Chronic administration of a norepinephrine antagonist prevents and partially reverses escalation of cocaine self-administration.

Anxiety is a critical component of the development and maintenance of drug addiction; however, anti-anxiety medications such as benzodiazepines and beta-blockers (ß-adrenergic receptor antagonists) are not used for the treatment of substance use disorder, except for the management of acute withdrawal syndrome. Preclinical studies have shown that beta-blockers may reduce stress-induced relapse; however, the effect of beta blockers on the escalation and maintenance of drug intake has not been tested. To address this issue, we chronically administered the ß-adrenergic receptor antagonist propranolol during the escalation or maintenance of cocaine intake in a model of extended access (6 h) to cocaine self-administration (0.5 mg/kg). The behavioural specificity of propranolol was tested using a non-drug reward (saccharin). Daily administration of propranolol (15 mg/kg) prevented the development of escalation of cocaine self-administration and partially reversed self-administration after the establishment of escalation of intake. Moreover, propranolol dose-dependently decreased the motivation for cocaine tested under a progressive ratio schedule of reinforcement during the development of escalation and after maintenance. Finally, propranolol administration had no effect on the escalation and maintenance of saccharin self-administration. These results demonstrate that chronic treatment with propranolol provides therapeutic efficacy in reducing cocaine self-administration during the development and after the establishment of escalation of cocaine self-administration in an animal model relevant to cocaine use disorder. These results suggest that beta blockers should be further investigated as a target for medication development for the treatment of cocaine use disorder.

Morphine Exposure Reduces Nicotine-Induced Upregulation of Nicotinic Receptors and Decreases Volitional Nicotine Intake in a Mouse Model.

INTRODUCTION: Nicotine addiction remains a primary health concern as tobacco smoking remains the number one cause of preventable death in America. At the same time, America is still facing the threat of the opioid epidemic. While the prevalence of smoking combustible cigarettes or electronic nicotine delivery systems in the United States varies between 12% and 35%, the smoking rates among the opioid use dependent (OUD) population is 74%-97%. We examined changes in brain reward mechanisms in which co-use of nicotine and opioids may result in enhanced reward and reinforcement. AIMS AND METHODS: Adult male and female α4-mCherryα6-GFP mice (C57BL/6J) were used in conditioned place preference (CPP) and microscopy assays to examine reward-related behavior and nicotinic acetylcholine receptor (nAChR) upregulation following treatments with saline, nicotine, morphine, or nicotine plus morphine. Following this, separate mice were trained in e-Vape self-administration assays to examine morphine's impact on nicotine reinforcement. RESULTS: We observed that nicotine and morphine coexposure in a CPP assay did not produce enhanced reward-related behavior when compared with nicotine or morphine alone. In parallel we observed coexposure reduced nicotine-induced upregulation of nAChRs on ventral tegmental area dopamine and GABA neurons. Additionally, we observed that concurrent morphine exposure reduced nicotine (plus menthol) vapor self-administration in male and female mice. CONCLUSIONS: While nicotine use is high among OUD individuals, our CPP assays suggest coexposure not only fails to enhance reward-related behavior but also reduces nicotine-induced changes in ventral tegmental area neurobiology. Our self-administration assays suggest that morphine exposure during nicotine acquisition reduces nicotine reinforcement-related behavior. IMPLICATIONS: While some may postulate that the co-use of opioids and nicotine may be driven by reward-related mechanisms, our data indicate that opioid exposure may hinder nicotine intake due to reduced upregulation of nAChRs critical for nicotine reward and reinforcement. Thus, the high co-use in OUD individuals may be a result of other mechanisms and this warrants further investigations into nicotine and opioid co-use.

Upregulation of nAChRs and Changes in Excitability on VTA Dopamine and GABA Neurons Correlates to Changes in Nicotine-Reward-Related Behavior.

