Drug combination of topical ripasudil and brimonidine enhances neuroprotection in a mouse model of optic nerve injury.

PURPOSE: To determine whether combination of topical ripasudil and brimonidine has more effective neuroprotection on retinal ganglion cells (RGCs) following injury to axons composing the optic nerve. METHODS: Topical ripasudil, brimonidine, or mixture of both drugs were administered to adult mice after optic nerve injury (ONI). The influence of drug conditions on RGC health were evaluated by the quantifications of surviving RGCs, phosphorylated p38 mitogen-activated protein kinase (phospho-p38), and expressions of trophic factors and proinflammatory mediators in the retina. RESULTS: Topical ripasudil and brimonidine suppressed ONI-induced RGC death respectively, and mixture of both drugs further stimulated RGC survival. Topical ripasudil and brimonidine suppressed ONI-induced phospho-p38 in the whole retina. In addition, topical ripasudil suppressed expression levels of TNFα, IL-1ß and monocyte chemotactic protein-1 (MCP-1), whereas topical brimonidine increased the expression level of basic fibroblast growth factor (bFGF). CONCLUSIONS: Combination of topical ripasudil and brimonidine may enhance RGC protection by modulating multiple signaling pathways in the retina.

Microglia sense astrocyte dysfunction and prevent disease progression in an Alexander disease model.

Alexander disease (AxD) is an intractable neurodegenerative disorder caused by GFAP mutations. It is a primary astrocyte disease with a pathological hallmark of Rosenthal fibres within astrocytes. AxD astrocytes show several abnormal phenotypes. Our previous study showed that AxD astrocytes in model mice exhibit aberrant Ca2+ signals that induce AxD aetiology. Here, we show that microglia have unique phenotypes with morphological and functional alterations, which are related to the pathogenesis of AxD. Immunohistochemical studies of 60TM mice (AxD model) showed that AxD microglia exhibited highly ramified morphology. Functional changes in microglia were assessed by Ca2+ imaging using hippocampal brain slices from Iba1-GCaMP6-60TM mice and two-photon microscopy. We found that AxD microglia showed aberrant Ca2+ signals, with high frequency Ca2+ signals in both the processes and cell bodies. These microglial Ca2+ signals were inhibited by pharmacological blockade or genetic knockdown of P2Y12 receptors but not by tetrodotoxin, indicating that these signals are independent of neuronal activity but dependent on extracellular ATP from non-neuronal cells. Our single-cell RNA sequencing data showed that the expression level of Entpd2, an astrocyte-specific gene encoding the ATP-degrading enzyme NTPDase2, was lower in AxD astrocytes than in wild-type astrocytes. In situ ATP imaging using the adeno-associated virus vector GfaABC1D ATP1.0 showed that exogenously applied ATP was present longer in 60TM mice than in wild-type mice. Thus, the increased ATP level caused by the decrease in its metabolizing enzyme in astrocytes could be responsible for the enhancement of microglial Ca2+ signals. To determine whether these P2Y12 receptor-mediated Ca2+ signals in AxD microglia play a significant role in the pathological mechanism, a P2Y12 receptor antagonist, clopidogrel, was administered. Clopidogrel significantly exacerbated pathological markers in AxD model mice and attenuated the morphological features of microglia, suggesting that microglia play a protective role against AxD pathology via P2Y12 receptors. Taken together, we demonstrated that microglia sense AxD astrocyte dysfunction via P2Y12 receptors as an increase in extracellular ATP and alter their morphology and Ca2+ signalling, thereby protecting against AxD pathology. Although AxD is a primary astrocyte disease, our study may facilitate understanding of the role of microglia as a disease modifier, which may contribute to the clinical diversity of AxD.

Severity of Peripheral Infection Differentially Affects Brain Functions in Mice via Microglia-Dependent and -Independent Mechanisms.

