Voltammetry In Brain Slices Research Articles

Patterns of ethanol intake in male rats with partial dopamine transporter deficiency.

Mesolimbic dopamine signaling plays a major role in alcohol and substance use disorders as well as comorbidities such as anxiety and depression. Growing evidence suggests that alcohol drinking is modulated by the function of the dopamine transporter (DAT), which tightly regulates extracellular dopamine concentrations. Adult male rats on a Wistar Han background (DAT+/+) and rats with a partial DAT deletion (DAT+/-) were used in this study. First, using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core from ethanol-naïve subjects, we measured greater evoked dopamine concentrations and slower dopamine reuptake in DAT+/- rats, consistent with increased dopamine signaling. Next, we measured ethanol drinking using the intermittent access two-bottle choice paradigm (20% v/v ethanol vs. water) across 5 weeks. DAT+/- rats voluntarily consumed less ethanol during its initial availability (the first 30 min), especially after longer periods of deprivation. In addition, DAT+/- males consumed less ethanol that was adulterated with the bitter tastant quinine. These findings suggest that partial DAT blockade and concomitant increase in brain dopamine levels has potential to reduce drinking and ameliorate alcohol use disorder (AUD).

Dopamine Dysregulation and Altered Responses to Drugs Affecting Dopaminergic Transmission in a New Dopamine Transporter Knockout (DAT-KO) Rat Model

Under normal conditions, dopamine (DA) clearance after release largely depends on uptake by the DA transporter (DAT). DAT expression/activity is reduced in some neuropsychiatric and neurological disorders. Our aim was to characterize the behavioral, neurochemical and electrophysiological effects of eliminating DAT in a novel knockout rat model we generated using CRISPR/Cas9. Consistent with existing DAT-KO models, our DAT-KO rats displayed increased locomotion, paradoxical calming by amphetamine, and reduced kinetics of DA clearance after stimulated release. Reduced DA kinetics were demonstrated using fast-scan cyclic voltammetry in brain slices containing the striatum or substantia nigra pars compacta (SNc) and in the dorsal striatum in vivo. Cocaine enhanced DA release in wild-type (WT) but not DAT-KO rats. Basal extracellular DA concentration measured with fast-scan controlled-adsorption voltammetry was higher in DAT-KO rats both in the striatum and SNc and was enhanced by L-DOPA (particularly after pharmacological block of monoamine oxidase), confirming that DA release after L-DOPA is not due to DAT reversal. The baseline firing frequency of SNc neurons was similar in both genotypes. However, D2 receptor-mediated inhibition of firing (by quinpirole or L-DOPA) was blunted in DAT-KO rats, while GABAB-mediated inhibition was preserved. We have also provided new data for the DAT-KO rat regarding the effects of slowing DA diffusion with dextran and blocking organic cation transporter 3 with corticosterone. Together, our results validate our DAT-KO rat and provide new insights into the mechanisms of chronic dysregulation of the DA system by addressing several unresolved issues in previous studies with other DAT-KO models.

Enhanced Transient Striatal Dopamine Release and Reuptake in Lphn3 Knockout Rats.

Latrophilin-3 (LPHN3) is an adhesion G protein coupled receptor involved in regulating neuroplasticity. Variants of LPHN3 are associated with increased risk of attention-deficit hyperactivity disorder. Data from mouse, zebrafish, Drosophila, and rat show that disruption of LPHN3 results in hyperactivity, and in the Sprague-Dawley Lphn3 knockout rat, exhibit deficits in learning and memory and changes in dopamine (DA) markers in the neostriatum. To determine the effects of Lphn3 deletion on DA neurotransmission, we compared the concentration, duration, and frequency of DA transients in KO and wild-type rats using fast-scan cyclic voltammetry in brain slices. Lphn3 KO rats showed higher release of DA, and the duration and interevent time were markedly decreased compared with wild-type rats. The data demonstrate that LPHN3 plays a heretofore unrecognized role in DA signaling and may represent a new target for small molecule regulation of DA neurotransmission with translational implications.

