Long-lasting silencing of orexin/hypocretin neurons using archaerhodopsin induces slow-wave sleep in mice

• New transgenic mice, TetO archaerhodopsin (ArchT) BAC mice, were generated.
• Specific expression of ArchT was confirmed by breeding with orexin-tTA mice.
• Slice patch clamp confirmed that green light illumination silenced orexin neurons.
• One hour silencing of orexin neurons induced slow-wave sleep in the dark period.

Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. Recent optogenetic studies revealed that the activation or inhibition of orexin neuronal activity affects the probability of sleep/wakefulness transition in the acute phase. To expand our understanding of how orexin neurons maintain wakefulness, we generated new transgenic mice in which orexin neurons expressed archaerhodopsin from Halorubrum strain TP009 (ArchT), a green light-driven neuronal silencer, using the tet-off system (orexin-tTA; TetO ArchT mice). Slice patch clamp recordings of ArchT-expressing orexin neurons demonstrated that long-lasting photic illumination was able to silence the activity of orexin neurons. We further confirmed that green light illumination for 1 h in the dark period suppressed orexin neuronal activity in vivo using c-Fos expression. Continuous 1 h silencing of orexin neurons in freely moving orexin-tTA; TetO ArchT mice during the night (the active period, 20:00–21:00) significantly increased total time spent in slow-wave sleep (SWS) and decreased total wake time. Additionally, photic inhibition increased sleep/wakefulness state transitions, which is also evident in animals lacking the prepro-orexin gene, orexin neurons, or functional orexin-2 receptors. However, continuous 1 h photic illumination produced little effect on sleep/wakefulness states during the day (the inactive period, 12:00–13:00). These results suggest that orexin neuronal activity plays a crucial role in the maintenance of wakefulness especially in the active phase in mice.