Lack of dopamine D4 receptor participation in mouse hyperdopaminergic locomotor response
Introduction
Drug addiction is a debilitating brain disorder and mental illness characterized by compulsive drug use, drug seeking and frequent relapses despite negative consequences [1]. Initial drug contact encodes a robust trigger for later acquisition and expression of drug addiction, ultimately resulting in relapse following environmental exposure to stress or cues [2]. Animal models that mimic the various stages of drug addiction serve as valuable tools to study stage-specific cellular mechanisms and treatments for drug addiction and relapse. Repetitive drug administration in rodents induces a progressive enhancement in locomotor activity and stereotypy, also referred to as behavioral sensitization, reflecting an enduring addiction-like state of drug wanting [3,4]. The initiation of behavioral sensitization is encoded by neural events in the brain’s reward center, the ventral tegmental area (VTA), while the later expression of sensitization is subsequently encoded in one of the main VTA outputs, the nucleus accumbens (or ventral striatum) [5]. Another model of drug addiction, dopamine transporter (DAT) knockout (KO) or knockdown (KD) mice, is thought to be either ‘genetically sensitized’ or ‘manic’, as the animals exhibit addiction-like genetic and physiological responses to ethanol treatment, cocaine treatment and anti-manic therapy [6,7].
Chronic psychostimulant (i.e. (meth)amphetamine and cocaine) consumption not only induces drug addiction, but also psychotic symptoms such as hallucinations and delusions; as such, antipsychotic remedies are routinely used to alleviate such symptoms in drug abusers [[8], [9], [10], [11]]. The atypical antipsychotic, clozapine (Clz), is an antagonist of dopamine D4 (D4R) and D2 (D2R) receptors (with higher binding affinity for D4R), in addition to serotonin 5-HT1A, 5-HT2 and muscarinic M4 receptors [[12], [13], [14]]. Clz has been extensively studied in the management of drug addiction in rodents using the behavioral sensitization model [15,16]. In particular, Clz is effective at preventing acute cocaine-, phencyclidine- or methamphetamine (METH)-induced hyperlocomotor activity, and it also mitigates the initiation and expression of cocaine sensitization [[17], [18], [19]]. Moreover, Clz is more efficacious than typical antipsychotics for improving both positive (psychotic) and negative (emotional) symptoms of schizophrenia, and it also does not have extrapyramidal and dystonia side effects, potentially due to fast-OFF drug actions on D2R [13,20]. Previous studies have suggested that the antipsychotic effect of Clz is mainly mediated through dual dopamine D2R and serotonin 5-HT2 antagonism, while D4R blockade may participate to a lesser extent [21,22].
The expression of D4R is enriched in limbic structures [23], and altered function or expression of the receptor has been linked with schizophrenia and psychostimulant addiction [24]. Postmortem analyses revealed an approximately 6-fold increase of D4R in the putamen of schizophrenic patients [20]. Furthermore, D4R KO mice exhibit decreased locomotor activity in response to a novel environment [25], and D4R-selective antagonist L-745,870 suppresses locomotor activity in rats [26], suggesting that D4R-mediated cellular signaling positively regulates novelty-induced locomotor activity. In light of the high comorbidity between psychosis and drug addiction [27], it is intriguing that D4R KO mice show enhanced locomotor responses to ethanol, cocaine and METH with increased dopamine synthesis [28]. However, L-745,870 attenuates nicotine reinstatement and blocks morphine withdrawal symptoms precipitated by naloxone [29]. These lines of evidence imply that D4R may differentially regulate the distinct properties and stages of drug addiction elicited by the dysregulation of various neurotransmitter systems. Yet, the precise role(s) of D4R in drug addiction and/or psychosis remain largely undefined [23,30].
To understand the complex role(s) of D4R in drug addiction and/or psychosis encoded by hyperdopaminergia, we tested the effects of D4R pharmacological manipulations on addiction-like and/or psychotic-like behaviors induced by repetitive METH treatment (behavioral sensitization) and genetic KD of DAT (spontaneous hyperactivity) in mice. Outbred ICR and inbred B6 mouse strains were used to assess potential strain differences in drug response [31]. Clz, but not D4R-selective antagonist L-745,870, effectively suppressed METH behavioral activation, as well as the initiation and expression of METH sensitization. Similar differential therapeutic efficacies of Clz and L-745,870 were observed in mitigating DAT KD spontaneous hyperactivity in a novel open field environment. Therefore, our results suggest that D4R signaling does not participate in hyperdopaminergia-induced behavioral sensitization, and blockade of D4R is likely dispensable in the therapeutic effects of Clz.
Section snippets
Animals
Six- to nine-week-old male Bltw:CD1 (ICR) (BioLasco Taiwan Co. Ltd), C57BL/6JNarl (B6) (National Laboratory Animal Center, Academia Sinica, Taiwan) and dopamine transporter knockdown (DAT KD) mice (gift from Dr. Xiaoxi Zhuang, University of Chicago, USA; DAT expression level is 10 % of wild-type (WT) mice [32]) were used in the study. DAT KD mice were maintained on the B6 background. Mice were group-housed in a room temperature (25 °C), 50–70 % humidity and 12 h light-dark cycle (lights on at
Clz treatment alleviates METH locomotor sensitization and DAT KD spontaneous hyperactivity
To assess the effects of the atypical antipsychotic Clz on METH behavioral sensitization, individual groups of ICR mice were given Clz (1, 2.5, or 5 mg/kg) 10 min before daily SA or METH (2 mg/kg) injection for 7 consecutive days and tested for locomotor activity on treatment days 1 (Fig. 2A) and 7 (Fig. 2B). Consistent with a previous study showing that systemic injection of Clz up to 10 mg/kg does not produce sedation [19], the basal activity of Clz and SA co-treated mice was unaltered
Discussion
In the present study, we demonstrate that Clz effectively suppressed the activation and initiation of METH sensitization. Clz treatment during sensitization initiation further suppressed sensitization expression upon METH challenge during the drug withdrawal stage in both ICR and B6 mice. Furthermore, Clz treatment alleviated the expression of DAT KD hyperactivity. In contrast, the D4R-selective antagonist L-745,870 was ineffective at alleviating the addiction-like and/or psychotic-like
Conclusions
Our experiments suggest a minor or non-existent role for D4R in regulating hyperdopaminergic behaviors induced by METH sensitization or DAT KD. Moreover, the development of tolerance for Clz dosages within the therapeutic window should be carefully considered when designing treatment programs for drug addiction and comorbid psychotic symptoms. Based on the lack of effect by D4R-selective antagonist in our behavioral experiments, we speculate that the therapeutic effects of Clz on drug seeking
Author statement
I-Mei Liao designed, performed the experiments, and analyzed the data. Jin-Chung Chen conceptualized, supervised, and funded the study. Both authors wrote, reviewed, and edited the manuscript.
Funding
The work was supported by Chang Gung Memorial Hospital [CMRPD1D0293]; Chang Gung University Healthy Ageing Research Center [EMRPD1F0251]; and the Ministry of Science and Technology [MOST104-2320-B-182-009], Taiwan.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgments
We thank Drs. Ching-Ping Tseng, Hui-Ju Tsai and Hsing-Ying Lee (Chang Gung University, Taiwan) for discussions and advice on the unpublished dot blot experiments of this study, and Dr. Marcus Calkins (Academia Sinica, Taiwan) for English editing of the manuscript.
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