European Journal of Neuroscience

European Journal of Neuroscience, Vol. 29, pp. 1108–1118, 2009 doi:10.1111/j.1460-9568.2009.06659.x

MOLECULAR AND DEVELOPMENTAL NEUROSCIENCE

Glutamate induces directed chemotaxis of microglia

Guo Jun Liu,1,2,3 Rajini Nagarajah,1 Richard B. Banati2,3 and Max R. Bennett1 1The Neurobiology Laboratory, Brain and Mind Research Institute, University of Sydney, NSW 2050, Australia 2Medical Radiation Sciences, Imaging Laboratories, Brain and Mind Research Institute, Faculty of Health Sciences, University of Sydney, NSW, Australia 3Life Sciences, Australian Nuclear Science and Technology Organisation, PMB 1 Menai, NSW, Australia

Keywords: GFP transgenic mice, glia, neurotransmitters, receptors, spinal cord slice, tissue culture

Abstract Microglia in the brain possess dynamic processes that continually sample the surrounding parenchyma and respond to local insults by rapidly converging on the site of an injury. One of the chemotaxic agents responsible for this response is ATP. Here we show that the transmitter glutamate is another such chemotaxic agent. Microglia exposed to glutamate increase their cell membrane ruffling and migrate to a source of glutamate in cell culture and in spinal cord slices. This chemotaxis is meditated by a-amino-3-hydroxy-5- methylisoxazole-4-propionic acid (AMPA) and metabotropic glutamate receptors on the microglia. Chemotaxis is dependent on redistribution of actin filaments in the cells and on tubulin following receptor activation. Thus glutamate, which is released at synapses as well as from damaged cells, can mediate rapid chemotaxic responses from microglial cells.

Introduction Ramified (resting) microglia are present in large numbers in the brain et al., 2008). With concomitant release of UDP there is phagocytosis parenchyma, amounting to between 10% and 20% of all glial cells to of neuronal debris following activation of microglial P2Y6 purinergic be found there (Vaughan & Peters, 1974; Banati, 2003). These glia receptors (Koizumi et al., 2007). However, the most ubiquitous are highly motile with very active extension and retraction of their transmitter in the brain is glutamate that is at very high concentration extensive processes that can occur half a dozen times in 40 min in neurons and glial cells. Microglia possess glutamate receptors, in (Nimmerjahn et al., 2005), indicating that far from resting they are particular group III metabotropic receptors (mGlu4,6,8), which reduce continually surveying their microenvironment. Perturbations to this microglia neurotoxicity (Taylor et al., 2003), and group II metabo- environment, such as occurs if neurons are injured (Koizumi et al., tropic receptors (mGlu2,3), which enhance microglia neurotoxicity 2007) and die (Marin-Teva et al., 2004) or if there is loss of the (Taylor et al., 2002, 2005; Biber et al., 2007). It is possible that integrity of a vascular wall (Nimmerjahn et al., 2005), leads to the glutamate acting on these and other receptors might also affect the ramified microglia becoming reactive, increasing motility and motility and direction of migration of microglial cells, for instance chemotaxis to the site of the injured cells, which may then be towards regions of high glutamate release, whether due to high levels phagocytized. of synaptic transmission or the direct release of glutamate from The question arises as to the principal substances released from the neurons following ischaemia or exocytotic insults. We have investi- injured cells that are responsible for the conversion of nearby gated this possibility in the present work. microglia from the ramified to the reactive state, their increase in motility and subsequent chemotaxis to the site of injury. Three of these that have received a lot of attention are fractalkine, chemokine (C-C Materials and methods motif) ligand 21 (CCL21) and ATP. Both fractalkine and CCL21 are Microglial culture chemokines, released from nerve terminals, that diffuse to engage receptors on microglia, increasing their motility and inducing Protocols for handling animals in this study were reviewed and chemotaxis (de Jong et al., 2005; Re & Przedborski, 2006). ATP, approved by the Animal Ethics Committee at the University of released from injured cells, also increases the motility and chemotaxis Sydney, Australia. Microglia were cultured from 1 to 2-day-old of the processes of nearby microglia to the injured site by acting on neonatal Sprague–Dawley rats according to methods described previously (Liu et al., 2006). Briefly, the rats were anaesthetized microglia P2Y12 purinergic receptors (Honda et al., 2001; Haynes et al., 2006), which also mediate ATP-facilitated transmission of and killed by i.p. injection with 0.2 mL of Lethabarb (325 mg ⁄ mL calcium waves between cells (Farber & Kettenmann, 2005; Bennett pentobarbital; Virbac, Peakhurst, Australia), and the thoracolumbar regions of the spinal cords were dissected and placed in ice-cold Hank’s balanced salt solution (in mm: NaCl, 137; KCl, 5; KH2PO4, Correspondence: Dr M. R. Bennett, as above. 0.4; NaHCO3,4;Na2HPO4, 0.3; d-glucose, 5; pH 7.4). After E-mail: [email protected] removing the meninges, the spinal cord segments were treated with Received 26 April 2008, revised 13 December 2008, accepted 12 January 2009 0.25% trypsin (Sigma-Aldrich, St Louis, MO, USA) at 37C for

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd Glutamate-induced chemotaxis of microglia 1109

