Cocaine-induced Changes Research Articles

Vagus Nerve Stimulation (VNS) Modulates Synaptic Plasticity in the Infralimbic Cortex via Trk-B Receptor Activation to Reduce Drug-Seeking in Male Rats.

Drugs of abuse cause changes in the prefrontal cortex (PFC) and associated regions that impair inhibitory control over drug-seeking. Breaking the contingencies between drug-associated cues and the delivery of the reward during extinction learning reduces relapse. Vagus nerve stimulation (VNS) has previously been shown to enhance extinction learning and reduce drug-seeking. Here we determined the effects of VNS-mediated release of brain-derived neurotrophic factor (BDNF) on extinction and cue-induced reinstatement in male rats trained to self-administer cocaine. Pairing 10 d of extinction training with VNS facilitated extinction and reduced drug-seeking behavior during reinstatement. Rats that received a single extinction session with VNS showed elevated BDNF levels in the medial PFC as determined via an enzyme-linked immunosorbent assay. Systemic blockade of tropomyosin receptor kinase B (TrkB) receptors during extinction, via the TrkB antagonist ANA-12, decreased the effects of VNS on extinction and reinstatement. Whole-cell recordings in brain slices showed that cocaine self-administration induced alterations in the ratio of AMPA and NMDA receptor-mediated currents in Layer 5 pyramidal neurons of the infralimbic cortex (IL). Pairing extinction with VNS reversed cocaine-induced changes in glutamatergic transmission by enhancing AMPAR currents, and this effect was blocked by ANA-12. Our study suggests that VNS consolidates the extinction of drug-seeking behavior by reversing drug-induced changes in synaptic AMPA receptors in the IL, and this effect is abolished by blocking TrkB receptors during extinction, highlighting a potential mechanism for the therapeutic effects of VNS in addiction.

Cannabidiol (CBD) facilitates cocaine extinction and ameliorates cocaine-induced changes to the gut microbiome in male C57BL/6JArc mice

Cocaine use disorder (CUD) is a global health problem with no approved medications. One potential treatment target is the gut microbiome, but it is unknown if cocaine induces long-lasting effects on gut microbes. A novel therapeutic candidate for CUD, cannabidiol (CBD), can improve gut function in rodent models. It is possible that protective effects of CBD against cocaine use are mediated by improving gut health. We examined this question in this experiment. Cocaine conditioned place preference (CPP) was conducted in adult male C57BL/6JArc mice. Mice were treated with vehicle or 20 mg/kg CBD prior to all cocaine CPP sessions (N = 11–13/group). Mice were tested drug free 1, 14 and 28 days after cessation of cocaine and CBD treatment. Fecal samples were collected prior to drug treatment and after each test session. Gut microbiome analyses were conducted using 16 s rRNA sequencing and correlated with behavioural parameters. We found a persistent preference for a cocaine-environment in mice, and long-lasting changes to gut microbe alpha diversity. Cocaine caused persistent changes to beta diversity which lasted for 4 weeks. CBD treatment reduced cocaine-environment preference during abstinence from cocaine and returned gut beta diversity measures to control levels. CBD treatment increased the relative abundance of Firmicutes phyla and Oscillospira genus, but decreased Bacteroidetes phyla and Bacteroides acidifaciens species. Preference score in cocaine-treated mice was positively correlated with abundance of Actinobacteria, whereas in mice treated with CBD and cocaine, the preference score was negatively correlated with Tenericutes abundance. Here we show that CBD facilitates cocaine extinction memory and reverses persistent cocaine-induced changes to gut microbe diversity. Furthermore, CBD increases the abundance of gut microbes which have anti-inflammatory properties. This suggests that CBD may act via the gut to reduce the memory of cocaine reward. Our data suggest that improving gut health and using CBD could limit cocaine abuse.

Impact of cannabidiol on brain glucose metabolism of C57Bl/6 male mice previously exposed to cocaine.