Alterations in Nucleus Accumbens Dopamine Terminals Following Dopamine Tolerance‐ or Sensitization‐Inducing Cocaine Self‐Administration Protocols

Cocaine has diverse effects on the function of the mesolimbic dopamine system, depending on the schedule and timing of self‐administration behavior. In cocaine self‐administering rats, sensitization as well as tolerance are phenomena that are not only observable behaviorally, but also at the molecular level, specifically at the dopamine transporter (DAT). When rats are allowed long access to cocaine (LgA), in which they can receive an unlimited number of infusions (1.5 mg/kg, i.v.) over the course of six hours per day for 14 days, cocaine intake moderately escalates and the DAT shows robust tolerance to cocaine in vitro. In a short access paradigm (ShA), during which rats have unlimited access to cocaine but only for two hours per day for 14 days, cocaine intake is constant and the DAT is unchanged, showing cocaine responsiveness that is the same as their drug naïve counterparts. However, in contrast, when rats are allowed intermittent access to cocaine (IntA), during which rats are allowed unlimited access to cocaine with no time‐outs for 5 minutes out of every 30 minutes, six hours per day for 14 days, cocaine intake during the 5 min bout is drastically increased and the DAT shows sensitization to the effects of cocaine in vitro. DAT sensitization occurs even though the animals undergoing the IntA paradigm have the same levels of cocaine intake as animals on the ShA paradigm. While the consequences of these self‐administration paradigms have been well characterized at the DAT, because opposite DAT changes occur with two schedules that produce escalation in dopamine‐dependent cocaine self‐administration behaviors, it is clear that there are other neurobiological changes contributing to these behaviors. It is not known how LgA, ShA, and IntA affect other aspects of the dopamine terminal, and so the purpose of this study is to characterize additional regulatory mechanisms on dopamine terminals. Using voltammetry in brain slices, we found that after an LgA protocol, there are no differences in the sensitivity of D2 or D3 dopamine release‐inhibiting autoreceptors compared to control animals. This juxtaposes previous findings from our lab that showed that autoreceptors became subsensitive following a limited access 24 hour paradigm. We are interested in the effects of IntA cocaine self‐administration on autoreceptor function, and the regulation of the strongly inhibitory kappa opioid receptors that are on dopamine terminals. By studying how other dopamine‐regulating processes, other than the DAT, respond to different self‐administrations paradigms, we will be able to gain a better understanding of how cocaine affects the dopamine system as a whole.Support or Funding InformationR01DA014030R01DA011697T32AA007565

Dopamine D3 autoreceptor inhibition enhances cocaine potency at the dopamine transporter.

Cocaine is a commonly abused central nervous system stimulant that enhances dopamine (DA) neurotransmission through its ability to block dopamine transporters (DATs). Recent evidence suggests there may be an interaction between DATs and D2/D3 autoreceptors that modulates cocaine's effects. The purpose of this study was to explore how D2/D3 autoreceptors modulate the ability of cocaine to inhibit DA uptake through DATs on pre-synaptic DA terminals. Using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core from male and female C57BL/6J mice, we first sought to examine the effects of global autoreceptor blockade using the non-selective D2/D3 autoreceptor antagonist, raclopride. We found that the ability of cocaine to inhibit DA uptake was increased by raclopride and that this effect was consistent across sexes. Furthermore, using D2 (L-741,626) or D3 (SB-277011-A) autoreceptor selective antagonists, we discovered that blockade of D3, but not D2, autoreceptors was responsible for the increased cocaine potency. Alterations in cocaine potency were attributable to alterations in uptake inhibition, rather than cocaine effects on vesicular DA release, suggesting that these results may be a product of a functional D3/DAT interaction apart from the canonical inhibitory actions of D3 autoreceptors on DA release. In addition, application of D2 (sumanirole) and D3 (PD 128907) autoreceptor-specific agonists had inverse effects, whereby D2 autoreceptor activation decreased cocaine potency and D3 autoreceptor activation had no effect. Together, these data show that DA autoreceptors dynamically regulate cocaine potency at the DAT, which is important for understanding cocaine's rewarding and addictive properties. We propose a model whereby presynaptic dopamine autoreceptors dynamically modulate cocaine potency through two separate mechanisms. We demonstrate that D2 agonists decrease cocaine potency, whereas D3 antagonists increase cocaine potency, likely through an allosteric mechanism outside of their canonical actions on dopamine release. These findings give important and novel insight into the contribution of D2/D3 autoreceptors to dopamine transporter function.