20 min. The neurons and glial cells were suspended in Dulbecco’s Observing microglial cell membrane ruffling modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented The purified microglia grown on poly-D-lysine-pre-coated glass with 10% cosmic calf serum (HyClone, Logan, UT, USA) and 1% coverslips (20 lg ⁄ mL; Sigma-Aldrich) in the supplemented DMEM penicillin ⁄ streptomycin ⁄ glutamine (Invitrogen, Carlsbad, CA, USA), for 1–2 days were used for the experiments. After the microglia were and placed into culture flasks pre-coated with poly-d-lysine washed with bath solution (in mm: NaCl, 137; KCl, 5; CaCl2, 2.5; (20 lg ⁄ mL; Sigma-Aldrich). Culture flasks were placed at 37Cin MgCl2, 1.2; HEPES, 10; pH 7.4) at room temperature (22–24C) for a5%CO2 atmosphere, and culture medium was exchanged with 1:00 h, they were transferred to the recording chamber. After taking fresh supplemented DMEM every 2–3 days. Ten–14 days after initial controls for 5 min, they were perfused (3 mL ⁄ min) with the bath plating, neurons disappeared and astrocytes formed a confluent solution (negative control), with the bath solution plus glutamate monolayer on the flask surface, whereas microglia and oligodendro- (varied concentration), and with the bath solution plus ATP (100 lm, cytes grew loosely attached on the surface of the astrocyte positive control). The microglia were viewed under an Olympus monolayer and were detached by gentle shaking of the culture flask microscope (BX50WI; Olympus, Tokyo, Japan), and transmitted light using a rotating shaker (Heidolph Unimax, Germany) at 350 rpm and images from the cultured microglia and fluorescent images from GFP- 37C for 15 min. Detached cells from the astrocyte monolayer in the microglia spinal cord slice were captured with a digital camera medium were removed and pelleted by centrifugation. The pellet of (Cascade 650; Photometrics, Roper Scientific, Tucson, AZ, USA) at cells was re-suspended in the supplemented DMEM and then placed intervals of 10 s for 20 min, which was controlled by a software (QED d onto poly- -lysine-coated 13-mm round glass coverslips in a 24-well in vivo, Media Cybernetics, Silver Spring, MD, USA). plate for experiments of cell membrane ruffling and immunohisto- chemistry, and 18 · 18 mm square (no. 2 thickness) coverslips for experiments on chemotaxis using a Dunn chamber. The cells were Chemotaxis accessed using pressure injection gently washed twice at 15 min after plating because microglia selectively adhere to the poly-d-lysine coating, whereas other cell Chemotaxis of microglia in culture and in spinal cord slices induced types that may be present, such as astrocytes and oligodendrocytes, by glutamate was assessed by microinjection of bath solution and bath m take a longer period of time to adhere (Dobrenis, 1998). The purity solution plus 1 m glutamate (1–2 p.s.i.) through a micropipette with of the microglial cultures was greater than 98%, which was a picospritzer (General Valve, Fairfield, NJ, USA). For cultures the tip confirmed by immunostaining with microglial marker CD11b openings (about 2 lm) of the micropipettes were positioned to areas monoclonal antibody (against Fab fragment of MRC OX42; lacking cell bodies and their processes; these pipettes were placed Robinson et al., 1986; Liu et al., 2006; Werry et al., 2006; about 5 lm above the coverslips to avoid any direct mechanical Invitrogen). Purified microglial cultures of 1–2 days were used for stimulation. In spinal cord slices the area of measurement was also the experiments. about 25 lm in diameter around the tip of the pipette, which showed a shadow under fluorescent. Transmitted light images were captured from cultured microglia and Spinal cord slice preparation fluorescent images were captured from spinal cord slices where GFP- Spinal cord slices were prepared from 10 to 15-days-old B6.129p- labelled microglia fluorescence green. The fluorescent images from the Cx3cr1tm1Litt ⁄ J green fluorescence protein (GFP) mice where mono- slices were directly processed without digitization and the background cytes including microglia are green-fluorescent (Jung et al., 2000). fluorescence, as determined by a lack of cells, was subtracted. The The mice were purchased from the Jackson’s Laboratory (Bar Harbor, changes of fluorescent intensity were then plotted over the period of ME, USA). After mice were decapitated, the dorsal skin was removed observation. Coverslips and slices were only used once and results and the spine from sacral to thoracic vertebral column was exposed, obtained were from at least three repeats for both cultures and spinal transected, dissected free and transferred into ice-cold Krebs solution cord slices. The spinal cord slices used for the experiments were preparations between 2 and 6:00 h, counted from the time of killing (3C) bubbled with 95% O2–5% CO2. This solution contained (in the animals. mm): NaCl, 117; KCl, 3.6; CaCl2, 2.5; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 25; glucose, 11; pH 7.3. The thoracolumbar spinal cord was then dissected from the spine and was transferred to an ice-cold Chemotaxis assessed using Dunn chamber solution bubbled with 95% O2–5% CO2. Due to a high sensitivity of spinal cord cells to anoxia, the time between the decapitation and the Glutamate-induced chemotaxis was assessed by the Dunn chemotaxis transfer of the spinal cord to the ice-cold bubbled Krebs solution was chamber (Hawksley, Sussex, UK), which allows direct observation of kept as short as possible (< 60 s). A thoracolumbar segment of about cell movement. The assay was performed according to the method 2 cm was isolated and transverse slices were obtained. Meninges were described previously (Webb et al., 1996). Briefly, microglia grown on not removed. the poly-d-lysine-pre-coated 18 · 18 mm square coverslips (no. 2 The spinal cord segment merged in 2% agar was glued vertically thickness) for 1–2 days were washed with DMEM at 37C for 1:00 h. with cyanoacrylate glue onto the platform of a home-made vibratome The coverslips were then placed over the chamber, whose outer and chamber filled with oxygenated ice-cold Krebs solution. Transverse inner wells were filled with DMEM pre-bubbled for at least 30 min. slices with a thickness of 200–300 lm were prepared with a vibratome The coverslip was sealed with wax around three edges leaving a slit (Campden, UK). Slices were transferred to a storage chamber for exchange of the medium in the outer well. To observe the containing the Krebs solution and bubbled with 95% O2–5% CO2. chemically directed cell migration (chemotaxis), the medium in the After a recovery period of at least 1:00 h at 35C, slices were outer well was exchanged through a fine plastic tip for DMEM transferred to the recording chamber and fixed by nylon threads glued (negative control), for DMEM plus different concentrations of on a U-shaped platinum wire. Slices were continuously superfused at a glutamate, for DMEM plus glutamate receptor agonists, for DMEM rate of 3–4 mL ⁄ min with the Krebs solution bubbled with 95% plus glutamate (100 ll) and plus glutamate receptor antagonists or O2–5% CO2 at 35C. The slices prepared in 2:00–5:00 h were used for plus intracellular pathway inhibitors, and for DMEM plus ATP the experiments. (positive control). The edge of the coverslip was then sealed

