Nicotine stimulation of extracellular glutamate levels in the nucleus accumbens: neuropharmacological characterization

In the present study, we have characterized the neuropharmacological regulation of nicotine-induced increases in extracellular nucleus accumbens glutamate levels. Sprague-Dawley rats were stereotaxically implanted with 2 mm microdialysis probes in the nucleus accumbens and on the following day in vivo microdialysis experiments were performed in awake, freely moving animals. An acute dose of nicotine (0.3-0.6 mg/kg, s.c.) produced an increase in nucleus accumbens glutamate levels with a maximal increase of approximately 50% following the higher dose. No changes in nucleus accumbens aspartate levels were found. The increase in glutamate levels following nicotine (0.3 mg/kg, s.c.) was blocked by mecamylamine (1 mg/kg, i.p. ) but not by haloperidol (0.2 mg/kg, i.p.) pretreatment. Local perfusion of artificial cerebrospinal fluid (CSF) without calcium did not alter nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. Local perfusion with a selective blocker for the GLT-1 glutamate transporter, dihydrokainic acid (DHKA) (10(-4) M), had no effect, while local perfusion with a nonselective glutamate transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (10(-4) M), blocked nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. In animals previously dopamine denervated by local injections of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens, nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels was enhanced vs. sham-lesioned animals. These findings demonstrate a novel form of nucleus accumbens glutamate release that is dopamine- and calcium-independent. The ability of PDC to block the effects of nicotine suggest that a glutamate transporter may be involved in mediating the stimulation of glutamate release.     

Involvement of glial cell line-derived neurotrophic factor in inhibitory effects of a hydrophobic dipeptide Leu-Ile on morphine-induced sensitization and rewarding effects

There are few efficacious medications for drug dependence at present. We have previously demonstrated that Leu-Ile, which induces the expression of not only tumor necrosis factor-alpha (TNF-alpha) but also glial cell line-derived neurotrophic factor (GDNF), inhibits methamphetamine (METH) and morphine (MOR)-induced sensitization and rewarding effects by regulating extracellular dopamine levels via the induction of TNF-alpha expression, and indicated the potential of Leu-Ile as a novel therapeutic agent for METH and MOR-induced dependence. In the present study, we investigated the involvement of GDNF in inhibitory effects of Leu-Ile on MOR-induced sensitization and rewarding effects. Repeated treatment with MOR for 9 days, which results in an enhancement of the locomotor-stimulating effects (sensitization) of MOR, increased GDNF levels in the nucleus accumbens compared with those in saline-treated mice. Repeated pre-treatment with Leu-Ile for 9 days potentiated the MOR-induced increase in GDNF levels. MOR at a low dose (3mg/kg) produced place preference in GDNF heterozygous knockout (GDNF-(+/-)) mice, but not in littermate GDNF-(+/+) mice. No inhibitory effect of Leu-Ile on MOR-induced place preference was observed in GDNF-(+/-) mice. These results suggest that GDNF is involved in the inhibitory effects of Leu-Ile on MOR-induced sensitization and rewarding effects.     

A dopamine‐μ1 opioid link in the rat ventral tegmentum shared by palatable food (Fonzies) and non‐psychostimulant drugs of abuse

The role of μ₁ opioid receptors in the stimulation of dopamine transmission in the rat nucleus accumbens by an unusual palatable food (Fonzies) and non-psychostimulant drugs of abuse was investigated by the use of naloxonazine, a pseudo-irreversible antagonist of μ₁ opioid receptors. Feeding of Fonzies stimulated dopamine release in the medial prefrontal cortex and in the shell, but not in the core of the nucleus accumbens. Pretreatment with naloxonazine given systemically (15 mg/kg i.p. 20 h before) completely prevented the stimulation of dopamine release in the shell of the nucleus accumbens by Fonzies without affecting that in the prefrontal cortex. Systemic pretreatment with naloxonazine reduced or, depending on the dose, abolished, the stimulation of dopamine release in the nucleus accumbens shell by morphine, nicotine and ethanol, but did not affect that by haloperidol. Naloxonazine also prevented the stimulatory effects of Fonzies, nicotine and morphine on nucleus accumbens dopamine transmission when infused bilaterally in the ventral tegmental area. The results indicate that μ₁ opioid receptors in the ventral tegmentum play a major role in the stimulant effects of food and drugs of abuse on mesolimbic dopamine transmission.     

