Male Sprague-Dawley rats weighing 250-275 g on arrival were obtained from Harlan Sprague-Dawley (Madison, WI) and housed individually in a reverse cycle room (12-h light/12-h dark) with food and water freely available. Rats were given 4-5 days to acclimate and subsequently tested during the dark period of the light cycle. For experiments involving intra-NAcc infusions, rats were anesthetized with a mix of ketamine (100 mg/kg, i.p.) and xylazine (6 mg/kg, i.p.), placed in a stereotaxic apparatus with the incisor bar positioned 5.0mm above the interaural line (Pellegrino et al. 1979
), and chronically implanted with bilateral guide cannulae (22 gauge, Plastics One, Roanoke, VA) aimed at the NAcc core (A/P, +3.4mm; L, ± 1.5mm; DV, -7.5mm from bregma and skull). Cannulae were angled at 10° to the vertical and positioned either 1 mm (for biochemistry and behavioral testing with amphetamine and PF) or 4 mm (for Lentiviral infection and subsequent testing with amphetamine) above the final injection site. Guide cannulae were secured with dental acrylic anchored to stainless steel skull screws. After surgery, 28 gauge obturators were placed into the guide cannulae (extending 1 mm for guide cannulae positioned 1 mm and flush for guide cannulae positioned 4 mm above the final injection site) and rats returned to their home cage for a 7 day recovery period. All surgical procedures were conducted using aseptic techniques according to approved Institutional Animal Care and Use Committee and Institutional Biosafety Committee protocols.
In the behavioral and biochemistry experiments testing the effects of PF, microinjections were made in the freely moving rat in a volume of 0.5μl/side over 30 seconds through 28 gauge injection cannulae extending 1 mm beyond the guide cannula tips. In the experiment testing the effect of NAcc GluR1(S845A) expression, amphetamine microinjections were made using the same guide cannulae used to deliver the Lentiviral vectors. Thus, injection cannulae extended 4 mm beyond the guide cannula tips in this case. Injection cannulae were connected via polyethylene tubing (PE20) to Hamilton syringes and left in place for 1 minute after the injection to allow for diffusion. Microinjection cannulae were then removed, the obturators replaced, and rats returned either to the activity boxes or their home cages.
Rats were placed individually in locomotor monitoring chambers for 30-60 minutes, administered their respective NAcc infusion, and returned to the locomotor chambers for 60-minutes. In the experiments testing the effects of PF, PF (1.0, 5.0, or 10 μg/side) was microinjected into the NAcc either alone or in cocktail with amphetamine (2.5 μg/side). In each of three experiments, saline, amphetamine, and amphetamine+PF microinjections were made in counterbalanced order with at least three days between injections. In a fourth experiment, rats in different groups received microinjections into the NAcc of saline or PF alone (1.0, 5.0, or 10 μg/side) again in counterbalanced order. In the experiment testing the effect of NAcc GluR1(S845A) expression, Lenti-GFP and Lenti-GluR1(S845A) infected rats were administered amphetamine (2.5 μg/side) into the NAcc 3 weeks following infection. Locomotor activity was measured using a bank of 12 activity monitoring boxes. Each box (22 × 43 × 33 cm) was constructed of opaque plastic (rear and two side walls), a plexiglas front-hinged door, and a tubular stainless steel ceiling and floor. Two photocell beams, positioned 2.5 cm above the floor and spaced evenly along the longitudinal axis of each box, estimated horizontal locomotion. Separate interruptions of photocell beams were detected and recorded via an electrical interface by a computer situated in an adjacent room using locally developed software.
