Neurochemical and Cardiovascular Effects of 4-Chloro Ring-Substituted Synthetic Cathinones in Rats.

Synthetic cathinones are a class of new psychoactive substances that display psychomotor stimulant properties, and novel cathinone analogs continue to emerge in illicit drug markets worldwide. The aim of the present study was to characterize the pharmacology of 4-chloro ring-substituted cathinones that are appearing in illicit drug markets compared with the effects of 4-methylmethcathinone (mephedrone). Synaptosomes were prepared from rat caudate for dopamine transporter (DAT) assays or from whole brain minus caudate and cerebellum for norepinephrine transporter (NET) and serotonin transporter (SERT) assays. Findings from transporter uptake inhibition and release assays showed that mephedrone and 4-chloromethcathinone (4-CMC) function as substrates at DAT, NET, and SERT, with similar potency at all three transporters. In contrast, 4-chloro-α-pyrrolidinopropiophenone (4-CαPPP) was an uptake inhibitor at DAT and NET, with similar potency at each site, but had little activity at SERT. 4-Chloroethcathinone (4-CEC) was a low-potency uptake inhibitor at DAT and NET but a substrate at SERT. In rats implanted with telemetry transmitters, mephedrone and 4-CMC increased blood pressure, heart rate, and locomotor activity to a similar extent. 4-CEC and 4-CαPPP were less potent at increasing blood pressure and had modest stimulatory effects on heart rate and activity. 4-CMC also transiently decreased temperature at the highest dose tested. All three 4-chloro ring-substituted cathinones are biologically active, but only 4-CMC has potency comparable to mephedrone. Collectively, our findings suggest that 4-CMC and other 4-chloro cathinones may have abuse potential and adverse effects in humans that are analogous to those associated with mephedrone. SIGNIFICANCE STATEMENT: The 4-chloro ring-substituted cathinones all produced significant cardiovascular stimulation, with 4-chloromethcathinone (4-CMC) showing potency similar to mephedrone. All of the drugs are likely to be abused given their effects at the dopamine transporter, particularly 4-CMC.

Amphetamine-like Neurochemical and Cardiovascular Effects of α-Ethylphenethylamine Analogs Found in Dietary Supplements.

Dietary supplements often contain additives not listed on the label, including α-ethyl homologs of amphetamine such as N,α-diethylphenethylamine (DEPEA). Here, we examined the neurochemical and cardiovascular effects of α-ethylphenethylamine (AEPEA), N-methyl-α-ethylphenethylamine (MEPEA), and DEPEA as compared with the effects of amphetamine. All drugs were tested in vitro using uptake inhibition and release assays for monoamine transporters. As expected, amphetamine acted as a potent and efficacious releasing agent at dopamine transporters (DAT) and norepinephrine transporters (NET) in vitro. AEPEA and MEPEA were also releasers at catecholamine transporters, with greater potency at NET than DAT. DEPEA displayed fully efficacious release at NET but weak partial release at DAT (i.e., 40% of maximal effect). In freely moving, conscious male rats fitted with biotelemetry transmitters for physiologic monitoring, amphetamine (0.1-3.0 mg/kg, s.c.) produced robust dose-related increases in blood pressure (BP), heart rate (HR), and motor activity. AEPEA (1-10 mg/kg, s.c.) produced significant increases in BP but not HR or activity, whereas DEPEA and MEPEA (1-10 mg/kg, s.c.) increased BP, HR, and activity. In general, the phenethylamine analogs were approximately 10-fold less potent than amphetamine. Our results show that α-ethylphenethylamine analogs are biologically active. Although less potent than amphetamine, they produce cardiovascular effects that could pose risks to humans. Given that MEPEA and DEPEA increased locomotor activity, these substances may also have significant abuse potential. SIGNIFICANCE STATEMENT: The α-ethyl homologs of amphetamine have significant cardiovascular, behavioral, and neurochemical effects in rats. Given that these compounds are often not listed on the ingredient labels of dietary supplements, these compounds could pose a risk to humans using these products.

Stereoselective neurochemical, behavioral, and cardiovascular effects of α-pyrrolidinovalerophenone enantiomers in male rats.

