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. 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. A total of 107 rats were used in this study.
Prior to surgery, the rats were anesthetized with 80 mg/kg ketamine and 12 mg/kg xylazine (i.p.). An indwelling silastic catheter was placed into the right jugular vein (side opposite the heart) and sutured in place. The catheter was routed to a screw-on mount (Plastics One, Roanoke, VA) that was sutured below the skin between the shoulder blades. The catheters were sealed with plastic obturators when not in use. Following catheter implantation, some rats were mounted in a stereotaxic apparatus (Kopf Instruments, CA) and a viral vector (2 μl/side) was administered at the borders between the infralimbic/prelimbic cortices (+2.5 mm A/P, ±0.5 mm M/L, ±4.5 D/V) and prelimbic cortex/anterior cingulate (+2.5 mm A/P, ±0.5 mm M/L, ±3.0 D/V) via 33-gauge cannulae. All coordinates were relative to bregma according to the atlas of Paxinos and Watson (Paxinos and Watson, 1997
). Thus, each rat was microinjected with a 4 μl of virus bilaterally into the infralimbic cortex, prelimbic cortex and anterior cingulate, which collectively form the mPFC in the rat. Each 2 μl microinjection was made over 10 minutes and the microinjector was left in place for 2 minutes after the infusion.
Following a 10 to 14-day (for shRNA) or 7-day (all other experiments) recovery period from surgery, the rats were placed in operant chambers and allowed to lever press for intravenous cocaine infusions (0.25 mg cocaine/56 μl saline/infusion over 4 sec) for 14 days. Rats initially were trained using a fixed ratio (FR1) schedule of reinforcement with each daily self-administration session initiated by an intravenous priming injection of cocaine (0.25 mg). When the animals achieved stable responding with the FR1 schedule (i.e. less than 15% variation in response rates over three consecutive days) they were switched to an FR5 schedule for 14 consecutive days of self-administration. A 60-sec timeout period during which responses have no scheduled consequences followed each cocaine infusion. The rats were limited to a maximum of 30 cocaine infusions per daily 2-hour self-administration session.
For those animals in cocaine reinforcement experiments (using the progressive ratio schedule), when stable responding with the FR5 schedule was achieved, as defined by a less than 15% change in the number of responses on two consecutive days, the rats were switched to a progressive ratio (PR) schedule of reinforcement. The progressive ratio schedule is based on that of Richardson and Roberts (Richardson and Roberts, 1996
). The response requirement for the i
th reinforcement was given by: R(i)=[5e0.2i
– 5] where the brackets indicate the rounding function. Using this exponential function, the response requirement for the first ten injections was as follows: 1, 2, 4, 6, 9, 11, 15, 19, 25, 31 (etc.). Thus, under a PR schedule the response requirement for each subsequent drug delivery increased until the subject failed to meet a requirement. The session expired when an animal took more than one hour to receive an injection. The break point was operationally defined as the number of cocaine infusions administered prior to the termination of the session.
AAV cloning and packaging
The EGFP-U6-pACP plasmid contains AAV2 inverted terminal repeats flanking a CMV promoter, the coding sequence for EGFP, an intron and polyadenylation signal derived from SV40, and further downstream, a murine U6 pol III promoter. To obtain each AAV-shRNA, synthetic oligos encoding the shRNA and its respective complement (Integrated DNA Technologies, Coralville, IA) were annealed and ligated into unique BbsI
sites in the AAV plasmid downstream of the U6 promoter. The BDNF mRNA target sequence used, ACCATAAGGACGCGGACTTGT (from NCBI reference sequence NM_012513), was selected following in vitro
screening of multiple candidates (Bass and Terwilliger, unpublished). Control vectors included AAV10 encoding either EGFP alone, or AAV10 encoding EGFP plus a scrambled (scr) shRNA, (CTGTTACGCTGGCTCTATCGA)
which does not correspond to any known rat mRNA sequence. Packaging of all AAV was carried out using a standard triple transfection protocol to generate helper virus-free pseudotyped AAV10 virus (Xiao et al., 1998
). An AAV2/10 rep/cap plasmid provided AAV2 replicase and AAV10 capsid functions (Gao et al., 2002
; De et al., 2006