The rewarding properties of MDMA are preserved in mice lacking mu-opioid receptors

The involvement of mu-opioid receptors in the rewarding properties of MDMA was explored in mu-opioid receptor knockout mice using the conditioning place preference paradigm. The associated release of dopamine in the nucleus accumbens was investigated by in vivo microdialysis. A significant rewarding effect of MDMA (10 mg/kg, i.p.) was observed in both wild-type and mu-opioid receptor knockout mice. MDMA (10 mg/kg, i.p.) also induced similar increases in dopamine and decreases in 3,4-dihydroxyphenylacetic acid and homovanillic acid in the nucleus accumbens dialysates of both wild-type and mu-opioid receptor knockout mice. No significant differences in basal levels of dopamine, 3,4-dihydroxyphenylacetic or homovanillic acids between wild-type and mu-opioid receptor knockout mice were observed. In summary, the present results suggest that, in contrast to what has been reported for other drugs of abuse such as opioids, ethanol, nicotine and Delta(9)-tetrahydrocannabinol, mu-opioid receptors do not play a major role in the rewarding properties of MDMA. These differences could be due to distinct mechanisms controlling dopamine release in the nucleus accumbens and suggest that the effects of MDMA on dopaminergic neurons are independent of micro -opioid receptors.     

Putative role of presynaptic alpha7* nicotinic receptors in nicotine stimulated increases of extracellular levels of glutamate and aspartate in the ventral tegmental area

We have previously provided evidence that the stimulatory action of systemic nicotine on dopamine release in the rat nucleus accumbens is initiated in the ventral tegmental area (VTA), and that it appears to be mediated partly through an indirect, presynaptic mechanism. Thus, it was found that blockade of N-methyl-D-aspartate (NMDA) receptors in the VTA attenuates the enhancing effect of nicotine on extracellular levels of dopamine in the nucleus accumbens. Moreover, the nicotine-induced dopamine output in the nucleus accumbens was found to be blocked by pretreatment with methyllycaconitine (MLA) in the VTA, indicating a role for alpha7* nicotinic acetylcholine receptors (nAChRs) in this mechanism. Thus, nicotine may exert its effects in the VTA through stimulation of alpha7* nAChRs localized on excitatory amino acid (EAA)ergic afferents. To test this hypothesis, we here measured extracellular concentrations of glutamate and aspartate in the VTA in response to systemic nicotine, with or without concurrent infusion of MLA in the VTA, using microdialysis in anaesthetized rats. Since the medial prefrontal cortex is an important source of EAA input to the VTA, we also assessed the density of alpha-bungarotoxin binding sites in the VTA in rats lesioned bilaterally in the prefrontal cortex with ibotenic acid and in sham-lesioned rats by means of quantitative autoradiography. Nicotine (0.5 mg/kg, s.c.) significantly increased extracellular levels of both aspartate and glutamate in the VTA. MLA (0.3 mM) infused locally in the VTA prevented the nicotine-induced increase in glutamate and aspartate levels. Ibotenic acid lesions of the prefrontal cortex decreased the density of alpha-bungarotoxin binding sites in the VTA by about 30%. These data indicate that nicotine increases the extracellular levels of excitatory amino acids in the VTA through stimulation of nAChRs in the VTA and that part of the alpha7* nAChR population in the VTA is localized on neurons originating in the prefrontal cortex.     

Rewarding and aversive effects of nicotine are segregated within the nucleus accumbens

Forebrain dopamine plays a critical role in motivated behavior. According to the classic view, mesolimbic dopamine selectively guides behavior motivated by positive reinforcers. However, this has been challenged in favor of a wider role encompassing aversively motivated behavior. This controversy is particularly striking in the case of nicotine, with opposing claims that either the rewarding or the aversive effect of nicotine is critically dependent on mesolimbic dopamine transmission. In the present study, the effects of 6-hydroxydopamine lesions of nucleus accumbens core vs. medial shell on intravenous nicotine conditioned place preference and conditioned taste aversion were examined in male adult rats. Dopaminergic denervation in accumbens medial shell was associated with decreased nicotine conditioned place preference. Conversely, denervation in accumbens core was associated with an increase in conditioned place preference. In addition, dopaminergic denervation of accumbens core but not medial shell abolished conditioned taste aversion for nicotine. We conclude that nucleus accumbens core and medial shell dopaminergic innervation exert segregated effects on rewarding and aversive effects of nicotine. More generally, our findings indicate that dopaminergic transmission may mediate or enable opposing motivational processes within functionally distinct domains of the accumbens.     

Interactions between imidazoline binding sites and dopamine levels in the rat nucleus accumbens

Imidazoline binding sites are present in the striatal complex and in the extended amygdala and have been implicated in mood disorders. In this report we analysed the influence of these sites on the functional activity of the mesolimbic dopaminergic transmission, one of the major brain systems involved in the regulation of motivation and reward. We studied the effects of two imidazoline ligands, S23229 and S23230 (respectively S(+) and R(-) enantiomers of the S22687 or (5-[2-methyl phenoxy methyl] 1,3-oxazolin-2-yl) amine), on extracellular dopamine in the nucleus accumbens using microdialysis in freely moving rats. We compared these imidazoline ligands to cocaine, a dopamine uptake blocker known to increase extracellular dopamine concentrations. S23229 dose-dependently increased extracellular dopamine and locomotor activity. S23230 dose-dependently increased extracellular dopamine and produced a near-significant dose-effect on locomotor activity. S23229 had a stronger efficacy than S23230 and increased dopamine levels in the nucleus accumbens at an extent similar to the one of cocaine. These results suggest that central imidazoline binding sites could contribute to the functional regulation of the mesolimbic dopaminergic system.     

Cyclazocine-induced sleep disruptions in nondependent addicts

After one adaptation night, the sleep of seven male nondependent opiate addicts was studied following intramuscular cyclazocine (0.125; 0.25; 0.50 mg/70 kg) or placebo at weekly intervals in a randomized double-blind crossover design. Drug effects were measured on sleep stages and several episodic phenomena. Cyclazocine caused dose-related increases in sleep latency, REMS latency, percent spindle sleep, and a marked increase over placebo in wakefulness, drowsiness, and shifts in sleep-waking states. Cyclazocine produced a dose-related decrease in all measures of delta sleep, and some measures of REMS, and a marked decrease below placebo of sleep efficiency and total REMS. All doses of cyclazocine caused sustained periods of waking with little muscle tension. Cyclazocine (0.5mg) consistently caused urination during periods of extended arousal; urination has not been seen after morphine or other opioids of the mu type. These studies indicate that cyclazocine has effects on human sleep which are in some ways similar and other ways dissimilar to morphine type analgesics. The results are consistent with the concept that cyclazocine is a mixed agonist-antagonist of the opioid type with agonist actions at the kappa receptor.     