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 1110 G. J. Liu et al. immediately, and the chamber was transferred to a heated stage (35C) cells were counted from at least three transwells and three fields of on the Olympus microscope (BX50WI). Microglia were pre-incubated each transwell with Image J. at least for 40 min with purinergic receptor antagonists, glutamate receptor antagonists and intracellular pathways inhibitors. Microglia were also pre-incubated with ATP degrading enzyme apyrase (Grade Immunohistochemistry III; Sigma-Aldrich) for at least 10 min. Immunohistochemistry was carried out to investigate the effect of A region close to the outer edge of the bridge of the Dunn chamber glutamate on cell division cycle (CDC) 42 and cytoskeleton actin of was viewed and images captured via the cascade 650 camera microglia. (Photometrics, Pleasanton, CA, USA) at intervals of 30 s and Cells grown on the coverslips were incubated with the bath solution 120 min. The camera was controlled by the QED in vivo software at room temperature (22–24C) for 60 min. The cells were then (Media Cybernetics, Bethesda, MD, USA). The captured digital incubated with fresh bath solution (negative control), 1 mm glutamate images were further analyzed using public-shared software provided and 0.1 mm ATP (positive control) at room temperature for another by Image J (WCIF Image J; Wright Cell Image Facility, Toronto, 5 min. The cells were fixed with 4% paraformaldehyde at room Canada). Images of fields of cells were smoothed and then subtracted temperature for 10 min followed by incubation with 1% Triton X-100 from a background field where there were no cell bodies or processes at room temperature for 1 min to increase cell membrane permeability. present. The images were then digitized with the threshold set to The cells were washed three times (10 min ⁄ wash) with phosphate- exclude any remaining background. Each digitized image was then buffered saline (PBS; in mm: NaCl, 137; KCl, 2; Na HPO ,8; subtracted from the previous image using the plug-in function (Delta- 2 4 KH PO , 1) and were then incubated in 2% bovine serum albumin up). The total distance travelled between the starting point and the 2 4 (BSA; Sigma-Aldrich) dissolved in PBS for 60 min to block non- point of a cell reached after 120 min was measured by using the specific binding sites. The cells were then incubated overnight at 4C MTrack2 plug-in of the WCIF program. The distance and direction with mouse anti-rat CD11b antibody (microglial marker; Chemicon, were shown as x-, y-axes on scatter diagrams whose x-axis was Temecula, CA, USA) and rabbit anti-rat CDC42 antibody (a small positioned parallel to the outer edge of the bridge of the Dunn Rho-family GTPase implicated in localized actin dynamics; a purified chamber, while the y-axis was at right angles to this outer edge. rabbit antibody against synthetic peptides surrounding amino acid 144 Results were averaged from more than five microglia per coverslip and of human CDC42 that has species reactivity for human, mouse, rat and from at least three different coverslips. bovine; BioVision, Mountain View, CA, USA; Kumanogoh et al., 2001; Schwamborn & Puschel, 2004; Garcia et al., 2006) diluted in PBS containing 2% BSA (1 : 100 and 1 : 10, respectively). After Chemotaxis assessed using Boyden chamber overnight incubation with the primary antibodies the cells were Chemotaxis of microglia were also performed in transwell (also washed three times (10 min ⁄ wash) with PBS, and then were incubated called Boyden chamber) with cell culture polyethylene terephthalate with the secondary antibodies diluted in PBS containing 2% BSA for membrane with 24-well 8-lm pore size (Falcon, Franklin Lakes, 1:00 h at room temperature. The secondary antibodies were Alexa NJ, USA). One hundred thousand cells in DMEM containing 1 mm Fluor (AF) 594-conjugated goat anti-rabbit (1 : 100) antibody (Invi- glutamine were added to the upper reservoir, and 0.7 mL DMEM trogen) to label CDC42 polyclonal antibody and AF488-conjugated with glutamine with 1 mm glutamine was placed to the lower goat anti-mouse (1 : 200) antibody (Invitrogen) for CD11b monoclo- reservoir. After a 16-h incubation of cells in 5% CO2 at 37C, the nal antibody. After three washes, the cells on the coverslips were lower reservoir was replaced with fresh DMEM in the absence and mounted on glass slides with ProLong Gold antifade reagent presence of chemotaxis reagents (100 lm glutamate and 100 lm containing DAPI (Invitrogen) and sealed with nail polish. The cells ATP). In experiments investigating the effects of P2 receptor were viewed under an Axiovert 200M microscope (Zeiss, Jena, antagonists on glutamate- and ATP-induced chemotaxis of micro- Germany), and images were acquired with a digital camera (AxioCam, glia, 100 lm suramin (Sigma-Aldrich) and 100 lm PPADS (pyri- Zeiss). Specificity of CD11b antibody (1 : 100) was determined by doxal-phosphate-6-azophenyl-2¢,4¢-disulphonic acid; Sigma-Aldrich) positive staining in microglia but negative staining in oligondendro- in DMEM were placed on both upper and lower reservoirs. After cytes and astrocytes. CDC42 antibody (1 : 10 dilution or 20 lg ⁄ mL) 40 min incubation, the lower reservoir was replaced with glutamate was used according to manufacturer’s instructions, which shows only or ATP plus suramin and PPADS. After 4:00 h of migration, cells CDC42 protein labelled in Western blots. To minimize cross- remaining on the top side of the membrane were scraped off with a reactivity, the AF594-conjugated goat anti-rabbit whole antibody has cotton swab. Cells migrated to the bottom of the membrane were been adsorbed against human IgG, human serum, mouse IgG and fixed with 4% paraformaldehyde for 10 min and washed three times bovine serum; while the AF488-conjugated goat anti-mouse whole (5 min each wash). The migrated cells on the bottom of the antibody has been adsorbed against bovine IgG, goat IgG, rabbit IgG, membrane were stained with 4¢,6-diamidino-2-phenylindolePI rat IgG, human IgG and human serum. Specificity of secondary (DAPI; Invitrogen). Images were captured under an Olympus antibodies [AF594-conjugated goat anti-rabbit (1 : 100) antibody and microscope (BX61WI, Olympus) mounted with 4· objective and a AF488-conjugated goat anti-mouse (1 : 200) antibody] was deter- CCD camera (QICAM; Q-Imaging, Surrey, BC, Canada). Migrated mined by incubating both antibodies alone (without primary antibod-

Fig. 1. Glutamate-induced microglial cell membrane ruffling and chemotaxis in cultured rat spinal cord microglia. (A) Representative images of four experiments showing that glutamate (glu) reversibly induced cell membrane ruffling (white arrows in the middle panel). (B) Typical images of microglial chemotaxis in four experiments after constant microinjection of 1 mm glutamate from a micropipette, in which cell bodies and their processes move towards the tip of the pipette. (C) Typical images of microglial chemotaxis in a Dunn chamber after 4 min and 40 min in which the numbered cells 1–4 moved toward (upward) to the source of glutamate. (D and E) Scatter plots of the movement of 22 microglia in Dunn chambers in the Dulbecco’s modified Eagle’s medium (DMEM) (D) and DMEM + 100 lm glutamate (E); there was spontaneous movement but without clear direction in DMEM; in glutamate, the microglia moved towards the glutamate source (high). (F) A dose–response curve of glutamate-induced maximum distance migrated in Dunn chamber with ED50 =26lm (n = 25–182). Scale bars: 10 lm (A–C). Values in (E) shows mean ± SEM.

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 Glutamate-induced chemotaxis of microglia 1111

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 1112 G. J. Liu et al.

