Investigation On The Behavioral Effects And Related Neuro-Electrochemistry Of Potential Medications For The Treatment Of Substance Use Disorders.

National Institute On Drug Abuse

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Abstract

We have investigated the roles of dopamine (DA) D3 receptors (D3R) in the rewarding effects of cocaine and opioids, which actions at the cellular and neural circuit mechanisms are still unclear. We have employed Cre-LoxP techniques to selectively delete D3R from presynaptic dopamine neurons or postsynaptic dopamine D1 receptor (D1R)-expressing neurons in male and female mice. We utilized RNAscope in situ hybridization, immunohistochemistry, real-time polymerase chain reaction, voltammetry, optogenetics, microdialysis, and behavioral assays to functionally characterize the roles of presynaptic versus postsynaptic D3R in cocaine and opioid actions. Our results revealed D3R expression in about 25% of midbrain dopamine neurons and about 70% of D1R expressing neurons in the nucleus accumbens. While dopamine D2 receptors (D2R) were expressed in about 80% dopamine neurons, we found no D2R and D3R colocalization among these cells. Selective deletion of D3R from dopamine neurons increased exploratory behavior in novel environments and enhanced pulse-evoked nucleus accumbens dopamine release. Conversely, deletion of D3R from D1R-expressing neurons attenuated locomotor responses to D1-like and D2-like agonists. Strikingly, deletion of D3R from either cell type reduced oxycodone self-administration and oxycodone-enhanced brain-stimulation reward. In contrast, neither of these D3R deletions impacted cocaine self-administration, cocaine-enhanced brain-stimulation reward, or cocaine-induced hyperlocomotion. Furthermore, D3R knockout in dopamine neurons reduced oxycodone-induced hyperactivity and analgesia, while deletion from D1R-expressing neurons potentiated opioid-induced hyperactivity without affecting analgesia. Our results suggest different presynaptic versus postsynaptic D3R function in the mesolimbic dopamine system. D2R and D3R are expressed in different populations of midbrain dopamine neurons, regulating dopamine release. Mesolimbic D3R are critically involved in the actions of opioids but not cocaine. The extracellular synaptic levels of DA are regulated by several mechanisms, including metabolic pathways and neuronal membrane reuptake. Dopamine reuptake is provided by a protein, the DA transporter (DAT), which is the main pharmacologic target of addictive psychostimulants like cocaine and also of prescribed medications like modafinil (MOD), which has shown little, if any, propensity for misuse in clinical or preclinical studies. We have explored the neurochemical and behavioral actions of MOD to better characterize its atypical psychostimulant profile. We found that MOD had a lower potency and efficacy than cocaine in stimulating nucleus accumbens shell (NAS) and core (NAC) DA levels. Our results indicate that MOD shares mechanisms with cocaine and methylphenidate but has a unique pharmacological profile that includes facilitation of electrotonic coupling and lower potential for misuse, which may be exploited in future therapeutic drug design for cocaine use disorder. Though MOD might prove useful as a treatment for specific addicted populations (e.g. heavy crack-cocaine users, or cocaine and methamphetamine addicts without alcohol use disorder comorbidity), broader effective medications for psychostimulant use disorders are still an unmet medical need. To this end, several newly synthesized analogs of MOD, for example JJC8-091 and JJC8-088, have been tested in our preclinical models as potential medications for methamphetamine (METH) and cocaine use disorder. JJC8-091 and JJC8-088, were assessed alone and in combination with cocaine to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088, but not JJC8-091, produced cocaine-like effects, increasing extracellular DA concentrations in the NAS with greater efficacy and more potently than JJC8-091. In contrast, JJC8-091 was not self-administered and was effective in blocking cocaine-induced reinstatement to drug seeking. Computational models predicted that while JJC8-088 prefers or stabilizes an outward facing conformation of DAT, like cocaine, JJC8-091 stabilizes DAT towards a more occluded conformation. Further, we found that cocaine and cocaine-like typical psychostimulants elicited changes in DA dynamics