), while adenoviral helper functions were supplied by the pHelper plasmid (Stratagene, La Jolla, CA). Briefly, AAV-293 cells were transfected with 1.33 pmol of pHelper, and 1.15 pmol each of AAV2/10 and the shRNA-AAV vector plasmids, via calcium phosphate precipitation. The cells were harvested 48 hours later and the pellets resuspended in DMEM, freeze-thawed three times and centrifuged to produce a clarified viral lysate. The vector stocks were titered by real-time PCR using the ABI Prism 7700 Sequence Detection System from Perkin-Elmer Applied Biosystems (Foster City, CA) as previously described (Clark et al., 1999
). The average titer of the preparations was approximately 1 × 1012
10 days after rats received unilateral microinjections with AAV-viral vector containing EGFP-linked scrambled BDNF shRNA, animals were perfused with formalin, brains were post-fixed in 10% formalin for 24 h and then cryoprotected in sucrose prior to freezing at -80°C. 40 μm sections were obtained using a cryostat and stored in a solution of 7% sucrose/20%ethylene glycol in 0.1 M PBS. For BDNF immunohistochemistry, free-floating sections were washed in 0.1 M PBS for 1 h. Antigen retrieval was carried out by heating sections in 10 mM sodium citrate (pH 6.0) at 80°C for 15 min. The sections were permeabilized in 0.5% Triton X-100 for 20 min and then incubated with 10% normal goat serum for 1 hr prior to overnight incubation at 4°C with a 1:250 dilution of anti-BDNF polyclonal antibody (ab1779, Chemicon International Inc., CA) prepared in 1X GDB buffer (1% gelatin solution, 5% Triton X-100). After washing in 0.5% Triton X-100 for 1 h, sections were incubated in a 1:1000 dilution of Cy3 goat-anti rabbit IgG in 1X GDB for 1 h at room temperature. Sections were washed in 0.1 M PBS for 1 hr and mounted with Vectashield (Vector). BDNF immunoreactivity and GFP expression were examined with an Olympus BX60 fluorescence microscope and images were acquired and intensity of staining was measured using ImageJ software.
For Western blot analysis histone extracts from dissected rat mPFC were used and the experiments were performed as described previously (Sadri-Vakili et al., 2007
). Briefly, mPFC from both hemispheres of a cocaine or yoke rat was homogenized in 200 μl of lysis buffer (1% Nonidet P-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM PMSF, sodium butyrate 1mM, and protease inhibitors) at 4°C. After removal of cellular debris by centrifugation, the supernatant was collected and protein levels in the lysates were measured by the Bradford assay (Bio- Rad Laboratories). 10-20 μg of each sample was boiled in the presence of sample buffer for 5 min before separation on 10-20% SDS- polyacrylamide gel, and proteins were transferred to nitrocellulose membranes. The immunoblots were blocked with 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.2% Tween 20 (TBST) for 60 min. The membranes were then incubated overnight at 4°C with specific antibodies that included: di acetyl lysine 9 and lysine 14 histone H3 (AcH3) antibody (Upstate, Lake Placid, NY) at a dilution of 1:1500, anti-histone H3 antibody (Upstate, Lake Placid, NY) at a dilution of 1:500, anti-phospho-CREB (Abcam, Cambridge, MA) 1:500, and anti-MeCP2 (Abcam, Cambridge, MA). Primary antibody incubation was followed by 6 washes (10 min, rocking, RT) in TTBS before incubation with the secondary antibody (HRP-conjugated goat anti-rabbit IgG, Jackson ImmunoResearch Laboratories, West Grove, PA), 6 washes, and visualization using the ECL detection system (NEN, Boston, MA).
Enzyme-linked Immunosorbant Assay
Ten days after rats received unilateral microinjections in the medial prefrontal cortex with AAV10 vector containing EGFP-linked shRNA targeting BDNF, 4 animals were sacrificed and bilateral medial prefrontal cortices were dissected. Tissues were lysed and homogenized and 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 (Cat. No. CYT306, Chemicon International Inc., CA). The brain tissue samples and serial dilutions of BDNF standards were loaded in triplicate onto a microplate coated with rabbit anti-human BDNF polyclonal antibodies and incubated overnight at 4°C. After 4 washes, biotinylated mouse anti-human BDNF monoclonal antibody (1:1000) was added to the microplate for 2.5 h at room temperature. The plates were washed 4 times and the strepavidin-enzyme conjugate was added to the plate and allowed to incubate for 1 h. After further washing, tetramethylbenzidine chromagenic substrate was added and then 15 min later the reaction was stopped. The absorbance at 450 nm was measured with a plate reader. BDNF concentration in the brain tissue samples was measured by comparing values to the prepared standard curve.
