Experimental animals and surgery
Male Sprague-Dawley rats, weighing 275–300 gm on arrival (Charles River, Kingston, NY, USA), were housed individually in a climate-controlled environment (21°C) on a 12-hr light-dark cycle. All animals were maintained according to the guidelines of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. To facilitate acquisition of cocaine self-administration, animals were temporarily maintained on a restricted diet of lab chow at 85% of their original body weight, and trained to lever-press for 45 mg sucrose pellets on a fixed ratio 1 (FR1) reinforcement schedule until acquisition criteria were achieved (100 pellets self-administered for 3 consecutive days). Animals were then fed ad libitum for at least 1 day before surgery.
Under sodium pentobarbital anesthesia (60 mg/kg, i.p.), a catheter composed of SILASTIC tubing (Green Rubber, Woburn, MA, USA) and treated with tridodecylmethyl ammonium chloride (TDMAC) heparin (Polysciences Inc., Warrington, PA), was surgically placed in the animal’s jugular vein. The catheter was secured with Mersilene surgical mesh (General Medical, New Haven, CT) at the jugular vein, and passed subcutaneously to exit the animals’ back through a 22 gauge cannula (Plastics One, Roanoke, VA) imbedded in dental cement on a Marlex surgical mesh (Bard Inc., Cranston, RI). For experiments with brain infusions, animals also underwent stereotaxic surgery to implant 26 gauge bilateral guide cannulae (Plastics One, Roanoke, VA) in the VTA or the substantia nigra (SN). Stereotaxic coordinates for the VTA and the SN were: −5.6 mm posterior to bregma, ±0.8 mm (VTA) or ±1.5 mm (SN) lateral, and −7.0 mm ventral to dura (Paxinos and Watson, 1998
). Dummy cannulae (33 gauge) were left in place throughout the experiment. Animals received a prophylactic injection of penicillin (60,000 IU/0.2 ml, i.m.) and antibiotic ointment to the catheter exit wound daily. Catheters were flushed daily with 0.2 ml of heparinized (20 U/ml), bacteriostatic saline containing gentamycin sulfate (0.33mg/ml).
Operant chambers (Med Associates Inc., St. Albans, VT) for cocaine and sucrose self-administration were contextually different from the animals’ home cage, and located in different rooms. Each chamber was equipped with an infusion pump (Razel Model A pump, Stamford, CT) and 10 ml glass syringe connected to a fluid swivel (Instech, Plymouth Meeting, PA) by Teflon tubing. Tygon® tubing enclosed by a metal spring connected the swivel to the animal’s catheter exit port and was secured to Teflon threads on the catheter assembly. Following 1 week of recovery from surgery, animals were trained to self-administer cocaine in 4 hr sessions for 5–6 days/week. A single lever-press response at the active lever produced a 0.5 mg/kg intravenous (iv) injection of cocaine (NIDA, Research Triangle Park, NC) delivered in 0.05 ml saline over 2.5 sec, concurrent with illumination of a cue light located above the active lever while the house light was extinguished. Each injection was followed by an additional 12.5 sec time-out (TO) period when the house light remained off, and active lever-press responses had no scheduled consequence during this period. Responses on the inactive lever were recorded but had no consequences.
Western blot and quantitative PCR
Animals were trained to self-administer cocaine for 3 weeks as described above, and brain tissue was collected at different time points (immediately after the final session, 1 day or 3 weeks withdrawal). Self-administering rats were paired with yoked partners that received an identical amount and temporal pattern of cocaine injections but not contingent upon lever-press behavior. An additional group of animals received yoked cocaine injections for the first time during the final session after receiving yoked saline injections in previous sessions to compare with the chronic cocaine groups with no withdrawal. Another group of animals self-administered saline throughout all sessions to control for potential surgical or other influences relating to the testing procedures.
