Serotonin 4 receptor (5-HT4R) internalization is isoform-specific: effects of 5-HT and RS67333 on isoforms A and B.

Serotonin 4 receptors (5-HT4Rs) are particularly abundant within the limbic system, where they constitute potential targets for the development of novel, rapid acting antidepressants. However, the population of limbic 5-HT4Rs is not homogenous, comprising various isoforms of which 5-HT4(a) and 5-HT4(b) are among the most abundant variants. Sequence divergence at their C-termini is predictive of specificity in isoform signalling and regulation, but the differences, if any, remain ill-defined. The present study compared isoforms 5-HT4(a) and 5-HT4(b) in their ability to undergo endocytic regulation following exposure to 5-HT and to the putatively fast acting antidepressant RS67333. Both ligands differed in their ability to induce internalization of either isoform, 5-HT being more effective than RS67333 in HEK293 cells and in neurons. In contrast, trafficking induced by 5-HT was isoform-specific. In particular, while PKC, GRK2 and betaarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on betaarrestin. After endocytosis, isoform (b) appeared scattered throughout the intracellular compartment and efficiently recycled to the membrane upon agonist removal. Isoform (a) accumulated in the perinuclear compartment and displayed little recycling. Isoform-specific subcellular distribution was present in HEK293 cells and in neurons. In neurons, where internalization by RS67333 was more pronounced than in HEK293 cells, receptors internalized by this ligand followed the same distribution pattern as observed with 5-HT. These results point to isoform-related differences in the way that 5-HTRs respond to different ligands. Such diversity should be taken into account when developing therapeutic agents that target 5-HT4Rs.

Disruption of dopaminergic neurotransmission in nucleus accumbens core inhibits the locomotor stimulant effects of nicotine and D-amphetamine in rats.

The locomotor stimulant effects of nicotine and amphetamine appear to be dependent on dopamine transmission in the nucleus accumbens. The present aim was to elucidate the contributions of the accumbens core and medial shell to these effects. In the first experiment, rats received bilateral intra-accumbens infusion of the dopaminergic antagonist eticlopride (or saline) prior to saline or nicotine (0.2 mg/kg s.c.) challenge. Eticlopride inhibited basal and nicotine-induced locomotor activity more effectively when infused into the core (0.0625--0.5 microg/side) than into the medial shell (0.5--1 microg/side). In a second experiment, rats received 6-hydroxydopamine infused into the core or medial shell, and were subsequently tested with saline, nicotine (0.2 mg/kg s.c.) and D-amphetamine (0.75 mg/kg s.c.). Residual dopaminergic innervation was assessed by autoradiographic [(125)I]RTI-55 labelling of the dopamine transporter. [(125)I]RTI-55 labelling in the accumbens core was positively correlated with the locomotor stimulant effects of both nicotine and D-amphetamine. In contrast, [(125)I]RTI-55 labelling in the medial shell was associated negatively with amphetamine-induced activity. Recent evidence suggests that dopamine release in the medial shell may mediate the reinforcing effect of nicotine and D-amphetamine. In contrast, the present findings suggest that dopamine release in the core subregion contributes preferentially to the locomotor stimulant effects of nicotine and D-amphetamine.

Enhancement of haloperidol-induced catalepsy by nicotine: an investigation of possible mechanisms.

Nicotine has been reported to potentiate the cataleptic effect of the dopamine receptor antagonist haloperidol in rats. This effect is paradoxical, since nicotine alone tends to increase nigrostriatal dopamine release. In the present experiments, a pro-cataleptic effect of nicotine was confirmed statistically but was small and variable. Three potential mechanisms underlying this effect were investigated. (i) Desensitization of brain nicotinic receptors appears to make little if any contribution to the pro-cataleptic effect of nicotine, insofar as the latter was not mimicked by two centrally active nicotinic antagonists (mecamylamine and chlorisondamine). (ii) Depolarization inactivation resulting from combined treatment with haloperidol and nicotine does not appear to be critical, since the pro-cataleptic effect of nicotine was not enhanced by chronic haloperidol administration, a treatment designed to enhance depolarization inactivation. (iii) The slow emergence and persistence of the acute pro-cataleptic effect of nicotine suggested possible mediation by a nicotine metabolite. However, neither cotinine nor nornicotine, the principal pharmacologically-active metabolites of nicotine, exerted a significant pro-cataleptic effect. In conclusion, the pro-cataleptic effect of nicotine was weak and variable in the present study, and its mechanism remains obscure.

Behavioral evidence of depolarization block of dopamine neurons after chronic treatment with haloperidol and clozapine.

Electrophysiological studies have shown that chronic treatment with haloperidol causes depolarization block (DB) of dopamine cells in anesthetized and paralyzed rats. It has been proposed that the emergence of DB underlies the therapeutic and side effects of this drug. However, the relevance of DB to the clinical actions of haloperidol has been questioned on the grounds that chronic drug-induced DB has not yet been demonstrated in freely moving animals. In this study, responding for rewarding electrical brain stimulation was used to assess the occurrence of DB in rats chronically treated with haloperidol or clozapine. The time course of the effects of acute haloperidol (7.8-500 microg/kg) and clozapine (5-40 mg/kg) and of withdrawal from chronic drug treatment on reward and performance measures were also characterized. Haloperidol and clozapine dose-dependently attenuated reward and performance, haloperidol producing a predominant suppression of performance, and clozapine preferentially attenuating reward. Chronic (21 d) treatment with haloperidol (500 microg/kg) caused responding to cease in the six rats tested, and repeated injection with apomorphine restored the behavior in all of them; such an effect of apomorphine was observed in only two of six rats treated acutely with the same dose of haloperidol. Chronic treatment with clozapine (20 mg/kg) increased reward thresholds, an effect that was reversed by apomorphine in chronically, but not acutely, treated rats. The times at which chronic haloperidol-treated rats resumed responding was positively correlated with indices of behavioral supersensitivity after withdrawal, suggesting that the effect of apomorphine was not caused by direct stimulation of upregulated postsynaptic receptors. These findings constitute the first behavioral evidence of DB in unanesthetized, freely moving animals treated chronically with antipsychotics. They also demonstrate that the neural substrates mediating reward and performance are functionally independent and differentially sensitive to haloperidol and clozapine.

Activation of neurotensin receptors in the prefrontal cortex stimulates midbrain dopamine cell firing.

The effects of medial prefrontal cortex microinjections of 3 nmol/0.5 microl of neurotensin-(1-13), the inactive fragment neurotensin-(1-8), or vehicle on the firing rate of midbrain dopamine neurons were studied in anesthetized rats. Twelve of 19 cells tested with neurotensin-(1-13) showed an average 20-25% increase in firing rate between 10 and 20 min after the injection. This effect was not mimicked by neurotensin-(1-8) (9 cells), nor by a control injection (10 cells) suggesting that it is mediated by high-affinity neurotensin receptors. These results suggest that activation of neurotensin receptors in the medial prefrontal cortex can modulate neural activity of a subpopulation of midbrain dopamine neurons.

Effect of pimozide on self-stimulation threshold under a continuous and fixed-interval schedule of reinforcement.

Rats implanted with a monopolar electrode in the medial mesencephalon and trained to press a lever to self-administer trains of stimulating pulses were tested under a continuous (CRF) and a fixed interval (FI-1s) schedule of reinforcement before and after systemic pimozide injection (0.35 mg/kg). As previously shown, this dose of pimozide caused reductions in maximal response rates and in the rewarding effectiveness of the stimulation. The magnitude of the attenuation in rewarding effectiveness was significantly greater under CRF than under FI conditions, an effect that can be attributed to the decrease in the number of earned rewards concomitant to a decrease in response rates. The present results show that the tighter control over reward density provided by the use of a FI schedule of reinforcement reduces the probability of artifactually measuring increases in reward thresholds following treatments that suppress response rates.

Mesencephalic substrate of reward: axonal connections.

The behavioral version of the collision technique was used to study the existence of axonal linkage between reward-relevant sites in the ventral tegmental area (VTA) and posterior mesencephalon (PM) in six rats trained to self-administer trains of electrical brain stimulation. The combined use of fixed and moveable stimulation electrodes allowed us to carry out collision tests at a total of 46 different combinations of VTA-PM sites, and collision-like effects were observed at 24 of these. Stimulation of the VTA and the most caudal PM sites generally resulted in collision curves that were characterized by a single increase in paired-pulse effectiveness (E-values), whereas recovery in those collision curves obtained from stimulation of the VTA and more rostral PM sites was generally slower, and often characterized by a double rise. Despite little variability in interelectrode distances (1.0-3.8 mm), collision intervals varied widely, ranging from 1.5 to 7.3 msec. Recovery from refractoriness (initial 25%) was also estimated and ranged from 0.7 to 1.0 msec, resulting in conduction-time estimates of 0.7-6.3 msec. The lack of correspondence between interelectrode distances and conduction times suggests the presence of axonal branching. Results of this study constitute the first behavioral evidence of a reward-relevant axonal link between the VTA and the PM. In addition, the finding that in one animal the anterior electrode was located within the posterior portion of the lateral hypothalamus (LH) suggests that the reward-relevant axonal bundle linking the LH and VTA may extend as far back as the caudal regions of the PM.

Opposite effects of mesencephalic microinjections of cholecystokinin octapeptide and neurotensin-(1-13) on brain stimulation reward.

Changes in operant responding for brain stimulation reward were studied before and after a microinjection of 1 nmol of sulphated cholecystokinin octapeptide, neurotensin or saline into the ventral tegmental area. Neurotensin produced a significant and long lasting decrease in the stimulation frequency required to produce a half-maximal rate of responding; cholecystokinin had the opposite effect, attenuating the rewarding efficacy the stimulation during the first 30 min post-injection. It is suggested that the opposite effects of the peptides on reward are due to their differential modulatory effects on a subpopulation of mesencephalic dopamine neurones.