Despite evidence of the beneficial effects of cannabidiol (CBD) in animal models of cocaine use disorder (CUD), CBD neuronal mechanisms remain poorly understood. This study investigated the effects of CBD treatment on brain glucose metabolism, in a CUD animal model, using [18F]FDG positron emission tomography (PET). Male C57Bl/6 mice were injected with cocaine (20 mg/kg, i.p.) every other day for 9 days, followed by 8 days of CBD administration (30 mg/kg, i.p.). After 48 h, animals were challenged with cocaine. Control animals received saline/vehicle. [18F]FDG PET was performed at four time points: baseline, last day of sensitization, last day of withdrawal/CBD treatment, and challenge. Subsequently, the animals were euthanized and immunohistochemistry was performed on the hippocampus and amygdala to assess the CB1 receptors, neuronal nuclear protein, microglia (Iba1), and astrocytes (GFAP). Results showed that cocaine administration increased [18F]FDG uptake following sensitization. CBD treatment also increased [18F]FDG uptake in both saline and cocaine groups. However, animals that were sensitized and challenged with cocaine, and those receiving only an acute cocaine injection during the challenge phase, did not exhibit increased [18F]FDG uptake when treated with CBD. Furthermore, CBD induced modifications in the integrated density of NeuN, Iba, GFAP, and CB1R in the hippocampus and amygdala. This is the first study addressing the impact of CBD on brain glucose metabolism in a preclinical model of CUD using PET. Our findings suggest that CBD disrupts cocaine-induced changes in brain energy consumption and activity, which might be correlated with alterations in neuronal and glial function.

Microglia-mediated calcium-permeable AMPAR accumulation in the nucleus accumbens drives hyperlocomotion during cocaine withdrawal

During withdrawal from cocaine, calcium permeable-AMPA receptors (CP-AMPAR) progressively accumulate in nucleus accumbens (NAc) synapses, a phenomenon linked to behavioral sensitization and drug-seeking. Recently, it has been suggested that neuroimmune alterations might promote aberrant changes in synaptic plasticity, thus contributing to substance abuse-related behaviors. Here, we investigated the role of microglia in NAc neuroadaptations after withdrawal from cocaine-induced conditioned place preference (CPP). We depleted microglia using PLX5622-supplemented diet during cocaine withdrawal, and after the place preference test, we measured dendritic spine density and the presence of CP-AMPAR in the NAc shell. Microglia depletion prevented cocaine-induced changes in dendritic spines and CP-AMPAR accumulation. Furthermore, microglia depletion prevented conditioned hyperlocomotion without affecting drug-context associative memory. Microglia displayed fewer number of branches, resulting in a reduced arborization area and microglia control domain at late withdrawal. Our results suggest that microglia are necessary for the synaptic adaptations in NAc synapses during cocaine withdrawal and therefore represent a promising therapeutic target for relapse prevention.

Cocaine Exposure Increases Excitatory Synaptic Transmission and Intrinsic Excitability in the Basolateral Amygdala in Male and Female Rats and across the Estrous Cycle

Introduction: Sex and ovarian hormones influence cocaine seeking and relapse vulnerability, but less is known regarding the cellular and synaptic mechanisms contributing to these behavioral sex differences. One factor thought to influence cue-induced seeking behavior following withdrawal is cocaine-induced changes in the spontaneous activity of pyramidal neurons in the basolateral amygdala (BLA). However, the mechanisms underlying these changes, including potential sex or estrous cycle effects, are unknown. Methods: Ex vivo whole-cell patch clamp electrophysiology was conducted to investigate the effects of cocaine exposure, sex, and estrous cycle fluctuations on two properties that can influence spontaneous activity of BLA pyramidal neurons: (1) frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and (2) intrinsic excitability. Recordings of BLA pyramidal neurons were conducted in adult male and female rats and across the estrous cycle following 2–4 weeks of withdrawal from extended-access cocaine self-administration (6 h/day for 10 days) or drug-naïve conditions. Results: In both sexes, cocaine exposure increased the frequency, but not amplitude, of sEPSCs and neuronal intrinsic excitability. Across the estrous cycle, sEPSC frequency and intrinsic excitability were significantly elevated only in cocaine-exposed females in the estrus stage of the cycle, a stage when cocaine-seeking behavior is known to be enhanced. Conclusions: Here, we identify potential mechanisms underlying cocaine-induced alterations in the spontaneous activity of BLA pyramidal neurons in both sexes along with changes in these properties across the estrous cycle.

Evidence for Phosphorylation-Dependent, Dynamic, Regulation of mGlu5 and Homer2 in Expression of Cocaine Aversion in Mice.