Fig. 2. Glutamate-induced microglial cell membrane ruffling and chemotaxis in GFP-transgenic mouse spinal cord slices where microglia fluoresce green. (A) Representative images of four similar experiments showing membrane ruffling before (control), during (glu 5 min) and washout (washout 5 min) of glutamate (1 mm); the cell ruffles are marked by white arrows in the middle panel. (B) Representative images of chemotaxis induced by glutamate applied by microinjection from the micropipette with the tip at the centre of a circle diameter 25 lm; the green fluorescent intensity becomes stronger within the circle during injection of glutamate, indicating microglia movement towards the source of glutamate. (C) Time course of changes in fluorescent intensity within a circle like that in (B) for injection of glutamate (1 mm, n = 4) and for bath solution as control (n = 3), indicating the glutamate-induced chemotaxis was not due to mechanical stimulation that might be generated during injection. The values shown in (C) are the relative values. The relative values are fluorescence intensity after application of glutamate was relative to the average fluorescence intensity prior to injection of glutamate. Scale bars: 10 lm (A and B). ies) with the microglia (Liu et al., 2005, 2006). There was not positive antibody using the protocol as described above except using 0.05% staining observed, which suggests both secondary antibodies (goat Saponin (Sigma-Aldrich) instead of Triton X-100. The cells were anti-rabbit and goat anti-mouse) do not recognize each other. incubated for 1:00 h with AF595-conjugated phalloidin (1 : 40; To view cell membrane ruffling caused by glutamate, AF594- Invitrogen) at the same time that the cells were incubated with conjugated phalloidin was used to label cytoskeletal F-actin using the AF488-conjugated goat-anti-mouse antibody. The remaining protocols one-step immunohistochemical method. The cells grown on coverslips are the same as those for the two-step staining with CD11b antibody. were incubated in the bath solution at room temperature and DMEM at The distribution of CDC42 within cells in the bath solution 37C and 5% CO2–95% O2 for 60 min. The cells were then incubated (negative control), bath plus glutamate and bath plus ATP (positive with fresh bath and DMEM solutions (negative control), 1 mm control) were calculated according to the area of CDC42 vs. the glutamate and 100 lm ATP (positive control) for 5 min at room total area of a cell marked by CD11b antibody. The number of cell temperature for incubations with bath solutions (bath, bath + membrane ruffles marked with phalloidin was compared with the glutamate, bath + ATP) and DMEM (DMEM, DMEM + glutamate, total number of cells marked with CD11b antibody. The relative area DMEM + ATP) at 37C. The microglia were labelled with CD11b of cell membrane ruffling was calculated as a percentage of the total

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 Glutamate-induced chemotaxis of microglia 1113

amplitudes are presented as mean ± SEM. Statistical significance was determined with the use of analysis of variance (anova) with a post hoc analysis, where P < 0.05 was considered significant. The microglia described in the Results refer to spinal microglia unless otherwise specified.

Results Glutamate increases cell membrane ruffling and chemotaxis of cultured microglia Microglia cell processes are increased by the addition of 1 mm glutamate to cultures (cell membrane ruffles; Fig. 1A, n = 15). There is an increase in cell membrane ruffles, during a 2-min period of exposure to different concentrations of glutamate, returning to baseline conditions on removal of the glutamate (Fig. 1A). Pressure injection of glutamate (1 mm) from a micropipette with a 2 lm diameter tip, placed in a microglia culture, leads to the invasion of the field by microglial cell bodies and their processes over a 30-min period (Fig. 1B, n = 4). Microglial cells grown on glass coverslips were placed in Dunn chambers for identifying chemotaxis migration up glutamate concen- tration gradients (Fig. 1C). With just DMEM in the Dunn chamber there is spontaneous movement of microglia but no preferred migrating direction (Fig. 1D), whereas there is a directional migration of microglia towards the source of glutamate (100 lm gradient; Fig. 1C and E). The dose–response curve for different size gradients of glutamate showed an ED50 of 26 lm for the chemotaxis response, measured as the distance migrated up the gradient in 120 min after exposure to glutamate (Fig. 1F, n = 25–182). Chemotaxis migration of spinal cord microglia induced by gluta- mate and ATP (positive control) was also investigated with transwells (Boyden chambers). A random microglia migration in DMEM was found (control, 67 ± 4 cells, n = 12 fields). The number of migrated cells was increased with 100 lm glutamate (263 ± 24 cells, n =12 fields, F2,37 = 60.3, P < 0.001 compared with the control) and m Fig. 3. Glutamate-induced microglial chemotaxis in a Dunn chamber is 100 l ATP (311 ± 14 cells, n = 15 fields, F2,37 = 60.3, P < 0.001 mediated by multiple glutamate receptor subtypes. (A) Chemotactic effects of compared with the control). glutamate receptor agonists, except for N-methyl-d-aspartic acid (NMDA; Glutamate and ATP also induced chemotaxis migration of microglia n = 126), all other glutamate receptor agonists significantly induced chemotaxis, cultured from the hippocampus, the other region of the CNS for which including a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; n = 123), kainate (KA; n = 71) and trans-d,l-1-aminocyclopentane-1,3-dicarbox- we used Boyden chambers. The number of random microglia ylic acid (ACPD; a group I and II metabotropic glutamate receptor agonist, migration in DMEM (control) was 79 ± 5 (n = 9 fields). The migrated n = 126) compared with control [Dulbecco’s modified Eagle’s medium cells increased to 250 ± 22 (n = 15 fields) under 100 lm glutamate (DMEM), n = 84]. (B) Effects of glutamate receptor antagonists on gluta- (F2,32 = 28.4, P < 0.001 compared with the control) and to 316 ± 23 mate-induced microglia chemotaxis; glutamate-induced chemotaxis was not m significantly reduced by NMDA receptor antagonist d())-2-amino-5-phospho- (n = 12 fields) under 100 l ATP (F2,32 = 28.4, P < 0.001 compared nopentanoic acid (AP5; n = 37), but was significantly inhibited by the AMPA with the control). and kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; n = 96), by the specific AMPA receptor antagonist GYKI 52466 [1-(4- aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydro- Glutamate acts as a chemotactic agent for microglia in spinal chloride; GYKI, n = 55] and by metabotropic glutamate receptor antagonists a-methyl-(4-carboxyphenyl)glycine (MCPG; for group I and II) and a-methyl- cord slices (4-sulphonophenyl)glycine (MSPG; for group II and III, n = 127); blockade of Transgenic mice homozygous for GFP+ ⁄ + in microglia provide a dl b glutamate transporters with -threo- -benzyloxyaspartate (TBOA; n = 60) did means for evaluating the extent to which the glutamate-induced not affect glutamate-induced microglia chemotaxis. Vertical error bars in (A) and (B) show mean ± SEM. *P < 0.001. chemotaxis of these cells observed in culture also occurs in situ. Spinal cord slices show highly motile GFP+ ⁄ + microglia, with spontaneous movement of cell bodies as well as processes. Bath perfusion of area of a cell marked by CD11b antibody. All results were from cells glutamate (1 mm) to such spinal cord slices leads to increases in on at least four different coverslips for each condition. microglia cell membrane ruffles (Fig. 2A, n = 4). Placing a micropi- pette tip (2 lm diameter) immediate above (5 lm) a slice and pressure-injecting 1 mm glutamate (at 1 p.s.i.) leads to the migration Chemicals and statistics of microglia cell bodies and processes into the region of the All generic chemicals were purchased from Sigma-Aldrich. All micropipette tip over 30 min (Fig. 2B). This effect was quantitated experiments were repeated at least three times, and values of peak by measuring the increase in fluorescence in a 25-lm-diameter circle

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 1114 G. J. Liu et al.

