Animals and treatment
Adult, C57BL/6 male mice (Charles River Laboratories, Wilmington, MA, USA) weighing 20–30g were housed 2 to the cage and given food and water ad libitum. Once treatment regimens began, the diet of all mice was supplemented daily with a hydrating gel (Harlan Sprague Dawley, Inc., Indianapolis, IN). The protocols described below were conducted in accordance with the National Institutes of Health, “Guidelines for the Humane Care and Use of Laboratory Animals” (NIH Publications No. 80-23) and were approved by in-house Institutional Animal Care and Use Committees at the Chicago Medical School and the Portland VA Medical Center.
Mice were treated with 10 doses (2 ×week; 3.5 days apart) of MPTP hydrochloride (25 mg/kg in saline, s.c., Sigma-Aldrich, St Louis, MO) and probenecid (250 mg/kg in dimethyl sulfoxide, i.p., Sigma), or with probenecid (equal volume injected i.p.) or saline (equal volume injected s.c.) alone (control groups). The probenecid-treated animals were used as a control group, since previous studies have shown no change in the number of DA neurons or in the level of striatal DA after probenecid treatment alone (Lau et al., 1990
, Petroske et al., 2001
The functional impact of the toxin or vehicle was assessed using a novel grid test (Tillerson and Miller, 2003
), which assesses coordination and rigidity. The grid was constructed of a horizontal square (15 cm2 with openings in the mesh of 0.5 cm2) attached to 4 cardboard walls. Each mouse was placed in the center of the grid and the grid was rotated 180o suspending the mouse approximately 30 cm above the floor. The mouse was allowed to move freely while upside down and was videotaped for 60 seconds. All steps with the forepaws were counted and each unsuccessful forepaw step was declared a fault. A ratio of total forepaw foot faults/total steps was established for each mouse. Animals had to take a minimum of 10 total steps to be included in the data set and must have stayed on the Grid for 10s for the trial to be recorded. A mean of 3 trials/mouse was used in the analysis. Data for each group was pooled and tested statistically with a one-way ANOVA followed by a Student-Newman-Keuls post hoc test.
Immunohistochemistry and stereology
Mice were deeply anesthetized with pentobarbital (135 mg/kg) and transcardially perfused with physiologically buffered saline followed by fixative (3% paraformaldehyde in 0.1M phosphate buffer (PB) and 2.5% sucrose). Brains were sunk in 20% sucrose, blocked and then cut at 50µm on a freezing microtome. Sections were collected serially in well dishes for stereological studies of tyrosine hydroxylase (TH) -immunoreactive, Nissl-stained neurons in the substantia nigra pars compacta (SNc). Sections were incubated overnight at 4oC in mouse anti-TH sera (Diasorin, Stillwater, MN, USA), diluted 1:2000, using reagents from the Mouse-on-Mouse kit (Vector Laboratories, Burlingame, CA). This was followed by a room temperature incubation in biotinylated goat anti-mouse IgG, diluted 1:250, for 2 h. Sections were then incubated for 45 min in the reagents from the avidin-biotin complex (elite kit, Vector Laboratories) and reacted with 3,3’ diaminobenzidine hydrochloride (DAB). Sections were mounted serially on gelatinized slides, left to dry overnight, stained with cresyl violet, dehydrated and covered with coverslips.