The synthetic cathinone α-pyrrolidinovalerophenone (α-PVP) continues to be abused despite being banned by regulatory agencies. The abused formulation of α-PVP is a racemic mixture consisting of two enantiomers, S-α-PVP and R-α-PVP. In this study, we investigated the neurochemical, behavioral, and cardiovascular effects of racemic α-PVP and its enantiomers in male rats. Racemic α-PVP blocked the uptake of both dopamine and norepinephrine ex vivo, but did not block the uptake of serotonin (5-HT), at their respective transporters. S-α-PVP was slightly more potent than racemic α-PVP, while R-α-PVP was 10 to 20 times less potent at blocking dopamine and norepinephrine uptake. In microdialysis studies, racemic and S-α-PVP increased extracellular dopamine levels in the nucleus accumbens, but not levels of 5-HT. Racemic and S-α-PVP also increased locomotor activity. When tested at the same doses, S-α-PVP produced larger effects than racemic α-PVP. R-α-PVP also increased extracellular dopamine levels and locomotor activity, but only at 30 times higher doses than S-α-PVP. Racemic and S-α-PVP were self-administered by rats at 0.03 mg/kg/injection, whereas R-α-PVP was self-administered at a 10 times higher dose. Dose-effect determinations following acquisition suggested that R-α-PVP was at least 30 times less potent than S-α-PVP. Finally, racemic and S-α-PVP increased blood pressure and heart rate at doses approximately 30 times less than was required for R-α-PVP to produce similar effects. These results show that the neurochemical, behavioral, and cardiovascular effects of racemic α-PVP most likely reflect the actions of S isomer.

The Supplement Adulterant ß-Methylphenethylamine Increases Blood Pressure by Acting at Peripheral Norepinephrine Transporters.

ß-Methylphenethylamine [(BMPEA), 2-phenylpropan-1-amine] is a structural isomer of amphetamine (1-phenylpropan-2-amine) that has been identified in preworkout and weight loss supplements, yet little information is available about its pharmacology. Here, the neurochemical and cardiovascular effects of BMPEA and its analogs, N-methyl-2-phenylpropan-1-amine (MPPA) and N,N-dimethyl-2-phenylpropan-1-amine (DMPPA), were compared with structurally related amphetamines. As expected, amphetamine and methamphetamine were potent substrate-type releasing agents at dopamine transporters (DATs) and norepinephrine transporters (NETs) in rat brain synaptosomes. BMPEA and MPPA were also substrates at DATs and NETs, but they were at least 10-fold less potent than amphetamine. DMPPA was a weak substrate only at NETs. Importantly, the releasing actions of BMPEA and MPPA were more potent at NETs than DATs. Amphetamine produced significant dose-related increases in blood pressure (BP), heart rate (HR), and locomotor activity in conscious rats fitted with surgically implanted biotelemetry transmitters. BMPEA, MPPA, and DMPPA produced increases in BP that were similar to the effects of amphetamine, but the compounds failed to substantially affect HR or activity. The hypertensive effect of BMPEA was reversed by the α-adrenergic antagonist prazosin but not the ganglionic blocker chlorisondamine. Radioligand binding at various G protein-coupled receptors did not identify nontransporter sites of action that could account for cardiovascular effects of BMPEA or its analogs. Our results show that BMPEA, MPPA, and DMPPA are biologically active. The compounds are unlikely to be abused due to weak effects at DATs, but they could produce adverse cardiovascular effects via substrate activity at peripheral NET sites.

Newly Developed Dopamine D3 Receptor Antagonists, R-VK4-40 and R-VK4-116, Do Not Potentiate Cardiovascular Effects of Cocaine or Oxycodone in Rats.

