Dopamine D2 receptor over-expression alters behavior and physiology in Drd2-EGFP mice

doi:10.1523/JNEUROSCI.4287-10.2011

J Neurosci. Author manuscript; available in PMC 2011 July 5.

Published in final edited form as:

J Neurosci. 2011 January 5; 31(1): 126–132.

doi: 10.1523/JNEUROSCI.4287-10.2011

PMCID: PMC3074337

NIHMSID: NIHMS261994

Dopamine D2 receptor over-expression alters behavior and physiology in Drd2-EGFP mice

Paul F. Kramer,¹ Christine H. Christensen,¹ Lisa A. Hazelwood,² Alice Dobi,¹ Roland Bock,¹ David R. Sibley,² Yolanda Mateo,³ and Veronica A. Alvarez¹

¹Section on Neuronal Structure, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD 20892

²Molecular Neuropharmacology Section, National Institute on Neurological Disorders and Stroke, NIH, Bethesda, MD 20892

³Section on Synaptic Pharmacology, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD 20892

Corresponding author: Veronica A. Alvarez at ; Email: alvarezva/at/mail.nih.gov

The publisher's final edited version of this article is available free at J Neurosci

See other articles in PMC that cite the published article.

Abstract

BAC transgenic mice expressing the fluorescent reporter protein EGFP under the control of the D1 and D2 dopamine receptor promoters (Drd1-EGFP and Drd2-EGFP) have been widely used to study striatal function and have contributed to our understanding of the physiological and pathological function of the basal ganglia. These tools were produced and promptly made available to address questions in a cell-specific manner that has transformed the way we frame hypotheses in neuroscience. However, these mice have not been fully characterized until now. We found that Drd2-EGFP mice display a ~40% increase in membrane expression of the dopamine D2 receptor (D2R) and a two-fold increase in D2R mRNA levels in the striatum when compared to wild-type and Drd1-EGFP mice D2R over-expression was accompanied by behavioral hypersensitivity to D2R-like agonists, as well as enhanced electrophysiological responses to D2R activation in midbrain dopaminergic neurons. DA transients evoked by stimulation in the nucleus accumbens showed slower clearance in Drd2-EGFP mice and cocaine actions on DA clearance were impaired in these mice. Thus, it was not surprising to find that Drd2-EGFP mice were hyperactive when exposed to a novel environment and locomotion was suppressed by acute cocaine administration. All together, this study demonstrates that Drd2-EGFP mice over-express D2R and have altered dopaminergic signaling that fundamentally differentiates them from wild-type and Drd1-EGFP mice.

Keywords: cocaine, dorsal striatum, ventral tegmental area, nucleus accumbens, ankyrin repeat and kinase domain-containing 1 (Ankk1), tetratricopeptide repeat domain 12

Introduction

The use of Bacteria Artificial Chromosome (BAC) transgenic mice has become commonplace in neuroscience research as they are critical for the identification of specific cell-types and for the cell-specific expression of CRE recombinase that, when combined with flox genes, can produce targeted gene expression or deletion. BAC transgenic mice carry large DNA clones containing one to two hundred thousand base pairs of genetic code that include complete regulatory sequences for the gene of interest and thus are able to achieve expression patterns that better mimic those of endogenous genes (Heintz, 2001; Gong et al., 2003). In recent years, BAC transgenic mice that express transgenes under the control of the D1 and D2 dopamine receptor (D1R and D2R) regulatory sequences have become very useful tools for studying striatal function and have improved our understanding of the physiological and pathological function of the basal ganglia circuits (Surmeier et al., 2007; Kreitzer and Malenka, 2008; Surmeier et al., 2009; Valjent et al., 2009) (Tian et al., 2010). Specifically, Drd1-EGFP and Drd2-EGFP transgenic mice that express the fluorescent reporter protein EGFP in neurons containing D1R or D2R, respectively, have eased the identification of the two subpopulations of medium spiny neurons (MSNs) in the striatum. MSNs are the main output of the striatum and project to the medial globus pallidus and the pars reticulata region of the substantia nigra (SNr) by way of two distinct and parallel pathways, the indirect and the direct pathways. MSNs of direct pathway mainly express D1R and send monosynaptic projections directly to the medial globus pallidus and SNr, while those of the indirect pathway mainly express D2R and send projections to the same regions via the lateral globus pallidus and the sub-thalamic nucleus. Using Drd1 and Drd2-EGFP BAC transgenic mice, several studies have demonstrated cell-specific morphological and cell-membrane properties that distinguish each population of MSNs, as well as cell-specific synaptic plasticity and changes induced by cocaine and other psychostimulants (Lee et al., 2006; Kreitzer and Malenka, 2007; Day et al., 2008; Gertler et al., 2008; Shen et al., 2008).

