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Parkinson’s disease is a neurodegenerative disease affecting around 10 million people worldwide. The death of dopaminergic neurons in the substantia nigra and the axonal fibers that constitute the nigrostriatal pathway leads to a loss of dopamine in the striatum that causes the motor symptoms of this disease. Traditional treatments have focused on reducing symptoms, while therapies with human fetal or stem cell-derived neurons have centered on implanting these cells in the striatum to restore its innervation. An alternative approach is pathway reconstruction, which aims to rebuild the entire structure of neurons and axonal fibers of the nigrostriatal pathway in a way that matches its anatomy and physiology. This type of repair could be more capable of reestablishing the signaling mechanisms that ensure proper dopamine release in the striatum and regulation of other motor circuit regions in the brain. In this manuscript, we conduct a review of the literature related to pathway reconstruction as a treatment for Parkinson’s disease, delve into the limitations of these studies, and propose the requisite design criteria to achieve this goal at a human scale. We then present our tissue engineering-based platform to fabricate hydrogel-encased dopaminergic axon tracts in vitro for later implantation into the brain to replace and reconstruct the pathway. These tissue-engineered nigrostriatal pathways (TE-NSPs) can be characterized and optimized for cell number and phenotype, axon growth lengths and rates, and the capacity for synaptic connectivity and dopamine release. We then show original data of advances in creating these constructs matching clinical design criteria using human iPSC-derived dopaminergic neurons and a hyaluronic acid hydrogel. We conclude with a discussion of future steps that are needed to further optimize human-scale TE-NSPs and translate them into clinical products.

A major obstacle in treating opioid use disorder is the persistence of drug seeking or craving during periods of abstinence, which is believed to contribute to relapse. Dopamine transmission in the mesolimbic pathway is posited to contribute to opioid reinforcement, but the processes by which dopamine influences drug seeking have not been completely elucidated. To examine whether opioid seeking during abstinence is associated with alterations in dopamine transmission, female and male rats self-administered oxycodone under an intermittent access schedule of reinforcement. Following self-administration, rats underwent a forced abstinence period, and cue-induced seeking tests were conducted to assess oxycodone seeking. One day following the final seeking test, rats were sacrificed to perform ex vivo fast scan cyclic voltammetry and western blotting in the nucleus accumbens. Rats displayed reduced dopamine uptake rate on abstinence day 2 and abstinence day 15, compared to oxycodone-naïve rats. Further, on abstinence day 15, rats had reduced phosphorylation of the dopamine transporter. Additionally, local application of oxycodone to the nucleus accumbens reduced dopamine uptake in oxycodone-naïve rats and in rats during oxycodone abstinence, on abstinence day 2 and abstinence day 15. These observations suggest that abstinence from oxycodone results in dysfunctional dopamine transmission, which may contribute to sustained oxycodone seeking during abstinence.

Parkinson’s disease (PD) affects 1–2% of people over 65, causing significant morbidity across a progressive disease course. The classic PD motor deficits are caused by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in the loss of their long-distance axonal projections that modulate striatal output. While contemporary treatments temporarily alleviate symptoms of this disconnection, there is no approach able to replace the nigrostriatal pathway. We applied microtissue engineering techniques to create a living, implantable tissue-engineered nigrostriatal pathway (TE-NSP) that mimics the architecture and function of the native pathway. TE-NSPs comprise a discrete population of dopaminergic neurons extending long, bundled axonal tracts within the lumen of hydrogel micro-columns. Neurons were isolated from the ventral mesencephalon of transgenic rats selectively expressing the green fluorescent protein in dopaminergic neurons with subsequent fluorescent-activated cell sorting to enrich a population to 60% purity. The lumen extracellular matrix and growth factors were varied to optimize cytoarchitecture and neurite length, while immunocytochemistry and fast-scan cyclic voltammetry (FSCV) revealed that TE-NSP axons released dopamine and integrated with striatal neurons in vitro. Finally, TE-NSPs were implanted to span the nigrostriatal pathway in a rat PD model with a unilateral 6-hydroxydopamine SNpc lesion. Immunohistochemistry and FSCV established that transplanted TE-NSPs survived, maintained their axonal tract projections, extended dopaminergic neurites into host tissue, and released dopamine in the striatum. This work showed proof of concept that TE-NSPs can reconstruct the nigrostriatal pathway, providing motivation for future studies evaluating potential functional benefits and long-term durability of this strategy. This pathway reconstruction strategy may ultimately replace lost neuroarchitecture and alleviate the cause of motor symptoms for PD patients.