Opioid and cocaine abuse are major public health burdens. Existing medications for opioid use disorder are limited by abuse liability and side effects, whereas no treatments are currently approved in the United States for cocaine use disorder. Dopamine D3 receptor (D3R) antagonists have shown promise in attenuating opioid and cocaine reward and mitigating relapse in preclinical models. However, translation of D3R antagonists to the clinic has been hampered by reports that the D3R antagonists GSK598,809 (5-(5-((3-((1S,5R)-1-(2-fluoro-4-(trifluoromethyl)phenyl)-3-azabicyclo[3.1.0]hexan-3-yl)propyl)thio)-4-methyl-4H-1,2,4-triazol-3-yl)-4-methyloxazole) and SB-277,011A (2-(2-((1r,4r)-4-(2-oxo-2-(quinolin-4-yl)ethyl)cyclohexyl)ethyl)-1,2,3,4-tetrahydroisoquinoline-6-carbonitrile) have adverse cardiovascular effects in the presence of cocaine. Recently, we developed two structurally novel D3R antagonists, R-VK4-40 and R-VK4-116, which are highly selective for D3R and display translational potential for treatment of opioid use disorder. Here, we tested whether R-VK4-40 ((R)-N-(4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) and R-VK4-116 ((R)-N-(4-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-carboxamide) have unwanted cardiovascular effects in the presence of oxycodone, a prescription opioid, or cocaine in freely moving rats fitted with surgically implanted telemetry transmitters. We also examined cardiovascular effects of the D3R antagonist, SB-277,011A, and L-741,626 (1-((1H-indol-3-yl)methyl)-4-(4-chlorophenyl)piperidin-4-ol), a dopamine D2 receptor-selective antagonist, for comparison. Consistent with prior reports, SB-277,011A increased blood pressure, heart rate, and locomotor activity alone and in the presence of cocaine. L-741,626 increased blood pressure and heart rate. In contrast, R-VK4-40 alone dose-dependently reduced blood pressure and heart rate and attenuated oxycodone-induced increases in blood pressure and oxycodone or cocaine-induced increases in heart rate. Similarly, R-VK4-116 alone dose-dependently reduced cocaine-induced increases in blood pressure and heart rate. These results highlight the safety of new D3R antagonists and support the continued development of R-VK4-40 and R-VK4-116 for the treatment of opioid and cocaine use disorders. SIGNIFICANCE STATEMENT: Opioid and cocaine abuse are major public health challenges and new treatments that do not adversely impact the cardiovascular system are needed. Here, we show that two structurally novel dopamine D3 receptor antagonists, R-VK4-40 and R-VK4-116, do not potentiate, and may even protect against, oxycodone- or cocaine-induced changes in blood pressure and heart rate, supporting their further development for the treatment of opioid and/or cocaine use disorders.

Persistent palatable food preference in rats with a history of limited and extended access to methamphetamine self-administration.

Recent studies have shown that when given a mutually exclusive choice between cocaine and palatable foods, most rats prefer the non-drug rewards over cocaine. Here, we used a discrete choice procedure to assess whether palatable food preference generalizes to rats with a history of limited (3 hours/day) or extended (6 or 9 hours/day) access to methamphetamine self-administration. On different daily sessions, we trained rats to lever-press for either methamphetamine (0.1-0.2 mg/kg/infusion) or palatable food (five pellets per reward delivery) for several weeks; regular food was freely available. We then assessed food-methamphetamine preference either during training, after priming methamphetamine injections (0.5-1.0 mg/kg), following a satiety manipulation (palatable food exposure in the home cage) or after 21 days of withdrawal from methamphetamine. We also assessed progressive ratio responding for palatable food and methamphetamine. We found that independent of the daily drug access conditions and the withdrawal period, the rats strongly preferred the palatable food over methamphetamine, even when they were given free access to the palatable food in the home cage. Intake of methamphetamine and progressive ratio responding for the drug, both of which increased or escalated over time, did not predict preference in the discrete choice test. Results demonstrate that most rats strongly prefer palatable food pellets over intravenous methamphetamine, confirming previous studies using discrete choice procedures with intravenous cocaine. Results also demonstrate that escalation of drug self-administration, a popular model of compulsive drug use, is not associated with a cardinal feature of human addiction of reduced behavioral responding for non-drug rewards.

Reinforcing effects of phenethylamine analogs found in dietary supplements.

