Behavioral Pharmacological Studies Research Articles

Metabotropic glutamate receptors and drug addiction

Historically, brain catecholamine systems have been the primary focus of studies examining the neural substrates of drug addiction. In the past two decades, however, a wealth of evidence has accumulated indicating a pivotal role for glutamatergic neurotransmission in mediating addictive behaviors as well as long-term neuroplasticity associated with chronic drug use. As a result, there has been increased interest in developing glutamate-based therapies for the treatment of addictive disorders. Metabotropic glutamate (mGlu) receptors are classified into subcategories designated as Group I (mGlu1 and mGlu5), Group II (mGlu2 and mGlu3), and Group III (mGlu4, mGlu6, mGlu7, and mGlu8), and have received a great deal of attention due to their mediation of slower modulatory excitatory neurotransmission. Pharmacological ligands targeting these receptors have demonstrated reduced incidences of excitotoxicity or severe adverse side effects as compared to those targeting ionotropic glutamate (iGlu) receptors. Behavioral genetic and pharmacological studies have explored the role of individual mGlu receptor subtypes in regulating various addiction-related behaviours and several mGlu receptor ligands have been the subject of clinical testing for other medical conditions.

Group 1 metabotropic glutamate receptors and schizophrenia

AbstractSchizophrenia is a prevalent neuropsychiatric disorder associated with perturbations in medial prefrontal cortex (PFC) excitability and connectivity. As such, research into the neurobiology of schizophrenia and related psychotic disorders has focused on understanding the possible role played by the major excitatory neurotransmitter glutamate in mediating the cognitive and psychosocial impairments characteristic of this disease. Historically, the ionotropic NMDA glutamate receptor has received the greatest experimental attention in this regard. However, converging data point to Group 1 metabotropic glutamate receptors (mGluRs), as well as the Group 1 mGluR scaffolding protein Homer, as critical for normal psychomotor and cognitive function. Moreover, data derived primarily from behavioral pharmacological and genetic studies conducted in animal models of psychosis, suggest that dysregulation of Group 1 mGluR signaling may contribute to anomalies in neuronal excitation putatively underpinning the manifestation of cognitive abnormalities and psychotic‐like behavior. These data implicate targeting Group 1 mGluRs as viable pharmacotherapeutic strategies for the treatment of schizophrenia and related disorders. WIREs Membr Transp Signal 2012, 1:94–103. doi: 10.1002/wmts.15For further resources related to this article, please visit the WIREs website.

Identification of a Dopamine Receptor-Mediated Opiate Reward Memory Switch in the Basolateral Amygdala–Nucleus Accumbens Circuit

The basolateral amygdala (BLA), ventral tegmental area (VTA), and nucleus accumbens (NAc) play central roles in the processing of opiate-related associative reward learning and memory. The BLA receives innervation from dopaminergic fibers originating in the VTA, and both dopamine (DA) D1 and D2 receptors are expressed in this region. Using a combination of in vivo single-unit extracellular recording in the NAc combined with behavioral pharmacology studies, we have identified a double dissociation in the functional roles of DA D1 versus D2 receptor transmission in the BLA, which depends on opiate exposure state; thus, in previously opiate-naive rats, blockade of intra-BLA D1, but not D2, receptor transmission blocked the acquisition of associative opiate reward memory, measured in an unbiased conditioned place preference procedure. In direct contrast, in rats made opiate dependent and conditioned in a state of withdrawal, intra-BLA D2, but not D1, receptor blockade blocked opiate reward encoding. This functional switch was dependent on cAMP signaling as comodulation of intra-BLA cAMP levels reversed or replicated the functional effects of intra-BLA D1 or D2 transmission during opiate reward processing. Single-unit in vivo extracellular recordings performed in neurons of the NAc confirmed an opiate-state-dependent role for BLA D1/D2 transmission in NAc neuronal response patterns to morphine. Our results characterize and identify a novel opiate addiction switching mechanism directly in the BLA that can control the processing of opiate reward information as a direct function of opiate exposure state via D1 or D2 receptor signaling substrates.