These BAC transgenic mice were generated by the GENSAT project, which is funded by the National Institutes of Health and aims at producing these valuable tools on a large scale for general use within the neuroscience community. Although the benefits of the GENSAT project are vast, one intrinsic limitation is that the BAC transgenic lines were not characterized in depth before becoming available to researchers, who then became responsible for this task. Here we characterize Drd2-EGFP mice and show that they display an elevated D2R expression pattern and have altered behavioral and physiological responses to D2R-like agonists and cocaine.

Material and Methods

Animals

All experiments were performed in accordance with guidelines from the National Institute on Alcohol Abuse and Alcoholism (NIAAA) Animal Care and Use Committee. Homozygote mice of both genders were used all experiments, unless otherwise stated, and were housed on a 12-hour light/dark cycle (0630 – 1830 light) with food and water ad libitum.Drd1-EGFP and Drd2-EGFP BAC transgenic mice were generated by the GENSAT project (Gong et al., 2003) and wild-type Swiss Webster mice were obtained from Taconic. Two colonies derived from the same founder Drd2-EGFP mouse line were used and compared in this study: the original colony established at NIAAA in 2003 from mice received from Dr. Charles Gerfen at the National Institute of Mental Health and a newer colony (Drd2-EGFP/M) established in 2009 from breeder mice obtained from the Mutant Mouse Regional Resource Center (stock: 000230-UNC). Drd2-EGFP heterozygote mice were generated by crossing homozygote Drd2-EGFP mice to Swiss Webster wild-type mice and were used only in the F1 generation.

Drugs

Cocaine (Sigma-Aldrich) was dissolved in sterile saline for injections. Quinelorane dihydrochloride (Sigma-Aldrich) stock solution (3 mg/ml) was prepared in 99% EtOH and diluted accordingly in sterile saline before the injections (final EtOH concentration < 0.1%). Tetrodotoxin (TTX, Ascent), quinpirole (Sigma-Aldrich), sulpiride (Tocris), ketanserin (Tocris), and [³H]methylspiperone (Perkin Elmer) were used for in vitro experiments.

Locomotor assessment

Locomotor activity was recorded in cages (10 × 6.5 inches) constructed out of clear polycarbonate walls with stainless steel or polycarbonate floors under dim illumination (~100 lux). Horizontal activity was detected as infrared beam crosses (1 inch spacing, 10 beams per cage) using Opto M3 activity monitors (Columbus Instruments, Columbus, OH). Prior to each experiment, mice were assigned to sex and age-matched groups and the different genotypes were run in parallel, whenever possible. For assessing acute and chronic responses to cocaine, mice (6 – 11 weeks old) were weighed and allowed to run freely for five minutes. They were then given an intra-peritoneal (i.p.) injection of either vehicle or cocaine (30 mg/kg) and locomotion was measured for the next 20 minutes. For the cocaine dose response curve, mice (naive to cocaine) received a vehicle injection for three consecutive days and, on the fourth day, were injected with one of three cocaine doses (5, 15 or 30 mg/kg). Experiments for the dose response curve to D2R agonist were performed following a previous protocol (Ralph and Caine, 2005). Adult mice (2 – 10 months old) were allowed to run freely in the cage for one hour, during which time baseline locomotor activity was determined. After this time, mice received an injection of either the vehicle or one of five doses of quinelorane (1, 3, 10, 30, 300 µg/kg). Mice were then immediately returned to the cage and horizontal locomotion was measured for three more hours. In all cases, locomotor activity is expressed as either infrared beam breaks per min, or normalized to the mean baseline value obtained for each genotype after vehicle injection. Data were fit using the Hill equation: min + (max − min) / [ 1 + (EC₅₀ / x)^rate ].

Membrane Radioligand Binding Assays

Brains were removed from mice (5 – 12 weeks old) and immediately transferred to cold ACSF. Striatal samples (20 – 40 mg) were obtained from thick coronal brain slices (1 mm) by microdissection under a dissection scope and immediately frozen in liquid nitrogen. For each assay, striatum from four mice of each genotype were pooled and stored at −80°C to allow for parallel processing. Tissue was suspended in lysis buffer (5 mM Tris-HCl, 5 mM MgCl₂, pH 7.4), incubated on ice for ten minutes, and homogenized using a glass dounce tissue grinder. The suspension was centrifuged at 20,000 × g for 30 min and the membranes resuspended in binding buffer (50 mM Tris, pH 7.4) to yield a protein concentration of ~50 µg/ml (Bradford protein assay, Bio-Rad, Hercules, CA). Membrane preparations were incubated for 90 min at RT with [³H]methylspiperone in a final reaction volume of 1 ml containing the 5HT₂ receptor antagonist ketanserin (50 nM). Non-specific binding was determined in the presence of (+)-butaclamol (4 µM). Bound ligand was separated by filtration through polyethyleneimine-soaked GF/C filters using a Brandel cell harvester and analyzed via liquid scintillation spectroscopy (60% efficiency). B_max (pmol receptor/mg protein) was estimated as the asymptote value of the isotherm, and the K_d calculated as the concentration of radioligand required to occupy half of the available receptors.