RATIONALE: Synthetic phenethylamine (PEA) analogs, such as ß-methylphenethylamine (BMPEA) and N,α-diethylphenethylamine (DEPEA), are often found in dietary supplements, despite regulations prohibiting their sale. PEA analogs are structurally related to amphetamine, and we have shown that BMPEA and DEPEA produce cardiovascular stimulation mimicking the effects of amphetamine. However, few studies have examined behavioral effects of BMPEA, DEPEA, and other PEA analogs. OBJECTIVES: Here, we examined the reinforcing effects of α-ethylphenethylamine (AEPEA, 1 mg/kg/injection), DEPEA (1 mg/kg/injection), and BMPEA (3 mg/kg/injection) as compared to amphetamine (0.1 mg/kg/injection) using a fixed-ratio 1 self-administration paradigm in male rats. METHODS: Male rats were trained in self-administration chambers containing 2 nose-poke holes. A nose-poke response in the active hole delivered drug or saline, whereas a nose-poke response in the inactive hole had no programmed consequence. Four groups of rats were initially trained for 10 days with the doses noted above. Upon acquisition of drug self-administration, a dose-effect function was determined by training rats on 3 additional doses for 3 days each. A separate group of rats was trained with saline. RESULTS: Male rats self-administered each PEA analog and amphetamine, as shown by significant increases in active responses versus inactive responses. Subsequent dose-response testing showed clear differences in potency of the compounds. Amphetamine showed a typical inverted U-shaped dose-effect function, peaking at 0.1 mg/kg/injection. AEPEA and DEPEA also showed inverted dose-effect functions, with each peaking at 0.3 mg/kg/injection. BMPEA did not show an inverted U-shaped dose-effect function, but active responding slowly increased up to a dose of 6 mg/kg/injection. CONCLUSIONS: Taken together, our findings indicate that dietary supplements containing PEA analogs may have significant abuse liability when used recreationally.

A stimulus-control account of regulated drug intake in rats.

RATIONALE: Patterns of drug self-administration are often highly regular, with a consistent pause after each self-injection. This pausing might occur because the animal has learned that additional injections are not reinforcing once the drug effect has reached a certain level, possibly due to the reinforcement system reaching full capacity. Thus, interoceptive effects of the drug might function as a discriminative stimulus, signaling when additional drug will be reinforcing and when it will not. OBJECTIVE: This hypothetical stimulus control aspect of drug self-administration was emulated using a schedule of food reinforcement. MATERIALS AND METHODS: Rats' nose-poke responses produced food only when a cue light was present. No drug was administered at any time. However, the state of the light stimulus was determined by calculating what the whole-body drug level would have been if each response in the session had produced a drug injection. The light was only presented while this virtual drug level was below a specific threshold. A range of doses of cocaine and remifentanil were emulated using parameters based on previous self-administration experiments. RESULTS: Response patterns were highly regular, dose-dependent, and remarkably similar to actual drug self-administration. CONCLUSION: This similarity suggests that the emulation schedule may provide a reasonable model of the contingencies inherent in drug reinforcement. Thus, these results support a stimulus control account of regulated drug intake in which rats learn to discriminate when the level of drug effect has fallen to a point where another self-injection will be reinforcing.

Differential involvement of dopamine receptors in conditioned suppression induced by cocaine.

Cocaine-paired stimuli can suppress food-reinforced operant behavior in rats, providing an animal model of conditioned drug effects. To study the neuropharmacological basis of this phenomenon, we examined the effects of various dopamine receptor antagonists on the acquisition and expression of cocaine-induced conditioned suppression in rats. Superimposed on an ongoing baseline of food-reinforced operant responding, a stimulus was paired with response-independent cocaine (3.0 mg/kg, i.v.) during each of 8 training sessions. To study acquisition, independent groups of rats were given saline, the dopamine D(1)-like receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390) (0.001-0.03 mg/kg, i.p.), or the dopamine D(2)-like receptor antagonist eticlopride (0.001-0.03 mg/kg, i.p.) prior to each training session. To study expression, independent groups of rats were trained first, then given saline, SCH 23390, eticlopride, or N-[4-(4-(2-methoxyphenyl)piperazinyl)butyl]-2-naphthamide (BP 897) (a dopamine D(3) partial receptor agonist; 0.1-1.0 mg/kg, i.p.) before test sessions in which the stimulus was presented without cocaine. Pre-treatment with either SCH 23390 or eticlopride during acquisition reduced the direct suppressant effects of cocaine, but conditioning was blocked only in rats that were treated with SCH 23390 during acquisition training. Expression of conditioning was attenuated only by eticlopride. Thus, dopamine at least partially mediates both the acquisition and expression of cocaine-induced conditioned suppression, with activation of dopamine D(1)- and D(2)-like receptors underlying these respective processes.

