Metabotropic glutamate receptor type 5 contributes to the spontaneous Ca2+ rhythms in the striatal neurons and astrocytes

Abstracts / Neuroscience Research 71S (2011) e108–e415

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endogenous adenosine produced via extracellular breakdown from ATP in the hippocampus of the rats.

P4-d01 Induction of inflammatory and anti-inflammatory cytokines in microglia

doi:10.1016/j.neures.2011.07.1427

Tomohiro Saito , Kenji Sudo, Hiroaki Miyamori, Kazuyuki Nakajima

P4-c20 Microglia regulates oligodendrocyte progenitor cell responses and remyelination Tatsuhide Tanaka , Koichi Murakami, Yoshio Bando, Shigetaka Yoshida Dept. of Functional of Anatomy and Neuroscience, Asahikawa Medical University, Asahikawa, Japan Disruption of myelin causes severe neurological diseases. Understanding of the molecular mechanisms that control myelination or remyelination is needed to develop therapeutic strategies for demyelinating diseases. However, details of the process of myelination and remyelination remain largely unknown. Feeding mice the copper chelator cuprizone (bis(cyclohexanone) oxalyldihydrazone) causes CNS demyelination. After 6 weeks of exposure of C57BL/6 mice to cuprizone, myelin basic protein (MBP) and 2 ,3 -cyclic nucleotide-3 -phosphodiesteeae (CNPase), myelin components, drastically reduced in the corpus callosum. On the other hand, the numbers of astrocytes and microglia in the corpus callosum were increased and these cells expressed various inflammatory cytokines and neurotrophic factors in the demyelinating and remyelinating corpus callosum. These data indicate that a dynamically regulated glial network plays a role during demyelination and remyelination. To explore the role of microglia on remyelination, we examined the effect of minocycline, which inhibits microglial activation, on the remyelination process in this model. Although decreased myelin components by cuprizone treatment were recovered in the remyelinating corpus callosum, minocycline suppressed the recovery of these myelin components in the remyelinating process. Furthermore, minocycline decreases olig1 and sox10, which are essential for differentiation of oligodendrocytes, in the remyelinating process. These results indicate that microglia activation is an important process for the response of oligodendrocyte progenitor cells (OPC) during remyelination. doi:10.1016/j.neures.2011.07.1428

Toshiaki

, Shinichi

Kohsaka 2 ,

Kazuyuki

Nakajima 1,2

1

Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo, Japan 2 Dept. of Neurochemistry, National Institute of Neurosciece, Tokyo, Japan When the brain is injured or affected by neurological disorders, inflammatory cytokines including tumor necrosis factor alpha (TNF␣) are released from activated microglia. However, it is poorly known how the production of TNF␣ in activated microglia is modulated in vivo. In this study, we investigated the modulation of TNF␣ produced in microglia in vitro from the viewpoint of intercellular interaction with astrocytes. Microglia and astrocytes were prepared from neonatal rat brain-derived primary culture. Both cell-type did not produce any TNF␣ in non-stimulated condition. However, microglia induced the cytokine when stimulated with lipopolysaccharide (LPS), and the amounts were promoted in mixed-culture with astrocytes, suggesting that astrocytes stimulate microglia. Thus, to separate the contact effects and the soluble factor-mediated effects of astrocytes, we used transwell that has double bottoms. Microglia and astrocytes were seeded on the outer well and the inner well, respectively, and the cultures were stimulated with LPS. Although astrocytes alone did not produce TNF␣, and microglia alone produced some amounts of TNF␣, abundant TNF␣ was induced in the combined wells, suggesting the mediation of astrocytes-derived soluble factor. The astrocytic conditioned medium actually promoted the production of TNF␣ in LPS-stimulated microglia in dose-dependent manner. Therefore, the promoting effects of astrocytes on the production of TNF␣ in microglia can be explained by the mediation of astrocytes-derived soluble factors. doi:10.1016/j.neures.2011.07.1429

doi:10.1016/j.neures.2011.07.1430

P4-d02 Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia Yohei Okubo 1 , Masato Mashimo 1 , Toshiko Yamazawa 1 , Miwako Yamasaki 2 , Masahiko Watanabe 2 , Toshihiko Murayama 3 , Masamitsu Iino 1 1