Quantitative PCR (qPCR)

Total RNA was purified from microdissected striatal tissue from two adult mice from each genotype (treated as independent samples) using RNeasy Micro (Qiagen, Valencia, CA) and cDNA was synthesized using iScript cDNA Synthesis Kit (BioRad, Hercules, CA). Primers for D2R amplification (forward: 5’-ATC TCT TGC CCA CTG CTC TTT GGA-3’; reverse: 5’-ATA GAC CAG CAG GGT GAC GAT GAA-3’) and probes containing a 5’ 6-FAM and a 3’-Black Hole Quencher-1 (5’-TG TAT CAT TGC CAA CCC TGC CTT CGT-3’) were synthesized by Integrated DNA Technologies (Coralville, IA). qPCR runs were performed using TaqMan Fast Polymerase (Applied Biosystems, Carlsbad, CA) in a StepOnePlus Real-Time PCR system. Cycling conditions were: initial hold at 95°C for 20 sec, 40 cycles of step 1 (95°C for one sec) and set 2 (60°C for 20 sec). Samples were run in quadruplicates and negative controls run in parallel. Relative quantification was calculated using the ΔΔCt method (StepOne System Software, Applied Biosystems).

Fast-scan cyclic voltammetry

Coronal brain slices (400 µm) containing the nucleus accumbens core from adult mice (2 – 4 months old) prepared as described previously (Mateo et al., 2005). Slices were kept in oxygenated modified Kreb’s buffer (in mM): NaCl 126, KCl 2.5, NaH₂PO₄ 1.2, CaCl₂ 2.4, MgCl₂ 1.2, NaHCO₃ 25, glucose 11, HEPES 20, L-ascorbic acid 0.4; at RT until required. Recordings were made at 34°C in a chamber perfused at 1 ml/min rate. Cylindrical carbon-fiber microelectrodes (50 – 100 µm exposed fiber) were prepared with T650 fibers (6 µm diameter, Goodfellow, Oakdale, PA) and inserted into a glass pipette (Cahill et al., 1996). The carbon-fiber electrode was held at −0.4 V and the potential was ramped to +1.2 V and back at 400 V/s every 100 ms. DA released was evoked by a rectangular, electrical pulse stimulation (50 – 600 µA, 0.6 ms/phase, biphasic) applied every 5 min. Data collection was done using LabVIEW software (National Instruments, Austin, TX). Ten cyclic voltammograms of charging currents were recorded as background before stimulation and the average subtracted from data collected during and after stimulation. Baseline DA signals were collected for five minutes before drug application. The decay of mean DA transient for each condition was fit using a single exponential and the decay time constant (τ) was calculated as k₋₁ (k = first order rate constant).

Sequencing

Clone GENSAT-BX37 was obtained from the Children’s Hospital Oakland Research Institute and grown in media with chloramphenicol (Sigma). DNA was extracted using a Large-Construct Kit (Qiagen). Sequencing was performed by Macrogen USA (Rockville, MD) using the forward primer 5’-CAT GGT CCT GCT GGA GTT CGT G-3’ and the reverse primer 5’-CGT CGC CGT CCA GCT CGA CCA G-3’.

Electrophysiology

Horizontal slices (250 µm) containing the VTA were prepared using a vibrating tissue slicer (Leica VT-1200S, Germany) and allowed to recover for 30 min at 33°C in oxygenated standard artificial cerebrospinal fluid containing (in mM): NaCl 124, NaH₂PO₄ 1, KCl 2.5, MgCl₂ 1.3, CaCl₂ 2.5, glucose 20, and NaHCO₃ 26.2. Interpependuncular fossa and medial terminal nucleus were used as landmarks to locate midbrain dopaminergic (DA) neurons. Their identity was further confirmed by the characteristic spontaneous activity at 2 – 5 Hz (in cell-attached) and the presence of I_h current (Zhang et al., 2010). Whole-cell voltage-clamp recordings were carried out using patch electrodes (4 – 6MΩ) filled with (in mM) KMeSO₄ 138, MgCl₂ 2, CaCl₂ 2, HEPES 10, EGTA 10, GTP-Na 0.3, Na₂ATP 4, pH 7.2- 7.4 (295 – 300 mOsm) at 35°C. Cells were held at −55 mV and series resistance (6 – 25mΩ) and input resistance were monitored throughout the recording. Data were collected using a Multiclamp 700B amplifier (Axon Instruments, Sunnyvale, CA) and currents were low-pass-filtered at 2 kHz and digitized at 5 kHz. The D2-like receptor agonist quinpirole (30, 90, 200 or 1000 nM) was added to the ACSF solution. Data were fit using the Hill equation: min + (max − min) / [ 1 + (EC₅₀ / x)^rate ], where min was set to zero.

