Negative feedback control of neuronal activity by microglia.

Microglia, the brain's resident macrophages, help to regulate brain function by removing dying neurons, pruning non-functional synapses, and producing ligands that support neuronal survival1. Here we show that microglia are also critical modulators of neuronal activity and associated behavioural responses in mice. Microglia respond to neuronal activation by suppressing neuronal activity, and ablation of microglia amplifies and synchronizes the activity of neurons, leading to seizures. Suppression of neuronal activation by microglia occurs in a highly region-specific fashion and depends on the ability of microglia to sense and catabolize extracellular ATP, which is released upon neuronal activation by neurons and astrocytes. ATP triggers the recruitment of microglial protrusions and is converted by the microglial ATP/ADP hydrolysing ectoenzyme CD39 into AMP; AMP is then converted into adenosine by CD73, which is expressed on microglia as well as other brain cells. Microglial sensing of ATP, the ensuing microglia-dependent production of adenosine, and the adenosine-mediated suppression of neuronal responses via the adenosine receptor A1R are essential for the regulation of neuronal activity and animal behaviour. Our findings suggest that this microglia-driven negative feedback mechanism operates similarly to inhibitory neurons and is essential for protecting the brain from excessive activation in health and disease.

The transcriptional response to acute cocaine is inverted in male mice with a history of cocaine self-administration and withdrawal throughout the mesocorticolimbic system.

A large body of work has demonstrated that cocaine-induced changes in transcriptional regulation play a central role in the onset and maintenance of cocaine use disorder. An underappreciated aspect of this area of research, however, is that the pharmacodynamic properties of cocaine can change depending on an organism's previous drug-exposure history. In this study, we utilized RNA sequencing to characterize how the transcriptome-wide effects of acute cocaine exposure were altered by a history of cocaine self-administration and long-term withdrawal (30 days) in the ventral tegmental area (VTA), nucleus accumbens (NAc), and prefrontal cortex (PFC) in male mice. First, we found that the gene expression patterns induced by a single cocaine injection (10 mg/kg) were discordant between cocaine-naïve mice and mice in withdrawal from cocaine self-administration. Specifically, the same genes that were upregulated by acute cocaine in cocaine-naïve mice were downregulated by the same dose of cocaine in mice undergoing long-term withdrawal; the same pattern of opposite regulation was observed for the genes downregulated by initial acute cocaine exposure. When we analyzed this dataset further, we found that the gene expression patterns that were induced by long-term withdrawal from cocaine self-administration showed a high degree of overlap with the gene expression patterns of acute cocaine exposure - even though animals had not consumed cocaine in 30 days. Interestingly, cocaine re-exposure at this withdrawal time point reversed this expression pattern. Finally, we found that this pattern was similar across the VTA, PFC, NAc, and within each brain region the same genes were induced by acute cocaine, re-induced during long-term withdrawal, and reversed by cocaine re-exposure. Together, we identified a longitudinal pattern of gene regulation that is conserved across the VTA, PFC, and NAc, and characterized the genes constituting this pattern in each brain region.

From Circuits to Chromatin: The Emerging Role of Epigenetics in Mental Health.

A central goal of neuroscience research is to understand how experiences modify brain circuits to guide future adaptive behavior. In response to environmental stimuli, neural circuit activity engages gene regulatory mechanisms within each cell. This activity-dependent gene expression is governed, in part, by epigenetic processes that can produce persistent changes in both neural circuits and the epigenome itself. The complex interplay between circuit activity and neuronal gene regulation is vital to learning and memory, and, when disrupted, is linked to debilitating psychiatric conditions, such as substance use disorder. To develop clinical treatments, it is paramount to advance our understanding of how neural circuits and the epigenome cooperate to produce behavioral adaptation. Here, we discuss how new genetic tools, used to manipulate neural circuits and chromatin, have enabled the discovery of epigenetic processes that bring about long-lasting changes in behavior relevant to mental health and disease.

Granulocyte-Colony Stimulating Factor Reduces Cocaine-Seeking and Downregulates Glutamatergic Synaptic Proteins in Medial Prefrontal Cortex.