Synthesis and Pharmacological Evaluation of Fluorine-Containing D3 Dopamine Receptor Ligands

A series of fluorine-containing N-(2-methoxyphenyl)piperazine and N-(2-fluoroethoxy)piperazine analogues were synthesized, and their affinities for human dopamine D(2), D(3), and D(4) receptors were determined. Radioligand binding studies identified five compounds, 18a, 20a, 20c, 20e, and 21e, which bind with high affinity at D(3) (K(i) = 0.17-5 nM) and moderate to high selectivity for D(3) vs D(2) receptors (ranging from ∼25- to 163-fold). These compounds were also evaluated for intrinsic activity at D(2) and D(3) receptors using a forskolin-dependent adenylyl cyclase assay. This panel of compounds exhibits varying receptor subtype binding selectivity and intrinsic activity at D(2) vs D(3) receptors. These compounds may be useful for behavioral pharmacology studies on the role of D(2)-like dopamine receptors in neuropsychiatric and neurological disorders. Furthermore, compound 20e, which has the highest binding affinity and selectivity for the D(3) receptor (K(i) = 0.17 nM for D(3), 163-fold selectivity for D(3) vs D(2) receptors), represents a candidate fluorine-18 radiotracer for in vivo PET imaging studies on the regulation of D(3) receptor expression.

The role of serotonin receptor subtypes in treating depression: a review of animal studies

Serotonin reuptake inhibitors (SSRIs) are effective in treating depression. Given the existence of different families and subtypes of 5-HT receptors, multiple 5-HT receptors may be involved in the antidepressant-like behavioral effects of SSRIs. Behavioral pharmacology studies investigating the role of 5-HT receptor subtypes in producing or blocking the effects of SSRIs were reviewed. Few animal behavior tests were available to support the original development of SSRIs. Since their development, a number of behavioral tests and models of depression have been developed that are sensitive to the effects of SSRIs, as well as to other types of antidepressant treatments. The rationale for the development and use of these tests is reviewed. Behavioral effects similar to those of SSRIs (antidepressant-like) have been produced by agonists at 5-HT(1A), 5-HT(1B), 5-HT(2C), 5-HT(4), and 5-HT(6) receptors. Also, antagonists at 5-HT(2A), 5-HT(2C), 5-HT(3), 5-HT(6), and 5-HT(7) receptors have been reported to produce antidepressant-like responses. Although it seems paradoxical that both agonists and antagonists at particular 5-HT receptors can produce antidepressant-like effects, they probably involve diverse neurochemical mechanisms. The behavioral effects of SSRIs and other antidepressants may also be augmented when 5-HT receptor agonists or antagonists are given in combination. The involvement of 5-HT receptors in the antidepressant-like effects of SSRIs is complex and involves the orchestration of stimulation and blockade at different 5-HT receptor subtypes. Individual 5-HT receptors provide opportunities for the development of a newer generation of antidepressants that may be more beneficial and effective than SSRIs.

Human behavioral pharmacology, past, present, and future: symposium presented at the 50th annual meeting of the Behavioral Pharmacology Society.

A symposium held at the 50th annual meeting of the Behavioral Pharmacology Society in May 2007 reviewed progress in the human behavioral pharmacology of drug abuse. Studies on drug self-administration in humans are reviewed that assessed reinforcing and subjective effects of drugs of abuse. The close parallels observed between studies in humans and laboratory animals using similar behavioral techniques have broadened our understanding of the complex nature of the pharmacological and behavioral factors controlling drug self-administration. The symposium also addressed the role that individual differences, such as sex, personality, and genotype play in determining the extent of self-administration of illicit drugs in human populations. Knowledge of how these factors influence human drug self-administration has helped validate similar differences observed in laboratory animals. In recognition that drug self-administration is but one of many choices available in the lives of humans, the symposium addressed the ways in which choice behavior can be studied in humans. These choice studies in human drug abusers have opened up new and exciting avenues of research in laboratory animals. Finally, the symposium reviewed behavioral pharmacology studies conducted in drug abuse treatment settings and the therapeutic benefits that have emerged from these studies.

