Generation of Nac1 mutant mice
The mouse Nac1
gene was cloned from a 129SvJ BAC library (Research Genetics, Inc., Huntsville, AL, USA) as described previously [15
]. The targeting vector containing the Escherichia coli
lacZ gene encoding β-galactosidase and the neomycin-resistance gene driven by the mouse phosphoglycerate kinase promoter [10
] was inserted into exon 2 (). Exon 2 was chosen as it encodes for the POZ/BTB functional domain of the NAC1 protein. Thus, deletion of exon 2 of the Nac1
gene produces a functional null allele. The construct was electroporated into mouse 129SvTac embryonic stem (ES) cells (a gift from Dr. P. Labosky, Vanderbilt University) and neomycin resistant colonies were selected for Southern blot analysis. 198 resistant clones were analyzed by PCR using a common 5' primer (5'-GGCCGCTAGTAGCTCTTACTT-3'), and specific 3' primers derived from exon 2 (5'-GTCGCAACTTGGAGAGCTAAC-3') for the wild type allele, resulting in an amplified fragment of 243 bp and the lacZ gene (5'-CAAAGCGCCATTCGCCATTCA-3') for the mutant allele resulting in an amplified fragment of 340 bp. Targeted ES cells were injected into C57BL/6N blastocycts to obtain 8 germ line chimeras. Chimeric mice were mated with C57BL/6N mice (Taconic, USA) to obtain heterozygote mice, which were mated to give rise to mice of all three genotypes ().
Figure 1 Generation of Nac1 mutant mice. A. The targeting vector of Nac1 a 5kb lacZ-PGK-neo cassette was inserted into exon 2; Enzyme restriction sites are designated as X;Xba and E; EagI, the positions of primersets used to detect wild-type and mutant alleles (more ...)
RNA Isolation and cDNA Synthesis
All RNA was isolated using TRIzol (GIBCO-BRL, Gaithersburg, MD) according to manufacturer's instructions. The quality of the RNA samples was determined by ethidium bromide staining of 18S and 28S rRNAs following fractionation on denaturing agarose gels. Contaminating genomic DNA was removed using 1 μL RNase-free DNase I (Boehringer)/10 μg of RNA at 37°C for 30 min. cDNA was synthesized using MMLV-RT (GIBCO-BRL) with deoxyribonucleotides (dNTPs) and random hexamer primers RT (GIBCO-BRL) at 42°C for 30 min.
Reverse Transcription Polymerase Chain Reaction (RT-PCR)
Conditions used for RT-PCR followed the method of Wilson and Melton (1994) with minimal modifications. The mouse HPRT (hypoxanthine guanine phosphoribosyl transferase) gene was used as an internal control, as the mRNA of this gene is expressed in most tissues in roughly equal amounts. cDNA synthesized from total brain RNA provided templates for PCRs under the following conditions: one cycle of 95°C for 5 min, followed by 25 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 2 min, and one cycle of 72°C for 5 min in a buffer containing 1.5 mmol/L MgCl2, 10 μmol/L primers , 0.05 μCi 32P-dATP, and 200 μmol/L dNTPs. Primer sequence available upon request. PCR products were separated on 5% acrylamide gels that were dried and exposed to phosphorimager screens overnight.
Western blot analysis
Tissues from individual animals were homogenized in 200 μL of ice-cold extraction buffer containing phosphate buffered saline (PBS), 1 mM EGTA, 1 mM EDTA, 1% SDS, and 1 mM PMSF. Protein concentrations were determined using a Bradford assay, with bovine serum albumin as the standard. Equivalent amounts of protein (50 μg) for each sample were resolved in 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, proteins were transferred to Immobilon-P membranes (Millipore). Membranes were incubated in PBS with 0.5% Tween 20 (PBS-T) containing 5% non-fat milk for 1 hr at room temperature to block non-specific binding. The blots were reacted with primary antibodies) overnight at 4 °C. After washing in PBS-T, the blots were incubated in secondary antibody in PBS-T for 1 hr. Membranes were then washed three times with PBS-T. Immunolabeling was detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). The blots were stripped using the standard protocol, washed and reprobed with the reference antibody (α-tubulin). The antibody for NAC1 was generated using a C-terminal peptide (TASHDGEAGPSAEVLQ) (Pro-Sci Inc., Poway, CA). Primary antibodies used were Nac-1 and α-tubulin (1:1000) (Sigma, St. Louis, MO). The secondary antibodies used were goat anti-rabbit IgG-HRP (1:500) and goat anti-mouse IgG-HRP (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA). Densitometric analysis was conducted to quantify the immunoreactivity in Western blotting with a scanner and ImageQuant software (Amersham Biosciences, Piscataway, NJ). The NAC1 bands were detected at ~59 kDa, α-tubulin bands at 50 kDa.