Psychostimulant use disorder is a major public health issue, and despite the scope of the problem there are currently no Food and Drug Administration (FDA)-approved treatments. There would be tremendous utility in development of a treatment that could help patients both achieve and maintain abstinence. Previous work from our group has identified granulocyte-colony stimulating factor (G-CSF) as a neuroactive cytokine that alters behavioral response to cocaine, increases synaptic dopamine release, and enhances cognitive flexibility. Here, we investigate the role of G-CSF in affecting extinction and reinstatement of cocaine-seeking and perform detailed characterization of its proteomic effects in multiple limbic substructures. Male Sprague Dawley rats were injected with PBS or G-CSF during (1) extinction or (2) abstinence from cocaine self-administration, and drug seeking behavior was measured. Quantitative assessment of changes in the proteomic landscape in the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) were performed via data-independent acquisition (DIA) mass spectrometry analysis. Administration of G-CSF during extinction accelerated the rate of extinction, and administration during abstinence attenuated cue-induced cocaine-seeking. Analysis of global protein expression demonstrated that G-CSF regulated proteins primarily in mPFC that are critical to glutamate signaling and synapse maintenance. Taken together, these findings support G-CSF as a viable translational research target with the potential to reduce drug craving or seeking behaviors. Importantly, recombinant G-CSF exists as an FDA-approved medication which may facilitate rapid clinical translation. Additionally, using cutting-edge multiregion discovery proteomics analyses, these studies identify a novel mechanism underlying G-CSF effects on behavioral plasticity.SIGNIFICANCE STATEMENT Pharmacological treatments for psychostimulant use disorder are desperately needed, especially given the disease's chronic, relapsing nature. However, there are currently no Food and Drug Administration (FDA)-approved pharmacotherapies. Emerging evidence suggests that targeting the immune system may be a viable translational research strategy; preclinical studies have found that the neuroactive cytokine granulocyte-colony stimulating factor (G-CSF) alters cocaine reward and reinforcement and can enhance cognitive flexibility. Given this basis of evidence we studied the effects of G-CSF treatment on extinction and reinstatement of cocaine seeking. We find that administration of G-CSF accelerates extinction and reduces cue-induced drug seeking after cocaine self-administration. In addition, G-CSF leads to downregulation of synaptic glutamatergic proteins in medial prefrontal cortex (mPFC), suggesting that G-CSF influences drug seeking via glutamatergic mechanisms.

Cocaine-related DNA methylation in caudate neurons alters 3D chromatin structure of the IRXA gene cluster.

Epigenetic mechanisms, like those involving DNA methylation, are thought to mediate the relationship between chronic cocaine dependence and molecular changes in addiction-related neurocircuitry, but have been understudied in human brain. We initially used reduced representation bisulfite sequencing (RRBS) to generate a methylome-wide profile of cocaine dependence in human post-mortem caudate tissue. We focused on the Iroquois Homeobox A (IRXA) gene cluster, where hypomethylation in exon 3 of IRX2 in neuronal nuclei was associated with cocaine dependence. We replicated this finding in an independent cohort and found similar results in the dorsal striatum from cocaine self-administering mice. Using epigenome editing and 3C assays, we demonstrated a causal relationship between methylation within the IRX2 gene body, CTCF protein binding, three-dimensional (3D) chromatin interaction, and gene expression. Together, these findings suggest that cocaine-related hypomethylation of IRX2 contributes to the development and maintenance of cocaine dependence through alterations in 3D chromatin structure in the caudate nucleus.

Ascorbate deficiency decreases dopamine release in gulo-/- and APP/PSEN1 mice.

Dopamine (DA) has important roles in learning, memory, and motivational processes and is highly susceptible to oxidation. In addition to dementia, Alzheimer's disease (AD) patients frequently exhibit decreased motivation, anhedonia, and sleep disorders, suggesting deficits in dopaminergic neurotransmission. Vitamin C (ascorbate, ASC) is a critical antioxidant in the brain and is often depleted in AD patients as a result of disease-related oxidative stress and dietary deficiencies. To probe the effects of ASC deficiency and AD pathology on the DAergic system, gulo-/- mice, which like humans depend on dietary ASC to maintain adequate tissue levels, were crossed with APP/PSEN1 mice and provided sufficient or depleted ASC supplementation from weaning until 12 months of age. Ex vivo fast-scan cyclic voltammetry showed that chronic ASC depletion and APP/PSEN1 genotype both independently decreased dopamine release in the nucleus accumbens, a hub for motivational behavior and reward, while DA clearance was similar across all groups. In striatal tissue containing nucleus accumbens, low ASC treatment led to decreased levels of DA and its metabolites 3,4-dihydroxyohenyl-acetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA). Decreased enzyme activity observed through lower pTH/TH ratio was driven by a cumulative effect of ASC depletion and APP/PSEN1 genotype. Together the data show that deficits in dopaminergic neurotransmission resulting from age and disease status are magnified in conditions of low ASC which decrease DA availability during synaptic transmission. Such deficits may contribute to the non-cognitive behavioral changes observed in AD including decreased motivation, anhedonia, and sleep disorders.