Statistics

Data are presented as mean ± standard error of the mean (SEM), and differences were determined using a t-test or an ANOVA followed by a Tukey Test for multiple comparisons.

Results

Mice carrying the BAC GENSAT-BX37, Drd2-EGFP mice, bred normally and were indistinguishable in appearance from wild-type Swiss Webster (weight at 7 – 10 weeks old for Drd2-EGFP female = 28.1 ± 0.5 g, males = 33.9 ± 0.7 g and wild-type female = 27.5 ± 0.6 g, males = 35.9 ± 0.7 g). The genetic background of Drd2-EGFP mice is mainly Swiss Webster, an outbred strain, with some contribution from the FVB/N strain because the transgene was injected in FVB/N fertilized oocytes and backcrossed to Swiss Webster mice. Drd1-EGFP mice were generated in the same fashion and share the same mixed background as Drd2-EGFP. Thus, Drd1-EGFP mice are a useful control for issues concerning the strain genetic background and they have been frequently compared to Drd2-EGFP mice (Lee et al., 2006; Surmeier et al., 2007; Day et al., 2008; Gertler et al., 2008). Throughout this study, wild-type Swiss Websters and Drd1-EGFP mice were used and compared to Drd2-EGFP mice in order to help determine differences among transgenic lines and deviations from the unmodified background strain.

Increased D2R membrane expression and mRNA levels in Drd2-EGFP mice

Membrane expression of D2R was determined using radioligand binding assay on membrane extracts from the striatum of Drd2-EGFP and wild-type mice. The saturation binding isotherm for these genotypes showed no significant changes in receptor affinity (K_d) (F_(3,11) = 0.21, p = 0.88), but did show a significant increase in maximum binding concentration (B_max), which was confirmed by a one-way ANOVA (F_(3,13) = 6.7, p < 0.01). Specifically, Drd2-EGFP mice displayed an increase in B_max that was ~40% higher than wild-type (B_max = 1.32 ± 0.06 pmol/mg for wild-type and 1.80 ± 0.04 pmol/mg for Drd2-EGFP, q = 4.9, p < 0.05 Tukey test) with no corresponding change in affinity (K_d = 0.21 ± 0.03 nM for wild-type and 0.20 ± 0.02 nM for Drd2-EGFP, p = 0.99 Tukey test) (Fig. 1A–C). In order to address whether this effect was a unique property of this particular mouse colony housed at the NIAAA an independent colony of Drd2-EGFP mice was set up with outside breeders and named Drd2-EGFP/M (also derived from the only founder line). These mice also showed an increase in B_max compared to wild-type that was equivalent to that seen in our original colony, with no change in affinity (B_max = 1.75 ± 0.13 pmol/mg, q = 4.7, p < 0.05; K_d = 0.22 nM, p = 0.97 Tukey test) (Fig. 1A–C). To determine whether EGFP expression in the striatum was responsible for this change, Drd1-EGFP mice were tested and no difference was detected in B_max or K_d compared to wild-type mice (B_max = 1.36 ± 0.10 pmol/mg, q = 0.4, p = 0.99; K_d = 0.22 nM, p = 0.93 Tukey test) (Fig. 1A–C). (Fig. 1A–C).

Figure 1

Over-expression of dopamine D2R subunit in the striatum of Drd2-EGFP mice

To determine whether altered D2R gene expression could account for the increased surface expression, quantitative PCR was performed to measure D2R mRNA levels in all genotypes. Primers were designed to amplify mRNA for both short and long isoforms of D2R from striatal total RNA. The assay revealed that mice from Drd2-EGFP and Drd2-EGFP/M had significantly higher levels of D2R mRNA compared to wild-type mice (Fig. 1D–F). D2R mRNA levels were normalized to the levels of β-Actin mRNA in each sample as an endogenous control. Drd2-EGFP mice showed two times higher levels of D2R mRNA, and Drd2-EGFP/M one and a half times higher levels, over those of wild-type mice (F_(3,24) = 102, p < 0.01 ANOVA and Tukey Test q = 7.75, p < 0.01).