Intrinsic membrane properties of pre-oromotor neurons in the intermediate zone of the medullary reticular formation

Neurons in the lower brainstem that control consummatory behavior are widely distributed in the reticular formation (RF) of the pons and medulla. The intrinsic membrane properties of neurons within this distributed system shape complex excitatory and inhibitory inputs from both orosensory and central structures implicated in homeostatic control to produce coordinated oromotor patterns. The current study explored the intrinsic membrane properties of neurons in the intermediate subdivision of the medullary reticular formation (IRt). Neurons in the IRt receive input from the overlying (gustatory) nucleus of the solitary tract and project to the oromotor nuclei. Recent behavioral pharmacology studies as well as computational modeling suggest that inhibition in the IRt plays an important role in the transition from a taste-initiated oromotor pattern of ingestion to one of rejection. The present study explored the impact of hyperpolarization on membrane properties. In response to depolarization, neurons responded with either a tonic discharge, an irregular/burst pattern or were spike-adaptive. A hyperpolarizing pre-pulse modulated the excitability of most (82%) IRt neurons to subsequent depolarization. Instances of both increased (30%) and decreased (52%) excitability were observed. Currents induced by the hyperpolarization included an outward 4-aminopyridine (4-AP) sensitive K+ current that suppressed excitability and an inward cation current that increased excitability. These currents are also present in other subpopulations of RF neurons that influence the oromotor nuclei and we discuss how these currents could alter firing characteristics to impact pattern generation.

GABAergic modulation of human social interaction in a prisoner's dilemma model by acute administration of alprazolam

Recent work in neuroeconomics has used game theory paradigms to examine neural systems that subserve human social interaction and decision making. Attempts to modify social interaction through pharmacological manipulation have been less common. Here we show dose-dependent modification of human social behavior in a prisoner's dilemma model after acute administration of the γ-aminobutyric acid (GABA)-A modulating benzodiazepine alprazolam. Nine healthy adults received doses of placebo, 0.5, 1.0, and 2.0 mg alprazolam in a counterbalanced within-subject design, while completing multiple test blocks per day on an iterated prisoner's dilemma game. During test blocks in which peak subjective effects of alprazolam were reported, cooperative choices were significantly decreased as a function of dose. Consistent with previous reports showing that high acute doses of GABA-modulating drugs are associated with violence and other antisocial behavior, our data suggest that at sufficiently high doses, alprazolam can decrease cooperation. These behavioral changes may be facilitated by changes in inhibitory control facilitated by GABA. Game theory paradigms may prove useful in behavioral pharmacology studies seeking to measure social interaction, and may help inform the emerging field of neuroeconomics.

Spaced initial stimulus familiarization enhances novelty preference in Long-Evans rats

Berlyne [Berlyne, D.E., 1950. Novelty and curiosity as determinants of exploratory behaviour. Brit. J. Psychol. 41, 68-80] first illustrated that rats prefer to explore novel objects over ones with which they have had previous experience. Recently, variants on this novel object recognition (NOR) task have become widely popular and have been employed in numerous neuroscience and behavioral pharmacological studies investigating memory processes. Given this popularity, a thorough understanding of the various behavioral processes involved in novelty reaction and preference is essential. The current study compared the effects of spaced and massed initial stimulus exposures upon later object exploration and novel stimulus preference in Long-Evans rats. Results illustrated that a distributed initial stimulus familiarization procedure promoted greater novel object preference than did a massed procedure, and suggest that the novel object recognition task is sensitive to spacing effects in a similar fashion to more traditional learning paradigms. The mechanisms underlying such spacing effects are briefly discussed.

Mechanistic model for the acute effect of fluvoxamine on 5-HT and 5-HIAA concentrations in rat frontal cortex

A mechanistic model is proposed to predict the time course of the concentrations of 5-HT and its metabolite 5-hydroxyindolacetic acid (5-HIAA) in rat frontal cortex following acute administration of SSRIs. In the model, SSRIs increase synaptic 5-HT concentrations by reversible blockade of the SERT in a direct concentration-dependent manner, while the 5-HT response is attenuated by negative feedback via 5-HT autoreceptors. In principle, the model allows for the description of oscillatory patterns in the time course of 5-HT and 5-HIAA concentrations in brain extracellular fluid. The model was applied in a pharmacokinetic–pharmacodynamic (PK/PD) investigation on the time course of the microdialysate 5-HT and 5-HIAA response in rat frontal cortex following a 30-min intravenous infusion of 3.7 and 7.3 mg/kg fluvoxamine. Directly after administration of fluvoxamine, concentrations of 5-HT were increased to approximately 450–600% of baseline values while 5-HIAA concentrations were decreased. Thereafter 5-HT and 5-HIAA concentrations gradually returned to baseline values in 6–10 h, respectively. The PK/PD analysis revealed that inhibition of 5-HT reuptake was directly related to the fluvoxamine concentration in plasma, with 50% inhibition of 5-HT reuptake occurring at a plasma concentration of 1.1 ng/ml (EC 50). The levels of 5-HT at which 50% of the inhibition of the 5-HT response was reached (IC 50) amounted to 272% of baseline. The model was unable to capture the oscillatory patterns in the individual concentration time curves, which appeared to occur randomly. The proposed mechanistic model is the first step in modeling of complex neurotransmission processes. The model constitutes a useful basis for prediction of the time course of median 5-HT and 5-HIAA concentrations in the frontal cortex in behavioral pharmacology studies in vivo.