Synaptic Microtubule-Associated Protein EB3 and SRC Phosphorylation Mediate Structural and Behavioral Adaptations During Withdrawal From Cocaine Self-Administration.

Addictive behaviors, including relapse, are thought to depend in part on long-lasting drug-induced adaptations in dendritic spine signaling and morphology in the nucleus accumbens (NAc). While the influence of activity-dependent actin remodeling in these phenomena has been studied extensively, the role of microtubules and associated proteins remains poorly understood. We report that pharmacological inhibition of microtubule polymerization in the NAc inhibited locomotor sensitization to cocaine and contextual reward learning. We then investigated the roles of microtubule end-binding protein 3 (EB3) and SRC kinase in the neuronal and behavioral responses to volitionally administered cocaine. In synaptoneurosomal fractions from the NAc of self-administering male rats, the phosphorylation of SRC at an activating site was induced after 1 d of withdrawal, while EB3 levels were increased only after 30 d of withdrawal. Blocking SRC phosphorylation during early withdrawal by virally overexpressing SRCIN1, a negative regulator of SRC activity known to interact with EB3, abolished the incubation of cocaine craving in both male and female rats. Conversely, mimicking the EB3 increase observed after prolonged withdrawal increased the motivation to consume cocaine in male rats. In mice, the overexpression of either EB3 or SRCIN1 increased dendritic spine density and altered the spine morphology of NAc medium spiny neurons. Finally, a cocaine challenge after prolonged withdrawal recapitulated most of the synaptic protein expression profiles observed at early withdrawal. These findings suggest that microtubule-associated signaling proteins such as EB3 cooperate with actin remodeling pathways, notably SRC kinase activity, to establish and maintain long-lasting cellular and behavioral alterations following cocaine self-administration.SIGNIFICANCE STATEMENT Drug-induced morphological restructuring of dendritic spines of nucleus accumbens neurons is thought to be one of the cellular substrates of long-lasting drug-associated memories. The molecular basis of these persistent changes has remained incompletely understood. Here we implicate for the first time microtubule function in this process, together with key players such as microtubule-bound protein EB3 and synaptic SRC phosphorylation. We propose that microtubule and actin remodeling cooperate during withdrawal to maintain the plastic structural changes initially established by cocaine self-administration. This work opens new translational avenues for further characterization of microtubule-associated regulatory molecules as putative drug targets to tackle relapse to drug taking.

Direct dopamine terminal regulation by local striatal microcircuitry.

Regulation of axonal dopamine release by local microcircuitry is at the hub of several biological processes that govern the timing and magnitude of signaling events in reward-related brain regions. An important characteristic of dopamine release from axon terminals in the striatum is that it is rapidly modulated by local regulatory mechanisms. These processes can occur via homosynaptic mechanisms-such as presynaptic dopamine autoreceptors and dopamine transporters - as well heterosynaptic mechanisms such as retrograde signaling from postsynaptic cholinergic and dynorphin systems, among others. Additionally, modulation of dopamine release via diffusible messengers, such as nitric oxide and hydrogen peroxide, allows for various metabolic factors to quickly and efficiently regulate dopamine release and subsequent signaling. Here we review how these mechanisms work in concert to influence the timing and magnitude of striatal dopamine signaling, independent of action potential activity at the level of dopaminergic cell bodies in the midbrain, thereby providing a parallel pathway by which dopamine can be modulated. Understanding the complexities of local regulation of dopamine signaling is required for building comprehensive frameworks of how activity throughout the dopamine system is integrated to drive signaling and control behavior.

ß-catenin mediates stress resilience through Dicer1/microRNA regulation.