The results suggest that higher D2R mRNA levels could account for the increased membrane expression in both mouse colonies of Drd2-EGFP, yet the molecular mechanism remains unclear. One possibility considered was the presence of additional copies of the Drd2 gene in the transgene. The original BAC RP23-161H15 contains a segment (217,489 bp) of mouse Chromosome 9 (49,082,781 – 49,300,270) that includes three genes: Drd2 (dopamine receptor D2), Ankk1 (ankyrin repeat and kinase domain-containing 1) and Ttc12 (tetratricopeptide repeat domain 12) (Fig. 1G). This BAC was modified to express EGFP downstream of the D2R start codon and named GENSAT-BX37 (Fig. 1H) (note: only A-box 3’primer sequence is reported at http://www.gensat.org/bacreport.jsp). We sequenced the flanking regions around EGFP in GENSAT-BX37 using primers anchored in EGFP and proceeding upstream and downstream (supplementary material). The sequencing results using the upstream-directed primer confirmed that EGFP was inserted directly after intron 1 of the Drd2 gene (the start codon is located in exon 2) (Fig. 1H–I) which is in agreement with the reported 3’primer used for EGFP insertion. The sequence obtained for the downstream flanking region corresponded to intergenic sequence between the Ankk1 and Ttc12 genes, suggesting that the Drd2 and Ankk1 genes were removed when EGFP was inserted. These results rule out the possibility of additional functional copies of the Drd2 present in the BAC, but also suggest that the Ttc12 gene was left behind in GENSAT-BX37.

Enhanced potency of D2R agonist at the cellular and behavioral level

We next sought to investigate the functional consequences for D2R over-expression. With this purpose, whole-cell voltage-clamp recordings were performed from midbrain dopaminergic neurons in acute brain slices prepared from Drd2-EGFP and Drd1-EGFP transgenic mice. Midbrain dopaminergic neurons express somatic D2R that couple to G-protein-gated inwardly rectifying K⁺ channels and, when activated, produce an outward current (Beckstead et al., 2004) that can be used to assess the functional consequences of D2R over-expression. DA neurons were identified by their characteristic I_h current and low firing frequency, and in Drd2-EGFP mice were also identified by their green fluorescence (Fig. 2A). The D2R like agonist quinpirole (200nM) evoked an outward current that was roughly 2 fold larger in Drd2-EGFP mice (wild-type = 54.3 pA and Drd1-EGFP = 83.8 pA, while Drd2-EGFP = 161 pA; F_(2,18)= 4.78, p < 0.05, Tukey Test Drd2-EGFP vs wild-type q = 4.18, p < 0.05) (Fig. 2D). A dose response curve was built to compare the currents evoked by multiple concentrations of quinpirole between the genotypes. Quinpirole evoked currents had a lower EC₅₀ in Drd2-EGFP mice than in wild-type and Drd1- EGFP mice, indicating an enhanced cellular potency for D2R agonist in the Drd2-EGFP genotype (EC₅₀ wild-type = 392 nM and Drd1-EGFP = 260 nM while Drd2-EGFP = 60 nM) (Fig. 2B).

Figure 2

Increased behavioral and cellular sensitivity to D2R agonist in Drd2-EGFP mice

We next tested whether enhanced cellular potency of a D2R-like agonist was also reflected in enhanced behavioral potency. While rats typically show a biphasic locomotor response to D2R-like agonists, mice display a dose-dependent decrease in locomotor activity (Halberda et al., 1997; Ralph and Caine, 2005). Mice were allowed to habituate to the activity cage for one hour, they then received i.p. injections of saline or different doses of quinelorane, and locomotion was measured during the next 3 hours (post saline locomotor activity for wild-type = 1174 ± 222 counts/3hrs; Drd1-EGFP = 714 ± 104 counts/3hrs; Drd2-EGFP = 5939 ± 873 counts/3hrs; Drd2-EGFP/M = 2354 ± 595 counts/3hrs). Drd2-EGFP and Drd2-EGFP/M mice showed potentiated responses to low doses of D2R-like agonist (at 3 µg/kg F_(3,19) = 4.67, p < 0.05, Tukey Test wild-type vs Drd2-EGFP and vs Drd2-EGFP/M, q = 4.5 and 4.4 , respectively and p < 0.05). These responses were reflected in a leftward shift of the dose response curve and lower EC₅₀ compared to wild-type and Drd1-EGFP (EC₅₀ Drd2-EGFP = 1.5 µg/kg, Drd2-EGFP/M = 1.2 µg/kg, Drd1-EGFP = 6 µg/kg and wild-type = 65 µg/kg) (Fig. 2E–F). Note that Drd2-EGFP mice exhibited hyperactivity, which is shown in more detail later in Fig. 4 A – B. As a result, locomotor activity after saline injection was lower in control mice than in Drd2-EGFP mice, but all control mice still displayed sufficiently high counts to allow for the detection of the inhibitory effects of D2R-like agonist on locomotion.

