REM sleep changes in rats induced by siRNA‐mediated orexin knockdown

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Abstract
Short interfering RNAs (siRNA) targeting prepro‐orexin mRNA were microinjected into the rat perifornical hypothalamus. Prepro‐orexin siRNA‐treated rats had a significant (59%) reduction in prepro‐orexin mRNA compared to scrambled siRNA‐treated rats 2 days postinjection, whereas prodynorphin mRNA was unaffected. The number of orexin‐A‐positive neurons on the siRNA‐treated side decreased significantly (23%) as compared to the contralateral control (scrambled siRNA‐treated) side. Neither the colocalized dynorphin nor the neighbouring melanin‐concentrating hormone neurons were affected. The number of orexin‐A‐positive neurons on the siRNA‐treated side did not differ from the number on the control side 4 or 6 days postinjection. Behaviourally, there was a persistent (∼ 60%) increase in the amount of time spent in rapid eye movement (REM) sleep during the dark (active) period for 4 nights postinjection, in rats treated with prepro‐orexin siRNA bilaterally. This increase occurred mainly because of an increased number of REM episodes and decrease in REM‐to‐REM interval. Cataplexy‐like episodes were also observed in some of these animals. Wakefulness and NREM sleep were unaffected. The siRNA‐induced increase in REM sleep during the dark cycle reverted to control values on the 5th day postinjection. In contrast, the scrambled siRNA‐treated animals only had a transient increase in REM sleep for the first postinjection night. Our results indicate that siRNA can be usefully employed in behavioural studies to complement other loss‐of‐function approaches. Moreover, these data suggest that the orexin system plays a role in the diurnal gating of REM sleep.