Lack of dopamine D4 receptor participation in mouse hyperdopaminergic locomotor response

Highlights

• Clozapine effectively suppresses methamphetamine sensitization and relapse.

• Chronic clozapine produces tolerance to methamphetamine sensitization suppression.

• Basal locomotor activity does not require dopamine D4 receptor signaling.

• Dopamine D4 receptor antagonism is dispensable for hyperdopaminergia pathogenesis.

Abstract

Chronic methamphetamine (METH) treatment induces behavioral sensitization in rodents. During this process, hyperactivation of the mesolimbic dopamine system plays a central role, and dopamine D2-like receptor-based antipsychotics are known to alleviate the behavioral hyperactivity. The atypical antipsychotic, clozapine (Clz), acts partially as a dopamine D4 receptor (D4R) antagonist and mitigates hyperdopaminergic drug addiction and/or comorbid psychotic symptoms; however, it remains unclear whether D4R blockade contributes to the therapeutic effects of Clz. Here, we evaluated the potential role of D4R in regulating hyperdopaminergia-induced behavioral hyperactivity in METH behavioral sensitization and dopamine transporter (DAT) knockdown (KD) mice. Clz or a D4R-selective antagonist, L-745,870, were co-administered to mice with daily METH in a METH sensitization model, and Clz or L-745,870 were administered alone in a DAT KD hyperactivity model. Locomotor activity and accumbal D4R expression were analyzed. Clz suppressed both the initiation and expression of METH behavioral sensitization, as well as DAT KD hyperactivity. However, repetitive Clz treatment induced tolerance to the suppression effect on METH sensitization initiation. In contrast, D4R inhibition by L-745,870 had no effect on METH sensitization or DAT KD hyperactivity. Accumbal D4R expression was similar between METH-sensitized mice with and without Clz co-treatment. In sum, our results suggest the mesolimbic D4R does not participate in behavioral sensitization encoded by hyperdopaminergia, a finding which likely extends to the therapeutic effects of Clz. Therefore, molecular targets other than D4R should be prioritized in the development of future therapeutics for treatment of hyperdopaminergia-dependent neuropsychiatric disorders.

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.