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We describe here the design, synthesis and characterization of a series of 4,5,6,7-tetrahydrooxazolo[4,5- c ]pyridines as metabotropic glutamate receptor (mGluR) 5 negative allosteric modulators (NAMs). Optimization of the substituents led to the identification of several compounds with good pharmacokinetic profiles, including long half life and high oral bioavailability, in both rats and monkeys. The receptor occupancy test in the rat cortex revealed favorable brain penetration of these compounds. The reprsentative compound 13 produced oral antidepressant-like effect in the rat forced swimming test (MED: 0.3 mg/kg, q.d.).

BACKGROUND AND PURPOSE Selective 5-hydroxytryptamine (5-HT, serotonin) reuptake inhibitors (SSRIs) are widely used antidepressants and their therapeutic effect requires several weeks of drug administration. The delayed onset of SSRI efficacy is due to the slow neuroadaptive changes of the 5-hydroxytryptaminergic (5-HTergic) system. In this study, we examined the acute and chronic effects of SSRIs on the 5-HTergic system using rat raphe slice cultures. EXPERIMENTAL APPROACH For organotypic raphe slice cultures, mesencephalic coronal sections containing dorsal and median raphe nuclei were prepared from neonatal Wistar rats and cultured for 14–16 days. KEY RESULTS Acute treatment with citalopram, paroxetine or fluoxetine (0.1–10 µM) in the slice cultures slightly increased extracellular 5-HT levels, while sustained exposure for 4 days augmented the elevation of 5-HT level in a time-dependent manner. Sustained exposure to citalopram had no effect on tissue contents of 5-HT and its metabolite, expression of tryptophan hydroxylase or the membrane expression of 5-HT transporters. The augmented 5-HT release was attenuated by Ca2+-free incubation medium or treatment with tetrodotoxin. Experiments with 5-HT1A/B receptor agonists and antagonists revealed that desensitization of 5-HT1 autoreceptors was not involved in the augmentation of 5-HT release. Finally, co-treatment with an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, but not an N-methyl-d-aspartate, receptor antagonist, suppressed this augmentation. CONCLUSION AND IMPLICATIONS These results suggest that sustained exposure to SSRIs induces the augmentation of exocytotic 5-HT release, which is caused, at least in part, by the activation of AMPA/kainate receptors in the raphe slice cultures.

Most clinically-used antidepressants acutely increase monoamine levels in synaptic clefts, while their therapeutic effects often require several weeks of administration. Slow neuroadaptive changes in serotonergic neurons are considered to underlie this delayed onset of beneficial actions. Recently, we reported that sustained exposure of rat organotypic raphe slice cultures containing abundant serotonergic neurons to selective serotonin (5-HT) reuptake inhibitors (citalopram, fluoxetine and paroxetine) caused the augmentation of exocytotic serotonin release. However, the ability of other classes of antidepressants to evoke a similar outcome has not been clarified. In this study, we investigated the sustained actions of two tricyclic antidepressants (imipramine and desipramine), one tetracyclic antidepressant (mianserin), three 5-HT and noradrenaline reuptake inhibitors (milnacipran, duloxetine and venlafaxine) and one noradrenergic and specific serotonergic antidepressant (mirtazapine) on serotonin release in the slice cultures. For seven of nine antidepressants, sustained exposure to the agents at concentrations of 0.1–100 µm augmented the level of increase in extracellular serotonin. The rank order of their potency was as follows: milnacipran>duloxetine>citalopram>venlafaxine>imipramine>fluoxetine>desipramine. Neither mirtazapine nor mianserin caused any augmentation. The highest augmentation by sustained exposure to milnacipran was partially attenuated by an α1-adrenoceptor antagonist, benoxathian, while the duloxetine-, venlafaxine- and citalopram-mediated increases were not affected. These results suggest that inhibition of the 5-HT transporter is required for the enhancement of serotonin release. Furthermore, the potent augmentation by milnacipran is apparently due to the accompanied activation of the α1-adrenoceptor.

Repeated intermittent exposure to psychostimulants and morphine leads to progressive augmentation of its locomotor activating effects in rodents. Accumulating evidence suggests the critical involvement of the mesocorticolimbic dopaminergic neurons, which project from the ventral tegmental area to the nucleus accumbens and the medial prefrontal cortex, in the behavioral sensitization. Here, we examined the acute and chronic effects of psychostimulants and morphine on dopamine release in a reconstructed mesocorticolimbic system comprised of a rat triple organotypic slice co-culture of the ventral tegmental area, nucleus accumbens and medial prefrontal cortex regions. Tyrosine hydroxylase-positive cell bodies were localized in the ventral tegmental area, and their neurites projected to the nucleus accumbens and medial prefrontal cortex regions. Acute treatment with methamphetamine (0.1-1000 µM), cocaine (0.1-300 µM) or morphine (0.1-100 µM) for 30 min increased extracellular dopamine levels in a concentration-dependent manner, while 3,4-methylenedioxyamphetamine (0.1-1000 µM) had little effect. Following repeated exposure to methamphetamine (10 µM) for 30 min every day for 6 days, the dopamine release gradually increased during the 30-min treatment. The augmentation of dopamine release was maintained even after the withdrawal of methamphetamine for 7 days. Similar augmentation was observed by repeated exposure to cocaine (1-300 µM) or morphine (10 and 100 µM). Furthermore, methamphetamine-induced augmentation of dopamine release was prevented by an NMDA receptor antagonist, MK-801 (10 µM), and was not observed in double slice co-cultures that excluded the medial prefrontal cortex slice. These results suggest that repeated psychostimulant- or morphine-induced augmentation of dopamine release, i.e. dopaminergic sensitization, was reproduced in a rat triple organotypic slice co-cultures. In addition, the slice co-culture system revealed that the NMDA receptors and the medial prefrontal cortex play an essential role in the dopaminergic sensitization. This in vitro sensitization model provides a unique approach for studying mechanisms underlying behavioral sensitization to drugs of abuse.