Cocaine-induced changes in the expression of the glutamate-related scaffolding protein Homer2 influence this drug's psychostimulant and rewarding properties. In response to neuronal activity, Homer2 is phosphorylated on S117/S216 by calcium-calmodulin kinase IIα (CaMKIIα), which induces a rapid dissociation of mGlu5-Homer2 scaffolds. Herein, we examined the requirement for Homer2 phosphorylation in cocaine-induced changes in mGlu5-Homer2 coupling, to include behavioral sensitivity to cocaine. For this, mice with alanine point mutations at (S117/216)-Homer2 (Homer2AA/AA ) were generated, and we determined their affective, cognitive and sensorimotor phenotypes, as well as cocaine-induced changes in conditioned reward and motor hyperactivity. The Homer2AA/AA mutation prevented activity-dependent phosphorylation of S216 Homer2 in cortical neurons, but Homer2AA/AA mice did not differ from wild-type (WT) controls with respect to Morris maze performance, acoustic startle, spontaneous or cocaine-induced locomotion. Homer2AA/AA mice exhibited signs of hypoanxiety similar to the phenotype of transgenic mice with a deficit in signal-regulated mGluR5 phosphorylation (Grm5AA/AA ). However, opposite of Grm5AA/AA mice, Homer2AA/AA mice were less sensitive to the aversive properties of high-dose cocaine under both place-conditioning and taste-conditioning procedures. Acute injection with cocaine caused dissociation of mGluR5 and Homer2 in striatal lysates from WT, but not Homer2AA/AA mice, suggesting a molecular basis for the deficit in cocaine aversion. These findings indicate that CaMKIIα-dependent phosphorylation of Homer2 gates the negative motivational valence of high-dose cocaine via regulation of mGlu5 binding, furthering an important role for dynamic changes in mGlu5-Homer interactions in addiction vulnerability.

A network-based analysis detects cocaine-induced changes in social interactions in Drosophila melanogaster.

Addiction is a multifactorial biological and behavioral disorder that is studied using animal models, based on simple behavioral responses in isolated individuals. A couple of decades ago it was shown that Drosophila melanogaster can serve as a model organism for behaviors related to alcohol, nicotine and cocaine (COC) addiction. Scoring of COC-induced behaviors in a large group of flies has been technologically challenging, so we have applied a local, middle and global level of network-based analyses to study social interaction networks (SINs) among a group of 30 untreated males compared to those that have been orally administered with 0.50 mg/mL of COC for 24 hours. In this study, we have confirmed the previously described increase in locomotion upon COC feeding. We have isolated new network-based measures associated with COC, and influenced by group on the individual behavior. COC fed flies showed a longer duration of interactions on the local level, and formed larger, more densely populated and compact, communities at the middle level. Untreated flies have a higher number of interactions with other flies in a group at the local level, and at the middle level, these interactions led to the formation of separated communities. Although the network density at the global level is higher in COC fed flies, at the middle level the modularity is higher in untreated flies. One COC specific behavior that we have isolated was an increase in the proportion of individuals that do not interact with the rest of the group, considered as the individual difference in COC induced behavior and/or consequence of group influence on individual behavior. Our approach can be expanded on different classes of drugs with the same acute response as COC to determine drug specific network-based measures and could serve as a tool to determinate genetic and environmental factors that influence both drug addiction and social interaction.

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.

Rewiring of Prelimbic Inputs to the Nucleus Accumbens Core Underlies Cocaine-Induced Behavioral Sensitization