Figure 4

Hyperactivity and deficient acute and chronic response to cocaine in Drd2-EGFP mice

Altered dopaminergic signaling and impaired cocaine effect on DA uptake

Fast-scan cyclic voltammetry was used to investigate the levels and kinetics of DA release and uptake evoked by electrical stimulation in the nucleus accumbens core (Fig. 3). DA transients for wild-type and Drd1-EGFP mice did not differ in any parameters and hence data from these two genotypes were pooled together as controls for statistical purposes. This singular value was then compared against Drd2-EGFP mice in all voltammetry analyses. The peaks of the evoked DA transients were similar among genotypes (mean control = 1.16 ± 0.08 µM and Drd2-EGFP = 1.08 ± 0.1 µM, n = 13-10). However, the descending phase of the transient was much slower in Drd2-EGFP mice (Fig. 3B, C). Quantification of the mean decay time constant (τ) confirmed a significant increase in the τ in Drd2-EGFP mice (526 ± 47 ms) compared to control mice (375 ± 23 ms; t(12) = 2.8, p < .05) (Fig. 3C). As a consequence of the severe slowdown of the DA clearance in Drd2-EGFP mice, the area of the DA transient was increased by ~2.5 times in these mice (area controls = 0.67 ± 0.08 and Drd2-EGFP = 1.2 ± 0.16, n = 13-10, t(23) = 3.0, p < 0.01) (Fig. 3D).

Figure 3

Altered DA release and uptake in the nucleus accumbens core

The kinetics of the descending portion are dependent on the rate of DA uptake (Wightman et al., 1988). Cocaine blocks the DA transporter and slows down the clearance of DA in control mice so we asked whether cocaine actions were affected in Drd2-EGFP mice. Figure 3E–F compares the effect of cocaine (10 µM) on the evoked DA transients of control and Drd2-EGFP mice and displays the τ value obtained after cocaine application normalized to their own baseline τ obtained before drug application. The results showed that the cocaine effects on τ are greatly reduced in Drd2-EGFP mice indicating that cocaine, at this concentration, did not slow down DA clearance noticeably in Drd2-EGFP mice (norm τ = 6.7 ± 0.93 and 2.22 ± 0.70 for controls and Drd2-EGFP mice, respectively; t(6) = 3.9, p < 0.01).

DA release is modulated by presynaptic D2R and given our findings of D2R over-expression and enhanced potency, it became important to address the effect of D2R antagonist on evoked DA release. In agreement with previous data (Kennedy et al., 1992; Phillips et al., 2002), sulpiride (2 µM) had minimal or no effect on the peak of the evoked DA transient in control mice. However, sulpiride increased the peak of evoked DA release in Drd2 mice by ~60% (Fig. 3E, t(7) = 5.5, p < 0.01) revealing some degree of tonic inhibition mediated by D2R in these mice.

Hyperlocomotion and paradoxical response to cocaine in Drd2-EGFP mice

When locomotor behavior was measured in naïve animals placed in a novel environment, Drd2-EGFP and Drd2-EGFP/M mice showed increased activity compared both control genotypes (Fig. 4A, B) (beam breaks/hour Drd1-EGFP = 1479 ± 113, wild-type = 1660 ± 157, Drd2-EGFP/M = 2533 ± 181 and Drd2-EGFP = 4126 ± 248, n=16–24, F_(3,137) = 41, p < 0.01 ANOVA, *, p < 0.01 and NS, p = 0.89 Tukey Test). Additionally, Drd2-EGFP mice showed no increase and even a slight dose-dependent decrease in locomotion after acute cocaine administration (Fig. 4C). In control mice, chronic cocaine administration caused locomotor sensitization, which was characterized by a further increase in locomotor response over the acute effect of roughly five folds, relative to saline, with repeated cocaine injections (Drd1-EGFP day 1 = 73.4 ± 11.4, day 10 = 161 ± 36.9, q = 8.8, p < 0.01, n = 17, Tukey Test) (Fig. 4D, 4E). However, Drd2-EGFP and Drd2-EGFP/M failed to show a sensitized response to cocaine (Drd2-EGFP day 1 = 117 ± 24.1, day 10 = 67.0 ± 12.9, n = 12; Drd2-EGFP/M day 1 = 59.8 ± 12.9, day 10 = 83.0 ± 16.7, n = 12, all p > 0.05, Tukey Test) (Fig. 4F, 4G). Furthermore, heterozygote mice for the BAC transgene also failed to show locomotor sensitization to cocaine (F_{(9, 84)} = 10.4, p < 0.01, Drd2-EGFP^+/− day 1 = 48.4 ± 8.8, day 10 = 36.6 ± 21.7, n = 3; Drd2-EGFP/M^+/− day 1 = 61.6 ± 11.9, day 10 = 68.8 ± 12.3, n = 9, all p > 0.05, Tukey Test; Fig. 4H).

Discussion

This study provides an extensive characterization of the BAC transgenic Drd2-EGFP and Drd1-EGFP mice and reveals that Drd2-EGFP mice are not comparable to controls with regards to their dopaminergic system. D2-BAC transgenic mice, a common tool used to study the physiological and morphological properties of medium spiny neurons in the striatum and nucleus accumbens, have altered D2R expression and signaling. High levels of D2R mRNA in the dorsal striatum are likely to explain the receptor over-expression seen at striatal membranes as well as the hypersensitivity to D2R-like agonists observed in these mice The data also suggest that the receptor over-expression extends beyond the striatum to midbrain dopaminergic neurons that also express D2R. Additionally, neurochemical analysis of DA terminal function showed slower clearance of extracellular DA in Drd2-EGFP mice that led to prolonged DA transients and could account for the increased locomotor activity observed in these mice.

