Chemogenetic Manipulation of Dopamine Neurons Dictates Cocaine Potency at Distal Dopamine Transporters.

The reinforcing efficacy of cocaine is largely determined by its capacity to inhibit the dopamine transporter (DAT), and emerging evidence suggests that differences in cocaine potency are linked to several symptoms of cocaine use disorder. Despite this evidence, the neural processes that govern cocaine potency in vivo remain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Using ex vivo fast scan cyclic voltammetry, pharmacological probes of the DAT, biochemical assessments of DAT membrane availability and phosphorylation, and cocaine self-administration, we tested the effects of chemogenetic manipulations on cocaine potency at distal DATs in the nucleus accumbens as well as the behavioral economics of cocaine self-administration. We discovered that chemogenetic manipulation of dopamine neurons produced rapid, bidirectional modulation of cocaine potency at DATs in the nucleus accumbens. We then provided evidence that changes in cocaine potency are associated with alterations in DAT affinity for cocaine and demonstrated that this change in affinity coincides with DAT conformation biases and changes in DAT phosphorylation state. Finally, we showed that chemogenetic manipulation of dopamine neurons alters cocaine consumption in a manner consistent with changes in cocaine potency at distal DATs. Based on the spatial and temporal constraints inherent to our experimental design, we posit that changes in cocaine potency are driven by alterations in dopamine neuron activity. When considered together, these observations provide a novel mechanism through which GPCRs regulate cocaine's pharmacological and behavioral effects.SIGNIFICANCE STATEMENT Differences in the pharmacological effects of cocaine are believed to influence the development and progression of cocaine use disorder. However, the biological and physiological processes that determine sensitivity to cocaine remain unclear. In this work, we use a combination of chemogenetics, fast scan cyclic voltammetry, pharmacology, biochemistry, and cocaine self-administration with economic demand analysis to demonstrate a novel mechanism by which cocaine potency is determined in vivo These studies identify a novel process by which the pharmacodynamics of cocaine are derived in vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.

Chemogenetic Manipulations of Ventral Tegmental Area Dopamine Neurons Reveal Multifaceted Roles in Cocaine Abuse.

Ventral tegmental area (VTA) dopamine (DA) neurons perform diverse functions in motivation and cognition, but their precise roles in addiction-related behaviors are still debated. Here, we targeted VTA DA neurons for bidirectional chemogenetic modulation during specific tests of cocaine reinforcement, demand, and relapse-related behaviors in male rats, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of Gq signaling, but not Gs signaling, in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory Gi/o signaling in DA neurons blunted the reinforcing and priming effects of cocaine, reduced stress-potentiated reinstatement, and altered behavioral strategies for cocaine seeking and taking. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein-signaling cascades, and DA neuron efferent targets, highlighting their multifaceted roles in addiction.SIGNIFICANCE STATEMENT G-protein-coupled receptors are crucial modulators of ventral tegmental area (VTA) dopamine neuron activity, but how this metabotropic signaling impacts the complex roles of dopamine in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs (designer receptors exclusively activated by designer drugs) during a variety of cocaine-seeking behaviors, revealing nuanced, pathway-specific roles in cocaine reward, effortful seeking, and relapse-like behaviors. Gq and Gs stimulation activated dopamine neurons, but only Gq stimulation robustly enhanced cocaine seeking. Gi/o inhibitory signaling reduced some, but not all, types of cocaine seeking. Results show that VTA dopamine neurons modulate numerous distinct aspects of cocaine addiction- and relapse-related behaviors, and point to potential new approaches for intervening in these processes to treat addiction.

Pharmacologically increasing microtubule acetylation corrects stress-exacerbated effects of organophosphates on neurons.

Many veterans of the 1990-1991 Gulf War contracted Gulf War Illness (GWI), a multisymptom disease that primarily affects the nervous system. Here, we treated cultures of human or rat neurons with diisopropyl fluorophosphate (DFP), an analog of sarin, one of the organophosphate (OP) toxicants to which the military veterans were exposed. All observed cellular defects produced by DFP were exacerbated by pretreatment with corticosterone or cortisol, which, in rat and human neurons, respectively, serves in our experiments to mimic the physical stress endured by soldiers during the war. To best mimic the disease, DFP was used below the level needed to inhibit acetylcholinesterase. We observed a diminution in the ratio of acetylated to total tubulin that was correctable by treatment with tubacin, a drug that inhibits HDAC6, the tubulin deacetylase. The reduction in microtubule acetylation was coupled with deficits in microtubule dynamics, which were correctable by HDAC6 inhibition. Deficits in mitochondrial transport and dopamine release were also improved by tubacin. Thus, various negative effects of the toxicant/stress exposures were at least partially correctable by restoring microtubule acetylation to a more normal status. Such an approach may have therapeutic benefit for individuals suffering from GWI or other neurological disorders linked to OP exposure.

