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 (Kd) (F(3,11) = 0.21, p = 0.88), but did show a significant increase in maximum binding concentration (Bmax), which was confirmed by a one-way ANOVA (F(3,13) = 6.7, p < 0.01). Specifically, Drd2-EGFP mice displayed an increase in Bmax that was ~40% higher than wild-type (Bmax = 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 (Kd = 0.21 ± 0.03 nM for wild-type and 0.20 ± 0.02 nM for Drd2-EGFP, p = 0.99 Tukey test) (). 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 Bmax compared to wild-type that was equivalent to that seen in our original colony, with no change in affinity (Bmax = 1.75 ± 0.13 pmol/mg, q = 4.7, p < 0.05; Kd = 0.22 nM, p = 0.97 Tukey test) (). To determine whether EGFP expression in the striatum was responsible for this change, Drd1-EGFP mice were tested and no difference was detected in Bmax or Kd compared to wild-type mice (Bmax = 1.36 ± 0.10 pmol/mg, q = 0.4, p = 0.99; Kd = 0.22 nM, p = 0.93 Tukey test) (). ().
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 (). 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) (). This BAC was modified to express EGFP downstream of the D2R start codon and named GENSAT-BX37 () (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) () 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 Ih
current and low firing frequency, and in Drd2-EGFP mice were also identified by their green fluorescence (). 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) (). 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) ().
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) (). Note that Drd2-EGFP mice exhibited hyperactivity, which is shown in more detail later in . 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.
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 (). 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 (). 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) (). 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) ().
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. 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
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% (, 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 () (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 (). 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) (). 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) (). 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; ).