Animals and housing
Male Sprague-Dawley rats (Rattus norvegicus) weighing 250–300 g were obtained from Taconic Laboratories (Germantown, NY). Animals were individually housed with food and water available ad libitum in their home cage. A 12/12 hr light/dark cycle was used with the lights on at 7:00 a.m. All experimental procedures were performed during the light cycle. The experimental protocols were all consistent with the guidelines issued by the U.S. National Institutes of Health and were approved by the University of Pennsylvania School of Medicine and the University of Pennsylvania School of Medicine’s Institutional Animal Care and Use Committee.
Prior to surgery, the rats were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine (i.p.; Sigma-Aldrich, St. Louis, MO). An indwelling silastic catheter (inner diameter 0.33 mm, outer diameter 0.64 mm) was inserted into the right jugular vein and sutured in place. The catheter was then passed subcutaneously over the shoulder blade and routed to a mesh backmount platform (CamCath, Cambridge, UK) that was sutured below the skin between the shoulder blades. Catheters were flushed daily with 0.3 ml of the antibiotic Timentin (ticarcillin disodium/potassium clavulanate, 0.93 mg/ml; Henry Schein, Melville, NY) dissolved in heparinized saline (10 U/ml). The catheters were sealed with plastic obturators when not in use.
Rats were allowed 7 days to recover from surgery before cocaine self-administration commenced. Rats were assigned to one of two groups: cocaine self-administering animals and yoked saline controls. Within each individual experiment, rats were randomly assigned to experimental and control groups. Each rat trained to respond for contingent cocaine infusions was paired with a yoked subject that received infusions of saline. Lever pressing for the saline-yoked rats had no scheduled consequences, but these animals received the same number and temporal pattern of infusions as self-administered by the paired cocaine-experimental rat. Rats responded for infusions of cocaine in the absence of cues (i.e., lights and tones).
Initially, cocaine-experimental rats were placed in the modular operant chambers (Med Associates, St. Albans, VT) and allowed to lever press for intravenous cocaine infusions (0.25 mg cocaine/59 µl saline, infusion over 5 s) on a fixed-ratio 1 (FR1) schedule of reinforcement. Once a cocaine-experimental rat achieved at least 20 infusions of cocaine in a single operant session under the FR1 schedule, the response requirement was switched to a FR5 schedule of reinforcement. For responding on both FR1 and FR5 schedules, the maximum number of cocaine infusions was limited to 30 per daily self-administration session and a 20 s time-out period followed each cocaine infusion, during which time active lever responses were tabulated but had no scheduled consequences. Daily 2 h operant sessions (7 days/week) were conducted for a total of 14 days. Responses made on the inactive lever, which had no scheduled consequences, were also recorded during both the FR1 and FR5 training sessions.
After the 14th
daily operant session, cocaine-experimental and yoked saline control rats were returned to their home cages where they underwent 7 days of forced drug abstinence. On the 7th
day of cocaine abstinence, brains were removed and the VTA was dissected on ice. Seven days of cocaine abstinence were chosen in order to draw comparisons to our previously published study examining cocaine-induced changes in transcriptional regulation of BDNF promoters in the prefrontal cortex (Sadri-Vakili et al. 2010
). Furthermore, cocaine-induced changes in chromatin structure at striatal BDNF promoters persist for at least 7 days of forced drug abstinence (Kumar et al. 2005
). VTA dissections were based upon standard anatomical landmarks and stereotaxic coordinates commonly used in studies examining biochemical changes in this brain region (Choi et al. 2011
, Fitzgerald et al. 1996
, Russo et al. 2007
). Each VTA was dissected from a coronal slice that spanned approximately −5.2 to −6.3 mm relative to bregma (Paxinos & Watson 1997
). VTA tissue samples were stored at -80°C until assayed using Western blot, ELISA, and ChIP methods.