Rats in different groups were infused intracranially with saline (0.5μl/side), amphetamine (2.5μg/side), PF (10μg/side) or amphetamine+PF into the NAcc and sacrificed 20 minutes later. Brains were rapidly removed for subsequent assessment of protein levels and PP1 activity. For immunoblotting, brains were flash-frozen on dry ice, 2mm thick sections obtained with a brain matrix, and 2mm diameter punches of the NAcc taken around the injection cannula tips. Tissue punches were homogenized in ice-cold lysis buffer (50mM Tris, 150mM NaCl, 1mM EDTA, 1% NP-40 ,1% Sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitor cocktails (1 and 2; Sigma-Aldrich, St. Louis, MO). A total of 20 μg of protein was loaded per lane and separated by 10% SDS-PAGE. Following transfer, membranes were incubated in blocking solution (5% milk in Tris-buffered saline containing Tween 20), incubated for 16 hours at 4°C in primary antibody for Darpp32 (1:1000; Cell Signaling Technologies, Beverly, MA), pDarpp32(T34; 1:500, Millipore, Billerica, MA), pDarpp32(S137; 1:5000; generously provided by Dr. Paul Greengard, Rockefeller University, New York, NY), GluR1(1:1000; Millipore), pGluR1 (S831; 1:500, Millipore), pGluR1(S845; 1:500, Millipore), Csnk1δ/ε (1:5000; BD Transduction Laboratories, Franklin Lake, NJ), CopGFP (1:1000; Evrogen, Moscow, Russia), β-actin (1:2000; Sigma-Aldrich), or Tubulin (1:10,000; Santa Cruz) and washed in TBS-T. Membranes were then incubated in a HRP-conjugated anti-rabbit or anti-mouse IgG and visualized using the ECL detection system (ECL Advanced, GE Healthcare). Blots were digitally imaged using the GeneSnap Bio Imaging System and quantified using Genetool software (Syngene, MD, USA). PP1 activity was assayed using the Protein Serine/Threonine Phosphatase (PSP) assay system (BioLabs) by measuring the release of inorganic phosphate from labeled protein. Myelin basic protein (MyBP) labeled with Γ-32P was used as a substrate. Fresh NAcc tissue was homogenized in lysis buffer (50mM Tris pH7.4, 150mM NaCl, 0.1mM EDTA, 0.1mM EGTA, 1mM MnCl2, 5mM DTT, 5% glycerol) containing a protease inhibitor cocktail but without phosphatase inhibitors. 10μg of tissue lysate was added to 30 μl of assay buffer containing PP2A and PP2B inhibitors, 2nM okadaic acid, and 2μM FK-506. After 5 minutes of preincubation at 30°C, the reaction was started by adding 10μl of substrate. Following 10 minutes of incubation at 30°C, the reaction was terminated by adding 200μl of cold 20% trichoroacetic acid. After centrifugation at 12000g for 5 min, radioactivity was counted in 200μl of supernatant by an LS6500 multi-purpose scintillation counter (Beckman, CA, USA). Data were expressed as pmol/min/μg protein.
Construct assembly and Lentiviral vectors
cDNAs for rat GluR1 (GenBank accession No. X17184) were constructed in a pcDNA1/Amp vector at the EcoRI and BamHI sites (Invitrogen) to obtain a pcDNA1/GluR1 plasmid. Mutagenesis was performed using the Quickchange II XL site-Directed mutagenesis kit. The following primers were used to produce the GluR1 S845A mutants. Forward primer: 5'-G ACC CTC CCC CGG AAC GCT GGG GCA GGA GCC AGC -3'. Reverse primer: 5'- GCT GGC TCC TGC CCC AGC GTT CCG GGG GAG GGT C-3'. The full length of the GluR1(S845A) cDNA was subcloned into a pCDH1-CMV-MCS-EF1-copGFP vector (System Bioscience). Recombinant Lentivirus was packaged in HEK TN cells using a pPACKH Lentivector Packaging Kit as described by the manufacturer (System Bioscience). The Lentivirus was purified and concentrated by PEG-8000. The average titer of the recombinant viral stocks was 1×106
infectious units/ml. The following recombinant Lentiviral vectors were used: Lenti-GluR1(S845A) and Lenti-GFP as a control. The viability of the Lentiviral vectors was confirmed in HEK cells infected either with Lenti-GFP, Lenti-GluR1, or Lenti-GluR1(S845A). GFP positive cells were observed in all cases 48 hours post-infection but GluR1 transgene expression was increased only in the Lenti-GluR1 and Lenti-GluR1(S845A) infected HEK cells (see Fig. S1
in Supplementary Information).
For the behavioral experiment, rats were transferred to a biosafety level 2 facility following one week of recovery from implant surgery and administered bilateral NAcc microinjections of the viral vectors in a fume hood (Loweth et al. 2010
). Microinjections were made in freely moving rats in a volume of 2.0 μl/side at a rate of 0.1μl/30 sec through 28 gauge cannulae extending 4 mm beyond the guide cannulae tips. Microinjection cannulae loaded with Lentivirus were connected via polyethelyne tubing (PE20) to Hamilton syringes and left in place for 5 minutes after the injection to allow for diffusion. Rats were returned to the housing room 72 hours later.