Peripheral infection induces inflammation in peripheral tissues and the brain, impacting brain function. Glial cells are key players in this process. However, the effects of peripheral infection on glial activation and brain function remain unknown. Here, we showed that varying degrees of peripheral infection had different effects on the regulation of brain functions by microglia-dependent and -independent mechanisms. Acute mild infection (one-day LPS challenge: 1LPS) exacerbated middle cerebral artery occlusion (MCAO) injury, and severe infection (four-day LPS challenge: 4LPS) for one week suppressed it. MCAO injury was assessed by triphenyltetrazolium chloride staining. We observed early activation of microglia in the 1LPS and 4LPS groups. Depleting microglia with a colony-stimulating factor-1 receptor (CSF1R) antagonist had no effect on 1LPS-induced brain injury exacerbation but abolished 4LPS-induced protection, indicating microglial independence and dependence, respectively. Microglia-independent exacerbation caused by 1LPS involved peripheral immune cells including macrophages. RNA sequencing analysis of 4LPS-treated microglia revealed increased factors related to anti-inflammatory and neuronal tissue repair, suggesting their association with the protective effect. In conclusion, varying degrees of peripheral inflammation had contradictory effects (exacerbation vs. protection) on MCAO, which may be attributed to microglial dependence. Our findings highlight the significant impact of peripheral infection on brain function, particularly in relation to glial cells.

G protein-coupled receptor 55 activated by palmitoylethanolamide is associated with the development of nocturia associated with circadian rhythm disorders.

AIMS: Bladder function is regulated by clock genes and dysregulation of circadian bladder function can cause nocturia. The blood concentration of palmitoylethanolamide (PEA), a fatty acid metabolite, changes with circadian rhythm. Clock gene abnormalities demonstrate the highest PEA levels during the sleep phase. PEA is a GPR55 agonist that influences urination; therefore, increased PEA during the sleep phase may cause nocturia. Herein, we investigated the function of GPR55 to evaluate the relationship between GPR55 and nocturia that evoked higher PEA during the sleep phase in patients with circadian rhythm disorders. MAIN METHODS: Male C57BL/6 mice were used. GPR55 localization was evaluated by immunofluorescence staining, qRT-PCR, and western blotting. Variations in PEA-induced intracellular Ca2+ concentrations were measured in primary cultured mouse urothelial cells (UCs) using Ca2+ imaging. PEA-induced NGF and PGI2 release in UCs was measured by ELISA. The micturition reflex pathway after PEA administration was evaluated using immunofluorescence staining. KEY FINDINGS: GPR55 was predominant in the UC layer. PEA induced release of Ca2+ from the endoplasmic reticulum into the UC cytoplasm. ELISA and immunofluorescence staining revealed that NGF and PGI2 were released from bladder UCs, stimulated the pontine micturition center in mice, and induced nocturia. SIGNIFICANCE: The loss of regular circadian metabolizing rhythm in fatty acids causes higher blood PEA levels during the sleep phase. Binding of PEA to GPR55 in UC may activate the downstream processes of the micturition reflex, leading to nocturia. These findings suggest a new mechanism for nocturia and its potential as a therapeutic target.

Neuroprotection and axon regeneration by novel low-molecular-weight compounds through the modification of DOCK3 conformation.

Dedicator of cytokinesis 3 (DOCK3) is an atypical member of the guanine nucleotide exchange factors (GEFs) and plays important roles in neurite outgrowth. DOCK3 forms a complex with Engulfment and cell motility protein 1 (Elmo1) and effectively activates Rac1 and actin dynamics. In this study, we screened 462,169 low-molecular-weight compounds and identified the hit compounds that stimulate the interaction between DOCK3 and Elmo1, and neurite outgrowth in vitro. Some of the derivatives from the hit compound stimulated neuroprotection and axon regeneration in a mouse model of optic nerve injury. Our findings suggest that the low-molecular-weight DOCK3 activators could be a potential therapeutic candidate for treating axonal injury and neurodegenerative diseases including glaucoma.

Astrocyte Immune Functions and Glaucoma.

Astrocytes, a non-neuronal glial cell type in the nervous system, are essential for regulating physiological functions of the central nervous system. In various injuries and diseases of the central nervous system, astrocytes often change their phenotypes into neurotoxic ones that participate in pro-inflammatory responses (hereafter referred to as "immune functions"). Such astrocytic immune functions are not only limited to brain diseases but are also found in ocular neurodegenerative diseases such as glaucoma, a retinal neurodegenerative disease that is the leading cause of blindness worldwide. The eye has two astrocyte-lineage cells: astrocytes and Müller cells. They maintain the physiological environment of the retina and optic nerve, thereby controlling visual function. Dysfunction of astrocyte-lineage cells may be involved in the onset and progression of glaucoma. These cells become reactive in glaucoma patients, and animal studies have suggested that their immune responses may be linked to glaucoma-related events: tissue remodeling, neuronal death, and infiltration of peripheral immune cells. In this review, we discuss the role of the immune functions of astrocyte-lineage cells in the pathogenesis of glaucoma.

Ocular P2 receptors and glaucoma.

Adenosine triphosphate (ATP), an energy source currency in cells, is released or leaked to the extracellular space under both physiological and pathological conditions. Extracellular ATP functions as an intercellular signaling molecule through activation of purinergic P2 receptors. Ocular tissue and cells release ATP in response to physiological stimuli such as intraocular pressure (IOP), and P2 receptor activation regulates IOP elevation or reduction. Dysregulated purinergic signaling may cause abnormally elevated IOP, which is one of the major risk factors for glaucoma. Glaucoma, a leading cause of blindness worldwide, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs), which are essential retinal neurons that transduce visual information to the brain. An elevation in IOP may stress RGCs and increase the risk for glaucoma pathogenesis. In the aqueous humor of human patients with glaucoma, the ATP level is significantly elevated. Such excess amount of ATP may directly cause RGC death via a specific subtype of P2 receptors. Dysregulated purinergic signaling may also trigger inflammation, oxidative stress, and excitotoxicity via activating non-neuronal cell types such as glial cells. In this review, we discussed the physiological roles of extracellular nucleotides in the ocular tissue and their potential role in the pathogenesis of glaucoma. This article is part of the Special Issue on 'Purinergic Signaling: 50 years'.

Astrocytic dysfunction induced by ABCA1 deficiency causes optic neuropathy.

Astrocyte abnormalities have received great attention for their association with various diseases in the brain but not so much in the eye. Recent independent genome-wide association studies of glaucoma, optic neuropathy characterized by retinal ganglion cell (RGC) degeneration, and vision loss found that single-nucleotide polymorphisms near the ABCA1 locus were common risk factors. Here, we show that Abca1 loss in retinal astrocytes causes glaucoma-like optic neuropathy in aged mice. ABCA1 was highly expressed in retinal astrocytes in mice. Thus, we generated macroglia-specific Abca1-deficient mice (Glia-KO) and found that aged Glia-KO mice had RGC degeneration and ocular dysfunction without affected intraocular pressure, a conventional risk factor for glaucoma. Single-cell RNA sequencing revealed that Abca1 deficiency in aged Glia-KO mice caused astrocyte-triggered inflammation and increased the susceptibility of certain RGC clusters to excitotoxicity. Together, astrocytes play a pivotal role in eye diseases, and loss of ABCA1 in astrocytes causes glaucoma-like neuropathy.

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route.

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

Transient astrocytic mGluR5 expression drives synaptic plasticity and subsequent chronic pain in mice.

Activation of astrocytes has a profound effect on brain plasticity and is critical for the pathophysiology of several neurological disorders including neuropathic pain. Here, we show that metabotropic glutamate receptor 5 (mGluR5), which reemerges in astrocytes in a restricted time frame, is essential for these functions. Although mGluR5 is absent in healthy adult astrocytes, it transiently reemerges in astrocytes of the somatosensory cortex (S1). During a limited spatiotemporal time frame, astrocytic mGluR5 drives Ca2+ signals; upregulates multiple synaptogenic molecules such as Thrombospondin-1, Glypican-4, and Hevin; causes excess excitatory synaptogenesis; and produces persistent alteration of S1 neuronal activity, leading to mechanical allodynia. All of these events were abolished by the astrocyte-specific deletion of mGluR5. Astrocytes dynamically control synaptic plasticity by turning on and off a single molecule, mGluR5, which defines subsequent persistent brain functions, especially under pathological conditions.

The Mlc1 Promoter Directs Müller Cell-specific Gene Expression in the Retina.

Purpose: Because the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain have become essential and have made great progress. However, it is unknown whether the same strategy that is used in the brain can be applied to the retina because retinal glia differs from glia in the brain. Here, we aimed to find a method for selective gene expression in Müller cells (characteristic glial cells in the retina) and identified Mlc1 as a specific promoter of Müller cells. Methods: Mlc1-tTA::Yellow-Cameleon-NanotetO/tetO (YC-Nano) mice were used as a reporter line. YC-Nano, a fluorescent protein, was ectopically expressed in the cell type controlled by the Mlc1 promotor. Immunofluorescence staining was used to identify the cell type expressing YC-Nano protein. Results: YC-Nano-positive (+) signals were observed as vertical stalks in the sliced retina and spanned from the nerve fiber layer through the outer nuclear layer. The density of YC-Nano+ cells was higher around the optic nerve head and lower in the peripheral retina. The YC-Nano+ signals colocalized with vimentin, a marker of Müller cells, but not with the cell markers for blood vessels, microglia, neurons, or astrocytes. Conclusions: The Mlc1 promoter allows us to manipulate gene expression in Müller cells without affecting astrocytes in the retina. Translational Relevance: Gene manipulation under control of Mlc1 promoter offers novel technique to investigate the role of Müller cells.

Adenosine A2B receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development.

Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.

Loss of P2Y1 receptors triggers glaucoma-like pathology in mice.

BACKGROUND AND PURPOSE: Glaucoma, the leading cause of blindness, damages the retinal ganglion cells. Elevated intraocular pressure (IOP) is a high-risk factor for glaucoma, so topical hypotensive drugs are usually used for treatment. Because not all patients do not respond adequately to current treatments, there is a need to identify a new molecular target to reduce IOP. Here, we have assessed the role of P2Y1 receptors in mediating elevated IOP. EXPERIMENTAL APPROACH: P2Y1 receptor agonist was instilled into the eyes of mice, and the IOP changes were measured by a rebound-type tonometer. Expression of P2Y1 receptors was estimated by immunohistochemistry. Ocular function was measured by a multifocal electroretinogram. KEY RESULTS: A single dose of the P2Y1 receptor agonist transiently reduced IOP and such effects were absent in P2Y1 receptor-deficient (P2Y1 KO) mice. P2Y1 receptors were functionally expressed in the ciliary body, trabecular meshwork and Schlemm's canal. Activation of P2Y1 receptors negatively regulated aquaporin 4 (AQP4) function but up-regulated endothelial NOS (eNOS). P2Y1 KO mice showed chronic ocular hypertension regardless of age. P2Y1 KO mice at 3 months old showed no damage to retinal ganglion cells, whereas 12-month-old mice showed a significant loss of these cells and impairment of ocular functions. Damage to retinal ganglion cells was attenuated by chronic administration of an IOP-reducing agent. CONCLUSION AND IMPLICATIONS: Activation of P2Y1 receptors reduced IOP via dual pathways including AQP4 and eNOS. Loss of P2Y1 receptors resulted in glaucomatous optic neuropathy, suggesting that P2Y1 receptors might provide an effective target in the treatment of glaucoma.