Blocking of conditioning to a cocaine-paired stimulus: testing the hypothesis that cocaine perpetually produces a signal of larger-than-expected reward.

According to a recent account of addiction, dopaminergic effects of drugs like cocaine mimic the neuronal signal that occurs when a natural reward has a larger value than expected. Consequently, the drug's expected reward value increases with each administration, leading to an over-selection of drug-seeking behavior. One prediction of this hypothesis is that the blocking effect, a cornerstone of contemporary learning theory, should not occur with drug reinforcers. To test this prediction, two groups of rats were trained to self-administer cocaine with a nose-poking response. For 5 sessions, a tone was paired with each self-administered injection (blocking group), or no stimulus was paired with injection (non-blocking group). Then, in both groups, the tone and a light were both paired with each injection for 5 sessions. In subsequent testing, the light functioned as a conditioned reinforcer for a new response (lever-pressing) in the non-blocking group, but not the blocking group. Thus, contrary to prediction, pre-training with the tone blocked conditioning to the light. Although these results fail to support a potentially powerful explanation of addiction, they are consistent with the fact that most conditioning and learning phenomena that occur with non-drug reinforcers can also be demonstrated with drug reinforcers.

Synthetic cannabinoids found in "spice" products alter body temperature and cardiovascular parameters in conscious male rats.

BACKGROUND: The misuse of synthetic cannabinoids is a persistent public health concern. Because these drugs target the same cannabinoid receptors as the active ingredient of marijuana, Δ9-tetrahydrocannabinol (THC), we compared the effects of synthetic cannabinoids and THC on body temperature and cardiovascular parameters. METHODS: Biotelemetry transmitters for the measurement of body temperature or blood pressure (BP) were surgically implanted into separate groups of male rats. THC and the synthetic cannabinoids CP55,940, JWH-018, AM2201 and XLR-11 were injected s.c., and rats were placed into isolation cubicles for 3h. RESULTS: THC and synthetic cannabinoids produced dose-related decreases in body temperature that were most prominent in the final 2h of the session. The rank order of potency was CP55,940>AM2201=JWH-018>THC=XLR-11. The cannabinoid inverse agonist rimonabant antagonized the hypothermic effect of all compounds. Synthetic cannabinoids elevated BP in comparison to vehicle treatment during the first h of the session, while heart rate was unaffected. The rank order of potency for BP increases was similar to that seen for hypothermia. Hypertensive effects of CP55,940 and JWH-018 were not antagonized by rimonabant or the neutral antagonist AM4113. However, the BP responses to both drugs were antagonized by pretreatment with either the ganglionic blocker hexamethonium or the α1 adrenergic antagonist prazosin. CONCLUSIONS: Our results show that synthetic cannabinoids produce hypothermia in rats by a mechanism involving cannabinoid receptors, while they increase BP by a mechanism independent of these sites. The hypertensive effect appears to involve central sympathetic outflow.

Attenuating Nicotine Reinforcement and Relapse by Enhancing Endogenous Brain Levels of Kynurenic Acid in Rats and Squirrel Monkeys.

