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...

We previously reported that extracellular ATP induces membrane ruffling and chemotaxis of microglia and suggested that their induction is mediated by the Gi/o-protein coupled P2Y(12) receptor (P2Y(12)R). Here we report discovering that the P2X(4) receptor (P2X(4)R) is also involved in ATP-induced microglial chemotaxis. To understand the intracellular signaling pathway downstream of P2Y(12)R that underlies microglial chemotaxis, we examined the effect of two phosphatidylinositol 3'-kinase (PI3K) inhibitors, wortmannin, and LY294002, on chemotaxis in a Dunn chemotaxis chamber. The PI3K inhibitors significantly suppressed chemotaxis without affecting ATP-induced membrane ruffling. ATP stimulation increased Akt phosphorylation in the microglia, and the increase was reduced by the PI3K inhibitors and a P2Y(12)R antagonist. These results indicate that P2Y(12)R-mediated activation of the PI3K pathway is required for microglial chemotaxis in response to ATP. We also found that the Akt phosphorylation was reduced when extracellular calcium was chelated, suggesting that ionotropic P2X receptors are involved in microglial chemotaxis by affecting the PI3K pathway. We therefore tested the effect of various P2X(4)R antagonists on the chemotaxis, and the results showed that pharmacological blockade of P2X(4)R significantly inhibited it. Knockdown of the P2X(4) receptor in microglia by RNA interference through the lentivirus vector system also suppressed the microglial chemotaxis. These results indicate that P2X(4)R as well as P2Y(12)R is involved in ATP-induced microglial chemotaxis.

Extracellular nucleotides, including ATP, have been demonstrated to transmit important physiological signals in the brain through either G-protein-coupled P2Y receptors or P2X receptors, which are ligand-gated ion channels. In this study, we performed a detailed analysis of the expression of the Gi/o-coupled receptor P2Y12 in the brain. Northern blot analysis demonstrated that P2Y12 is expressed predominantly in the brain, and to a lesser extent in the spleen. The cellular localization of P2Y12 was investigated by in situ hybridization, and P2Y12 mRNA was detected in small cells distributed throughout the brain, including the hippocampus. Expression of P2Y12 was also observed in naive and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy. Selective expression of P2Y12 mRNA in microglia was confirmed by double-label in situ hybridization and immunohistochemistry with antibodies against NeuN and Iba1 as an immunohistochemical marker for neurons and microglia, respectively. Hardly any P2Y12 mRNA was detected in macrophages obtained from the spleen and abdominal cavity, which share many surface molecules with microglia.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABA A Rs). Although neurosteroids are accepted allosteric modulators of GABA A Rs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABA A Rs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABA A R subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABA A R subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABA A Rs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.PKC | tonic current | receptor insertion | current rundown N eurosteroids are synthesized de novo in the brain from cholesterol, or steroid hormone precursors. Raising neurosteroid levels in the CNS causes anxiolysis, sedation/hypnosis, anticonvulsant action, and anesthesia and reduces depressivelike behaviors (1-3). Accordingly, dysregulation of neurosteroid signaling is associated with premenstrual dysphoric disorder, panic disorder, depression, schizophrenia, and bipolar disorder. Neurosteroids exert the majority of their actions via potentiating the activity of γ-aminobutyric acid receptors (GABA A Rs), which mediate the majority of fast synaptic inhibition in the adult brain. Accordingly, at low nanomolar concentrations they potentiate GABA-dependent currents, whereas at micromolar concentrations they directly activate GABA A Rs (4-8).GABA A Rs are Cl − -preferring pentameric ligand-gated ion channels that assemble from eight families of subunits: α(1-6), β(1-3), γ(1-3), δ, e, ө, π, and ρ(1-3) (9, 10). Receptor subtypes composed of α1-3βγ subunits largely mediate synaptic or phasic inhibition, whereas those constructed from α4-6β1-3, with or without γ/δ subunits, are principal determinants of tonic inhibition (11-13). Neurosteroids have been shown to bind GABA A Rs at an allosteric site distinct from that of GABA, benzodiazepines, or barbiturates (9, 14). Hosie et al. identified residues located within the transmembrane domain of GABA A R α and β subunits that are critical for the direct activation (α1-6; Threonine 236, β1-3; Tyrosine 284) and allosteric potentiation (α1-6 Asparagine 407, and α1-6 Glutamine 246) of neurosteroids (15-17). Accordingly, mutation of glutamine 241 (Q241) within the α1-6 subunits prevents allosteric potentiation of GABA A R composed of αβγ and αβδ subunits by neurosteroids (15,16).In addition to modulating channel gating, neurosteroids exert...

γ-Aminobutyric acid type A receptors (GABAARs) are the principal mediators of fast synaptic inhibition in the brain as well as the low persistent extrasynaptic inhibition, both of which are fundamental to proper brain function. Thus unsurprisingly, deficits in GABAARs are implicated in a number of neurological disorders and diseases. The complexity of GABAAR regulation is determined not only by the heterogeneity of these receptors but also by its posttranslational modifications, the foremost, and best characterized of which is phosphorylation. This review will explore the details of this dynamic process, our understanding of which has barely scratched the surface. GABAARs are regulated by a number of kinases and phosphatases, and its phosphorylation plays an important role in governing its trafficking, expression, and interaction partners. Here, we summarize the progress in understanding the role phosphorylation plays in the regulation of GABAARs. This includes how phosphorylation can affect the allosteric modulation of GABAARs, as well as signaling pathways that affect GABAAR phosphorylation. Finally, we discuss the dysregulation of GABAAR phosphorylation and its implication in disease processes.