Our first immunoblotting study was aimed to investigate the influence of a history of cocaine self-administration experience upon the protein expression of mGluR1/5 within PFC subregions at early versus protracted withdrawal. For this study, within each cohort of rats, separate groups of Sal1h, Sal6h and Coc6h rats underwent a 2-h cue test session under extinction conditions at either 3 or 30 days withdrawal (n=12 per group at the start of the experiment) to assay for cue-reinforced lever-pressing behavior. Immediately upon completion of this cue test, animals were killed by rapid decapitation. The dmPFC (anterior cingulate and dorsal prelimbic cortices) and vmPFC (ventral prelimbic and infralimbic cortices) were dissected out over ice in a manner identical to that described by others (Ghasemzadeh et al., 2009 (link)). The tissue derived from animals in this study was immunoblotted in 2 separate ways. First, comparisons were made across the tissue from the 3 different self-administration groups separately for each time-point (i.e., Sal1h, Sal6h & Coc6h rats sacrificed at 3 days withdrawal were compared in one assay, and a parallel assay compared Sal1h, Sal6h & Coc6h rats sacrificed at 30 days withdrawal in a separate assay). This “single time-point” analysis was done to enable a comparison of protein expression between the SAL1h and SAL6h rats at each time-point, as well as to determine whether or not a history of cocaine self-administration influenced protein expression relative to a history of saline self-administration at that particular withdrawal time-point. For these assays, the immunoreactivity of the mGluR1 and mGluR5 bands from the Sal6h and Coc6h animals was normalized to the average of that for the Sal1h animals run on the corresponding gel (n=4–5/gel) and the data expressed as a percentage of that averaged Sal1h signal for each gel. The data for the 3-day and for the 30-day assays were analyzed separately using an univariate analysis of variance (ANOVA) across the 3 different self-administration groups to determine the presence/absence of a cocaine effect.
The next analysis examined for proteomic correlates of the increased lever-pressing exhibited by both saline and cocaine self-administering animals at 30 days of withdrawal, by comparing tissue from the Sal6h and Coc6h animals sacrificed at both withdrawal time-points on the same gel. For this “time-course” assay, the immunoreactivity of the mGluR1 and mGluR5 bands were normalized to the average of the Sal6h-3 day withdrawal group run on the corresponding gel (n=3–4/gel) and the data expressed as a percentage of that averaged signal for each gel. The data were analyzed using orthogonal comparisons, within each self-administration group, to determine the presence/absence of time-dependent changes in protein expression. A second, follow-up, study assayed vmPFC and dmPFC tissue from Sal1h and Coc6h rats, sacrificed at 3 versus 30 days withdrawal, in the absence of any cue testing. This study was conducted to determine the extent to which our cocaine-induced changes in mGluR1/5 protein expression reflected alterations in the basal expression pattern of these receptors or some interaction between cocaine experience and the cue-reinforced testing conditions of our animals. The data analysis for this study was identical to that employed for the “time-course” study above.
The immunblotting procedures for detection of total mGluR1 and mGluR5 levels in brain tissue homogenate were identical to those described recently by our group (e.g., Cozzoli et al., 2009 (link), 2012 ; Goulding et al., 2011 (link)). In brief, tissue was homogenized in a solution consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 1 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, 1 mM orthovanadate, and 2 μM microcystin LR were included to inhibit phosphotases. After centrifugation at 10,000 g for 20 min, the supernatant of the homogenates were quantified for protein content using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) and stored at −80 °C. Protein samples (20 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis on Tris-Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA). Wet polyvinylidene difluoride (PVDF) (Bio-Rad) membrane transfer was employed, and membranes were pre-blocked with either phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dried milk powder for a minimum of 2 hrs before overnight incubation with primary antibody. An anti-mGluR5 rabbit polyclonal antibody (Millipore, Billerica, MA; 1:1000 dilution) and an anti-mGluR1a mouse polyclonal antibody (Millipore; 1:500 dilution) were used for receptor detection. A rabbit primary anti-calnexin antibody (Stressgen, Ann Arbor, MI; 1:1000 dilution) was used as a control to ensure even protein loading and transfer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Millipore; 1:5,000–1:10,000 dilution) or anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA; 1:10,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). Image J (NIH, Bethesda, MD) was used to quantify immunoreactivity levels.
The next analysis examined for proteomic correlates of the increased lever-pressing exhibited by both saline and cocaine self-administering animals at 30 days of withdrawal, by comparing tissue from the Sal6h and Coc6h animals sacrificed at both withdrawal time-points on the same gel. For this “time-course” assay, the immunoreactivity of the mGluR1 and mGluR5 bands were normalized to the average of the Sal6h-3 day withdrawal group run on the corresponding gel (n=3–4/gel) and the data expressed as a percentage of that averaged signal for each gel. The data were analyzed using orthogonal comparisons, within each self-administration group, to determine the presence/absence of time-dependent changes in protein expression. A second, follow-up, study assayed vmPFC and dmPFC tissue from Sal1h and Coc6h rats, sacrificed at 3 versus 30 days withdrawal, in the absence of any cue testing. This study was conducted to determine the extent to which our cocaine-induced changes in mGluR1/5 protein expression reflected alterations in the basal expression pattern of these receptors or some interaction between cocaine experience and the cue-reinforced testing conditions of our animals. The data analysis for this study was identical to that employed for the “time-course” study above.
The immunblotting procedures for detection of total mGluR1 and mGluR5 levels in brain tissue homogenate were identical to those described recently by our group (e.g., Cozzoli et al., 2009 (link), 2012 ; Goulding et al., 2011 (link)). In brief, tissue was homogenized in a solution consisting of 0.32 M sucrose, 2 mM EDTA, 1% w/v sodium dodecyl sulfate, 50 μM phenyl methyl sulfonyl fluoride and 1 μg/ml leupeptin (pH=7.2) and 1 mM sodium fluoride, 50 mM sodium pyrophosphate, 20 mM 2-glycerol phosphate, 1 mM p-nitrophenyl phosphate, 1 mM orthovanadate, and 2 μM microcystin LR were included to inhibit phosphotases. After centrifugation at 10,000 g for 20 min, the supernatant of the homogenates were quantified for protein content using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) and stored at −80 °C. Protein samples (20 μg/lane) were subjected to SDS-polyacrylamide gel electrophoresis on Tris-Acetate gradient gels (3–8%) (Invitrogen, Carlsbad, CA). Wet polyvinylidene difluoride (PVDF) (Bio-Rad) membrane transfer was employed, and membranes were pre-blocked with either phosphate-buffered saline containing 0.1% (v/v) Tween 20 and 5% (w/v) nonfat dried milk powder for a minimum of 2 hrs before overnight incubation with primary antibody. An anti-mGluR5 rabbit polyclonal antibody (Millipore, Billerica, MA; 1:1000 dilution) and an anti-mGluR1a mouse polyclonal antibody (Millipore; 1:500 dilution) were used for receptor detection. A rabbit primary anti-calnexin antibody (Stressgen, Ann Arbor, MI; 1:1000 dilution) was used as a control to ensure even protein loading and transfer. Membranes were washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Millipore; 1:5,000–1:10,000 dilution) or anti-mouse secondary antibody (Jackson Immuno Research Laboratories, West Grove, PA; 1:10,000) for 90 min, washed again, and immunoreactive bands were detected by enhanced chemiluminescence using either ECL Plus (Amersham Biosciences) or Pierce SuperSignal West Femto (Thermo Fisher Scientific, Rockford, IL). Image J (NIH, Bethesda, MD) was used to quantify immunoreactivity levels.