ß-catenin is a multi-functional protein that has an important role in the mature central nervous system; its dysfunction has been implicated in several neuropsychiatric disorders, including depression. Here we show that in mice ß-catenin mediates pro-resilient and anxiolytic effects in the nucleus accumbens, a key brain reward region, an effect mediated by D2-type medium spiny neurons. Using genome-wide ß-catenin enrichment mapping, we identify Dicer1-important in small RNA (for example, microRNA) biogenesis--as a ß-catenin target gene that mediates resilience. Small RNA profiling after excising ß-catenin from nucleus accumbens in the context of chronic stress reveals ß-catenin-dependent microRNA regulation associated with resilience. Together, these findings establish ß-catenin as a critical regulator in the development of behavioural resilience, activating a network that includes Dicer1 and downstream microRNAs. We thus present a foundation for the development of novel therapeutic targets to promote stress resilience.

Activity-Dependent Epigenetic Remodeling in Cocaine Use Disorder.

Substance use disorder (SUD) is a behavioral disorder characterized by cycles of abstinence, drug seeking, and relapse. SUD is characterized by aberrant learning processes which develop after repeated exposure to drugs of abuse. At the core of this phenotype is the persistence of symptoms, such as craving and relapse to drug seeking, long after the cessation of drug use. The neural basis of these behavioral changes has been linked to dysfunction in neural circuits across the brain; however, the molecular drivers that allow for these changes to persist beyond the lifespan of any individual protein remain opaque. Epigenetic adaptations - where DNA is modified to increase or decrease the probability of gene expression at key genes - have been identified as a mechanism underlying the long-lasting nature of drug-seeking behavior. Thus, to understand SUD, it is critical to define the interplay between neuronal activation and longer-term changes in transcription and epigenetic remodeling and define their role in addictive behaviors. In this review, we discuss the current understanding of drug-induced changes to circuit function, recent discoveries in epigenetic mechanisms that mediate these changes, and, ultimately, how these adaptations drive the persistent nature of relapse, with emphasis on adaptations in models of cocaine use disorder. Understanding the complex interplay between epigenetic gene regulation and circuit activity will be critical in elucidating the neural mechanisms underlying SUD. This, with the advent of novel genetic-based techniques, will allow for the generation of novel therapeutic avenues to improve treatment outcomes in SUD.

Amphetamine Reverses Escalated Cocaine Intake via Restoration of Dopamine Transporter Conformation.

Cocaine abuse disrupts dopamine system function, and reduces cocaine inhibition of the dopamine transporter (DAT), which results in tolerance. Although tolerance is a hallmark of cocaine addiction and a DSM-V criterion for substance abuse disorders, the molecular adaptations producing tolerance are unknown, and testing the impact of DAT changes on drug taking behaviors has proven difficult. In regard to treatment, amphetamine has shown efficacy in reducing cocaine intake; however, the mechanisms underlying these effects have not been explored. The goals of this study were twofold; we sought to (1) identify the molecular mechanisms by which cocaine exposure produces tolerance and (2) determine whether amphetamine-induced reductions in cocaine intake are connected to these mechanisms. Using cocaine self-administration and fast-scan cyclic voltammetry in male rats, we show that low-dose, continuous amphetamine treatment, during self-administration or abstinence, completely reversed cocaine tolerance. Amphetamine treatment also reversed escalated cocaine intake and decreased motivation to obtain cocaine as measured in a behavioral economics task, thereby linking tolerance to multiple facets of cocaine use. Finally, using fluorescence resonance energy transfer imaging, we found that cocaine tolerance is associated with the formation of DAT-DAT complexes, and that amphetamine disperses these complexes. In addition to extending our basic understanding of DATs and their role in cocaine reinforcement, we serendipitously identified a novel therapeutic target: DAT oligomer complexes. We show that dispersion of oligomers is concomitant with reduced cocaine intake, and propose that pharmacotherapeutics aimed at these complexes may have potential for cocaine addiction treatment.SIGNIFICANCE STATEMENT Tolerance to cocaine's subjective effects is a cardinal symptom of cocaine addiction and a DSM-V criterion for substance abuse disorders. However, elucidating the molecular adaptions that produce tolerance and determining its behavioral impact have proven difficult. Using cocaine self-administration in rats, we link tolerance to cocaine effects at the dopamine transporter (DAT) with aberrant cocaine-taking behaviors. Further, tolerance was associated with multi-DAT complexes, which formed after cocaine exposure. Treatment with amphetamine deconstructed DAT complexes, reversed tolerance, and decreased cocaine seeking. These data describe the behavioral consequence of cocaine tolerance, provide a putative mechanism for its development, and suggest that compounds that disperse DAT complexes may be efficacious treatments for cocaine addiction.

