Animals and genotyping
All experiments detailed are in accord with the Northwestern University Animal Care and Use Committee, and are in compliance with the NIH Guide to the Care and Use of Laboratory animals. Unless otherwise noted, all experiments were conducted with Drd2-eGFP BAC transgenic mice on an FVB/NJ (inbred; Jackson Laboratory), C57BL/6J (inbred; Jackson Laboratory), and Swiss Webster (outbred; Taconic Farms) backgrounds. The original founder animals were a gift from Drs. N. Heintz and P. Greengard (The Rockefeller University). They were generated by injection of a modified BAC (containing eGFP under the regulatory elements of the Drd2 gene) into pronuclei of FVB/N fertilized oocytes. Subsequently, zygotes were injected into pseudo-pregnant female Swiss Webster (an outbred strain from Taconic) (Gong et al., 2010
). All Drd2-eGFP BAC transgenic mice in this study were back-crossed with wild-type inbred breeders for at least 7-9 generations to create hemizygote FVB/N and C57BL/6 animals. The FVB/N line was outcrossed with the Swiss Webster animals for up to two generations for this study. All animals included in this study were males (unless otherwise indicated) between 3-5 weeks of age and housed and bred in the same environment. Transgene copy number was confirmed with tail biopsy using real-time quantitative PCR (with primers direct against the chloramphenicol-resistance gene of the BAC vector and Gapdh). Comparison was made using □□CT method (see below) with known standards.
Chronic DA depletion
Unilateral lesion of the nigrostriatal system was produced by 6-hydroxydopamine (6-OHDA) injection into the medial forebrain bundle (MFB). In brief, mice at postnatal day 28-32 were anesthetized with a mixture of ketamine (50 mg/kg i.p.) and xylazine (4.5 mg/kg i.p.). After immobilization on a stereotaxic frame (David Kopf Instruments) with a Cunningham adaptor (Harvard Apparatus), a hole was drilled (~1 mm diameter) at 0.7 mm posterior and 1.1 mm lateral to bregma for injection into the MFB (0.7 AP = anterior-posterior, 1.1 ML = medial-lateral, 4.8 DV = dorsal-ventral). 1 μL of 6-OHDA HCl (Sigma Chemical Co.) was dissolved at a concentration of 7.5 μg/μl saline with 0.02% ascorbate and injected using a calibrated glass micropipette (Drumond Scientific Co.), at a rate of 0.02 μl/min and at a depth of 4.8 mm from the surface of the skull. The micropipette was left in situ for another 30 minutes after the injection to maximize tissue retention of 6-OHDA and decrease capillary spread upon pipette withdrawal. Electrophysiological experiments were performed 3-4 wks later.
Drd2 antagonist treatment
Eticlopride HCl (1 mg/kg; Tocris Bioscience) was dissolved in sterile PBS and stored at 4°C. Homozygous Drd2-eGFP FVB/N mice (3-4 week) were injected intraperitoneally (i.p.) once daily for 14 days. Animals were killed for recording 24 hours after the last injection to ensure the clearance of eticlopride and its metabolites from the brain.
An open field arena (56 × 56 cm) was used for measuring non-reinforced ambulatory behavior of animals (4-5 weeks of age). Each arena was cleaned with disinfectant prior to the testing of each subject. Behavioral assessment of subjects were performed between 3:00-6:00 pm with the ambient light set at 35 lux. An individual mouse was placed in the center of the arena and its ambulation and exploratory activity was recorded by a CCD camera connected to a personal computer. Video tracking of animals was performed for 20 minutes using LimeLight2 (Coulbourn Instruments) at rate of 3.75 frames per second. The arena contained a 10×10 grid block on the bottom surface to allow off-line analysis of the animals’ movement in specific regions of the arena. In each session, the total distance traveled as well as the percentage of time/distance within different parts of the arena were recorded. Behavioral assays were performed at the Northwestern University Behavioral Phenotyping Core Facility.
Western blotting procedures were similar to that previously described (Chan et al., 2011
). In brief, membrane protein fractions were prepared by lysis of tissue in buffer containing 10 mM HEPES, pH 7.4, and 320 mM sucrose, followed by brief centrifugation to remove nuclei and insoluble material. After centrifugation at 16,000x g, the pellet was resuspended by gentle rocking at 4 °C in TEEN-Tx (0.1 M Tris, 1 mM EDTA, 1 mM EGTA, and 1% Triton X-100). Protein extracts were resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Western blotting was performed using Drd2 primary antibody at a concentration of 1μg/ml (a gift from Ryuichi Shigemoto, National Institute for Physiological Sciences, Okazaki, Japan).