Drd2-EGFP mice are hyperactive and acute cocaine administration decreases the locomotor activity. A similar paradoxical response to acute psychostimulants has been described in a mouse model of attention deficit/hyperactivity disorder in which 6-OHDA lesions were performed at neonatal age resulting in hyperactivity at puberty and an inhibitory locomotor response to methylphenidate and amphetamine (Avale et al., 2004). Furthermore, Drd2-EGFP mice failed to display locomotor sensitization, which has been observed, to different degrees, in all reported mouse strains.

It is important to note that this characterization has mainly been performed on homozygous BAC transgenic mice and thus these results and conclusions are limited to Drd2-EGFP homozygote mice. While most previous studies have used homozygote Drd2-EGFP mice, a recent study used heterozygote mice (Lerner et al., 2010). Heterozygote mice might display a milder phenotype (however, see Fig. 4H) and their use, in combination with appropriate controls, could be recommended over the use of homozygote mice. Nevertheless, further characterization of heterozygote Drd2-EGFP mice would be needed if used.

Two different mouse colonies carrying the GENSAT-BX37 clone showed similar alterations in the behavioral, neurochemical and physiological responses to DA and D2R agonist. At the same time, mice with a similar genetic background but carrying a different BAC clone that produces EGFP expression in a separate subset of medium spiny neurons in the striatum (Drd1-EGFP mice) did not show this phenotype. This result rules out a potential contribution of the mixed genetic background of these BAC transgenic mice. However, the results cannot rule out that EGFP expression selectively in D2R-expressing striatal neurons is responsible for the altered gene expression.

The results of this study seem to indicate that the BAC clone itself is responsible for the altered gene expression either because of a positional effect caused by the clone insertion event or the presence of the transgene in one or more copies. Multiple mechanisms can account for the altered gene expression, including positional effects related to the random process of the transgene insertion and the random number of copies that are integrated in each pedigree. The random insertion process may lead to the interruption of a gene or regulatory sequence, or it might position the transgene under the control of a transcriptional enhancer which, in turn, would modify the expression levels of the reporter gene or its expression pattern. For these reasons, the scientific community has always recommended the generation of at least three to four founder lines with independent insertion sites of the same transgene. Unfortunately, there is only one founder line for Drd2-GFP mice and this precludes further investigation of the plausible positional effects.

Sequencing analysis of the BAC clone GENSAT-BX37 seemed to confirm that indeed the coding sequence for D2R was removed and replaced by EGFP. However, the Ttc12 gene might still be present and this could account for the altered gene expression. Indeed, a previous study reported that mice carrying a modified version of the GENSAT-BX37 showed a 1,200 fold higher mRNA level for Ttc12 in D2R-expressing neurons of the striatum compared to other neurons in wild-type mice or Drd1-GFP mice (Heiman et al., 2008). The function of this gene product is unknown but allelic variations in this region of the genome have been linked to alcohol dependence (Yang et al., 2007) and could possibly be involved in the regulation of the dopaminegic system in adults and/or during development. Other transgenic mice generated from a modified RP23-161H15 clone or GENSAT-BX37, such as the Drd2-Cre transgenic mice, could express similar alterations. Thus, it is important to perform similar analyses and to quantify D2R expression levels if using them for experiments. The conclusions from this study highlight the importance of thorough characterization of the transgenic mice used in neuroscience research and call the community to re-examine the results obtained using Drd2-EGFP mice.

Supplementary Material

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Acknowledgements

We thank J.M. Urban and H. Puhl for their valuable technical advice with the qPCR and sequencing and M. Rubinstein and D.M. Lovinger for their helpful comments and discussions of the manuscript. This study was funded by the intramural programs of the NIAAA and NINDS.

Footnotes

Supplementary Figure 1: Sequencing results

Upstream and downstream sequences obtained from the sequencing analysis of GENSAT-BX37 using primers anchored in EGFP.

