C57BL/6J mice (Janvier Breeding Center, Le Genest St Isle, France), DAP12-mutated mice (DAP12KI) on C57BL/6 genetic background  and CD11b-deficient mice (B6.129S4-Itgamtm1Myd/J on C57BL/6 background obtained from the Jackson Laboratory) were used. For in vitro experiments, primary mesencephalic neurons were prepared from gestational age 13 days C57BL/6J mice embryos whereas primary microglial cells were obtained from newborn C57BL/6J, DAP12KI or CD11b−/− mice. For in vivo experiments, ten- to twelve week-old male C57BL/6J, DAP12KI and CD11b−/− mice, weighing 25 to 30 g were used. Mice were kept in a temperature-controlled room (23°C ± 1°C) under a 12-hour light/dark cycle and had ad libitum access to food and water. All animals were further genotyped after their sacrifice. Animal handling was carried out according to ethical regulations and guidelines (Guide for the care and use of laboratory animals, NIH publication no. 85–23, revised 1985) and the European Communities Council Directive 86/609/EEC. Experiments using vertebrates were approved by the Services Vétérinaires de Paris.
Lipopolysaccharide (LPS),1-methyl-4-phenylpyridinium (MPP+) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP-HCl) were purchased from Sigma-Aldrich (St Quentin-Fallavier, France). Dihydrorhodamine-123 (DHR-123), the cell permeant probes used for the detection of ROS was purchased from Molecular Probes (Invitrogen, CergyPontoise, France).
Midbrain cell cultures
Cultures were prepared from the ventral mesencephalon of gestational age 13 days C57BL/6J mice embryos. Dissociated cells in suspension obtained by mechanical trituration of midbrain tissue pieces were seeded at a density of 1.2 to 1.5 × 105 cells/cm2 onto tissue culture supports pre-coated with 1 mg/mL polyethylenimine (Sigma-Aldrich, St Quentin-Fallavier, France). The cultures were then maintained in N5 medium supplemented with 5 mM glucose, 5% horse serum, and 0.5% fetal calf serum, except for the first 3 days in vitro (DIV) during which the concentration of fetal calf serum was set at 2.5% to favor initial maturation of the cultures . Note that tyrosine hydroxylase-positive (TH+) neurons represent approximately 1 to 2% of the total number of neuronal cells present in these cultures.
Highly enriched microglial cell cultures
Microglial cells were derived from the cerebral cortex of newborn C57BL/6J, DAP12KI or CD11b−/− mice, according to procedures described previously . Pure ameboid microglial cells (>99%) were isolated from 2-week-old primary glial cultures grown in DMEM supplemented with 10% fetal calf serum. The cells were washed three times in DMEM and plated in CDM (1 to 1.5 × 106 cells per 35 mm dish).
Mixed neuron/microglia cultures
Freshly isolated microglial cells (1 to 2.105 cells/well) were added to 4-DIV primary neuronal cultures grown in 24-well plates. Control neuronal cultures were supplemented with an equal volume of cell-free medium. LPS (serotype 026:B6, Escherichia coli, at 100 ng/mL) or MPP+, at 0.1 μM, were applied directly to the mixed cultures at 5-DIV, that is, one day after the seeding of microglial cells on primary neurons.
Immunofluorescent detection protocols
The cultures were fixed for 12 minutes using 4% formaldehyde in Dulbecco’s (PBS), and then washed twice with PBS before an incubation step at 4°C for 24 to 72 h with the following antibodies. A monoclonal anti-TH antibody diluted 1/5000 (Diasorin, Stillwater, MN, USA) was used to assess the survival of DN. Microglial cells were characterized using a mouse anti-CD11b antibody (1/50; clone MRC OX-42; Serotec, Oxford, England). All antibodies were diluted in PBS containing 0.2% Triton X-100 except the mouse anti-CD11b antibody, which was diluted in PBS only. Detection of the primary antibodies was performed with a cyanin-3 conjugate of an anti-mouse IgG antibody (1/500, Sigma Aldrich) or with an Alexa Fluor 488 conjugate of an anti-rabbit antibody (1/500; Invitrogen, Carlsbad, CA, USA). Cell counting was performed at 200× magnification using a 20× objective matched with a 10× ocular. The number of TH+ neurons in each culture well was estimated after counting 20 visual fields distributed along the X and Y axes. Note that counts of neuronal cells were performed at 13-DIV, that is, at a stage when the death process affecting DA neurons is almost fully complete.
Quantification of superoxide ions
Superoxyde ion (O2
.-) levels were measured using dihydroethidium (Invitrogen) as fluorescent probes at 6-DIV, that is, when oxidative stress is at its peak. Mixed microglia-neuron cultures were exposed for 30 minutes to 1 μM dihydroethidium, washed three times, and then maintained in serum-free supplemented medium. The fluorescent signal, visualized by epifluorescence microscopy (excitation at 520 nm, emission at 610 nm), was quantified using the Simple-PCI software from C-Imaging Systems (Cranberry Township, PA, USA) and a Nikon TE-300 inverted microscope equipped with an ORCA-ER digital camera (Hamamatsu Photonics, Massy, France). Fluorescent images of randomly chosen fields (six to eight in each culture condition) were acquired with a 20× fluorescent objective. The average pixel intensity over the surface of each cell body was determined under the different test conditions. Background fluorescence was subtracted from raw data, and the results were expressed as a percentage of the mean fluorescence intensity per cell in control cultures. A minimum of 60 cells was analyzed under each test condition.
