Dermal macrophages set pain sensitivity by modulating the amount of tissue NGF through an SNX25-Nrf2 pathway.

Cross-talk between peripheral neurons and immune cells is important in pain sensation. We identified Snx25 as a pain-modulating gene in a transgenic mouse line with reduced pain sensitivity. Conditional deletion of Snx25 in monocytes and macrophages, but not in peripheral sensory neurons, in mice (Snx25cKO mice) reduced pain responses in both normal and neuropathic conditions. Bone marrow transplantation using Snx25cKO and wild-type mice indicated that macrophages modulated pain sensitivity. Expression of sorting nexin (SNX)25 in dermal macrophages enhanced expression of the neurotrophic factor NGF through the inhibition of ubiquitin-mediated degradation of Nrf2, a transcription factor that activates transcription of Ngf. As such, dermal macrophages set the threshold for pain sensitivity through the production and secretion of NGF into the dermis, and they may cooperate with dorsal root ganglion macrophages in pain perception.

Juvenile social isolation affects the structure of the tanycyte-vascular interface in the hypophyseal portal system of the adult mice.

Accumulating evidence indicates that social stress in the juvenile period affects hypothalamic-pituitary-adrenal (HPA) axis activity in adulthood. The biological mechanisms underlying this phenomenon remain unclear. We aimed to elucidate them by comparing adult mice that had experienced social isolation from postnatal day 21-35 (juvenile social isolation (JSI) group) with those reared normally (control group). JSI group mice showed an attenuated HPA response to acute swim stress, while the control group had a normal response to this stress. Activity levels of the paraventricular nucleus in both groups were comparable, as shown by c-Fos immunoreactivities and mRNA expression of c-Fos, Corticotropin-releasing factor (CRF), Glucocorticoid receptor, and Mineralocorticoid receptor. We found greater vascular coverage by tanycytic endfeet in the median eminence of the JSI group mice than in that of the control group mice under basal condition and after acute swim stress. Moreover, CRF content after acute swim stress was greater in the median eminence of the JSI group mice than in that of the control group mice. The attenuated HPA response to acute swim stress was specific to JSI group mice, but not to control group mice. Although a direct link awaits further experiments, tanycyte morphological changes in the median eminence could be related to the HPA response.

Olig2-astrocytes express neutral amino acid transporter SLC7A10 (Asc-1) in the adult brain.

We have reported that the transcription factor Olig2 labels a subpopulation of astrocytes (Olig2-astrocytes), which show distribution patterns different from those of GFAP-expressing astrocytes (GFAP-astrocytes) in the adult brain. Here, to uncover the specific functions of Olig2-astrocytes, we first analyzed public single-cell RNA-seq databases of adult mouse brains. Unbiased classification of gene expression profiles and subsequent gene ontology analyses revealed that the majority of Olig2-astrocytes belonged to an astrocytic cluster that is enriched for transporter-related genes. SLC7A10 (also known as ASC-1) was one of the representative neutral amino acid transporter genes in the cluster. To complement the in silico data analyses, we differentially isolated Olig2- and GFAP-astrocytes from the same frozen section of the lateral globus pallidus using laser microdissection and compared their gene expression by quantitative reverse transcription PCR. We confirmed that Olig2 and GFAP mRNAs were preferentially expressed in the Olig2- and GFAP-astrocytes, respectively, indicating that the laser microdissection method yielded minimal cross-contamination between two types of cells. The Olig2-astrocytes expressed significantly higher levels of SLC7A10 mRNA than the GFAP-astrocytes, corroborating the in silico data. We next localized SLC7A10 protein by immunohistochemistry in the lateral globus pallidus, which was also genetically labeled for Olig2. SLC7A10 co-localized with Olig2-genetic labeling, especially on the fine processes of Olig2-astrocytes. These results are consistent with the recent discovery that SLC7A10 is expressed not only in neurons but also in a subset of astrocytes. Taken together, our findings suggest that SLC7A10 exerts specific functions in Olig2-astrocytes of the adult brain.

Large-scale electron microscopic volume imaging of interfascicular oligodendrocytes in the mouse corpus callosum.

