Wobbler mice and homozygous healthy littermates (NFR/wr strain, NIH, Animal Resources, Bethesda, USA) were bred at Charles River Italia (Calco, Lecco, Italy). Mice were maintained at 21 ± 1°C with relative humidity 55 ± 10% and a 12-hour light/dark cycle. Food (standard pellets) and water were available ad libitum.
Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n° 116, G.U. suppl. 40, 18 Febbraio 1992, circolare n° 8, G.U. 14 Luglio 1994) and international laws and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996).
In order to identify wr/wr homozigous mice, healthy homozygous +/+ and healthy heterozygous wr/+ littermates, a genotyping analysis was performed. An Alu I restriction polymorphism of a Cct 4 amplification product was used for testing the allelic status at the wr locus (Rathke-Hartlieb 1999).
Early ymptomatic wobbler mice can be easily recognized by their phenotypical features. From 3rd week of age, wobbler mice begin to growth slower than healthy littermates and, only a week later, they are 40–50% (also depending on the strain) smaller that their age-matched healthy mice. At the 3rd–4th week wobbler mice already show an altered position of fingers, wrists and paws. This alteration derives from muscular atrophy and produces unsteady gait with a discrete tremor. Afterwards, instability and wobbling of the gait develop progressively thus producing alteration in walking that is a typical feature of motor impairment occurring in the wobbler mice. As previously proposed by Kozachuk and colleagues, these two parameters of abnormalities can be useful to determine the degree of clinical worsening during this phase of the disease .
To ensure optimal quality of spinal cord tissues for histochemical determinations, tissues were fixed following the transcardial perfusion method using a solution of 4% paraformaldehyde (w/v) in 0.1 M phosphate buffered saline (PBS) (pH 7.4). After perfusion, the isolated backbone was postfixed for 3 hours at 4°C in the same solution. After postfixation the spinal cord was gently removed from the vertebral column and cryoprotected by three serial 2-h incubations at 4°C, in 0.1 M PBS, containing increasing concentrations of sucrose (10%, 20% and 30%), then dipped in cooled isopentane (-35°C to -45°C) to quickly freeze them.
For free-floating immunohistochemistry, micrometric sections (30 μm thick) were placed in plate wells containing PBS, then rinsed three times (10 min each) to remove the Tissue-Tek® O.C.T.™ solution used to surround and cover spinal tissues and ensure optimal cutting.
Before incubation with specific antibodies directed against the GluR-1, 2, 4 subunits, the same experimental procedure was used. After three rinses in 0.1 M PBS, sections were preincubated in PBS containing 5% foetal bovine serum and then incubated in PBS containing 0.5% Triton-X100 for 1 hour at room temperature. For GluR3 immunostaining, preincubation in PBS containing 0.5% albumin for 24 hours at 4°C was done to reduce non-specific staining.
For all AMPA receptor subunits, sections were incubated overnight at 4°C in a solution of PBS containing 0.1% Triton-X100 and 3% FBS with the specific antibodies: anti-rabbit polyclonal antibody raised against GluR-1 (AB1504, Chemicon International, Temecula, CA, US; 1:200); anti-mouse monoclonal antibody raised against GluR-2 (MAB397, Chemicon International, Temecula, CA, US; 1:1000); anti-goat polyclonal antibody raised against GluR-3 (sc-7613, Santa Cruz Biotechnology, Santa Cruz, CA, US; 1:400) and anti-rabbit polyclonal antibody raised against GluR-4 (AB1508, Chemicon International, Temecula, CA, US; 1:50). For the NMDA receptor we used an anti-mouse monoclonal NR1 antibody (Catatolg No 32-0500, Zymed, Invitrogen, Carlsbad, CA, US; 1:100) and an anti-rabbit polyclonal NR2A antibody (Molecular Probes, Invitrogen, Carlsbad, CA, US; 1:100).
After incubation with the primary antibody and three rinses in PBS at room temperature, all the sections were incubated for 2 hours at room temperature in PBS containing 1% FBS and the appropriate secondary antibody (1:100).
For each AMPA and NMDA receptor subunit, after incubation with the secondary antibody the sections were rinsed three times (5 min) then incubated for 1 hour in a solution containing 1% avidin and biotinylated horseradish peroxidase (ABC kit) in PBS 0.1 M, pH 7.4. After one rinse in PBS and two rinses in Tris buffered saline (TBS) 0.1 M, pH 7.8–8.3, the sections were incubated for a few minutes in TBS containing 0.5% diamminobenzidine (DAB) (w/v) and H2O2 (0.6 μL for 1 mL of solution, was added just before application). As DAB is a photosensitive molecule, it was dissolved and stored in a dark vial until incubation.
In order to avoid possible false positive results control experiments were done using the primary or the secondary antibodies alone. All experiments did not produce staining in tissues examined.