Chromatin immunoprecipitation (ChIP) assay
We have adapted the ChIP technique to the analysis of brain tissue and have recently published detailed methodology for performing ChIP experiments (Braveman et al., 2004
; Chen-Plotkin et al., 2006
: Sadri-Vakili et al., 2007
). Briefly, mPFC from both hemispheres of an individual cocaine or yoke rat was cut into pieces and the pieces were weighed (< 30mg) and deposited into tubes. Formaldehyde was added (10 μl of 1% formaldehyde to 1 mg tissue) and brain pieces were incubated for 10 min at 37°C to cross-link DNA to associated proteins. Brain tissue was washed twice with ice-cold PBS containing protease inhibitors (PI, Complete Mini protease inhibitor cocktail tablets, Roche, Indianapolis, IN), and then suspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1) containing PI at a ratio of 10μl buffer for each mg of brain. After incubating on ice for 10 min, brain lysates were sonicated to shear lengths of 200-1000 base pair DNA fragments using 10 sets of 10-15 sec pulses at setting 3 of a sonicator (Branson Cell Disruptor 350). We have found that this regimen yields DNA fragments appropriate for ChIP (200 – 1000 bp) (Braveman et al., 2004
). The resulting homogenates from one brain were pooled and then centrifuged for 10 min at 13,000g
at 4°C to remove debris, and 200 μl aliquots of the suspension were placed into separate sample tubes. Each sample was diluted 10-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl), and 20 μl (1%) of the diluted sample was set aside and designated ‘Input’ DNA. Samples were then either processed immediately or stored at –80°C.
For immunoprecipitation, 2000 μl of each sample was pre-cleared with 80 μl of salmon sperm DNA/Protein A-agarose 50% slurry (Upstate Biotechnology, Lake Placid, NY) by incubating at 4°C for 30 min with gentle agitation before overnight incubation (4°C) with 5 μg of each of the following antibodies: phospho-CREB, MeCP2, di-acetyl lysine 9 and lysine 14 histone H3 (AcH3), or 5-methyl-cytosine (Upstate, Lake Placid, NY). Negative controls included no antibody mock and IgG (Jackson, West Grove, PA). Mock immunoprecipitation conditions (mouse IgG) were also included as a control. After immunoprecipitation, 80 μl of the salmon sperm DNA/protein A-agarose 50% slurry was added to samples, and immunocomplexes were collected for 1 h at 4°C on a rocking platform. After pelleting agarose (1000 rpm, 4°C, 2 min), the chromatin-antibody/protein A-agarose complexes were washed sequentially (4 min each on a rotating platform) with 1 ml each of: a low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), a high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), a LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and a TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Elution was performed by incubation in 250 μl of freshly made elution buffer (1% SDS, 0.1 M NaHCO3) for 15 min at room temperature; elution was repeated and eluates combined. Cross-links were then reversed by addition of 20 μl 5 M NaCl to the pooled eluates and heating of the mixture to 65°C for 4 h. The saved input DNA was diluted to a volume of 500 μl with sterile water, and cross-link reversal was performed in the same manner as for the ChIP samples. After cross-link reversal, all samples (ChIP and Input) were digested with 20 μg proteinase K (1 h, 45°C), and DNA was recovered with phenol/chloroform extraction and ethanol precipitation. DNA pellets were resuspended in 25 μl of sterile water.
One μl (1 or 4 μl for human samples) of ChIP-dreived DNA was used as template in 20 μl reactions containing 10 μl 2X SYBR Green Master Mix (Applied Biosystems, Foster City, CA) and 0.5 μM of each primer. Real-time thermal cycling was performed using an iCycler thermal cycler (Bio-Rad, Hercules, CA), with continuous SYBR Green monitoring according to the manufacturer's recommendations, using iCycler software. Cycling parameters for all amplifications were as follows: 60 cycles of 95°C for 30 sec, 57°C for 30 sec, and 72°C for 45 sec, followed by melt-curve analysis (55°C+ for 10 sec × 80 cycles). All PCR reactions were performed in triplicate and included negative controls (no DNA) as well as positive controls (serial dilutions of known amounts of genomic DNA).
Target DNA sequence quantities were estimated from threshold amplification cycle numbers (Tc) using iCycler software. For every gene sequence studied, a ΔTc value was calculated for each sample by subtracting the Tc value for the immunoprecipitated sample from the Tc value for the corresponding Input DNA to normalize for differences in ChIP sample aliquots before immunoprecipitation. DNA quantities were then expressed as percentages of corresponding Input using the following equation: (Antibody ChIP as a percentage of Input) = 2(ΔTc) × 100. Finally, DNA quantities (normalized to Input) were compared for immunoprecipitated vs. mock-immunoprecipitated samples; only when immunoprecipitated samples contained >1.5 times as much DNA were they considered to have sufficient DNA for analysis.