References

Avale ME, Falzone TL, Gelman DM, Low MJ, Grandy DK, Rubinstein M. The dopamine D4 receptor is essential for hyperactivity and impaired behavioral inhibition in a mouse model of attention deficit/hyperactivity disorder. Mol Psychiatry. 2004;9:718–726. [PubMed]
Beckstead MJ, Grandy DK, Wickman K, Williams JT. Vesicular dopamine release elicits an inhibitory postsynaptic current in midbrain dopamine neurons. Neuron. 2004;42:939–946. [PubMed]
Cahill PS, Walker QD, Finnegan JM, Mickelson GE, Travis ER, Wightman RM. Microelectrodes for the measurement of catecholamines in biological systems. Anal Chem. 1996;68:3180–3186. [PubMed]
Day M, Wokosin D, Plotkin JL, Tian X, Surmeier DJ. Differential excitability and modulation of striatal medium spiny neuron dendrites. J Neurosci. 2008;28:11603–11614. [PMC free article] [PubMed]
Gertler TS, Chan CS, Surmeier DJ. Dichotomous anatomical properties of adult striatal medium spiny neurons. J Neurosci. 2008;28:10814–10824. [PMC free article] [PubMed]
Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N. A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature. 2003;425:917–925. [PubMed]
Halberda JP, Middaugh LD, Gard BE, Jackson BP. DAD1- and DAD2-like agonist effects on motor activity of C57 mice: differences compared to rats. Synapse. 1997;26:81–92. [PubMed]
Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, Suarez-Farinas M, Schwarz C, Stephan DA, Surmeier DJ, Greengard P, Heintz N. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 2008;135:738–748. [PMC free article] [PubMed]
Heintz N. BAC to the future: the use of bac transgenic mice for neuroscience research. Nat Rev Neurosci. 2001:861–870. [PubMed]
Kennedy RT, Jones SR, Wightman RM. Dynamic observation of dopamine autoreceptor effects in rat striatal slices. J Neurochem. 1992;59:449–455. [PubMed]
Kreitzer AC, Malenka RC. Endocannabinoid-mediated rescue of striatal LTD and motor deficits in Parkinson's disease models. Nature. 2007:643–647. [PubMed]
Kreitzer AC, Malenka RC. Striatal plasticity and basal ganglia circuit function. Neuron. 2008;60:543–554. [PMC free article] [PubMed]
Lee KW, Kim Y, Kim AM, Helmin K, Nairn AC, Greengard P. Cocaine-induced dendritic spine formation in D1 and D2 dopamine receptor-containing medium spiny neurons in nucleus accumbens. Proc Natl Acad Sci U S A. 2006;103:3399–3404. [PubMed]
Lerner TN, Horne EA, Stella N, Kreitzer AC. Endocannabinoid signaling mediates psychomotor activation by adenosine A2A antagonists. J Neurosci. 2010;30:2160–2164. [PMC free article] [PubMed]
Mateo Y, Lack CM, Morgan D, Roberts DC, Jones SR. Reduced dopamine terminal function and insensitivity to cocaine following cocaine binge self-administration and deprivation. Neuropsychopharmacology. 2005;30:1455–1463. [PubMed]
Phillips PE, Hancock PJ, Stamford JA. Time window of autoreceptor-mediated inhibition of limbic and striatal dopamine release. Synapse. 2002;44:15–22. [PubMed]
Ralph RJ, Caine SB. Dopamine D1 and D2 agonist effects on prepulse inhibition and locomotion: comparison of Sprague-Dawley rats to Swiss-Webster, 129X1/SvJ, C57BL/6J, and DBA/2J mice. J Pharmacol Exp Ther. 2005;312:733–741. [PubMed]
Shen W, Flajolet M, Greengard P, Surmeier DJ. Dichotomous dopaminergic control of striatal synaptic plasticity. Science. 2008;321:848–851. [PMC free article] [PubMed]
Surmeier DJ, Plotkin J, Shen W. Dopamine and synaptic plasticity in dorsal striatal circuits controlling action selection. Curr Opin Neurobiol. 2009;19:621–628. [PMC free article] [PubMed]
Surmeier DJ, Ding J, Day M, Wang Z, Shen W. D1 and D2 dopamine-receptor modulation of striatal glutamatergic signaling in striatal medium spiny neurons. Trends Neurosci. 2007;30:228–235. [PubMed]
Tian X, Kai L, Hockberger PE, Wokosin DL, Surmeier DJ. MEF-2 regulates activity-dependent spine loss in striatopallidal medium spiny neurons. Mol Cell Neurosci. 2010;44:94–108. [PMC free article] [PubMed]
Valjent E, Bertran-Gonzalez J, Herve D, Fisone G, Girault JA. Looking BAC at striatal signaling: cell-specific analysis in new transgenic mice. Trends Neurosci. 2009;32:538–547. [PubMed]
Wightman RM, Amatore C, Engstrom RC, Hale PD, Kristensen EW, Kuhr WG, May LJ. Real-time characterization of dopamine overflow and uptake in the rat striatum. Neuroscience. 1988;25:513–523. [PubMed]
Yang BZ, Kranzler HR, Zhao H, Gruen JR, Luo X, Gelernter J. Association of haplotypic variants in DRD2, ANKK1, TTC12 and NCAM1 to alcohol dependence in independent case control and family samples. Hum Mol Genet. 2007;16:2844–2853. [PubMed]
Zhang TA, Placzek AN, Dani JA. In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area. Neuropharmacology. 2010 [PMC free article] [PubMed]