Fig. 5. Glutamate-induced chemotaxis in a Dunn chamber is mediated by intracellular actin and microtubules. Actin-interrupter cytochalasin D (cyto-D, n = 75) significantly inhibited glutamate-induced chemotaxis and spontaneous microglia movement. Microtubule inhibitor nocodazole (nocod, n = 97) abolished glutamate-induced chemotaxis but not spontaneous movement. PKC inhibitors calphostin C (cal-C, n = 54) and bisindolylmaleimide (bis, n = 73) did not significantly alter glutamate-induced chemotaxis. DMEM, Dulbecco’s modified Eagle’s medium. *P < 0.001.

Glutamate acts on a-amino-3-hydroxy-5-methylisoxazole-4- propionic acid (AMPA) and metabotropic glutamate receptors of cultured microglial cells to induce their chemotaxis Glutamate receptor agonists and antagonists were next used to identify the glutamate receptors on the microglia that mediate the glutamate- induced chemotaxis in the Dunn chamber. Gradients (100 lm)of glutamate (n = 99), AMPA (n = 123), kainate (n = 71) and trans-d, l-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD, an agonist for metabotropic glutamate receptor group I and II, n = 126) produced significantly (F5,623 = 28.8, P < 0.001) more chemotaxis of microglia over that of the DMEM control (n = 84, Fig. 3A), with no significant difference (F5,623 = 28.8, P > 0.05) between the chemotaxis in response to AMPA, kainate and ACPD compared with that due to glutamate alone. N-methyl-d-aspartic acid (NMDA, n = 126) did not Fig. 4. Glutamate-induced chemotaxis migration of microglia in Dunn produce any chemotaxis over controls (Fig. 3A; F5,623 = 28.8, chamber and Boyden chamber is not secondary to ATP release. (A) In Dunn P > 0.05). chamber, there was spontaneous microglial movement in Dulbecco’s modified Consistent with the suggestion that glutamate acts on these Eagle’s medium (DMEM), and the movement becomes greater and directed to receptors are the observations that chemotaxis in response to m m the source of glutamate (100 l , n = 99) and ATP (100 l , positive control, glutamate (100 lm gradient) was significantly reduced n = 26). Glutamate-induced microglial chemotaxis was not significantly altered by purinergic receptor (P2X and P2Y) inhibitors suramin and pyridoxal- (F6,557 = 22.5, P < 0.001 compared with glutamate) by the specific phosphate-6-azophenyl-2¢,4¢-disulphonic acid (PPADS; 100 lm, n = 35), nor AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methy- altered by ATP-degrading enzyme apyrase (n = 37). (B) In the Boyden lenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466; chamber, both ATP (100 lm, n = 15) and glutamate (100 lm, n = 13) 23 lm, n = 55), AMPA and kainate receptor antagonist 6-cyano- significantly increased the number of migrated microglia when compared with m control (DMEM, n = 12). Purinergic receptor antagonists suramin and PPADS 7-nitroquinoxaline-2,3-dione (CNQX; 100 l ; n = 96), combined significantly decreased ATP-induced microglia migration while they did not metabotropic receptor antagonist a-methyl-(4-carboxyphenyl)glycine alter the effect of glutamate (n = 9). *P < 0.001. (MCPG, for group I and II) and a-methyl-(4-sulphonophenyl)glycine (MSPG, for group II and III, n = 127, Fig. 3B), whereas the NMDA about the tip over time. Increases in fluorescence occur within 1 min receptor antagonist d(–)-2-amino-5-phosphonopentanoic acid (AP5; after glutamate injection commences and continue over at least a 100 lm, n = 37) had no effect (F6,557 = 22.5; Fig. 3B). The possi- further 30 min (Fig. 2C, n = 4). The pressure injection of bath bility that the glutamate transporter triggers chemotaxis was examined solution had no effect on chemotaxis of microglia in the slice (Fig. 2C, with the transporter inhibitor dl-threo-b-benzyloxyaspartate (TBOA; n = 3). This suggests that the glutamate effect is not caused by 20 lm, n = 60); this did not (F6,557 = 22.5, P > 0.05) significantly mechanical stimulation due to the pressure injection. reduce the chemotaxis response to glutamate (Fig. 3B). The chemo-

