Distinct accumbal subareas are involved in place conditioning of amphetamine and cocaine

In considering the heterogeneous function of the nucleus accumbens (NAC), the present work evaluated the conditioned place preference (CPP) after local infusion of d-amphetamine (AMP; 10, 15 microg/side) or cocaine (COC; 50, 100 microg/side) into two subareas of NAC, core and shell. A regular two-compartment CPP apparatus was used to test the place conditioning effects after 6 pairings of drug in one compartment and 6 pairings of vehicle in the other one. Significant CPP was observed with either AMP infused in the core area or COC infused into the shell area. Neither AMP in shell nor COC in core significantly produced CPP. These results indicate important differences between two neural substrates within NAC for the rewarding effects of AMP and COC on the CPP task.     

Isolation of a novel tetrapeptide with opiate and antiopiate activity from human brain cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1).

A novel tetrapeptide, Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), was purified from extracts of frontal cortex of human brain tissue by several consecutive reversed-phase high performance liquid chromatographic steps followed by a radioimmunoassay originally developed for Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1). Sequencing, mass spectrometric analysis, and comparison of its chromatographic behavior with that of the synthetic peptide confirmed the structure. Like Tyr-MIF-1, which was previously isolated from human brain tissue, Tyr-W-MIF-1 can inhibit the binding of 3H-DAMGO (selective for mu opiate receptors) to rat brain and can act as an opiate agonist as well as antagonist. Tyr-W-MIF-1 was a more potent opiate agonist than Tyr-MIF-1, the free acid of Tyr-W-MIF-1, and the structurally related hemoglobin-derived opiate peptide hemorphin-4 (Tyr-Pro-Trp-Thr) in the guinea pig ileum. Each of these peptides acted as opiate antagonists on the ileum from morphine-tolerant guinea pigs; the free acid of Tyr-W-MIF-1 was the most potent antagonist in inhibiting the activity of DAMGO. The results demonstrate the presence in human brain of a new member of the Tyr-MIF-1 family of biologically active peptides.     

Tamoxifen produces conditioned taste avoidance in male rats: An analysis of microstructural licking patterns and taste reactivity

Estrogen receptor activation has been shown to reduce body weight and produce a conditioned reduction in food intake in male rats that is putatively mediated by estradiol's suggested aversive effects. Evidence has shown that the selective estrogen receptor modulator tamoxifen used in the prevention and treatment of breast cancer may also produce changes in food intake and body weight, which are known to impact cancer development and survival. The purpose of the present study was to examine whether tamoxifen produces a conditioned reduction in intake similar to estradiol by producing a conditioned aversion. A one bottle lickometer test was used to examine conditioned changes in sucrose drinking, while the taste reactivity test was used to measure rejection reactions, which serve to index aversion in rats. A backward conditioning procedure that consisted of 3 conditioning days and one vehicle test day was used to examine conditioned changes in 0.3 M sucrose intake and taste reactivity. Our results show that tamoxifen produced a conditioned reduction in sucrose drinking in a one bottle fluid intake test that was similar to the effects produced by estradiol (positive control); however, no active rejection reactions were produced by either tamoxifen (1 and 10 mg/kg) or estradiol. The present results suggest that tamoxifen, at the doses used in the present study, acts as an estrogen receptor agonist to regulate food intake and that the conditioned reduction in intake produced by tamoxifen and estradiol reflects conditioned taste avoidance rather than conditioned taste aversion.     

Dissociation of analgesic and rewarding effects of endomorphin-1 in rats

The μ-receptor is the primary mediator of the effects of morphine and the endogenous opiates, endomorphin-1 and endomorphin-2. Here we demonstrate a dissociation of the analgesic and rewarding effects of endomorphin-1 in rats. Tail-flick results revealed that endomorphin-1 produced significant analgesic effects within 10-min after injection. However, it failed to show reward properties in the standard 45- min conditioned place preference (CPP) paradigm or in an abbreviated 10-min pairing which paralleled the time frame of the tail-flick findings. Morphine induced both analgesia and reward. Endomorphin-1 therefore is the first mu opiate shown to produce potent analgesia in the absence of reward behavior, and thus may have significant clinical potential.     

MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding

Behavioral evidence in laboratory animals and human beings indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems, especially with regard to antagonistic properties. To assess possible interactions between endogenous opiate antagonists and benzodiazepine receptor binding, we evaluated the effects of the peptides MIF-1 and Tyr-MIF-1 on benzodiazepine receptor binding in mouse brain membranes. Neither peptide affected receptor binding in cortex over a broad dose range, but both peptides significantly augmented GABA-stimulated benzodiazepine receptor binding at GABA concentrations of 10(-8) and 10(-7) M. Rosenthal-Scatchard analysis indicated that the increase in binding was largely due to increased apparent affinity. Both peptides augmented GABA-enhanced binding at low doses (MIF-1 10(-11) M, Tyr-MIF-1 10(-13) M) with decreased effects at higher doses. In cerebellum and brainstem, MIF-1 tended to enhance GABA-stimulated binding but Tyr-MIF-1 was inactive. These results indicate benzodiazepine-opiate and benzodiazepine-peptide interactions.     

Method for estimating a compound’s opiate activity based on a versatile three-dimensional model of nonselective opiate pharmacophore

A versatile three-dimensional (3D) model for a nonselective opiate pharmacophore was constructed using an alternative strategy for searching the structural similarity of molecules. The method for calculating the qualitative evaluation of agonistic and antagonistic properties of opiate receptor ligands was developed on this basis. Examples of using this method for evaluating the opiate activity of compounds which essentially differ in their structure from opiates and the traditional opioids are given.     

Repeated-testing of place preference expression for evaluation of anti-craving-drug effects

Summary. In addiction research, the conditioned place preference (CPP) paradigm is a widely used animal model of conditioned reward. Usually, CPP development is studied, while only few studies examine CPP expression. In the present study, the suitability of a schedule allowing repeated testing of CPP expression was evaluated. Two groups of rats were either conditioned with cocaine or morphine then the repeated-testing-schedule was applied. This schedule consisted of four repeated applications of a sequence of drug- (i.e. cocaine or morphine), saline- and anti-craving-drug- (i.e. acamprosate, naloxone, their joint administration or saline as internal control) tests. Methodologically, the repeated-testing-schedule produced stable CPP expression in both groups over 12 subsequent tests. In conclusion, it is suggested as a useful method to study effects of anti-craving-drugs on CPP expression, thereby reducing the overall number of experimental animals. The evaluation of the anti-craving-drug effects revealed that neither acamprosate and naloxone given separately nor their combined administration significantly reduced cocaine- or morphine-CPP expression. Thus, we suggest that these anti-craving-drugs are unlikely to be effective for relapse prevention in cocaine- or morphine-addicts.     

Augmented cocaine conditioned place preference in rats pretreated with systemic ghrelin

The physiological mechanism through which food restriction (FR) enhances the biobehavioral actions of psychostimulants is unknown but may involve the gut peptide ghrelin. Plasma levels of ghrelin are increased by FR and reduced by eating. Moreover, systemically administered ghrelin crosses into the brain and is known to augment the locomotor-stimulating effects of cocaine [COC: Wellman et al., 2005]. This study sought to determine whether pretreatment with ghrelin (5 nmol) would enhance the rewarding properties of COC (0.0, 0.312, 0.625, or 1.25 mg/kg i.p.) as measured by conditioned place preference (CPP). Adult male Sprague–Dawley rats were given free access to both sides of a CPP chamber to determine initial side preference. The rats were then confined for 30 min to either their preferred side or non-preferred side on 8 consecutive days. When rats were confined to the least preferred side, each was injected with 0.5 ml (i.p.) of either ghrelin (5 nmol) or saline 1 h before the conditioning trial and then injected (i.p.) with one of the COC doses immediately prior to the conditioning trial. On alternate days, rats were injected with vehicle one hour before and again immediately before the conditioning trial. Place preference scores were computed as the differences in time (min) spent on the least preferred side of the chamber for the pre-test and the postconditioning test, covaried by the initial degree of preference (% time spent on the black side during the pre-test). These analyses indicated a significant interaction between ghrelin pretreatment and COC dose on changes in preference scores. Significantly higher place preference scores were noted for rats treated with either 0.312 or 0.625 mg/kg COC doses, but only when these COC doses were preceded by administration of 5 nmol ghrelin. In contrast, saline pretreated rats exhibited significant CPP at the 1.25 mg/kg COC dose, but the ghrelin pretreated group did not. These results provide partial support for the contention that ghrelin pretreatment can augment the rewarding effects of sub-threshold doses of COC in a CPP procedure. Moreover, these findings are consistent with the view that ghrelin may play a role in the capacity of FR to augment psychostimulant action.     