For colocalisation experiments to characterize AMPA and NR2A receptor subunit expression in astrocytes and neurons in the cervical spinal cord of four-week-old wobbler mice and healthy littermates perfused samples were sectioned at a thickness of 15 μm. As either anti-GFAP antibody or anti-NR1 antibody are monoclonal anti-mouse we could not perform colocalisation experiments among these two proteins. For immunofluorescence experiments the following antibodies were used: (rabbit anti-GluR1 polyclonal antibody AB-10129, Immunological Sciences, Roma, IT, 1:200), (rabbit anti-GluR2 polyclonal antibody AB-10699, Immunological Sciences, Roma, IT, 1:200), (goat anti-GluR3 polyclonal antibody sc-7613, Santa Cruz Biotechnology, Santa Cruz, CA, US; 1:400) (rabbit anti-GluR4 polyclonal antibody AB-10122, Immunological Sciences, Roma, IT, 1:50), (rabbit anti-NR2A polyclonal antibody AB-10675, Immunological Sciences, Roma, IT, 1:200). All antibodies were incubated as reported in the immunohistochemical methods section. Secondary antibodies (Alexa-488, Molecular Probes, 1:1000) were incubated for 2 h at room temperature. All sections were incubated using a specific marker for astrocytes (anti-mouse monoclonal GFAP antibody, MAB-12029 Immunological Sciences, Roma, IT, 1:5000) at a dilution of. The secondary antibody (Alexa-Cy5 conjugated, Molecular Probes) was used to visualize astrocyte staining. For Nissl immunofluorescence, sections were incubated 30 min with 530–615 NeuroTrace Fluorescent Nissl reagent (Molecular Probes, 1:100) at room temperature.
Sections were observed with an Olympus Fluoview microscope BX61 with confocal system FV500. Images were pseudocolored (red for the GFAP-associated staining and green for the AMPA and NMDA receptor associated staining, purple for Nissl-associated staining) and the signal obtained from the three different channels was automatically merged by Olympus fluoview software.
Western blot analysis
Subcellular fractionation of spinal cord tissue was done as previously reported, with minor modifications . Two different preparations, whole homogenate and TIF, were obtained using four pooled animals for each group. Cervical and lumbar spinal cord were homogenized in ice-cold 0.32 M sucrose containing 1 mM Hepes, 1 mM MgCl2, 1 mM EDTA, 1 mM NaHCO3, 0.1 mM PMSF, pH 7.4, with complete sets of protease inhibitors (Complete™, Roche Diagnostics, Basel, Switzerland) and phosphatase inhibitors (Sigma-Aldrich; Saint Louis, MO, US). The homogenized tissue was centrifuged at 1,000 × g for 10 min. The supernatant (S1) was centrifuged at 13,000 × g for 15 min to obtain a crude membrane fraction (P2 fraction). The pellet was re-suspended in 1 mM Hepes + Complete™ in a glass-glass Potter apparatus and centrifuged at 100,000 × g for 1 h. The pellet (P3) was resuspended in buffer containing 75 mM KCl and 1% Triton-X100 and centrifuged at 100,000 × g for 1 h. The final pellet (P4) was homogenized in a glass-glass Potter apparatus in 20 mM Hepes. Then an equal volume of glycerol was added and this fraction, referred to as TIF, was stored at -80°C until processing. TIF was used instead of the classical post-synaptic density (PSD) because the amount of starting material was very limited. The protein composition of this preparation was, however, carefully tested for the absence of presynaptic markers (e.g. synaptophysin) . Nitrocellulose papers were blocked with 10% albumin in TBS, and incubated for 2 h at room temperature with the primary antibodies NR1 (diluted 1:1000), NR2A (1:1000), GluR1 (1:2000), GluR2 (1:1000), GluR3 (1:2000), GluR4 (1:2000), alphaCaMKII (Chemicon International, Temecula, CA, US; 1:3000) in TBS containing 3% albumin. To avoid any difference of interpretation from the results of immunohistochemical experiments and western blot analysis the same batches for AMPA and NMDA receptor antibodies were used for both. After extensive rinsing in 0.1% TBS/Tween, the nitrocellulose papers were incubated with horseradish peroxidase-conjugated secondary antibodies [goat anti-rabbit, for polyclonal antibodies, dilution 1:10000 (Pierce Biotechnology Inc., Rockford, IL, U.S.); goat anti-mouse, for monoclonal antibodies, dilution 1:20000 (Pierce Biotechnology Inc., Rockford, IL, U.S.)] and the antigen-antibody complex was revealed by enhanced chemiluminescence (ECL; Amersham International, Little Chalfont, Buckinghamshire, UK).
Optical density was quantified using the AIS image analyzer (Imaging Research Inc., Ontario, Canada).