Single oligodendrocytes produce myelin sheaths around multiple axons in the central nervous system. Interfascicular oligodendrocytes (IOs) facilitate nerve conduction, but their detailed morphologies remain largely unknown. In the present study, we three-dimensionally reconstructed IOs in the corpus callosum of adult mouse using serial block face scanning electron microscopy. The cell bodies of IOs were morphologically polarized and extended thick processes from the cytoplasm-rich part of the cell. Processes originating from the cell body of each IO can be classified into two types: one myelinates an axon without branching, while the other type branches and each branch myelinates a distinct axon. Myelin sheaths originating from a particular IO have biased thicknesses, wrapping axons of a limited range of diameters. Consistent with this finding, IOs transduced and visualized with a rabies viral vector expressing GFP showed statistically significant variation in their myelination patterns. We further reconstructed the sheath immediately adjacent to that derived from each of the analyzed IOs; the thicknesses of the pair of sheaths were significantly correlated despite emanating from different IOs. These results suggest that a single axon could regulate myelin sheath thicknesses, even if the sheaths are derived from distinct IOs. Collectively, our results indicate that the IOs have their own myelin profiles defined by myelin thickness and axonal diameter although axons may regulate thickness of myelin sheath.

SNX25 regulates proinflammatory cytokine expression via the NF-κB signal in macrophages.

Innate immunity is the first line of defense against bacterial infection and is initiated by macrophages. Sorting nexin 25 (SNX25) is an SNX family member and is reported to negatively regulate TGF-ß signaling by enhancing TGF receptor degradation. However, few studies have focused on the relationship between SNX25 and the immune system. We knocked down SNX25 expression in macrophages and examined inflammatory cytokine expression, a hallmark of innate immunity, after lipopolysaccharide stimulation. SNX25 knockdown increased proinflammatory cytokine expression in RAW 264.7 cells. In addition, SNX25 knockdown activated the NF-κB signal by promoting ubiquitination of IκBα. These results suggest that SNX25 inhibits the NF-κB signal and thereby regulates proinflammatory cytokine expression in macrophages.

Neural expression of sorting nexin 25 and its regulation of tyrosine receptor kinase B trafficking.

Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. Recent reports point to critical roles for SNX25 as a negative regulator of transforming growth factor ß signaling, but the expression patterns of SNX25 in the central nervous system (CNS) remain almost uncharacterized. Here, we show widespread neuronal expression of SNX25 protein and Snx25 mRNA using immunohistochemistry and in situ hybridization. As an exception, SNX25 was present in the Bergmann glia of the cerebellum. SNX25 immunoreactivity was found in cholinergic and catecholaminergic neurons. Moreover, SNX25 colocalized with tropomyosin receptor kinase B (TrkB) in the neurons of the cortex and hippocampus. In vitro, SNX25 can interact with full-length TrkB, but not with its C-terminal-truncated isoform. Overexpression of SNX25 accelerated degradation of full-lengh TrkB, indicating that SNX25 promotes the trafficking of TrkB for lysosomal degradation. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the regulation of BDNF-TrkB signaling in the CNS.

Circadian rhythms of sorting nexin 25 in the mouse suprachiasmatic nucleus.

Entrainment of mammalian circadian rhythms requires receptor-mediated signaling in the hypothalamic suprachiasmatic nucleus (SCN), the site of the master circadian pacemaker. Receptor-mediated signaling is regulated by endocytosis, indicating that endocytosis-related proteins contribute to SCN pacemaking. Sorting nexin 25 (SNX25) belongs to the sorting nexin superfamily, whose members are responsible for membrane attachment to organelles of the endocytic system. In this study, we showed that Snx25 mRNA and SNX25 protein are highly expressed and exhibit remarkable circadian rhythms in the SCN of adult mice. Expression was maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. These findings suggest that SNX25 is a new actor in endocytic signaling, perhaps contributing to the circadian pacemaking system.

Blood-to-brain communication in the hypothalamus for energy intake regulation.