BackgroundUnbalanced activity of medium spiny neurons (MSNs) of the direct and indirect pathways mediates reward-related behaviors induced by addictive drugs. Prelimbic (PL) input to MSNs in the nucleus accumbens core (NAcC) plays a key role in cocaine-induced early locomotor sensitization (LS). However, the adaptive plastic changes at PL-to-NAcC synapses underlying early LS remain unclear. MethodsUsing transgenic mice and retrograde tracing, we identified NAcC-projecting pyramidal neurons (PNs) in the PL cortex based on the expression of dopamine receptor types (D1R or D2R). To examine cocaine-induced alterations in PL-to-NAcC synapses, we measured excitatory postsynaptic current amplitudes evoked by optostimulation of PL afferents to MSNs. Riluzole was chosen to test the effects of PL excitability on cocaine-induced changes of PL-to-NAcC synapses. ResultsNAcC-projecting PNs were segregated into D1R- and D2R-expressing PNs (D1- and D2-PNs, respectively), and their excitability was opposingly regulated by respective dopamine agonists. Both D1- and D2-PNs exhibited balanced innervation of direct MSNs and indirect MSNs in naïve animals. Repeated cocaine injections resulted in biased synaptic strength toward direct MSNs through presynaptic mechanisms in both D1- and D2-PNs, although D2R activation reduced the D2-PN excitability. Under group 1 metabotropic glutamate receptors coactivation, however, D2R activation enhanced the D2-PN excitability. The cocaine-induced rewiring accompanied LS, and both rewiring and LS were precluded by PL infusion of riluzole, which reduced the intrinsic excitability of PL neurons. ConclusionsThese findings indicate that cocaine-induced rewiring of PL-to-NAcC synapses correlates well with early behavioral sensitization and that rewiring and LS can be prevented by riluzole-induced reduction of excitability of PL neurons.

The medial preoptic area and acute cocaine's stimulant effects in rats: Potential influences of estradiol and biological sex

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA's role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either ‘low-estradiol’ or ‘high-estradiol’ based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA's role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Impact of Perineuronal Net Removal in the Rat Medial Prefrontal Cortex on Parvalbumin Interneurons After Reinstatement of Cocaine Conditioned Place Preference.

Parvalbumin (PV)-positive cells are GABAergic fast-spiking interneurons that modulate the activity of pyramidal neurons in the medial prefrontal cortex (mPFC) and their output to brain areas associated with learning and memory. The majority of PV cells within the mPFC are surrounded by a specialized extracellular matrix structure called the perineuronal net (PNN). We have shown that removal of PNNs with the enzyme chondroitinase-ABC (Ch-ABC) in the mPFC prevents the consolidation and reconsolidation of cocaine-associated conditioned place preference (CPP) memories. Here we examined the extent to which retrieval of a CPP memory during cocaine-primed reinstatement altered the levels and function of PV neurons and their surrounding PNNs during the reconsolidation period. We further determined the extent to which PNN removal prior to reinstatement altered PV intensity levels and PV cell function. Male Sprague-Dawley rats were trained for cocaine-induced conditioned place preference (CPP) followed by extinction training, microinjection of Ch-ABC in the prelimbic PFC, and cocaine-induced reinstatement. Rats were sacrificed immediately prior to reinstatement or at 2 h, 6 h, or 48 h after reinstatement for immunohistochemistry or 2 h later for electrophysiology. Our findings indicate that PNN removal only partially diminished reinstatement. Cocaine-primed reinstatement produced only minor changes in PNN or PV intensity in vehicle controls. However, after PNN removal, the intensity of remaining PNN-surrounded PV cells was decreased at all times except at 2 h post-reinstatement, at which time cocaine increased PV intensity. Consistent with this, in vehicle controls, PV neurons naturally devoid of PNNs showed a similar pattern to Ch-ABC-treated rats prior to and after cocaine reinstatement, suggesting a protective effect of PNNs on cocaine-induced changes in PV intensity. Using whole-cell patch-clamp, cocaine-primed reinstatement in Ch-ABC-treated rats decreased the number of elicited action potentials but increased excitatory synaptic transmission, which may have been compensatory. These findings suggest that without PNNs, cocaine-induced reinstatement produces rapid changes in PV intensity and PV cell excitability, which may in turn regulate output of the mPFC post-memory retrieval and diminish the maintenance of cocaine memory during reconsolidation.

18 F]FDG brain uptake of C57Bl/6 male mice is affected by locomotor activity after cocaine use: A small animal positron emission tomography study.

