3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in nonmedical contexts that pose risk for cardiovascular and neurologic complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines 5-hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) compared with MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT and partial releasers at NET but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties. SIGNIFICANCE STATEMENT: Despite the clinical utility of 3,4-methylenedioxymethamphetamine (MDMA), the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.

Two common constituents of psychoactive "bath salts", 3,4-methylenedioxypyrovalerone (MDPV) and α-pyrrolidinoipentiophenone (α-PVP) belong to a novel class of synthetic chemicals structurally related to the psychostimulant drug, cathinone. Recreational use of MDPV and α-PVP pose serious health risks, which may be exacerbated by concomitant use of both substances. Preclinical psychopharmacology studies have established that MDPV and α-PVP have high abuse liabilities, comparable to that of cocaine and methamphetamine. Whereas polysubstance use is common among recreational users of synthetic cathinones, preclinical behavioral assays can serve to inform potential behavioral health risks of drug mixtures. This study employed a rodent model of conditioned drug reward, conditioned place preference (CPP), to determine if concurrent treatment with MDPV (1 mg/kg) and α-PVP (1 mg/kg) produced stronger locomotor activation or CPP compared to each individual substance. A secondary aim of this study was to assess sex as variable in the behavioral effects of these substances. Females exhibited a stronger response than males to the locomotor stimulant effects of α-PVP and the α-PVP + MDPV mixture. Additionally, the α-PVP + MDPV mixture produced significantly greater increases in activity compared to either drug alone in females. MDPV and the α-PVP + MDPV mixture established CPP in both sexes, whereas α-PVP alone failed to produce CPP in either sex. These results are consistent with previous preclinical study findings that females may be more susceptible to the psychostimulant effects of these synthetic cathinones. Further investigation is warranted to determine the mechanisms responsible for sex differences in the behavioral effects of these drugs.

Recent clinical trials indicate favorable therapeutic outcomes of psychedelic-assisted psychotherapy for anxiety and treatment-resistant depression. Whereas the neurobiological systems underlying these effects are not well understood, animal behavioral models can serve to investigate these mechanisms. For the current study, we implemented 2 rodent models predictive of anxiolytic drug effects, a light/dark test and an elevated plus maze (EPM), to investigate the acute and subchronic effects of LSD, respectively. Forty-eight, adult male Sprague-Dawley rats were randomly assigned to receive LSD (0.00, 0.02, 0.04, 0.08 mg/kg) and assessed in the light/dark test 15 min after the first injection. Five additional injections were given, once every 48 hours, after which rats were assessed in the EPM either 48 (n = 24) or 72 hours (n = 24) after the last injection. A dose-dependent and statistically significant decrease was observed in number of entries into, F(3, 44) = 12.79, p < .001,η2 = .47, and time spent, F(3, 48) = 14.15, p < .001, ηp2 = .47, in the brightly lit compartment. In the EPM, closed arm entries, F(1, 17) = 28.85, p < .001, ηp2 =.36, and time spent in closed arms, F(1, 17) = 20.14, p < .001, ηp2=.99, were significantly higher compared to entries or time in open arms by rats assessed 48 hours after the last LSD injection, but not by rats tested 72 hours after the last injection. These findings indicate that acute and subchronic LSD treatment produce transient anxiogenic effects. In consideration of positive therapeutic outcomes of psychedelic-assisted psychotherapy, alternative preclinical models may be warranted to discern the mechanisms underlying the putative therapeutic effects of serotonergic hallucinogens.

