The main findings of the current study were that C57BL/6J-congenic D5R KO showed normal acute and sensitized locomotor responses to cocaine and developed normal cocaine CPP, whereas C57BL/6J-congenic D1R KO showed no acute cocaine response but failed to sensitize to cocaine in a dose-dependent fashion.
Analysis of locomotor exploration during undrugged exposure to a novel open field showed that while genotypes did not differ in initial activity, D1R KO were generally more active than WT over the latter part of the session. D1R KO mice also showed markedly more exploration of the aversive center of the open field than WT controls, further indicating the disorganization of exploratory behavior in D1R KO. This basal open field profile is largely consistent with previous studies of D1R KO, although some discrepancies exist (reviewed in (Holmes et al. 2004
; Waddington et al. 2005
)). In contrast to the D1R phenotype, D5R KO showed marginally quicker habituation to the open field as evidenced by lesser activity than WT during the latter part of the test, but were otherwise normal. This concurs with previous observations of moderately depressed locomotor activity in D5R KO mice (Holmes et al. 2001
; O'Sullivan et al. 2005
A well-replicated finding is that D1R KO mice (Drago et al. 1996
; Xu et al. 1994
), including those tested on a congenic C57BL/6J background (Karasinska et al. 2005
), fail to respond to the hyperactivity-inducing effects of acute treatment with D1R/D5R agonists or psychostimulants including cocaine. D1R KO consistently showed negligible locomotor responses to acute cocaine treatment across three separate experiments in our study. On the other hand, D5R KO responded to acute cocaine in a manner indistinguishable from WT controls. This latter finding contrasts somewhat with an earlier observation of a modestly diminished locomotor response to acute cocaine in D5R KO mice (Elliot et al. 2003
). The use of a 129/SvJ1 × C57BL/6J hybrid genetic background in the earlier study, versus a congenic C57BL/6J background in the present study, provides the most likely explanation for this difference given the ability of genetic background to influence dopamine receptor KO phenotypes (Holmes et al. 2004
; Kelly et al. 1998
; Waddington et al. 2005
), although other methodological factors may also have contributed. Notwithstanding, the finding that, unlike loss of D1R, loss of D5R does not abolish the acute locomotor response to cocaine extends evidence that D5R KO does not affect the discriminative-stimulus effects of cocaine (Elliot et al. 2003
). Thus, D5R appears to play a dispensable role in mediating various acute behavioral effects of cocaine. By extension this suggests that the ability of mixed D1R/D5R antagonists to attenuate the reinforcing properties of cocaine across various experimental animal paradigms (Barrett et al. 2004
; Self 2004
) and in human volunteers (Romach et al. 1999
) are likely principally D1R mediated.
Loss of D5R failed to alter the development of cocaine sensitization in three replications using two different sensitizing doses - i.e., after receiving repeated cocaine injections, D5R KO showed an augmented locomotor response to cocaine challenge that was no different from the augmented response produced in WT controls. Extending these data to another model of cocaine's reinforcing effects D5R KO also developed normal cocaine CPP. In this context, previous studies have demonstrated intact cocaine CPP in mice with D1R KO or loss of Fos in D1R-expressing neurons (Karasinska et al. 2005
; Miner et al. 1995
; Zhang et al. 2006
). This contrasts with the ability of systemic or intra-accumbel administration of the D1R/D5R antagonist SCH23390 to prevent cocaine CPP (Baker et al. 1998
; Cervo and Samanin 1995
; Hnasko et al. 2007
; Pruitt et al. 1995
). Taken together with recent evidence that constitutive loss of dopamine itself does not prevent cocaine CPP (Hnasko et al. 2007
), these data indicate that KO of dopamine signaling through either D1R or D5R is not sufficient to prevent cocaine CPP.
As noted in the Introduction a previous study had demonstrated that D1R KO did not develop locomotor sensitization to cocaine (Xu et al. 2000
). Subsequent studies found that mice lacking function of either the NR1 N
-methyl-D-aspartate receptor or the immediate-early gene Fos in D1R-expressing neurons showed attenuated cocaine locomotor sensitization (Heusner and Palmiter 2005
; Zhang et al. 2006
). Consistent with these studies, D1R KO failed to display sensitization after repeated treatment with 30 mg/kg cocaine. Nonetheless, the possibility remained that the absence of a sensitization effect in D1R KO had actually been a false negative caused by increased
sensitization to this relatively high sensitizing dose. This could have resulted in stereotypies in D1R KO and a consequent reduction in the principle measure of sensitization, total distance traveled. We addressed this issue in a replicate experiment in which stereotypies were measured, and found that although D1R KO again failed to show sensitization-related increase in total distance travelled there was no indication of excessive stereotypies.
