Search

Dopamine D2 receptors (D2Rs) in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) are associated with vulnerability to addiction; however, whether D2Rs in these two brain regions play differential roles in regulation of drug intake is unknown. Here, we compared the effect of decreased mRNA level of Drd2 in each region on cocaine self-administration in a dose-response function. Drd2 mRNA levels in rat VTA or NAc were knocked down by bilateral microinjection of lentivirus coding shRNAs against rat Drd2 or scrambled shRNA. Drd2 knockdown was persistent and stable between 20 and 90 days after lentiviral infection. Animals were trained to self-administer cocaine 20 days after Drd2 shRNA treatment. Compared to scrambled shRNA treated rats, Drd2 knockdown in the VTA increased cocaine self-administration at all tested doses (0.02-0.56 mg/kg/infusion) producing an upward shift (both the ascending and descending limb) in the dose-response curve of cocaine self-administration. In contrast, intra-NAc knockdown increased cocaine self-administration only on the ascending limb of the dose-response curve (0.02-0.07 mg/kg/infusion). These data suggest that D2Rs in the VTA, not in the NAc, regulate high-dose cocaine intake. The present study not only demonstrates that low levels of D2Rs in either region increase low doses of cocaine intake, but also reveals for the first time their dissociable roles in limiting high doses of cocaine self-administration., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Concurrent use of cocaine and heroin (speedball) has been shown to exert synergistic effects on dopamine neurotransmission in the nucleus accumbens (NAc), as observed by significant increases in extracellular dopamine levels and compensatory elevations in the maximal reuptake rate of dopamine. The present studies were undertaken to determine whether chronic self-administration of cocaine, heroin or a combination of cocaine:heroin led to compensatory changes in the abundance and/or affinity of high- and low-affinity DAT binding sites. Saturation binding of the cocaine analog [(125) I] 3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester ([(125) I]RTI-55) in rat NAc membranes resulted in binding curves that were best fit to two-site binding models, allowing calculation of dissociation constant (Kd ) and binding density (Bmax ) values corresponding to high- and low-affinity DAT binding sites. Scatchard analysis of the saturation binding curves clearly demonstrate the presence of high- and low- affinity binding sites in the NAc, with low-affinity sites comprising 85 to 94% of the binding sites. DAT binding analyses revealed that self-administration of cocaine and a cocaine:heroin combination increased the affinity of the low-affinity site for the cocaine congener RTI-55 compared to saline. These results indicate that the alterations observed following chronic speedball self-administration are likely due to the cocaine component alone; thus further studies are necessary to elaborate upon the synergistic effect of cocaine:heroin combinations on the dopamine system in the NAc., (© 2014 Wiley Periodicals, Inc.)