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 potencyin vivoremain unclear. In male rats, we used chemogenetics with intra-VTA microinfusions of the agonist clozapine-n-oxide to bidirectionally modulate dopamine neurons. Usingex vivofast 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 STATEMENTDifferences 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 determinedin vivo. These studies identify a novel process by which the pharmacodynamics of cocaine are derivedin vivo, and thus this work has widespread implications for understanding the mechanisms that regulate cocaine consumption across stages of addiction.

The dopamine transporter (DAT) has been implicated in a variety of arousal-related processes including the regulation of motor activity, learning, motivated behavior, psychostimulant abuse, and, more recently, sleep/wake state. We previously demonstrated that DAT uptake regulates fluctuations in extracellular dopamine (DA) in the striatum across the light/dark cycle with DA levels at their highest during the dark phase and lowest during the light phase. Despite this evidence, whether fluctuations in DA uptake across the light/dark cycle are associated with changes in sleep/wake has not been tested. To address this, we employed a combination of sleep/wake recordings, fast scan cyclic voltammetry, and western blotting to examine whether sleep/wake state and/or light/dark phase impact DA terminal neurotransmission in male rats. Further, we assessed whether variations in plasma membrane DAT levels and/or phosphorylation of the threonine 53 site on the DAT accounts for fluctuations in DA neurotransmission. Given the extensive evidence indicating that psychostimulants increase DA through interactions with the DAT, we also examined to what degree the effects of cocaine at inhibiting the DAT vary across sleep/wake state. Results demonstrated a significant association between individual sleep/wake states and DA terminal neurotransmission, with higher DA uptake rate, increased phosphorylation of the DAT, and enhanced cocaine potency observed after periods of sleep. These findings suggest that sleep/wake state influences DA neurotransmission in a manner that is likely to impact a host of DA-dependent processes including a variety of neuropsychiatric disorders.

A highly challenging question in neuroscience is to understand how aminergic neural circuits contribute to the planning and execution of behaviors, including the generation of olfactory memories. In this regard, electrophysiological techniques like Local Field Potential or imaging methods have been used to answer questions relevant to cell and circuit physiology in different animal models, such as the fly Drosophila melanogaster. However, these techniques do not provide information on the neurochemical identity of the circuits of interest. Different approaches including fast scan cyclic voltammetry, allow researchers to identify and quantify in a timely fashion the release of endogenous neuroactive molecules, but have been only used in in vitro Drosophila brain preparations. Here, we report a procedure to record for the first time the release of endogenous amines -dopamine, serotonin and octopamine- in adult fly brain in vivo, by fast scan cyclic voltammetry. As a proof of principle, we carried out recordings in the calyx region of the Mushroom Bodies, the brain area mainly associated to the generation of olfactory memories in flies. By using principal component regression in normalized training sets for in vivo recordings, we detect an increase in octopamine and serotonin levels in response to electric shock and olfactory cues respectively. This new approach allows the study of dynamic changes in amine neurotransmission that underlie complex behaviors in Drosophila and shed new light on the contribution of these amines to olfactory processing in this animal model.

Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine release and clearance throughout the brain, especially the striatum where dopamine terminals are abundant and signals are heavily regulated by release machinery and the dopamine transporter (DAT). Peak height measurement is perhaps the most common method for measuring dopamine release, but it is influenced by changes in clearance. Michaelis-Menten-based modeling has been a standard in measuring dopamine clearance, but it is problematic in that it requires experimenter fitted modeling subject to experimenter bias. This study presents the use of the first derivative (velocity) of evoked dopamine signals as an alternative approach for measuring and distinguishing dopamine release from clearance. Maximal upward velocity predicts reductions in dopamine peak height due to D

Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine (DA) release and clearance throughout the brain, including the ventral and dorsal striatum. Striatal DA terminals are abundant with signals heavily regulated by release machinery and the dopamine transporter (DAT). Peak height is a common method for measuring release but can be affected by changes in clearance. The Michaelis-Menten model has been a standard in measuring DA clearance, but requires experimenter fitted modeling subject to experimenter bias. The current study presents the use of the first derivative (velocity) of evoked DA signals as an alternative approach for measuring dopamine release and clearance and can be used to distinguish the two measures. Maximal upwards velocity predicts reductions in DA peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT numbers, predicted maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wildtype C57 (WT) and DAT knock out (DATKO) mice. In contrast, downward velocity was considerably reduced and exponential decay (tau) was increased in DATKO mice, supporting use of both measures for changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23 and PTT on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these measures for DA release and clearance.