3,4-Methylenedioxypyrovalerone (MDPV), one of several synthetic cathinones, is a popular constituent of illicit 'bath salts'. In preclinical studies utilizing drug discrimination methods with male rodents, MDPV has been characterized as similar to both cocaine and 3,4-methylenedioxymethamphetamine-hydrochloride (MDMA). Whereas few drug discrimination studies have utilized female rats, the current study evaluated the discriminative stimulus effects of MDPV in 12 adult female Sprague-Dawley rats trained to discriminate 0.5 mg/kg MDPV from saline under a fixed ratio 20 schedule of food reinforcement. Stimulus substitution was assessed with MDPV and its enantiomers, other synthetic cathinones [alpha pyrrolidinopentiophenone-hydrochloride(α-PVP), 4-methylmethcathinone (4-MMC)], other dopamine agonists (cocaine, [+)-methamphetamine] and serotonin agonists [MDMA, lysergic acid diethylamide (LSD)] Stimulus antagonism was assessed with the dopamine D1 receptor antagonist, Sch 23390 and the D2 receptor antagonist, haloperidol. Cocaine and (+)-methamphetamine engendered full stimulus generalization to MDPV with minimal effects on response rate. LSD produced partial substitution, whereas MDMA and 4-MMC produced complete substitution, and all these serotonergic compounds produced dose-dependent response suppression. (S)-MDPV and α-PVP engendered full substitution with similar potency to the racemate, while (R)-MDPV failed to substitute up to 5 mg/kg. Both Sch 23390 and haloperidol attenuated the discrimination of low MDPV doses and essentially shifted the dose-response curve to the right but failed to block discrimination of the training dose. These findings are generally consistent with previous reports based exclusively on male rodents. Moreover, they confirm the contribution of dopaminergic mechanisms but do not rule out the possible contribution of other neurotransmitter actions to the interoceptive stimulus effects of MDPV.

Synthetic cathinones gained initial popularity on the illicit drug market as a result of attempts to evade legal restrictions on other commonly abused psychostimulants. A body of published research has determined that the psychopharmacology of the synthetic cathinone 3, 4-methylenedioxypyrovalerone (MDPV) is comparable to cocaine and methamphetamine (METH). Few preclinical studies have systematically investigated concurrent use of synthetic cathinones with other psychostimulant drugs. The present study utilized conditioned place preference (CPP), a rodent model of conditioned drug reward, to evaluate the effects of concurrent treatment with MDPV and METH. Male (N = 72) and female (N = 105) Sprague-Dawley rats underwent a two-compartment biased CPP procedure, with one trial per day for eight consecutive days. Subjects were randomly assigned to the following treatment groups: saline, METH (1 mg/kg), MDPV (1, 3.2, 5.6 mg/kg) or a mixture consisting of METH (1 mg/kg) and MDPV (1, 3.2, 5.6 mg/kg). All treatments increased locomotor activity during drug conditioning trials, and most treatments produced higher activity increases in females compared to males. Although the level of CPP established by MDPV and MDPV + METH mixtures varied between males and females, sex differences were not statistically significant. Although none of the MDPV+METH mixtures produced stronger CPP than either substance alone, some mixtures of MDPV and METH produced higher increases in locomotor activity compared to either drug alone. Further studies with higher doses may be warranted to determine if concurrent use of MDPV and METH pose an enhanced risk for abuse.

Recent discoveries from clinical trials with psychedelic-assisted therapy have led to a resurgence of interest in the psychopharmacology of lysergic acid diethylamide (LSD). Preclinical drug discrimination is an invaluable tool to investigate the neurochemical mechanisms underlying subjective drug effects. The current study extends previous drug discrimination research by including both sexes. Adult female (n = 8) and male (n = 8) Sprague-Dawley rats were trained to discriminate 0.08 mg/kg LSD from saline under a fixed ratio 20 schedule of food reinforcement. Substitution tests were conducted with several substances, including other serotonergic hallucinogens, psychostimulants, mixed psychedelic-stimulants and synthetic cathinones. Stimulus antagonist tests were conducted with selected serotonin and dopamine antagonists. LSD-substitution with serotonergic hallucinogens was comparable between sexes. Modest but intriguing differences were observed between male and female rats in the extent of partial substitution by 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyamphetamine enantiomers and the synthetic cathinones, 3,4-methylenedioxypyrovalerone and 4-methylmethcathinone. Dopamine antagonists failed to block the LSD cue in both sexes and exerted stronger rate suppressant effects in male rats. The 5-hydroxytryptamine antagonist, (R)-(+)-a-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenyl) ethyl]-4-piperidinemethanol (MDL 100 907) blocked LSD discrimination in both sexes, although complete blockade was evident at lower doses in male rats. These results support previous findings regarding the prominent role of serotonergic activities underlying LSDs discriminative stimulus effects in male rats and generalize these findings to female rats. In consideration of the rising popularity in psychedelic-assisted psychotherapy, further research may be warranted to evaluate possible sex differences in the behavioral and subjective effects of LSD.