The arcuate nucleus (Arc) integrates circulating hormonal and metabolic signals to control energy expenditure and intake. One of the most important routes that enables the Arc to sense circulating molecules is through the median eminence (ME), which lacks a typical blood-brain barrier. However, the mechanism by which circulating molecules reach the Arc neurons remains unclear. This review focuses on what is known to date regarding the special structure and permeability of the ME vasculature and active transport of circulating molecules from the ME to the Arc. Recent studies have demonstrated that the ME displays angiogenic behavior that is expected to provide high vascular permeability. Parenchymal diffusion of circulating molecules from the ME vasculature is size-dependent, and tanycytes actively transport circulating molecules from the ME to the Arc. Finally, we highlight structural plasticity of the Arc and ME as playing an important role in maintaining energy balance homeostasis.

The Role of the Wnt Signaling Pathway in Upper Jaw Development of Chick Embryo.

Cleft lip with or without cleft palate (CLP) usually results from a failure of the medial nasal prominences to fuse with the lateral and maxillary prominences. This failure inhibits facial morphogenesis regulated by several major morphogenetic signaling pathways. We hypothesized that CLP results from the failure of the Wnt signaling pathway. To examine whether Wnt signaling can influences upper jaw development, we applied beads soaked with Dickkopf-1 (Dkk-1), Alsterpaullone (AL) or Wnt3a to the right side of the maxillary prominence of the chick embryo. The embryo showed a defect of the maxilla on the treated side, and skeletal staining revealed hypoplasia of the premaxilla and palatine bone as a result of Dkk-1-soaked bead implantation. 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers in the treated maxillary prominence were significantly lower at both 24 and 48 hr after implantation. Down-regulation of the expression of Bmp4, Tbx22, Sox9, and Barx1 was confirmed in the maxillary prominence treated with Dkk-1, which indicated that the deformity of the maxillary bone was controlled by gene targets of the Wnt signaling pathway. Expression of N-cadherin was seen immunohistochemically in the maxillary prominences of embryos at 6 hr and increased at 24 hr after AL treatment. Wnt signaling enhanced by AL or Wnt3a up-regulated the expression levels of Msx1, Bmp4, Tbx22, Sox9, and Barx1. Our data suggest that the Wnt signaling pathway regulates maxillary morphogenesis and growth through Bmp4, Tbx22, Sox9, and Barx1. Wnt signaling might regulate N-cadherin expression via Msx1, resulting in cell aggregation for osteochondrogenesis.

Responses of perivascular macrophages to circulating lipopolysaccharides in the subfornical organ with special reference to endotoxin tolerance.

BACKGROUND: Circulating endotoxins including lipopolysaccharides (LPS) cause brain responses such as fever and decrease of food and water intake, while pre-injection of endotoxins attenuates these responses. This phenomenon is called endotoxin tolerance, but the mechanisms underlying it remain unclear. The subfornical organ (SFO) rapidly produces proinflammatory cytokines including interleukin-1ß (IL-1ß) in response to peripherally injected LPS, and repeated LPS injection attenuates IL-1ß production in the SFO, indicating that the SFO is involved in endotoxin tolerance. The purpose of this study is to investigate features of the IL-1ß source cells in the SFO of LPS-non-tolerant and LPS-tolerant mice. METHODS: We first established the endotoxin-tolerant mouse model by injecting LPS into adult male mice (C57BL/6J). Immunohistochemistry was performed to characterize IL-1ß-expressing cells, which were perivascular macrophages in the SFO. We depleted perivascular macrophages using clodronate liposomes to confirm the contribution of IL-1ß production. To assess the effect of LPS pre-injection on perivascular macrophages, we transferred bone marrow-derived cells obtained from male mice (C57BL/6-Tg (CAG-EGFP)) to male recipient mice (C57BL/6N). Finally, we examined the effect of a second LPS injection on IL-1ß expression in the SFO perivascular macrophages. RESULTS: We report that perivascular macrophages but not parenchymal microglia rapidly produced the proinflammatory cytokine IL-1ß in response to LPS. We found that peripherally injected LPS localized in the SFO perivascular space. Depletion of macrophages by injection of clodronate liposomes attenuated LPS-induced IL-1ß expression in the SFO. When tolerance developed to LPS-induced sickness behavior in mice, the SFO perivascular macrophages ceased producing IL-1ß, although bone marrow-derived perivascular macrophages increased in number in the SFO and peripherally injected LPS reached the SFO perivascular space. CONCLUSIONS: The current data indicate that perivascular macrophages enable the SFO to produce IL-1ß in response to circulating LPS and that its hyporesponsiveness may be the cause of endotoxin tolerance.