Previous reports indicate that nicotine reward is mediated through α4ß2*, α6ß2*, and α4α6ß2* nicotinic acetylcholine receptors (nAChRs; * indicates that additional nAChR subunits may be present). Little is known about α4α6ß2* nAChR involvement in reward and reinforcement because of a lack of methods that allow the direct investigation of this particular nAChR subtype. Here, we use male and female mice that contain α4-mCherry and α6-GFP nAChR subunits to show that concentrations of nicotine sufficient to evoke reward-related behavior robustly upregulate α4* and α4α6* nAChRs on midbrain dopamine (DA) and GABA neurons. Furthermore, the extent of α4α6* nAChR upregulation on ventral tegmental area (VTA) DA neurons aligns with the magnitude of nicotine reward-related behavior. We also show that the upregulation of nAChRs is accompanied by a functional change in firing frequency of both DA and GABA neurons in the VTA that is directly linked to nicotine reward-related behavior.

Why flavored vape products may be attractive: Green apple tobacco flavor elicits reward-related behavior, upregulates nAChRs on VTA dopamine neurons, and alters midbrain dopamine and GABA neuron function.

While nicotine is the primary addictive component in tobacco products, additional flavors have become a concern with the growing popularity of electronic nicotine delivery systems (ENDS). For this reason, we have begun to investigate popular tobacco and ENDS flavors. Here, we examined farnesol, a chemical flavorant used in green apple and fruit flavors in ENDS e-liquids, for its ability to produce reward-related behavior. Using male and female 3-6 month old C57BL/6 J mice and farnesol doses of 0.1, 1, and 10 mg/kg we identified a sex-dependent effect in a conditioned place preference assay: farnesol-alone produces reward-related behavior in only male mice. Despite this sex-dependent effect, 1.0 mg/kg farnesol enhances locomotor activity in both male and female mice. To understand farnesol's effect on reward-related behavior, we used whole-cell patch-clamp electrophysiology and confocal microscopy to investigate changes in putative dopamine and GABA neurons. For these approaches, we utilized genetically modified mice that contain fluorescent nicotinic acetylcholine receptors (nAChRs). Our electrophysiological assays with male mice revealed that farnesol treatment increases ventral tegmental area (VTA) dopamine neuron firing frequency and this may be due to a decrease in inhibitory tone from GABA neurons. Our microscopy assays revealed that farnesol treatment produces a significant upregulation of α6* nAChRs in male mice but not female mice. This was supported by an observed increase in α6* nAChR function in additional electrophysiology assays. These data provide evidence that popular tobacco flavorants may alter smoking-related behavior and promote the need to examine additional ENDS flavors.

Brain Region Specific Single-Molecule Fluorescence Imaging.

We developed an approach utilizing nanoscale vesicles extracted from brain regions combined with single molecule imaging to monitor how an animal's physiological condition regulates the dynamics of protein distributions in different brain regions. This method was used to determine the effect of nicotine on the distribution of receptor stoichiometry in different mouse brain regions. Nicotine-induced upregulation of α4ß2 nicotinic acetylcholine receptors (nAChRs) is associated with changes in their expression, trafficking, and stoichiometry. The structural assembly of nAChRs has been quantified in cell culture based systems using single molecule techniques. However, these methods are not capable of quantifying biomolecule assembly that takes place in a live animal. Both nicotine-induced upregulation and changes in nAChR stoichiometry differ across brain regions. Our single molecule approach revealed that nicotine acts differentially across brain regions to alter assembly in response to exposure and withdrawal.

Translating the atypical dopamine uptake inhibitor hypothesis toward therapeutics for treatment of psychostimulant use disorders.

