An approach for prioritizing candidate genes from RNA-seq using preclinical cocaine self-administration datasets as a test case.

RNA-sequencing (RNA-seq) technology has led to a surge of neuroscience research using animal models to probe the complex molecular mechanisms underlying brain function and behavior, including substance use disorders. However, findings from rodent studies often fail to be translated into clinical treatments. Here, we developed a novel pipeline for narrowing candidate genes from preclinical studies by translational potential and demonstrated its utility in 2 RNA-seq studies of rodent self-administration. This pipeline uses evolutionary conservation and preferential expression of genes across brain tissues to prioritize candidate genes, increasing the translational utility of RNA-seq in model organisms. Initially, we demonstrate the utility of our prioritization pipeline using an uncorrected P-value. However, we found no differentially expressed genes in either dataset after correcting for multiple testing with false discovery rate (FDR < 0.05 or <0.1). This is likely due to low statistical power that is common across rodent behavioral studies, and, therefore, we additionally illustrate the use of our pipeline on a third dataset with differentially expressed genes corrected for multiple testing (FDR < 0.05). We also advocate for improved RNA-seq data collection, statistical testing, and metadata reporting that will bolster the field's ability to identify reliable candidate genes and improve the translational value of bioinformatics in rodent research.

HIV gp120 impairs nucleus accumbens neuroimmune function and dopamine D3 receptor-mediated inhibition of cocaine seeking in male rats.

Cocaine Use Disorders (CUDs) are associated with an increased risk of human immunodeficiency virus (HIV) infection. Cocaine and the HIV envelope protein gp120 each induce distinct deficits to mesocorticolimbic circuit function and motivated behavior; however, little is known regarding how they interact to dysregulate these functions or how such interactions impact pharmacotherapeutic efficacy. We have previously shown that the selective, weak partial agonist of the dopamine D3 receptor (D3R), MC-25-41, attenuates cocaine-seeking behavior in male rats. Here, we sought to characterize changes in striatal neuroimmune function in gp120-exposed rats across abstinence from operant access to cocaine (0.75 mg/kg, i.v.) or sucrose (45 mg/pellet), and to examine the impact of gp120 exposure on MC-25-41-reduced cocaine seeking. After establishing a history of cocaine or sucrose self-administration, rats received intracerebroventricular gp120 infusions daily the first 5 days of abstinence and were sacrificed either on day 6 or after 21 days of forced abstinence and a cue-induced cocaine seeking test. We demonstrated that MC-25-41 treatment attenuated cue-induced cocaine seeking among control rats but not gp120-exposed rats. Moreover, postmortem analysis of nucleus accumbens (NAc) core neuroimmune function indicated cocaine abstinence- and gp120-induced impairments, and the expression of several immune factors within the NAc core significantly correlated with cocaine-seeking behavior. We conclude that cocaine abstinence dysregulates striatal neuroimmune function and interacts with gp120 to inhibit the effectiveness of a D3R partial agonist in reducing cocaine seeking. These findings highlight the need to consider comorbidities, such as immune status, when evaluating the efficacy of novel pharmacotherapeutics.

Synthesis and Pharmacological Characterization of a Difluorinated Analogue of Reduced Haloperidol as a Sigma-1 Receptor Ligand.

Reduced haloperidol (1) was previously reported to act as a potent sigma-1 receptor (S1R) ligand with substantially lower affinity to the dopamine D2 receptor (D2R) compared to haloperidol. It was also found to facilitate brain-derived neurotrophic factor (BDNF) secretion from astrocytic glial cell lines in a sigma-1 receptor (S1R)-dependent manner. Although an increase in BDNF secretion may have beneficial effects in some neurological conditions, the therapeutic utility of reduced haloperidol (1) is limited because it can be oxidized back to haloperidol in the body, a potent dopamine D2 receptor antagonist associated with well-documented adverse effects. A difluorinated analogue of reduced haloperidol, (±)-4-(4-chlorophenyl)-1-(3,3-difluoro-4-(4-fluorophenyl)-4-hydroxybutyl)piperidin-4-ol (2), was synthesized in an attempt to minimize the oxidation. Compound (±)-2 was found to exhibit high affinity to S1R and facilitate BDNF release from mouse brain astrocytes. It was also confirmed that compound 2 cannot be oxidized back to the corresponding haloperidol analogue in liver microsomes. Furthermore, compound 2 was distributed to the brain following intraperitoneal administration in mice and reversed the learning deficits in active avoidance tasks. These findings suggest that compound 2 could serve as a promising S1R ligand with therapeutic potential for the treatment of cognitive impairments.