Transnasal transplantation of human induced pluripotent stem cell-derived microglia to the brain of immunocompetent mice.

Microglia are the resident immune cells of the brain, and play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are relatively different from mouse microglia. However, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To elucidate the in vivo characteristics of human microglia, methods have been developed to generate and transplant induced pluripotent or embryonic stem cell-derived human microglia into neonatal or adult mouse brains. However, its widespread use remains limited by the technical difficulties of generating human microglia, as well as the need to use immune-deficient mice and conduct invasive surgeries. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia and transplant them into the brain via a transnasal route in immunocompetent mice, in combination with a colony stimulating factor 1 receptor antagonist. We found that human microglia were able to migrate through the cribriform plate to different regions of the brain, proliferate, and become the dominant microglia in a region-specific manner by occupying the vacant niche when exogenous human cytokine is administered, for at least 60 days.

Reactive astrocyte-driven epileptogenesis is induced by microglia initially activated following status epilepticus.

Extensive activation of glial cells during a latent period has been well documented in various animal models of epilepsy. However, it remains unclear whether activated glial cells contribute to epileptogenesis, i.e., the chronically persistent process leading to epilepsy. Particularly, it is not clear whether interglial communication between different types of glial cells contributes to epileptogenesis, because past literature has mainly focused on one type of glial cell. Here, we show that temporally distinct activation profiles of microglia and astrocytes collaboratively contributed to epileptogenesis in a drug-induced status epilepticus model. We found that reactive microglia appeared first, followed by reactive astrocytes and increased susceptibility to seizures. Reactive astrocytes exhibited larger Ca2+ signals mediated by IP3R2, whereas deletion of this type of Ca2+ signaling reduced seizure susceptibility after status epilepticus. Immediate, but not late, pharmacological inhibition of microglial activation prevented subsequent reactive astrocytes, aberrant astrocyte Ca2+ signaling, and the enhanced seizure susceptibility. These findings indicate that the sequential activation of glial cells constituted a cause of epileptogenesis after status epilepticus. Thus, our findings suggest that the therapeutic target to prevent epilepsy after status epilepticus should be shifted from microglia (early phase) to astrocytes (late phase).

Potential roles of astrocytes and Müller cells in the pathogenesis of glaucoma.

Glaucoma, a progressive optic neuropathy and the leading cause of blindness, is characterized by impairment or degeneration of retinal ganglion cells (RGCs), which transmit visual information to the brain. Currently, 70 million people worldwide are affected by glaucoma. Elevated intraocular pressure (IOP), a major risk factor of glaucoma, directly damages RGCs. However, a substantial proportion of glaucoma patients have a normal IOP level. In particular, over 90% of Japanese glaucoma patients are reported to have normal IOP levels. Thus, a new focus for glaucoma pathology has emerged. Glial cells contribute to tissue homeostasis. Under pathological conditions, glial cells become reactive, lose their homeostatic functions, and gain neurotoxic functions, which trigger neurodegeneration in several diseases including glaucoma. Reactive glial cells have been identified in the eyes of glaucoma patients. In a glaucoma animal model, reactive glial cells are observed at early stages of the disease when RGCs are intact, indicating the possible role of glial cells in the pathogenesis of glaucoma. In this review, we introduce potential roles of glial cells in the pathogenesis of glaucoma. We focus on the roles of the ocular macroglial cells such as astrocytes and Müller cells, and discuss their roles in the pathogenesis of glaucoma.

[Pathogenic roles of retinal glia in glaucoma].