Supersensitive Kappa Opioid Receptors Promotes Ethanol Withdrawal-Related Behaviors and Reduce Dopamine Signaling in the Nucleus Accumbens.

Background:Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined.Methods:Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens.Results:Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group.Conclusions:These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety/compulsive-like behaviors may be driven by greater kappa opioid receptor sensitivity and a hypodopaminergic state of the nucleus accumbens.

Chronic ethanol self-administration in macaques shifts dopamine feedback inhibition to predominantly D2 receptors in nucleus accumbens core

BackgroundGiven the high level of homology between nonhuman primates and humans in regard to anatomy, physiology and ethanol drinking patterns, nonhuman primates represent an unparalleled preclinical model for examining the neurobiological basis of ethanol abuse. MethodsHere we examined the neurochemical consequences of chronic daily ethanol use using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core or dorsolateral caudate taken from male cynomolgus macaques following ethanol drinking. ResultsWe found that in both regions the ability of ethanol to decrease dopamine release was unchanged, indicating that ethanol self-administration does not produce tolerance or sensitization to ethanol effects on dopamine release at the dopamine terminal at this time point. We also found that in the nucleus accumbens core, autoregulation of dopamine release was shifted from equal D2 and D3 receptor involvement in control animals to primarily D2 receptor-mediated in drinkers. Specifically, the effect quinpirole, a D2/D3 receptor agonist, on dopamine release was equal across groups; however, dopamine signals were reversed to a greater extent by the selective D3 receptor antagonist SB-277,011A in control animals, indicating a greater contribution of D2 receptors in quinpirole-induced inhibition following ethanol self-administration. In the dorsolateral caudate, the effects of quinpirole and reversal with SB-277,011A was not different between ethanol and control slices. ConclusionsThis work provides novel insight into the dopaminergic adaptations resulting from chronic ethanol use in nonhuman primates and indicates that alterations in D2/D3 dopamine autoreceptor signaling may be an important neurochemical adaptation to ethanol consumption during early use.

Social isolation rearing increases dopamine uptake and psychostimulant potency in the striatum

Social isolation rearing (SI) is a model of early life stress that results in neurobiological alterations leading to increased anxiety-like behaviors. These animals also exhibit an increased propensity to administer psychostimulants, such as cocaine; however, the mechanisms governing this increased addiction vulnerability remain to be elucidated. Long-term stressors have been shown to produce important alterations in nucleus accumbens core (NAc) function. The NAc regulates motivated and goal-directed behaviors, and individual differences in NAc function have been shown to be predictive of addiction vulnerability. Rats were reared in group (GH; 4/cage) or SI (1/cage) conditions from weaning (PD 28) into early adulthood (PD 77) and dopamine release was assessed using voltammetry in brain slices containing the NAc and dorsomedial striatum. SI rats exhibited enhanced dopamine release and uptake in both regions compared to GH rats. In regard to psychostimulant effects directly at the dopamine transporter (DAT), methylphenidate and amphetamine, but not cocaine, inhibited uptake more in SI than GH rats. The increased potencies were positively correlated with uptake rates, suggesting that increased potencies of amphetamine-like compounds are due to changes in DAT function. Cocaine's effects on uptake were similar between rearing conditions, however, cocaine enhanced evoked dopamine release greater in SI than GH rats, suggesting that the enhanced cocaine reinforcement in SI animals involves a DAT independent mechanism. Together, the results provide the first evidence that greater psychostimulant effects in SI compared to GH rats are due to effects on dopamine terminals related to uptake dependent and independent mechanisms.

Voluntary ethanol intake predicts κ-opioid receptor supersensitivity and regionally distinct dopaminergic adaptations in macaques.

The dopaminergic projections from the ventral midbrain to the striatum have long been implicated in mediating motivated behaviors and addiction. Previously it was demonstrated that κ-opioid receptor (KOR) signaling in the striatum plays a critical role in the increased reinforcing efficacy of ethanol following ethanol vapor exposure in rodent models. Although rodents have been used extensively to determine the neurochemical consequences of chronic ethanol exposure, establishing high levels of voluntary drinking in these models has proven difficult. Conversely, nonhuman primates exhibit similar intake and pattern to humans in regard to drinking. Here we examine the effects of chronic voluntary ethanol self-administration on dopamine neurotransmission and the ability of KORs to regulate dopamine release in the dorsolateral caudate (DLC) and nucleus accumbens (NAc) core. Using voltammetry in brain slices from cynomolgus macaques after 6 months of ad libitum ethanol drinking, we found increased KOR sensitivity in both the DLC and NAc. The magnitude of ethanol intake predicted increases in KOR sensitivity in the NAc core, but not the DLC. Additionally, ethanol drinking increased dopamine release and uptake in the NAc, but decreased both of these measures in the DLC. These data suggest that chronic daily drinking may result in regionally distinct disruptions of striatal outputs. In concert with previous reports showing increased KOR regulation of drinking behaviors induced by ethanol exposure, the strong relationship between KOR activity and voluntary ethanol intake observed here gives further support to the hypothesis that KORs may provide a promising pharmacotherapeutic target in the treatment of alcoholism.

A Single Amphetamine Infusion Reverses Deficits in Dopamine Nerve-Terminal Function Caused by a History of Cocaine Self-Administration.

There are ∼ 1.6 million people who meet the criteria for cocaine addiction in the United States, and there are currently no FDA-approved pharmacotherapies. Amphetamine-based dopamine-releasing drugs have shown efficacy in reducing the motivation to self-administer cocaine and reducing intake in animals and humans. It is hypothesized that amphetamine acts as a replacement therapy for cocaine through elevation of extracellular dopamine levels. Using voltammetry in brain slices, we tested the ability of a single amphetamine infusion in vivo to modulate dopamine release, uptake kinetics, and cocaine potency in cocaine-naive animals and after a history of cocaine self-administration (1.5 mg/kg/infusion, fixed-ratio 1, 40 injections/day × 5 days). Dopamine kinetics were measured 1 and 24 h after amphetamine infusion (0.56 mg/kg, i.v.). Following cocaine self-administration, dopamine release, maximal rate of uptake (Vmax), and membrane-associated dopamine transporter (DAT) levels were reduced, and the DAT was less sensitive to cocaine. A single amphetamine infusion reduced Vmax and membrane DAT levels in cocaine-naive animals, but fully restored all aspects of dopamine terminal function in cocaine self-administering animals. Here, for the first time, we demonstrate pharmacologically induced, immediate rescue of deficits in dopamine nerve-terminal function in animals with a history of high-dose cocaine self-administration. This observation supports the notion that the DAT expression and function can be modulated on a rapid timescale and also suggests that the pharmacotherapeutic actions of amphetamine for cocaine addiction go beyond that of replacement therapy.

Varenicline enhances dopamine release facilitation more than nicotine after long-term nicotine treatment and withdrawal.

An important factor contributing to the high relapse rates among smokers is nicotine withdrawal symptoms. Multiple studies suggest that decreased dopamine release in nucleus accumbens plays a key role in withdrawal. However, recent reports showed that long-term nicotine exposure itself also decreases accumbal dopamine release, suggesting that additional mechanisms are involved in withdrawal. Here, we used real-time cyclic voltammetry in brain slices containing the nucleus accumbens to further elucidate the changes in dopamine release linked to nicotine withdrawal. Rats received vehicle or nicotine via the drinking water for 2–3 months. Studies assessing the expression of somatic signs in vehicle-treated, nicotine-treated, and 24-h nicotine withdrawn rats showed that nicotine withdrawal led to a significant increase in somatic signs. Subsequent voltammetry studies showed that long-term nicotine decreased single-pulse-stimulated dopamine release via an interaction at α6β2* receptors. Nicotine withdrawal led to a partial recovery in α6β2* receptor-mediated release. In addition, long-term nicotine treatment alone increased dopamine release paired-pulse ratios and this was partially reversed with nicotine removal. We then evaluated the effect of bath-applied nicotine and varenicline on dopamine release. Nicotine and varenicline both decreased single-pulse-stimulated release in vehicle-treated, nicotine-treated, and nicotine withdrawn rats. However, bath-applied varenicline increased paired-pulse ratios to a greater extent than nicotine during long-term nicotine treatment and after its withdrawal. Altogether these data suggest that nicotine withdrawal is associated with a partial restoration of dopamine release measures to control levels and that varenicline's differential modulation of dopamine release may contribute to its mechanism of action.