For determination of AMPA and NMDA receptor subunit levels and their phosphorylation status, brain tissue from the VTA and the SN was dissected following brief (1.6 sec) microwave irradiation (5kW) aimed at the head (Muromachi, Kikai Co. Ltd., Tokyo, Japan) as previously described (Edwards et al., 2007
). Following microwave fixation, VTA and SN tissue was obtained from chilled coronal brain slices (~−4.8 to −6.3 mm posterior to bregma) using a 16-gauge punch. Immediately following brain dissection, tissue was homogenized by sonication and boiled for 5 min in lysis buffer (320 nM sucrose, 5 nM HEPES, 50nM NaF, 1 mM EGTA, 1 mM EGTA, 1% SDS containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktails I and II; Sigma, St. Louis, MO), and stored at −80°C until further analysis.
Following protein determination by the Lowry method, 20–40 µg protein aliquots were separated by SDS-PAGE on 7.5–10% acrylamide using Tris/Glycine/SDA buffer (Bio-Rad, Hercules, CA) and electrophoretically transferred to PVDF membranes. Membranes were blocked with 5% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 overnight at 4°C, washed and incubated in affinity-purified rabbit polyclonal anti-pGluR1S831
(1:2500; Millipore, Billerica, MA), and stripped and re-probed with antibodies for total GluR1 protein (1:5,000; Millipore). Other blots were probed with antibodies for GluR2, NR2A, NR2B or mouse monoclonal anti-GAD (1:5,000; Millipore,), or anti-pTHS40
(1:1500; Cell Signaling Technology, Inc., Danvers MA) followed by mouse monoclonal anti-TH (1:200,000; Millipore). All blots were stripped and re-probed for (3-tubulin as a protein loading control. Following labeling with primary antibodies, blots were washed and labeled with the appropriate species-specific peroxidase-conjugated secondary antibodies (1:25,000; Vector Laboratories, Burlingame, CA, USA), labeled proteins were detected by enhanced chemiluminescence, and densitized using the NIH image 1.57 as described previously (Edwards et al., 2007
). Under these conditions, target protein amounts were linear over a 3–4 fold range. Each blot contained tissue from 5–6 age- and group-matched untreated controls that remained in their home cages but were handled daily to allow normalization of data between blots.
In separate study groups, tissue was dissected without microwave fixation in animals euthanized immediately or 1 day after withdrawal from chronic yoked or self-administered cocaine. Total RNA was extracted from the VTA tissue using Trizol reagent (Invitrogen, Carlsbad, CA), precipitated with isopropanol and treated with DNase to remove genomic DNA (Ambion, Austin, TX). RNA was reverse transcribed to cDNA using a first-strand synthesis kit (Invitrogen, Carlsbad, CA). Cycle thresholds (Ct) were determined in triplicates of a same sample by the ΔΔCt method using the following primer sequences for GluR1: 5'-GTCCGCCCTGAGAAATCCAG-3', 5'-CTCGCCCTTGTCGTACCAC-3', GluR2: 5'-GCCGAGGCGAAACGAATGA-3', 5'-CACTCTCGATGCCATATACGTTG-3', and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-AACGACCCCTTCATTGAC-3', 5'-TCCACGACATACTCAGCAC-3'.
Characterization of HSV-GluR1 vectors in vitro and in vivo
PC12 cells (Clontech, Palo Alto, CA) were plated at a density of 106
cells per well on 35 mm culture plates maintained at 37°C, 5% CO2 in DMEM supplemented with 10% fetal calf serum and 1% solution of 5000 U/ml penicillin and 5000 µg/ml streptomycin (Invitrogen, Carlsbad, CA) as previously described (Kumar et al., 2005
). Cells were infected with 1 µl/well HSV vectors (4.0 × 107
infectious units/ml) after 70–80% confluence in 35-mm wells as described previously (Bachtell et al., 2008
). After 24 hr, cells were incubated for 30 min with either 5 µM forskolin, phorbol-12-myristate-13-acetate (PMA) or fresh medium. Cells were harvested using a lysis buffer (1% SDS, 20 mm HEPES, pH 7.4, 100 mm NaCl, 20% glycerol, 1 mm EDTA, 1 mm EGTA, 10 µg/ml leupeptin, 10 µg/ml aprotenin, 10 µg/ml pepstatin, 1 mm phenylmethylsulphonyl fluoride, 50 mm NaF, 0.1 mm sodium orthovanadate, and 10 mm sodium pyrophosphate), briefly sonicated and centrifuged, and levels of GluR1, pGluR1S845
were determined in 20 µg protein aliquots by western blot as described above. Uniformity of protein loading concentration was confirmed with Ponceau S solution staining (Sigma, St. Louis, MO).