Diazepam Concurrently Increases the Frequency and Decreases the Amplitude of Transient Dopamine Release Events in the Nucleus Accumbens.

Benzodiazepines are commonly prescribed anxiolytics that pose abuse liability in susceptible individuals. Although it is well established that all drugs of abuse increase brain dopamine levels, and benzodiazepines are allosteric modulators of the GABAA receptor, it remains unclear how they alter dopamine release. Using in vivo fast-scan cyclic voltammetry, we measured diazepam-induced changes in the frequency and amplitude of transient dopamine release events. We found that diazepam concurrently increases the frequency and decreases the amplitude of transient dopamine release events in the awake and freely moving rat. The time course during which diazepam altered the frequency and amplitude of dopamine release events diverged, with the decreased amplitude effect being shorter lived than the increase in frequency, but both showing similar rates of onset. We conclude that diazepam increases the frequency of accumbal dopamine release events by disinhibiting dopamine neurons, but also decreases their amplitude. We speculate that the modest abuse liability of benzodiazepines is due to their ability to decrease the amplitude of dopamine release events in addition to increasing their frequency.

Hypocretin/orexin knock-out mice display disrupted behavioral and dopamine responses to cocaine.

The hypocretin/orexin (HCRT) system is implicated in reward and reinforcement processes through actions on the mesolimbic dopamine (DA) system. Here we provide evidence for the relationship between HCRT and DA in vivo in anesthetized and freely moving mice. The ability of cocaine to elicit reward-related behaviors in mice lacking the HCRT prepro-peptide (HCRT knock-out; KO) and wild-type controls was determined using conditioned place preference. Using a combination of microdialysis and in vivo fast scan cyclic voltammetry in anesthetized and freely moving mice, we investigated the underlying role of HCRT in the regulation of DA release and uptake. We show that, unlike wild-type mice, HCRT KO mice fail to develop characteristic conditioned place preference for cocaine. These mice also demonstrated reduced DA release and uptake under baseline conditions in both anesthetized and freely moving experiments. Further, diminished DA signaling in HCRT KO mice persists following administration of cocaine. These findings indicate that HCRT is essential for the expression of behaviors associated with the rewarding effects of cocaine, and suggest that HCRT regulation of reward and reinforcement may be related to disruptions to DA neurotransmission.

Chemogenetic Signaling in Space and Time: Considerations for Designing Neuroscience Experiments Using DREADDs.

The use of designer receptors exclusively activated by designer drugs (DREADDs) has led to significant advances in our understanding of the neural circuits that govern behavior. By allowing selective control over cellular activity and signaling, DREADDs have become an integral tool for defining the pathways and cellular phenotypes that regulate sleep, pain, motor activity, goal-directed behaviors, and a variety of other processes. In this review, we provide a brief overview of DREADDs and discuss notable discoveries in the neurosciences with an emphasis on circuit mechanisms. We then highlight methodological approaches to achieve pathway specific activation of DREADDs. Finally, we discuss spatial and temporal constraints of DREADDs signaling and how these features can be incorporated into experimental designs to precisely dissect circuits of interest.

Spinal Dopaminergic Mechanisms Regulating the Micturition Reflex in Male Rats with Complete Spinal Cord Injury.