GABA(B) modulation of dopamine release in the nucleus accumbens core.

Modulation of the concentration of dopamine (DA) released from dopaminergic terminals in the nucleus accumbens (NAc) influences behaviours such as the motivation to obtain drugs of abuse. γ-Aminobutyric acid type B (GABAB ) receptors are expressed throughout the mesolimbic circuit, including in the NAc, and baclofen, an agonist of GABAB receptors, can decrease drug-seeking behaviours. However, the mechanism by which GABAB receptors modulate terminal DA release has not been well studied. We explored how baclofen modulates the concentration of DA released from terminals in the NAc core using fast-scan cyclic voltammetry in brain slices from adult male C57BL/6J mice. We found that baclofen concentration-dependently decreased single pulse-evoked DA release. This effect was blocked by the GABAB antagonist, CGP 52432, but not by a nicotinic acetylcholine receptor antagonist. Suppression of DA release by a saturating concentration of baclofen was sustained for up to 1 h. The effect of baclofen was reduced with electrical stimulations mimicking burst firing of DA neurons. Similar to the D2 receptor agonist, quinpirole, baclofen reduced the probability of DA release, supporting a mechanistic overlap with D2 receptors. Baclofen-mediated suppression of DA release persisted after a locomotor-sensitizing cocaine treatment, indicating that GABAB receptors on DA terminals were not altered by cocaine exposure. These data suggest that baclofen-mediated suppression of terminal DA release is due to GABAB activation on DA terminals to reduce the probability of DA release. This effect does not readily desensitize, and persists regardless of chronic cocaine treatment.

Biphasic mechanisms of amphetamine action at the dopamine terminal.

In light of recent studies suggesting that amphetamine (AMPH) increases electrically evoked dopamine release ([DA]o), we examined discrepancies between these findings and literature that has demonstrated AMPH-induced decreases in [DA]o. The current study has expanded the inventory of AMPH actions by defining two separate mechanisms of AMPH effects on [DA]o at high and low doses, one dopamine transporter (DAT) independent and one DAT dependent, respectively. AMPH concentrations were measured via microdialysis in rat nucleus accumbens after intraperitoneal injections of 1 and 10 mg/kg and yielded values of ∼10 and 200 nM, respectively. Subsequently, voltammetry in brain slices was used to examine the effects of low (10 nM), moderate (100 nM), and high (10 μM) concentrations of AMPH across a range of frequency stimulations (one pulse; five pulses, 20 Hz; 24 pulses, 60 Hz). We discovered biphasic, concentration-dependent effects in WT mice, in which AMPH increased [DA]o at low concentrations and decreased [DA]o at high concentrations across all stimulation types. However, in slices from DAT-KO mice, [DA]o was decreased by all concentrations of AMPH, demonstrating that AMPH-induced increases in [DA]o are DAT dependent, whereas the decreases at high concentrations are DAT independent. We propose that low AMPH concentrations are insufficient to disrupt vesicular sequestration, and therefore AMPH acts solely as a DAT inhibitor to increase [DA]o. When AMPH concentrations are high, the added mechanism of vesicular depletion leads to reduced [DA]o. The biphasic mechanisms observed here confirm and extend the traditional actions of AMPH, but do not support mechanisms involving increased exocytotic release.

Biphasic mechanisms of amphetamine action at the dopamine terminal (661.4)

In light of recent studies suggesting that amphetamine (AMPH) increases spontaneous and electrically evoked dopamine release ([DA]o), we examined discrepancies between these findings and previous literature which has demonstrated marked decreases in [DA]o after AMPH. The current study has refined and expanded the mechanisms of AMPH actions by defining two mechanisms of AMPH effects at high and low doses, one dopamine transporter (DAT)‐independent and one DAT‐dependent, respectively. Using voltammetry in brain slices, we examined the effects of low, moderate, and high concentrations of AMPH across a range of tonic and phasic stimulation parameters (1 pulse; 5 pulse 20 Hz; 24 pulse 60 Hz) in wild‐type (WT) and DAT knockout (KO) mice. We discovered novel, biphasic, concentration‐dependent effects in WT mice, where AMPH increased [DA]o at low concentrations and decreased [DA]o at high concentrations. We then used DAT‐KO animals to examine the mechanisms by which AMPH exerts these effects. In DAT‐KO slices, [DA]o was decreased by all concentrations of AMPH, demonstrating that AMPH‐induced increases in [DA]o are entirely DAT‐dependent, while the decreases at high concentrations are DAT‐independent. We propose that when AMPH concentrations are low, they are insufficient to deplete dopamine vesicles, and therefore AMPH acts solely as a DAT blocker, increasing peak [DA]o. When AMPH concentrations are high, the added mechanism of vesicular depletion leads to reduced [DA]o. The biphasic mechanisms observed here support and extend the established actions of AMPH.Grant Funding Source: Supported by NIH grants DA021325, DA030161, DA006634, DA007246, DA031533, DA031791 and AA007565

