Effects of rimonabant, a cannabinoid CB1 receptor ligand, on energy expenditure in lean rats.

OBJECTIVE: To determine the effect of rimonabant on energy expenditure (O2 consumption) in rats at different metabolic states and in cannabinoid CB1 receptor-deficient (CB1R-/-) mice. DESIGN: Animals were exposed to light-dark cycles and fed only during dark cycles. Rimonabant or vehicle was administered together with food (absorptive), following overnight feeding (postabsorptive) or following a whole day of no food (fasting). Indirect calorimetric measurements, physical activity and food intake were measured continuously. RESULTS: Compared with vehicle-treated rats, rats administered 3 and 10 mg kg(-1) rimonabant showed an 18 and 49% increase in O2 consumption, respectively after 3 h. A second dose of rimonabant administered 9-14.5 h after the first one failed to affect O2 consumption, suggesting the development of tolerance. Similarly, stereotypic behaviors and ambulatory activity increased following the first dose but these effects were not observed after the second dose. Respiratory quotients revealed no effect of rimonabant on rates of carbohydrate and fat oxidation. Analysis of the correlation between O2 consumption and physical activity indicated that factors other than increased physical activity may contribute to the increase in O2 consumption. Similar studies in mice demonstrated that wild type but not CB1R-/- mice showed a change in O2 consumption and physical activity following rimonabant administration, suggesting that these effects are mediated by the cannabinoid CB1 receptor. CONCLUSION: Previous studies suggested that reduced food intake alone may not explain the weight reduction observed with rimonabant. Our studies suggest that rimonabant stimulates significant acute energy expenditure in non-obese rodents, which could not be completely accounted for by an increase in physical activity. However, with the observation that there is rapid development of tolerance, these results suggest that there may be additional mechanism(s) that lead to weight loss in these rodents.

Identification of 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT2C receptor agonists.

Synthesis and evaluation of the activity of new 4-methyl-1,2,3,4,10,10a-hexahydropyrazino[1,2-a]indoles as 5-HT(2C) receptor agonists are described. Appropriately substituted, several analogs displayed selectivity against the other 5-HT(2) receptor subtypes of 1 order of magnitude or more. Selectivity was improved for several compounds versus the lead 1, increasing the therapeutic interest in this series of 5-HT(2C) receptor agonists.

Pharmacological characterization of Ro 63-1908 (1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol), a novel subtype-selective N-methyl-D-aspartate antagonist.

Ro 63-1908, 1-[2-(4-hydroxy-phenoxy)-ethyl]-4-(4-methyl-benzyl)-piperidin-4-ol, is a novel subtype-selective N-methyl-D-aspartate (NMDA) antagonist that has been characterized in vitro and in vivo. Ro 63-1908 inhibited [(3)H]dizocilpine ((3)H-MK-801) binding in a biphasic manner with IC(50) values of 0.002 and 97 microM for the high- and low-affinity sites, respectively. Ro 63-1908 selectively blocked recombinant receptors expressed in Xenopus oocytes containing NR1C + NR2B subunits with an IC(50) of 0.003 microM and those containing NR1C + NR2A subunits with an IC(50) of >100 microM, thus demonstrating greater than 20,000-fold selectivity for the recombinant receptors expressing NR1C + NR2B. Ro 63-1908 blocked these NMDA NR2B-subtype receptors in an activity-dependent manner. Ro 63-1908 was neuroprotective against glutamate-induced toxicity and against oxygen/glucose deprivation-induced toxicity in vitro with IC(50) values of 0.68 and 0.06 microM, respectively. Thus, the in vitro pharmacological characterization demonstrated that Ro 63-1908 was a potent and highly selective antagonist of the NR2B subtype of NMDA receptors. Ro 63-1908 was active against sound-induced seizures (ED(50) = 4.5 mg/kg i.p. when administered 30 min beforehand) in DBA/2 mice. The dose required to give a full anticonvulsant effect did not produce a deficit in the Rotarod test. NMDA-induced seizures were also inhibited by Ro 63-1908 with an ED(50) of 2.31 mg/kg i.v. when administered 15 min before testing. Ro 63-1908 gave a dose-related neuroprotective effect against cortical damage in a model of permanent focal ischemia. Maximum protection of 39% was seen at a plasma concentration of 450 ng/ml. There were, however, no adverse cardiovascular or CNS side-effects seen at this dosing level.