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.

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 naive 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.

Parkinson’s disease (PD) affects 10 million patients worldwide, making it the second most prevalent neurodegenerative disease. Motor symptoms emerge from the loss of dopamine in the striatum after the death of dopaminergic neurons and the long-projecting axons of the nigrostriatal pathway. Current treatments, such as dopamine replacement, deep brain stimulation or cell therapies, disregard the loss of this pathway at the core of symptoms. We sought to address this by improving our tissue-engineered nigrostriatal pathway (TE-NSP) technology, which consists of a tubular hydrogel with a collagen/laminin core that encases an aggregate of dopaminergic neurons and their axons in a way that resembles the nigrostriatal pathway. These constructs can be implanted to replace the lost neurons and axons with fidelity to the pathway, and thus provide dopamine according to feedback from the host circuitry. While TE-NSPs have been traditionally fabricated with agarose, here we utilized a hyaluronic acid (HA) hydrogel to expand the functionality of the encasement and our control over its properties. Using rat ventral midbrain neurons, we found that TE-NSPs exhibited longer and faster neurite growth with HA relative to agarose, with no differences observed in electrically-evoked dopamine release. When transplanted, HA hydrogels reduced host neuron loss and inflammation around the implant compared to agarose, and the cells and axons within TE-NSPs survived and maintained their cytoarchitecture for at least 2 weeks.HighlightsWe fabricated engineered dopaminergic axons encased in a tubular hydrogel.We made hydrogels from methacrylated hyaluronic acid and compared them to agarose.Axons in HA hydrogels had longer and faster axon growth and displayed evoked dopamine release.HA hydrogels reduced the host inflammatory response and supported neuron and axon survival in vivo.This platform may be used to reconstruct the nigrostriatal pathway to treat Parkinson’s disease.

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 D(1)-like receptor (DR(1)) 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 D(2)-like receptors (DR(2)) 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 DR(1) tonically suppress tonic EUS activity to enable voiding and activation of DR(2) facilitates voiding.

A major theme of addiction research has focused on the neural substrates of individual differences in the risk for addiction; however, little is known about how vulnerable populations differ from those that are relatively protected. Here, we prospectively measured dopamine (DA) neurotransmission prior to cocaine exposure to predict the onset and course of cocaine use. Using in vivo voltammetry, we first generated baseline profiles of DA release and uptake in the dorsomedial striatum (DMS) and nucleus accumbens of drug-naive male rats prior to exposing them to cocaine using conditioned place preference (CPP) or operant self-administration. We found that the innate rate of DA uptake in the DMS strongly predicted motivation for cocaine and drug-primed reinstatement, but not CPP, responding when “price” was low, or extinction. We then assessed the impact of baseline variations in DA uptake on cocaine potency in the DMS using ex vivo voltammetry in naive rats and in rats with DA transporter (DAT) knockdown. DA uptake in the DMS of naive rats predicted the neurochemical response to cocaine, such that rats with innately faster rates of DA uptake demonstrated higher cocaine potency at the DAT and rats with DAT knockdown displayed reduced potency compared to controls. Together, these data demonstrate that inherent variability in DA uptake in the DMS predicts the behavioral response to cocaine, potentially by altering the apparent potency of cocaine.

D-amphetamine maintenance therapy shows promise as a treatment for people with cocaine addiction. Preclinical studies using Long Access (LgA) cocaine self-administration procedures suggest D-amphetamine may act by preventing tolerance to cocaine’s effects at the dopamine transporter (DAT). However, Intermittent Access (IntA) cocaine self-administration better reflects human patterns of use, is especially effective in promoting addiction-relevant behaviors, and instead of tolerance, produces psychomotor, incentive, and neural sensitization. We asked, therefore, how D-amphetamine maintenance during IntA influences cocaine use and cocaine’s potency at the DAT. Male rats self-administered cocaine intermittently (5 min ON, 25 min OFF x10; 5-h/session) for 14 sessions, with or without concomitant D-amphetamine maintenance therapy during these 14 sessions (5 mg/kg/day via s.c. osmotic minipump). We then assessed responding for cocaine under a progressive ratio schedule, responding under extinction and cocaine-primed reinstatement of drug seeking. We also assessed the ability of cocaine to inhibit dopamine uptake in the nucleus accumbens core using fast scan cyclic voltammetry ex vivo. IntA cocaine self-administration produced psychomotor (locomotor) sensitization, strong motivation to take and seek cocaine, and it increased cocaine’s potency at the DAT. D-amphetamine co-administration suppressed the psychomotor sensitization produced by IntA cocaine experience. After cessation of D-amphetamine treatment, the motivation to take and seek cocaine was also reduced, and sensitization of cocaine’s actions at the DAT was reversed. Thus, treatment with D-amphetamine might reduce cocaine use by preventing sensitization-related changes in cocaine potency at the DAT, consistent with an incentive-sensitization view of addiction.

