Abstract
The pineal hormone melatonin was extracted in 1958 from the bovine pineal gland. Ever since it has received vast popular attention as a hormone with beneficial effects on sleep disorders, jet lag and longevity. Recent attention has focused on its role in circadian physiology. The suprachiasmatic nucleus (SCN) is the circadian pacemaker in all mammals. Melatonin phase advances the circadian rhythm of the activity of the SCN neurons when administered at specific times of the day. This effect is more pronounced at dusk. Also melatonin inhibits spontaneous firing of SCN neurons. However the cellular mechanisms underlying the effects of melatonin in the SCN have not yet been determined. Whether these effects are dependent on the circadian time or not is also still unclear. Melatonin has two G-protein coupled receptors in the mammalian brain, MT1 and MT2. These receptors are believed to be indirectly coupled to ion channels. One current that could be modulated by melatonin is the hyperpolarization-activated current(Ih). Ih is a mixed Na+/K+ inward current that plays an important role in the regulation of SCN neurons excitability by limiting the duration of hyperpolarizing events. The mammalian hyperpolarization-activated and cyclic-nucleotide gated non selective cation channel that generates Ih is encoded by four members of a gene family (HCN1- 4). Whole cell voltage clamp, as well as immunohistochemistry, in rat hypothalamic brain slices, was used to study the effect of melatonin on Ih in SCN neurons. The results indicated first, that melatonin inhibits Ih. When the receptor specificity of this effect was examined using the putative MT2 receptors antagonists 4P-PDOT and luzindole, it was found that these antagonists behaved as partial agonists for melatonin. To test the hypothesis that the circadian effect of melatonin on the SCN was due to receptor availability, the localization of MT1 and MT2 receptors was examined at different circadian times using receptor specific antibodies. It was shown that MT1 receptors exhibited a circadian pattern of expression that coincided with the known window of time for melatonin effects on the SCN (dusk). MT2 receptors were not detected in the SCN at any of the circadian times tested. Further, as the majority of SCN neurons exhibit Ih, the channel subunits composition underlying this conductance was examined using antibodies against the subunits HCN1, 2, and 4. The results showed that only HCN1 and HCN2 subunits were present in the SCN. However, the subunits exhibited a partially overlapping pattern of distribution. The kinetic properties of the observed Ih current were consistent with heteromeric ion channels formed by a combination of HCN1 and HCN2 subunits.|In conclusion, melatonin may regulate SCN neurons by activating MT1 receptors which in turn inhibit Ih. MT2 receptors appear to play at most a minor role in melatonin circadian effects on the SCN. In addition, this is the first demonstration that both luzindole and 4P-PDOT act as partial agonists for melatonin. Consequently, care in the use of these putative antagonists in behavioral studies is warranted. This is a further step toward the understanding of melatonin’s role in circadian physiology which in turn is helpful in pointing the way to the treatment of jet lag and sleep disorders.