Abrogated Caveolin-1 expression via histone modification enzyme Setdb2 regulates brain edema in a mouse model of influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is a serious complication that can follow influenza virus infection. Once a cytokine storm is induced during influenza virus infection, tight junction protein disruption occurs, which consequently leads to blood-brain barrier (BBB) breakdown. However, the details of IAE pathogenesis are not well understood. Here, we established a murine IAE model by administration of lipopolysaccharide following influenza virus infection. Brains from IAE model mice had significantly higher expression of type I interferons and inflammatory cytokines. In addition, the expression of Caveolin-1, one of the key proteins that correlate with protection of the BBB, was significantly lower in brains from the IAE group compared with the control group. We also found that, among 84 different histone modification enzymes, only SET domain bifurcated 2 (Setdb2), one of the histone methyltransferases that methylates the lysine 9 of histone H3, showed significantly higher expression in the IAE group compared with the control group. Furthermore, chromatin immunoprecipitation revealed that methylation of histone H3 lysine 9 was correlated with repression of the Caveolin-1 promoter region. These studies identify Caveolin-1 as a key regulator of BBB permeability in IAE and reveal that it acts through histone modification induced by Setdb2.

The inner mitochondrial membrane protein ANT1 modulates IL-6 expression via the JNK pathway in macrophages.

Mitochondria are increasingly associated with inflammation. Here, we focus on the relationship between inflammation and adenine nucleotide translocator type 1 (ANT1), which is localized in the mitochondrial inner membrane. ANT1 plays an important role in oxidative phosphorylation, and mutations in the ANT1 gene are responsible for mitochondrial diseases. Ample studies have demonstrated that ANT1 has a critical role in cardiomyocytes and neurons, but little has been reported on its functions in immune cells. We knocked down ANT1 expression in macrophages and examined inflammatory cytokine expression after lipopolysaccharide stimulation. ANT1 knockdown reduces the expression of IL-6. JNK, upstream of IL-6, is downregulated, but other MAP kinases and the NF-κB signaling remain unchanged. These results suggest that ANT1 modulates IL-6 expression through JNK in macrophages.

NGF and BDNF expression in mouse DRG after spared nerve injury.

Neuropathic pain is initiated by a primary lesion in the peripheral nervous system and spoils quality of life. Neurotrophins play important roles in the development and transmission of neuropathic pain. There are conflicting reports that the dorsal root ganglion (DRG) in an injured nerve contribute to neuropathic pain, whereas several studies have highlighted the important contribution of the DRG in a non-injured nerve. Clarifying the role of neurotrophins in neuropathic pain is problematic because we cannot distinguish injured and intact neurons in most peripheral nerve injury models. In the present study, to elicit neuropathic pain, we used the spared nerve injury (SNI) model, in which injured DRG neurons are distinguishable from intact ones, and mechanical allodynia develops in the intact sural nerve skin territory. We examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRGs of SNI model mice. NGF and BDNF levels increased in the injured L3 DRG, while NGF decreased in the intact L5 DRG. These data offer a new point of view on the role of these neurotrophins in neuropathic pain induced by peripheral nerve injury.

Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells.

Oxidative stress contributes to myocardial ischemia-reperfusion injury, which causes cardiomyocyte death and precipitate life-threatening heart failure. Propofol has been proposed to protect cells or tissues against oxidative stress. However, the mechanisms underlying its beneficial effects are not fully elucidated. In the present study, we employed an in vitro oxidative injury model, in which rat cardiac H9c2 cells were treated with H2O2, and investigated roles of propofol against oxidative stress. Propofol treatment reduced H2O2-induced apoptotic cell death. While H2O2 induced expression of the antioxidant enzyme HO-1, propofol further increased HO-1 mRNA and protein levels. Propofol also promoted nuclear localization of Nrf2 in the presence of H2O2. Knockdown of Nrf2 using siRNA suppressed propofol-inducible Nrf2 and expression of Nrf2-downstream antioxidant enzyme. Knockdown of Nrf2 suppressed the propofol-induced cytoprotection. In addition, Nrf2 overexpression induced nuclear localization of Nrf2 and HO-1 expression. These results suggest that propofol exerts antioxidative effects by inducing nuclear localization of Nrf2 and expression of its downstream enzyme in cardiac cells. Finally, we examined the effect of propofol on cardiomyocytes using myocardial ischemia-reperfusion injury models. The expression level of Nrf2 protein was increased at 15 min after reperfusion in the ischemia-reperfusion and propofol group compared with ischemia-reperfusion group in penumbra region. These results suggest that propofol protects cells or tissues from oxidative stress via Nrf2/HO-1 cascade.