All mice (3−5 months old, in a mixed genetic background of 129SvEvTac;C57BL/6N F2 generation; 23−40g) were housed in groups of 4 per cage and maintained on a 12 hr light-dark cycle with food and water available ad libitum in accordance with the University of Pennsylvania Animal Care and Use Committee. Mice of both sexes were used in all studies. All experimental testing sessions were conducted between 12.00 and 18.00 hours, with animals randomly assigned to treatment conditions and tested in counterbalanced order.
All drug doses were calculated as mg/kg base weight and were dissolved in 0.9% saline. Cocaine hydrochloride and amphetamine was obtained from NIDA Drug Supply (Research Triangle Park, NC). Saline (0.9%) and dopamine hydrochloride were obtained from Sigma Aldrich, St Louis, MO.
Locomotor activity in response to intraperitoneal drug administration was analyzed in a “home cage” activity monitoring system (MedAssociates, St. Albans, VT). The home cage (28.9 × 17.8 × 12 cm) was placed in a photo-beam frame (30 × 24 × 8 cm) with sensors arranged in an 8-beam array strip. To avoid effects of novelty during testing, mice were injected i.p. with saline (0.9% sodium chloride) and individually placed in the cages for 3 days prior to drug administration. Beam break data were read into MedAssociates personal computer-designed software and monitored for 30 min. For acute studies, mice were injected i.p. with either saline, cocaine (5.0 mg/kg, 10.0 mg/kg or 20.0 mg/kg) or amphetamine (2.0 mg/kg) and individually placed in the cages. For behavioral sensitization studies, baseline activity was recorded for 3 days to reduce novelty induced hyperlocomotion similar to the procedure for acute administration. Drugs were administered on day 4 and repeated once a day for 7 days (day 4−10, cocaine) or 4 days (day 4−7, amphetamine) and locomoter activity was recorded. All mice remained in their home cages with no drug administration during the development of sensitization. To evaluate cocaine induced sensitization, mice were tested on day 29 with an i.p. injection of either saline (saline control groups) or a challenge dose of cocaine (5 mg/kg). A second cocaine challenge was administered on day 40 to evaluate long lasting effects of sensitization. During the course of the amphetamine study, compliance with animal facility regulation on moving mice prevented us from testing on day 29, therefore mice were tested on day 90 with saline only and then on day 91 with an i.p. injection of a challenge dose of amphetamine (2.0 mg/kg). Beam break data was monitored and recorded for 30min.
Conditioned Place Preference
10-day Conditioned Place Preference
To facilitate adaptation to novel surroundings, mice were transported to the testing room at least one hour prior to testing. Briefly, for preconditioning, day 1, mice were allowed to explore both sides of a 2-chambered CPP apparatus. The place conditioning boxes consisted of two distinct plastic sides (20×20×20 cm) differing in lighting intensity; one with stripes on the walls and a metal grid over cage bedding (referred to as striped side) and the other with gray walls and smooth flooring (referred to as solid side). A partition separated the two sides with an opening that allowed access to either side of the chamber during preconditioning and test days. This partition was closed off during pairing days. Preconditioning phase: On day 1 mice were placed randomly on the striped or solid side and allowed to explore both sides of the place conditioning boxes for 900 sec; time spent in each side was determined using the criteria of placement of head and front paws in the chamber. These data were used to separate mice into groups with approximately equal biases for each side. None of the mice exhibited a significant preference for one side over the other. Conditioning phase: Mice were paired for 8 days (days 2−9), with the saline group receiving injections (0.9% sodium chloride) on both sides of the boxes, while the drug paired group received cocaine (10mg//kg) or amphetamine (2mg/kg) on one side and saline on the other side. For cocaine experiments, drug was paired for 30min, while amphetamine was paired for 45 min. Drug-paired sides were randomized among all groups. Testing phase: On day 10, all animals were given a saline injection and placed on the same side they were originally introduced on pre-conditioning day (striped or solid). Because of the randomization, approximately half of the drug-paired mice were introduced into the side that was paired with drug, and the other half were introduced into the side that was paired with saline. Animals were allowed to roam freely between the two sides, and time spent on each side was recorded.