Traumatic spinal cord injury (SCI) often causes micturition dysfunction. We recently discovered a low level of spinally-derived dopamine (DA) that regulates recovered bladder and sphincter reflexes in SCI female rats. Considering substantial sexual dimorphic features in the lower urinary tract, it is unknown if the DA-ergic mechanisms act in the male. Histological analysis showed a similar distribution of tyrosine hydroxylase (TH)+ neurons in the lower cord of male rats and the number increased following thoracic SCI. Subsequently, focal electrical stimulation in slices obtained from L6/S1 spinal segments of SCI rats elicited detectable DA release with fast scan cyclic voltammetry. Using bladder cystometrogram and external urethral sphincter (EUS) electromyography in SCI male rats, intravenous (i.v.) administration of SCH 23390, a D1-like receptor (DR1) antagonist, induced significantly increased tonic EUS activity and a trend of increased residual volume, whereas activation of these receptors with SKF 38393 did not influence the reflex. Meanwhile, blocking spinal D2-like receptors (DR2) with remoxipride had no effect but stimulating these receptors with quinpirole elicited EUS bursting to increase voiding volume. Further, intrathecal delivery of SCH 23390 and quinpirole resulted in similar responses to those with i.v. delivery, respectively, which indicates the central action regardless of delivery route. In addition, metabolic cage assays showed that quinpirole increased the voiding frequency and total voiding volume in spontaneous micturition. Collectively, spinal DA-ergic machinery regulates recovered micturition reflex following SCI in male rats; spinal DR1 tonically suppress tonic EUS activity to enable voiding and activation of DR2 facilitates voiding.

Accelerated development of cocaine-associated dopamine transients and cocaine use vulnerability following traumatic stress.

Post-traumatic stress disorder and cocaine use disorder are highly co-morbid psychiatric conditions. The onset of post-traumatic stress disorder generally occurs prior to the development of cocaine use disorder, and thus it appears that the development of post-traumatic stress disorder drives cocaine use vulnerability. We recently characterized a rat model of post-traumatic stress disorder with segregation of rats as susceptible and resilient based on anxiety-like behavior in the elevated plus maze and context avoidance. We paired this model with in vivo fast scan cyclic voltammetry in freely moving rats to test for differences in dopamine signaling in the nucleus accumbens core at baseline, in response to a single dose of cocaine, and in response to cocaine-paired cues. Further, we examined differences in the acquisition of cocaine self-administration across groups. Results indicate that susceptibility to traumatic stress is associated with alterations in phasic dopamine signaling architecture that increase the rate at which dopamine signals entrain to cocaine-associated cues and increase the magnitude of persistent cue-evoked dopamine signals following training. These changes in phasic dopamine signaling correspond with increases in the rate at which susceptible rats develop excessive cocaine-taking behavior. Together, our studies demonstrate that susceptibility to traumatic stress is associated with a cocaine use-vulnerable phenotype and suggests that differences in phasic dopamine signaling architecture may contribute to the process by which this vulnerability occurs.

Hypocretin receptor 1 involvement in cocaine-associated behavior: Therapeutic potential and novel mechanistic insights.

Since its discovery in 1998, the hypocretin/orexin system has been identified as a critical modulator of behavior. Through interactions with dopamine neurons of the ventral tegmental area, this system is poised to regulate motivation for drug rewards by impacting dopamine neurotransmission in target structures including the nucleus accumbens. Across numerous experiments, we and others have identified a critical influence of hypocretin receptor 1 in mediating the behavioral and physiological effects of cocaine which positions this receptor as a potential target for the treatment of cocaine addiction. Here we discuss evidence for hypocretin receptor 1 involvement in driving cocaine-associated behavior and how hypocretin receptor 1 in the ventral tegmental area are critical for supporting dopamine neuron activity and dopamine neurotransmission. We then present new data supporting the novel hypothesis that in addition to exerting acute actions on dopamine systems, pharmacological hypocretin manipulations also produce lasting adaptations to dopamine terminals that impact sensitivity to cocaine, and ultimately, future behavior.

Local GABAA Receptor-Mediated Suppression of Dopamine Release within the Nucleus Accumbens.

Benzodiazepines make up a class of psychoactive drugs that act as allosteric co-activators of the inhibitory GABAA receptor. These drugs are useful for the treatment of several psychiatric disorders but also hold considerable abuse liability. Despite the common use and misuse of benzodiazepines, the mechanisms through which these drugs exert their reinforcing effects remain incompletely understood. Transient phasic increases in dopamine levels are believed to play an important role in defining the reinforcing properties of drugs of abuse, and we recently demonstrated that systemic administration of benzodiazepines increased the frequency of these events but concomitantly reduced their amplitude. This observation provides insight into the pharmacological effects of benzodiazepines on dopamine signaling, but the processes through which benzodiazepines drive changes in phasic dopamine signals remain unclear. In these studies, we investigated the mechanisms through which benzodiazepines may reduce the phasic dopamine transient amplitude. We tested the effect of the benzodiazepine diazepam and the GABAA agonist muscimol on evoked dopamine release from nucleus accumbens brain slices using fast scan cyclic voltammetry. We found that both diazepam and muscimol reduce dopamine release and that reductions in dopamine release following GABAA receptor activation can be blocked by co-application of a GABAB receptor antagonist. These results suggest that activation of GABAA receptors in the nucleus accumbens decreases dopamine release by disinhibition of local GABA signaling and subsequent activation of GABAB receptors. Overall, this work provides a putative mechanism through which benzodiazepines reduce the amplitude of phasic dopamine release in vivo.