Quantitative polymerase chain reaction (qPCR) was used to determine the abundance of transcripts of interest with procedures similar to that described previously (Chan et al., 2011
). In brief, striata were microdissected in ice-cold aCSF from a series of 300 μm tissue slices. Total RNA was isolated using RNeasy micro kit (Qiagen). cDNA was synthesized using qScript cDNA Supermix (Quanta Biosciences). Real-time PCR was performed using Fast SYBR Mastermix (Applied Biosystems) on a StepOnePlus thermocycler (Applied Biosystems). The thermal cycling conditions comprised of an initial denaturing step at 95°C for 20 s, and 40 cycles at 95°C for 3 s, 60°C for 30 s. The PCR cycle threshold (Ct) values were measured within the exponential phase of the PCR reaction using StepOnePlus software version 2.1 (Applied Biosystems). A correction was performed using a passive reference dye (Rox) present in the PCR master mix. Reactions with any evidence of nonspecificity (i.e. low melting temperatures or multiple peaks in melting point analysis) were excluded from the analysis. A relative quantification method (i.e. □□CT method) was used to quantify differences in gene expression level (Schmittgen and Livak, 2008
). To increase accuracy of the gene expression analysis, a panel of reference genes (Atp5b
) were included in order to establish an ensemble reference — weighted Cts based on the stability of each reference gene were calculated from established algorithms (Vandesompele et al., 2002
; Andersen et al., 2004
; Pfaffl et al., 2004
; Silver et al., 2006
), as described in equations 1
R is a relative value that incorporates the rank values of all the reference genes for a particular algorithm. αRG
represents the average R value calculated using 4 different algorithms. CtRG
is the Ct value of a reference gene for an individual sample. Experiments for each gene of interest were run in triplicates. Desalted primers were custom synthesized (Invitrogen) and intron-spanning whenever possible. No-template and no-reverse-transcriptase control assays produced negligible signals, suggesting that primer dimer formation and genomic DNA contamination effects were small. The mRNA levels in each subgroup of samples were characterized by their median values. Results were presented as fold difference relative to their respective wild-type controls. Data are presented as median fold difference and median experimental error (standard deviation) (Bookout et al., 2006
). Statistical analysis (Mann-Whitney U test) was performed. Differences between the genotypes were judged significant at confidence levels of 95% (P<0.05).
Mice (males, 3-5 weeks old) were anesthetized with ketamine/xylazine and perfused transcardially with ice cold artificial cerebrospinal fluid (aCSF) containing in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2.0 CaCl2, 1.0 MgCl2, 25 NaHCO3, and 12.5 glucose, bubbled continuously with carbogen (95% O2 and 5% CO2). The brains were rapidly removed, glued to the stage of a slicer (Leica), and immersed in ice-cold aCSF. Striatal slices were cut at a thickness of 240-275 μm and transferred to a holding chamber, where they were submerged in aCSF at 35°C for 30 minutes, and returned to room temperature before recording. SPNs within the dorsal striatum were identified by their somatic morphological characteristics under IR-DIC or Dodt-contrast optics. Somatic eGFP expression was verified using epifluorescence microscopy to confirm cell identity before breaking into whole-cell mode. Recordings were made at room temperature (20-22°C) with patch electrodes (1.5 mm outer diameter) fabricated from filamented, thick-wall borosilicate-glass (Sutter Instruments) pulled on a Flaming-Brown puller (Sutter) and fire polished immediately before use. Pipette resistance was typically ~3-5 MΩ. For current-clamp recordings, the internal solution consisted of (in mM): 135 KMeSO4, 5 KCl, 10 Na2-phosphocreatine, 5 EGTA, 0.5 CaCl2, 2 Mg-ATP, 0.5 Na3-GTP, 5 HEPES, adjusted to pH 7.25-7.30 with KOH, 300 mOsm. The liquid junction potential in recordings was ~7 mV and was not corrected for. For voltage-clamp experiments, pipettes were filled with a Cs+-based internal solution containing (in mM): 125 CsMeSO3, 5 TEA-Cl, 10 Na2-phosphocreatine, 5 HEPES, 0.25 EGTA, 2 Mg-ATP, 0.5 Na-GTP, 1 QX-314-Cl, adjusted to pH 7.25-7.30 with CsOH, 300 mOsm. Electrical stimulation (200-400 μs) was performed using parallel bipolar tungsten electrodes (Frederick Haer & Co) placed in the layer V-VI of the cortex. Somatic whole-cell patch-clamp recordings were obtained with an amplifier (Molecular Devices). The signal for voltage clamp recordings was filtered at 1 kHz and digitized at 10 kHz with a digitizer (Molecular Devices). For current-clamp recordings, the amplifier bridge circuit was adjusted to compensate for electrode resistance and subsequently monitored.
SPN in tissue slices (as described above) were loaded with Alexa Fluor 594 (50 micromolar) through the patch pipette. All experiments were performed at room temperature. Images were acquired with a 60X/1.00 NA water-immersion lens (Olympus). The two-photon excitation source was a Chameleon-Ultra2 tunable laser system (680 to 1080 nm) using titanium:sapphire gain medium with all-solid-state active components and a computer-optimized algorithm to ensure reproducible excitation wavelength, average power, and peak power (Coherent Laser Group). Optical signals were acquired using 810 nm excitation beam (80-MHz pulse repetition frequency and 250 fs pulse duration) to excite Alexa 594. The fluorescence emission was collected by external or non-descanned photomultiplier tubes (PMTs). The red fluorescence (580-640 nm) was collected by a multi-alkali-cathode (S-20) PMT. Measurements were taken in a sample plane along dendritic segments (>50 μm from the soma). On average, ~80 spines were counted per dendritic segments; up to 3 measurements were performed on each cell.
Data and statistical analyses
Curve fitting and data analyses were done with ClampFit 9 (Molecular Devices), Igor Pro 6.0 (Wavemetrics), MATLAB 7.12 (MathWorks), MiniAnalysis 6.0.3 (Synaptosoft), and Prism 5 (GraphPad). Box plots were used for graphic representation: the central line represents the median, the edges represent the interquartile ranges, and the whiskers represent the overall distribution. Asterisks indicate 95% confidence (p<0.05) unless otherwise noted. Normal distributions were not assumed regardless of sample size or variance. Pairwise comparisons for unrelated samples were performed using a Mann-Whitney U test with a threshold of p<0.05 for significance. Kruskal–Wallis one-way analysis of variance was performed for group comparisons with a threshold of p<0.05. Frequency-intensity (F-I) curves were analyzed with two-way repeated measures analysis of variance, with SPN type or treatment group and current injection as independent variables. Group main effect between SPN type or treatment group is reported. Significance was set as α = 0.05.