P4-c21 Up-regulation of tumor necrosis factor alpha (TNF␣) production in microglia through the intercellular interaction with astrocytes Masuda 1

Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo In this study, we investigated whether or not inflammatory and antiinflammatory cytokines are induced in microglia in vivo, and how their induction is regulated. To induce inflammatory and anti-inflammatory cytokines in the brain, lipopolysaccharide (LPS) was injected into rat cerebral cortex. Immunoblotting analysis revealed that the inflammatory cytokines (TNF␣ and IL-1␤) and anti-inflammatory cytokines (IL-4 and IL-10) are induced in LPS-injected region, but not in the control area. The time course experiment indicated that the amounts of the inflammatory and antiinflammatory cytokines peak at 1 day after LPS-injection and decrease thereafter. The inflammatory cytokines (TNF␣ and IL-1␤) were immunohistochemically detected in activated microglia. The anti-inflammatory cytokine (IL-10) was also stained in activated microglia. The cell-type inducing IL-4 was not identified in brain parenchyma. As above, we demonstrated that the inflammatory cytokines (TNF␣ and IL-1␤) and anti-inflammatory cytokine (IL-10) were induced in LPS-stimulated microglia in vivo. We next examined their regulation in microglia prepared from rat neonatal brainderived primary culture. In nonstimulated microglia, TNF␣, IL-1␤ and IL-10 were not detected. However, in microglia stimulated with LPS, these three cytokines were induced and their amounts increased depending on the concentration of LPS and culture time. Accordingly, the inflammatory and anti-inflammatory cytokines detected in LPS-injected brain were suggested to be derived from microglia activated by LPS.

Dept. Pharmacol., Grad. Sch. Med., Univ. of Tokyo, Tokyo, Japan 2 Dept. Anatomy, Hokkaido Univ. Sch. of Med., Sapporo, Japan 3 Lab. Chem. Pharmacol., Grad. Sch. Pharm. Sci., Chiba Univ., Chiba, Japan

The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we report a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5trisphosphate (IP3 ) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber (PF)-Purkinje cell (PC) synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP3 signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum. doi:10.1016/j.neures.2011.07.1431

P4-d03 Metabotropic glutamate receptor type 5 contributes to the spontaneous Ca2+ rhythms in the striatal neurons and astrocytes Atsushi Tamura 1,2 , Naohiro Yamada 3 , Yuichi Yaguchi 3 , Yoshio Machida 1 , Issei Mori 1 , Makoto Osanai 1,2 1 3

Grad. Sch. of Med., Tohoku Univ., Sendai, Japan 2 JST, CREST, Tokyo, Japan Grad. Sch. Eng., Osaka Univ., Suita, Japan

Calcium ion (Ca2+ ) is an important messenger for intracellular signal transduction processes. We conducted the Ca2+ imaging study on striatal slices of GFAP-GFP mice, which expressed GFP in astrocytes. Long lasting spontaneous intracellular Ca2+ ([Ca2+ ]i ) transients (spontaneous Ca2+ rhythms), which lasted up to about 300 s, were observed in both GFP positive cells (astrocytes) and GFP negative cells (putative neurons). We found the significant correlated pairs of the spontaneous Ca2+ rhythms both between neurons, and between a neuron and an astrocyte. Tetrodotoxin (TTX) blocked little the Ca2+ rhythms in both neurons and glial cells. On the other hand, the frequencies of the spontaneous Ca2+ rhythms in the both types of cells were greatly

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Abstracts / Neuroscience Research 71S (2011) e108–e415

reduced by administration of the intracellular Ca2+ store depletor, thapsigargin or IP3 receptor antagonist, 2-APB. Therefore, these Ca2+ rhythms were not induced by action potential, and mainly due to Ca2+ release from an intracellular Ca2+ store (i.e. endoplasmic reticulum) via IP3 receptor.Activation of some kind of metabotropic receptors induces the Ca2+ release from an intracellular Ca2+ store. It was reported that metabotropic glutamate receptor type 5 (mGluR5) was one of therapeutic targets for Parkinson’s disease. Thus, we investigated effects of mGluR5 on the spontaneous Ca2+ rhythms. Antagonist of mGluR5, MPEP, suppressed the spontaneous Ca2+ rhythms. Thus, mGluR5PLC-IP3 signal cascade might be concerned with the Ca2+ rhythms. This result suggested that mGluR5 might contribute to the information processing in striatum by means of regulating intracellular Ca2+ concentration. Research fund: JST, CREST.