The currently available antismoking medications have limited efficacy and often fail to prevent relapse. Thus, there is a pressing need for newer, more effective treatment strategies. Recently, we demonstrated that enhancing endogenous levels of kynurenic acid (KYNA, a neuroinhibitory product of tryptophan metabolism) counteracts the rewarding effects of cannabinoids by acting as a negative allosteric modulator of α7 nicotinic receptors (α7nAChRs). As the effects of KYNA on cannabinoid reward involve nicotinic receptors, in the present study we used rat and squirrel monkey models of reward and relapse to examine the possibility that enhancing KYNA can counteract the effects of nicotine. To assess specificity, we also examined models of cocaine reward and relapse in monkeys. KYNA levels were enhanced by administering the kynurenine 3-monooxygenase (KMO) inhibitor, Ro 61-8048. Treatment with Ro 61-8048 decreased nicotine self-administration in rats and monkeys, but did not affect cocaine self-administration. In rats, Ro 61-8048 reduced the ability of nicotine to induce dopamine release in the nucleus accumbens shell, a brain area believed to underlie nicotine reward. Perhaps most importantly, Ro 61-8048 prevented relapse-like behavior when abstinent rats or monkeys were reexposed to nicotine and/or cues that had previously been associated with nicotine. Ro 61-8048 was also effective in monkey models of cocaine relapse. All of these effects of Ro 61-8048 in monkeys, but not in rats, were reversed by pretreatment with a positive allosteric modulator of α7nAChRs. These findings suggest that KMO inhibition may be a promising new approach for the treatment of nicotine addiction.

Cocaine self-administration under variable-dose schedules in squirrel monkeys.

Squirrel monkeys self-administered cocaine under a variable-dose schedule, with the dose varied from injection to injection. As in earlier studies with rats, post-injection pauses varied as a monotonic function of dose, allowing a cocaine dose-effect curve to be obtained during each session. These curves were shifted by pretreatment with dopamine antagonists, demonstrating that this procedure may provide an efficient means of evaluating treatments that affect drug self-administration. However, drug intake eventually became "dysregulated" after extensive training (100-300 sessions), with relatively short pauses following all doses. Dose-sensitivity was restored by adding a 60-s timeout period after each injection, suggesting that dysregulation occurred because the monkeys developed a tendency to self-administer another injection before the previous injection had been adequately distributed. Finally, when the response requirement under the variable-dose schedule was increased from 1 to 10, both the post-injection pause and the rate of responding following the pause ("run rates") were found to vary with dose. The dose-dependency of run rates suggests that post-injection pauses reflect not only motivational factors, such as satiety, but also the direct effects of cocaine on leverpressing.

Pharmacological mechanisms underlying the cardiovascular effects of the "bath salt" constituent 3,4-methylenedioxypyrovalerone (MDPV).

BACKGROUND AND PURPOSE: 3,4-Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone with stimulatory cardiovascular effects that can lead to serious medical complications. Here, we examined the pharmacological mechanisms underlying these cardiovascular actions of MDPV in conscious rats. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats had telemetry transmitters surgically implanted for the measurement of BP and heart rate (HR). On test days, rats were placed individually in standard isolation cubicles. Following drug treatment, cardiovascular parameters were monitored for 3 h sessions. KEY RESULTS: Racemic MDPV (0.3-3.0 mg·kg-1 ) increased BP and HR in a dose-dependent manner. The S(+) enantiomer (0.3-3.0 mg·kg-1 ) of MDPV produced similar effects, while the R(-) enantiomer (0.3-3.0 mg·kg-1 ) had no effects. Neither of the hydroxylated phase I metabolites of MDPV altered cardiovascular parameters significantly from baseline. Pretreatment with the ganglionic blocker chlorisondamine (1 and 3 mg·kg-1 ) antagonized the increases in BP and HR produced by 1 mg·kg-1 MDPV. The α1 -adrenoceptor antagonist prazosin (0.3 mg·kg-1 ) attenuated the increase in BP following MDPV, while the ß-adrenoceptor antagonists propranolol (1 mg·kg-1 ) and atenolol (1 and 3 mg·kg-1 ) attenuated the HR increases. CONCLUSIONS AND IMPLICATIONS: The S(+) enantiomer appeared to mediate the cardiovascular effects of MDPV, while the metabolites of MDPV did not alter BP or HR significantly; MDPV increased BP and HR through activation of central sympathetic outflow. Mixed-action α/ß-adrenoceptor antagonists may be useful as treatments in counteracting the adverse cardiovascular effects of MDPV.

Reinforcing and neurochemical effects of the "bath salts" constituents 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy-N-methylcathinone (methylone) in male rats.