The action of dietary phytochemicals quercetin, catechin, resveratrol and naringenin on estrogen-mediated gene expression

Hepatic expression of apolipoprotein (apo) II is in part modulated by estrogen-mediated stabilization of its mRNA. This stabilization is due to the estrogen-regulated mRNA stabilizing factor (E-RmRNASF) expressed in the liver in response to estrogen (Ratnasabapathy, 1995, Cell. Mol. Biol. Res, 41: 583-594). E-RmRNASF protects the RNA from targeted endonucleolytic degradation. The hepatic expression of E-RmRNASF is modulated by certain estrogenic and antiestrogenic nonsteroidal environmental xenobiotics (Ratnasabapathy et al. 1997, Biochem. Pharmacol., 53: 1425-1434). To determine whether dietary phytochemicals purported to prevent hormone-dependent breast and prostate cancers, and atherosclerosis, acted via the estrogen-cell-signaling pathway, roosters were administered increasing doses up to 1 mmole/kg of resveratrol, quercetin, catechin or naringenin parenterally and tested for hepatic expression of E-RmRNASF. Besides estrogen, the expression of E-RmRNASF in the liver was stimulated by resveratrol and catechin, indicating these agents to be estrogenic. A lack of E-RmRNASF expression was seen with the roosters treated with the vehicle, naringenin or quercetin. To determine whether the agents exerted partial agonistic or antagonistic effects, roosters were administered combinations of estrogen and increasing doses of the above phytochemicals. Resveratrol showed agonistic activity at all concentrations (10-1000 micromol/kg) tested. Catechin showed partial agonistic activity, while quercetin and naringenin appeared to be antagonistic.     

Expression of the FOS proto-oncogene protein in brain after ICV administration of Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2)

Tyr-W-MIF-1 is a tetrapeptide recently isolated from brain that has opiate modulating activity. In this study, we used immunocytochemical (ICC) detection of FOS proto-oncogene protein to map brain areas activated by an ICV injection of Tyr-W-MIF-1 (200 μg). The analgesic effect of the peptide, which lasted 1 h, was confirmed in each rat with the tail flick test. FOS was activated in several limbic structures, including the cingulate and infralimbic cortex, nucleus accumbens, and central nucleus of the amygdala. FOS activation also occurred in several diencephalic nuclei, including the supraoptic, paraventricular, and periventricular nuclei of the hypothalamus, and the paraventricular nucleus of the thalamus. Several activated areas contained mu-opiate receptors. However, despite the known selectivity of Tyr-W-MIF-1 for mu receptors, FOS immunoreactivity was also induced in nuclei of the amygdala, hypothalamus, and thalamus, where concentrations of kappa receptors were high but those of mu and delta receptors were not detected. The results show that Tyr-W-MIF-1 induces FOS activation in several brain areas, including but not limited to, areas associated with nociception and stress-induced analgesia.     

Supraspinal anti-allodynic and rewarding effects of endomorphins in rats

Two potent endogenous opioid peptides, endomorphin-1 (EM-1) and -2 (EM-2), which are selective micro-opioid agonists, have been identified from bovine and human brain. These endomorphins were demonstrated to produce a potent anti-allodynic effect at spinal level. In the present study, we further investigated their supraspinal anti-allodynic effects and rewarding effects. In a neuropathic pain model (sciatic nerve crush in rats), EM-1 and -2 (15 microg, i.c.v.) both showed significant suppressive effects in the cold-water allodynia test, but EM-1 showed a longer duration than EM-2. Naltrexone (NTX; 15 microg) and naloxonazine (NLZ; 15 microg) were both able to completely block the anti-allodynic effects of EM-1 and -2. In the tests of conditioned place preference (CPP), only EM-2 at the dose of 30 microg showed significant positive rewarding effect, whereas both endomorphins did not induce any reward at the dose of 15 microg. Due to the low solubility and the undesired effect (barrel rotation of the body trunk), EM-1 was not tested for the dose of 30 microg in the CPP tests. It was also found that acute EM-2 (30 microg) administration increased dopamine turnover in the shell of nucleus accumbens in the microdialysis experiments. From these results, it may suggest that EM-1 and -2 could be better supraspinal anti-allodynic agents compared with the other opioid drugs, although they may also induce rewarding.     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Modulation of neuropeptide FF (NPFF) receptors influences the expression of amphetamine-induced conditioned place preference and amphetamine withdrawal anxiety-like behavior in rats

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.     

Attenuation of morphine dependence and withdrawal in rats by venlafaxine, a serotonin and noradrenaline reuptake inhibitor.

The effects of venlafaxine, a novel serotonin and adrenaline reuptake inhibitor, on the morphine withdrawal and activation of morphine conditioned place preference (CPP), were investigated in rats. Our results showed that the most morphine withdrawal signs, including jumping, writhing, shakes, exploring, lacrimation, piloerection, irritability, and diarrhea, were attenuated by pretreatment with 10 or 20 mg/kg venlafaxine. To investigate the effects of venlafaxine on relapse to opiate dependence, the morphine CPP was used and a dopamine D2 antagonist sulpiride was selected as a control drug. The morphine CPP disappeared following a 28-day drug-free period and appeared again after given a single injection of 1 mg/kg morphine. Acute treatment with sulpiride (25 or 50 mg/kg, i.p.) 30 min prior to 1 mg/kg morphine injection significantly blocked the reacquisition of CPP, while venlafaxine (10 or 20 mg/kg, i.p.) did not show significant effect. However, chronic treatment with venlafaxine (5 or 10 mg/kg, i.p. twice, daily, for seven consecutive days) significantly attenuated the reacquisition of morphine CPP, whereas chronic treatment with sulpiride (10 or 20 mg/kg, i.p.) have no significant effect. Our results demonstrated for the first time that venlafaxine strongly attenuates morphine withdrawal and morphine-induced reaquisition of     

Different doses of estradiol benzoate induce conditioned place preference after paced mating

The ovarian hormones estrogen and progesterone are required for the complete display of sexual behavior in female rats. Paced mating produces a reward state in intact cycling and ovariectomized (OVX), hormonally primed females as evaluated by the conditioned place preference (CPP) paradigm. Most of the studies that have evaluated CPP induced by paced mating in OVX females have used relatively high doses of estradiol benzoate (EB). In the present study we determined if different doses of EB, combined with progesterone (P), could induce CPP after paced mating. For this purpose OVX female rats were divided in five groups that received one of different doses of estradiol benzoate (5, 2.5, 1.25 or 0.625 μg estradiol + 0.5 mg of progesterone) before being allowed to pace the sexual interaction and conditioned in a CPP paradigm. We found that the lowest dose of EB used (0.625 μg) significantly reduced the lordosis quotient and the lordosis coefficient. Even though these females paced the sexual interaction, they didn't change its original preference, suggesting that sexual interaction did not induce a positive affective, reward state. Females allowed to pace the sexual interaction with higher doses of EB developed CPP after paced mating. These results indicate that a threshold of estradiol is required for paced mating to induce CPP.     

The effect of paragigantocellularis lateralis lesion on conditioned place preference (CPP) in presence or absence of alpha2 adrenergic agonist (clonidine) in male rats

The nucleus paragigantocellularis lateralis (LPGi) is located in the rostral ventrolateral medulla (RVLM), a brain stem region that regulates homeostatic functions such as blood pressure and cardiovascular reflexes, respiration, pain and opiate withdrawal syndrome. LPGi has many anatomical relationships with important nuclei such as arcute nucleus, caudal raphe nucleus, periaqueductal gray (PAG), locus coeruleus (LC), and dentate. In this study we have examined the role of LPGi in the conditioned place preference (CPP) induced by morphine in the presence and absence of clonidine in the rat. We used 49 male N-MRI rats which were divided into 7 groups randomly: 1: Control, 2: Control+saline, 3: sham control, 4: lesion, 5: lesion +0.02 mg/kg clonidine, 6: lesion +0.2 mg/kg clonidine, 7: lesion +2 mg/kg clonidine. Animals were anaesthetized with ketamine (110 mg/kg) and rampune (Xylazine) (3 mg/kg) mixture. In the process of surgery LPGi nucleus has been destroyed bilaterally by DC electrical current (1 mA, 6 second), with stainless steel electrode placed in stereotaxic coordinates of (AP = 11.8, Lat +/- 1.86 and Depth = 10.5). After the recovery period, they were treated with clonidine one hour before the application of Hand's method to induce CPP. We have not found any significant differences between the results of control, control+saline and sham groups in the CPP test but there is a significant increase in the CPP time between sham and LPGi lesion+saline groups (P < 0.019). Clonidine at different doses (0.02, 0.2 and 2 mg/kg) have decreased CPP time in LPGi lesioned group in comparison with lesioned+saline group as well (p < 0.002). In this study we have also demonstrated that clonidine has not any effects on the CPP time in the intact animals. Our results indicate that LPGi lesion induces CPP. It seems that LPGi is involved in drug reinforcements and also LPGi lesion induces sensitivity to alpha2 adrenergic agonist.     

