The initial microglial responses that occur after brain injury and in various neurological diseases are characterized by microglial accumulation in the affected sites of brain that results from the migration and proliferation of these cells. The early-phase signal responsible for this accumulation is likely to be transduced by rapidly diffusible factors. In this study, the possibility of ATP released from injured neurons and nerve terminals affecting cell motility was determined in rat primary cultured microglia. Extracellular ATP and ADP induced membrane ruffling and markedly enhanced chemokinesis in Boyden chamber assay. Further analyses using the Dunn chemotaxis chamber assay, which allows direct observation of cell movement, revealed that both ATP and ADP induced chemotaxis of microglia. The elimination of extracellular calcium or treatment with pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulphonic acid, suramin, or adenosine-3Ј-phosphate-5Ј-phosphosulfate did not inhibit ATP-or ADP-induced membrane ruffling, whereas AR-C69931MX or pertussis toxin treatments clearly did so. As an intracellular signaling molecule underlying these phenomena, the small G-protein Rac was activated by ATP and ADP stimulation, and its activation was also inhibited by pretreatment with pertussis toxin. These results strongly suggest that membrane ruffling and chemotaxis of microglia induced by ATP or ADP are mediated by G i/o -coupled P2Y receptors. Key words: microglia; ATP; ADP; membrane ruffling; chemotaxis; G i/o -coupled P2Y receptorsAccumulated evidence suggests that extracellular ATP functions in various tissues and cells (Dubyak and El-Moatassim, 1993). The roles of extracellular ATP as a neurotransmitter and neuromodulator in the CNS have been well documented. For example, ATP induces excitation and increases in calcium in various neurons in the brain (Edwards et al., 1992;Shen and North, 1993;Chen et al., 1994;Inoue et al., 1995;Nabekura et al., 1995). In addition to the role played by ATP in neurons, effects of ATP on glial cells have also been demonstrated. In astrocytes, for example, DNA synthesis, process formation, and the increase in the expression of glial fibrillary acidic protein (Neary et al., 1994), arachidonic acid release (Chen and Chen, 1998), Erk activation (Neary et al., 1999), and calcium wave propagation (Scemes et al., 2000) were reported to be stimulated by ATP. Ca 2ϩ release from internal stores by ATP stimulation was also reported in oligodendrocytes (Kirischuk et al., 1995). This evidence suggests diverse roles of extracellular ATP in the CNS.Reports have shown that ATP stimulates microglia, another kind of glial cell in the CNS, to release various biologically active substances, such as interleukin-1␤ (Ferrari et al., 1996(Ferrari et al., , 1997, plasminogen (Inoue et al., 1998), and tumor necrosis factor-␣ (Hide et al., 2000). Microglial cell death was also demonstrated after stimulation with high-dose ATP (Ferrari et al., 1999). After neuronal damage, microglia migrate to the affected sites, where the...

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.

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A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons. Keywords: a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, development, GluR1 subunit, post-synaptic density-95/ Dlg/ ZO-1 domain, Shank3, synapse. Transmission at excitatory synapses is primarily mediated by glutamate acting on three classes of ligand-gated ion channels, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and NMDA receptors (Wisden and Seeburg 1993;Hollmann and Heinemann 1994). In addition to their role in synaptic transmission, these glutamate receptors (GluRs) have been thought to play a crucial role in many brain functions, including activity-dependent synaptogenesis during development and synaptic plasticity (McDonald and Johnston 1990;Bliss and Collingridge 1993).Many excitatory synapses in young developing neurons have been found to express only NMDA receptors, which are continuously blocked by magnesium at resting membrane potentials. As no evoked transmission is observed even when glutamate is present, these synapses are referred to as 'silent synapses'. During later development, AMPA receptors are delivered and clustered on the synaptic membrane in an activity-dependent manner, and the synapses subsequently become functionally active (Durand et al. 1996;Wu et al. 1996;Pickard et al. 2000;Liao et al. 2001;Isaac 2003). Thus, the clustering of AMPA receptors on the synaptic membrane is an essential event during synaptogenesis. Address correspondence and reprint requests to S. Kohsaka, Department of Neurochemistry, National Institute of Neu...

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