Glaucoma, progressive optic neuropathy, is the first cause of blindness in Japan. Blindness in this disease is induced by damages or degeneration of retinal ganglion cells (RGCs), retinal neurons transmit visual information to brain. An elevated intraocular pressure (IOP) is widely recognized as one of the most important risk factors and that IOP directly damages RGCs by mechanical stress, however, accumulating evidences have shown that a majority of Japanese patients for primary open angle glaucoma shows normal level of IOP. Thus, new target for glaucoma pathology is emerged. In this issue, we introduce potential roles of glial cells for pathogenesis of glaucoma. In the CNS, reactive gliosis has been recognized in a variety of neurodegenerative diseases. Such glial activation is also found in retinae of human glaucoma patients and animal models. Importantly, glial activation precedes RGS degeneration, indicating the possibility that reactive glial cells actively contribute to pathogenesis of glaucoma. In this issue, we will focus on macroglial cells such as Muller cells and astrocytes, and discuss their roles in glaucoma.

Author Correction: Intermittent restraint stress induces circadian misalignment in the mouse bladder, leading to nocturia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microglial ROCK is essential for chronic methylmercury-induced neurodegeneration.

Methylmercury (MeHg), an environmental pollutant, causes serious damage to many organs. Effects on the CNS were initially thought to arise from MeHg acting directly on neurons, but it also has significant effects on non-neuronal cells such as microglia. Microglia, which are very sensitive to changes in the brain environment, show various phenotypes. We previously reported that upon short exposure to MeHg (MeHgshort ) at low concentration, microglia exhibited a neuroprotective phenotype; whereas, long-term exposure (MeHglong ) induced a neurotoxic phenotype of microglia. However, contributions of microglia to MeHg-induced CNS damage remain unknown. Even at very low concentrations, MeHglong but not MeHgshort caused significant neuronal damage associated with an increased number of reactive microglia in cortical slices from wild-type (WT) mice. Two-photon imaging of cortical slices from Iba1-GFP mice revealed that microglia in control conditions exhibited elongated and complex processes with high motility. MeHglong caused a significant reduction in process motility, retraction of processes, and hypertrophic cell bodies, indicating activated microglia. Moreover, MeHglong -treated microglia upregulated pro-inflammatory molecues, suggesting a change into a neurotoxic phenotype of microglia. As a molecular target, Rho-kinase (ROCK) was found to be key for controlling microglial reactivity and neurotoxicity. Expression level of ROCK was increased by MeHglong in WT slices, which was abolished by minocycline or Y-27632. We confirmed that MeHg directly activates microglial ROCK pathways prepared from WT mice. In addition, MeHg-evoked damage of primary neurons was significantly enhanced by the presence of microglia from WT mice, but offset by minocycline or Y-27632. Taken together, our data demonstrate that MeHg causes neurodegeneration by inducing a neurotoxic microglia phenotype via a ROCK-mediated mechanism.

Evaluation of M1-microglial activation by neurotoxic metals using optimized organotypic cerebral slice cultures.

M1-microglia (neurotoxic microglia) regulate neuronal development and cell death and are involved in many pathologies in the brain. Although organotypic brain slice cultures are widely used to study the crosstalk between neurons and microglia, little is known about the properties of microglia in the mouse cerebral cortex slices. Here, we aimed to optimize the mouse cerebral slice cultures that reflect microglial functions and evaluate the effects of neurotoxic metals on M1-microglial activation. Most microglia in the cerebral slices prepared from postnatal day (P) 7 mice were similar to mature microglia in adult mice brains, but those in the slices prepared from P2 mice were immature, which is a conventional preparation condition. The degree of expression of M1-microglial markers (CD16 and CD32) and inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) by lipopolysaccharide, a representative microglia activator, in the cerebral slices of P7 mice were higher than that in the slices of P2 mice. These results indicate that M1-microglial activation can be evaluated more accurately in the cerebral slices of P7 mice than in those of P2 mice. Therefore, we next examined the effects of various neurotoxic metals on M1-microglial activation using the cerebral slices of P7 mice and found that methylmercury stimulated the activation to M1-microglia, but arsenite, lead, and tributyltin did not induce such activation. Altogether, the optimized mouse cerebral slice cultures used in this study can be a helpful tool to study the influence of various chemicals on the central nervous system in the presence of functionally mature microglia.