Effects of selective dopamine D1-like and D2-like agonists on prepulse inhibition of startle in inbred C3H/HeJ, SPRET/EiJ, and CAST/EiJ mice

Prepulse inhibition (PPI) and locomotor activity have been used to investigate the effects of antipsychotic and stimulant drugs and their underlying dopaminergic mechanisms. Whereas D2-like agonists consistently decreased PPI and increased locomotion in rats in previous studies, we recently reported that these hallmark behavioral effects were not observed in several mouse strains. Nevertheless, we recently identified three mouse strains (C3H/HeJ, SPRET/EiJ, and CAST/EiJ) that exhibited locomotor hyperactivity after administration of a selective D2-like agonist. We hypothesized that, similar to rats, C3H/HeJ, SPRET/EiJ, and CAST/EiJ mice would exhibit decreased PPI after administration of a D2-like agonist. Administration of the D2-like agonist quinelorane dose-dependently decreased PPI in C3H/HeJ and SPRET/EiJ mice. In agreement with previous reports in rats and other strains of mice, the D1-like agonist R-6-Br-APB also decreased PPI in C3H/HeJ and SPRET/EiJ mice. In contrast, CAST/EiJ mice had low levels of baseline PPI in our standard test session and quinelorane and R-6-Br-APB had no effect on PPI under those conditions. Through the optimization of session parameters, we obtained higher baseline PPI in CAST/EiJ mice and found that quinelorane but not R-6-Br-APB decreased PPI. In summary, similar to rats and unlike previous published reports on several strains of mice, we have now identified three strains of mice in which a D2-like agonist decreased PPI. The C3H/HeJ, SPRET/EiJ, and CAST/EiJ mice may more closely mirror the Sprague Dawley rat than most other mouse strains and may confer advantages in cross-species behavioral pharmacology studies related to D2 receptor function.

Lack of Association of 5-HTTLPR Genotype with Smoking Cessation in a Nicotine Replacement Therapy Randomized Trial

Cigarette smoking is the leading preventable cause of death worldwide, accounting for at least 30% of all cancer deaths and over three quarters (87%) of lung cancer deaths in developed countries; however, despite progress made in the treatment of tobacco dependence, available Food and Drug

Haploinsufficiency of the arginine–vasopressin gene is associated with poor spatial working memory performance in rats

Behavioral pharmacological studies have implicated a role for the neurophysin arginine–vasopressin in learning and memory. Vasopressin, and its analogues, can produce either improvements or impairments in mnemonic functions, effects that depend upon the agent administered, the memory process measured and the task employed. As recent data have implicated vasopressin in regulating the cognitive functions of the prefrontal cortex, we sought to determine whether changes in vasopressinergic tone would affect a form of memory that is dependent upon this brain region. To that end, we used a genetic approach to examine how haploinsufficiency of the vasopressin gene affects working memory performance. Specifically, we tested a naturally occurring null-mutant rat on an operant delayed-non-match-to-position task. Male and female heterozygous and wild-type rats were trained to perform this working memory task, and the effects of varying the delay across which they had to maintain task information were systematically varied. Although vasopressin-deficient rats omitted fewer trials and completed trials more quickly, they exhibited delay-dependent deficits of choice accuracy. The genotype effects were not modified by sex. Collectively, these data indicate that even partial vasopressin deficiency can trigger deficits of spatial working memory performance and add to the growing body of results supporting a regulatory control of neocortical-dependent cognitive functions by this neurohormone.