Low-threshold spiking interneurons (LTSIs) in the dorsomedial striatum are potent modulators of goal-directed learning. Here, we uncover a novel function for LTSIs in locally and directly gating striatal dopamine, using in vitro fast scan cyclic voltammetry as well as in vivo GRAB-DA sensor imaging and pharmacology during operant learning. We demonstrate that LTSIs, acting via GABAB signaling, attenuate dopamine release, thereby serving as local coordinators of striatal plasticity.

Some clinical trials are investigating modafinil (Mod) as a treatment for attentional deficit and hyperactivity disorder (ADHD) in children and adolescents. Mod increases dopamine (DA) levels in the reward system by blocking dopamine transporter (DAT). Social interactions are rewarding behaviors and evidence reveals the importance of reward circuitry in social interactions. Chronic psychostimulant treatments alter DA neurotransmission and associated behaviors. The aim of this work was to evaluate the effects of chronic Mod treatment during preadolescence on social play behavior, locomotor activity, and DA in nucleus accumbens (NAc). Preadolescent male Sprague-Dawley rats were injected with Mod (75 mg/kg i.p.) or vehicle for 14 days (PND22 to PND35). After that, we measured social play behavior, content and DA release in NAc by HPLC coupled to electrochemical detection, protein levels of DA type 2 receptor (D2) by Western blot and DA kinetic by fast-scan cyclic voltammetry (FSCV) in NAc. Regarding social play, the total number of pinning events decreased in the Mod group compared with the vehicle. The K+-stimulated DA release in NAc was significantly lower in Mod-treated rats compared with vehicle group. Also, Mod increases locomotor activity at the first injection, but this effect is almost completely lost at day 14 of Mod treatment. Chronic Mod treatment during preadolescence in rats impairs dopaminergic neurotransmission in NAc and decreases the capacity of rats to perceive rewarding effects of social play. Importantly, as Mod is being evaluated to treat ADHD in children and adolescents, potential effects on social behavior should be considered since this kind of behavior in this particular stage is crucial for neurodevelopment.

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.

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.

Summary The dorsomedial striatum (DMS) is a central hub supporting goal-directed learning and motor performance. Recent evidence has revealed unexpected roles for local inhibitory GABAergic networks in modulating striatal output and behavior.1 The sparse low-threshold spiking interneuron subtype (LTSI), which exhibits robust reward-circumscribed population activity, is a bidirectional regulator of initial goal-directed learning.2 Striatal dopamine signaling is a central reward-related neuromodulatory system mediating goal-directed action and performance, serving as a teaching signal,3 facilitating synaptic plasticity,4 and invigorating motor behaviors.5 Given the dynamic modulation of LTSIs during goal-directed behavior, we hypothesized that they could provide a novel GABAergic mechanism of local striatal dopaminergic regulation to shape early learning. We provide anatomical evidence for close proximation of LTSI terminals and dopaminergic processes in striatum, suggesting that LTSIs directly control dopaminergic axon activity. Using in vitro fast scan cyclic voltammetry, we demonstrate that LTSIs directly attenuate optogenetically evoked dopamine via GABAB receptor signaling. In vivo, GRABDA dopamine sensor imaging shows that LTSIs strongly modulate striatal dopamine dynamics during operant learning, while pharmacological stabilization of dopamine via intra-striatal aripiprazole microinjection suppresses the effects of LTSI inhibition on learning. Together, these results uncover an unexpected function for LTSIs in gating striatal dopamine to facilitate goal-directed learning.

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.