Input and IP samples were interrogated with gene promoter-specific primers in triplicate reactions in real-time PCR analysis as previously described (Braveman et al., 2004
; Chen-Plotkin et al., 2006
; Sadri-Vakili et al., 2007
). 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’-GCAGTTGGACAGTCATTGGTAACC-3’ and reverse 5’-ACGCAAACGCCCTCATTCTG-3’; BDNF exonII (a, b, c)
: forward 5’-GCAGAGTCCATTCAGCACCTTG-3’ and reverse 5’-TGGCTTGACAGCGAGGAAAAG-3’; BDNF exon IV
(CREB and MeCP2 binding sites): forward 5’-AACAAGAGGCTGTGACACTATGCTC-3’ and reverse 5’-CAGTAAGTAAAGGCTAGGGCAGGC-3’, forward 5’-AAAGCATGCAATGCCCT-3 and reverse 5’-GAGATTTCATGCTAGCTCGC-3’, 5’-GGCTTCTGTGTGCGTGAATTTGC-3’ and reverse 5’-AAAGTGGGTGGGAGTCCACGAG-3’; BDNF exon IV
: forward 5’-TTTGGGGCAGACGAGAAAGC-3’ and reverse 5’-GGCAGT GGAGTCACATTGTTGTC-3’.
ChIP products were quantified using the luciferase- and T4 DNA polymerase-based DNA Quantitation System (Promega, Madison, WI) according to manufacturer's instructions. The light output was immediately measured in a luminometer (TD-20/20 Luminometer, Turner Designs, Sunnyvale, CA). ChIP products were also quantified using Quant-iT PicoGreen dsDNA reagent (Invitrogen, Carlsbad, CA) and assayed on the Wallac Victor 1420 Multilabel Counter (PerkinElmer, Waltham, MA) with excitation and emission wavelengths of 485 nm and 535 nm, respectively.
RNA extraction and reverse transcription
RNA was extracted from dissected brain regions from one hemisphere of cocaine or yoke rats using RNeasy kit (Qiagen, Valencia, CA) according to manufacturer's instructions and as described previously (Sadri-Vakili et al., 2007
). Reverse transcription reactions were performed using the Superscript First Strand Synthesis System for RT-PCR reactions (Invitrogen, Carlsbad, CA) using specific primers to quantitate the amount of gene expression as compared to a standard curve. The following primers were used for real-time PCR in order to quantitate the amount of gene expression. The exon specific BDNF primers were designed based on previously published results (Nakayama et al., 1994
; Chen et al., 2003
; Martinowich et al., 2003
: Jiang et al., 2008
). BDNF exon I
: forward 5’-AAGCCGAACTTCTCACATGATGA -3’ and reverse 5’-TGCAACCGAAGTATGAAATAACCATAG -3’; BDNF exon II (a, b, c)
: forward 5’-GCAGAGTCCATTCAGCACCTT G-3’ and reverse 5’-TGGCTTGACAGCGAGGAAAAG-3’; BDNF exon IV
: forward, 5’-CTGCCTAGATCAAATGGAGCTTCT-3’ and reverse 5’-GGAAATTGCATGGCGGAGGTAA-3’; BDNF exon IV
: forward 5’-TTTGGGGCAGACGAGAAAGC-3’ and reverse 5’-GGCAGT GGAGTCACATTGTTGTC-3’; CB1
: forward 5’- CCTCTACGTGGG CTCAAATG-3’ and reverse 5’-GGAAGGGACTACCCCTGAAG-3’; DRD2
: forward 5’-GGTCTGCAAAGCCTTCTCTC-3’ and reverse 5’-TACTAT GCCATGCTGCTCAC-3’; GAPDH
: forward 5’-AACAGCAAC TCCCATTC TTC-3’ and reverse 5’-TGGTCCAGGGTTTCTTACTC-3’; GluR1
: forward 5’-ATGCTGACCTCCTT CTGTGG-3’ and reverse 5’-TCCTGTAGTTCCGGGCGTAG-3’; GluR2
: forward 5’-ATTTCGGGTAGGGATGGTTC-3’ and reverse 5’-GCGAAACTGTTGGCTACCTC; mGluR5
: forward 5’-GCACAGTCCAGTGAGAGG AG-3’ and reverse 5’-TTGTCCACAGTTGGTTGGTG-3’; NR1
: forward 5’-TAGTTAGCCACCCGCCTAC-3’ and reverse 5’-GACATTCGGGTAGTCAGTCC-3’; PPD
: forward 5’-CTGCACACAGGGAACACAAG-3’ and reverse 5’-TGATACAGAATGGCGTGGTC; PPE
: forward 5’-GCTTGGGTGTTCTGCTTCTC-3’ and reverse 5’- TCCAGGGTTCT TAGTGCTGG-3’; SAP97
: forward 5’-ACCCGTGGACATTCTCAATC-3’ and reverse 5’-CGGTATCAGGACGAAGAGG-3’. Quantitative real time-PCR was performed in an iCycler (Bio-Rad) with the use of SYBR-green PCR Master Mix (Applied Biosystems, Foster City, CA) through 50 PCR cycles (95°C for 30 sec, 57°C for 60 sec, 72°C for 90 sec). 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. The mRNA levels were normalized for each well to the GAPDH mRNA levels. Single PCR products were verified by assessing that the melting temperature of the product had a single value.