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 Glutamate-induced chemotaxis of microglia 1115 taxis response to glutamate is therefore mediated by AMPA and phalloidin (Fig. 6C), was significantly increased on individual cells metabotropic glutamate receptors on the microglial cells rather than (F1,92 = 250, P < 0.001 for bath; F1,55 = 67.9, P < 0.001 for DMEM; NMDA receptors and glutamate transporters. Fig. 6D), as were the number of cells producing ruffles compared with that in bath solution (F1,25 = 32.1, P < 0.001, P < 0.001) or DMEM (F1,55 = 64.7, P < 0.001; Fig. 6E). Glutamate-induced chemotaxis is not mediated by ATP Microglial cells release ATP in response to glutamate through activation of their AMPA receptors (Liu et al., 2006), and as ATP is Discussion a chemotaxic agent for microglia (see Introduction and also positive Reactive microglia possess group I (mGluR ), group II (mGluR ) control in Fig. 4; n = 26) it is possible that the cells are sensing ATP 1,5 2,3 and group III (mGluR ) metabotropic glutamate receptors (Biber for chemotaxis but not glutamate, although it is not clear how this 4,6,8 et al., 1999; Farber & Kettenmann, 2005; Matute et al., 2006). In would lead to a gradient of ATP in the Dunn and Boyden chambers. addition, reactive microglia possess ionotropic receptors (with In order to eliminate this possibility, microglia were plated in a Dunn subunits GluR ; Noda et al., 2000; Hagino et al., 2004), but chamber with a glutamate gradient (100 lm) and the addition of the 2,3,4,5 not NMDA receptors (Gilabert & McNaughton, 1997; Farber & purinergic receptor antagonists suramin (100 lm) and PPADS Kettenmann, 2005). In the present work the chemotaxis induced by (100 lm). These substances are antagonists for P2Y and 1,2,11,12 glutamate in the Dunn chamber was reproduced when glutamate was P2X (in the case of suramin), and P2Y and P2X (in the 1,2,3,5 4 1–5,7 replaced with AMPA, kainate or the metabotropic glutamate receptor case of PPADS). Present evidence suggests that P2X and P2Y are 4 12 agonist ACPD, but not with NMDA. As AMPA receptors are the the main ATP receptors involved in microglial chemotaxis (Ohsawa main functional ionotropic glutamate receptor rather than kainate et al., 2007). Blocking these purinergic receptors with suramin and receptors (Pocock & Kettenmann, 2007), it seems likely that PPADS (n = 35) had no antagonistic effects on glutamate-driven glutamate’s action on AMPA receptors and metabotropic glutamate chemotaxis of microglia (F = 16.7, P > 0.05; Fig. 4A), nor did 4,264 receptors is primarily responsible for initiating chemotaxis. This was the ATP degrading enzyme apyrase (n = 37, F = 16.7, P > 0.05; 4,264 confirmed by experiments using both glutamate receptor agonists and Fig. 4A). antagonists. Boyden chambers were also used to assess if concentrations of Reactive microglial cells also possess purinergic receptors that suramin and PPADS used in combination with glutamate were induce chemotaxis (Farber & Kettenmann, 2006). P2Y receptors sufficient to antagonize the effect of ATP. ATP at 100 lm increased 12 mediate an increase in motility and chemotaxis of microglia in an ATP the number of migrated cells (n = 15 compared with control n = 12, concentration gradient (Ohsawa et al., 2007). We therefore examined F = 16.7, P < 0.001). The effect was significantly antagonized by 4,55 the possibility that, as glutamate triggers ATP release from microglial 100 lm suramin plus 100 lm PPADS (n = 12, F = 16.7, 4,55 cells (Liu et al., 2006), increases in motility of microglia induced by P < 0.001; Fig. 4B). However, the effect of glutamate-induced glutamate may actually be due to the evoked release of ATP chemotaxis migration of microglia was not significantly altered by autocatalytically acting on P2Y receptors. However, ATP-degrading purinergic receptor antagonists suramin and PPADS (n =9, 12 enzyme apyrase and suramin (100 lm), which blocks P2Y recep- F = 16.7, P > 0.05; Fig. 4B). This suggests that microglia 12 4,55 tors, did not block the chemotaxis or motility of microglia exposed to chemotaxis is due to a direct effect of glutamate on AMPA and glutamate. metabotropic glutamate receptors rather than indirectly through ATP The cell bodies of ramified microglia in vivo are relatively stable, released from microglia induced by glutamate. although their ramifying processes are continually sampling the parenchyma (Nimmerjahn et al., 2005). However, when the cells are reactive they can migrate rapidly at peak velocity ‡10 lm ⁄ min Glutamate-induced chemotaxis is mediated by intracellular (Carbonell et al., 2005). It has been shown that the glutamate receptor actin and microtubules agonist kainate, probably acting on AMPA-preferring receptors (Noda Chemotaxis of microglia induced by glutamate (100 lm) in a Dunn et al., 2000; Hagino et al., 2004), induces dramatic morphological and chamber as well as spontaneous movement are significantly arrested, cytoskeletal changes in microglia, involving rearrangement of action and glutamate-induced chemotaxis was significantly inhibited by pre- filaments thought to be involved in locomotion (Christensen et al., incubation with actin interrupter cytochalasin D (100 lm, n = 75, 2006), as well as instigating microglia proliferation such that they F5,476 = 39.5, P < 0.001 compared with glutamate, Fig. 5). Inhibition increase in numbers between two- and threefold (Tikka et al., 2001). of microtubules with nacodazole (100 lm, n = 97) significantly In the present work we have shown that the reactive microglia in reduced glutamate-induced chemotaxis but not spontaneous move- culture respond to concentration gradients of glutamate, AMPA, ment (Fig. 5), whereas blocking protein kinase C (PKC) with kainate or ACPD (metabotropic glutamate receptor agonist) by calphostin C (500 nm, n = 54, F5,476 = 39.5, P > 0.05 compared with chemotaxis of their cell bodies up the concentration gradients, glutamate) and bisindolylmaleimide (100 nm, n = 73) had no effect whether in a two-dimensional Dunn chamber or Boyden chamber or on either (F5,476 = 39.5, P > 0.05 compared with glutamate; Fig. 5). towards a point source provided by a micropipette in a spinal cord Glutamate leads to a redistribution of CDC42 (a small Rho-family slice. The concentration gradients were effective in the Dunn chamber GTPase) accompanied by an increase in cell membrane ruffles and down to levels as small as 1 lm, indicating a probable physiological staining with phalloidin. CDC42 is found localized to the cell nuclear function for this glutamate-induced chemotaxis. This was supported area before application of glutamate, as shown by the distribution of by further experiments in which glutamate was released from a CDC42 antibody compared with the total area of microglia marked micropipette placed at least 5 lm above a spinal cord slice. The with CD11b antibody (Fig. 6A), whereas after exposure to glutamate nearby ramifying GFP+ ⁄ + microglia were observed to transform into (1 mm) CDC42 is distributed throughout the cell body (Fig. 6A and B; reactive microglia and undergo chemotaxis towards the glutamate F1,140 = 268, P < 0.001). The increase in cell membrane ruffles in the source showing that the chemotaxis effect is not confined to presence of glutamate (1 mm), identified with the actin marker dissociated and cultured microglia. Ambient levels of glutamate are

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 1116 G. J. Liu et al.

Fig. 6. Immunohistochemical evidence for glutamate-induced microglial membrane ruffling that involves CDC42 and actin. (A) Images taken from the cells in both bath solution (left column) and bath solution with 1 mm glutamate (right column). CDC42 distribution was localized to the area of the nucleus [marked with 4¢,6-diamidino-2-phenylindolePI (DAPI) on the bottom panel] in the bath solution (control), and the area covers almost the same as that of the whole cell as indicated with CD11b (microglial marker) in the presence of 1 mm glutamate. (B) Statistical data showing that glutamate significantly increases the area (relative to total area of cells marked by CD11b on the top panel of A) occupied by CDC42 when compared with the control. (C) Typical images of microglial membrane ruffling marked with phalloidin under bath solution (top panel) and Dulbecco’s modified Eagle’s medium (DMEM; lower panel); the area of cell membrane ruffling was increased under glutamate (right column, 1 mm) when compared with control (left column). The morphology of microglia in the bath and DMEM without glutamate (left column) is different; microglia in DMEM show more processes, a smaller body and irregular shape. (D) Glutamate significantly increased the area of cell membrane ruffling relative to total area of cells in both bath solution and DMEM. (E) Glutamate (1 mm) significantly increased the number of cells with membrane ruffles (relative to total number of cells) in bath solution and DMEM. Scale bars: 10 lm (A and C). *P < 0.001. between 0.5 lm and 2.0 lm in the extracellular space of unstimulated spinal cord injury, or exocytotic insults, large amounts of glutamate parenchyma (Meldrum, 2000). The present work shows that gradients can be released from neurons, astrocytes and microglia. The released of this order of concentration can lead to the chemotaxis of microglia. glutamate can strongly attract microglia migrating to the area of Under pathological conditions, like ischaemia, or damage due to pathology. Therefore, the glutamate can act as another chemotaxis