kept at low level. Similarly, those in GDNF-administered facial nucleus did not recover. These results indicate that GDNF provided to cut facial nerve is able to suppress microglial activation/proliferation in the nucleus, but cannot restore the functionally depressed state of injured motoneurons.

doi:10.1016/j.neures.2011.07.1432

The mesencephalic trigeminal nucleus (Me5) receives proprioceptive sensory afferent of the trigeminal nerve from the jaw-closing muscle spindles and Ruffini nerve ending of the periodontal ligaments, and also send their axons to the motor trigeminal nucleus. Also in recent years, elucidation of glial cell-neuron network advances, the fact that the glia cells in the hippocampus and the cerebellum have responded variously in neural network has been shown. But, detailed research concerning the distribution and role of the glia cells in the brain stem such as the pons and the medulla has not been shown. In this study, we used two types of the glial marker, glial fibrillary acidic protein (GFAP) and S100 calcium binding protein (S100). We recently reported that S100-like immunoreactivity was observed in the trigeminal motor nucleus (Mo5), on the other hand, GFAP-like immunoreactivity was observed around the Mo5. The aim of this study was to investigate the distribution of the glia cell in the Me5. As a result, GFAP and S100-like immunoreactivity was observed in many regions of the brain stem including the Me5. They made contacts with Me5 neuronal cell body on the same focal place. Recently, it has been reported S100 protein worked as a neurotransmitter to the neuron from astrocyte, and adjusted neural activity in the brain. Thus, these results suggest possibility the S100 positive cells in the Me5 participate in the jaw-jerk reflex.

P4-d04 Regulation of microglial proliferation by cell cycleassociated proteins in the transected rat facial nucleus Shinichi Yamamoto 1 , Shinichi Kohsaka 2 , Kazuyuki Nakajima 1,2 1 2

Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo Dept. of Neurochemistry, National Institute of Neuroscience

Transection of rat facial nerve leads to an increase of microglial cell number in the ipsilateral facial nucleus. In the previous study, we demonstrated that up-regulated macrophage-colony stimulating factor (M-CSF) in the transected facial nucleus triggers the induction of cFms (receptor for M-CSF) and proliferating cell nuclear antigen (PCNA) in microglia and causes the microglia to divide. However, little is known about the relationship between the microglial proliferation and cell cycle-associated proteins in the transected facial nucleus. In the present study, we analyzed a change in the amounts of cell cycle-associated proteins including cyclins, cyclin-dependent protein kinase (Cdk) and Cdk inhibitors in the axotomized facial nucleus. Immunoblotting revealed that cyclin A is induced 3 days after transection and the high levels were maintained for 5–7 days. The cyclin A was immunohistochemically recognized in activated microglia in transected facial nucleus. Cyclin D was observed to increase in transected facial nucleus during 3–7 days after transection. Cyclin E was found to decrease at early time after injury. Likewise, analysis of Cdks indicated that Cdk2 and Cdk4 are present in the transected facial nucleus, suggesting that the Cdks serve as mitotic promoting factor by forming complex with suitable cyclin. In addition, a Cdk inhibitor p21 turned out to increase 5 days after axotomy. These results strongly suggest that cyclin (A, D and E), Cdks (2 and 4) and Cdk inhibitor (p21) are involved in the regulation of microglial proliferation in transected rat facial nucleus. doi:10.1016/j.neures.2011.07.1433

P4-d05 Glial cell line-derived neurotrophic factor attenuates microglial activation/proliferation, but not restore functionally down-regulated motoneurons Yoshinaru Honda 1 , Toshihumi Ichimiya 1 , Shinichi Kohsaka 2 , Kazuyuki Nakajima 1,2 1 Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo, Japan 2 Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo, Japan