An interesting and somewhat surprisingly finding was that, when tested with a 20 mg/kg sensitizing dose, D1R KO displayed evidence of a partial sensitization to cocaine. Specifically, despite again showing no acute locomotor response to cocaine, D1R KO clearly showed a cocaine-induced increase in locomotor activity following repeated treatment with a 20 mg/kg dose. However, unlike the pre- versus post-sensitization locomotor increase in WT and D5R KO the cocaine-induced locomotor response in D1R KO was not significantly increased across the phases. Nonetheless, these data suggest that D1R KO can exhibit locomotor sensitization under certain conditions and as such are seemingly at odds with current assumptions regarding the necessary contribution of D1R to cocaine sensitization. The reasons for the apparent discrepancy between our findings and those of Xu et al (Xu et al. 2000
) remain to be ascertained. Genetic background is one obvious difference which, as discussed above in the context of D5R KO, could also be an influence on phenotypic outcomes in the D1R KO. Sensitizing dose per se
does not appear to be the defining difference, as divergent effects were found at the same 20 mg/kg dose. Of possible critical importance however is the fact that our study used a sensitization protocol in which mice were treated once daily, while Xu et al treated twice daily. Therefore, the sensitizing regimen used by Xu et al may have more closely approximated to our 30 mg/kg cocaine sensitizing experiment, in which D1R KO showed no sensitization. This is one way to reconcile the findings of the two studies. In turn, it suggests a refinement of the D1R sensitization model in which the effects of D1R KO are dependent upon the intensity of sensitization regimen employed, with higher daily exposure to cocaine associated with a clear loss-of-sensitization phenotype.
A number of caveats to this and the other conclusions of the present study should be acknowledged. These mainly relate to the inherent limitations of the constitutive KO approach. First, irreversible receptor KO cannot dissociate a failure to develop
sensitization from a failure to express
sensitization to the higher dose of cocaine in D1R KO. Suggesting that a failure to show sensitization in D1R KO may be due an inability to express the behavior, treatment with a D1R/D5R antagonist during sensitizing phase does not typically prevent the subsequent expression of sensitization (Fontana et al. 1993
; Kalivas and Stewart 1991
; Mattingly et al. 1996
; Steketee 1998
; White et al. 1998
). On the other hand, the sensitization process is clearly paralleled by changes in D1R/D5R function (Kalivas and Stewart 1991
). The second limitation stems from the potential for constitutive KO of a gene to produce compensatory alterations, especially during development, that mask the normal function of a dopamine receptor (Holmes et al. 2004
; Waddington et al. 2005
). In this context, it would be premature to completely exclude a role for D5R in reward-related behavioral adaptations until additional techniques for selectively inactivating D5R (e.g., RNA interference) can be brought to bear upon the question. The same qualification applies to our findings regarding extant low-dose locomotor sensitization in D1R KO. For example, compensatory changes may have somehow mitigated the consequences of D1R loss for sensitization to the low, but not high, sensitizing cocaine dose. Of note in this context is that while attenuated D1R KO locomotor sensitization to amphetamine has been found in some but not all studies (Crawford et al. 1997
; Karper et al. 2002
; McDougall et al. 2005
; Xu et al. 2000
), Karper et al found that the D1R/D5R antagonist SCH23390 blocked amphetamine sensitization in WT but not D1R KO (Karper et al. 2002
). This suggests the uncoupling of the D1R contribution to amphetamine sensitization in the KO mice via the recruitment of alternative mechanisms (Karper et al. 2002
). Indeed, there is evidence of abnormal cortical neuronal morphology and changes in gene expression of for example G-protein signaling related (Rgs family) proteins in D1R KO (Stanwood et al. 2005
; Zhang et al. 2005
). Whether these or other changes support the partial maintenance of cocaine sensitization observed in D1R KO remains to be determined.
In summary, present findings further extend the phenotypic characterization of the consequences of KO of D1R or D5R for basal locomotor exploration, acute responses to cocaine and sensitization to repeated cocaine administration. During undrugged exposure to a novel open field, congenic D1R KO mice showed locomotor hyperactivity relative to WT, while congenic D5R KO were normal with the exception of marginally quicker habituation. The locomotor hyperactivity-inducing effects of acute cocaine treatment were unaltered in D5R KO but lost in D1R KO. Locomotor sensitization to repeated cocaine treatment was normal in D5R KO, as was conditioned place preference to cocaine. D1R KO prevented sensitization to a high but not low sensitizing dose of cocaine, without causing excessive locomotor stereotypies. Taken together, these data demonstrate that constitutive KO of D5R does not abolish two behavioral measures of cocaine's reinforcement-related effects, and that KO of D1R is not sufficient to block cocaine sensitization under all conditions.