Once dry, Nissl-stained, TH-immunopositive neurons were counted in the SNc following optical fractionator rules (see e.g., Petroske et al., 2001
; Chan et al., 2007
). Briefly, after a random selection of a starting section, neurons were counted in every third section for a total of 7 sections through the entire midbrain. Neurons were counted using dedicated software (StereoInvestigator, MicroBrightfield, Williston, VT) and hardware (Nikon E400 microscope with a motorized stage in 3 axes driven by the software. Using the optical disector, the total number of Nissl-stained, TH-immunoreactive neurons was estimated. The volume of the SNc was estimated following the Cavalieri method (Sterio, 1984
In vivo microdialysis
Approximately four weeks after the last MPTP/p or vehicle injection, animals (n = 6/group) were anesthetized (1 ml/100 g of 2.5% ketamine, 1% xylazine and 0.5% acepromazine in normal saline), and placed in a mouse stereotaxic apparatus (Cartesian Research, Inc., Oregon). A small hole was drilled on the left side of the skull anterior: −3.2 mm; lateral: +1.7 mm (Franklin, 1997
) and a stainless steel guide cannula (8 mm long, 21-gauge; Small Parts, Miami Lakes, FL) was lowered 1.5 mm from the surface of the skull. The animals were allowed to recover for one week prior to the start of the microdialysis experiment. Dialysis probes, 210 µm in diameter and 1 mm long, were prepared as previously described (Meshul et al., 1999
). Details of the microdialysis procedure have been reported (Holmer et al., 2005
). Briefly, the tip of the guide cannula was positioned within the cortex and the probe lowered so that the tip reaches the most ventral aspect of the SN. The probe was secured to the guide cannula and the other end was attached to a syringe containing artificial cerebrospinal fluid (aCSF). A pump (Orion, Model 365, Thermo Electron Corp, UK) pushed the aCSF through the probe overnight at a rate of 0.2 µl/min. The following morning, the pump speed was increased to 2 µl/min for 1 hour and then 4 samples were collected every 15 minutes. The mean probe recovery was 10.4 ± 1.2%.
Glutamate concentration in the dialysate samples was determined using a Hewlett Packard HPLC 1090 interfaced with a Hewlett Packard 1046A Programmable Fluorescence Detector. Dialysates were derivatized with o-phthalaldehyde and chromatographed as previously reported (Meshul et al., 1999
, Meshul et al., 2002
). Assay sensitivity was in the sub-picomole range. After collecting baseline glutamate levels, mice were injected with 15mg/kg levodopa (l-dopa ,i.p.) and then 6 samples were collected every 15 minutes. The animals were freely moving during the entire microdialysis procedure. At the conclusion of the experiment, the animals were transcardially perfused with glutaraldehyde fixative (2.5% glutaraldehyde/0.5% paraformaldehyde in 0.1 M HEPES, pH 7.3, containing 0.1% picric acid). Vibratome sections (100µm) were cut and stained with thionin to verify the site of the probe placement within the SN. Data were discarded from animals with incorrect placements.
Glutamate transporter assays
To establish possible alterations in glutamate transporter function, mice (n = 20/group) were treated chronically with MPTP/p or vehicle. All mice were killed 3 weeks later by cervical dislocation and decapitation. Following rapid removal of the brains, the midbrain was blocked and the paired SN were dissected. Crude synaptosomes were prepared from the midbrains of these animals and the SN from 5 mice were pooled for each group in each experiment.
The SN from each side of the brain was isolated within 2 min of decapitation. The SN from 5 mice per group (MPTP/p and vehicle groups) were pooled and homogenized in ice-cold medium of 0.32M sucrose in 50mM Tris, buffered to pH 7.5. The homogenate was centrifuged 5 min at 1200g at 4oC (Sorvall centrifuge, SS-34 rotor). The supernatant was then transferred to a fresh tube, centrifuged 12 min at 17,000g at 4oC and the pellet washed in ice-cold gradient medium and collected by centrifugation. The crude pellet was re-suspended in a final volume of 750 µl of homogenization medium. High affinity Na+-dependent transport was assayed at 4 different concentrations of D-[3H]aspartate increasing from 0.4 to 40 ×10-5M, as described previously (De Souza et al., 1999
). D-aspartate was used instead of L-glutamate in these experiments, because D-aspartate is not metabolized by glutamate transporters (Davies and Johnston, 1976
; Fyske et al., 1992
). For the assay, resuspended synaptosomes were added to duplicate microcentrifuge tubes containing Krebs solution, pH 7.4. Following protein determination, the remaining synaptosomes were incubated at 37°C with 15 nCi of D -[2,3-3H]-aspartic acid (New England Nuclear, Downers Grove, IL). The reaction was stopped with 200 µl of ice cold 1 mM D-aspartate, followed by microcentrifugation at 15,700g at 4°C. The pellets were washed twice without re-suspending using Krebs medium followed by microcentrifugation. The washed pellets were dissolved by overnight incubation in 200 µl of 2% sodium dodecyl sulfate and suspended in 5 ml of Ecoscint A (National Diagnostics, Atlanta, GA, USA). The quantity of radioactivity was determined by liquid scintillation spectroscopy and the kinetic parameters Km and Vmax were determined by non-linear regression analysis. Data gathered were the means of results obtained from determination performed as triplicates in 4 experiments and the rate of transport was plotted as a function of substrate concentration.