Selective activation of Dopamine D3 receptors and norepinephrine transporter blockade enhances sustained attention.

Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of ∼500 nM and behaves as a NET substrate. Systemic dosing of SK609 (4 mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4 mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05 mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25 mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8 mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.

Tissue engineered nigrostriatal pathway for treatment of Parkinson's disease.

The classic motor deficits of Parkinson's disease are caused by degeneration of dopaminergic neurons in the substantia nigra pars compacta, resulting in the loss of their long-distance axonal projections that modulate the striatum. Current treatments only minimize the symptoms of this disconnection as there is no approach capable of replacing the nigrostriatal pathway. We are applying microtissue engineering techniques to create living, implantable constructs that mimic the architecture and function of the nigrostriatal pathway. These constructs consist of dopaminergic neurons with long axonal tracts encased within hydrogel microcolumns. Microcolumns were seeded with dopaminergic neuronal aggregates, while lumen extracellular matrix, growth factors, and end targets were varied to optimize cytoarchitecture. We found a 10-fold increase in axonal outgrowth from aggregates versus dissociated neurons, resulting in remarkable axonal lengths of over 6 mm by 14 days and 9 mm by 28 days in vitro. Axonal extension was also dependent upon lumen extracellular matrix, but did not depend on growth factor enrichment or neuronal end target presence. Evoked dopamine release was measured via fast scan cyclic voltammetry and synapse formation with striatal neurons was observed in vitro. Constructs were microinjected to span the nigrostriatal pathway in rats, revealing survival of implanted neurons while maintaining their axonal projections within the microcolumn. Lastly, these constructs were generated with dopaminergic neurons differentiated from human embryonic stem cells. This strategy may improve Parkinson's disease treatment by simultaneously replacing lost dopaminergic neurons in the substantia nigra and reconstructing their long-projecting axonal tracts to the striatum.

L-Tyrosine availability affects basal and stimulated catecholamine indices in prefrontal cortex and striatum of the rat.

We previously found that L-tyrosine (L-TYR) but not D-TYR administered by reverse dialysis elevated catecholamine synthesis in vivo in medial prefrontal cortex (MPFC) and striatum of the rat (Brodnik et al., 2012). We now report L-TYR effects on extracellular levels of catecholamines and their metabolites. In MPFC, reverse dialysis of L-TYR elevated in vivo levels of dihydroxyphenylacetic acid (DOPAC) (L-TYR 250-1000 µM), homovanillic acid (HVA) (L-TYR 1000 µM) and 3-methoxy-4-hydroxyphenylglycol (MHPG) (L-TYR 500-1000 µM). In striatum L-TYR 250 µM elevated DOPAC. We also examined L-TYR effects on extracellular dopamine (DA) and norepinephrine (NE) levels during two 30 min pulses (P2 and P1) of K+ (37.5 mM) separated by t = 2.0 h. L-TYR significantly elevated the ratio P2/P1 for DA (L-TYR 125 µM) and NE (L-TYR 125-250 µM) in MPFC but lowered P2/P1 for DA (L-TYR 250 µM) in striatum. Finally, we measured DA levels in brain slices using ex-vivo voltammetry. Perfusion with L-TYR (12.5-50 µM) dose-dependently elevated stimulated DA levels in striatum. In all the above studies, D-TYR had no effect. We conclude that acute increases within the physiological range of L-TYR levels can increase catecholamine metabolism and efflux in MPFC and striatum. Chronically, such repeated increases in L-TYR availability could induce adaptive changes in catecholamine transmission while amplifying the metabolic cost of catecholamine synthesis and degradation. This has implications for neuropsychiatric conditions in which neurotoxicity and/or disordered L-TYR transport have been implicated.

Reinforcing Doses of Intravenous Cocaine Produce Only Modest Dopamine Uptake Inhibition.