Cocaine/heroin combinations (speedball) exert synergistic neurochemical and behavioral effects that are thought to contribute to the increased abuse potential and subjective effects reported by polydrug users. In vivo fast-scan cyclic voltammetry was used to examine the effects of chronic intravenous self-administration (25 consecutive sessions) of cocaine (250 μg/inf), heroin (4.95 μg/inf) and speedball (250/4.95 μg/inf cocaine/heroin) on changes in electrically evoked dopamine (DA) efflux, maximal rate of DA uptake (V(max)) and the apparent affinity (K(m)) of the DA transporter in the nucleus accumbens. The increase in electrically evoked DA was comparable following cocaine and speedball injection; however, heroin did not increase evoked DA. DA transporter K(m) values were similarly elevated following cocaine and speedball, but unaffected by heroin. However, speedball self-administration significantly increased baseline V(max), while heroin and cocaine did not change baseline V(max), compared with the baseline V(max) values of drug-naïve animals. Overall, elevated DA clearance is a likely consequence of synergistic elevations of nucleus accumbens extracellular DA concentrations by chronic speedball self-administration, as reported previously in microdialysis studies. The present results indicate neuroadaptive processes that are unique to cocaine/heroin combinations and cannot be readily explained by simple additivity of changes observed with cocaine and heroin alone., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Cocaine/heroin combinations (speedball) induce a synergistic elevation in extracellular dopamine concentrations ([DA](e)) in the nucleus accumbens (NAc) that can explain the increased abuse liability of speedball. To further delineate the mechanism of this neurochemical synergism, in vivo fast-scan cyclic voltammetry (FSCV) was used to compare NAc DA release and reuptake kinetic parameters following acute administration of cocaine, heroin and speedball in drug-naïve rats. These parameters were extracted from accumbal DA overflow induced by electrical stimulation of the ventral tegmental area. Evoked DA efflux was increased following both cocaine and speedball delivery, whereas heroin did not significantly change evoked DA release from baseline. DA efflux was significantly greater following cocaine compared to speedball. However, DA transporter (DAT) apparent affinity (K(m)) values were similarly elevated following cocaine and speedball administration, but unaffected by heroin. Neither drug induced substantial changes in the maximal reuptake rate (V(max)). These data, combined with published microdialysis and electrophysiological results, indicate that the combination of cocaine-induced competitive inhibition of DAT and the increase in the DA release elicited by heroin is responsible for the synergistic increase in ([DA](e)) induced by speedball., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The reinforcing effects and long-term consequences of cocaine self-administration have been associated with brain regions of the mesolimbic dopamine pathway, namely the nucleus accumbens (NAc). Studies of cocaine-induced biochemical adaptations in rodent models have advanced our knowledge; however, unbiased detailed assessments of intracellular alterations in the primate brain are scarce, yet essential, to develop a comprehensive understanding of cocaine addiction. To this end, two-dimensional difference in gel electrophoresis (2D-DIGE) was used to compare changes in cytosolic protein abundance in the NAc between rhesus monkeys self-administering cocaine and controls. Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser-desorption ionization time-of-flight time-of-flight (MALDI-TOF-TOF). In total, 1098 spots were subjected to statistical analysis with 22 spots found to be differentially abundant of which 18 proteins were positively identified by mass spectrometry. In addition, approximately 1000 protein spots were constitutively expressed of which 21 proteins were positively identified by mass spectrometry. Increased levels of proteins in the cocaine-exposed monkeys include glial fibrillary acidic protein, syntaxin-binding protein 3, protein kinase C isoform, adenylate kinase isoenzyme 5 and mitochondrial-related proteins, whereas decreased levels of proteins included beta-soluble N-ethylmaleimide-sensitive factor attachment protein and neural and non-neural enolase. Using a complimentary proteomics approach, the differential expression of phosphorylated proteins in the cytosolic fraction of these subjects was examined. Two-dimensional gel electrophoresis (2DGE) was followed by gel staining with Pro-Q Diamond phosphoprotein gel stain, enabling differentiation of approximately 150 phosphoprotein spots between the groups. Following excision and trypsin digestions, MALDI-TOF-TOF was used to confirm the identity of 15 cocaine-altered phosphoproteins. Significant increased levels were detected for gamma-aminobutyric acid type A receptor-associated protein 1, 14-3-3 gamma-protein, glutathione S-transferase and brain-type aldolase, whereas significant decreases were observed for beta-actin, Rab GDP-dissociation inhibitor, guanine deaminase, peroxiredoxin 2 isoform b and several mitochondrial proteins. Results from these studies indicate coordinated dysregulation of proteins related to cell structure, signaling, metabolism and mitochondrial function. These data extend and compliment previous studies of cocaine-induced biochemical alterations in human postmortem brain tissue, using an animal model that closely recapitulates the human condition and provide new insight into the molecular basis of the disease and potential targets for pharmacotherapeutic intervention.

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in the nucleus accumbens (NAc), a brain region associated with drug reinforcement. Two-dimensional gel electrophoresis was used to compare protein alterations in the NAc between cocaine overdose (COD) victims (n=10) and controls (n=10). Following image normalization, spots with significantly differential image intensities (P<0.05) were identified, excised, trypsin digested and analyzed by matrix-assisted laser desorption ionization-time of flight-time of flight. A total of 1407 spots were found to be present in a minimum of five subjects per group and the intensity of 18 spots was found to be differentially abundant between the groups, leading to positive identification of 15 proteins by peptide mass fingerprinting (PMF). Of an additional 37 protein spots that were constitutively expressed, 32 proteins were positively identified by PMF. Increased proteins in COD included beta-tubulin, liprin-alpha3 and neuronal enolase, whereas decreased proteins included parvalbumin, ATP synthase beta-chain and peroxiredoxin 2. The present data provide a preliminary protein profile of COD, suggesting the involvement of novel proteins and pathways in the expression of this complex disease. Additional studies are warranted to further characterize alterations in the differentially regulated proteins. Understanding the coordinated involvement of multiple proteins in cocaine abuse provides insight into the molecular basis of the disease and offers new targets for pharmacotherapeutic intervention for drug abuse-related disorders.