Role of the Dopaminergic System in the Acquisition, Expression and Reinstatement of MDMA-Induced Conditioned Place Preference in Adolescent Mice

Background

The rewarding effects of 3,4-methylenedioxy-metamphetamine (MDMA) have been demonstrated in conditioned place preference (CPP) procedures, but the involvement of the dopaminergic system in MDMA-induced CPP and reinstatement is poorly understood.

Methodology/Principal Findings

In this study, the effects of the DA D1 antagonist SCH 23390 (0.125 and 0.250 mg/kg), the DA D2 antagonist Haloperidol (0.1 and 0.2 mg/kg), the D2 antagonist Raclopride (0.3 and 0.6 mg/kg) and the dopamine release inhibitor CGS 10746B (3 and 10 mg/kg) on the acquisition, expression and reinstatement of a CPP induced by 10 mg/kg of MDMA were evaluated in adolescent mice. As expected, MDMA significantly increased the time spent in the drug-paired compartment during the post-conditioning (Post-C) test, and a priming dose of 5 mg/kg reinstated the extinguished preference. The higher doses of Haloperidol, Raclopride and CGS 10746B and both doses of SCH 23390 blocked acquisition of the MDMA-induced CPP. However, only Haloperidol blocked expression of the CPP. Reinstatement of the extinguished preference was not affected by any of the drugs studied. Analysis of brain monoamines revealed that the blockade of CPP acquisition was accompanied by an increase in DA concentration in the striatum, with a concomitant decrease in DOPAC and HVA levels. Administration of haloperidol during the Post-C test produced increases in striatal serotonin, DOPAC and HVA concentrations. In mice treated with the higher doses of haloperidol and CGS an increase in SERT concentration in the striatum was detected during acquisition of the CPP, but no changes in DAT were observed.

Conclusions/Significance

These results demonstrate that, in adolescent mice, the dopaminergic system is involved in the acquisition and expression of MDMA-induced CPP, but not in its reinstatement.     

Galnon Facilitates Extinction of Morphine-Conditioned Place Preference but Also Potentiates the Consolidation Process

Learning and memory systems are intimately involved in drug addiction. Previous studies suggest that galanin, a neuropeptide that binds G-protein coupled receptors, plays essential roles in the encoding of memory. In the present study, we tested the function of galnon, a galanin receptor 1 and 2 agonist, in reward-associated memory, using conditioned place preference (CPP), a widely used paradigm in drug-associated memory. Either before or following CPP-inducing morphine administration, galnon was injected at four different time points to test the effects of galanin activation on different reward-associated memory processes: 15 min before CPP training (acquisition), immediately after CPP training (consolidation), 15 min before the post-conditioning test (retrieval), and multiple injection after post-tests (reconsolidation and extinction). Galnon enhanced consolidation and extinction processes of morphine-induced CPP memory, but the compound had no effect on acquisition, retrieval, or reconsolidation processes. Our findings demonstrate that a galanin receptor 1 and 2 agonist, galnon, may be used as a viable compound to treat drug addiction by facilitating memory extinction process.     

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone: new additions to the 6,3'-disubstituted flavone family of high-affinity ligands of the brain benzodiazepine binding site with agonistic properties

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.     

Low dose domoic acid in neonatal rats abolishes nicotine induced conditioned place preference during late adolescence

In this study, neonatal rats were chronically exposed to low, non-convulsive doses of the kainate receptor agonist domoic acid (DOM), or saline. Later, as adolescents, all animals were tested in a nicotine-induced conditioned place preference (CPP) paradigm. As expected, a nicotine-induced CPP was evident in the adolescent control rats, but surprisingly, not in the DOM animals. This study demonstrates the importance of KA receptors in the development of normal adolescent behaviors manifested in response to the rewarding properties of nicotine.