The reinforcing efficacy of cocaine is thought to stem from inhibition of the dopamine transporter (DAT) and subsequent increases in extracellular dopamine concentrations in the brain. In humans, this hypothesis has generally been supported by positron emission tomography imaging studies where the percent of DATs occupied by cocaine is used as a measure of cocaine activity in the brain. Interpretation of these studies, however, often relies on the assumption that measures of DAT occupancy directly correspond with functional DAT blockade. In the current studies, we used in vivo and in vitro fast scan cyclic voltammetry in mice to measure dopamine uptake inhibition following varying doses of cocaine as well as two high affinity DAT inhibitors. We then compared dopamine clearance rates following these drug treatments to dopamine clearance obtained from DAT knockout mice as a proxy for complete DAT blockade. We found that administration of abused doses of cocaine resulted in approximately 2% of maximal DAT blockade. Overall, our data indicate that abused doses of cocaine produce a relatively modest degree of DA uptake inhibition, and suggest that the relationship between DAT occupancy and functional blockade of the DAT is more complex than originally posited.

Susceptibility to traumatic stress sensitizes the dopaminergic response to cocaine and increases motivation for cocaine.

Patients with post-traumatic stress disorder have a heightened vulnerability to developing substance use disorders; however, the biological underpinnings of this vulnerability remain unresolved. We used the predator odor stress model of post-traumatic stress disorder with segregation of subjects as susceptible or resilient based on elevated plus maze behavior and context avoidance. We then determined behavioral and neurochemical differences across susceptible, resilient, and control populations using a panel of behavioral and neurochemical assays. Susceptible subjects showed a significant increase in the motoric and dopaminergic effects of cocaine, and this corresponded with heightened motivation to self-administer cocaine. Resilient subjects did not show differences in the motoric effects of cocaine, in dopamine signaling in vivo, or in any measure of cocaine self-administration. Nonetheless, we found that these animals displayed elevations in both the dopamine release-promoting effects of cocaine and dopamine autoreceptor sensitivity ex vivo. Our results suggest that the experience of traumatic stress may produce alterations in dopamine systems that drive elevations in cocaine self-administration behavior in susceptible subjects, but may also produce both active and passive forms of resilience that function to prevent gross changes in cocaine's reinforcing efficacy in resilient subjects.

Dopamine uptake dynamics are preserved under isoflurane anesthesia.

Fast scan cyclic voltammetry is commonly used for measuring the kinetics of dopamine release and uptake. For experiments using an anesthetized preparation, urethane is preferentially used because it does not alter dopamine uptake kinetics compared to freely moving animals. Unfortunately, urethane is highly toxic, can induce premature death during experiments, and cannot be used for recovery surgeries. Isoflurane is an alternative anesthetic that is less toxic than urethane, produces a stable level of anesthesia over extended periods, and is often used for recovery surgeries. Despite these benefits, the effects of isoflurane on dopamine release and uptake have not been directly characterized. In the present studies, we assessed the utility of isoflurane for voltammetry experiments by testing dopamine signaling parameters under baseline conditions, after treatment with the dopamine uptake inhibitor cocaine, and after exposure to increasing concentrations of isoflurane. Our results indicate that surgical levels of isoflurane do not significantly alter terminal mechanisms of dopamine release and uptake over prolonged periods of time. Consequently, we propose that isoflurane is an acceptable anesthetic for voltammetry experiments, which in turn permits the design of studies in which dopamine signaling is examined under anesthesia prior to recovery and subsequent experimentation in the same animals.

Hypocretin receptor 1 blockade preferentially reduces high effort responding for cocaine without promoting sleep.

Recent evidence suggests that blockade of the hypocretin receptor 1 may act as a useful pharmacotherapy for cocaine abuse. Here we investigated the extent to which various doses of a hypocretin receptor 1 antagonist, SB-334867, affect cocaine self-administration at varying doses of cocaine and across a range of effort requirements, and tested if these SB-334867 doses produce sedative effects. First, we trained animals to self-administer one of three doses of cocaine on a progressive ratio schedule, and then tested the effects of three doses of SB-334867. Responding for cocaine was then analyzed to segregate features of relatively high and low effort requirements across the progressive ratio session. In another set of experiments, we tested potential sleep-promoting effects of the same doses of SB-334867. Our data indicate that blockade of hypocretin receptor 1 preferentially reduces high effort responding for cocaine at levels that do not promote sedation.