and reveal the behavior of further differentiated myeloid-derived mononuclear cell subsets in parasite control and immune regulation of the CNS.

Voluntary physical exercise (wheel running, RUN) and environmental enrichment both stimulate adult hippocampal neurogenesis but do so by different mechanisms. RUN induces precursor cell proliferation, whereas ENR exerts a survival-promoting effect on newborn cells. In addition, continued RUN prevented the physiologically occurring age-related decline in precursor cell in the dentate gyrus but did not lead to a corresponding increase in net neurogenesis. We hypothesized that in the absence of appropriate cognitive stimuli the potential for neurogenesis could not be realized but that an increased potential by proliferating precursor cells due to RUN could actually lead to more adult neurogenesis if an appropriate survival-promoting stimulus follows the exercise. We thus asked whether a sequential combination of RUN and ENR (RUNENR) would show additive effects that are distinct from the application of either paradigm alone. We found that the effects of 10 days of RUN followed by 35 days of ENR were additive in that the combined stimulation yielded an approximately 30% greater increase in new neurons than either stimulus alone, which also increased neurogenesis. Surprisingly, this result indicates that although overall the amount of proliferating cells in the dentate gyrus is poorly predictive of net adult neurogenesis, an increased neurogenic potential nevertheless provides the basis for a greater efficiency of the same survival-promoting stimulus. We thus propose that physical activity can "prime" the neurogenic region of the dentate gyrus for increased neurogenesis in the case the animal is exposed to an additional cognitive stimulus, here represented by the enrichment paradigm.

Neuroinflammation is discussed to play a role in specific subgroups of different psychiatric disorders, including anxiety disorders. We have previously shown that a mouse model of trait anxiety (HAB) displays enhanced microglial density and phagocytic activity in key regions of anxiety circuits compared to normal-anxiety controls (NAB). Using minocycline, we provided causal evidence that reducing microglial activation within the dentate gyrus (DG) attenuated enhanced anxiety in HABs. Besides pharmacological intervention, "positive environmental stimuli", which have the advantage of exerting no side-effects, have been shown to modulate inflammation-related markers in human beings. Therefore, we now investigated whether environmental enrichment (EE) would be sufficient to modulate upregulated neuroinflammation in high-anxiety HABs. We show for the first time that EE can indeed attenuate enhanced trait anxiety, even when presented as late as adulthood. We further found that EE-induced anxiolysis was associated with the attenuation of enhanced microglial density (using Iba-1 as the marker) in the DG and medial prefrontal cortex. Additionally, EE reduced Iba1 + CD68+ microglia density within the anterior DG. Hence, the successful attenuation of trait anxiety by EE was associated in part with the normalization of neuro-inflammatory imbalances. These results suggest that pharmacological and/or positive behavioral therapies triggering microglia-targeted anti-inflammatory effects could be promising as novel alternatives or complimentary anxiolytic therapeutic approaches in specific subgroups of individuals predisposed to trait anxiety.

Toll-like receptors (TLR) constitute a highly conserved class of receptors through which the innate immune system responds to both pathogen- and host-derived factors. Although TLRs are involved in a wide range of central nervous system (CNS) disorders including neurodegenerative diseases, the molecular events leading from CNS injury to activation of these innate immune receptors remain elusive. The stress protein heat shock protein 60 (HSP60) released from injured cells is considered an endogenous danger signal of the immune system. In this context, the main objective of the present study was to investigate the impact of extracellular HSP60 on the brain in vivo.

The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis.

Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain's immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences.

Adult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Delta9-tetrahydrocannabinol (THC) and Cannabidiol (CBD) fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1) deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail.