transgenic rats (Kasof et al., 1995
; Koya et al., 2009
) and female Sprague-Dawley rats (Charles River, Raleigh, NC, USA) were housed individually in standard plastic cages in a temperature- and humidity-controlled room. They were maintained on a 12:12 h reverse light:dark cycle (lights on at 20.00 h), and allowed free access to food and water. They were acclimatized to these housing conditions for a minimum of 7 days prior to drug treatments. Experimental procedures were approved by the NIDA Animal Care and Use Committee.
For acute cocaine experiments, rats were injected with cocaine (30 mg/kg, i.p.; n=8) or saline (1 ml/kg; n=6) and placed in a round Plexiglass chamber (38 cm diameter). Rats were sacrificed 90 minutes after injections to obtain brain tissue. This treatment was repeated three times on separate days to obtain three biological replicates for microarray analysis. Additionally, three independent biological replicates were similarly produced for quantitative PCR (qPCR). For qPCR analyses of arc, pdyn, adora2a, only these three independent biological replicates were used. For qPCR analyses of fosB, nr4a3, kcnc1, and map2k6, we also used remaining RNA from each of the microarray samples.
For repeated cocaine experiments, female rats were sensitized with four injections of cocaine (15 mg/kg i.p.) administered once every second day. On injection days, each rat was placed in a 43 × 43 cm square Plexiglas locomotor activity chamber (Med Associates, St Albans, VT, USA) to habituate for 30 minutes. Rats were injected with cocaine and locomotor activity was recorded as distance traveled for 60 minutes. Following the fourth repeated cocaine injection, rats remained in their home cage for 7–8 days. On test day, rats were habituated for 30 min in the locomotor activity chambers and then injected with cocaine (30 mg/kg i.p.) or saline vehicle. Rats were decapitated and brains were extracted ninety minutes after injections. The entire sensitization treatment was repeated three times over separate weeks to obtain three biological replicates of each group for qPCR analysis.
Striatal tissue was dissociated to obtain a single-cell suspension. Rats were decapitated and their striata extracted within two minutes. Each striatum was minced with razor blades on an ice-cold glass plate and placed in 1 ml of Hibernate A (Cat# HALF; Brain Bits, Springfield, IL). Striata were enzymatically digested in 1 ml of Accutase per striatum (Cat# SCR005; Chemicon, Billerica, MA) with end-over-end mixing for 30 min at 4°C. Tissue was centrifuged for two min at 425×g and resuspended in 300 μl of ice-cold Hibernate A. All subsequent centrifugation steps were performed at 4°C.
Digested striatal tissue was mechanically dissociated by trituration. Four striata were combined into one Eppendorf tube and triturated ten times with a large diameter Pasteur pipet (~1.3mm). Tubes were briefly placed on ice for large tissue pieces to settle; 600 μl of cloudy suspension containing dissociated cells were transferred to a 15 ml Falcon tube on ice. 600μL of fresh Hibernate A were added to the original Eppendorf tube and then trituration steps were repeated as above with medium and small diameter pipets (~0.8mm and ~0.4mm). Each cloudy suspension containing dissociated cells was pooled with the previous suspension.
Remaining cell clusters in the pooled cell suspension were removed by filtration through pre-wetted 100 μm and 40 μm cell strainers (Falcon brand, BD Biosciences, San Jose, CA). Small cellular debris in the suspension was reduced by centrifuging the filtrate for 3 min at 430×g through a three-step density gradient of Percoll (Cat# P1644; Sigma, St. Louis, MO). The cloudy top layer (approximately 2 ml) containing debris was discarded. Cells in the remaining layers were resuspended in the remaining Percoll solution and centrifuged for five min at 550xg. The pellet was resuspended in 1 ml of Hibernate A.
Immunolabeling and FACS
Dissociated cells were fixed and permeabilized by adding an equal volume of ethanol for a final concentration of 50% ethanol and kept on ice for 15 minutes with occasional mixing. Cells were centrifuged for two min at 425xg and resuspended in RNase-free phosphate-buffered saline (PBS). Cells were incubated with a biotinylated primary antibody against NeuN (1:1000 dilution, Cat# MAB377B, Chemicon) and a primary antibody against β-galactosidase ((β-gal; 1:10000 dilution, Cat# 4600-1409, Biogenesis, Poole, United Kingdom). Cells were rotated end-over-end in primary antibody for 30 minutes at 4°C and centrifuged for three min at 425xg. Cells were washed with 800 μl of PBS and resuspended in 700 μl of PBS. Cells were incubated with fluorescently-labeled streptavidin (Streptavidin-phycoerythrin, 1:1000 dilution, Cat# SA1004-1, Invitrogen, Carlsbad, CA) and secondary antibody (Alexa Fluor 488-labeled anti-goat IgG, 1:1000 dilution, Cat# A-11055, Invitrogen) and rotated end-over-end for 15 min. Cells were washed with 800 μl of PBS, centrifuged for three min at 425×g, and resuspended in 1 ml of PBS.
Immunolabeled cells were scanned and sorted at the Johns Hopkins Bayview Campus flow cytometry core facility. A FACS Aria (BD, Franklin Lakes, NJ) was used for cell sorting. Control samples were analyzed first to determine optimal criteria for sorting test samples. Specifically, fixed cells without antibody treatment were used to set the light scatter gate. Fixed cells treated with fluorescent secondary antibody, but no primary antibody, were then used to set thresholds for endogenous tissue fluorescence and non-specific binding by streptavidin or secondary antibody. Next, control samples single-labeled with primary and secondary antibody for each protein (NeuN or β-gal) were used to compensate for fluorescent overlap in neighboring channels. Finally, test samples labeled with both fluorescent markers were analyzed and sorted. Sorted cells were collected into low-binding tubes (Cat# 022431081, Eppendorf, Westbury, NY) with 100 μl of PBS in each tube.
After sorting, samples containing either βgal-positive or βgal-negative cells from cocaine-injected rats or all NeuN-positive cells from saline-injected rats were centrifuged for 8 min at 2650×g at 18°C. NeuN-negative cells from saline-injected rats were centrifuged for 8 min at 6000×g at 18°C. For microarray experiments, RNA was extracted with Trizol Reagent (Cat# 15596-026, Invitrogen) according to manufacturer’s instructions. Quality and quantity of RNA were assessed using the Bioanalyzer Picochip (Cat# 5067-1513, Agilent Technologies, Palo Alto, CA). For qPCR experiments, RNA was extracted with RNEasy Micro kit (Cat# 74004; Qiagen, Valencia, CA) according to manufacturer’s instructions with DNase treatment. Quantity and purity of RNA was assessed using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE).
Microarrays were used to analyze RNA from NeuN-positive and NeuN-negative cells from saline-injected rats, and βgal-positive and βgal-negative neurons from cocaine-injected rats. As described above in the drug treatment section, microarrays contained three biological replicates for each of the four sample types. Five μl of total RNA from each sample were labeled using the Illumina TotalPrep RNA Amplification Kit (Cat# IL1791; Ambion, Austin, TX) in a two-step process of cDNA synthesis followed by in vitro RNA transcription with biotin-16-UTP. 0.75 μg of biotin-labeled cRNA was hybridized for 16 hours to Illumina Sentrix Rat Ref12_v1 BeadChips (Cat# BD-27-303; Illumina, San Diego, CA). Biotinylated cRNA hybridized to the chip was detected with Cy3-labeled streptavidin and quantified using Illumina’s BeadStation 500GX Genetic Analysis Systems scanner. All labeling and analysis was done at the Johns Hopkins Bayview Medical Campus Lowe Family Genomics Core.
Real time quantitative PCR
Real-time quantitative PCR was used to validate FACS and microarray results. RNA was reverse-transcribed into cDNA using the RETROscript kit (Cat# AM1710, Ambion) with an oligo(dT) primer. Each 25 μl PCR reaction included 12.5 μl of Taqman Gene Expression Master Mix (Cat #4369514; Applied Biosystems, Foster City, CA), 1.1 μl each of 20 μM forward and reverse primers, 0.25 μl of 100 μM FAM-labeled probe, 5 μl of water, and 5 μl of 1 ng/μl cDNA. Primer and probe combinations  were designed using the Roche Universal Probe Library Assay Design Center to be intron-spanning. PCR reactions were monitored using the Opticon Light Cycler (Biorad, Hercules, MD). The program began with 20 plate reads at 50°C for photobleaching, followed by 5 min at 95°C, and then 40 cycles of 20 sec at 94°C, 1 min at 60°C, and a plate read.