We verified if cocaine-induced peripheral activation might disrupt [18 F]FDG brain uptake after a cocaine challenge and suggested an optimal protocol to measure cocaine-induced brain metabolic alterations in mice. C57Bl/6 male mice were injected with [18 F]FDG and randomly separated into three groups. Groups 1 and 2 were kept conscious after [18 F]FDG administration and after 5 min received saline or cocaine (20 mg/kg). The animals in group 1 (n= 5) were then evaluated in the open field for 30 min and those from group 2 (n= 6) were kept alone in a home cage for the same period. Forty-five minutes after [18 F]FDG administration, images were acquired for 30 min. Group 3 (n= 6) was kept anesthetized and image acquisition started immediately after tracer injection, for 75min. Saline (Day 1) or cocaine (Day 2) was injected 5 min after starting acquisition. Another set of animals (n= 5) were treated with cocaine every other day for 10 days or saline (n= 6) and were scanned with the dynamic protocol to verify its efficacy. [18 F]FDG uptake increased after cocaine administration when compared to baseline only in animals kept under anesthesia. No brain effect of cocaine was observed in animals submitted to the open field or kept in the home cage. The use of anesthesia is essential to visualize cocaine-induced changes in brain metabolism by [18 F]FDG PET, providing an interesting preclinical approach to investigate naïve subjects and enabling a bidirectional translational science approach for better understanding of cocaine use disorder.

Cocaine-Induced Changes in Sperm Cdkn1a Methylation Are Associated with Cocaine Resistance in Male Offspring.

Paternal environmental perturbations can influence the physiology and behavior of offspring. For example, our previous work showed reduced cocaine reinforcement in male, but not female, progeny of rat sires that self-administered cocaine. The information transfer from sire to progeny may occur through epigenetic marks in sperm, encompassing alterations in small noncoding RNAs, including microRNAs (miRNAs) and/or DNA methylation. Here, no reliable changes in miRNAs in the sperm of cocaine- relative to saline-experienced sires were identified. In contrast, 272 differentially methylated regions were observed in sperm between these groups. Two hypomethylated promoter regions in the sperm of cocaine-experienced rats were upstream of cyclin-dependent kinase inhibitor 1a (Cdkn1a). Cdkn1a mRNA also was selectively increased in the NAc of cocaine-sired male (but not female) offspring. Cocaine self-administration also enhanced Cdkn1a expression in the accumbens of cocaine-sired rats. These results suggest that changes in Cdkn1a may play a role in the reduced cocaine reinforcing efficacy observed in cocaine-sired male rats. Introducing a 90 d delay between sire self-administration and breeding reversed both cocaine resistance and the increase in accumbens Cdkn1a mRNA in male offspring, indicating that cocaine-induced epigenetic modifications are eliminated with sperm turnover. Collectively, our results indicate that cocaine self-administration produces hypomethylation of Cdkn1a in sperm and a selective increase in the expression of this gene in the NAc of male offspring, which is associated with blunted cocaine reinforcement.SIGNIFICANCE STATEMENT The relatively new field of transgenerational epigenetics explores the effects of environmental perturbations on offspring behavior and physiology. Our prior work in rats indicated that male, but not female, progeny of sires that self-administered cocaine displayed reduced cocaine reinforcement. The information transfer from sire to progeny may occur through heritable epigenetic marks in sperm, including DNA methylation. The present findings revealed two hypomethylated promoter regions upstream of the Cdkn1a gene in sire sperm. Remarkably, Cdkn1a expression was selectively decreased in offspring NAc, a brain region that regulates cocaine reinforcement.

Ih blockade reduces cocaine-induced firing patterns of putative dopaminergic neurons of the ventral tegmental area in the anesthetized rat