Transection of adult rat facial nerve led to the increased number of activated microglia in the ipsilateral facial nucleus. We tested whether or not glial cell line-derived neurotrophic factor (GDNF) is able to alter the microglial activation/proliferation in the injured facial nucleus. GDNF-containing gelfoam was administered at the cut facial nerve, and the facial nucleus in brainstem was removed at the desired time point. The effects on microglial activation/proliferation were evaluated by determining the amounts of ionized Ca2+ binding adapter molecule 1 (Iba1) as a microglial marker, cFms as a receptor of macrophage-colony stimulating factor (M-CSF) and proliferating cell nuclear antigen (PCNA) as a proliferation marker. Immunoblotting revealed that the amounts of Iba1, cFms and PCNA in GDNF-administered facial nucleus are significantly less than those in PBS-administered facial nucleus (control side). In support of this result, immunohistochemical experiments indicated that the number of cells stained with Iba1, cFms and PCNA antibodies in the GDNF-administered facial nucleus was significantly lower than that in PBS-administered facial nucleus, suggesting that GDNF prevents microglial activation/proliferation. We next investigated the effects of GDNF on the injured motoneurons. As reported previously, the amounts of vesicular acetylcholine transporter (VAChT) severely decrease in the transected facial nucleus. In PBS-administered facial nucleus, the amounts of VAChT were

doi:10.1016/j.neures.2011.07.1434

P4-d06 S100-immunoreactive cells contacting with rat mesencephalic trigeminal neurons Akira Kawata , Tomohisa Sugiyama, Tadaomi Akagi, Seiji Akaike, Hideko Tsuzuki, Kazuyoshi Higashi, Osamu Takahashi Div. of Histology, Dept. of Anatomy, Kanagawa Dental College, Yokosuka, Japan

doi:10.1016/j.neures.2011.07.1435

P4-d07 Fabp7, a brain specific fatty acid binding protein, regulates proliferation of oligodendrocyte precursor cells Ryuichi Kimura 1 , Kaichi Yoshizaki 1,2 , Youko Matsumoto 1 , Yuji Tsunekawa 1,2 , Noriko Osumi 1,2 1 2

Div. of Dev. Neurosci., Grad. Sch. of Med., Tohoku Univ., Sendai, Japan Tohoku Neuroscience Global COE

Oligodendrocyte precursor cells (OPCs) are considered as the fourth type of glial cells in the central nervous system in addition to astrocytes, oligodendrocytes and microglia. We have previously revealed that proliferation of OPCs in the hippocampus followed circadian rhythm, in which the proliferation increased in the dark period and decreased in the light period. However, the molecular mechanisms of diurnal proliferation of OPCs remain unclear. The expression of some protein in the brain is modulated by circadian rhythm. Fabp7, a brain specific fatty acid binding protein, is one of such proteins, the expression of which increases in the dark period and decreases in the light period. To know the role of Fabp7 on the diurnal proliferation of OPCs, we first examined whether OPCs were expressed Fabp7 in the hippocampus of the wild type C57BL6 mice at 8 weeks. We found that the expression of Fabp7 was observed in 97/98 OPCs marked with PDGFR␣. Next, in order to investigate the proliferation of hippcampal OPCs, we injected BrdU three times per day into the wild type and Fabp7 knockout mice (Owada et al., 2006). The number of BrdU+PDGFR␣+ cells was reduced by 71.0% in Fabp7 knockout mice compared to the wild type littermates. These results suggest that Fabp7 promotes the proliferation of OPCs. In the further study we will examine whether oscillated expression of Fabp7 is relate with OPCs proliferation during the day. doi:10.1016/j.neures.2011.07.1436

P4-d08 Involvement of adenosine A3 receptor in microglial process extension Keiko Ohsawa , Tomomi Sanagi, Yasuko Nakamura, Eri Suzuki, Shinichi Kohsaka Dept. Neurochem., Natl. Inst. Neurosci., Tokyo, Japan Microglia have highly branched and motile cell processes and monitor the brain parenchyma under physiological conditions. In response to pathological stimuli microglia show morphological change and migrate toward the