RATIONALE: 3,4-Methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxy-N-methylcathinone (methylone) are synthetic drugs found in so-called "bath salts" products. Both drugs exert their effects by interacting with monoamine transporter proteins. MDPV is a potent uptake blocker at transporters for dopamine and norepinephrine while methylone is a non-selective releaser at transporters for dopamine, norepinephrine, and serotonin (5-HT). OBJECTIVES: We hypothesized that prominent 5-HT-releasing actions of methylone would render this drug less reinforcing than MDPV. METHODS: To test this hypothesis, we compared behavioral effects of MDPV and methylone using intravenous (i.v.) self-administration on a fixed-ratio 1 schedule in male rats. Additionally, neurochemical effects of the drugs were examined using in vivo microdialysis in nucleus accumbens, in a separate cohort of rats. RESULTS: MDPV self-administration (0.03 mg/kg/inj) was acquired rapidly and reached 40 infusions per session, similar to the effects of cocaine (0.5 mg/kg/inj), by the end of training. In contrast, methylone self-administration (0.3 and 0.5 mg/kg/inj) was acquired slowly, and response rates only reached 20 infusions per session by the end of training. In dose substitution studies, MDPV and cocaine displayed typical inverted U-shaped dose-effect functions, but methylone did not. In vivo microdialysis revealed that i.v. MDPV (0.1 and 0.3 mg/kg) increased extracellular dopamine while i.v. methylone (1 and 3 mg/kg) increased extracellular dopamine and 5-HT. CONCLUSIONS: Our findings support the hypothesis that elevations in extracellular 5-HT in the brain can dampen positive reinforcing effects of cathinone-type drugs. Nevertheless, MDPV and methylone are both self-administered by rats, suggesting these drugs possess significant abuse liability in humans.

Effects of fatty acid amide hydrolase (FAAH) inhibitors on working memory in rats.

RATIONALE: Manipulations of the endocannabinoid system could potentially produce therapeutic effects with minimal risk of adverse cannabis-like side effects. Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of the cannabinoid-receptor agonist, anandamide, and show promise for treating a wide range of disorders. However, their effects on learning and memory have not been fully characterized. OBJECTIVES: We determined the effects of five structurally different FAAH inhibitors in an animal model of working memory known to be sensitive to impairment by delta-9 tetrahydrocannabinol (THC). METHODS: A delayed nonmatching-to-position procedure was used in rats. Illuminated nosepoke holes were used to provide sample cues (left versus right) and record responses (correct versus incorrect) after delays ranging from 0 to 28 s. Various test drugs were given acutely up to two times per week before daily sessions. RESULTS: One FAAH inhibitor, AM3506 (3 mg/kg), decreased accuracy in the memory task. Four other FAAH inhibitors (URB597, URB694, PF-04457845, and ARN14633) and a monoacylglycerol lipase inhibitor (JZL184, which blocks the degradation of the endocannabinoid 2-arachidonoylglycerol) had no effect. Testing of AM3506 in combination with antagonists for receptors known to be affected by anandamide and other fatty acid amides indicated that the impairment induced by AM3506 was mediated by cannabinoid CB1 receptors, and not by alpha-type peroxisome proliferator-activated receptors (PPAR-alpha) or vanilloid transient receptor potential cation channels (TRPV1). CONCLUSIONS: FAAH inhibitors differ with respect to their potential for memory impairment, abuse liability, and probably other cannabis-like effects, and they should be evaluated individually for specific therapeutic and adverse effects.

Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing effect of adenosine A2A agonists is most probably mediated in the periphery.

Lorazepam reinstates punishment-suppressed remifentanil self-administration in rats.

RATIONALE: We recently described a reinstatement procedure that models relapse to drug abuse in cases where abstinence results from aversive consequences of drug use. The potential value of this punishment-based model of relapse depends on its sensitivity to relapse-inducing events that are ineffective in the widely used extinction-based model. OBJECTIVES: It is known that certain drugs can have anti-punishment effects, but these drugs have not been tested in the punishment-based reinstatement procedure. Therefore, the effects of the benzodiazepine, lorazepam, were examined using punishment-based and extinction-based reinstatement procedures. METHODS: Rats self-administered the opioid, remifentanil (4 microg/kg per infusion). Two punishment groups were trained with response-contingent footshock that suppressed baseline rates of responding to zero. In an extinction group, remifentanil delivery was discontinued, and baseline responding stabilized at a low rate (mean=0.06 responses/min). Lorazepam (0.08-10 mg/kg, IP) was given during test sessions with the shock contingency discontinued for both punishment groups. Remifentanil delivery was maintained during testing in one punishment group but not the other. RESULTS: Lorazepam reinstated self-administration responding in both punishment groups but not in the extinction group. Priming injections of heroin reinstated responding in both the punishment and extinction groups, but combining heroin and lorazepam did not enhance reinstatement. CONCLUSIONS: This is the first demonstration that a trigger for relapse may have different effects depending on whether aversive conditioning contributed to the achievement of abstinence. It may be important to consider potential anti-punishment effects of both abused drugs and therapeutic agents in the treatment of individuals with a history of drug abuse.