5-HT1B receptor agonist enhances breakpoint for cocaine on a progressive ratio (PR) schedule during maintenance of self-administration in female rats but reduces breakpoint for sucrose.

Background: Previous research showed that the 5-HT1B receptor agonist CP94253 enhanced cocaine reinforcement rate during maintenance of daily self-administration (SA), but inhibited reinforcement rate after 21 days of abstinence in male rats. Here we examined whether female rats show similar effects of CP94253 during maintenance as males across estrous cycle phases. Methods: Female rats trained on a fixed ratio 5 (FR5) cocaine reinforcement schedule were tested for the effects of CP94253 (5.6 mg/kg, s.c.) on cocaine reinforcement rate during each phase of the estrous cycle, with access to either low (0.075 and 0.1875) or high (0.375 and 0.75) cocaine doses available for 1 h sequentially in descending dose order. Other female and male rats trained on a progressive ratio (PR) schedule of cocaine or sucrose reinforcement were tested for CP94253 (0, 3.2, 5.6, and 10 mg/kg, s.c.) effects on reinforcement rate in 3-h sessions. CP94253 effects on responding during sucrose cue-reactivity were also examined post-abstinence. Results: Regardless of sex, CP94253 enhanced breakpoints on the PR schedule during maintenance of cocaine SA but attenuated breakpoints for sucrose reinforcement and decreased responding during sucrose cue-reactivity. FR results showed that CP94253 attenuated cocaine reinforcement rate during all estrous cycle phases except metestrus. Conclusions: Overall, we suggest that CP94253 increased incentive motivation for cocaine during maintenance of SA in female and male rats, yet decreased motivation for sucrose. We also suggest that 5-HT1BRs modulate motivation similarly across sexes except when females are in metestrus.

Nuclear factor kappa B signaling within the rat nucleus accumbens core sex-dependently regulates cue-induced cocaine seeking and matrix metalloproteinase-9 expression.

Chronic drug self-administration and withdrawal are associated with distinct neuroimmune adaptations that may increase drug craving and relapse vulnerability in humans. The nuclear factor kappa-B (NF-κB) pathway is a critical regulator of many immune- and addiction-related genes such as the extracellular matrix enzyme matrix metalloproteinase-9 (MMP-9), which is a known modulator of learning, memory, and synaptic plasticity. While some studies suggest striatal NF-κB signaling may regulate drug-conditioned behavior, no studies to date have examined whether NF-κB signaling within the nucleus accumbens core (NAc core) alters downstream neuroimmune function and cue-motivated cocaine seeking following a period of forced abstinence, whether any effects are specific to cocaine over other reinforcers, or whether sex differences exist. Here, we examined whether viral-mediated knockdown of the p65 subunit of NF-κB within the NAc core would alter MMP-9 expression and cue-induced cocaine- and sucrose-seeking behavior following a period of forced abstinence in male and female rats. We demonstrate that NAc core p65 knockdown results in a significant decrease in cue-induced cocaine seeking in males but not females. This effect was specific to cocaine, as p65 knockdown did not significantly affect cue-induced sucrose seeking in either males or females. Moreover, we demonstrate that males express higher levels of MMP-9 within the NAc core and nucleus accumbens shell (NAcSh) compared to females, and that p65 knockdown significantly decreases MMP-9 in the NAc core of males but not females among cocaine cue-exposed animals. Altogether, these results suggest that NAc core NF-κB signaling exerts modulatory control over cue-motivated drug-seeking behavior and downstream neuroimmune function in a sex-specific manner. These findings highlight the need to consider sex as an important biological variable when examining immunomodulatory mechanisms of cocaine seeking.

HuD Regulates mRNA-circRNA-miRNA Networks in the Mouse Striatum Linked to Neuronal Development and Drug Addiction.