Influence of δ-Opioid Receptors in the Behavioral Effects of Nicotine

Multiple studies in animal models and humans suggest that the endogenous opioid system is an important neurobiological substrate for nicotine addictive properties. In this study, we evaluated the participation of δ-opioid receptors in different behavioral responses of nicotine by using δ-opioid receptor knockout mice. Acute nicotine administration induced hypolocomotion and antinociception in wild-type mice, which were similar in knockout animals. The development of tolerance to nicotine-induced antinociception was also similar in both genotypes. In agreement, the expression and functional activity of δ-opioid receptors were not modified in the different layers of the spinal cord and brain areas evaluated after chronic nicotine treatment. The somatic manifestation of the nicotine withdrawal syndrome precipitated by mecamylamine was also similar in wild-type and δ-opioid receptor knockout mice. In contrast, nicotine induced a conditioned place preference in wild-type animals that was abolished in knockout mice. Moreover, a lower percentage of acquisition of intravenous nicotine self-administration was observed in mice lacking δ-opioid receptors as well as in wild-type mice treated with the selective δ-opioid receptor antagonist naltrindole. Accordingly, in-vivo microdialysis studies revealed that the enhancement in dopamine extracellular levels induced by nicotine in the nucleus accumbens was reduced in mutant mice. In summary, the present results show that δ-opioid receptors are involved in the modulation of nicotine rewarding effects. However, this opioid receptor does not participate either in several acute effects of nicotine or in the development of tolerance and physical dependence induced by chronic nicotine administration.     

Baclofen antagonizes nicotine-, cocaine-, and morphine-induced dopamine release in the nucleus accumbens of rat

Evidence recently provided has suggested a specific involvement of the GABAergic system in modulating positive reinforcing properties of several drugs of abuse through an action on mesolimbic dopaminergic neurons. The GABA(B) receptor agonist baclofen has been proposed as a potential therapeutic agent for the clinical treatment of several forms of drug addiction. In the present study, using the in vivo microdialysis technique, we investigated the effect of baclofen on nicotine, cocaine, and morphine-induced increase in extracellular dopamine (DA) levels in the shell of the nucleus accumbens, a brain area supposedly involved in the modulation of the central effects of several drugs of abuse, of freely moving rats. As expected, nicotine (0.6 mg/kg s.c.), morphine (5 mg/kg s.c.), and cocaine (7.5 mg/kg i.p.) administration in rats induced a marked increase in extracellular DA concentrations in the nucleus accumbens, reaching a maximum value of +205 +/- 8.4%, +300 +/- 22.2%, and +370 +/- 30.7%, respectively. Pretreatment with baclofen (1.25 and 2.5 mg/kg i.p.) dose-dependently reduced the nicotine-, morphine-, and cocaine-evoked DA release in the shell of the nucleus accumbens. Furthermore, baclofen alone did not elicit changes in basal DA extracellular levels up to 180 min. Taken together, our data are in line with previous reports demonstrating the ability of baclofen to modulate the mesolimbic DAergic transmission and indicate baclofen as a putative candidate in the pharmacotherapy of polydrug abuse.     

Chronic bupropion attenuated the anhedonic component of nicotine withdrawal in rats via inhibition of dopamine reuptake in the nucleus accumbens shell

Bupropion, a dopamine reuptake inhibitor, is an effective therapy for smoking cessation, but the behavioral and neurochemical mechanisms mediating its antismoking properties are relatively unknown. To explore the hypothesis that bupropion ameliorates nicotine withdrawal partly by a dopamine-dependent mechanism, we investigated the effects of chronic bupropion on potassium-stimulated dopamine overflow in the nucleus accumbens shell in nicotine-withdrawing rats. We also assessed the effects of chronic bupropion on behavioral aspects of nicotine withdrawal measured by elevations in brain reward thresholds and somatic signs of withdrawal. Rats were treated with nicotine or saline for 7 days and then coadministration of bupropion or saline was initiated. After 14 days of coadministration of bupropion/saline and nicotine/saline, nicotine/saline administration was terminated, whereas bupropion/saline administration continued. These conditions mimic bupropion administration in human smokers. Cessation of nicotine administration in non-bupropion-treated rats elevated reward thresholds reflecting a reward deficit, increased somatic signs and diminished potassium-evoked dopamine overflow in the nucleus accumbens shell. Chronic bupropion lowered reward thresholds and increased potassium-evoked dopamine release regardless of previous nicotine exposure, possibly by inhibition of dopamine reuptake, and thus attenuated the anhedonic and neurochemical effects of nicotine withdrawal. Chronic bupropion blocked withdrawal-associated increased somatic signs. Finally, acute experimenter-administered nicotine enhanced brain reward function equally in all groups, indicating that bupropion does not alter the reward-facilitating effects of experimenter-administered nicotine. In conclusion, the bupropion-induced increase in extracellular dopamine in the nucleus accumbens shell may ameliorate the anhedonia associated with nicotine withdrawal, which in turn may facilitate smoking cessation.     

SR141716A reduces the reinforcing properties of heroin but not heroin-induced increases in nucleus accumbens dopamine in rats

The present experiments tested the hypothesis that the selective CB1 receptor antagonist SR141716A alters heroin self-administration by attenuating heroin-induced increases in nucleus accumbens dopamine levels. SR141716A pretreatment dose-dependently (0.3-3 mg/kg, i.p.) reduced operant heroin self-administration by male Wistar rats under a fixed ratio schedule of reinforcement, and significantly lowered the breaking point of responding for heroin under a progressive ratio schedule of reinforcement. These observations are consistent with recent reports that CB1 receptor inactivation reduces the rewarding properties of opiates. Operant responding for water reinforcement by water-restricted rats was unaltered by these SR141716A doses. Microdialysis tests revealed that heroin self-administration significantly increases interstitial dopamine levels in the nucleus accumbens shell of vehicle-pretreated control rats. However, whereas SR141716A pretreatment dose-dependently reduced heroin self-administration, it did not alter the heroin-associated increase in nucleus accumbens dopamine. These findings suggest that the CB1 antagonist-induced attenuation of heroin reward does not involve dopaminergic mechanisms in the nucleus accumbens shell.     

Dynamic increases in dopamine during paced copulation in the female rat

The role of dopamine in the rewarding aspects of sexual behaviour in female rats was investigated. This is a unique model because sexual behaviour is only rewarding when copulatory stimuli are experienced at the preferred rate of copulation for a female rat. In addition, increases in dopamine in the nucleus accumbens occur during sexual behaviour only when the female achieves this preferred rate of copulation. In this study, minute-by-minute changes in nucleus accumbens dopamine were monitored using in vivo microdialysis. We report here that extracellular dopamine in the nucleus accumbens increases before coital stimulation only when sexual behaviour is occurring under conditions that are rewarding to the female rat. We conclude that increases in dopamine in the nucleus accumbens are involved in anticipation, not consummation, of sexual behaviour in the female rat.     

Suppressive effects of trihexyphenidyl on methamphetamine-induced dopamine release as measured by in vivo microdialysis

Abuse of methamphetamine (MAP) and cocaine causes severe medical and social problems throughout the world. Our previous study found that trihexyphenidyl (THP), a muscarinic cholinergic receptor antagonist, specifically suppressed the rewarding properties of MAP but not of cocaine, as measured by conditioned place preference in mice. The present study examined using in vivo microdialysis whether THP differentially affects the extracellular dopamine (DA) levels in the nucleus accumbens and striatum of mice injected with MAP and cocaine in comparison with another antimuscarinic agent, scopolamine (SCP). In addition, locomotor activity was simultaneously measured during microdialysis. In vivo microdialysis experiments revealed that during the initial hour after injection of MAP (1 mg/kg) DA levels increased up to 698% in the nucleus accumbens and 367% in the striatum as compared to the basal level. These increases were reduced to 293% in the nucleus accumbens and 207% in the striatum by treatment with 5 mg/kg THP. However, SCP (3 mg/kg) had no effect on the increases in extracellular DA levels in both regions after MAP injection. Cocaine (10 mg/kg) increased DA levels during the initial hour to 254% in the nucleus accumbens and 220% in the striatum as compared to the basal level. These increases were unaffected by treatment with either THP or SCP. On the contrary, both THP and SCP enhanced the locomotor-stimulant action of MAP and cocaine. These results, together with our previous finding, suggest that THP may specifically antagonize the rewarding properties of MAP through suppression of DA release in the mesolimbic area without retarding locomotor activity.     

