r-bPiDI, an α6ß2* Nicotinic Receptor Antagonist, Decreases Nicotine-Evoked Dopamine Release and Nicotine Reinforcement.

α6ß2* nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons mediate nicotine-evoked dopamine (DA) release and nicotine reinforcement. α6ß2* antagonists inhibit these effects of nicotine, such that α6ß2* receptors serve as therapeutic targets for nicotine addiction. The present research assessed the neuropharmacology of 1,10-bis(3-methyl-5,6-dihydropyridin-1(2H)-yl)decane (r-bPiDI), a novel small-molecule, tertiary amino analog of its parent compound, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI). bPiDI was previously shown to inhibit both nicotine-evoked DA release and the reinforcing effects of nicotine. In the current study, r-bPiDI inhibition of [(3)H]nicotine and [(3)H]methyllycaconitine binding sites was evaluated to assess interaction with the recognition binding sites on α4ß2* and α7* nAChRs, respectively. Further, r-bPiDI inhibition of nicotine-evoked DA release in vitro in the absence and presence of α-conotoxin MII and following chronic in vivo nicotine administration were determined. The ability of r-bPiDI to decrease nicotine self-administration and food-maintained responding was also assessed. Results show that r-bPiDI did not inhibit [(3)H]nicotine or [(3)H]methyllycaconitine binding, but potently (IC50 = 37.5 nM) inhibited nicotine-evoked DA release from superfused striatal slices obtained from either drug naïve rats or from those repeatedly treated with nicotine. r-bPiDI inhibition of nicotine-evoked DA release was not different in the absence or presence of α-conotoxin MII, indicating that r-bPiDI acts as a potent, selective α6ß2* nAChR antagonist. Acute systemic administration of r-bPiDI specifically decreased nicotine self-administration by 75 %, and did not alter food-maintained responding, demonstrating greater specificity relative to bPiDI and bPiDDB, as well as the tertiary amino analog r-bPiDDB. The current work describes the discovery of r-bPiDI, a tertiary amino, α-conotoxin MII-like small molecule that acts as a potent and selective antagonist at α6ß2* nAChRs to specifically decrease nicotine self-administration in rats, thus, establishing r-bPiDI as a lead compound for development as a treatment for nicotine addiction.

Discriminative stimulus effects of NMDA, AMPA, and mGluR5 glutamate receptor ligands in methamphetamine-trained rats.

Glutamate contributes to the reinforcing and stimulant effects of methamphetamine, yet its potential role in the interoceptive stimulus properties of methamphetamine is unknown. In this study, adult male Sprague-Dawley rats were trained to discriminate methamphetamine [1.0 mg/kg, intraperitoneally] from saline in a standard operant discrimination task. The effects of methamphetamine (0.1-1.0 mg/kg, intraperitoneally); N-methyl-D-aspartate (NMDA) receptor channel blockers, MK-801 (0.03-0.3 mg/kg, intraperitoneally) and ketamine (1.0-10.0 mg/kg, intraperitoneally); polyamine site NMDA receptor antagonist, ifenprodil (1-10 mg/kg); α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (1-10 mg/kg, intraperitoneally); and metabotropic 5 glutamate receptor antagonist, 6-methyl-2-(phenylethynyl)pyridine (1-10 mg/kg), given alone were determined in substitution tests. The effects of MK-801 (0.03 and 0.1 mg/kg), ketamine (1.0 and 3.0 mg/kg), ifenprodil (5.6 mg/kg), 6-cyano-7-nitroquinoxaline-2,3-dione (5.6 mg/kg), and 6-methyl-2-(phenylethynyl)pyridine (5.6 mg/kg) were also tested in combination with methamphetamine to assess for alterations in the methamphetamine cue. In substitution tests, none of the test drugs generalized to the methamphetamine cue. However, ketamine and ifenprodil produced significant leftward shifts in the methamphetamine dose-response curve. In addition, the potention by MK-801 nearly attained significance. These results suggest that blockade of the NMDA receptor augments the interoceptive stimulus properties of methamphetamine.

Methylphenidate and fluphenazine, but not amphetamine, differentially affect impulsive choice in spontaneously hypertensive, Wistar-Kyoto and Sprague-Dawley rats.

Impulsivity is one of the core symptoms of attention-deficit/hyperactivity disorder (ADHD). The spontaneously hypertensive rat (SHR), a putative animal model of ADHD, has been used to investigate the neurobiology of impulsivity, although this model has been questioned over concerns that use of Wistar-Kyoto rats (WKY) as a comparison strain may exaggerate effects. The present study compared SHR, WKY and standard, outbred Sprague-Dawley (SD) rats on a delay discounting task where the primary measure was mean adjusted delay (MAD), or the indifference point (in sec) between choice of an immediate delivery of 1 grain-based pellet versus 3 pellets delivered after varying delays. The acute dose effects of the ADHD medications amphetamine (0.1-1.0 mg/kg) and methylphenidate (1.0-10 mg/kg) were then determined; in addition, the effect of the dopamine receptor antagonist fluphenazine (0.1-1.0 mg/kg) was also assessed for comparison with the indirect agonists. While there were no strain differences in the rate of task acquisition or stabilization of baseline MAD scores, SHR had significantly lower MAD scores than WKY but not SD due to the greater individual variability of MAD scores in SD. Although amphetamine did not alter MAD scores in any strain, methylphenidate selectively increased MAD scores in WKY and fluphenazine selectively increased MAD scores in SHR. WKY were also more sensitive than SHR and SD to the response-impairing effects of each drug. The finding that SHR showed a decrease in impulsivity following fluphenazine, but not following either amphetamine or methylphenidate, suggests that delay discounting in SHR may not represent a valid predictive model for screening effective ADHD medications in humans.

bPiDI: a novel selective α6ß2* nicotinic receptor antagonist and preclinical candidate treatment for nicotine abuse.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (nAChRs) containing α6ß2 subunits expressed by dopamine neurons regulate nicotine-evoked dopamine release. Previous results show that the α6ß2* nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB) inhibits nicotine-evoked dopamine release from dorsal striatum and decreases nicotine self-administration in rats. However, overt toxicity emerged with repeated bPiDDB treatment. The current study evaluated the preclinical pharmacology of a bPiDDB analogue. EXPERIMENTAL APPROACH: The C10 analogue of bPiDDB, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI), was evaluated preclinically for nAChR antagonist activity. KEY RESULTS: bPiDI inhibits nicotine-evoked [³H]dopamine overflow (IC50= 150 nM, I(max)=58%) from rat striatal slices. Schild analysis revealed a rightward shift in the nicotine concentration-response curve and surmountability with increasing nicotine concentration; however, the Schild regression slope differed significantly from 1.0, indicating surmountable allosteric inhibition. Co-exposure of maximally inhibitory concentrations of bPiDI (1 µM) and the α6ß2* nAChR antagonist α-conotoxin MII (1 nM) produced inhibition not different from either antagonist alone, indicating that bPiDI acts at α6ß2* nAChRs. Nicotine treatment (0.4 mg·kg⁻¹·da⁻¹, 10 days) increased more than 100-fold the potency of bPiDI (IC50=1.45 nM) to inhibit nicotine-evoked dopamine release. Acute treatment with bPiDI (1.94-5.83 µmol·kg⁻¹, s.c.) specifically reduced nicotine self-administration relative to responding for food. Across seven daily treatments, bPiDI decreased nicotine self-administration; however, tolerance developed to the acute decrease in food-maintained responding. No observable body weight loss or lethargy was observed with repeated bPiDI. CONCLUSIONS AND IMPLICATIONS: These results are consistent with the hypothesis that α6ß2* nAChR antagonists have potential for development as pharmacotherapies for tobacco smoking cessation.

Effect of environmental enrichment on methylphenidate-induced locomotion and dopamine transporter dynamics.

Rats raised in an enriched condition (EC) are less sensitive to the locomotor effects of stimulant drugs than rats raised in an impoverished condition (IC). Methylphenidate (MPD), a primary pharmacotherapy for attention-deficit/hyperactivity disorder, has abuse potential. This study determined whether environmental enrichment differentially altered the effects of MPD on locomotor activity and dopamine (DA) transporter (DAT) function. Acute and repeated MPD (3 or 10 mg/kg, s.c.) increased locomotion in EC, IC and social condition (SC) rats; however, EC rats showed a blunted response to repeated MPD (3 mg/kg). The maximal velocity (V(max)) of [(3)H]DA uptake in the presence of the combination of phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator and okadaic acid, a protein phosphatase inhibitor was decreased in EC and IC rats by 68% and 40%, respectively, indicating that DAT in prefrontal cortex (PFC) is more sensitive to PKC-mediated down-regulation in EC rats. Acute MPD (10 mg/kg) administration decreased the V(max) of [(3)H]DA uptake in PFC and striatum in EC rats, but not in IC rats. Furthermore, [(3)H]WIN 35,428 binding density was decreased in PFC of EC and IC rats, and in striatum of EC rats given repeated MPD (10 mg/kg). These results demonstrate that environmental enrichment modulates DAT dynamics in PFC. However, since the change in DAT function was observed only following the high dose of MPH (10 mg/kg), the attenuated locomotor response to repeated MPD (3 mg/kg) in EC rats is not likely due to a specific DAT alteration in the brain regions examined.

Oral methylphenidate establishes a conditioned place preference in rats.

Emerging data suggest that illicit methylphenidate abuse is a growing problem. Although abuse of the drug typically occurs by the intranasal route, oral (per os; p.o.) methylphenidate also has abuse potential. The present study compared the effects of p.o. and intraperitoneal (i.p.) methylphenidate in rats using the conditioned place preference (CPP) procedure. Young adult male Sprague-Dawley rats were trained to consume oyster crackers injected initially with saline. Next, rats were randomly assigned to receive p.o. or i.p. methylphenidate (3 or 10mg/kg) or saline immediately or 30min prior to 30min conditioning trials. Methylphenidate or saline were each paired 4 times with an end compartment; preference for the methylphenidate-paired compartment was then assessed on a drug-free session. When given immediately prior to conditioning, significant CPP was obtained with both 3 and 10mg/kg of i.p. methylphenidate, but only with 10mg/kg of p.o. methylphenidate. When given 30min prior to conditioning, there was no evidence of CPP for any dose of i.p. or p.o. methylphenidate. These findings are the first demonstration that p.o. methylphenidate has rewarding effects, although i.p. methylphenidate is obtained at a 3mg/kg dose which did not establish CPP with p.o. administration. The lack of CPP following 30min pretreatment also suggests that conditioning may require the CS to be associated with a US of ascending, rather than descending, brain levels of methylphenidate. These results are consistent with clinical evidence of the reduced abuse liability of p.o. methylphenidate relative to methylphenidate taken by other (e.g., intranasal) routes.

Repeated nicotine administration robustly increases bPiDDB inhibitory potency at alpha6beta2-containing nicotinic receptors mediating nicotine-evoked dopamine release.

The novel nicotinic receptor (nAChR) antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), and its chemically reduced analog, r-bPiDDB, potently inhibit nicotine-evoked dopamine (DA) release from rat striatal slices. Since tobacco smokers self-administer nicotine repeatedly, animal models incorporating repeated nicotine treatment allow for mechanistic evaluation of therapeutic candidates following neuroadaptive changes. The current study determined the ability of bPiDDB, r-bPiDDB and alpha-conotoxin MII (alpha-CtxMII), a peptide antagonist selective for alpha6beta2-containing nAChRs, to inhibit nicotine-evoked [(3)H]DA release from striatal slices from rats repeatedly administered nicotine (0.4mg/kg for 10 days) or saline (control). Concomitant exposure to maximally effective concentrations of r-bPiDDB (1nM) and alpha-CtxMII (1nM) resulted in inhibition of nicotine-evoked [(3)H]DA release no greater than that produced by either antagonist alone, suggesting that r-bPiDDB inhibits alpha6beta2-containing nAChRs. Repeated nicotine treatment increased locomotor activity, demonstrating behavioral sensitization. Concentration-response curves for nicotine-evoked [(3)H]DA release were not different between nicotine-treated and control groups. Maximal inhibition for alpha-CtxMII was greater following repeated nicotine compared to control (I(max)=90% vs. 62%), with no change in potency. bPiDDB was 3-orders of magnitude more potent in inhibiting nicotine-evoked [(3)H]DA release in nicotine-treated rats compared to control rats (IC(50)=5pM vs. 6nM), with no change in maximal inhibition. Neither a shift to the left in the concentration response nor a change in maximal inhibition was observed for r-bPiDDB following repeated nicotine. Thus, repeated nicotine treatment may differentially regulate the stoichiometry, conformation and/or composition of alpha6beta2-containing nAChRs mediating nicotine-evoked striatal DA release. Therefore, bPiDDB and r-bPiDDB appear to target different alpha6beta2-containing nAChR subtypes.

Nicotinic receptor-based therapeutics and candidates for smoking cessation.

Tobacco dependence is the most preventable cause of death and is a chronic, relapsing disorder in which compulsive tobacco use persists despite known negative health consequences. All currently available cessation agents (nicotine, varenicline and bupropion) have limited efficacy and are associated with high relapse rates, revealing a need for more efficacious, alternative pharmacotherapies. The major alkaloid in tobacco, nicotine, activates nicotinic receptors (nAChRs) which increase brain extracellular dopamine producing nicotine reward leading to addiction. nAChRs are located primarily presynaptically and modulate synaptic activity by regulating neurotransmitter release. Subtype-selective nAChR antagonists that block reward-relevant mesocorticolimbic and nigrostriatal dopamine release induced by nicotine may offer advantages over current therapies. An innovative approach is to provide pharmacotherapies which are antagonists at nAChR subtypes mediating nicotine evoked dopamine release. In addition, providing multiple medications with a wider array of targets and mechanisms should provide more treatment options for individuals who are not responsive to the currently available pharmacotherapies. This review summarizes the currently available smoking cessation therapies and discusses emerging potential therapeutic approaches employing pharmacological agents which act as antagonists at nicotinic receptors.

Nicotinic receptors differentially modulate the induction and expression of behavioral sensitization to methylphenidate in rats.

RATIONALE: Nicotinic acetylcholine receptors (nAChRs) regulate sensitization to stimulant drugs such as d-amphetamine and cocaine. OBJECTIVES: The current study determined if nAChRs modulate the induction and/or expression of behavioral sensitization to high methylphenidate doses. METHODS: In experiment 1, rats received saline or mecamylamine (3 mg/kg, sc), followed by saline or methylphenidate (5.6 or 10 mg/kg, sc) during 10 daily sessions; the effect of methylphenidate (1-17 mg/kg, sc) alone was determined 14 days later. In experiment 2, rats received saline or dihydro-beta-erythroidine (DHbetaE; 3 mg/kg, sc), followed by saline or 5.6 mg/kg of methylphenidate. In experiment 3, rats received saline or methylphenidate (5.6 or 10 mg/kg, sc) alone for 10 days; the effect of acute mecamylamine (3 mg/kg, sc) on the response to methylphenidate (1-17 mg/kg, sc) was determined 14 days later. Locomotor activity, sniffing, rearing, grooming, and stereotypy ratings were dependent measures. RESULTS: Methylphenidate produced dose-dependent increases in locomotor activity, sniffing, and stereotypy on day 1 and these effects were enhanced on day 10, indicative of sensitization. Mecamylamine attenuated methylphenidate-induced stereotypy only on day 1, but reduced locomotor activity, sniffing, rearing, and stereotypy on day 10 and during the methylphenidate challenge phase; similar results were obtained with DHbetaE. However, acute mecamylamine did not alter the effects of the methylphenidate challenge following the induction of sensitization to methylphenidate alone. CONCLUSIONS: Although nAChRs do not appear to regulate the expression of methylphenidate-induced behavioral sensitization, inhibition of high-affinity beta2 subunit nAChRs attenuates the induction of behavioral sensitization to high doses of methylphenidate.

Neuropharmacology of the interoceptive stimulus properties of nicotine.

Preclinical drug discrimination techniques play a significant role in advancing our knowledge of the receptor mechanisms underlying the interoceptive effects of nicotine. Early reports confirmed that nicotinic acetylcholine receptors (nAChRs) are critical for transduction of the nicotine cue. In recent years, advances in molecular biology and the discovery of novel ligands with greater selectively for specific nAChR subtypes have furthered our understanding of these mechanisms. There is now evidence regarding the specific nAChR subtypes involved in nicotine discrimination; in addition, there is also evidence suggesting that other systems (i.e., adenosine, cannabinoid, dopamine, glutamate and serotonin) may play a modulatory role. The neuroanatomical structures mediating the nicotine cue have also begun to be elucidated. However, much remains to be learned about the predictive validity of the drug discrimination procedure, particularly with regard to the relation between interoceptive and reinforcing effects and individual differences in vulnerability to tobacco dependence. Recent data also suggests that the mechanisms involved in the conditional and discriminative stimulus properties of nicotine may be dissociable. Avenues for future research should include assessing the mechanisms of the subjective effects of nicotine withdrawal, factors contributing to individual differences in sensitivity to the nicotine cue, and the role of behavioral factors involved in drug cross-substitution.

N,N'-Alkane-diyl-bis-3-picoliniums as nicotinic receptor antagonists: inhibition of nicotine-evoked dopamine release and hyperactivity.

The current study evaluated a new series of N,N'-alkane-diyl-bis-3-picolinium (bAPi) analogs with C6-C12 methylene linkers as nicotinic acetylcholine receptor (nAChR) antagonists, for nicotine-evoked [3H]dopamine (DA) overflow, for blood-brain barrier choline transporter affinity, and for attenuation of discriminative stimulus and locomotor stimulant effects of nicotine. bAPi analogs exhibited little affinity for alpha4beta2* (* indicates putative nAChR subtype assignment) and alpha7* high-affinity ligand binding sites and exhibited no inhibition of DA transporter function. With the exception of C6, all analogs inhibited nicotine-evoked [3H]DA overflow (IC50 = 2 nM-6 microM; Imax = 54-64%), with N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB; C12) being most potent. bPiDDB did not inhibit electrically evoked [3H]DA overflow, suggesting specific nAChR inhibitory effects and a lack of toxicity to DA neurons. Schild analysis suggested that bPiDDB interacts in an orthosteric manner at nAChRs mediating nicotine-evoked [3H]DA overflow. To determine whether bPiDDB interacts with alpha-conotoxin MII-sensitive alpha6beta2-containing nAChRs, slices were exposed concomitantly to maximally effective concentrations of bPiDDB (10 nM) and alpha-conotoxin MII (1 nM). Inhibition of nicotine-evoked [3H]DA overflow was not different with the combination compared with either antagonist alone, suggesting that bPiDDB interacts with alpha6beta2-containing nAChRs. C7, C8, C10, and C12 analogs exhibited high affinity for the blood-brain barrier choline transporter in vivo, suggesting brain bioavailability. Although none of the analogs altered the discriminative stimulus effect of nicotine, C8, C9, C10, and C12 analogs decreased nicotine-induced hyperactivity in nicotine-sensitized rats, without reducing spontaneous activity. Further development of nAChR antagonists that inhibit nicotine-evoked DA release and penetrate brain to antagonize DA-mediated locomotor stimulant effects of nicotine as novel treatments for nicotine addiction is warranted.

Methylphenidate enhances the abuse-related behavioral effects of nicotine in rats: intravenous self-administration, drug discrimination, and locomotor cross-sensitization.

Stimulant drugs, including D-amphetamine, cocaine, and methylphenidate, increase cigarette smoking in controlled human laboratory experiments. Although the mechanism(s) underlying this effect are unknown, it is possible that stimulants may enhance directly the abuse-related effects of nicotine. In the present study, we characterized the behavioral pharmacological interactions between methylphenidate and nicotine in the intravenous self-administration, drug discrimination, and locomotor cross-sensitization procedures. Adult male Sprague-Dawley rats were trained to respond for intravenous nicotine (0.01 or 0.03 mg/kg/infusion) or sucrose, and the acute effects of methylphenidate (1.25-10 mg/kg) were determined; in addition, separate groups of rats were treated with methylphenidate (2.5 mg/kg) or saline before 12 consecutive nicotine (0.03 mg/kg/infusion) self-administration sessions. Next, the discriminative stimulus effects of nicotine (0.03-0.3 mg/kg) and methylphenidate (1.25-10 mg/kg), alone and in combination with a low nicotine dose (0.056 mg/kg), were tested in nicotine-trained rats. Finally, the locomotor effect of repeated methylphenidate (2.5 mg/kg) was tested in rats previously treated with nicotine (0.2-0.8 mg/kg). Results indicated that acute methylphenidate increased the rate of nicotine self-administration at doses that reduced sucrose-maintained responding; furthermore, tolerance to this effect was not apparent following repeated methylphenidate. Methylphenidate, while not substituting for nicotine alone, dose-dependently enhanced the discriminative stimulus effect of a low nicotine dose. In addition, repeated nicotine exposure promoted the development of locomotor sensitization to methylphenidate. Taken together with recent clinical findings, these results suggest that methylphenidate may enhance the abuse-related behavioral effects of nicotine, perhaps increasing vulnerability to tobacco dependence.

The monoamine oxidase inhibitor phenelzine enhances the discriminative stimulus effect of nicotine in rats.

In addition to delivering nicotine, tobacco smoke also inhibits monoamine oxidase (MAO). Although MAO inhibitors (MAOIs) can increase nicotine self-administration in rodents, the effects of MAOIs on the discriminative stimulus effect of nicotine are not known. This study examined the effects of three MAOIs (phenelzine, clorgyline and pargyline) with varying selectivity for MAOA and MAOB in the nicotine drug discrimination procedure in rats. Adult male Sprague-Dawley rats were trained to discriminate nicotine (0.3 mg/kg, subcutaneously) from saline in a standard, two-lever food-reinforced operant task. Once the discrimination was acquired, the ability of each MAOI to substitute for or alter the discriminative stimulus effect of nicotine was determined. In substitution tests, nicotine (0.03-0.3 mg/kg) produced full, dose-dependent substitution. Although the selective MAOA inhibitor clorgyline (3-56 mg/kg) and the selective MAOB inhibitor pargyline (3-56 mg/kg) did not elicit any nicotine-appropriate responding, partial substitution was obtained with the nonselective MAO inhibitor phenelzine (1-17 mg/kg). Phenelzine (10 mg/kg) also enhanced the discriminative stimulus effect of a low dose of nicotine (0.056 mg/kg) and prolonged the time course effect of the nicotine-training dose. These findings indicate that concomitant inhibition of MAOA and MAOB can enhance the discriminative stimulus effect of nicotine in rats.