Intermittent Cocaine Self-Administration Produces Sensitization of Stimulant Effects at the Dopamine Transporter

Previous literature investigating neurobiological adaptations following cocaine self-administration has shown that high, continuous levels of cocaine intake (long access; LgA) results in reduced potency of cocaine at the dopamine transporter (DAT), whereas an intermittent pattern of cocaine administration (intermittent access; IntA) results in sensitization of cocaine potency at the DAT. Here, we aimed to determine whether these changes are specific to cocaine or translate to other psychostimulants. Psychostimulant potency was assessed by fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens following IntA, short access, and LgA cocaine self-administration, as well as in brain slices from naive animals. We assessed the potency of amphetamine (a releaser), and methylphenidate (a DAT blocker, MPH). MPH was selected because it is functionally similar to cocaine and structurally related to amphetamine. We found that MPH and amphetamine potencies were increased following IntA, whereas neither was changed following LgA or short access cocaine self-administration. Therefore, whereas LgA-induced tolerance at the DAT is specific to cocaine as shown in previous work, the sensitizing effects of IntA apply to cocaine, MPH, and amphetamine. This demonstrates that the pattern with which cocaine is administered is important in determining the neurochemical consequences of not only cocaine effects but potential cross-sensitization/cross-tolerance effects of other psychostimulants as well.

Paradoxical tolerance to cocaine after initial supersensitivity in drug-use-prone animals.

There is great interest in outlining biological factors and behavioral characteristics that either predispose or predict vulnerability to substance use disorders. Response to an inescapable novel environment has been shown to predict a "drug-use-prone" phenotype that is defined by rapid acquisition of cocaine self-administration. Here, we showed that response to novelty can also predict the neurochemical and behavioral effects of acute and repeated cocaine in rats. We used cocaine self-administration under a fixed-ratio 1 schedule followed by fast-scan cyclic voltammetry in brain slices to measure subsecond dopamine (DA) release and uptake parameters in drug-use-prone and -resistant phenotypes. Despite no significant differences in stimulated release and uptake, animals with high responses to a novel environment had DA transporters that were more sensitive to cocaine-induced uptake inhibition, which corresponded to greater locomotor activating effects of cocaine. These animals also acquired cocaine self-administration more rapidly and, after 5 days of extended access cocaine self-administration, high-responding animals showed robust tolerance to DA uptake inhibition by cocaine. The effects of cocaine remained unchanged in animals with low novelty responses. Similarly, the rate of acquisition was negatively correlated with DA uptake inhibition by cocaine after self-administration. Thus, we showed that tolerance to the cocaine-induced inhibition of DA uptake coexists with a behavioral phenotype that is defined by increased preoccupation with cocaine as measured by rapid acquisition and early high intake.

Examining the Complex Regulation and Drug-Induced Plasticity of Dopamine Release and Uptake Using Voltammetry in Brain Slices

Fast scan cyclic voltammetry in brain slices (slice voltammetry) has been used over the last several decades to increase substantially our understanding of the complex local regulation of dopamine release and uptake in the striatum. This technique is routinely used for the study of changes that occur in the dopamine system associated with various disease states and pharmacological treatments, and to study mechanisms of local circuitry regulation of dopamine terminal function. In the context of this Review, we compare the relative advantages of voltammetry using striatal slice preparations versus in vivo preparations, and highlight recent advances in our understanding of dopamine release and uptake in the striatum specifically from studies that use slice voltammetry in drug-naïve animals and animals with a history of psychostimulant self-administration.