MPTP injection and tissue preparation
Groups of mice received MPTP under a subchronic or acute protocol. For subchronic MPTP intoxication, mice were given an intra-peritoneal (i.p.) injection of 30 mg/kg (free base) MPTP-HCl per day for 5 consecutive days and were then euthanized at 1, 2, 4, 7 or 21 days after the last MPTP injection. For acute MPTP intoxication, mice were i.p.-injected with 4 doses of 20 mg/kg (free base) MPTP-HCl at 2-h intervals and were then euthanized 2 or 7 days after the last MPTP injection. Control mice received an equivalent volume of 0.9% NaCl solution. For immunohistochemistry, mice were injected with a lethal dose of pentobarbital (100 mg/kg) and then transcardially perfused with 50 mL of heparin solution (5 U/mL) followed by 100 mL of ice-cold 4% paraformaldehyde (PFA) solution. After extraction from the skull, brains were further post-fixed overnight in fresh 4% PFA/PB solution, and cryoprotected with 30% sucrose in PB. Coronal free-floating striatal and mesencephalic sections (30-μm thick) were prepared using a freezing microtome (Leica) and collected in 10 regularly spaced series. For quantitative PCR, brains were rapidly removed from the skull and striata, ventral mesencephalon and cortex were dissected on humidified filters at 4°C. Tissues were then frozen in liquid nitrogen and kept at −80°C until use.
Real-time polymerase chain reaction (PCR)
Real-time quantitative (q)PCR was performed as described . The primer sequences were as follows: mouse Dat forward 5-CGC TGG AGG CAG TCG AA-3, and reverse 5-CGG AGC ATT TGC TTT TAC TCA TG-3; mouse Cd11b forward 5-GAT GCT TAC CTG GGT TAT GCT TCT-3, and reverse 5-CCG AGG TGC TCC TAA AAC CA-3; mouse Dap12 forward 5-TGG TGT TGA CTC TGC TGA TTG C-3, and reverse 5-CCT TCC GCT GTC CCT TGA C-3. Primer sequences of housekeeping gene were as follow: mouse Hprt forward 5-CTT CCT CCT CAG ACC GCT TTT-3, and reverse 5-AAC CTG GTT CAT CAT CGC TAA TC-3; mouse Gapdh forward 5-TGT GTC CGT CGT GGA TCT GA-3, and reverse 5-CCT GCT TCA CCA CCT TCT TGA-3.
Measurement of striatal MPP+ levels
Mice were euthanized 90 minutes after one i.p. injection of 30 mg/kg MPTP-HCl, and their striata were recovered and treated with 500 μL 0.1 N HClO4 before being processed for HPLC using UV detection (295-nm wavelength).
Measurement of striatal dopamine, DOPAC and HVA levels
Seven days after the last MPTP injection, mice were euthanized and their striata were dissected out and treated with 0.1 N perchloric acid containing 0.05% disodium ethylenediaminetetraacetic acid (EDTA) and 0.05% sodium metabisulfite. Striatal tissue content in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was assessed by high performance liquid chromatography (HPLC) using electrochemical detection with a potential set at +0.65 V.
Immunohistochemical staining on mouse brain sections was performed as previously described . The following primary antibodies were used: anti-TH (1:1000; Pel-Freez Biochemicals), anti-Iba1 (1:500; Wako Chemicals). Staining was revealed by the ABC method (Vector Laboratories) with 3,3-diaminobenzidine (DAB) as the peroxidase substrate. Mouse sections were counterstained with thionin solution (Nissl stain).
For double-staining experiments, brain sections were simultaneously incubated with two primary antibodies: anti-CD11b (rat - 1:250; Serotec, Oxford, England), anti-DAP12 (rabbit- 1:100; Chemicon International, Merk Millipore, Molsheim, France). Sections were then incubated in specific CY3- or Alexa488-conjugated secondary antibodies (Jackson ImmunoResearch Europe, Suffok, England) at 1:1000 dilution for 120 minutes at room temperature.
Image and data analysis
DAB-immunostained sections were analyzed by bright-field microscopy, using a Leitz microscope equipped with image analysis software (Mercator, ExploraNova, La Rochelle, France). TH+ and Nissl+ cell bodies were quantified stereologically on regularly spaced sections covering the whole substantia nigra pars compacta (SNpc) using the VisioScan stereology tool. The investigator performing the quantification was blinded to the treatment and genotype groups during the analysis. Fluorescent sections were analyzed on a Zeiss Axioplan 2 using ExploraNova FluoUp 1.0 software. Striatal TH optic densitometry was measured by image analysis software (Mercator, ExploraNova).
All values are expressed as the mean ± standard error of the mean (SEM). Differences in means between two groups were analyzed using the two-tailed Student’s t-test, or when data were not normally distributed, with the nonparametric Mann–Whitney U-test. Differences in means among multiple datasets were analyzed using one- or two-way analysis of variance (ANOVA) with time, treatment, or genotype as the independent factors. When ANOVA showed significant differences, pairwise comparisons between means were tested by the Tukey post hoc analysis. When data were not normally distributed, ANOVA on ranks was used (Kruskal-Wallis test followed by pairwise comparison using the Dunn test). In all analyses, P-values of less than 0.05 were considered significant (SigmaStat Statistical Software, Systat Software, Inc., San Jose, CA, USA).