The concurrent use of cocaine and opiate combinations (speedball) has increased since the 1970s and now represents a growing subset of intravenous drug abusers. An isobolographic analysis was applied to the ascending limb of the dose-effect curves for rat self-administration of cocaine, heroin, and their combination to determine the nature of the interaction. The addition of heroin to cocaine shifted the dose-effect curve for self-administration to the left, and the modulation in reinforcing efficacy of the combination of cocaine and heroin was found to be additive. A second experiment used microdialysis to determine the effects of this drug combination on nucleus accumbens (NAc) extracellular levels of dopamine ([DA](e)) in rats self-administering low doses of cocaine, heroin, or cocaine/heroin combinations. These doses of cocaine and cocaine/heroin combinations significantly increased NAc [DA](e), while heroin alone did not. The ratio of the % baseline of [DA](e) (or the dialysate concentrations of DA) to cocaine in the dialysate was higher during self-administration of cocaine/heroin combinations than with cocaine alone. These data indicate that although the interaction between cocaine and heroin in maintaining self-administration is additive, a potentiation of NAc dopaminergic neurotransmission is present, suggesting that NAc [DA](e) may not be a direct measure of reinforcing efficacy and/or it is not central to the mediation of the self-administration of this drug combination., (Neuropsychopharmacology (2006) 31, 139-150. doi:10.1038/sj.npp.1300786; published online 1 June 2005.)

Chronic cocaine and withdrawal induce significant alterations in nucleus accumbens (NAc) glutamatergic function in humans and rodent models of cocaine addiction. Dysregulation of glutamatergic function of the prefrontal cortical-NAc pathway has been proposed as a critical substrate for unmanageable drug seeking. Previously, we demonstrated significant up-regulation of NMDA, (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptor subunit mRNAs and protein levels in the ventral tegmental area (VTA), but not the substantia nigra, of cocaine overdose victims (COD). The present study was undertaken to examine the extent of altered ionotropic glutamate receptor (iGluR) subunit expression in the NAc and the putamen in cocaine overdose victims. Results revealed statistically significant increases in the NAc, but not in the putamen, of NMDA receptor subunit (NR)1 and glutamate receptor subunit (GluR)2/3 wit trends in GluR1 and GluR5 in COD. These results extend our previous finding and indicate pathway-specific alterations in iGluRs in COD. In order to determine that changes were related to cocaine intake and not to other factors in the COD victims, we examined the effects of cocaine intravenous self-administration in rhesus monkeys for 18 months (unit dose of 0.1 mg/kg/injection and daily drug intake of 0.5 mg/kg/session). Total drug intake for the group of four monkeys was 37.9 +/- 4.6 mg/kg. Statistically significant elevations were observed for NR1, GluR1, GluR2/3 and GluR5 (p < 0.05) and a trend towards increased NR1 phosphorylated at serine 896 (p = 0.07) in the NAc but not putamen of monkeys self-administering cocaine compared with controls. These results extend previous results by demonstrating an up-regulation of NR1, GluR2/3 and GluR5 in the NAc and suggest these alterations are pathway specific. Furthermore, these changes may mediate persistent drug intake and craving in the human cocaine abuser.

Chronic opiate administration induces a number of biochemical alterations within the mesolimbic dopamine system that may mediate various aspects of the addictive process. In the present study, rats were administered morphine (1.0 mg/infusion) for 20 days (17.6+/-3.0 infusions/day) based on infusion histories of self-administering rats. Calbindin-D28K immunoreactive neurons were microdissected from the nucleus accumbens (NAc) shell and core subregions and gene expression was assessed using cDNA macroarrays. Comparison of gene expression between the shell and core subregions of vehicle-treated rats revealed significantly higher relative abundance of GABA-A alpha1, Galphai2 and post-synaptic density protein 95 transcript (PSD-95) mRNA levels in the shell, whereas Ggamma2 and synuclein 1 were more abundant in the core of the NAc. In the NAc shell, morphine administration resulted in upregulation of caspace 9, NF-kappaB, NF-H, tau, GABA-A delta subunit, FGFR1, Ggamma2, synuclein 1, syntaxin 5 and 13, GRK5, and c-fos mRNAs. Caspace 1, D2 dopamine receptor, GABA-A alpha1 subunit, GRIA 1/3/4, Galphai2, PSD-95 and CREB were down-regulated in the NAc shell with morphine administration. In the core, neuronal apoptotic inhibitory protein (NAIP), GABA-A alpha1 subunit, GRIN2C, GRIA1, mGluR1, D4 dopamine receptor and PSD-95 were upregulated by morphine administration whereas bax, bcl-x, cox-1 and MAP2 were decreased. These data demonstrate that morphine administration alters gene expression differentially in NAc subregions. Specifically, GABA-A alpha1 subunit, GRIA1 subunit and PSD-95 mRNAs were decreased in the shell but increased in the core following morphine administration. In addition, these results provide potential targets for further evaluation in models of morphine reinforcement as well as novel mechanisms of action in morphine-induced pathophysiology.