The RNA-binding protein HuD (a.k.a., ELAVL4) is involved in neuronal development and synaptic plasticity mechanisms, including addiction-related processes such as cocaine conditioned-place preference (CPP) and food reward. The most studied function of this protein is mRNA stabilization; however, we have recently shown that HuD also regulates the levels of circular RNAs (circRNAs) in neurons. To examine the role of HuD in the control of coding and non-coding RNA networks associated with substance use, we identified sets of differentially expressed mRNAs, circRNAs and miRNAs in the striatum of HuD knockout (KO) mice. Our findings indicate that significantly downregulated mRNAs are enriched in biological pathways related to cell morphology and behavior. Furthermore, deletion of HuD altered the levels of 15 miRNAs associated with drug seeking. Using these sets of data, we predicted that a large number of upregulated miRNAs form competing endogenous RNA (ceRNA) networks with circRNAs and mRNAs associated with the neuronal development and synaptic plasticity proteins LSAMP and MARK3. Additionally, several downregulated miRNAs form ceRNA networks with mRNAs and circRNAs from MEF2D, PIK3R3, PTRPM and other neuronal proteins. Together, our results indicate that HuD regulates ceRNA networks controlling the levels of mRNAs associated with neuronal differentiation and synaptic physiology.

Neuroimmune Mechanisms as Novel Treatment Targets for Substance Use Disorders and Associated Comorbidities.

Recent studies examining the neurobiology of substance abuse have revealed a significant role of neuroimmune signaling as a mechanism through which drugs of abuse induce aberrant changes in synaptic plasticity and contribute to substance abuse-related behaviors. Immune signaling within the brain and the periphery critically regulates homeostasis of the nervous system. Perturbations in immune signaling can induce neuroinflammation or immunosuppression, which dysregulate nervous system function including neural processes associated with substance use disorders (SUDs). In this review, we discuss the literature that demonstrates a role of neuroimmune signaling in regulating learning, memory, and synaptic plasticity, emphasizing specific cytokine signaling within the central nervous system. We then highlight recent preclinical studies, within the last 5 years when possible, that have identified immune mechanisms within the brain and the periphery associated with addiction-related behaviors. Findings thus far underscore the need for future investigations into the clinical potential of immunopharmacology as a novel approach toward treating SUDs. Considering the high prevalence rate of comorbidities among those with SUDs, we also discuss neuroimmune mechanisms of common comorbidities associated with SUDs and highlight potentially novel treatment targets for these comorbid conditions. We argue that immunopharmacology represents a novel frontier in the development of new pharmacotherapies that promote long-term abstinence from drug use and minimize the detrimental impact of SUD comorbidities on patient health and treatment outcomes.

5-HT1B receptor agonist attenuates cocaine self-administration after protracted abstinence and relapse in rats.

BACKGROUND: The 5-HT1B receptor (5-HT1BR) agonist, CP94253, enhances cocaine intake during maintenance of self-administration (SA) but attenuates intake after 21 days of forced abstinence in male rats. AIMS: We examined whether CP94253 attenuates cocaine intake in female rats after a period of abstinence, and if these attenuating effects persist or revert to enhancing cocaine intake during resumption (i.e. relapse) of daily cocaine SA. METHODS: Male and female rats trained to lever press on a fixed ratio 5 schedule of cocaine reinforcement underwent ⩾21 days of forced abstinence. They were then tested for the effects of CP94253 (5.6 mg/kg, SC) or vehicle on cocaine SA. During the test session, rats had 1-h access to the training dose of cocaine (0.75 mg/kg, IV) followed by 1-h access to a lower cocaine dose (0.075 mg/kg, IV). Rats then resumed cocaine SA for 15 days to mimic relapse and were retested as done previously. Subsequently, rats underwent abstinence again (21-60 days) and were tested for CP94253 effects on locomotion and cue reactivity (i.e. responding for light/tone cues previously paired with cocaine infusions). RESULTS: Regardless of sex, CP94253 decreased cocaine intake after abstinence and during resumption of SA and decreased cue reactivity while having no effect on locomotion. CONCLUSIONS: CP94253 decreases cocaine intake and cocaine seeking in both males and females even after resumption of cocaine SA. These findings suggest that the inhibitory effects of CP94253 observed after abstinence are long-lasting, and therefore, 5-HT1BR agonists may have clinical efficacy as anti-relapse medications for cocaine use disorders.

microRNA regulation related to the protective effects of environmental enrichment against cocaine-seeking behavior.