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 Glutamate-induced chemotaxis of microglia 1117 attractant similarly to ATP and chemokines under pathological Biber, K., Neumann, H., Inoue, K. & Boddeke, H.W. (2007) Neuronal ‘On’ and conditions. ‘Off’ signals control microglia. Trends Neurosci., 30, 596–602. It is well established that if the transmitter substance c-aminobutyric Bouzigues, C., Morel, M., Triller, A. & Dahan, M. (2007) Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve acid (GABA) forms a concentration gradient in the vicinity of a growth cones analyzed by single quantum dot imaging. Proc. Natl. Acad. neuronal growth-cone it will reorient following rapid rearrangement of Sci. U.S.A., 104, 11251–11256. actin filaments in the cone (Zheng et al., 1994; Song & Poo, 2001; Carbonell, W.S., Murase, S., Horwitz, A.F. & Mandell, J.W. (2005) Migration Xiang et al., 2002; Bouzigues et al., 2007), which can be followed by of perilesional microglia after focal brain injury and modulation by CC chemokine receptor 5: an in situ time-lapse confocal imaging study. staining with phalloidin and is inhibited by cytochalasin B (Nolte J. Neurosci., 25, 7040–7047. et al., 1996). We showed phalloidin-stained rearrangement of actin, Christensen, R.N., Ha, B.K., Sun, F., Bresnahan, J.C. & Beattie, M.S. (2006) inhibited by cytochalasin D, following AMPA and metabotropic Kainate induces rapid redistribution of the actin cytoskeleton in ameboid glutamate receptor activation, which is to be expected given the microglia. J. Neurosci. Res., 84, 170–181. importance of actin assembly in the process of filopodia formation Dobrenis, K. (1998) Microglia in cell culture and in transplantation therapy for central nervous system disease. Methods, 16, 320–344. (Huber et al., 2003). The GABA receptor-induced reorganization of Farber, K. & Kettenmann, H. (2005) Physiology of microglial cells. Brain Res. the actin filaments subsequent to a turning response is mediated by Brain Res. Rev., 48, 133–143. phospholipase C activating two antagonistic pathways: an attractive Farber, K. & Kettenmann, H. (2006) Purinergic signaling and microglia. one mediated by IP3 and the release of calcium from internal stores; Pflugers Arch., 452, 615–621. Garcia, Z., Silio, V., Marques, M., Cortes, I., Kumar, A., Hernandez, C., Checa, and a repulsive one through PKC (Xiang et al., 2002). In the present A.I., Serrano, A. & Carrera, A.C. (2006) A PI3K activity-independent work we found that blocking PKC with calphostin C and bis- function of p85 regulatory subunit in control of mammalian cytokinesis. indolylmaleimide did not alter glutamate-induced chemotaxis. EMBO J., 25, 4740–4751. Low concentrations of glutamate (1 lm) can induce cultured Gilabert, R. & McNaughton, P. (1997) Enrichment of the fraction of microglia chemotaxis in Dunn chambers indicating that synaptically nociceptive neurones in cultures of primary sensory neurones. J. Neurosci. Methods, 71, 191–198. released glutamate might act as a chemotaxis attractant on microglia in Hagino, Y., Kariura, Y., Manago, Y., Amano, T., Wang, B., Sekiguchi, M., the spinal cord and hippocampus, from where we derived the microglia Nishikawa, K., Aoki, S., Wada, K. & Noda, M. (2004) Heterogeneity and used in these experiments. The glutamate-induced chemotaxis effect on potentiation of AMPA type of glutamate receptors in rat cultured microglia. microglia may also happen in other regions, besides the hippocampus Glia, 47, 68–77. Haynes, S.E., Hollopeter, G., Yang, G., Kurpius, D., Dailey, M.E., Gan, W.B. & and spinal cord, in the CNS. Recently, Wu & Zhuo (2008) have shown Julius, D. (2006) The P2Y12 receptor regulates microglial activation by that glutamate has no chemotaxic effect on microglia from adult extracellular nucleotides. Nat. Neurosci., 9, 1512–1519. (6–8 weeks old) hippocampus removed from heterozygous (fractalki- Honda, S., Sasaki, Y., Ohsawa, K., Imai, Y., Nakamura, Y., Inoue, K. & Kohsaka, ne ⁄ GFP+ ⁄ +) mice. The results on glutamate-induced chemotaxis in the S. (2001) Extracellular ATP or ADP induce chemotaxis of cultured microglia present experiments were obtained from postnatal (10–14 day old) through Gi ⁄ o-coupled P2Y receptors. J. Neurosci., 21, 1975–1982. ) ⁄ + Huber, A.B., Kolodkin, A.L., Ginty, D.D. & Cloutier, J.F. (2003) Signaling at spinal cords removed from homozygous (fractalkine ⁄ GFP ) mice. the growth cone: ligand-receptor complexes and the control of axon growth The different results might therefore be due to the different properties of and guidance. Annu. Rev. Neurosci., 26, 509–563. microglia from different aged animals. This possibility remains to be de Jong, D.J., Tielen, J., Habraken, C.M., Wetzels, J.F. & Naber, A.H. (2005) determined. The durations of glutamate application used to induce 5-Aminosalicylates and effects on renal function in patients with Crohn’s disease. Inflamm. Bowel Dis., 11, 972–976. chemotaxis of microglia were also different. We applied glutamate for Jung, S., Aliberti, J., Graemmel, P., Sunshine, M.J., Kreutzberg, G.W., Sher, A. minutes (> 10 min), while Wu & Zhuo (2008) briefly (100 ms) injected & Littman, D.R. (2000) Analysis of fractalkine receptor CX(3)CR1 function the glutamate. This might be a major reason for the different results. by targeted deletion and green fluorescent protein reporter gene insertion. Therefore, our results might be more applicable to pathological Mol. Cell. Biol., 20, 4106–4114. conditions where large amounts of glutamate may be released. Koizumi, S., Shigemoto-Mogami, Y., Nasu-Tada, K., Shinozaki, Y., Ohsawa, K., Tsuda, M., Joshi, B.V., Jacobson, K.A., Kohsaka, S. & Inoue, K. (2007) UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Abbreviations Nature, 446, 1091–1095. Kumanogoh, H., Miyata, S., Sokawa, Y. & Maekawa, S. (2001) Biochemical ACPD, trans-d,l-1-aminocyclopentane-1,3-dicarboxylic acid; AMPA, a-ami- and morphological analysis on the localization of Rac1 in neurons. Neurosci. no-3-hydroxy-5-methylisoxazole-4-propionic acid; AP5, d(-)-2-amino-5-phos- Res., 39, 189–196. phonopentanoic acid; BSA, bovine serum albumin; CDC42, cell division cycle Liu, G.J., Werry, E.L. & Bennett, M.R. (2005) Secretion of ATP from Schwann 42; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DAPI, 4¢,6-diamidino-2- cells in response to uridine triphosphate. Eur. J. Neurosci., 21, 151–160. phenylindolePI; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green Liu, G.J., Kalous, A., Werry, E.L. & Bennett, M.R. (2006) Purine release from fluorescence protein; GYKI 52466, 1-(4-aminophenyl)-4-methyl-7,8-methy- spinal cord microglia after elevation of calcium by glutamate. Mol. lenedioxy-5H-2,3-benzodiazepine hydrochloride; MCPG, a-methyl-(4-carb- Pharmacol., 70, 851–859. oxyphenyl)glycine; MSPG, a-methyl-(4-sulphonophenyl)glycine; NMDA, Marin-Teva, J.L., Dusart, I., Colin, C., Gervais, A., van Rooijen, N. & Mallat, N-methyl-d-aspartic acid; PBS, phosphate-buffered saline; PPADS, pyri- M. (2004) Microglia promote the death of developing Purkinje cells. Neuron, doxal-phosphate-6-azophenyl-2¢,4¢-disulphonic acid; TBOA, dl-threo-b-ben- 41, 535–547. zyloxyaspartate. Matute, C., Domercq, M. & Sanchez-Gomez, M.V. (2006) Glutamate-mediated glial injury: mechanisms and clinical importance. Glia, 53, 212–224. Meldrum, B.S. (2000) Glutamate as a neurotransmitter in the brain: review of References physiology and pathology. J. Nutr., 130, 1007S–1015S. Banati, R.B. (2003) Neuropathological imaging: in vivo detection of glial Nimmerjahn, A., Kirchhoff, F. & Helmchen, F. (2005) Resting microglial cells activation as a measure of disease and adaptive change in the brain. Br. Med. are highly dynamic surveillants of brain parenchyma in vivo. Science, 308, Bull., 65, 121–131. 1314–1318. Bennett, M.R., Buljan, V., Farnell, L. & Gibson, W.G. (2008) Purinergic Noda, M., Nakanishi, H., Nabekura, J. & Akaike, N. (2000) AMPA-kainate junctional transmission and propagation of calcium waves in cultured spinal subtypes of glutamate receptor in rat cerebral microglia. J. Neurosci., 20, cord microglial networks. Purinergic Signal., 4, 47–59. 251–258. Biber, K., Laurie, D.J., Berthele, A., Sommer, B., Tolle, T.R., Gebicke-Harter, Nolte, C., Moller, T., Walter, T. & Kettenmann, H. (1996) Complement 5a P.J., van Calker, D. & Boddeke, H.W. (1999) Expression and signaling of controls motility of murine microglial cells in vitro via activation of an group I metabotropic glutamate receptors in astrocytes and microglia. inhibitory G-protein and the rearrangement of the actin cytoskeleton. J. Neurochem., 72, 1671–1680. Neuroscience, 73, 1091–1107.

