Selective MT₂ melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms

This study demonstrates the involvement of the MT₂ (Mel(1b)) melatonin receptor in mediating phase advances of circadian activity rhythms by melatonin. In situ hybridization histochemistry with digoxigenin-labeled oligonucleotide probes revealed for the first time the expression of mt₁ and MT₂ melatonin receptor mRNA within the suprachiasmatic nucleus of the C3H/HeN mouse. Melatonin (0.9 to 30 μg/mouse, s.c.) administration during 3 days at the end of the subjective day (CT 10) to C3H/HeN mice kept in constant dark phase advanced circadian rhythms of wheel running activity in a dose-dependent manner [EC₅₀=0.72 μg/mouse; 0.98 ± 0.08 h (n=15) maximal advance at 9 μg/mouse]. Neither the selective MT₂ melatonin receptor antagonists 4P-ADOT and 4P-PDOT (90 μ/mouse, s.c.) nor luzindole (300 μg/mouse, s.c.), which shows 25-fold higher affinity for the MT₂ than the mt₁ subtype, affected the phase of circadian activity rhythms when given alone at CT 10. All three antagonists, however, shifted to the right the dose-response curve to melatonin, as they significantly reduced the phase shifting effects of 0.9 and 3 μg melatonin. This is the first study to demonstrate that melatonin phase advances circadian rhythms by activation of a membrane-bound melatonin receptor and strongly suggests that this effect is mediated through the MT₂ melatonin receptor subtype within the circadian timing system. We conclude that the MT₂ melatonin receptor subtype is a novel therapeutic target for the development of sub-type-selective analogs for the treatment of circadian sleep and mood-related disorders.