BACKGROUND: MicroRNAs (miRNAs) are "master post-transcriptional regulators" of gene expression. Here we investigate miRNAs involved in the incentive motivation for cocaine elicited by exposure to cocaine-associated cues. METHODS: We conducted NanoString nCounter analyses of microRNA expression in the nucleus accumbens shell of male rats that had been tested for cue reactivity in a previous study. These rats had been trained to self-administer cocaine while living in isolate housing, then during a subsequent 21-day forced abstinence period they either stayed under isolate housing or switched to environmental enrichment (EE), as this EE intervention is known to decrease cocaine seeking. This allowed us to create groups of "high" and "low" cocaine seekers using a median split of cocaine-seeking behavior. RESULTS: Differential expression analysis across high- and low-seekers identified 33 microRNAs that were differentially expressed in the nucleus accumbens shell. Predicted mRNA targets of these microRNAs are implicated in synaptic plasticity, neuronal signaling, and neuroinflammation signaling, and many are known addiction-related genes. Of the 33 differentially-expressed microRNAs, 8 were specifically downregulated in the low-seeking group and another set of 8 had expression levels that were significantly correlated with cocaine-seeking behavior. CONCLUSION: These findings not only confirm the involvement of previously identified microRNAs (e.g., miR-212, miR-495) but also reveal novel microRNAs (e.g., miR-3557, miR-377) that alter, or are altered by, processes associated with cocaine-seeking behavior. Further research examining the mechanisms involved in these microRNA changes and their effects on signaling may reveal novel therapeutic targets for attenuating drug craving.

Preclinical support for the therapeutic potential of zolmitriptan as a treatment for cocaine use disorders.

Serotonin 1B receptor (5-HT1BR) agonists enhance cocaine intake in rats during daily self-administration but attenuate cocaine intake after prolonged abstinence. Here we investigated whether the less selective but clinically available 5-HT1D/1BR agonist, zolmitriptan, produces similar effects. Male and free-cycling female Sprague-Dawley rats were trained to lever press for cocaine (0.75 mg/kg, i.v.) or sucrose (45 mg pellet) reinforcement until performance rates stabilized. Rats then received zolmitriptan (3.0, 5.6, and 10 mg/kg, s.c.) prior to testing for its effects on response and reinforcement rates. Under cocaine testing conditions, rats had access to the training dose for the first hour followed by a lower cocaine dose (0.075 mg/kg, i.v.) for the second hour. Zolmitriptan decreased cocaine intake at both cocaine doses and in both sexes even without a period of abstinence and without altering sucrose intake. A separate group of rats underwent identical training procedures and were tested for effects of the selective 5-HT1B and 5-HT1D receptor antagonists, SB224289 (3.2, 5.6, and 10 mg/kg, s.c.) and BRL15572 (0.3, 1.0, and 3.0 mg/kg, i.p.), respectively, alone or in combination with zolmitriptan (5.6 mg/kg, s.c.) under identical cocaine testing procedures as above. The zolmitriptan-induced decrease in cocaine intake was reversed by SB224289 and to a lesser extent by BRL15572, suggesting that the effects of zolmitriptan involve both 5-HT1B and 5-HT1D receptors. Neither zolmitriptan, SB224289, or BRL15572 altered locomotor activity at the doses effective for modulating cocaine intake. These findings suggest that zolmitriptan has potential for repurposing as a treatment for cocaine use disorders.

Environmental enrichment during forced abstinence from cocaine self-administration opposes gene network expression changes associated with the incubation effect.

Environmental enrichment (EE) is a robust intervention for reducing cocaine-seeking behaviors in animals when given during forced abstinence. However, the mechanisms that underlie these effects are not well-established. We investigated the adult male rat transcriptome using RNA-sequencing (RNA-seq) following differential housing during forced abstinence from cocaine self-administration for either 1 or 21 days. Enriched, 21-day forced abstinence rats displayed a significant reduction in cocaine-seeking behavior compared to rats housed in isolation. RNA-seq of the nucleus accumbens shell revealed hundreds of differentially regulated transcripts between rats of different forced abstinence length and housing environment, as well as within specific contrasts such as enrichment (isolated 21 days vs. enriched 21 days) or incubation (isolated 1 day vs. isolated 21 days). Ingenuity Pathway Analysis affirmed several pathways as differentially enriched based on housing condition and forced abstinence length including RELN, the Eif2 signaling pathway, synaptogenesis and neurogenesis pathways. Numerous pathways showed upregulation with incubation, but downregulation with EE, suggesting that EE may prevent or reverse changes in gene expression associated with protracted forced abstinence. The findings reveal novel candidate mechanisms involved in the protective effects of EE against cocaine seeking, which may inform efforts to develop pharmacological and gene therapies for treating cocaine use disorders. Furthermore, the finding that EE opposes multiple pathway changes associated with incubation of cocaine seeking strongly supports EE as a therapeutic intervention and suggests EE is capable of preventing or reversing the widespread dysregulation of signaling pathways that occurs during cocaine forced abstinence.