Olig2-Lineage Astrocytes: A Distinct Subtype of Astrocytes That Differs from GFAP Astrocytes.

Astrocytes are the most abundant glia cell type in the central nervous system (CNS), and are known to constitute heterogeneous populations that differ in their morphology, gene expression and function. Although glial fibrillary acidic protein (GFAP) is the cardinal cytological marker of CNS astrocytes, GFAP-negative astrocytes can easily be found in the adult CNS. Astrocytes are also allocated to spatially distinct regional domains during development. This regional heterogeneity suggests that they help to coordinate post-natal neural circuit formation and thereby to regulate eventual neuronal activity. Here, during lineage-tracing studies of cells expressing Olig2 using Olig2CreER; Rosa-CAG-LSL-eNpHR3.0-EYFP transgenic mice, we found Olig2-lineage mature astrocytes in the adult forebrain. Long-term administration of tamoxifen resulted in sufficient recombinant induction, and Olig2-lineage cells were found to be preferentially clustered in some adult brain nuclei. We then made distribution map of Olig2-lineage astrocytes in the adult mouse brain, and further compared the map with the distribution of GFAP-positive astrocytes visualized in GFAPCre; Rosa-CAG-LSL-eNpHR3.0-EYFP mice. Brain regions rich in Olig2-lineage astrocytes (e.g., basal forebrain, thalamic nuclei, and deep cerebellar nuclei) tended to lack GFAP-positive astrocytes, and vice versa. Even within a single brain nucleus, Olig2-lineage astrocytes and GFAP astrocytes frequently occupied mutually exclusive territories. These findings strongly suggest that there is a subpopulation of astrocytes (Olig2-lineage astrocytes) in the adult brain, and that it differs from GFAP-positive astrocytes in its distribution pattern and perhaps also in its function. Interestingly, the brain nuclei rich in Olig2-lineage astrocytes strongly expressed GABA-transporter 3 in astrocytes and vesicular GABA transporter in neurons, suggesting that Olig2-lineage astrocytes are involved in inhibitory neuronal transmission.

Microglia support ATF3-positive neurons following hypoglossal nerve axotomy.

Microglia are essential in developmental processes and maintenance of neuronal homeostasis. Experimental axotomy of motor neurons results in neurodegeneration, and microglia in motor nuclei become activated and migrate towards injured neurons. However, whether these activated microglia are protective or destructive to neurons remains controversial. In the present study, we transected the hypoglossal nerve in BALB/c mice, causing activating transcription factor 3 (ATF3) and growth associated protein 43 (GAP43) induction, and partial neuronal death. Inhibition of microglial accumulation by minocycline administration impaired microglial accumulation, decreased GAP43 mRNA expression, and reduced motor neuron survival. Expression of ATF3 contributed to nerve regeneration, and increased within 6 h after axotomy, prior to microglial migration. Further, microglial contact with neuronal cell bodies was associated with neuronal ATF3 expression. Colchicine administration blocked lesion-induced ATF3 transcription in axotomized neurons and microglial accumulation. In addition, perineuronal microglia-derived ciliary neurotrophic factor (CNTF) increased, indicating that perineuronal microglia in the hypoglossal nucleus protect axotomized motor neurons by releasing trophic factors. We also observed that microglia secrete CNTF and that neurons have CNTFRα and can respond to it in vitro. CNTF promote neurite elongation and neuronal survival of primary cultured neurons. Microglia make contact through unknown neuronal signals that are possibly regulated by ATF3 in hypoglossal nucleus. Moreover, they play important roles in regenerating motor neurons and are potential new therapeutic targets for motor neuron diseases.