Discovery of (R)-1-[2-hydroxy-3-(4-hydroxy-phenyl)-propyl]-4-(4-methyl-benzyl)-piperidin-4-ol: a novel NR1/2B subtype selective NMDA receptor antagonist.

Starting from Ro-25-6981 as a lead compound, highly potent and selective NR1/2B subtype selective NMDA receptor antagonists, with low activity at alpha(1) adrenergic receptors were developed.

Investigation of stretching behaviour induced by the selective 5-HT6 receptor antagonist, Ro 04-6790, in rats.

1. The present study examined the effects of the selective 5-HT6 receptor antagonist 4-amino-N-(2, 6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) on locomotor activity and unconditioned behaviour in male Sprague Dawley rats (230-300 g). 2. In non-quantified behavioural observations, animals treated with Ro 04-6790 (3, 10 or 30 mg kg(-1), i.p) showed no overt behavioural signs except a dose-dependent reduction in locomotor activity and a behavioural syndrome of stretching, yawning and chewing. The latter behaviour was most pronounced between 30 and 90 min following the administration of Ro 04-6790. 3. Detailed analysis of the stretching and yawning behaviour showed that Ro 04-6790 (3, 10 or 30 mg kg(-1), i.p.) dose-dependently induced stretching. The number of stretches observed following treatment with either Ro 04-6790 (10 mg kg(-1) i.p.) or Ro-04-6790 (30 mg kg(-1), i.p.) was significantly greater than that observed in saline-treated rats. The yawning behaviour, however, was not dose-dependent nor was the number of yawns in any of the drug treated groups significantly greater than in those treated with saline. 4. Pretreatment (30 min) with the non-selective muscarinic antagonists scopolamine (0.1, 0.3 or 1 mg kg(-1), i.p.) and atropine (0.3, 1 or 3 mg kg(-1), s.c.) but not methylatropine (1, 3 or 10 mg kg(-1), s.c) significantly inhibited stretching induced by Ro 04-6790 (30 mg kg(-1), i.p.). 5. The dopamine D2-like receptor antagonist, haloperidol (0.03, 0.1 or 0.3 mg kg(-1), s.c.) given at the same time as Ro 04-6790 (30 mg kg(-1), i.p.) had no effect on the stretching induced by the 5-HT6 antagonist. 6. These data suggest that systemic injection of the 5-HT6 antagonist, Ro 04-6790, produces a stretching behaviour that appears to be mediated by an increase in cholinergic neurotransmission in the CNS and which could be a useful functional correlate for 5-HT6 receptor blockade. There is no evidence for dopamine D2-like receptor involvement in this behaviour.

The 5-hydroxytryptamine6 receptor-selective radioligand [3H]Ro 63-0563 labels 5-hydroxytryptamine receptor binding sites in rat and porcine striatum.

Ro 63-0563 [4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulfonamide] is a high affinity 5-hydroxytryptamine6 (HT6) receptor antagonist with more than 100-fold selectivity for the 5-HT6 receptor compared with 69 other receptors and binding sites. The present study describes the properties of [3H]Ro 63-0563, the first selective 5-HT6 receptor radioligand. Specific binding of [3H]Ro 63-0563 (nonspecific binding defined in the presence of 10 microM methiothepin) to recombinant rat and human 5-HT6 receptors was saturable, rapid, and reversible with equilibrium dissociation constants (Kd) of 6.8 nM and 4.96 nM, respectively. The pharmacological profile of the rat 5-HT6 receptor labeled with [3H]Ro 63-0563 (methiothepin > D-lysergic acid diethylamide > clozapine approximately Ro 63-0563 > lisuride > ergotamine approximately Ro 04-6790 > 5-HT > amitriptyline approximately metergoline approximately mianserin approximately ritanserin > methysergide > mesulergine) was similar to that obtained by using either [3H]D-lysergic acide diethylamide or [3H]5-HT as radioligand. In equilibrium binding studies with rat striatal membranes, [3H]Ro 63-0563 labeled a single binding site with Kd and Bmax values of 11. 7 nM and 175 fmol/mg protein, respectively. In porcine striatal membranes, [3H]Ro 63-0563 also labeled a single binding site with Kd and Bmax values of 8.0 nM and 130 fmol/mg protein, respectively. The affinities of 14 5-HT6 receptor ligands at this binding site were similar to those found for the recombinant rat and human 5-HT6 receptor, which suggested the presence of 5-HT6 receptors in porcine striatum.