For immunocytochemistry and confocal microscopy of HSV-GluR1 over-expression, naïve rats received unilateral stereotaxic infusions of HSV-GluR1WT or HSV-GluR1S845A (1.0 µl/side) in the VTA, while the contralateral side (balanced left and right) received infusions of the PBS solution delivered through 26 gauge Hamilton microsyringes (Hamilton, Reno, NV) at 5.6 mm posterior to bregma, ±0.8 mm lateral, and −8.0 mm ventral to dura. After 2 days, rats were anesthetized with chloral hydrate and killed via intracardiac perfusion of PBS followed by 4% paraformaldehyde (20 min, 12 ml/min). The brains were post-fixed in 4% paraformaldehyde overnight and cryoprotected in 20% glycerol/PBS at 4 °C for 3 days. Coronal brain sections (30 µm) were blocked with 3% normal donkey serum and 0.3% Triton-X in PBS for 60 min prior to incubation with rabbit polyclonal anti-GluR1 (1:1,000; Millipore) and mouse monoclonal anti-TH (1:5000; Millipore) in 3% normal donkey serum and 0.3% Tween-20 for 18–20 h. After washing with PBS, sections were incubated with fluorescent-tagged secondary antibodies for 60 min (Cy2-conjugated donkey anti-mouse for TH; Cy3-conjugated donkey anti-rabbit for GluR1; Jackson Immunoresearch, West Grove, PA). After incubation with secondary antibodies, VTA sections were counterstained with DAPI (1:5000; Roche Applied Science, Mannheim, Germany) for 20 min at room temperature. VTA sections were sequentially dehydrated in 70%, 95% and 100% of ethanol and Citrosolv, and cover-slipped with DPX (Sigma-Aldrich). Negative controls for antibody labeling indicated a lack of specific staining when omitting or diluting the primary antibody.
Confocal microscopy was performed to quantify the relative percent of ectopic HSV-GluR1 expression in TH-positive neurons, and to determine the percentage of infected TH-positive neurons within a 0.5 mm diameter zone of highest HSV-mediated GluR1 expression in the VTA. Simultaneous epifluorescence (Cy2 and Cy3) images were obtained at 20X and 63X magnification with a laser-scanning confocal microscope (Zeiss Axiovert 200 and LSM510-META; Thornwood, New York) using three lasers comprised of argon (458, 477, 488 and 514 nm), HeNe1 (543 nm) and HeNe2 (633 nm). Laser-scanning and optical sectioning in the Z plane were performed using multitrack scanning with a section thickness of 1.45 µm for 20X or 0.45 µm for 63X magnification (Donovan et al., 2008
). Co-localization was assessed by analysis of adjacent Z sections, orthogonal sectioning through Z sections, and three-dimensional reconstruction with rotation. Confocal images were imported into Adobe Photoshop (Adobe Systems, Mountain View, CA) for composition of merged images. Single- and double-labeled GluR1WT
and TH-positive cells were counted within the infected region across 3 slices per animal and averaged to obtain an individual percent colocalization for each of 6 animals. The mean percentage of colocalization across animals was determined and expressed as both a percentage of TH-positive expression in ectopic GluR1WT
-expressing cells and the percentage of total TH-positive cells within the infected region that express GluR1WT
. Dendritic labeling of GluR1 in HSV-GluR1WT
expressing TH-positive neurons was quantified by measuring the length of GluR1-labeled processes from the soma in 3–5 cells/animal under blinded conditions and expressed as the mean process length (µm)/animal (3–4 animals per group).