Granulocyte Colony Stimulating Factor Enhances Reward Learning through Potentiation of Mesolimbic Dopamine System Function.

Deficits in motivation and cognition are hallmark symptoms of multiple psychiatric diseases. These symptoms are disruptive to quality of life and often do not improve with available medications. In recent years there has been increased interest in the role of the immune system in neuropsychiatric illness, but to date no immune-related treatment strategies have come to fruition. The cytokine granulocyte-colony stimulating factor (G-CSF) is known to have trophic and neuroprotective properties in the brain, and we recently identified it as a modulator of neuronal and behavioral plasticity. By combining operant tasks that assess discrete aspects of motivated behavior and decision-making in male mice and rats with subsecond dopamine monitoring via fast-scan cyclic voltammetry, we defined the role of G-CSF in these processes as well as the neural mechanism by which it modulates dopamine function to exert these effects. G-CSF enhanced motivation for sucrose as well as cognitive flexibility as measured by reversal learning. These behavioral outcomes were driven by mesolimbic dopamine system plasticity, as systemically administered G-CSF increased evoked dopamine release in the nucleus accumbens independent of clearance mechanisms. Importantly, sustained increases in G-CSF were required for these effects as acute exposure did not enhance behavioral outcomes and decreased dopamine release. These effects seem to be a result of the ability of G-CSF to alter local inflammatory signaling cascades, particularly tumor necrosis factor α. Together, these data show G-CSF as a potent modulator of the mesolimbic dopamine circuit and its ability to appropriately attend to salient stimuli.SIGNIFICANCE STATEMENT Emerging evidence has highlighted the importance of the immune system in psychiatric diseases states. However, the effects of peripheral cytokines on motivation and cognitive function are largely unknown. Here, we report that granulocyte-colony stimulating factor (G-CSF), a pleiotropic cytokine with known trophic and neuroprotective properties in the brain, acts directly on dopaminergic circuits to enhance their function. These changes in dopaminergic dynamics enhance reward learning and motivation for natural stimuli. Together, these results suggest that targeting immune factors may provide a new avenue for therapeutic intervention in the multiple psychiatric disorders that are characterized by motivational and cognitive deficits.

In vivo imaging identifies temporal signature of D1 and D2 medium spiny neurons in cocaine reward.

The reinforcing and rewarding properties of cocaine are attributed to its ability to increase dopaminergic transmission in nucleus accumbens (NAc). This action reinforces drug taking and seeking and leads to potent and long-lasting associations between the rewarding effects of the drug and the cues associated with its availability. The inability to extinguish these associations is a key factor contributing to relapse. Dopamine produces these effects by controlling the activity of two subpopulations of NAc medium spiny neurons (MSNs) that are defined by their predominant expression of either dopamine D1 or D2 receptors. Previous work has demonstrated that optogenetically stimulating D1 MSNs promotes reward, whereas stimulating D2 MSNs produces aversion. However, we still lack a clear understanding of how the endogenous activity of these cell types is affected by cocaine and encodes information that drives drug-associated behaviors. Using fiber photometry calcium imaging we define D1 MSNs as the specific population of cells in NAc that encodes information about drug associations and elucidate the temporal profile with which D1 activity is increased to drive drug seeking in response to contextual cues. Chronic cocaine exposure dysregulates these D1 signals to both prevent extinction and facilitate reinstatement of drug seeking to drive relapse. Directly manipulating these D1 signals using designer receptors exclusively activated by designer drugs prevents contextual associations. Together, these data elucidate the responses of D1- and D2-type MSNs in NAc to acute cocaine and during the formation of context-reward associations and define how prior cocaine exposure selectively dysregulates D1 signaling to drive relapse.

Histone arginine methylation in cocaine action in the nucleus accumbens.

