All procedures were approved by the Institutional Animal Care and Use Committee or the Emory Institutional Review Board. Mice expressing enhanced green fluorescent protein (eGFP) driven by an actin promoter (a gift from M. Okabe, Osaka University, Japan) were used to culture microglia. Cortical tissue from postnatal day 2–3 actin-eGFP
or wild-type C57Bl/6 mice (The Jackson Laboratory) was dissociated and plated with serum-supplemented DMEM (Gibco #11960) containing (mM): 25 glucose, 2 glutamine, and 1 Na pyruvate. After 2–3 weeks, floating microglia were isolated and plated onto Matrigel matrix-coated coverslips (BD Biosciences, thickness ~ 0.2 mm). Microglia adhered overnight and were confirmed ≥ 95% pure based on IB4 staining (data not shown). For astrocyte-microglia co-cultures, eGFP microglia were applied onto 50% confluent wild-type astrocytes and allowed to adhere overnight. Human microglia were cultured as previously described41
. Adult human microglial cells were obtained from two Emory University Hospital patients (ages 30–45) undergoing hippocampectomy due to seizures or resection due to a subarachnoid tumor. Human microglia were allowed to adhere for 5–7 days prior to imaging.
Adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), 5′-(N-ethylcarboxamido)adenosine (NECA), adenosine, A2a agonist CGS-21680, A2a antagonist SCH-58261, lipopolysaccharide (LPS), lipoteichoic acid from Staphylococcus aureus (LTA), Gαs inhibitor NF449, and the protein kinase A (PKA) inhibitor H89 were all purchased from Sigma. Nuclear factor-κB (NF-κB) inhibitors 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) and SN50, forskolin (FSK), adenylate cyclase inhibitor 2′,5′-dideoxyadenosine (ddAdo), Gαs activator cholera toxin (Vibrio cholerae, CLX), Rho GTPase inhibitor exoenzyme C3 (Clostridium botulinum), Rho kinase (ROCK) inhibitor Y27632, and adenosine deaminase (ADA) were all purchased from Calbiochem. We also utilized recombinant mouse complement component C5a, recombinant mouse TNF-α (R&D Systems), and unmethylated CpG motif-containing oligonucleotides (CpG, InvivoGen). Isolectin GS-IB4 (IB4) from Griffonia simplicifolia and human β-amyloid 1–42 (aggregated according to manufacturer instructions) was obtained from Invitrogen.
Four-dimensional confocal imaging
Cells were continuously perfused with imaging buffer (mM): 10 HEPES, 150 NaCl, 3 KCl, 22 Sucrose, 10 Glucose, 1 MgCl2, and 2 CaCl2, pH 7.4. The buffer was maintained at 32–33.5°C using TC-344B temperature controller (Warner Instruments). After establishing upper and lower z-axis limits, time-lapse confocal image stacks of eGFP microglia were acquired every 15–20 seconds. Each image stack spanned the entire z-plane of microglia at 0.5–2 µm steps (25–30 images per stack). Image stacks were acquired using IPlab software (BD Biosciences), Olympus IX51 microscope with DSU confocal unit, a MFC-2000 z-motor (ASI) and a Uniblitz shutter (Vincent Associates). Image stacks were collected continuously during 5 min periods of buffer perfusion, agonist perfusion, and washout with an ORCA-ER cooled CCD camera (Hamamatsu).
Image processing and analysis
Following acquisition, images were compiled and transformed into four-dimensional renderings of microglia throughout the course of a perfusion using Imaris 4.2 (Bitplane). After background noise subtraction with a 10 µm-width Gaussian filter, image stacks were reconstructed into three-dimensional surfaces at each time-point based on calibrated voxel sizes (x/y: 0.105 µm2, z: 0.5–2 µm), a signal intensity threshold, and a 0.2 µm-width Gaussian filter. All time-lapse surfaces were then split into objects to enable measurements of cell volume and surface area through time. Cell surface area-to-volume ratio (SA:V) at each time-point was then calculated to assess changes in cell structure.
Microglial cell process motility was assessed by comparing average speed of tracked regions of interest (ROIs) within cell processes during periods of buffer and agonist perfusion. ROIs, or discrete spherical volumes of fluorescence, were analyzed based on pre-defined criteria, such as minimum diameter, maximum travel distance between consecutive image stacks, and the type of tracking algorithm utilized. To assign ROIs to cell processes and enable tracking, we first optimized these criteria. While varying these parameter values, we compared sensitivity and reliability of the tracking analysis to agonist-induced changes in track speed, number of tracks, and track length (Supplementary Fig. 1
, and data not shown). Additionally, we minimized changes in the number of unassigned ROIs (i.e. regions not designated by the algorithm to any track). As determined empirically, the parameter values we held constant for all analyses of process motility were 2 µm minimum diameter, 3 µm maximum travel distance and GapClose Autoregressive tracking algorithm, a function which matches predicted ROI positions with actual ROI positions within image data and thereby models continuous motion. Tracks created within the cell body were omitted from analyses. Baseline process ramification of control group was lower than that of CpG- and TNF-α-treated groups (p < 0.01), but not different from LPS- and LTA-treated groups. Baseline process motility of control group was lower than that of CpG- and LPS-treated groups (p < 0.01), but not different from LTA- and TNF-α-treated groups.