VTA slice cultures were prepared from postnatal day 25–35 rats anaesthetized with isoflurane as described previously (Han et al., 2006
; Krishnan et al., 2007
; Cao et al., 2010b
). A tissue block containing midbrain was taken and sliced in ice-cold solution containing (in mM) 254 sucrose, 3 KCl, 1.25 NaH2
, 10 d
-glucose, 24 NaHCO3
, and 2 MgSO4
. Slices, 300 µm thick were transferred to a holding chamber in 34°C containing artificial cerebrospinal fluid (aCSF, in mM: 128 NaCl, 3 KCl, 1.25 NaH2
, 10 d
-glucose, 24 NaHCO3
, and 2 MgSO4
, pH 7.35, 295–305 mOsm). After 45–60 min recovery, slices were transferred onto the membrane of Millicell (Millipore) containing culture medium: MEM with 30 mM HEPES, 20 mM D-glucose, 5% B27, 5.0 mM L-glutamine, and 25 U/ml streptomycin/penicillin. After 60 min incubation, GFP-tagged HSV-GluR1WT
vectors were pipetted onto the VTA area of the slice surface. Slices were maintained overnight at 34°C, and then put into a recording chamber perfused with standard aCSF at a rate of 2.5 ml/min. All solutions, except for culture medium, were saturated with 95% O2
and 5% CO2
. GFP positive cells were visualized with an upright fluorescence microscope using infrared differential interference contrast (IR-DIC) illumination. Whole-cell voltage-clamp recordings were performed under continuous single-electrode voltage clamp mode (AxoClamp 2B, Axon Instruments Inc., Union City, CA, USA). Electrodes (2–4MΩ) were filled with pipette solution containing (in mM) 115 potassium gluconate, 20 KCl, 1.5 MgCl2
, 10 phosphocreatine, 10 Hepes, 2 ATP-Mg and 0.5 GTP (pH 7.2, 285 mOsm). Data acquisition was made using DigiData 1322A and pClamp 8 (Axon Instruments).
In these experiments, putative dopamine neurons in the VTA were identified by large hyperpolarization-activated currents (Ih
) as described previously (Ungless et al., 2003
; Cao et al., 2010a
current was evoked by a family of 10 mV voltage steps (duration 600 ms) from −60 mV to −140 mV holding potentials. In separate experiments, Ih
-positive neurons were recorded in 150 µm thick slices, and cells were filled with biocytin (1% internal recording solution) using 20 pA depolarized current injections. Slices were fixed immediately after recording in 4% formaldehyde for 2 hours and then stored at 4°C in PBS. Slices were processed for TH immunoreactivity as discussed above.
AMPA- and NMDA-mediated locomotion
Locomotor activity was recorded in the using 1.95 m circular test chambers with a 12 cm wide runway, and equipped with four pairs of photocells located at 90-degree intervals. Drug naïve animals with bilateral guide cannulae in the VTA were habituated to the locomotor apparatus for 2 hr prior to testing, and given 1.0 µl intra-VTA infusions of the HSV vectors through bilateral 33-gauge infusion cannulae extending 1 mm beyond the guide cannulae tip over a 5 min period. Infusion cannulae were left in place for an additional 2 min to allow for diffusion. Each subsequent test session incorporated a 2-hr habituation phase followed by an intra-VTA infusion of either a PBS vehicle, AMPA (10 ng/side, bilateral) or NMDA (500 ng/side, bilateral) in a volume of 0.5 µl/side over 100 sec through bilateral 33-gauge infusion cannulae in counter balanced order over 3 consecutive test days. The injectors were left in place for 30 sec, gently removed, and the animals were placed back into the locomotor apparatus where locomotor activity was recorded for 1 hr.