The Long-Acting D3 Partial Agonist MC-25-41 Attenuates Motivation for Cocaine in Sprague-Dawley Rats.

The dopamine D3 receptor is a prime target for developing treatments for cocaine use disorders (CUDs). In this study, we conducted a pre-clinical investigation of the therapeutic potential of a long-acting, D3 receptor partial agonist, MC-25-41. Male rats were pre-treated with MC-25-41 (vehicle, 1.0, 3.0, 5.6, or 10 mg/kg, intraperitoneal (IP)) five minutes prior to tests of cocaine or sucrose intake on either a progressive ratio schedule of reinforcement or a variable interval 60 s multiple schedule consisting of 4, 15-min components with sucrose or cocaine available in alternating components. A separate cohort of rats was tested on a within-session, dose-reduction procedure to determine the effects of MC-25-41 on demand for cocaine using a behavioral economics analysis. Finally, rats were tested for effects of MC-25-41 on spontaneous and cocaine-induced locomotion. MC-25-41 failed to alter locomotion, but reduced reinforcement rates for both cocaine and sucrose on the low-effort, multiple schedule. However, on the higher-effort, progressive ratio schedule of cocaine reinforcement, MC-25-41 reduced infusions, and active lever presses at doses that did not alter sucrose intake. The behavioral economics analysis showed that MC-25-41 also increased cocaine demand elasticity compared to vehicle, indicating a reduction in consumption as price increases. Together, these results suggest that similar to other D3-selective antagonists and partial agonists, MC-25-41 reduces motivation for cocaine under conditions of high cost but has the added advantage of a long half-life (>10 h). These findings suggest that MC-25-41 may be a suitable pre-clinical lead compound for development of medications to treat CUDs.

Dual Effect of 5-HT1B/1D Receptors on Dopamine Neurons in Ventral Tegmental Area: Implication for the Functional Switch After Chronic Cocaine Exposure.

BACKGROUND: Serotonin (5-HT) 1B/1D receptor (5-HT1B/1DR) agonists undergo an abstinence-induced switch in their effects on cocaine-related behaviors, which may involve changes in modulation of dopamine (DA) neurons in the ventral tegmental area (VTA). However, it is unclear how 5-HT1B/1DRs affect VTA DA neuronal function and whether modulation of these neurons mediates the abstinence-induced switch after chronic cocaine exposure. METHODS: We examined the ability of 5-HT1B/1DRs to modulate D2 autoreceptors (D2ARs) and synaptic transmission in the VTA by slice recording and single unit recording in vivo in naïve mice and in mice with chronic cocaine treatment. RESULTS: We report a bidirectional modulation of VTA DA neuronal firing through the interaction of VTA 5-HT1B/1DRs and D2ARs. In both VTA slices and the VTA of anesthetized mice, the 5-HT1B/1DR agonist CP94253 decreased DA neuronal firing rate and evoked excitatory postsynaptic currents to DA neurons in slice. Paradoxically, CP94253 decreased quinpirole-induced inhibition of DA neurons by reducing D2AR-mediated G protein-gated inwardly rectifying potassium current. This manifested decreased GABAA (gamma-aminobutyric acid A) receptor-mediated evoked inhibitory postsynaptic currents in slice, resulting in disinhibition of DA neurons, in opposition to the 5-HT1B/1DR-induced inhibition. This dual effect was verified in chronic cocaine-treated and mild stress-treated, male mice on days 1 and 20 posttreatment. CONCLUSIONS: This study revealed dual effects of CP94253 on VTA DA neurons that are dependent on D2AR sensitivity, with anti-inhibition under normal D2AR sensitivity and inhibition under low D2AR sensitivity. These dual effects may underlie the ability of CP94253 to both enhance and inhibit cocaine-induced behaviors.

Economic demand analysis of within-session dose-reduction during nicotine self-administration.