Characterization of Ro 04-6790 and Ro 63-0563: potent and selective antagonists at human and rat 5-HT6 receptors.

1. This study describes the in vitro characterization of two potent and selective 5-HT6 receptor antagonists at the rat and human recombinant 5-HT6 receptor. 2. In binding assays with [3H]-LSD, 4-amino-N-(2,6 bis-methylamino-pyrimidin-4-yl)-benzene sulphonamide (Ro 04-6790) and 4-amino-N-(2,6 bis-methylamino-pyridin-4-yl)-benzene sulphonamide (Ro 63-0563) had mean pKi values +/-s.e.mean at the rat 5-HT6 receptor of 7.35+/-0.04 and 7.83+/-0.01, respectively and pKi values at the human 5-HT6 receptor of 7.26+/-0.06 and 7.91+/-0.02, respectively. 3 .Both compounds were found to be over 100 fold selective for the 5-HT6 receptor compared to 23 (Ro 04-6790) and 69 (Ro 63-0563) other receptor binding sites. 4. In functional studies, neither compound had any significant effect on basal levels of cyclicAMP accumulation in Hela cells stably expressing the human 5-HT6 receptor, suggesting that the compounds are neither agonists nor inverse agonists at the 5-HT6 receptor. However, both Ro 04-6790 and Ro 63-0563 behaved as competitive antagonists with mean +/-s.e.mean pA2 values of 6.75+/-0.07 and 7.10+/-0.09, respectively. 5. In rats habituated to observation cages, Ro 04-6790 produced a behavioural syndrome similar to that seen following treatment with antisense oligonucleotides designed to reduce the expression of 5-HT6 receptors. This behavioural syndrome consisted of stretching, yawning and chewing. 6. Ro 04-6790 and Ro 63-0563 represent valuable pharmacological tools for the identification of 5-HT6 receptors in natural tissues and the study of their physiological function.

The putative 5-ht6 receptor: localization and function.

Until recently, the majority of actions of the neurotransmitter 5-hydroxytryptamine (5-HT) were generally believed to be mediated by members of the 5-HT1, 5-HT2, 5-HT3 and 5-HT4 receptor families. The application of molecular cloning techniques has revealed the existence of gene products encoding several novel, putative 5-HT receptors for which little or no prior pharmacological or functional data presently exist. The present challenge to pharmacologists is to determine the physiological relevance of these gene products, establish whether or not they function as endogenous receptors, find selective agents and determine potential therapeutic uses of these compounds. Here we review work detailing the cloning and characterization of the recombinant 5-ht6 receptor, its distribution and evidence for functional responses mediated by naturally occurring 5-ht6 receptors.

Involvement of 5-HT6 receptors in nigro-striatal function in rodents.

4-Amino-N-(2,4 bis-methylamino-pyrimidin-4-yl) benzene sulphonamide (Ro 04-6790) is a potent, selective and competitive antagonist for the 5-HT6 receptor which can be detected in the cerebro-spinal fluid (CSF) of rats following intraperitoneal administration. Since 5-HT6 receptor mRNA and 5-HT6 receptor-like immunoreactivity have been shown to be present in the striatum, the purpose of the present study was to evaluate the effect of 5-HT6 receptor antagonism on haloperidol- and SCH 23390-induced catalepsy in mice and on the turning behaviour of rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not induce catalepsy and had no effect on catalepsy induced by either haloperidol or SCH 23390. Ro 04-6790 (3, 10 and 30 mg kg(-1) i.p.) did not itself induce rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle nor did it affect the rotational behaviour induced by either L-Dopa or amphetamine. 5-HT6 receptor antagonism inhibited the rotational behaviour of 6-OHDA lesioned rats induced by treatment with the muscarinic antagonists scopolamine and atropine. The data support earlier conclusions from experiments with antisense oligonucleotides that the 5-HT6 receptor is involved in the control of acetylcholine neurotransmission in the rat brain.