Mephedrone (MEPH) is one of several synthetic cathinone derivatives and a common constituent of illicit 'bath salts'. Concomitant use of MEPH with other psychostimulants is common among recreational users, but their combined effects have not been evaluated rigorously. In experiment 1, 56 male Sprague-Dawley rats were administered saline, MEPH (1 or 5 mg/kg), COC (5 mg/kg), or a mixture of MEPH (1 or 5 mg/kg) + COC (5 mg/kg) for seven consecutive days. Following a 10-day drug washout, rats were given a challenge injection of COC (5 mg/kg). Locomotor activity was recorded for 60 minutes immediately before and for 60 minutes immediately after injections on days 1, 7, and 17. In experiment 2, an unbiased conditioned place preference procedure was implemented over a 10-day period with a separate group of 66 male Sprague-Dawley rats randomly assigned to similar drug treatments used in experiment. Results of experiment 1 indicated significant increases in horizontal activity after repeated treatment with MEPH+COC mixtures, but not with either drug alone. Additionally, rats pretreated with MEPH + COC mixtures exhibited an augmented response to cocaine following drug abstinence. Evidence for CPP was established in rats treated with 5 mg/kg MEPH, 5 mg/kg COC and the 5 mg/kg MEPH + 5 mg/kg COC mixture. In conclusion, cocaine and mephedrone may have additive locomotor stimulant effects, although further assessment with a wider range of dose combinations must be evaluated. As a precautionary note, concurrent use of these substances may pose an enhanced risk for abuse.

Recreational abuse of illicit synthetic cathinones is an ongoing public health concern. Recent studies indicate that the methcathinone derivative 4-methylmethcathinone (4-MMC) produces behavioral and neurochemical effects similar to the entactogen 3,4-methylenedioxymethamphetamine (MDMA). Whereas polysubstance abuse is common, most preclinical studies of drug abuse liability only evaluate the effects of single drugs. Utilizing the locomotor sensitization paradigm, the present study assessed the combined locomotor stimulant effects of 4-MMC and MDMA for induction of sensitization following repeated administration and for expression of sensitization to a challenge dose of either substance alone after a 10-day period of drug abstinence. Male Sprague-Dawley rats received once daily intraperitoneal injections of saline, 4-MMC (1.0 mg/kg or 5.0 mg/kg), MDMA (3.0 mg/kg), or a mixture containing 4-MMC (1.0 mg/kg or 5.0 mg/kg) + MDMA (3.0 mg/kg) for 7 consecutive days. Following a 10-day drug-free period, rats were given a single intraperitoneal injection of either saline, 4-MMC (1.0 or 5.0 mg/kg), or 3.0 mg/kg MDMA. Activity was recorded for 1 h immediately before and 1 h immediately after injections on days 1, 7, and 17. 4-MMC treatment failed to induce locomotor sensitization, but, when combined with MDMA, sensitization was induced to a greater extent than with MDMA alone. Furthermore, the expression of sensitization to a subsequent challenge dose of MDMA was observed only in animals previously exposed to MDMA or a 5.0 mg/kg 4-MMC + MDMA mixture. In consideration of these findings along with the fact that 4-MMC has similar neurochemical actions to MDMA, further research may be warranted to determine the abuse liability of drug mixtures including 4-MMC and MDMA.

3,4-Methylenedioxypyrovalerone (MDPV) is a popular synthetic cathinone reported to have a high abuse potential. Recent preclinical research indicates the psychopharmacology of MDPV is comparable to cocaine. Despite a recent influx of research on the psychopharmacology of MDPV, few studies have employed preclinical drug discrimination methods to discern the neurochemical mechanisms involved in its interoceptive stimulus effects.

Hallucinogens comprise a diverse collection of chemicals with multifarious receptor actions in the central nervous system. Preclinical drug screening methods have proven invaluable in the evaluation and characterization of hallucinogen psychopharmacology. Used in concert with structural chemistry and receptor pharmacology methods, preclinical drug discrimination research has informed our current understanding of hallucinogens and the neurochemical receptor mechanisms responsible for their interoceptive stimulus effects. This chapter summarizes the strengths and limitations of drug discrimination as an in vivo drug detection method and offers a brief review of historical and contemporary drug discrimination research with classical hallucinogens.

Synthetic cathinones, known as "bath salts" on the illicit drug market, pose a significant public health concern. 3,4-Methylenedioxypyrovalerone (MDPV), one of several popular constituents of illicit bath salts, produces similar pharmacological actions to cocaine, albeit with greater potency and efficacy. The present study sought to characterize behavioral and neurochemical effects of repeated exposure to MDPV alone and in combination with cocaine. Male Sprague-Dawley rats were randomly assigned to one the following four treatments, administered once daily for seven days: 1 mg/kg MDPV, 5 mg/kg cocaine, 1 mg/kg MDPV +5 mg/kg cocaine, or saline. Locomotor activity was assessed for 1 h immediately before and 1 h immediately after injections on days 1 and 6. Brains were harvested 20 min after the final injection on day 7 and brain tissue punches were obtained to determine monoamine content within the anterior striatum, medial prefrontal cortex, and nucleus accumbens using High-Performance Liquid Chromatography (HPLC). Drug-induced increases in horizontal activity were significantly greater on treatment day 6 compared to treatment day 1 in all three drug treatment groups in comparison to the saline control group. MDPV produced significantly higher increases in activity compared to either saline or cocaine, although concurrent treatment with MDPV and cocaine produced sub-additive effects. Neurochemical analyses provided no evidence of alterations in total monoamine content following repeated administration of MDPV, cocaine, or the MDPV + COC mixture. Further investigations targeting possible changes in DA receptor sensitivity following repeated exposure to MDPV may help elucidate the mechanistic changes responsible for MDPV-induced behavioral sensitization. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'

Psychopharmacology research has amassed substantial evidence for similarities between synthetic cathinones and other commonly abused psychostimulants. Few studies have utilized drug discrimination methods to investigate synthetic cathinones, and the precise neurochemical substrates underlying their interoceptive effects have not been examined. The present study assessed the involvement of D 1 and D 2 dopaminergic receptors in the stimulus effects of 3,4-methylenedioxypyrovalerone (MDPV) and mephedrone (MEPH) in rats trained to discriminate d -amphetamine. Eight male Sprague-Dawley rats were trained to discriminate 0.5 mg/kg d -amphetamine (AMPH) from saline. Dose–response curves were then generated with AMPH (0.0–1.0 mg/kg), MDPV (0.0–1.0 mg/kg), and MEPH (0.0–2.0 mg/kg). Subsequently, Sch 39166 (0.3 mg/kg) and haloperidol (0.5 mg/kg) were administered in combination with select doses of MDPV and MEPH. Both MDPV and MEPH produced full substitution for AMPH. Sch 39166 produced a downward shift in the MDPV and MEPH dose–response curves and haloperidol produced similar results with MDPV. These preliminary findings indicate that MDPV and MEPH produce interoceptive stimuli that are similar to those produced by AMPH and that D 1 and D 2 dopamine receptors contribute to these effects. Additional studies are warranted to investigate the contribution of other receptor mechanisms involved in the interoceptive stimuli produced by synthetic cathinones.

Although the synthetic cathinone 4-methylmethcathinone (4-MMC, mephedrone) has been a subject of intensive research investigation, the pharmacological mechanisms involved in its interoceptive stimulus effects have yet to be fully characterized.

Recreational use of 3,4-methylenedioxypyrovalerone (MDPV) in the early 2000s prompted numerous scientific investigations of its behavioral and neurochemical effects. The purpose of this study was to further characterize the interoceptive stimulus effects of MDPV using a validated in-vivo drug-detection assay. Male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg MDPV from saline under a fixed ratio 20 (FR 20) schedule of food reinforcement. After stimulus control was established with MDPV (∼35 training sessions), substitution tests were commenced with drugs from several chemical classes, including drugs with predominantly dopaminergic actions [MDPV, d-amphetamine, (+)-methamphetamine, (−)-cocaine], drugs with predominantly serotonergic actions [(+)-lysergic acid diethylamide, (+)-fenfluramine], and drugs with both serotonergic and dopaminergic actions (3,4-methylenedioxymethamphetamine, 4-methylmethcathinone). Full substitution for the 0.3 mg/kg MDPV cue was observed with d-amphetamine, (+)-methamphetamine, and (−)-cocaine. Surprisingly, the 5-HT releaser (+)-fenfluramine fully substituted in half the subjects, but completely suppressed responding in the remaining subjects. 3,4-Methylenedioxymethamphetamine, 4-methylmethcathinone, and (+)-lysergic acid diethylamide failed to fully substitute for MDPV. These results indicate that the MDPV cue is similar to cues produced by drugs with predominantly dopamine-increasing effects and perhaps serotonin-releasing effects among individual subjects. Given these findings, further research is warranted to directly assess the contributions of dopamine and serotonin receptor isoforms to the discriminative stimulus functions of MDPV.

Recent reports on the abuse of novel synthetic cathinone derivatives call attention to serious public health risks of these substances. In response to this concern, a growing body of preclinical research has characterized the psychopharmacology of these substances, particularly mephedrone (MEPH) or methylenedioxypyrovalerone (MDPV), noting their similarities to 3,4-methylenedioxymethamphetamine (MDMA) and cocaine. Few studies have utilized drug discrimination methodology to characterize the psychopharmacological properties of these substances.The present study employed a rodent drug discrimination assay to further characterize the stimulus effects of MEPH and MDPV in comparison to MDMA and to a drug mixture comprised of d-amphetamine and MDMA.Eight male Sprague-Dawley rats were trained to discriminate 1.5 mg/kg MDMA, and eight rats were trained to discriminate a mixture of 1.5 mg/kg MDMA and 0.5 mg/kg d-amphetamine (MDMA + AMPH) from vehicle. Substitution tests were conducted with MDMA, d-amphetamine, MDPV, MEPH, and cocaine.Dose-response curves generated with MDMA and MEPH were comparable between training groups. In contrast, AMPH, MDPV, and cocaine produced only partial substitution in animals trained to discriminate MDMA but produced full substitution in animals trained to discriminate the MDMA + AMPH mixture.These findings indicate that MDPV's effects may be more similar to those of traditional psychostimulants, whereas MEPH exerts stimulus effects more similar to those of MDMA. Additional experiments with selective DA and 5-hydroxytryptamine (5-HT) receptor antagonists are required to further elucidate specific receptor mechanisms mediating the discriminative stimulus effects of MDPV and mephedrone.

BACKGROUND:Synthetic cathinones, 4-methylmethcathinone (4-MMC) and 3,4-methylenedioxypyrovalerone (MDPV), serve as a substrate or blocker at monoaminergic transporters, respectively, and produce locomotor stimulant effects in rodents. The present study investigated in rats the effects of repeated exposure to 4-MMC, MDPV, or mixtures of the two on the induction of locomotor sensitization and expression of cross-sensitization to cocaine. METHODS:Seventy-two male Sprague-Dawley rats received daily intraperitoneal injections of saline, MDPV (0.5mg/kg), 4-MMC (0.5, 1.0, or 2.0mg/kg) or mixtures of 0.5mg/kg MDPV+4-MMC (0.5, 1.0, or 2.0mg/kg) for seven consecutive days. Locomotor activity was recorded on days 1 and 7 and again after an acute injection of 5mg/kg cocaine following a 10day drug washout period. RESULTS:Rats injected with 0.5mg/kg MDPV, 0.5, 1.0, or 2.0mg/kg 4-MMC, or 2.0mg/kg 4-MMC+0.5mg/kg MDPV displayed time-dependent increases in horizontal activity that were augmented on day 7 compared to day 1. In addition, rats pretreated with 0.5mg/kg MDPV, 2.0mg/kg 4-MMC, or mixtures of 4-MMC+MDPV displayed an enhanced response to cocaine. CONCLUSIONS:Locomotor responses sensitize to MDPV and to certain mixtures of MDPV and 4-MMC following repeated dosing. Furthermore, previous exposure to these substances may produce cross-sensitization to the locomotor stimulant effects of cocaine. Considered together with recent findings that 4-MMC and MDPV have different sites of action, but both influence monoaminergic functioning, further investigations utilizing a variety of behavioral assays may prove informative regarding the abuse liability of synthetic cathinone mixtures.

Modafinil is a wake-promoting drug with FDA approval for the treatment of excessive daytime sleepiness that has been prescribed for ADHD and recently assessed as a potential treatment for psychostimulant dependence. Previous research indicates that modafinil modestly increases locomotor activity and produces similar discriminative stimulus effects to psychostimulants in rodents, although the subjective effects of modafinil are reportedly distinct from those of cocaine or amphetamine in humans with a history of psychostimulant abuse.

This study sought to investigate the effects of environmentally relevant gestational followed by continued chronic exposure to the herbicide, atrazine, on motor function, cognition, and neurochemical indices of nigrostriatal dopamine (DA) activity in male rats. Dams were treated with 100 μg/kg atrazine, 10mg/kg atrazine, or vehicle on gestational day 1 through postnatal day 21. Upon weaning, male offspring continued daily vehicle or atrazine gavage treatments for an additional six months. Subjects were tested in a series of behavioral assays, and 24h after the last treatment, tissue samples from the striatum were analyzed for DA and 3,4-dihydroxyphenylacetic acid (DOPAC). At 10mg/kg, this herbicide was found to produce modest disruptions in motor functioning, and at both dose levels it significantly lowered striatal DA and DOPAC concentrations. These results suggest that exposures to atrazine have the potential to disrupt nigrostriatal DA neurons and behaviors associated with motor functioning.

Recreational use of illicit methcathinone derivatives is a public health concern in the USA and Europe. Recent reports indicate that mephedrone (MEPH) produces neurochemical and behavioral effects comparable with amphetamines. The present study investigated the effects of a mixture of MEPH and D-amphetamine (AMPH) on the induction and expression of behavioral sensitization. Thirty female CD-1 mice received seven subcutaneous injections of saline, AMPH (1.0 mg/kg), MEPH (3.0 mg/kg), or AMPH (1.0 mg/kg)+MEPH (3.0 mg/kg) over an 8-day period with 48 h between the first and second injection and 24 h between subsequent injections. Activity was assessed immediately following the first and seventh dose. After a 10-day washout, 1.0 mg/kg AMPH was administered to all animals and activity was assessed in a similar manner. Compared with mice treated with AMPH or MEPH, those treated with AMPH+MEPH displayed stronger indices of behavioral sensitization after the seventh dose and in response to AMPH after the washout period. These results suggest that MEPH may enhance sensitivity to the behavioral effects of AMPH. Considered in light of findings that MEPH has comparable neurochemical actions to the amphetamines and is commonly used with other stimulants, further research on the abuse liability of drug mixtures with methcathinones and other psychostimulants is warranted.

Modafinil is a novel wake-promoting drug with FDA approval for the treatment of sleep-related disorders that has recently been investigated as a potential agonist replacement therapy for psychostimulant dependence. Previous research in animals and humans indicates modafinil has a lower abuse liability than traditional psychostimulants, although few studies have carefully assessed modafinil's stimulus properties in combination with other psychostimulants. The current study trained male Sprague-Dawley rats to discriminate subcutaneous injections of 0.3 mg/kg (n=8) or 1.0 mg/kg d-amphetamine (n=8) from saline under an FR 20 schedule of food reinforcement and substitution tests were administered with d-amphetamine (0.03-1.0 mg/kg, s.c.), modafinil (32-256 mg/kg, i.g.), and a low modafinil dose (32 mg/kg, i.g.) in combination with d-amphetamine (0.03-1.0 mg/kg, s.c.) to determine if these drugs have additive effects. The selective D2 dopamine agonist, PNU-91356A, was also tested as a positive control and ethanol and morphine were tested as negative controls. Results indicate that modafinil produced dose-dependent and statistically significant d-amphetamine-lever responding in both groups and nearly complete substitution in animals trained to discriminate 1.0 mg/kg d-amphetamine. Modafinil pretreatment slightly increased the discrimination of low d-amphetamine doses in animals trained to discriminate 0.3 mg/kg d-amphetamine. These results support previous findings that modafinil and d-amphetamine may have additive effects. In consideration of recent interests in modafinil as an agonist treatment for psychostimulant dependence, additional preclinical investigations utilizing other methodologies to examine modafinil in combination with other stimulants, such as behavioral sensitization paradigms or drug self-administration, may be of interest.