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118 1118 G. J. Liu et al.

Ohsawa, K., Irino, Y., Nakamura, Y., Akazawa, C., Inoue, K. & Kohsaka, S. factor alpha-induced neurotoxicity in concert with microglial-derived Fas (2007) Involvement of P2X4 and P2Y12 receptors in ATP-induced ligand. J. Neurosci., 25, 2952–2964. microglial chemotaxis. Glia, 55, 604–616. Tikka, T., Fiebich, B.L., Goldsteins, G., Keinanen, R. & Koistinaho, J. (2001) Pocock, J.M. & Kettenmann, H. (2007) Neurotransmitter receptors on Minocycline, a tetracycline derivative, is neuroprotective against excitotox- microglia. Trends Neurosci., 30, 527–535. icity by inhibiting activation and proliferation of microglia. J. Neurosci., 21, Re, D.B. & Przedborski, S. (2006) Fractalkine: moving from chemotaxis to 2580–2588. neuroprotection. Nat. Neurosci., 9, 859–861. Vaughan, D.W. & Peters, A. (1974) Neuroglial cells in the cerebral cortex Robinson, A.P., White, T.M. & Mason, D.W. (1986) Macrophage heterogeneity of rats from young adulthood to old age: an electron microscope study. in the rat as delineated by two monoclonal antibodies MRC OX-41 and J. Neurocytol., 3, 405–429. MRC OX-42, the latter recognizing complement receptor type 3. Immunol- Webb, S.E., Pollard, J.W. & Jones, G.E. (1996) Direct observation and ogy, 57, 239–247. quantification of macrophage chemoattraction to the growth factor CSF-1. Schwamborn, J.C. & Puschel, A.W.(2004) The sequential activity of the GTPases J. Cell Sci., 109(Pt 4), 793–803. Rap1B and Cdc42 determines neuronal polarity. Nat. Neurosci., 7, 923–929. Werry, E.L., Liu, G.J. & Bennett, M.R. (2006) Glutamate-stimulated ATP Song, H. & Poo, M. (2001) The cell biology of neuronal navigation. Nat. Cell release from spinal cord astrocytes is potentiated by substance P. Biol., 3, E81–E88. J. Neurochem., 99, 924–936. Taylor, D.L., Diemel, L.T., Cuzner, M.L. & Pocock, J.M. (2002) Activation of Wu, L.J. & Zhuo, M. (2008) Resting microglial motility is independent group II metabotropic glutamate receptors underlies microglial reactivity and of synaptic plasticity in Mammalian brain. J. Neurophysiol., 99, 2026– neurotoxicity following stimulation with chromogranin A, a peptide up- 2032. regulated in Alzheimer’s disease. J. Neurochem., 82, 1179–1191. Xiang, Y., Li, Y., Zhang, Z., Cui, K., Wang, S., Yuan, X.B., Wu, C.P., Poo, Taylor, D.L., Diemel, L.T. & Pocock, J.M. (2003) Activation of microglial M.M. & Duan, S. (2002) Nerve growth cone guidance mediated by G group III metabotropic glutamate receptors protects neurons against protein-coupled receptors. Nat. Neurosci., 5, 843–848. microglial neurotoxicity. J. Neurosci., 23, 2150–2160. Zheng, J.Q., Felder, M., Connor, J.A. & Poo, M.M. (1994) Turning of Taylor, D.L., Jones, F., Kubota, E.S. & Pocock, J.M. (2005) Stimulation of nerve growth cones induced by neurotransmitters. Nature, 368, 140– microglial metabotropic glutamate receptor mGlu2 triggers tumor necrosis 144.

ª The Authors (2009). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience, 29, 1108–1118