A glass micropipette (inner diameter: 1.15 ± 0.05 mm, outer diameter: 1.65 ± 0.05 mm, glass type 8250, Garner Glass Co.) was pulled to a ~1.4 MΩ tip with a Narishige pipette puller, filled with agonist diluted in imaging buffer, and attached to a micromanipulator and a current generator (Model 6400, Dagan). The positive holding current value (+80 nA) was determined empirically by observing microglial morphological responses (occurrence of membrane ruffling) while in close proximity to an ATP-containing micropipette with different holding currents. Agonist application was performed by ejecting a −200nA current. In order to analyze cell chemotaxis in response to iontophoretic agonist application, cell body was tracked by setting ROI diameter to the size of the nucleus.
Actin filament staining
Cultured wild-type microglia were treated with agonists, fixed and stained with 1:100 Alexa Fluor-488 phalloidin (Invitrogen) and 1:100 IB4 to identify microglia.
In vivo injections and immunohistochemistry
Transgenic mice that encode eGFP in place of exon 2 within the Cx3cr1 gene were obtained from the Jackson Laboratory. Animals (1–3 month old) received a single intraperitoneal injection of either sterile PBS or LPS (2 mg/kg, 100 µl total volume). After 48 hours, mice were anesthetized with Avertin and placed into a stereotaxic apparatus for an intracortical injection of vehicle (50% DMSO in PBS, 2 µl) or the A2A antagonist SCH-58261 (1 mM in 2 µl) at 400 nl per minute at 1.0 mm caudal, 2.0 mm lateral, and 1.0 mm ventral from bregma. The needle was left in place for 5 minutes. After another 25 minutes, brain tissue was removed and drop-fixed overnight in 4% paraformaldehyde with 0.1% glutaraldehyde and cryopreserved in sucrose. 40 µm-thick sections were washed in PBS and placed on slides for microscopy. Adora-eGFP BAC transgenic mice were obtained from the Mutant Mouse Regional Resource Centers and received an intraperitoneal injection of either sterile PBS or LPS (2 mg/kg, 100 µl total volume). After 48–72 hours, tissue was processed and immunostained using anti-GFP antibody (1:1000, Invitrogen).
Total RNA was derived from homogenized primary mouse microglia according to manufacturer instructions using Purelink Micro-to-Midi total RNA purification system (Invitrogen). RNA (50 ng) was then reverse transcribed and amplified using SuperScript III One-Step RT-PCR System with Platinum Taq
(Invitrogen) and specific primers, as detailed previously5,42–50
. cDNA synthesis was carried out at 50°C for 30 min, followed by pre-denaturation at 94°C for 2 min, 30–40 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 30 sec (except for P2Y4: 51°C; hP1 receptors: 58°C; activation markers: 60°C), and finally elongation at 70°C for 1 min. To check for DNA contamination, reverse transcriptase was omitted from some reaction mixtures. For primer pairs that did not produce visible bands using isolated RNA, 2 ng cDNA encoding each receptor was used to run control reactions. Plasmids for mouse adenosine receptors and mP2Y2 were from Open Biosystems, while mP2Y4 and mP2Y6 were a gift from W. O’Neal at the University of North Carolina, Chapel Hill.
Microglial phagocytosis was analyzed using the Vybrant phagocytosis assay kit (Invitrogen) according to manufacturer instructions. Briefly, primary mouse microglia were isolated, allowed to adhere overnight onto 0.05 mg/ml poly-D-lysine-coated 96-well plates, and treated with LPS (100 ng/ml) at 37°C for 24 hours. After pre-exposure to ATP (50 µM), CGS-21680 (50 µM), or adenosine (50 µM) for 20 min, fluorescein-labeled Escherichia coli bioparticles were applied for 2 hours along with agonists. Cells were examined using the FlexStation II microplate reader (Molecular Devices) at 480 nm excitation and 520 nm emission. Results were normalized to empty wells and DMEM-treated cells.
Agonist-induced release of cAMP was measured using the CatchPoint Cyclic-AMP Fluorescence Assay kit (Molecular Devices) with some modification to manufacturer instructions. Briefly, HEK 293 cells were plated with DMEM-Glutamax supplemented with 10% fetal bovine serum, 10 U/ml penicillin, and 10 µg/ml streptomycin, and transfected with cDNA encoding the mouse A2A receptor (Open Biosystems) at 3:1 ratio using FuGene 6 (Roche). HEK cells were then incubated in 0.75 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma) for 10 minutes to inhibit phosphodiesterase activity. Indicated agonists or 30 µM forskolin (activator of adenylate cyclase) were then added for 15 minutes at 37°C. Fluorescence intensity in each well was detected using the FlexStation II plate reader (Molecular Devices) using 530 nm excitation and 590 nm emission.
All statistical tests were performed on raw data except for the phagocytosis analysis. Average cell ramification, process track speed, and migration during baseline were compared to periods of agonist exposure using a paired Student’s t-test. Data were normally distributed and had equal variances, as verified using Bartlett’s test. Analyses of different pre-treatment conditions or inhibitor effects on agonist responses in the confocal imaging experiments were performed using repeated-measures two-way ANOVA. cAMP release was analyzed with one-way ANOVA and Dunn’s multiple comparisons post-test. Phagocytosis analysis was performed with the Kruskal-Wallis test and Dunn’s multiple comparisons post-test.