Reinstatement of punishment-suppressed opioid self-administration in rats: an alternative model of relapse to drug abuse.

RATIONALE: Animal models of relapse to drug abuse typically assess the ability of various manipulations to reinstate responding that has ceased due to non-reinforcement (extinction). However, there is a lack of information concerning the reinstatement of responding that has ceased for reasons other than extinction. OBJECTIVES: This study examined the ability of response-independent reinforcer delivery (priming) to reinstate food- or drug-reinforced responding that had been suppressed by response-contingent footshock (punishment). METHODS: Nose-poke responding by separate groups of rats was reinforced with food (45 mg/delivery) or intravenous remifentanil (4 micro g/kg per infusion), a short-acting micro -opioid agonist. After either 3 or 27 days of training (with 100 reinforcers/day), a punishment contingency was introduced that rapidly suppressed responding. Then, the punishment contingency was discontinued, and half the rats received priming. RESULTS: Priming by non-contingent delivery of food or remifentanil significantly reduced the number of sessions required for responding to resume. There were no significant differences in this effect between short-term and long-term training or between food- and drug-trained groups. CONCLUSIONS: Self-administration responding that has been suppressed by punishment can be reinstated by priming, and it can eventually resume even without priming. Under the conditions studied here, priming after punishment had effects qualitatively similar to those typically seen after extinction. This punishment/reinstatement procedure may be useful for comparing the effects of other manipulations known to affect behavior in the extinction/reinstatement model of relapse.

Abuse liability and stimulant properties of dextromethorphan and diphenhydramine combinations in rats.

RATIONALE: The abuse of over-the-counter (OTC) medications has been widely reported. However, there are few preclinical studies examining the behavioral effects of OTC medications at higher, abused doses. OBJECTIVES: The objectives of the current study were to determine whether the anti-histamine diphenhydramine (DIP) and the antitussive dextromethorphan (DEX), either alone or in combination, would have stimulant properties and be self-administered in animals. METHODS: For drug self-administration, naive rats with no history of exposure to other drugs were trained to self-administer i.v. DEX+DIP (0.5+0.5, 1+1 or 2+2 mg/kg per injection), DEX alone (1 mg/kg) or DIP (1 mg/kg) alone under five-response fixed-ratio (FR) schedule with a 30-s time-out after each injection in 2-h sessions 3-5 days a week. Separate groups of rats were tested on locomotor activity. After 8 consecutive days of habituation, naive rats were injected with 3, 10, or 30 mg/kg DEX or DIP alone i.p., or in combination of 3+3 mg/kg, 10+10 mg/kg, or 30+30 mg/kg DEX+DIP i.p. Saline was injected i.p. during the intervening days. Locomotor activity was measured for each session. RESULTS: DEX+DIP combinations were self-administered, but the drugs alone were not. The acquisition of DEX+DIP self-administration was rapid with a majority of rats reaching the final FR5 schedule in 22-23 sessions. The total i.v. combination dose for each final scheduled session ranged from 40 mg/kg to 160 mg/kg DEX+DIP. Significant increases in locomotor activity were observed for DIP, an effect that was significantly enhanced when DIP was given in combination with DEX. Significant mortality was also observed for the drug combination at each dose tested when given i.v., with the highest mortality following the highest dose (2+2 mg/kg). When given i.p., no mortality was observed. CONCLUSIONS: These results show that combinations of DEX and DIP have stimulant properties and are self-administered by animals. Abuse of this combination in humans would be expected.