Primers and probes used for qPCR
Standard curve reactions were first run for each gene to determine amplification efficiency . Expression levels for each amplified gene were calculated using efficiency^ΔCq where ΔCq = Cq(experimental gene) − Cq(reference gene) for each biological replicate. Gorasp2 was chosen as a reference gene because it was not differently expressed between neurons and glia in microarray analyses. It was further validated as a reference gene due to equal qPCR amplification across all samples when loading the same amount of template. Technical assay triplicate Cq values were averaged before calculating ΔCq for each gene in a sample. For each mRNA measured in qPCR, gene expression values were averaged across biological replicates. Then to reflect fold-change values, we divided this average by the average gene expression values of NeuN-positive cells from saline-injected rats. These values are indicated as means and standard errors in the graphs and tables.
Brain tissue was obtained from wild-type female Spraque-Dawley rats following the same acute and repeated cocaine treatments described above for the microarray and qPCR experiments. However 90 minutes after test day injections, rats were deeply anesthetized with isoflurane and perfused with 100 ml of PBS followed by 400 ml of 4% paraformaldehyde (PFA). Brains were post-fixed in PFA for 2 hours and transferred to 30% sucrose solution at 4°C for 2–3 days. Brains were frozen on powdered dry ice and kept at −80°C until sectioning. Coronal sections were cut 40 μm thick between Bregma 2.5 and −0.5 (Paxinos and Watson, 1998
Sections were immunolabeled for Fos and Arc. Briefly, sections were washed three times in Tris-buffered saline (TBS) and permeabilized for 30 min in TBS with 0.2% Triton X-100. Sections were washed again in TBS and incubated in primary antibodies diluted in PBS with 0.3% Triton X-100 for 24 hours on a shaker at 4°C. We used a rabbit polyclonal against c-Fos antibody (1:500 dilution, Cat# sc-52, Santa Cruz Biotechnology, Santa Cruz, CA) and a mouse monoclonal Arc antibody (1:100 dilution, Cat# sc-17839, Santa Cruz Biotechnology). Sections were washed three times in TBS and incubated in secondary antibodies diluted in PBS for 1.5 hours on a shaker at room temperature. We used Alexa Fluor 488-labeled donkey anti-rabbit antibody (1:200 dilution, Cat# A21206, Invitrogen) and Alexa Fluor 568-labeled goat anti-mouse antibody (1:200 dilution, Cat# A11004, Invitrogen). Sections were washed in TBS, mounted onto chrom-alum coated slides, and coverslipped with VectaShield hard-set mounting media.
Fluorescent images of Fos and Arc immunoreactivity in caudate-putamen and NAc (approximately 2.0 mm anterior to Bregma) were captured using a CCD camera (Coolsnap Photometrics, Roper Scientific Inc., Trenton, NJ) attached to a Zeiss Axioskop 2 microscope. Images for counting labeled cells were taken at 200x magnification; images for determining Fos and Arc colocalization were taken at 400x magnification. Labeled cells from four hemispheres per rat were automatically counted using IPLab software for Macintosh, version 3.9.4 r5 (Scanalytics, Inc., Fairfax, VA). Counts from all four hemispheres were averaged to obtain a single value for each rat.
Locomotor sensitization data were analyzed using one-way ANOVA with repeated measures for the main effect of time over 4 injection days. Statistical significance was set at p < 0.05. Microarray data were analyzed using DIANE 6.0, a spreadsheet-based microarray analysis program based on SAS JMP 7.0, as previously described (Garg et al., 2009
). Fluorescence intensity data from microarrays were subject to filtering by detection p
-values and Z
normalization. Sample quality was analyzed using scatterplots, principal components analysis, and gene sample Z
-score-based hierarchical clustering to exclude possible outliers. ANOVA tests were then used to eliminate genes with larger variances within each comparison group. Genes were identified as differentially expressed if p <0.01, absolute Z
-ratio ≥ 1.5, and false discovery ratio (fdr) <0.07. For qPCR data, one-way ANOVA was used to compare data for each gene. Statistical significance was set at p <0.05. For Fos and Arc immunohistochemistry, a t-test assuming unequal variance was used to calculate statistical significance, set at p <0.05.