The abuse of cocaine/opiate combinations (speedball) represents a growing trend in illicit drug use. Delineation of neurobiological substrates mediating the reinforcing effects of the combination may increase our knowledge of reinforcement mechanisms and provide useful new information for the development of pharmacotherapies. Several studies suggest dopaminergic innervations of the nucleus accumbens (NAc) have a central role in the brain processes underlying drug reinforcement. The present study was undertaken to determine the relationship between the self-administration of cocaine/heroin combinations and NAc extracellular dopamine concentrations ([DA]e) using in vivo microdialysis and microbore high-pressure liquid chromatography. Rats were assigned randomly to one of three groups to self-administer i.v. cocaine (125, 250, and 500 micrograms/infusion; n = 5), heroin (4.5, 9, and 18 micrograms/infusion; n = 5), or cocaine/heroin combinations (125/4.5; 250/9, and 500/18 micrograms/infusion; n = 4) under a fixed ratio (FR) 10: 20-s time-out schedule of reinforcement/multicomponent dosing session. After stable rates of responding were engendered and maintained, microdialysis samples were collected in 10-min intervals during the self-administration session. Self-administration of cocaine/heroin combinations produced synergisitic elevations in NAc [DA]e (1000% baseline) compared with cocaine (400% baseline) and heroin (not significantly different from baseline levels). Neither the number of infusions nor the interinfusion intervals was significantly different between the groups across the self-administration session. Moreover, cocaine concentrations were not significantly different between the cocaine and cocaine/heroin groups. These results demonstrate that heroin interacts with cocaine to produce synergistic elevations in [DA]e, providing a neurochemical basis for understanding the abuse liability of cocaine/opiate combinations.

Studies indicate that nucleus accumbens (NAcc) dopamine neurotransmission is involved in the reinforcing and direct effects of cocaine. The present study was initiated to explore further the relationship of NAcc extracellular dopamine concentrations ([DA]e) and cocaine self-administration using a yoked littermate design. In the first experiment, one rat from each litter was trained to self-administer cocaine i.v. (SA: 0.33 mg/inf) under a fixed ratio 2 schedule, while a second rat received simultaneous infusions of cocaine yoked to the infusions of the SA (YC). NAcc [DA]e and cocaine concentrations ([COC]) were assessed during the test sessions using in vivo microdialysis combined with microbore HPLC procedures. [DA]e and [COC] were significantly elevated in the SA and YC groups during the self-administration session; however, [DA]e were greater in the SA group compared to the YC group in the first hour of the session, even though [COC] were not significantly different. On the following day, the rats previously allowed to self-administer cocaine were administered response-independent cocaine infusions yoked to the infusion pattern from the previous day. [DA]e were significantly elevated above baseline levels during the session but were significantly less than concentrations obtained when cocaine was self-administered by these subjects. [COC] during the sessions were not significantly different between the two days. Baseline [DA]e were not significantly different between the SA and YC groups or between Day 1 and Day 2. Furthermore, there was no significant difference in the in vitro probe recovery between one and two days following probe implantation. These results suggest that the context in which cocaine was administered significantly altered the neurochemical response to equivalent brain concentrations of cocaine. NAcc [DA]e was significantly increased when the delivery of cocaine infusions was contingent on the behavior of the rat, indicative of a role in the neural processes underlying cocaine reinforcement.

2 beta-propanoyl-3 beta-(4-tolyl) tropane (PTT) is a novel tropane that has been shown to be approximately 20 times more potent than cocaine in binding to the 3 beta-[4'-iodophenyl] tropane-2 beta-carboxylic acid methyl ester (RTI-55) site on the dopamine transporter, an effect partially attributable to the methyl constituent at the para position on the phenyl ring. In addition, PTT lacks the ester linkage of cocaine, thus increasing its metabolic stability. This study was undertaken to compare the quantitative and temporal effects of PTT and cocaine on in vivo neurochemical measures and motor behavior. The effects of PTT (0.3, 1.0 and 3.0 mg/kg; i.p.) and cocaine HCl (3.0, 10.0, & 30.0 mg/kg; i.p.) on nucleus accumbens extracellular dopamine concentrations [DA]e were evaluated using in vivo microdialysis. Locomotor activity and stereotypic behaviors were also assessed. PTT and cocaine increased [DA]e and total locomotor activity in a dose-dependent manner with PTT approximately 30 times more potent than cocaine. The relationship between [DA]e and locomotor activity was linear over the test session for cocaine, but not for PTT. In a subsequent experiment, pronounced stereotypic behaviors were evident in rats administered cocaine (10.0 and 30.0 mg/kg) or PTT (3.0 mg/kg). The stereotypy elicited by PTT was longer in duration and greater in intensity than that elicited by the highest dose of cocaine. These results extend previously published data by demonstrating similar in vivo potencies for PTT on nucleus accumbens [DA]e and locomotor activity. However, these data do not support the hypothesis that the time course of increased nucleus accumbens [DA]e and stimulated locomotor activity are related.