Correlation between 5-HT7 receptor affinity and protection against sound-induced seizures in DBA/2J mice.

Audiogenic seizures can be induced in DBA/2J mice following intense auditory stimulation. A number of neurotransmitters, including 5-hydroxytryptamine (5-HT), are believed to be involved in mediating this effect since it has been shown previously that depletion of 5-HT or blockade of 5-HT receptors protects DBA/2J mice from these audiogenic seizures. The present study was undertaken to determine whether antagonism of the newly identified 5-HT7 receptor may protect DBA/2J mice from audiogenic seizures by attempting to correlate in vivo potency of compounds with their affinity at the 5-HT7 receptor. All compounds used in the correlation were shown to be antagonists at the 5-HT7 receptor and a statistically significant correlation was observed between 5-HT7 affinity and doses for half-maximal response (ED50) for protection of DBA/2J mice from sound-induced seizures (r = 0.80; P < 0.05). No significant correlation was observed between in vivo activity and affinity at either 5-HT1A, 5-HT2A or 5-HT2C receptors. It is also unlikely that interactions between the 5-ht5 receptor will protect DBA/2J mice from audiogenic seizures since metergoline and mesulergine which are both active in this in vivo model have no affinity for the 5-ht5 receptor. There are similarities between the pharmacology of the 5-HT7 receptor and that of the 5-HT1A receptor, however the correlation between the in vivo potency in DBA/2J mice and 5-HT1A affinity was not significant. Furthermore, the 5-HT1A receptor antagonist WAY 100135 did not protect DBA/2J mice from audiogenic seizures at doses that antagonise 5-HT1A receptor-mediated effects in mice. These data suggest that antagonism of 5-HT7 receptors may protect against audiogenic seizures in DBA/2J mice although a definitive conclusion must await studies with selective 5-HT7 antagonists.

Pharmacologic evaluation of the discriminative stimulus of metachlorophenylpiperazine.

A pharmacologic analysis of the discriminative stimulus of metachlorophenylpiperazine (mCPP) is reported. mCPP and m-trifluoromethylphenylpiperazine generalised, whereas 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole, 6-chloro-2-(1-piperazinyl)-pyrazine, and mesulergine partially generalised to the mCPP discriminative cue. However, although mianserin, methiothepin, ritanserin, mesulergine and N-(1-methyl-5'-indolyl)-N'-(3-pyridyl)urea hydrochloride (SB 200646) all antagonised the effect of 5-hydroxytryptamine (5-HT) on IP3 formation in the rat choroid plexus, they failed to antagonise the mCPP response in the drug discrimination studies. The 5-HT3 receptor antagonist MDL 72222 neither generalised nor antagonised the mCPP cue. These data suggest that neither the 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5, 5-HT6, nor 5-HT7 receptors are involved. The response does appear to be mediated by a postsynaptic 5-HT receptor, however, because fenfluramine generalised to the cue. Haloperidol generalises, and amphetamine partially antagonises the mCPP discriminative cue and low doses of apomorphine partially generalises to the mCPP cue, which suggests that a decrease in dopamine neurotransmission may also be involved.

Effects of altered 5-ht6 expression in the rat: functional studies using antisense oligonucleotides.

The purpose of the present study was to determine whether the 5-ht6 receptor is functionally expressed in the rat brain by blocking its translation from mRNA with treatments of phosphorothioate antisense oligonucleotides. Rats were treated with either saline, antisense (AO) or scrambled oligonucleotides (SO) for 4 days. Treatment with AO reduced the number of [3H]LSD binding sites in the frontal lobes by 30% but had no significant effect on the number of 5-HT1A and 5-HT2A receptor binding sites in the cortex of the rats. A behavioural syndrome of yawning, stretching and chewing, however, was observed in AO treated rats but not in any of the other treatment groups. This AO-specific behaviour had returned to normal 5 days after cessation of the oligodeoxynucleotide treatment. These data suggest that the 5-ht6 receptor has a physiological function in the rat brain where it appears to be under the tonic control of endogenous 5-HT.

Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor.

A heptadecapeptide was identified and purified from porcine brain tissue as a ligand for an orphan heterotrimeric GTP-binding protein (G protein)-coupled receptor (LC132) that is similar in sequence to opioid receptors. This peptide, orphanin FQ, has a primary structure reminiscent of that of opioid peptides. Nanomolar concentrations of orphanin FQ inhibited forskolin-stimulated adenylyl cyclase activity in cells transfected with LC132. This inhibitory activity was not affected by the addition of opioid ligands, nor did the peptide activate opioid receptors. Orphanin FQ bound to its receptor in a saturable manner and with high affinity. When injected intracerebroventricularly into mice, orphanin FQ caused a decrease in locomotor activity but did not induce analgesia in the hot-plate test. However, the peptide produced hyperalgesia in the tail-flick assay. Thus, orphanin FQ may act as a transmitter in the brain by modulating nociceptive and locomotor behavior.

Determination of the role of the 5-ht6 receptor in the rat brain: a study using antisense oligonucleotides.

The purpose of the present study was to determine possible physiological functions of the 5-ht6 receptor using antisense oligonucleotides (AOs) in male rats. Repeated intracerebroventricular treatment with AOs but not with a scrambled form of the antisense sequence (SO) gave rise to a specific behavioral syndrome of yawning, stretching and chewing and caused a 30% reduction in the number of [3H]-lysergic acid diethylamide binding sites (measured in the presence of 300 nM spiperone). Neither sequence, however, had any effect on other parameters measured (e.g., locomotor activity, body weight, food intake, body temperature and nociception). The specific behavioral syndrome did not appear to be caused by modulation of dopaminergic neurotransmission since no changes in the tissue levels of either dopamine or its metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid were seen. Furthermore, haloperidol (0.03 mg/kg s.c.) did not reduce the number of yawns or stretches. An increase in cholinergic neurotransmission did appear to be involved since the behavioral syndrome was dose-dependently antagonized by atropine. The present study suggests that 5-ht6 receptors are functionally expressed in the rat brain, where one of their functions appears to be the control of cholinergic neurotransmission.

Identification of 5-hydroxytryptamine7 receptor binding sites in rat hypothalamus: sensitivity to chronic antidepressant treatment.

Due to the high level of expression of mRNA for the 5-hydroxytrytamine (5-ht7) receptor in the hypothalamus and the high affinity of 5-HT for this receptor, [3H]5-HT binding was performed in rat hypothalamus to determine whether 5-ht7 receptor binding sites are present in animal tissue. [3H]5-HT binding was performed in the presence of 100 nM pindolol, which is inactive at 5-ht7 receptors but prevents the binding of [3H]5-HT to 5-HT1A and 5-HT1B receptor binding sites. Under these conditions, [3H]5-HT bound to a binding site with an affinity of 1.94 nM. Displacement studies showed the pharmacology of the hypothalamic binding site to correlate well with the published pharmacology of the 5-ht7 receptor (r = 0.921). The treatment of rats with fluoxetine (5 mg/kg/day, orally) for 21 days caused a significant reduction in the number of hypothalamic 5-ht7 receptor binding sites. These data suggest that the 5-ht7 receptor binding site is expressed in rat hypothalamus and that this receptor binding site is down-regulated after a chronic increase in the synaptic level of 5-HT.

An investigation into the discriminative stimulus and reinforcing properties of the CCKB-receptor antagonist, L-365,260 in rats.

The discriminative stimulus properties of the selective CCKB-receptor antagonist, L-365,260 were evaluated in rats trained to discriminate diazepam (2 mg/kg) or morphine (5 mg/kg) from vehicle, using a two-lever food reinforced technique. In the diazepam drug discrimination, the benzodiazepine-receptor agonist FG8205 (0.063-2 mg/kg) produced dose-related drug associated responding, whereas L-365,260 (0.125-4 mg/kg) treated animals showed vehicle appropriate behaviour. In rats trained to discriminate morphine from saline, L-365,260 (0.063-4 mg/kg) produced saline lever responding. When a dose of 1 mg/kg L-365,260 was administered in combination with morphine, the dose response curve for drug lever responding was not significantly affected. This was in contrast to the effect produced by the opiate antagonist naloxone (0.3 mg/kg) which shifted the dose-response curve to the right. Another group of rats underwent training to discriminate a dose of 6 mg/kg L-365,260 from vehicle. None of the animals learned the discrimination within 50 daily training sessions. In addition, unlike morphine (3 mg/kg), or changing the training dose of cocaine, intravenous administration of L-365,260 (0.3-10 mg/kg) did not modify lever pressing or the number of injections received by rats trained to self administer cocaine (0.25 mg/injection). L-365,260 (0.1-3 mg/kg) produced a dose-related inhibition of pentagastrin-stimulated gastric acid secretion in vivo. When administered dissolved in a mixture of ethanol/propylene glycol/saline, the ID50 was 0.83 mg/kg, and when suspended in an ethanol/carboxymethylcellulose vehicle, it was 0.7 mg/kg. It was concluded: 1) that L-365,260 does not produce discriminative stimuli similar to either diazepam or morphine; 2) that the potentiation of morphine-induced behaviour by L-365,260 does not extend to the discriminative stimulus properties of morphine; 3) that L-365,260 itself does not produce readily discriminable interoceptive stimuli in rats; and 4) that L-365,260 does not substitute for the reinforcing drug cocaine.

Curative effects of the atypical antidepressant mianserin in the chronic mild stress-induced anhedonia model of depression.

This study was designed to validate a novel animal model of depression by testing the curative effects of the atypical antidepressant mianserin. In this paradigm, the hedonic state of rats was assessed using an intracranial self-stimulation (ICSS) procedure. The ICSS threshold was determined before, during and after a 38-day period of exposure to a variety of intermittent, unpredictable, mild stressors. After 11 days of this regimen, the ICSS threshold was significantly higher in the stressed rats, suggesting a gradual decrease of sensitivity to reward. This "anhedonia" lasted throughout the stress regimen and progressively diminished over a 20-day period after stress was terminated. When stressed animals exhibiting anhedonia were treated with mianserin, the stress-induced increase in the ICSS threshold was gradually reversed over ten days of treatment. These results provide further support for the value of this anhedonia paradigm in modelling an important aspect of human depressive disorders.

Yawning induced by apomorphine, physostigmine or pilocarpine is potentiated by dihydropyridine calcium channel blockers.

Previous studies have shown that dihydropyridine (DHP) calcium channel blockers can potentiate yawning induced by apomorphine in rats. The present study was undertaken to examine whether or not this interaction was seen with other compounds that induce yawning or if it represented a specific interaction with dopaminergic mechanisms. Yawning induced by apomorphine (40 micrograms/kg SC), physostigmine (50 micrograms/kg SC) or pilocarpine (1 mg/kg SC) was dose-dependently potentiated by the DHP calcium channel blocker nifedipine (1.25-10 mg/kg IP). Nimodipine (1.25-5 mg/kg IP) and nitrendipine (1.25-5 mg/kg IP) also significantly increased the yawning response. The DHP calcium channel blockers alone induced only a low incidence of yawning. The effects of nifedipine on physostigmine-induced yawning were reversed by the DHP calcium channel activator BAY K 8644 which also inhibited yawning induced by physostigmine (100 micrograms/kg SC) and pilocarpine (2 mg/kg SC). In contrast to the DHP compounds, diltiazem (2.5-10 mg/kg IP) and verapamil (2.5-10 mg/kg IP) failed to potentiate yawning. Sulpiride (10 mg/kg SC) antagonised the nifedipine potentiation of apomorphine-induced yawning but not that of physostigmine-induced yawning; atropine (2.5 mg/kg SC) antagonised both effects. These results support the hypothesis that this effect of dihydropyridine compounds is not dependent on, nor mediated through, dopaminergic mechanisms.