The hypocretin receptor 1 (HCRTr1) is a critical participant in the regulation of motivated behavior. Previous observations demonstrate that acute pharmacological blockade of HCRTr1 disrupts dopamine (DA) signaling and the motivation for cocaine when delivered systemically or directly into the ventral tegmental area (VTA). To further examine the involvement of HCRTr1 in regulating reward and reinforcement processing, we employed an adeno-associated virus to express a short hairpin RNA designed to knock down HCRTr1. We injected virus into the VTA and examined the effects of HCRTr1 knockdown on cocaine self-administration and DA signaling in the nucleus accumbens (NAc) core. We determined that the viral approach was effective at reducing HCRTr1 expression without affecting the expression of hypocretin receptor 2 or DA-related mRNAs. We next examined the effects of HCRTr1 knockdown on cocaine self-administration, observing delayed acquisition under a fixed-ratio schedule and reduced motivation for cocaine under a progressive ratio schedule. These effects did not appear to be associated with alterations in sleep/wake activity. Using fast-scan cyclic voltammetry, we then examined whether HCRTr1 knockdown alters DA signaling dynamics in the NAc core. We observed reduced DA release and slower uptake rate as well as attenuated cocaine-induced DA uptake inhibition in rats with knockdown of HCRTr1. These observations indicate that HCRTr1 within the VTA influence the motivation for cocaine, likely via alterations in DA signaling in the NAc.

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.

Many veterans of the 1990–1991 Gulf War contracted Gulf War Illness, a multi-symptom disease that primarily affects the nervous system. Here we treated cultures of human or rat neurons with diisopropylfluorophosphate (DFP), an analog of sarin, one of the organophosphate 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 the 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 organophosphate exposure.

Background Increased locomotor activity in response to the same stimulus is an index of behavioral sensitization observed in preclinical models of drug addiction and compulsive behaviors. Repeated administration of quinpirole, a D2/D3 dopamine agonist, induces locomotor sensitization. This effect is potentiated and accelerated by co-administration of U69593, a kappa opioid receptor agonist. The mechanism underlying kappa opioid receptor potentiation of quinpirole-induced locomotor sensitization remains to be elucidated. Methods Immunofluorescence anatomical studies were undertaken in mice brain slices and rat presynaptic synaptosomes to reveal kappa opioid receptor and D2R pre- and postsynaptic colocalization in the nucleus accumbens. Tonic and phasic dopamine release in the nucleus accumbens of rats repeatedly treated with U69593 and quinpirole was assessed by microdialysis and fast scan cyclic voltammetry. Results Anatomical data show that kappa opioid receptor and D2R colocalize postsynaptically in medium spiny neurons of the nucleus accumbens and the highest presynaptic colocalization occurs on the same dopamine terminals. Significantly reduced dopamine levels were observed in quinpirole, and U69593-quinpirole treated rats, explaining sensitization of D2R. Presynaptic inhibition induced by kappa opioid receptor and D2R of electrically evoked dopamine release was faster in U69593-quinpirole compared with quinpirole-repeatedly treated rats. Conclusions Pre- and postsynaptic colocalization of kappa opioid receptor and D2R supports a role for kappa opioid receptor potentiating both the D2R inhibitory autoreceptor function and the inhibitory action of D2R on efferent medium spiny neurons. Kappa opioid receptor co-activation accelerates D2R sensitization by contributing to decrease dopamine release in the nucleus accumbens.

Orexins ('hypocretins') are peptides produced by neurons of the hypothalamus that project to structures implicated in reward and emotion processing. Converging evidence demonstrates functional roles of orexin signaling in arousal, sleep/wakefulness and motivated behaviors for natural and drug rewards. Suvorexant, a dual orexin receptor antagonist, recently received approval from the US Food and Drug Administration to treat insomnia. In Experiment 1, rats self-administered cocaine under a progressive-ratio schedule of reinforcement and the effects of suvorexant on motivation to self-administer cocaine were measured. In Experiment 2, the effects of suvorexant on cocaine reward were assessed by using a place conditioning paradigm, and 50-kHz ultrasonic vocalizations were also recorded to track changes in hedonic reactivity to cocaine. To rule out potentially confounding effects of suvorexant-induced somnolence, locomotor activity was also measured. In Experiment 3, the effects of suvorexant on cocaine-evoked elevations in ventral striatal dopamine were examined. Data reveal that suvorexant (i) reduced the number of cocaine infusions earned during progressive-ratio self-administration; (ii) attenuated initial positive hedonic reactivity to cocaine and prevented cocaine place preference; (iii) did not affect cocaine-induced hyperlocomotion and (iv) reduced cocaine-induced elevations in extracellular ventral striatal dopamine. The present study examined the therapeutic potential of suvorexant in rodent models of cocaine use disorder. These results contribute toward a growing literature supporting therapeutic roles of orexin receptor antagonists in treating substance use disorders.

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.

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.

Amphetamine derivatives have been used in a wide variety of pathologies because of their pharmacological properties as psychostimulants, entactogens, anorectics, and antidepressants. However, adverse cardiovascular effects (sympathomimetics) and substance abuse problems (psychotropic and hallucinogenic effects) have limited their use. 4-Methylthioamphetamine (MTA) is an amphetamine derivative that has shown to inhibit monoamine uptake and monoamine oxidase. However, the pharmacological characterization (neurochemical, behavioral, and safety) of its derivatives 4-ethylthioamphetamine (ETA) and 4-methylthio-phenil-2-butanamine (MT-But) have not been studied. In the current experiments, we show that ETA and MT-But do not increase locomotor activity and conditioned place preference with respect to MTA. At the neurochemical level, ETA and MT-But do not increase in vivo DA release in striatum, but ETA and MT-But affect the nucleus accumbens bioaccumulation of DA and DOPAC. Regarding cardiovascular effects, the administration of MTA and ETA increased the mean arterial pressure and only ETA significantly increases the heart rate. Our results show that the pharmacological and safety profiles of MTA are modulated by changing the methyl-thio group or the methyl group of the aminoethyl chain.

Hypocretin- (also known as orexin) containing neurons of the lateral hypothalamus project widely throughout the brain and are implicated in a range of behaviors via signaling at hypocretin receptor 1 (HCRTr1) and hypocretin receptor 2. These neurons send a moderate, but influential, projection to the dopaminergic ventral tegmental area which—as part of the mesolimbic dopamine system—is highly implicated in motivated behavior and substance abuse. In this chapter, we will explore the contribution of HCRTr1 to dopamine neurotransmission and its consequential importance to addiction-related processes. Following, we review evidence for HCRTr1 involvement in supporting drug-associated behavior across behavioral models of addiction. We then discuss HCRTr1 regulation of ventral tegmental dopamine neuron activity and its impact on dopamine neurotransmission in the nucleus accumbens, and address hypocretin-dopamine interactions and their effects in females. Finally, we propose that HCRTr1 regulation of dopamine release influences behavioral responses to drug-associated cues.

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.

Benzodiazepines make up a class of psychoactive drugs that act as allosteric co-activators of the inhibitory GABA

Epigenetic remodeling contributes to synaptic plasticity via modification of gene expression, which underlies cocaine-induced long-term memory. A prevailing hypothesis in drug addiction is that drugs of abuse rejuvenate developmental machinery to render reward circuitry highly plastic and thus engender drug memories to be highly stable. Identification and reversal of these pathological pathways are therefore critical for cocaine abuse treatment. Previous studies revealed an interesting finding in which the mRNA of histone lysine demethylase, KDM6B, is upregulated in the medial prefrontal cortex (mPFC) during early cocaine withdrawal. However, whether and how it contributes to drug-seeking behavior remain unknown. Here we used a conditioned place preference paradigm to investigate the potential role of KDM6B in drug-associated memory. We found that KDM6B protein levels selectively increased in the mPFC during cocaine withdrawal. Notably, systemic injection of KDM6B inhibitor, GSK-J4, disrupted both reconsolidation of cocaine-conditioned memory and cocaine-primed reinstatement, suggesting dual effects of KDM6B in cocaine reward memory. In addition, we found that NMDAR expression and function were both enhanced during early cocaine withdrawal in mPFC. Injection of GSK-J4 selectively reversed this cocaine-induced increase of NR2A expression and synaptic function, suggesting that mal-adaptation of cocaine-induced synaptic plasticity in mPFC largely underlies KDM6B-mediated cocaine-associated memory. Altogether, these data suggest that KDM6B plays an essential role in cocaine-associated memory, which mainly acts through enhancing cocaine-induced synaptic plasticity in the mPFC. Our findings revealed a novel role of KDM6B in cocaine-associated memory and inhibition of KDM6B is a potential strategy to alleviate drug-seeking behavior.

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, querying the roles of DA neuron inhibitory and excitatory G-protein signaling in these processes. Designer receptor stimulation of Gq-, 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 cocaine’s reinforcing and priming effects, reduced stress-potentiated reinstatement, and altered cue-induced cocaine seeking strategy, but not the motivational impact of cocaine cues per se. Results demonstrate that DA neurons play several distinct roles in cocaine seeking, depending on behavioral context, G-protein signaling, and DA neuron efferent target, highlighting their multifaceted roles in addiction.Significance StatementG-protein coupled receptors are crucial modulators of VTA dopamine neuron activity, but how metabotropic signaling impacts dopamine’s complex roles in reward and addiction is poorly understood. Here, we bidirectionally modulate dopamine neuron G-protein signaling with DREADDs 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 altered the response strategy employed during cued reinstatement, and 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 indicate potential new approaches for intervening in these processes to treat addiction.

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 Gqsignaling, but not Gssignaling, in DA neurons enhanced cocaine seeking via functionally distinct projections to forebrain limbic regions. In contrast, engaging inhibitory Gi/osignaling 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 STATEMENTG-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. Gqand Gsstimulation activated dopamine neurons, but only Gqstimulation robustly enhanced cocaine seeking. Gi/oinhibitory 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.

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.

Dopamine from the ventral tegmental area and glutamate from several brain nuclei converge in the nucleus accumbens (NAc) to drive motivated behaviors. Repeated activation of D2 receptors with quinpirole (QNP) induces locomotor sensitization and compulsive behaviors, but the mechanisms are unknown. In this study, in vivo microdialysis and fast scan cyclic voltammetry in adult anesthetized rats were used to investigate the effect of repeated QNP on dopamine and glutamate neurotransmission within the NAc. Following eight injections of QNP, a significant decrease in phasic and tonic dopamine release was observed in rats that displayed locomotor sensitization. Either a systemic injection or the infusion of QNP into the NAc decreased dopamine release, and the extent of this effect was similar in QNP-sensitized and control rats, indicating that inhibitory D2 autoreceptor function is maintained despite repeated activation of D2 receptors and decreased dopamine extracellular levels. Basal extracellular levels of glutamate in the NAc were also significantly lower in QNP-treated rats than in controls. Moreover, the increase in NAc glutamate release induced by direct stimulation of medial prefrontal cortex was significantly lower in QNP-sensitized rats. Together, these results indicate that repeated activation of D2 receptors disconnects NAc from medial prefrontal cortex and ventral tegmental area. Repeated administration of the dopamine D2 receptor agonist quinpirole (QNP) induces locomotor sensitization. We found that the NAc of QNP-sensitized rats has reduced glutamate levels coming from prefrontal cortex together with a decreased phasic and tonic dopamine neurotransmission but a conserved presynaptic D2 receptor function. We suggest that locomotor sensitization is because of increased affinity state of D2 post-synaptic receptors.

Cocaine addiction is a chronic psychiatric disorder characterized by pathological motivation to obtain cocaine and behavioral and neurochemical hypersensitivity to cocaine-associated cues. These features of cocaine addiction are thought to be driven by aberrant phasic dopamine signaling. We previously demonstrated that blockade of the hypocretin receptor 1 (HCRTr1) attenuates cocaine self-administration and reduces cocaine-induced enhancement of dopamine signaling. Despite this evidence, the effects of HCRTr1 blockade on endogenous phasic dopamine release are unknown. In the current studies, we assessed whether blockade of HCRTr1 alters spontaneous and cue-evoked dopamine release in the nucleus accumbens core of freely moving rats. We first validated the behavioral and neurochemical effects of the novel, highly selective, HCRTr1 antagonist RTIOX-276 using cocaine self-administration and fast-scan cyclic voltammetry (FSCV) in anesthetized rats. We then used FSCV in freely moving rats to examine whether RTIOX-276 impacts spontaneous and cue-evoked dopamine release. Finally, we used ex vivo slice FSCV to determine whether the effects of RTIOX-276 on dopamine signaling involve dopamine terminal adaptations. Doses of RTIOX-276 that attenuate the motivation for cocaine reduce spontaneous dopamine transient amplitude and cue-evoked dopamine release. Further, these doses attenuated cocaine-induced dopamine uptake inhibition at the level of dopamine terminals. Our results provide support for the standing hypothesis that HCRTr1 blockade suppresses endogenous phasic dopamine signals, likely via actions at dopamine cell bodies. These results also elucidate a second process through which HCRTr1 blockade attenuates the effects of cocaine by reducing cocaine sensitivity at dopamine terminals.

Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine signaling or cocaine self-administration, particularly under high effort conditions. To address this, we examined the effects of hypocretin receptor 1, and/or hypocretin receptor 2 blockade on dopamine signaling and cocaine reinforcement. We used in vivo fast scan cyclic voltammetry to test the effects of hypocretin antagonists on dopamine signaling in the nucleus accumbens core and a progressive ratio schedule to examine the effects of these antagonists on cocaine self-administration. Results demonstrate that blockade of either hypocretin receptor 1 or both hypocretin receptor 1 and 2 significantly reduces the effects of cocaine on dopamine signaling and decreases the motivation to take cocaine. In contrast, blockade of hypocretin receptor 2 alone had no significant effects on dopamine signaling or self-administration. These findings suggest a differential involvement of the two hypocretin receptors, with hypocretin receptor 1 appearing to be more involved than hypocretin receptor 2 in the regulation of dopamine signaling and cocaine self-administration. When considered with the existing literature, these data support the hypothesis that hypocretins exert a permissive influence on dopamine signaling and motivated behavior via preferential actions on hypocretin receptor 1.

Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine andN-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long–Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuatedd-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated thed-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine andN-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effectsin vivoand is converted to clozapine andN-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Diphenylpyraline hydrochloride (DPP) is an internationally available antihistamine that produces therapeutic antiallergic effects by binding to histamine H1 receptors. The complete neuropharmacological and behavioral profile of DPP, however, remains uncharacterized. Here we describe studies that suggest DPP may fit the profile of a potential agonist replacement medication for cocaine addiction. Aside from producing the desired histamine reducing effects, many antihistamines can also elicit psychomotor activation and reward, both of which are associated with increased dopamine concentrations in the nucleus accumbens (NAc). The primary aim of this study was to investigate the potential ability of DPP to inhibit the dopamine transporter, thereby leading to elevated dopamine concentrations in the NAc in a manner similar to cocaine and other psychostimulants. The psychomotor activating and rewarding effects of DPP were also investigated. For comparative purposes cocaine, a known dopamine transporter inhibitor, psychostimulant and drug of abuse, was used as a positive control. As predicted, both cocaine (15 mg/kg) and an equimolar dose of DPP (14 mg/kg) significantly inhibited dopamine uptake in the NAc in vivo and produced locomotor activation, although the time-course of pharmacological effects of the two drugs was different. In comparison to cocaine, DPP showed a prolonged effect on dopamine uptake and locomotion. Furthermore, cocaine, but not DPP, produced significant conditioned place preference, a measure of drug reward. The finding that DPP functions as a potent dopamine uptake inhibitor without producing significant rewarding effects suggests that DPP merits further study as a potential candidate as an agonist pharmacotherapy for cocaine addiction.

The locus coeruleus-noradrenergic systems supplies norepinephrine throughout the central nervous system. State-dependent neuronal discharge activity of locus coeruleus noradrenergic neurons has long-suggested a role of this system in the induction of an alert waking state. Work over the past two decades provides unambiguous evidence that the locus coeruleus, and likely other noradrenergic nuclei, exert potent wake-promoting actions via an activation of noradrenergic β- and α1-receptors located within multiple subcortical structures, including the general regions of the medial septal area, the medial preoptic area and, most recently, the lateral hypothalamus. Conversely, global blockade of β- and α1-receptors or suppression of norepinephrine release results in profound sedation. The wake-promoting action of central noradrenergic neurotransmission has clinical implications for treatment of sleep/arousal disorders, such as insomnia and narcolepsy, and clinical conditions associated with excessive arousal, such as post-traumatic stress disorder.

The fast sampling rates of fast scan cyclic voltammetry make it a favorable method for measuring changes in brain monoamine release and uptake kinetics in slice, anesthetized, and freely moving preparations. The most common analysis technique for evaluating changes in dopamine signaling uses well-established Michaelis-Menten kinetic methods that can accurately model dopamine release and uptake parameters across multiple experimental conditions. Nevertheless, over the years, many researchers have turned to other measures to estimate changes in dopamine release and uptake, yet to our knowledge no systematic comparison amongst these measures has been conducted. To address this lack of uniformity in kinetic analyses, we have created the Demon Voltammetry and Analysis software suite, which is freely available to academic and non-profit institutions. Here we present an explanation of the Demon Voltammetry acquisition and analysis features, and demonstrate its utility for acquiring voltammetric data under in vitro, in vivo anesthetized, and freely moving conditions. Additionally, the software was used to compare the sensitivity of multiple kinetic measures of release and uptake to cocaine-induced changes in electrically evoked dopamine efflux in nucleus accumbens core slices. Specifically, we examined and compared tau, full width at half height, half-life, T₂₀, T₈₀, slope, peak height, calibrated peak dopamine concentration, and area under the curve to the well-characterized Michaelis-Menten parameters, dopamine per pulse, maximal uptake rate, and apparent affinity. Based on observed results we recommend tau for measuring dopamine uptake and calibrated peak dopamine concentration for measuring dopamine release.