Abstract
Objective: Identify neuronal substrates mediating sleep promoting effects of melatonin
Background: Melatonin promotes sleep, however the mechanism is unclear. Since the perifornical hypothalamic (PF-LH) orexin system is critical for wake promotion, we asked: Does melatonin promote sleep by inhibiting orexin neurons? We used C57BL/6J mice as our animal model and designed four experiments to address this question.
Experiment 1: examined the presence of melatonin receptors on orexin neurons by double-label immunofluorescence.
Experiment 2: examined the effects of melatonin on the activation of orexin neurons. Standard sterile surgical protocol was used to implant bilateral guide cannulas targeted toward the orexinergic PF-LH. Melatonin (500pmole/50 nl/side) was bilaterally infused into PF-LH at dark onset. The animals were euthanized two hour later to examine c-Fos expression (marker of neuronal activation) in orexin neurons.
Experiment 3: examined the effects of bilateral PF-LH infusion of melatonin on sleep-wakefulness. Animals were implanted with bilateral guide cannulas and sleep recording electrodes. At the onset of active (dark) period, melatonin (500pmole/50 nl/side) was infused into the PF-LH and its effects on spontaneous bouts of sleep-wakefulness were examined.
Experiment 4: examined the effects melatonin receptor blockade on spontaneous bouts of sleep-wakefulness. Surgical and experimental procedures used were as described in experiment 3 except at sleep (light) onset melatonin receptor antagonist, Luzindol, (10pmol/50 nL/side) was administered and its effects on sleep-wakefulness were examined
Results: Orexin neurons express MT1, but not MT2 receptors. Melatonin infusion into the orexinergic PF-LH significantly (p<0.05) reduced the number of orexin neurons with c-Fos immunoreactivity, increased NREM sleep and reduced wakefulness during normal active period. Blockade of melatonin receptors by local infusion of Luzindol into the orexinergic PF-LH, during normal sleep period significantly (p<0.05) increased wakefulness
Conclusions: We suggest that melatonin may act via the MT1 receptors to inhibit orexin neurons and promote sleep.
Disclosure: Dr. Thakkar has nothing to disclose. Dr. Sharma has nothing to disclose. Dr. Sahota has nothing to disclose.
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