Amphetamine augments action potential‐dependent dopaminergic signaling in the striatum in vivo

Amphetamine (AMPH) is thought to disrupt normal patterns of action potential-dependent dopaminergic signaling by depleting dopamine (DA) vesicular stores and promoting non-exocytotic DA efflux. Voltammetry in brain slices concurrently demonstrates these key drug effects, along with competitive inhibition of neuronal DA uptake. Here, we perform comparable kinetic and voltammetric analyses in vivo to determine whether AMPH acts qualitatively and quantitatively similar in the intact brain. Fast-scan cyclic voltammetry measured extracellular DA in dorsal and ventral striata of urethane-anesthetized rats. Electrically evoked recordings were analyzed to determine K(m) and V(max) for DA uptake and vesicular DA release, while background voltammetric current indexed basal DA concentration. AMPH (0.5, 3, and 10 mg/kg i.p.) robustly increased evoked DA responses in both striatal subregions. The predominant contributor to these elevated levels was competitive uptake inhibition, as exocytotic release was unchanged in the ventral striatum and only modestly decreased in the dorsal striatum. Increases in basal DA levels were not detected. These results are consistent with AMPH augmenting action potential-dependent dopaminergic signaling in vivo across a wide, behaviorally relevant dose range. Future work should be directed at possible causes for the distinct in vitro and in vivo pharmacology of AMPH.

Cocaine-Insensitive Dopamine Transporters with Intact Substrate Transport Produced by Self-Administration

Psychomotor stimulant drugs such as cocaine and amphetamine activate brain dopamine (DA) neurotransmission and support self-administration in humans and laboratory animals. Cocaine amplifies DA signaling by blocking the DA transporter (DAT), and this has been described as the most important mechanism underlying cocaine's reinforcing effects. Amphetamine has the added mechanism of reverse transport of intracellular DA through the DAT. We used cocaine and amphetamine self-administration under a fixed-ratio 1 schedule followed by microdialysis in freely moving rats to measure extracellular DA levels and fast scan cyclic voltammetry in brain slices to measure subsecond DA release and uptake parameters. Following a high dose (1.5 mg/kg intravenous) cocaine self-administration paradigm (40 injections/day × 5 days), the DAT was markedly less sensitive to cocaine, as measured by microdialysis and voltammetry in the nucleus accumbens core. In contrast, the DAT substrate amphetamine retained the same efficacy at the DAT in cocaine self-administering animals, and amphetamine did not mimic cocaine's effect on the DAT when self-administered. A single session of cocaine self-administration caused a significant decrease in the ability of cocaine to inhibit the DAT, a finding that may provide a neurochemical basis for rapid tolerance. The effects of cocaine returned to normal within a few weeks following cessation of self-administration. Here, we, for the first time, demonstrate an in vivo, pharmacologically induced alteration in the sensitivity of the DAT to cocaine that is specific to cocaine, spares DAT and substrate/releaser interactions, and is independent of maximal rate of DA uptake (V(max)).

Attenuation of basal and cocaine-enhanced locomotion and nucleus accumbens dopamine in cannabinoid CB1-receptor-knockout mice.

Effect of cannabinoid CB1 receptor deletion on cocaine's actions is controversial. This is partly based on findings in CB1-receptor-knockout (CB1(-/-)) mice with CD1 genetic background. In the present study, we used CB1(-/-) mice with a C57BL/6J genetic background to further investigate the role of CB1 receptors in cocaine's action. Locomotor activity was assessed using AccuScan locomotor chambers. Brain extracellular dopamine (DA) levels were measured by in vivo microdialysis and by fast-scan cyclic voltammetry in the nucleus accumbens (NAc). CB1(-/-) mice displayed a significant reduction in basal levels of locomotion and extracellular DA, as well as in cocaine-enhanced locomotion and extracellular DA, as compared to their wild-type (CB1(+/+)) littermates. The reduction in basal and cocaine-enhanced DA appears to be related to a reduction in basal DA release, not to an increase in DA clearance, as indicated by fast-scan cyclic voltammetry in brain slices. Pharmacological blockade of CB1 receptors by SR141716 inhibited locomotion and NAc DA release in CB1(+/+) mice. The present findings suggest an important role for CB1 receptors in mediating cocaine's behavioral and neurochemical effects.