The hyperpolarization-activated cation current (Ih) is a determinant of intrinsic excitability in various cells, including dopaminergic neurons (DA) of the ventral tegmental area (VTA). In contrast to other cellular conductances, Ih is activated by hyperpolarization negative to -55 mV and activating Ih produces a time-dependent depolarizing current. Our laboratory demonstrated that cocaine sensitization, a chronic cocaine behavioral model, significantly reduces Ih amplitude in VTA DA neurons. Despite this reduction in Ih, the spontaneous firing of VTA DA cells after cocaine sensitization remained similar to control groups. Although the role of Ih in controlling VTA DA excitability is still poorly understood, our hypothesis is that Ih reduction could play a role of a homeostatic controller compensating for cocaine-induced change in excitability. Using in vivo single-unit extracellular electrophysiology in isoflurane anesthetized rats, we explored the contribution of Ih on spontaneous firing patterns of VTA DA neurons. A key feature of spontaneous excitability is bursting activity; bursting is defined as trains of two or more spikes occurring within a short interval and followed by a prolonged period of inactivity. Burst activity increases the reliability of information transfer. To elucidate the contribution of Ih to spontaneous firing patterns of VTA DA neurons, we locally infused an Ih blocker (ZD 7288, 8.3 μM) and evaluated its effect. Ih blockade significantly reduced firing rate, bursting frequency, and percent of spikes within a burst. In addition, Ih blockade significantly reduced acute cocaine-induced spontaneous firing rate, bursting frequency, and percent of spikes within a burst. Using whole-cell patch-clamp, we determine the progressive reduction of Ih after acute and chronic cocaine administration (15 mg/k.g intraperitoneally). Our data show a significant reduction (~25%) in Ih amplitude after 24 but not 2 h of acute cocaine administration. These results suggest that a progressive reduction of Ih could serve as a homeostatic regulator of cocaine-induced spontaneous firing patterns related to VTA DA excitability.

Cocaine-Induced Changes in Tonic Dopamine Concentrations Measured Using Multiple-Cyclic Square Wave Voltammetry in vivo.

For over 40 years, in vivo microdialysis techniques have been at the forefront in measuring the effects of illicit substances on brain tonic extracellular levels of dopamine that underlie many aspects of drug addiction. However, the size of microdialysis probes and sampling rate may limit this technique’s ability to provide an accurate assessment of drug effects in microneural environments. A novel electrochemical method known as multiple-cyclic square wave voltammetry (M-CSWV), was recently developed to measure second-to-second changes in tonic dopamine levels at microelectrodes, providing spatiotemporal resolution superior to microdialysis. Here, we utilized M-CSWV and fast-scan cyclic voltammetry (FSCV) to measure changes in tonic or phasic dopamine release in the nucleus accumbens core (NAcc) after acute cocaine administration. Carbon-fiber microelectrodes (CFM) and stimulating electrodes were implanted into the NAcc and medial forebrain bundle (MFB) of urethane anesthetized (1.5 g/kg i.p.) Sprague-Dawley rats, respectively. Using FSCV, depths of each electrode were optimized by determining maximal MFB electrical stimulation-evoked phasic dopamine release. Changes in phasic responses were measured after a single dose of intravenous saline or cocaine hydrochloride (3 mg/kg; n = 4). In a separate group, changes in tonic dopamine levels were measured using M-CSWV after intravenous saline and after cocaine hydrochloride (3 mg/kg; n = 5). Both the phasic and tonic dopamine responses in the NAcc were augmented by the injection of cocaine compared to saline control. The phasic and tonic levels changed by approximately x2.4 and x1.9, respectively. These increases were largely consistent with previous studies using FSCV and microdialysis. However, the minimal disruption/disturbance of neuronal tissue by the CFM may explain why the baseline tonic dopamine values (134 ± 32 nM) measured by M-CSWV were found to be 10-fold higher when compared to conventional microdialysis. In this study, we demonstrated phasic dopamine dynamics in the NAcc with acute cocaine administration. M-CSWV was able to record rapid changes in tonic levels of dopamine, which cannot be achieved with other current voltammetric techniques. Taken together, M-CSWV has the potential to provide an unprecedented level of physiologic insight into dopamine signaling, both in vitro and in vivo, which will significantly enhance our understanding of neurochemical mechanisms underlying psychiatric conditions.

Neuroadaptations associated with cocaine sensitization in putative DA neurons of the VTA are reverted using an HDACi

Drugs of abuse are known to cause long-term changes in the brain that possibly contribute to the individuals vulnerability to develop addiction. These modifications of brain function are mediated by epigenetic modifications in gene expression, chromatin remodeling being one of them. One common type of chromatin remodeling is histone acetylation, which is under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs). It has been shown that cocaine sensitization alters the intrinsic properties of dopamine (DA) neurons of the ventral tegmental area (VTA), which in turn alter dopamine transmission in the mesocorticolimbic network. One intrinsic property of VTA neurons affected by exposure of drugs of abuse is the hyperpolarization-activated cation current (Ih), which is mediated by HCN channels. Previously, our laboratory has demonstrated a reduction in Ih in VTA DA neurons from cocaine-sensitized rats. Little is known about epigenetic regulation of ion channels by drug exposure, and no studies of epigenetic regulation of h-current have been performed. Using the whole-cell patch clamp technique, we investigated the effects of an HDAC inhibitor (SAHA) on cocaine-induced changes in Ih current. Additionally, we explored the effect of SAHA on rebound excitation in brain slices from cocaine-sensitized rats. In vitro incubation of midbrain slices with SAHA (3 µM for two hours) reversed the cocaine-induced reduction in VTA DA Ih to baseline levels. Current clamp traces also demonstrated SAHA reversal of cocaine-induced Ih reduction concomitant with a reduced action potential firing. These results support the idea that the decreased h-current in VTA DA neurons after cocaine sensitization is epigenetically regulated, and suggests the possibility that HDAC inhibitors could reverse cocaine-induced neuroadaptations in reward circuits.

Acute cocaine exposure occludes long-term depression in ventral tegmental area GABA neurons

The ventral tegmental area (VTA) in the midbrain is essential in incentive salience of reward behavior. Drugs of abuse increase midbrain dopamine cell activity and/or dopamine levels, and can alter endogenous VTA glutamate plasticity, leading to addiction or dependence. VTA dopamine cells are regulated by local inhibitory GABA cells, which exhibit a form of pre-synaptic cannabinoid receptor 1-dependent long-term depression of their glutamatergic inputs. Our current aim was to determine cocaine's influence on VTA GABA cell glutamate plasticity and circuity. Using whole cell voltage-clamp electrophysiology in VTA slices of GAD67-GFP knock-in mice, we recorded excitatory inputs on VTA GABA cells. Acute and chronic injections of cocaine were sufficient to occlude long-term depression. The plasticity could be reversed to the naïve state however, as long-term depression was again observed following a 7-day abstinence from acute cocaine exposure. Furthermore, chronic cocaine decreased AMPA/NMDA ratios at glutamate synapses onto VTA GABA cells, compared to vehicle injection controls, the opposite change noted in dopamine cells. Collectively, our data suggest the cellular mechanism of cocaine-mediated synaptic modification that may result in dependence/withdrawal could involve changes in glutamate input to VTA GABA circuitry in addition to VTA dopamine cells. Therefore VTA GABA cells may also play a role, possibly in a synergistic manner with the dopamine circuit, in cocaine-induced changes to the VTA reward pathway than previously known.

HDAC3 Activity within the Nucleus Accumbens Regulates Cocaine-Induced Plasticity and Behavior in a Cell-Type-Specific Manner

Epigenetic mechanisms regulate processes of neuroplasticity critical to cocaine-induced behaviors. This includes the Class I histone deacetylase (HDAC) HDAC3, known to act as a negative regulator of cocaine-associated memory formation within the nucleus accumbens (NAc). Despite this, it remains unknown how cocaine alters HDAC3-dependent mechanisms. Here, we profiled HDAC3 expression and activity in total NAc mouse tissue following cocaine exposure. Although chronic cocaine did not affect expression of Hdac3 within the NAc, chronic cocaine did affect promoter-specific changes in HDAC3 and H4K8Ac occupancy. These changes in promoter occupancy correlated with cocaine-induced changes in expression of plasticity-related genes. To causally determine whether cocaine-induced plasticity is mediated by HDAC3's deacetylase activity, we overexpressed a deacetylase-dead HDAC3 point mutant (HDAC3-Y298H-v5) within the NAc of adult male mice. We found that disrupting HDAC3's enzymatic activity altered selective changes in gene expression and synaptic plasticity following cocaine exposure, despite having no effects on cocaine-induced behaviors. In further assessing HDAC3's role within the NAc, we observed that chronic cocaine increases Hdac3 expression in Drd1 but not Drd2-cells of the NAc. Moreover, we discovered that HDAC3 acts selectively within D1R cell-types to regulate cocaine-associated memory formation and cocaine-seeking. Overall, these results suggest that cocaine induces cell-type-specific changes in epigenetic mechanisms to promote plasticity important for driving cocaine-related behaviors.SIGNIFICANCE STATEMENT Drugs of abuse alter molecular mechanisms throughout the reward circuitry that can lead to persistent drug-associated behaviors. Epigenetic regulators are critical drivers of drug-induced changes in gene expression. Here, we demonstrate that the activity of an epigenetic enzyme promotes neuroplasticity within the nucleus accumbens (NAc) critical to cocaine action. In addition, we demonstrate that these changes in epigenetic activity drive cocaine-seeking behaviors in a cell-type-specific manner. These findings are key in understanding and targeting cocaine's impact of neural circuitry and behavior.

Dopamine neurons gate the intersection of cocaine use, decision making, and impulsivity.

Gambling and substance use disorders are highly comorbid. Both clinical populations are impulsive and exhibit risky decision-making. Drug-associated cues have long been known to facilitate habitual drug-seeking, and the salient audiovisual cues embedded within modern gambling products may likewise encourage problem gambling. The dopamine neurons of the ventral tegmental area (VTA) are exquisitely sensitive to drugs of abuse, uncertain rewards, and reward-paired cues and may therefore be the common neural substrate mediating synergistic features of both disorders. To test this hypothesis, we first gained specific inhibitory control over VTA dopamine neurons by transducing a floxed inhibitory DREADD (AAV5-hSyn-DIO-hM4D(Gi)-mCherry) in rats expressing Cre recombinase in tyrosine hydroxylase neurons. We then trained rats in our cued rat gambling task (crGT), inhibiting dopamine neurons throughout task acquisition and performance, before allowing them to self-administer cocaine in the same diurnal period as crGT sessions. The trajectories of addiction differ in women and men, and the dopamine system may differ functionally across the sexes; therefore, we used male and female rats here. We found that inhibition of VTA dopamine neurons decreased cue-induced risky choice and reduced motor impulsivity in males, but surprisingly, enhanced risky decision making in females. Inhibiting VTA dopamine neurons also prevented cocaine-induced changes in decision making in both sexes, but nevertheless drove all animals to consume more cocaine. These findings show that chronic dampening of dopamine signalling can have both protective and deleterious effects on addiction-relevant behaviours, depending on biological sex and dependent variable of interest.

Serotonin2B receptor blockade in the rat dorsal raphe nucleus suppresses cocaine-induced hyperlocomotion through an opposite control of mesocortical and mesoaccumbens dopamine pathways

Serotonin2B receptor (5-HT2BR) antagonists inhibit cocaine-induced hyperlocomotion independently of changes of accumbal dopamine (DA) release. Given the tight relationship between accumbal DA activity and locomotion, and the inhibitory role of medial prefrontal cortex (mPFC) DA on subcortical DA neurotransmission and DA-dependent behaviors, it has been suggested that the suppressive effect of 5-HT2BR antagonists on cocaine-induced hyperlocomotion may result from an activation of mPFC DA outflow which would subsequently inhibit accumbal DA neurotransmission. Here, we tested this hypothesis by means of the two selective 5-HT2BR antagonists, RS 127445 and LY 266097, using a combination of neurochemical, behavioral and cellular approaches in male rats.The intraperitoneal (i.p.) administration of RS 127445 (0.16 mg/kg) or LY 266097 (0.63 mg/kg) potentiated cocaine (10 mg/kg, i.p.)-induced mPFC DA outflow. The suppressant effect of RS 127445 on cocaine-induced hyperlocomotion was no longer observed in rats with local 6-OHDA lesions in the mPFC. Also, RS 127445 blocked cocaine-induced changes of accumbal glycogen synthase kinase (GSK) 3β phosphorylation, a postsynaptic cellular marker of DA neurotransmission. Finally, in keeping with the location of 5-HT2BRs on GABAergic interneurons in the dorsal raphe nucleus (DRN), the intra-DRN perfusion of the GABAAR antagonist bicuculline (100 μM) prevented the effect of the systemic or local (1 μM, intra-DRN) administration of RS 127445 on cocaine-induced mPFC DA outflow. Likewise, intra-DRN bicuculline injection (0.1 μg/0.2 μl) prevented the effect of the systemic RS 127445 administration on cocaine-induced hyperlocomotion and GSK3β phosphorylation.These results show that DRN 5-HT2BR blockade suppresses cocaine-induced hyperlocomotion by potentiation of cocaine-induced DA outflow in the mPFC and the subsequent inhibition of accumbal DA neurotransmission.