Naltrexone reduces the relative reinforcing value of nicotine in a cigarette smoking choice paradigm

Human behavioral pharmacology studies can examine how medications that target different neurotransmitter systems influence different aspects of smoking. Naltrexone and bupropion have been shown to alter ad lib smoking behavior; however, medication effects on nicotine reward in a cigarette choice paradigm have yet to be investigated. This study explored the effects of an acute dose of naltrexone, bupropion, or placebo on the relative reinforcing value of nicotine from cigarette smoking using new nicotine and de-nicotinized (Quest, 0.6 and 0.05 mg = "denicotinized") cigarettes. In a double-blind, within-subjects design, 26 dependent smokers participated in three experimental cigarette smoking sessions following pretreatment with either naltrexone (50 mg), bupropion (300 mg), or placebo. After medication administration and 2 h of monitored deprivation from cigarettes and food, participants rated their responses to the initial exposure to the cigarettes and then participated in four choice sessions over a 2-h period during which they could take four puffs from either cigarette. The relative reinforcing value of nicotine, as measured by the number of nicotine puffs chosen out of 16, was significantly lower following naltrexone compared to placebo. There were no effects of an acute dose of bupropion on nicotine choices. These results suggest that naltrexone may reduce the relative reinforcing effects of nicotine via cigarette smoking and support ongoing investigation of opioid antagonists as potential smoking cessation pharmacotherapies.

Phosphodiesterase type 5 (PDE5) inhibition and cognitive enhancement

Drug development for the treatment of dementia and age-related cognitive decline has been slow to produce clinically viable alternatives to the two existing FDA approved drug treatments, AChE inhibitors and the noncompetitive NMDA receptor antagonist, memantine. Recent human and preclinical animal studies suggest that phosphodiesterase type 5 (PDE5) inhibitors, already in clinical use for the treatment of erectile dysfunction in men, may have central cognitive enhancing effects. Recent behavioral pharmacological studies with rodents demonstrate that PDE5 inhibitors improve memory consolidation in young animals using passive avoidance and object recognition tasks. In addition, we have shown that pretraining administration of the PDE5 inhibitor sildenafil citrate can attenuate a spatial learning impairment in the 14-unit T-maze. Research findings with aged animals show that pretraining administration of sildenafil can improve the long-term retention of spatial memory 1 week following maze acquisition. We propose that the available research findings provide preclinical support for a possible alternative use of PDE5 inhibitors as cognitive enhancing agents that improve different kinds of learning and memory and protect brain systems against neurodegeneration.

Novel analogs of the σ receptor ligand BD1008 attenuate cocaine-induced toxicity in mice

Previous studies have shown that BD1008 ( N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)ethylamine) and related analogs attenuate the toxicity and stimulant effects of cocaine through antagonism of σ receptors. In the present study, six analogs of BD1008 (UMB 98–103) were synthesized and evaluated in receptor binding and behavioral studies. Competition binding studies confirmed that all six compounds have high affinity for σ 1 receptors, moderate affinity for σ 2 receptors, and low to negligible affinity for monoamine transporters, opioid, N-methyl- d-aspartate, dopamine, and 5-HT receptors. In behavioral pharmacological studies, pretreatment of mice with UMB 100, UMB 101, or UMB 103 significantly attenuated cocaine-induced convulsions and lethality. Together with earlier studies, the data suggest that analogs of BD1008 are promising medication development leads for reducing the toxicity of cocaine.

Evidence for genetic linkage between a polymorphism in the adenosine 2A receptor and panic disorder.

Data from clinical and behavioral pharmacological studies have implicated adenosine in anxiety behaviors, while genetic studies have suggested that adenosine receptors may be associated with panic disorder. We have undertaken an analysis of several DNA sequence variations in the adenosine 2A receptor (ADORA2A) in a large sample of panic disorder pedigrees. Individuals from 70 panic disorder pedigrees, and 83 child-parent 'trios', were genotyped at five single-nucleotide polymorphisms (SNPs) in and near the ADORA2A gene and were analyzed for genetic linkage and association. Linkage analysis revealed elevated LOD scores for a silent substitution (1083C/T, SNP-4) in the second coding exon. This SNP has been previously reported to be associated with panic disorder. We observed a maximal heterogeneity LOD score of 2.98 (theta=0) under a recessive genetic model and narrow diagnostic model. Other SNPs showed no evidence for linkage. Association tests were not significant for any of the five ADORA2A SNPs. When SNP haplotypes were assessed in the triads with TRANSMIT, one 3-marker haplotype (SNPs 1, 4, 5) was nominally significantly associated with panic disorder (p=0.029). Pairwise estimations of linkage disequilibrium between the SNPs showed strong patterns of linkage disequilibrium across the ADORA2A locus. Analyses carried out by broadening the panic disorder phenotype to include agoraphobia continued to support linkage to ADORA2A. Our findings provide evidence for a susceptibility locus for panic disorder, and possibly including agoraphobia, either within the ADORA2A gene or in a nearby region of chromosome 22, and serves as the first successful candidate gene replication study in panic disorder.