Reduced operant ethanol self-administration and in vivo mesolimbic dopamine responses to ethanol in PKCepsilon-deficient mice

There is increasing evidence that individual protein kinase C (PKC) isozymes mediate specific effects of ethanol on the nervous system. In addition, multiple lines of evidence suggest that the mesoaccumbens dopamine reward system is critically involved in the rewarding and reinforcing effects of ethanol. Yet little is known about the role of individual PKC isozymes in ethanol reinforcement processes or in regulation of mesolimbic systems. In this study, we report that mice lacking the epsilon isoform of PKC (PKCepsilon) show reduced operant ethanol self-administration and an absence of ethanol-induced increase in extracellular dopamine levels in the nucleus accumbens. PKCepsilon null mice exhibited a 53% decrease in alcohol-reinforced operant responses under basal conditions, as well as following ethanol deprivation. Behavioural analysis revealed that while both genotypes had the same number of drinking bouts following deprivation, PKCepsilon null mice demonstrated a 61% reduction in number of ethanol reinforcers per bout and a 57% reduction in ethanol-reinforced response rate. In vivo microdialysis experiments showed that, in contrast to wild-type mice, PKCepsilon null mice exhibited no change in extracellular levels of dopamine in the nucleus accumbens following acute administration of ethanol (1 and 2 g/kg i.p.), while mesolimbic dopamine responses to cocaine (20 mg/kg i.p.) or high potassium (100 mM) in these mice were comparable with that of wild-types. These data provide further evidence that increases in extracellular mesolimbic dopamine levels contribute to the reinforcing effects of ethanol, and indicate that pharmacological agents inhibiting PKCepsilon may be useful in the treatment of alcohol dependence.     

Ascorbate reduces morphine-induced extracellular DOPAC level in the nucleus accumbens: A microdialysis study in rats

Most drugs of abuse increase dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) release in the shell of the nucleus accumbens. The effects of ascorbate, which is known to modulate dopamine neurotransmission, on the extracellular level of DOPAC in the nucleus accumbens of naive rats and of rats treated acutely with morphine were studied by using in vivo microdialysis and high performance liquid chromatography with electrochemical detection (HPLC-ECD). Acute morphine (20 mg/kg ip) treatment increased the level of DOPAC in the nucleus accumbens to approximately 170% of basal level. Acute treatment with ascorbate (500 mg/kg ip) alone did not alter nucleus accumbens' DOPAC level, but pretreatment with ascorbate (500 mg/kg ip) 30 min before morphine administration attenuated the effects of acute morphine on the level of DOPAC. These results suggest that ascorbate modulates the mesolimbic dopaminergic pathway.     

Nicotine infused into the nucleus accumbens increases synaptic dopamine as measured by in vivo microdialysis

It has been postulated that addiction to nicotine is mediated by dopamine release in the mesolimbic system. It is possible that nicotine might act directly on the dopamine terminals to release dopamine. This hypothesis was tested by infusing nicotine through a microdialysis probe into the nucleus accumbens of freely moving rats. Dopamine, dihydroxyphenylacetic acid, and homovanillic acid from the extracellular space were collected by microdialysis and measured by high pressure liquid chromatography. Nicotine increased extracellular dopamine in a dose-related manner. Systemic injection of the nicotine antagonist mecamylamine blocked the dopamine increase induced by local nicotine. These results suggest that nicotine releases dopamine by a local action in the nucleus accumbens terminal area of the mesolimbic system. Presynaptic induction of dopamine release might play a role in nicotine addiction.     

Chronic nicotine‐induced changes in gene expression of delta and kappa‐opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat

Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)‐derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real‐time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied.