Our study demonstrates that expression of mutant DISC1 was associated with impaired METH-induced CPP, delayed behavioral sensitization, decreased density of DA D2 receptors and altered AKT/GSK3 signaling in the ventral striatum in mutant DISC1 mice. The results indicate that perturbations in DISC1 functions could impact the signaling that may underlie reward and drug-induced neuroplasticity, contributing to comorbidity between substance abuse and major psychiatric disorders. Recent studies have suggested that substance abuse and schizophrenia may share genes that encode for proteins involved in neurodevelopment (Namkung et al., 2005; Uhl, 2008). We think that DISC1 is a promising gene for comorbidity research as it has been implicated in neurodevelopment and major psychiatric diseases and interacts with key signaling molecules involved in drug-induced neuroplasticity.
Our findings clearly indicate sex-dependent effects of METH in control and mutant DISC1 mice. We found that mutant female mice exhibited delayed sensitization and impaired METH-induced CPP. It is unlikely that the abnormal sensitization and CPP were due to non-specific alterations in locomotor activity or learning in female DISC1 mice as female mutant mice displayed robust fear conditioning but did not demonstrate CPP at the METH dose of 0.5 mg/kg. This double dissociation suggests that the lack of CPP in female mutant DISC1 mice may be due to abnormal reward processes in the ventral striatum (Chiang et al., 2009
). Since female DISC1 mice develop CPP at a higher METH dose (2 mg/kg), one can speculate about delayed rather than impaired sensitization in mutant mice. Repeated exposure to a number of psychoactive drugs facilitates development of CPP (Lett, 1989
) and it is possible that, with an increased duration of training, mutant female mice could become sufficiently sensitized to express CPP to 0.5 mg/kg METH. The state-dependent learning, whereby a METH-paired compartment becomes novel and more attractive in the drug-free test, may be excluded as the other groups continued to show the same preferences after injection of the training dose of METH.
The present data are in line with our prior findings about sex-dependent neurobehavioral abnormalities in mutant DISC1 mice (Pletnikov et al., 2008
; Ayhan et al., 2010
). In the context of METH addiction, our study is consistent with the wealth of animal and human data about gender-related responses to psychostimulants, mostly attributed to the influence of ovarian hormones on DA neurotransmission in the brain (Robinson and Becker, 1986
). One of the potential mechanisms whereby estrogens could influence METH-induced neuroadaptation includes interactions between Kalirin 7 and DISC1 (Hayashi-Takagi et al, 2010
). As Kalirin 7 has been shown to be involved in cocaine-induced and estrogen-stimulated dendritic spine maturation (Penzes, 2008
; Ma et al., 2010
; Kiraly et al., 2010
) and Kalirin 7-DISC1 interactions have been demonstrated to play an important role in dendritic spine formation (Hayashi-Takagi et al, 2010
), one could speculate that METH-induced behavioral alterations in DISC1 female mice could in part result from abnormal DISC1-Kalirin 7 interactions. Future research will address this intriguing possibility.
Although the use of a single concentration of [11
C]-raclopride does not allow us to interpret the present binding results as change in receptors numbers vs. affinity, the concentration of the ligand of 6 nM is expected to lead to occupation of a high percentage of receptors sufficient for a reliable analysis of the data (de Araujo et al, 2009
). We found that expression of mutant DISC1 in naïve mice were associated with a decrease in DA D2 receptor binding in the ventral striatum that play an important role in mediating the motor (Pijnenburg et al., 1976
) and rewarding (Ikemoto et al., 2005
; Selling et al., 2006
) effects of stimulants. It is conceivable that decreased density of D2 DA receptors may have contributed to delayed sensitization and impaired CPP in mutant DISC1 mice. We think that the observed regional differences in DA D2 receptor binding (i.e., decreased in the ventral striatum and somewhat increased in the VTA/SN area) may be related to the regional activity of the CAMKII promoter that has been shown to be predominantly active in forebrain neurons (Pletnikov et al, 2008
). The down-regulation of endogenous DISC1 found in striatum of mutant DISC1 mice corroborates our previous findings of the decreased expression in cortical samples (Ayhan et al, 2010
), supporting our general hypothesis that mutant DISC1 may have a dominant negative effect on the endogenous counterpart.
Decreased D2 DA receptors binding in the ventral but not dorsal striatum is a feature of impulsive rats that are susceptible to escalating doses of cocaine self-administration (Dalley et al., 2007
). This may seem opposite to our findings in mutant DISC1 mice that exhibited impaired METH-induced sensitization and CPP. Down-regulation of DA D2 receptors has been also observed after high repeated doses of psychostimulants (Chen et al., 1999
). Since neuroadaptation to drugs of abuse and the mechanisms that predispose to self-administration are different, one could interpret our results as a state of “adapted” DA D2 signaling due to disruption of endogenous DISC1, making mutant mice less sensitive to effects of METH. The opposite data, however, have been reported for mice with the point mutation in exon 2 of Disc1
(Clapcote et al., 2007
; Lipina et al., 2010
), i.e., increased sensitivity to low doses of amphetamine and up-regulation of DA D2 receptors in the striatum of male mice. It is conceivable that the missense mutation and mutant DISC1 may have different effects on neuronal plasticity.
The mechanisms of dominant-negative effects of mutant DISC1 may involve a disruption of interaction of endogenous DISC1 with GRB2, an adaptor protein for DA D2-like receptors (Oldenhof et al., 1998
). Alternatively, mutant DISC1 could affect interactions between endogenous DISC1 and a host of DA receptors interacting proteins involved in regulation of DA D2 receptor trafficking (Kabbani and Levenson, 2007
). For example, an agonist-induced D2 receptor internalization may be significantly reduced if the function of arrestin-2 becomes compromised (Macey et al., 2004
). Importantly, changes in endogenous DISC1 may also affect signaling pathways downstream of D2 DA receptor, including AKT and GSK-3β (Beaulieu et al., 2004
) that interact with DISC1 and may be involved in the pathophysiology of mental conditions. For example, recent studies have demonstrated the suppression of phosphorylation of AKT following knockdown of endogenous DISC1 protein in primary neuronal cultures (Hashimoto, 2006
), direct interactions of DISC1 with GSK-3β (Mao et al., 2009
) or KIAA1212, an AKT binding partner (Kim et al., 2009
). There have been also reports to suggest a role of AKT/GSK3β signaling in schizophrenia, depression, psychosis and responses to lithium (Beaulieu et al., 2009; Benedetti et al., 2004
; Mao et al., 2009
; Karege et al., 2007
; Serretti et al., 2008
; Feyberg, 2010) as well as in neurodevelopment (Hur and Zhou, 2010
), and NMDA-dependent synaptic plasticity (Peineau et al., 2008
) that can be affected by chronic exposure to drugs of abuse (Russo et al., 2010
We evaluated some aspects of AKT/GSK3β signaling to begin elucidating the mechanisms of abnormal CPP and sensitization in mutant DISC1 mice. We found that acute treatment with METH decreased levels of phosphorylated AKT and GSK-3β in naïve control mice without altering those in naïve mutant DISC1 mice at 90 min post-treatment. These results seem consistent with a previous study that has reported an amphetamine-induced decrease in phosphorylation of AKT and GSK-3β in the mouse striatum (Beaulieu et al., 2004
). One could hypothesize that alterations in DISC1 may render AKT/GSK-3β signaling less sensitive to psychostimulants, leading to delayed sensitization and impaired CPP. After METH ED treatments, a challenge with a single injection of METH decreased GSK-3β phosphorylation in control but not mutant mice, further suggesting alterations in GSK-3β signaling. This may in part explain the absence of sensitization in METH ED-treated mutant mice after a protracted period of abstinence (Fig. A5). Future studies with this model will evaluate the AKT/GSK-3 pathway in greater detail.
In a broad context, the current study is similar to those where METH-induced neuroadaptation has been studied in other genetic mouse models with mutations relevant to psychiatric disorders and/or DA signaling. For example, behavioral sensitization after repeated METH treatment has been shown to be significantly reduced in Sandy
mice, indicating that dysbindin may have a role in the development of behavioral sensitization (Nagai et al., 2010
). Mice with the deletion of FEZ1 (fasciculation and elongation protein zeta 1), another DISC1 partner, have been demonstrated to exhibit enhanced responses to METH (Sakae et al., 2008
), supporting the idea that dynamic changes in the balance between levels of anti-addictive and pro-addictive factors in the brain could determine susceptibility to substance dependence (Niwa et al., 2008
In conclusion, our study indicates that perturbations in DISC1 functions in the ventral striatum may impact the molecular mechanisms of reward and drug-induced neuroadaptation, contributing to between substance abuse and major mental illnesses. This seems to be a fertile area for the future research.