Mice (n = 5/group) were treated with MPTP/p or vehicle. Mice were killed by cervical dislocation and decapitation after 2, 3, 4 or 8 injections of MPTP/p or vehicle and at 3 days or 3 weeks post-treatment. Their brains were rapidly removed, frozen on dry ice (in isopentane) and the midbrains sectioned on a cryostat at a thickness of 20µm. Five sections (every 12th section) through the SN were directly mounted onto proteinase-resistant microscope slides, dried at room temperature, and stored in slide boxes containing desiccant at −80oC until processing. Slides were transferred from the freezer directly into a fixative solution (4% paraformaldehyde in 0.1M PB) for 30 min. They were then rinsed in PBS and labeled according to FD NeuroApop (FD NeuroTechnologies, Ellicott City, MD) kit instructions. Following the reaction in the chromogen, sections were rinsed, air dried, counterstained with methyl green or cresyl-violet, dehydrated and coverslipped. Apoptotic profiles were counted in all sections through the SNc and results were pooled for each group.
Electron microscopy studies of cell death
Mice (n = 5/group) treated with MPTP/p or vehicle, were anesthetized 1 or 3 weeks after treatment. Their brains were fixed by perfusion through the heart first with saline and then with a fixative containing 4% paraformaldehyde with 0.1% glutaraldehyde in 0.1 M PB. Coronal sections were cut through the midbrain at 70µm using a vibrating microtome (Leica, Milton Keynes, UK) and collected in vials containing the same buffer. Sections were flattened onto watch glasses then immersed in 1% osmium tetroxide in 0.1 M PB for 30 minutes. After a brief wash in water, they were dehydrated through an ascending series of alcohol concentrations, spending 40 minutes in a saturated solution of uranyl acetate in the 70% ethanol stage. Finally the sections were immersed in two changes of propylene oxide before being transferred to Durcupan resin (ACM Fluka, Sigma Aldrich, Poole UK) overnight. The resin was then gently warmed and the sections transferred to glass slides and a coverslip applied.
Areas of SNc were chosen randomly from each mouse to determine whether autophagosomes were present in intact neurons and whether MPTP/p treatment led to autophagic cell death. The coverslip was removed and a small piece of tissue excised and glued onto a pre-formed resin block using cyanoacrylate glue. Three blocks were cut from the SNc of each mouse. These blocks were then re-sectioned using an ultramicrotome (Ultracut E, Leica, Milton Keynes UK) at 60–70 nm and serial sections were mounted onto single slot, pioloform coated, copper grids. The sections were contrasted using lead stain and examined in a Philips 401 electron microscope. Images of dendrites showing signs of disruption and degenerating cell bodies were captured digitally (Multiscan, Gatan, Oxford UK).
Data were first checked to verify a normal distribution. Non-normally-distributed data (number of TH-positive, Nissl-stained neurons) were analysed using Kruskal-Wallis followed by Dunnett’s post-hoc tests. The four baseline data points from the microdialysis study were averaged at each time point and then a grand mean determined. Values are expressed as the mean + S.E.M. in picomoles/µl of extracellular SN glutamate within the dialysate sample. Baseline levels and levels following acute l-dopa administration were compared using a one-way ANOVA followed by the Tukey-Kramer post-hoc for comparison of multiple means. Glutamate transport was plotted as a nonlinear regression. Only two groups - MPTP/p and vehicle (combined saline and probenecid controls) - were used for statistical tests in the glutamate transport and behavioral (grid test) experiments. Therefore, an unpaired Student’s t-test was used for both data sets.