RO-10-5824 is a selective dopamine D4 receptor agonist that increases novel object exploration in C57 mice

Novelty seeking as a behavioral phenomenon emerges as a compromise between approach and avoidance behavior. Although novelty seeking is thought to play a role in drug abuse and in cognition, the biological basis for this construct is poorly understood. At a genetic level, dopamine D4 receptors (D4R) appear to be critical for the behavioral expression of novelty seeking. In humans, polymorphisms of D4R have been associated with novelty-seeking traits in general and attention deficit-hyperactivity disorder in particular. Similarly, D4R (−/−) mice exhibit less novel object exploration than D4R (+/+) mice. Due to of the paucity of selective D4R ligands for use in behavioral pharmacology studies, few studies have examined the behavioral effects of D4R compounds in animals. The present experiments characterized RO-10-5824, a new, selective D4R partial agonist with minimal affinity for dopamine D2 and D3 receptors, and tested the hypothesis that activation of D4R increases the investigation by mice of a novel object placed in the center of a familiar open field. C57BL/6J and DBA/1J male mice were used in a dose response study of the selective D4R partial agonist RO-10-5824 (0, 1.0, 3.0, or 10.0 mg/kg). While having no effect on the amount of locomotor activity in novel or familiar environments, RO-10-5824 (10.0 mg/kg) increased time spent in the center of the enclosure in the presence of a novel object in C57 but not DBA mice. These results support the hypothesis that stimulation of D4R can enhance novelty seeking in mice and that this effect may be dependent on subtle genetic differences.

Involvement of sigma receptors in the behavioral effects of cocaine: evidence from novel ligands and antisense oligodeoxynucleotides

Pharmacological and molecular biological tools were used to validate the involvement of σ receptors in the actions of cocaine. Radioligand binding studies demonstrated significant levels of σ receptors in the brain and heart, where cocaine interacts preferentially with the σ 1 subtype. In behavioral pharmacological studies using mice, nine novel σ receptor antagonists significantly attenuated cocaine-induced convulsions, while structural analogs with weak interactions with σ receptors were ineffective. In contrast to the protection provided by the antagonists, a classical σ receptor agonist exacerbated the convulsive effects of cocaine. The antagonists also attenuated cocaine-induced lethality, with the best compound protecting against death even when administered as a post-treatment. At doses where the antagonists had no effect on baseline locomotor activity, they significantly attenuated the locomotor stimulatory effects of cocaine, suggesting their ability to block the psychomotor as well as the toxic effects of cocaine. To further validate that the anti-cocaine effects were achieved by interfering with cocaine’s access to σ receptors, antisense oligodeoxynucleotides against σ 1 receptors were shown to attenuate the convulsive and locomotor stimulatory effects of cocaine. Together, the studies support the involvement of σ receptors, particularly the σ 1 subtype, in the behavioral effects of cocaine.

Discovery of a Novel Dopamine Transporter Inhibitor, 4-Hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-Methylphenyl Ketone, as a Potential Cocaine Antagonist through 3D-Database Pharmacophore Searching. Molecular Modeling, Structure−Activity Relationships, and Behavioral Pharmacological Studies

A novel, fairly potent dopamine transporter (DAT) inhibitor, 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3, K(i) values of 492 and 360 nM in binding affinity and inhibition of dopamine reuptake, respectively), with significant functional antagonism against cocaine and a different in vitro pharmacological profile from cocaine at the three transporter sites (dopamine, serotonin, and norepinephrine) was discovered through 3D-database pharmacophore searching. Through structure-activity relationships and molecular modeling studies, we found that hydrophobicity and conformational preference are two additional important parameters that determine affinity at the DAT site. Chemical modifications of the lead compound (3) led to a high affinity analogue (6, K(i) values of 11 and 55 nM in binding affinity and inhibition of dopamine reuptake, respectively). In behavioral pharmacological testing, 6 mimics partially the effect of cocaine in increasing locomotor activity in mice but lacks cocaine-like discriminative stimulus effect in rats. Taken together, these data suggest that 6 represents a promising lead for further evaluations as potential therapy for the treatment of cocaine abuse.