Changes in endothelial cell proliferation and vascular permeability after systemic lipopolysaccharide administration in the subfornical organ.

The subfornical organ (SFO) has highly permeable fenestrated vasculature and is a key site for immune-to-brain communications. Recently, we showed the occurrence of continuous angiogenesis in the SFO. In the present study, we found that systemic administration of bacterial lipopolysaccharide (LPS) reduced the vascular permeability and endothelial cell proliferation. In LPS-administered mice, the SFO vasculature showed a significant decrease in the immunoreactivity of plasmalemma vesicle associated protein-1, a marker of endothelial fenestral diaphragms. These data suggest that vasculature undergoes structural change to decrease vascular permeability in response to systemic LPS administration.

Voluntary Exercise Induces Astrocytic Structural Plasticity in the Globus Pallidus.

Changes in astrocyte morphology are primarily attributed to the fine processes where intimate connections with neurons form the tripartite synapse and participate in neurotransmission. Recent evidence has shown that neurotransmission induces dynamic synaptic remodeling, suggesting that astrocytic fine processes may adapt their morphologies to the activity in their environment. To illustrate such a neuron-glia relationship in morphological detail, we employed a double transgenic Olig2(CreER/WT); ROSA26-GAP43-EGFP mice, in which Olig2-lineage cells can be visualized and traced with membrane-targeted GFP. Although Olig2-lineage cells in the adult brain usually become mature oligodendrocytes or oligodendrocyte precursor cells with NG2-proteoglycan expression, we found a population of Olig2-lineage astrocytes with bushy morphology in several brain regions. The globus pallidus (GP) preferentially contains Olig2-lineage astrocytes. Since the GP exerts pivotal motor functions in the indirect pathway of the basal ganglionic circuit, we subjected the double transgenic mice to voluntary wheel running to activate the GP and examined morphological changes of Olig2-lineage astrocytes at both the light and electron microscopic levels. The double transgenic mice were divided into three groups: control group mice were kept in a cage with a locked running wheel for 3 weeks, Runner group were allowed free access to a running wheel for 3 weeks, and the Runner-Rest group took a sedentary 3-week rest after a 3-week running period. GFP immunofluorescence analysis and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology comparable to that of the Control group in the Runner-Rest group. Our results indicated that the fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively participate in motor functions.

Hedgehog Signaling Modulates the Release of Gliotransmitters from Cultured Cerebellar Astrocytes.

Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family, plays essential roles in the development of the central nervous system. Recent studies suggest that the Hh signaling pathway also functions in mature astrocytes under physiological conditions. We first examined the expression of genes encoding Hh signaling molecules in the adult mouse cerebellum by in situ hybridization histochemistry. mRNA for Patched homolog 1 (Ptch1), a receptor for Hh family members, was expressed in S100ß-positive astrocytes and Shh mRNA was expressed in HuC/D-positive neurons, implying that the Hh signaling pathway contributes to neuro-glial interactions. To test this hypothesis, we next examined the effects of recombinant SHH N-terminal protein (rSHH-N) on the functions of cultured cerebellar astrocytes. rSHH-N up-regulated Hh signal target genes such as Ptch1 and Gli-1, a key transcription factor of the Hh signaling pathway. Although activation of Hh signaling by rSHH-N or purmorphamine influenced neither glutamate uptake nor gliotransmitters release, inhibition of the Hh signaling pathway by cyclopamine, neutralizing antibody against SHH or intracellular Ca(2+) chelation decreased glutamate and ATP release from cultured cerebellar astrocytes. On the other hand, cyclopamine, neutralizing antibody against SHH or Ca(2+) chelator hardly affected D-serine secretion. Various kinase inhibitors attenuated glutamate and ATP release, while only U0126 reduced D-serine secretion from the astrocytes. These results suggested that the Hh signaling pathway sustains the release of glutamate and ATP and participates in neuro-glial interactions in the adult mouse brain. We also propose that signaling pathways distinct from the Hh pathway govern D-serine secretion from adult cerebellar astrocytes.