HSV-GluR1 over-expression in the VTA and cocaine self-administration
Animals were trained to self-administer cocaine (0.5 mg/kg/injection) for 3 weeks as described above, then the response requirement was raised over subsequent sessions from a fixed ratio (FR) 1 to FR5 and continued until cocaine intake stabilized to within 10% of the mean of 3 consecutive sessions. Following stabilization, animals were trained in a within-session FR5 dose-response procedure with each injection dose (1.0, 0.3, 0.1, 0.03, and 0 mg/kg) available in descending order in sequential 60 min components following an initial 30 min loading phase (0.5 mg/kg/injection). The unit dose/injection was adjusted by reducing the injection volume (0.2, 0.06, 0.02, 0.006, and 0 mls) to produce unit injection doses of 1, 0.3, 0.1, 0.03, and 0 mg/kg cocaine, respectively. Animals were trained until the injection dose producing peak self-administration rates remained constant for 3 consecutive sessions. Following stabilization of peak rate cocaine doses, animals received intra-VTA or intra-SN infusions of the HSV vectors (1 µl/side) through bilateral guide cannulae as described above. Animals were then tested in within-session dose-response tests during HSV-GluR1 over-expression of (post-infusion days 2–5), and 7–10 days after HSV infusions when HSV-mediated over-expression is diminished to undetectable levels in brain (Carlezon et al., 1997
; Sutton et al., 2003
; Bachtell et al., 2008
Following FR5 dose-response testing, rats were re-stabilized on the FR5 schedule (0.5 mg/kg/injection) in daily 4 hr sessions and then trained on a progressive ratio (PR) schedule at either 0.5 or 1.0 mg/kg/injection for 2 weeks. The number of active lever-presses for each successive cocaine injections increased according to the calculation [5e(injection number × 0.2)]−5; i.e., responses/injection increased as 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, etc.). The highest ratio of responses/injection achieved before a 1 hr period when no further injections were earned (break point) was determined in daily tests until they varied <10% for 3 consecutive days. Animals then received the same HSV infusions as in FR5 testing and break points were determined for post-injection days 2–5 (over-expression) and again on days 7–10 (post-expression).
HSV-GluR1 over-expression in the VTA and sucrose pellet self-administration
Bilateral guide cannulae were implanted in the VTA of drug-naïve rats as described above. Following a week of recovery, rats were food restricted to 85% body weight and trained to self-administer sucrose pellets on a FR1:TO 15 sec reinforcement schedule for a maximum of 50 sucrose pellets available. Following acquisition, the reinforcement schedule was increased to FR5 for at least 3 weeks and until the latency to consume 50 pellets stabilized to <10% variance from the mean of 3 consecutive sessions. Animals showing stable responses on the FR5 schedule received intra-VTA infusions of HSV- vectors and were tested during HSV-GluR1 over-expression (2–5 days post-infusion). Animals were given 1 week break from food self-administration and then trained on a progressive ratio schedule until break points stabilized on the following schedule progression (1, 2, 5, 9, 13, 19, 25, 33, 41, 51, 61, 76, 91, 111, etc.). Animals then received the same HSV vectors in the VTA and break points were determined during GluR1 over-expression (2–5 days post infusion).
Following completion of behavioral testing, animals were anesthetized with chloral hydrate (300 mg/kg, i.p.) and bilateral infusions of 0.5 µl cresyl violet were delivered in the VTA or SN through the guide cannulae. Five minutes after the cresyl violet infusions, animals were decapitated, brains dissected and infusion sites were identified in 1 mm coronal slices. Only animals with correct bilateral infusion sites in the VTA and the SN were included in the data analysis.
Biological and locomotor data were analyzed by 1-factor ANOVA across study groups, or unpaired T-tests if only 2 groups were compared. Self-administration data were analyzed by 2-factor ANOVA (dose × HSV treatment) with repeated measures on dose (FR only), and by 1-factor ANOVA at each dose, followed by post-hoc tests with Fisher's Least Significant Difference (LSD) test. Statistical significance was preset at p < 0.05.