Medication-assisted treatments are unavailable to patients with cocaine use disorders. Efforts to develop potential pharmacotherapies have led to the identification of a promising lead molecule, JJC8-091, that demonstrates a novel binding mode at the dopamine transporter (DAT). Here, JJC8-091 and a structural analogue, JJC8-088, were extensively and comparatively assessed to elucidate neurochemical correlates to their divergent behavioral profiles. Despite sharing significant structural similarity, JJC8-088 was more cocaine-like, increasing extracellular DA concentrations in the nucleus accumbens shell (NAS) efficaciously 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. Electrophysiology experiments confirmed that JJC8-091 was more effective than JJC8-088 at inhibiting cocaine-mediated enhancement of DA neurotransmission. Further, when VTA DA neurons in DAT-cre mice were optically stimulated, JJC8-088 produced a significant leftward shift in the stimulation-response curve, similar to cocaine, while JJC8-091 shifted the curve downward, suggesting attenuation of DA-mediated brain reward. Computational models predicted that JJC8-088 binds in an outward facing conformation of DAT, similar to cocaine. Conversely, JJC8-091 steers DAT towards a more occluded conformation. Collectively, these data reveal the underlying molecular mechanism at DAT that may be leveraged to rationally optimize leads for the treatment of cocaine use disorders, with JJC8-091 representing a compelling candidate for development.

Atypical dopamine transporter inhibitors R-modafinil and JHW 007 differentially affect D2 autoreceptor neurotransmission and the firing rate of midbrain dopamine neurons.

Abuse of psychostimulants like cocaine that inhibit dopamine (DA) reuptake through the dopamine transporter (DAT) represents a major public health issue, however FDA-approved pharmacotherapies have yet to be developed. Recently a class of ligands termed "atypical DAT inhibitors" has gained attention due to their range of effectiveness in increasing extracellular DA levels without demonstrating significant abuse liability. These compounds not only hold promise as therapeutic agents to treat stimulant use disorders but also as experimental tools to improve our understanding of DAT function. Here we used patch clamp electrophysiology in mouse brain slices to explore the effects of two atypical DAT inhibitors (R-modafinil and JHW 007) on the physiology of single DA neurons in the substantia nigra and ventral tegmental area. Despite their commonalities of being DAT inhibitors that lack cocaine-like behavioral profiles, these compounds exhibited surprisingly divergent cellular effects. Similar to cocaine, R-modafinil slowed DA neuron firing in a D2 receptor-dependent manner and rapidly enhanced the amplitude and duration of D2 receptor-mediated currents in the midbrain. In contrast, JHW 007 exhibited little effect on firing, slow DAT blockade, and an unexpected inhibition of D2 receptor-mediated currents that may be due to direct D2 receptor antagonism. Furthermore, pretreatment with JHW 007 blunted the cellular effects of cocaine, suggesting that it may be valuable to investigate similar DAT inhibitors as potential therapeutic agents. Further exploration of these and other atypical DAT inhibitors may reveal important cellular effects of compounds that will have potential as pharmacotherapies for treating cocaine use disorders.

Amphetamine augments vesicular dopamine release in the dorsal and ventral striatum through different mechanisms.

Amphetamine has well-established actions on pre-synaptic dopamine signaling, such as inhibiting uptake and degradation, activating synthesis, depleting vesicular stores, and promoting dopamine-transporter reversal and non-exocytotic release. Recent in vivo studies have identified an additional mechanism: augmenting vesicular release. In this study, we investigated how amphetamine elicits this effect. Our hypothesis was that amphetamine enhances vesicular dopamine release in dorsal and ventral striata by differentially targeting dopamine synthesis and degradation. In urethane-anesthetized rats, we employed voltammetry to monitor dopamine, electrical stimulation to deplete stores or assess vesicular release and uptake, and pharmacology to isolate degradation and synthesis. While amphetamine increased electrically evoked dopamine levels, inhibited uptake, and up-regulated vesicular release in both striatal sub-regions in controls, this psychostimulant elicited region-specific effects on evoked levels and vesicular release but not uptake in drug treatments. Evoked levels better correlated with vesicular release compared with uptake, supporting enhanced vesicular release as an important amphetamine mechanism. Taken together, these results suggested that amphetamine enhances vesicular release in the dorsal striatum by activating dopamine synthesis and inhibiting dopamine degradation, but targeting an alternative mechanism in the ventral striatum. Region-distinct activation of vesicular dopamine release highlights complex cellular actions of amphetamine and may have implications for its behavioral effects.