distinct from those elicited by atypical DAT blockers, as measured via voltammetry procedures. When given in combination, typical DUIs enhance the stimulatory effects of cocaine on evoked DA release while atypical DUIs blunt them. Pretreatments with an inhibitor of CaMKII, a kinase that interacts with DAT and that regulates synapsin phosphorylation and mobilization of reserve pools of DA vesicles, blunted the effects of cocaine on evoked DA release, suggesting a role for CaMKII in modulating the effects of cocaine on evoked DA release without affecting cocaine inhibition of DA reuptake. This effect is related to a specific DAT conformation stabilized by cocaine. Moreover, atypical DUIs, which prefer a distinct DAT conformation, blunt cocaine neurochemical and behavioral effects, indicating a unique mechanism underlying their potential as medications for treating psychostimulant use disorder. We have extended our studies to investigate the role of sex as a biological factor in the neurochemical effects of typical and atypical DAT blockers. We found that cocaine slowed DA clearance in both male and female mice but produced more robust increases in evoked NAS DA in female mice. R-modafinil produced mild increases in evoked NAS DA and slowed DA clearance across the sexes. The modafinil analog, JJC8-088, a typical DAT inhibitor, produced increases in evoked NAS DA in female and male mice. Finally, JJC8-091, an atypical DAT inhibitor, produced limited increases in evoked NAS DA and slowed DA clearance in both sexes. In this work we begin to tease out how sex differences may alter the effects of DAT targeting drugs and highlight how this may help focus research toward effective treatment options for psychostimulant use disorder. Previous studies have shown that dual inhibitors of DAT and sigma receptors, for example rimcazole, significantly reduce the behavioral/reinforcing effects of cocaine, which have been associated with stimulation of extracellular DA levels resulting from DAT inhibition. These rimcazole effects resemble those of some atypical DAT inhibitors, like JJC8-091 that, interestingly, has nanomolar affinity for sigma receptors. Thus, we have investigated the effects of dual inhibitors of DAT and sigma receptors in combination with cocaine on NAS DA dynamics in naïve male Sprague Dawley rats. In microdialysis studies, administration of rimcazole or its structural analog SH 3-24 significantly reduced NAS DA efflux stimulated by increasing doses of cocaine. Using the same experimental conditions, in FSCV tests we show that rimcazole pretreatments attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. Under the same conditions, JJC8-091, a modafinil analog and dual inhibitor of DAT and sigma receptors, similarly attenuated cocaine-induced stimulation of evoked NAS DA release but produced no additional effect on DA clearance rate. As a proof of concept, in a recently published study, we have tested a compound, CM699, synthesized to obtain dual inhibition of sigma receptors and DAT. CM699 showed affinities of 14.1 and 311 nM at sigma-1 receptors and DAT, respectively. In our microdialysis study in rats, CM699 after intraperitoneal administration significantly reduced the stimulatory effects of cocaine on extracellular NAS DA levels. CM699 also insurmountably antagonized cocaine self-administration behavior, indicating efficacy as a cocaine antagonist in vivo. When given alone, CM699 was not self-administered nor had significant effects on NAS DA levels, suggesting minimal if any abuse potential. Further, in a biochemical assay designed to probe the conformation of DAT, (+)-pentazocine potentiated cocaine-induced cysteine accessibility of DAT transmembrane domain 6a, suggesting a shift in conformational equilibrium of DAT towards outward-facing, whereas CM699 blocked this effect. These results provide preclinical proof of concept for dual sigma receptors/DAT inhibition as a novel DAT-conformational approach for the development of medications to treat SUD. Taken together, these results provide the neurochemical groundwork towards understanding actions of dual inhibitors of DAT and sigma receptors on DA dynamics that likely mediate the behavioral effects of psychostimulants like cocaine. Collectively, these data reveal underlying molecular mechanisms at DAT that may be leveraged to rationally optimize leads for the treatment of psychostimulant use disorder and other substance use disorders.