Dopaminergic innervations of the nucleus accumbens (NAcc) have a significant role in the brain processes underlying the reinforcing actions of abused drugs. In addition to the evidence from selective lesions and receptor antagonists, recent determinations of increased extracellular dopamine concentrations ([DA]e) in this structure during cocaine and ethanol self-administration have extended this notion. This study was undertaken to determine if the reinforcing effects of heroin were similarly correlated with changes in [DA]e in the NAcc using in vivo microdialysis. In the first experiment, naive rats were randomly divided into three groups and administered either two i.v. infusions of saline (n = 6) or heroin (5.4, 18 or 30 micrograms/infusion; n = 6, 8 and 6, respectively) 1 hr apart. [DA]e in the NAcc was significantly increased in a dose-dependent manner after response-independent heroin administration. In the second experiment, responding was engendered and maintained with 5.4, 18 or 30 micrograms/infusion of heroin (i.v.; n = 4, 5 and 5/group, respectively) under a fixed ratio 10 (FR10) schedule of reinforcement. After stable baselines of heroin intake were obtained, microdialysis samples were collected from the NAcc during the self-administration session. [DA]e did not significantly differ from baseline during self-administration in any of the groups. The increase in NAcc [DA]e following acute response-independent heroin administration is consistent with previously published reports. However, the latter results do not support the hypothesis that NAcc dopamine is critically involved in the processes underlying heroin self-administration. In summary, the results indicate that in vivo neurochemical data obtained from acute experimenter-administered heroin can not be equated with that obtained during heroin self-administration.

The effects of repeated systemic or intra-nucleus accumbens cocaine administration on locomotor activity were examined for environmental dependence. Repeated IP administration of cocaine (15 mg/kg) for 5 days in the context of a given environment increased the locomotor response to a subsequent IP cocaine challenge in that environment. However, there were no differences in the locomotor response to a subsequent IP cocaine challenge in the test chamber in subjects which had received prior repeated IP administration of cocaine in the home-cage. In a second experiment, cocaine (100 micrograms/side) was infused into the nucleus accumbens (NACC) daily for 5 days. This repeated administration produced increases in locomotor activity to subsequent intra-NACC cocaine infusions that were environmentally independent. In contrast to the effects of repeated IP cocaine administration, subjects which received administration of vehicle, acute cocaine, or repeated cocaine in the NACC did not differ following an IP cocaine challenge. The results from these experiments indicate that increases in the response to IP cocaine following repeated IP administration are in part environmentally dependent. Moreover, repeated intra-NACC cocaine infusions increase the responsiveness of the NACC to subsequent intra-NACC cocaine. However, local activation of the NACC alone does not appear to be adequate to produce sensitization to systemically administered cocaine.

In the first experiment, the conditioned place preference (CPP) paradigm was used to examine the rewarding properties of bilateral microinfusions of cocaine HCl into the nucleus accumbens (0, 12.5, 25, 50, or 100 micrograms). No dose of intra-accumbens cocaine induced a significant CPP. However, bilateral intra-accumbens infusions of d-amphetamine sulfate (10 micrograms) or intraperitoneal administration of cocaine HCl (5 or 10 mg/kg) both produced a significant preference for the drug-paired compartment. In the second experiment, the ability of bilateral intra-accumbens infusions of cocaine HCl (50 micrograms) to elicit conditioned locomotor activity (CLA) was examined. During the conditioning trials, intra-accumbens cocaine significantly increased locomotor activity. On the test day, when no drug was administered, the group that had previously received cocaine in the activity chamber showed significantly greater locomotor activity than the vehicle control group. This demonstration of CLA indicates that rats are able to associate the effects of intra-accumbens infusions of cocaine with environmental stimuli; however, these infusions are not rewarding as measured by the CPP paradigm. In addition, these results may indicate important differences between the neural substrates for cocaine and amphetamine reward and reveal a dissociation between CPP and CLA.