For the electrophysiology experiments, dissociated cultures of medium spiny neurons were prepared from E18 Sprague-Dawley rats. Under isoflurane anesthesia, embryos were removed and the NAcc separated from diencephalon, dissected free of meninges, and diced. Tissues were digested in 0.03% (wt/vol) trypsin. After dissociation by trituration, cells were counted, suspended in culture medium consisting of Neurobasal supplemented with 2% B-27 (both from Gibco-Invitrogen Corp), 0.5 mm GlutaMax I, 10% deactivated fetal bovine serum, and 1% penicillin/Streptomycin. Cells were plated on poly l-lysine-coated 25-mm cover slips at a density of 8 × 104 cells/cm2. Neurons were either left uninfected or infected with Lenti-GFP or Lenti-GluR1(S845A) at day 1 of culture. Recordings were performed 10-18 days following infection. Cells in these three groups were also assayed for evoked pGluR1(S845) following 15 minutes incubation with the PKA activating D1 dopamine receptor agonist SKF81297 (10 μM).
Cultured medium spiny neurons were plated on coverslips, placed in an experimental chamber, and visualized using an inverted microscope under phase-contrast illumination (Nikon, Tokyo, Japan). All experiments were performed at room temperature (~20°C). The chamber was flushed with bath solution for 5 min before recording began. During experiments, solution flow was ~0.5 ml/min. Bath solution contained (in mM/l) 140 NaCl, 1 MgCl2, 5 KCl, 10 Glucose, 10 Hepes, 2 CaCL2*2H2O at pH=7.4. Patch electrodes were pulled from borosilicate glass capillary tubing (TW150-4, World Precision Instruments Inc., Sarasota, FL) on a micropipette puller (Sutter P-97, Sutter Instrument Co., Novato, CA). They had a resistance of 2-4 M when filled with solution containing (in mM/l) 135 CsCl, 1MgCl2, 10 Hepes, 10 EGTA, 3.6 Mg-ATP, 0.1 Na-GTP, and 14 Na-Creatinephosphate. Whole-cell voltage-clamp was performed using an Axopatch 200B, 16-bit data Digidata 1332A acquisition system driven by Clampex 8.2 (Axon Instruments, Foster City, CA). Cells were held at -60 mV, and gap-free recordings were acquired for 2 min each. Multiple recordings were possible in most cells. Puffs of 0.5mM Na-glutamate dissolved in bath solution were applied for 50ms with a Picospritzer II (General Valve Corp., Fairfield, NJ). The tip of the infusion pipette was placed within 80-100 μm of the recorded cell. Current traces were filtered at 1 kHz and digitized at 2 kHz. For each cell, a representative glutamate response was chosen for group analysis and background activity was analyzed from 30 s of recording prior to glutamate application.
Histology and Immunofluorescence
After the completion of the behavioral experiments testing the effects of NAcc PF, rats were deeply anesthetized and perfused transcardially with 0.9% saline and 10% formalin. Brains were removed and stored in the formalin solution for at least 24 hours. 40-μm sections were then prepared, mounted on gelatin-coated slides, and stained with cresyl violet to identify rats with injection cannula tips located bilaterally in the NAcc core. Only rats with both cannula tips located in the NAcc core were retained for statistical analyses. Four rats failed to meet this criterion.
After completion of the behavioral experiment testing the effects of NAcc GluR1(S845A), rats were perfused with 0.9 % saline and 4% paraformaldehyde and brains transferred to a 25% sucrose solution. To detect GFP fluorescence 40 μm coronal sections were prepared, mounted on gelatin-coated slides with Fluoromount-G (southern Biotech), and analyzed using a fluorescence microscope. Injection cannula tips located in the NAcc core were identified and the distribution pattern of GFP positive cells around the cannula tips assessed. Entire brains (from midbrain to prefrontal cortex) were also examined for GFP positive cells to determine the extent of anterograde and retrograde transport of the virus from the NAcc core microinjection site. GFP positive cells were observed only in close proximity to the injection cannula tips in the NAcc and were not detected in any other brain region. Again, only rats with both cannula tips located in the NAcc core were retained for statistical analysis. Five rats failed to meet this criterion.
PF-670462 (4-(3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl) pyrimidin-2-ylamine) was generously provided by Pfizer (Groton, CT). S(+)-amphetamine sulfate was purchased from Sigma-Aldrich (St. Louis, MO). Both drugs were dissolved in physiological saline. SKF81297 was purchased from Tocris (Ellisville, MO) and dissolved in DMSO. Doses refer to the weight of the salt.
The PF-amphetamine locomotor and electrophysiology peak current data were analyzed with one way ANOVA. The immunoblot data were normalized to β-actin or tubulin and ratios analyzed with two way between ANOVA with amphetamine and PF as the factors. The GluR1(S845A) expression pGluR1(S845) and GluR1 protein data were analyzed with two way between ANOVA with infection and drug challenge as the factors. The NAcc GluR1(S845A) expression locomotor data were analyzed with between-within ANOVA with infection as the between factor and time as the within factor. In all cases, post hoc comparisons after ANOVA were made with the Scheffé test.