BACKGROUND: This study determined if a within-session dose-reduction design sufficiently captures elasticity of demand for nicotine in male and female rats using environmental enrichment to manipulate demand elasticity. METHODS: Male and female Sprague-Dawley rats were trained to self-administer nicotine (60 µg/kg/infusion). In Experiment 1, rats began daily dose-reduction for nine sessions following acquisition. Rats then underwent a minimum of five within-session dose-reduction sessions where each dose was available for 10 min. In Experiment 2, rats were reared in isolated, social, or enriched housing followed by acquisition of nicotine self-administration. Rats then underwent within-session dose-reduction. Housing environments were then switched, followed by additional testing sessions. Consumption was calculated for each dose and exponential demand curves were fit. RESULTS: No sex differences in acquisition of nicotine self-administration were detected for either experiment. In experiment 1, demand intensity (Q0; estimated intake if nicotine were freely available), was higher with between- compared to within-session dose-reduction, although elasticity of demand (α; rate of decline in nicotine intake as a function of increasing unit price), was lower. In Experiment 2, animals reared in enrichment had fewer infusions during acquisition compared to animals in isolation. Enriched males had reduced demand intensity compared to both isolated and social males, whereas isolated females had reduced intensity compared to enriched females. CONCLUSIONS: The within-session dose-reduction procedure for nicotine self-administration replicated effects of environmental enrichment on consumption behaviors. Additionally, this procedure captured differences in nicotine demand due to sex, laying important groundwork for future translational research on mechanisms of nicotine dependence.

Neural Circuits Associated with 5-HT1B Receptor Agonist Inhibition of Methamphetamine Seeking in the Conditioned Place Preference Model.

5-HT1B receptors (5-HT1BRs) modulate psychostimulant reward and incentive motivation in rodents. Here we investigated the effects of the 5-HT1BR agonist CP94253 (10 mg/kg, IP) on the acquisition and expression of methamphetamine (Meth) conditioned place preference (CPP) in C57BL/6 male mice. We subsequently examined the potential brain regions involved in CP94253 effects using FOS as a marker of neural activity. In the acquisition experiment, mice received the agonist 30 min before each of the Meth injections given during conditioning. In the expression experiment, mice that had acquired Meth-CPP were given either saline or CP94253 and were tested for CPP 30 min later. We found that CP94253 attenuated the expression of Meth-CPP, but had no effect on acquisition. Mice expressing Meth-CPP had elevated numbers of FOS+ cells in the ventral tegmental area (VTA) and basolateral amygdala (BlA) and reduced FOS+ cells in the central amygdala (CeA) compared to saline controls. CP94253 given before the expression test, but not acutely in drug-naive mice, enhanced FOS+ cells in the VTA, the nucleus accumbens (NAc) shell and core, and the dorsomedial striatum and reversed the Meth-conditioned changes in FOS in the BlA and CeA. Approximately 50-70% of FOS+ cells in the NAc and VTA were GABAergic regardless of group. By contrast, we did not observe FOS-labeling in dopamine neurons in the VTA. The findings suggest that CP94253 attenuates the motivational effects of the Meth-associated environment and highlight the amygdala, VTA, NAc, and dorsomedial striatum as potential regions involved in this effect.

Dopamine D3 receptor partial agonist LS-3-134 attenuates cocaine-motivated behaviors.

AIMS: The dopamine D3 receptor (D3R) is a pharmacotherapeutic target for drug dependence. We have successfully imaged human D3Rs using radiolabeled LS-3-134, an arylamide phenylpiperazine with moderate selectivity for the D3R over D2R and low efficacy at the D2 and D3R. In this study, we screened for effects of LS-3-134 as a potential anti-cocaine therapeutic. METHODS: Male rats were pretreated with LS-3-134 (0, 1.0, 3.2, or 5.6 mg/kg, IP) 15 min prior to tests for its effects on spontaneous and cocaine-induced locomotion. We next investigated the effects of LS-3-134 (0, 1.0, 3.2, 5.6, or 10.0 mg/kg, IP) on operant responding on a multiple variable-interval (VI) 60-second schedule with alternating cocaine (0.375 mg/kg, IV) and sucrose (45 mg) reinforcer components. Additionally, we tested LS-3-134 (5.6 mg/kg, IP) effects on a progressive ratio (PR) schedule of cocaine reinforcement, on extinction of cocaine-seeking behavior, and on reinstatement of extinguished cocaine-seeking behavior by cocaine-associated light/tone cues. RESULTS: LS-3-134 did not alter spontaneous locomotion, but reduced cocaine-induced locomotion, break points on the high-effort progressive ratio schedule of reinforcement, and responding during extinction and cue reinstatement. In contrast, LS-3-134 did not alter cocaine or sucrose reinforcement on the low-effort multiple VI 60-second schedule. CONCLUSIONS: The effects of LS-3-134 are similar to other dopamine D3 low efficacy partial agonists and antagonists in attenuating cocaine intake under high effort schedules of reinforcement and in attenuating cocaine-seeking behavior elicited by cocaine-associated cues. These findings are consistent with the anti-craving profile of other dopamine D3 drugs.