3-day Conditioned Place Preference
These studies were carried out essentially as described above with respect to preconditioning and testing days. However, conditioning took place on one day, in two sessions. Animals were exposed to one side of the box in the morning session and the opposite side in the afternoon session. Animals were paired with either a saline injection on each side (saline paired group), cocaine (20 mg/kg), or amphetamine (2.0mg/kg) on one of the sides (drug paired group) and saline on the opposite side. Drug-paired mice were randomized with respect to session time and box side among all groups.
Naïve mice were anesthetized with a combination of ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.). Using coordinates derived from Franklin and Paxinos (1997) atlas (in millimeters: AP, +1.42; ML, ± 0.8; DV, −2.8 relative to bregma) microdialysis guide cannulae (20 gauge, 10mm; Small Parts, Miami Lakes, FL) were implanted bilaterally above the nucleus accumbens. The animal was placed in a stereotaxic alignment system (David Kopf Instruments, Tujunga, CA)with a Cunnigham mouse adapter. The skull was exposed and two holes were drilled. Cannulae were positioned and the skull was prepared for resin application and a light-cured dental resin was used to secure guide cannulae in position (Kerr Corporation, Orange, CA). The incision was closed with a tissue adhesive. After surgery, mice were allowed to recover at least 7 days prior to microdialysis.
A microdialysis probe (24 gauge; including 1.5−2 mm of active membrane) was lowered into the guide cannula and perfused with a microdialysis buffer (5 mM glucose, 2.7 mM KCl, 140 mM NaCl, 1.4 mM CaCl2, 1.2 mM MgCl2, 0.15% phosphate-buffered saline [pH=7.4]) at a rate of 2 μl/min via a syringe pump (BAS Bioanalytical System, West Lafayette, IN). After four hours, dialysis samples were collected every 20 min, beginning 2 hours prior to an i.p. injection of 20 mg/kg cocaine (0.01 ml/g) and for 120 min thereafter. Samples were collected into vials containing 10 ml of 0.05 M HCl and were frozen at −80°C until analysis.
HPLC analysis of dopamine
Dopamine in the dialysate sample was measured using a HPLC system with electrochemical detection. The mobile phase consisted of 4.76 mM citric acid, 150 mM NaH2PO4, 50 μM EDTA, 2.5 mM sodium dodecyl sulfate, 10% methanol (v/v), 17% acetylnitrile (v/v) at a pH = 5.6. Dopamine was separated using a reversed-phase column (HR-80, ESA) and oxidized/reduced using coulometric detection (Coulochem II; ESA Inc.). Three electrodes were used: a guard cell (+0.40 V), a reduction analytical electrode (E1, −0.10 V), and an oxidation analytical electrode (E2, +0.22 V). The peak area was measured with ESA 501 Chromatography Data System and the dopamine values were compared with an external standard curve for quantification.
Histology and Statistical Analysis
After completion of microdialysis experiments, mice were deeply anesthetized using pentobarbital (50mg/kg, i.p.) and perfused transcardially with saline and 4% formaldehyde solution. The brains were stored in the latter solution until sectioning. Placement of the microdialysis probes within the nucleus accumbens were verified in cresyl violet-stained tissue sections (50 μm). Only those mice with probes located in the nucleus accumbens were included in the data analysis. For all data, statistical analyses were performed using StatView (SAS). Behavioral sensitization locomotor data and conditioned place preference data were analyzed for significance with ANOVAs using a Bonferroni–Dunn post hoc test. The microdialysis data were analyzed using a two-way ANOVA with repeated measures over time followed by an LSD multiple comparisons post hoc test.