Enzyme-linked Immunosorbant Assay
VTA tissue homogenates were diluted to 10 µg/µl and 20 µg/µl. The concentration of BDNF in the diluted lysates was quantified using the Chemikine™ Brain Derived Neurotrophic Factor (BDNF) Sandwich ELISA kit (Chemicon International Inc., CA). Diluted VTA tissue homogenates and serial dilutions of BDNF standards were loaded in triplicate onto a 96 well plate coated with rabbit anti-human BDNF polyclonal antibodies and incubated overnight at 4°C. Each microplate was washed 4 times and biotinylated mouse anti-human BDNF monoclonal antibody (1:1000) was added to each well for 2.5 h at room temperature. Subsequently, the plates were washed 4 times and a strepavidin-enzyme conjugate was added to each well and allowed to incubate for 1 h. After further washing, tetramethylbenzidine chromagenic substrate was added to each well. Fifteen minutes later the reaction was stopped. Absorbance at 450 nm was measured with a plate reader. BDNF concentration in the VTA tissue homogenates was measured by comparing values to the prepared standard curve.
Chromatin immunoprecipitation (ChIP) assay
A modified ChIP technique was adapted from previously published studies from our laboratory in order to analyze DNA/protein complexes in dissected brain tissue (Braveman et al. 2004
, Chen-Plotkin et al. 2006
). Briefly, the VTA underwent a formaldehyde cross-linking step to link the transcription factors or histone proteins to DNA. Samples then were homogenized and subjected to immunoprecipitation with antibodies specific for the CBP or di-acetyl lysine 9, lysine14 histone H3 (H3K9K14Ac2; AcH3). The antibody-transcription factor-DNA complexes were washed to reverse the crosslinks, and the DNA was detected by qPCR using specific primers for the multiple BDNF promoters. Threshold amplification cycle numbers (Tc
) using iCycler software were used to calculate IP DNA quantities as percentage of corresponding inputs. The following exon specific BDNF primers were designed based on previously published sequences (Chen et al. 2003
, Martinowich et al. 2003
, Jiang et al. 2008
) and used for real-time PCR analysis: BDNF exon I
: forward 5’-GCAGTTGGACAG TCATTGGTAACC-3’ and reverse 5’-ACGCAAACGCCCTCATTCTG-3’; BDNF exon IV
: forward 5’-AACAAGAGGCTGTGACAC TATGCTC-3’ and reverse 5’-CAGTAAGTAAAGGCTAGGGCAGGC-3’.
RNA extraction and reverse transcription
RNA was extracted from dissected VTA following 14 days of cocaine self-administration and 1 or 7 days of forced abstinence using a RNeasy kit (Qiagen, Valencia, CA) according to manufacturer’s instructions and as described previously (Sadri-Vakili et al. 2010
). Reverse transcription reactions were performed using specific primers in an iCycler (Bio-Rad) (25°C for 10 min, 42°C for 50 min, 70°C for 15 min) in an iCycler (Bio-Rad) to quantify the amount of gene expression as compared to a standard curve (Superscript First Strand Synthesis System; Invitrogen, Carlsbad, CA). The following primers were used: BDNF exon I
: forward 5’-GCGTTGAGAAAGCTGCTTCAG-3’ and reverse 5’-GAATGAGCGAGGTTACCAATGA-3’; BDNF exonII (a, b, c)
: forward 5’-GCAGAGTCCATTCAGCACCTTG-3’ and reverse 5’-TGGCTTGACAGCGAGGAAAAG-3’; BDNF exon IV
: forward 5’-TTCCACTATCAATAATTTAACTTCTTTGC-3’ and reverse 5’-CTCTTACTATATATTTCCCCTTCTCTTCAGT-3’; BDNF exon VI
: forward 5’- TTTGGGGCAGACGAGAAAGC-3’ and reverse 5’-GGCAGT GGAGTCACATTGTTGTC-3’; GAPDH
: forward 5’-AACAGCAACTCCCATTC TTC-3’ and reverse 5’-TGGTCCAGGGTTTCTTACTC-3’;. BDNF primers were based on previously published sequences (Aid et al. 2007
). Quantitative real time-PCR was performed using 50 PCR cycles (95°C for 30 s, 57°C for 60 s, 72°C for 90 s) in an iCycler (Bio-Rad) with the use of SYBR-green PCR Master Mix (Applied Biosystems, Foster City, CA). The threshold cycle for each sample was chosen from the linear range and converted to a starting quantity by interpolation from a standard curve run on the same plate for each set of primers. For each replicate, mRNA levels were normalized to their respective GAPDH mRNA levels.
In each experiment, we used data from 3 to 6 individual rats per treatment condition. Differences between groups were analyzed for statistical significance by using two-tailed Student’s t-test. When more than two groups of data were compared two-way ANOVA was used followed by Bonferroni post-hoc test.