Repeated cocaine exposure regulates transcriptional regulation within the nucleus accumbens (NAc), and epigenetic mechanisms-such as histone acetylation and methylation on Lys residues-have been linked to these lasting actions of cocaine. In contrast to Lys methylation, the role of histone Arg (R) methylation remains underexplored in addiction models. Here we show that protein-R-methyltransferase-6 (PRMT6) and its associated histone mark, asymmetric dimethylation of R2 on histone H3 (H3R2me2a), are decreased in the NAc of mice and rats after repeated cocaine exposure, including self-administration, and in the NAc of cocaine-addicted humans. Such PRMT6 down-regulation occurs selectively in NAc medium spiny neurons (MSNs) expressing dopamine D2 receptors (D2-MSNs), with opposite regulation occurring in D1-MSNs, and serves to protect against cocaine-induced addictive-like behavioral abnormalities. Using ChIP-seq, we identified Src kinase signaling inhibitor 1 (Srcin1; also referred to as p140Cap) as a key gene target for reduced H3R2me2a binding, and found that consequent Srcin1 induction in the NAc decreases Src signaling, cocaine reward, and the motivation to self-administer cocaine. Taken together, these findings suggest that suppression of Src signaling in NAc D2-MSNs, via PRMT6 and H3R2me2a down-regulation, functions as a homeostatic brake to restrain cocaine action, and provide novel candidates for the development of treatments for cocaine addiction.

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.

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.

Extended access of cocaine self-administration results in tolerance to the dopamine-elevating and locomotor-stimulating effects of cocaine.

Tolerance to the neurochemical and psychoactive effects of cocaine after repeated use is a hallmark of cocaine addiction in humans. However, comprehensive studies on tolerance to the behavioral, psychoactive, and neurochemical effects of cocaine following contingent administration in rodents are lacking. We outlined the consequences of extended access cocaine self-administration as it related to tolerance to the psychomotor activating, dopamine (DA) elevating, and DA transporter (DAT) inhibiting effects of cocaine. Cocaine self-administration (1.5 mg/kg/inj; 40 inj; 5 days), which resulted in escalation of first hour intake, caused reductions in evoked DA release and reduced maximal rates of uptake through the DAT as measured by slice voltammetry in the nucleus accumbens core. Furthermore, we report reductions in cocaine-induced uptake inhibition and a corresponding increase in the dose of cocaine required for 50% inhibition of DA uptake (Ki ) at the DAT. Cocaine tolerance at the DAT translated to reductions in cocaine-induced DA overflow as measured by microdialysis. In addition, cocaine-induced elevations in locomotor activity and stereotypy were reduced, while rearing behavior was enhanced in animals with a history of cocaine self-administration. Here, we demonstrate both neurochemical and behavioral cocaine tolerance in an extended-access rodent model of cocaine abuse, which allows for a better understanding of the neurochemical and psychomotor tolerance that develops to cocaine in human addicts. We demonstrate tolerance to the neurochemical and behavioral effects of cocaine following extended-access cocaine self-administration. With respect to neurochemistry, we show reduced cocaine-induced dopamine uptake inhibition, an increased dose of cocaine required for 50% inhibition of the dopamine transporter, and reduced cocaine-induced dopamine overflow. In addition, we show escalation of cocaine intake and reduced cocaine-induced locomotor activity following cocaine self-administration.

Amphetamine potency varies with dopamine uptake rate across striatal subregions.

Amphetamine is a central nervous system psychostimulant with a high potential for abuse. Recent literature has shown that genetic and drug-induced elevations in dopamine transporter (DAT) expression augment the neurochemical and behavioral potency of psychostimulant releasers. However, it remains to be determined if the well-documented differences in DAT levels across striatal regions drive regionally distinct amphetamine effects within individuals. DAT levels and dopamine uptake rates have been shown to follow a gradient in the striatum, with the highest levels in the dorsal regions and lowest levels in the nucleus accumbens shell; thus, we hypothesized that amphetamine potency would follow this gradient. Using fast scan cyclic voltammetry in mouse brain slices, we examined DAT inhibition and changes in exocytotic dopamine release by amphetamine across four striatal regions (dorsal and ventral caudate-putamen, nucleus accumbens core and shell). Consistent with our hypothesis, amphetamine effects at the DAT and on release decreased across regions from dorsal to ventral, and both measures of potency were highly correlated with dopamine uptake rates. Separate striatal subregions are involved in different aspects of motivated behaviors, such as goal-directed and habitual behaviors, that become dysregulated by drug abuse, making it critically important to understand regional differences in drug potencies.

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.