The prepro-orexin siRNA-injected (unilateral) side demonstrated a marked reduction in orexin-A immunoreactivity (; n = 4) on day 2 as compared to the side that received scrambled siRNA (control). Double immunofluorescence labelling of MCH and orexin-A revealed that there was a marked reduction in orexin-A immunoreactivity on the prepro-orexin siRNA-injected side () as compared with the side treated with scrambled siRNA; in contrast, there was no reduction in MCH immunoreactivity on the prepro-orexin siRNA-injected side as compared with scrambled siRNA side.
Fig. 1 A representative photomicrograph shows that the side (left) with unilateral injection of siRNA against prepro-orexin mRNA evinces reduced orexin-A immunoreactivity in the perifornical region of the hypothalamus as compared to the side (right) injected (more ...)
Fig. 2 Double immunofluorescence labelling of MCH (FITC, green) and orexin-A (Cy3, red) neurons in a representative photomicrograph revealed that the prepro-orexin siRNA-injected (unilateral) side (left) had fewer orexin-A-positive neurons than the scrambled (more ...)
Double immunofluorescence labelling of dynorphin B and orexin-A revealed that all orexin-A-immunoreactive neurons were double-labelled and showed dynorphin B immunoreactivity. Dynorphin B immunoreactivity was also found in many other nonorexin-positive neurons (). Similar findings were also reported by two previous studies (Bayer et al., 2002
and Harthoorn et al., 2005
). On the prepro-orexin siRNA-injected (unilateral) side, the number of orexin-A-labelled neurons was significantly reduced 2 days after receiving the prepro-orexin siRNA treatment, as compared with the scrambled siRNA side (23% reduction; n
= 5, mean ± SEM 219 ± 29 vs. 285 ± 21; P
< 0.05, t
= 3.3, paired t
-test; , ). In contrast, there was no reduction in dynorphin B neurons on the prepro-orexin siRNA side as compared with the scrambled siRNA side (n
= 3, mean ± SEM 1283 ± 143 vs. 1331 ± 153; ). The prepro-orexin siRNA-treated side did not show any significant change in orexin-A-immunoreactive neurons on days 4 (n
= 3, mean ± SEM 145 ± 21 vs. 144 ± 41) and 6 (n
= 3, mean ± SEM 213 ± 47 vs. 175 ± 42) postinjection as compared with scrambled siRNA (control) side.
Fig. 3 Double immunofluorescence labelling of orexin-A (FITC-labelled, green) and dynorphin B (Cy3-labelled, red) in a representative photomicrograph reveals that all orexin-A-immunoreactive cells show dynorphin B immunoreactivity but not vice versa. The prepro-orexin (more ...)
Number of orexin-A-positive neurons in the prepro-siRNA- and scrambled siRNA-treated sides on day 2
Number of dynorphin B-positive neurons in the siRNA- and scrambled siRNA-treated sides on day 2
Real-time PCR data showed that there was a 58.5% down-regulation of prepro-orexin mRNA (; n = 9, P < 0.05, Student's t-test) in the siRNA-treated (bilateral injections) group: mean, 41.5% of control; range, 29.8–57.9%; controls being the scrambled siRNA-treated (bilateral injections) group (mean, 100%; range, 81.4–122.8%). Although there was still a slight decrease (16.7%) in prodynorphin in the siRNA-treated group, the change was not statistically significant and its range of variation overlapped with that of the control group.
Fig. 4 Bilateral injection of siRNA against prepro-orexin into the PFH induced a significant decrease in prepro-orexin mRNA but not prodynorphin mRNA (n = 9) when compared to scrambled siRNA-treated (bilateral) rats (n = 9). Prepro-orexin and prodynorphin mRNA (more ...)
The prepro-orexin siRNA-treated animals (bilateral injections) showed a significant increase (n = 6, F6,30 = 6.1, P < 0.01, one-way RM anova; see , ) in the amount of time spent in REM sleep exclusively during the dark (active) period after prepro-orexin siRNA administration (bilateral) as compared to the baseline (preinjection night). The increase persisted over the first four postinjection nights (all P < 0.05, post hoc test). In contrast, the scrambled siRNA-treated (bilateral injections) rats (controls) had only a transient increase in REM sleep during the first postinjection night (n = 6, F3,15 = 6.9, P < 0.01, one-way RM anova; for the first night postinjection (P < 0.05, post hoc test) as compared to the baseline (preinjection night), perhaps due to cellular trauma from the microinjection and a consequent decrease in orexinergic neurotransmission.
Fig. 5 REM sleep percentages (filled bars) during the dark period after prepro-orexin siRNA injection (bilateral, n = 6, A) or scrambled siRNA injection (n = 6, B). There was a significant increase in full-criteria REM sleep in prepro-orexin siRNA-treated animals (more ...)
Sleep percentages in prepro-siRNA- and scrambled siRNA-treated rats
Further analysis of REM sleep characteristics revealed that the increase in the amount of time spent in REM sleep after prepro-orexin siRNA injection was mainly due to an increase in the number of REM sleep episodes (n = 6, F6,30 = 3.5, P < 0.05, one-way RM anova; ) and rapid REM-to-REM sleep cycling (F6,30 = 4.0, P < 0.01, one-way RM anova; ). There was no significant change in average REM duration or latency to REM sleep.
REM sleep and cataplexy or SOREM during the night
In scrambled siRNA-treated animals, the transient REM sleep increase was also due to an increased number of episodes and a shortened REM sleep interval (P < 0.05 for the first night postinjection; ). The transient increase in REM sleep was only observed with injections in the PFH; there were no REM sleep changes in rats (n = 2) where the injections were in nonorexinergic zone (see ).
Fig. 6 Percentage of REM sleep after prepro-siRNA injection (bilateral) into sites outside the orexinergic zone (mean of two rats). Note the percentage of REM on the two postinjection nights is about the same as that on the baseline night. The insert shows the (more ...)
In contrast to the REM sleep enhancement during the dark period, during the light (inactive) period neither the prepro-orexin siRNA- or scrambled siRNA-treated rats showed any change in REM sleep compared to the baseline during the first three postinjection days (). In addition, neither group of animals showed any statistically significant changes in wakefulness or NREM sleep during the dark or the light period ().
REM sleep percentage during the light period in animals treated with siRNA injection (bilateral, n = 6, A) or scrambled siRNA injection (bilateral, n = 6, B) showed no significant differences from baseline. Data are expressed as mean ± SEM.
Cataplexy or SOREM-like events were observed in three of the six prepro-orexin siRNA-treated rats and occurred almost exclusively during the dark period (only one cataplexy or SOREM-like episode was observed during the light period in one rat on postinjection day 2). The cataplexy or SOREM behaviour was always followed by wakefulness. Cataplexy or SOREM-like episodes were not present in: (i) rats before prepro-orexin siRNA injection; (ii) scrambled siRNA-treated animals, and (iii) in animals with prepro-orexin siRNA injections in nonorexinergic zones. The average percentage of time spent in cataplexy or SOREM-like episodes in the prepro-orexin siRNA-treated rats in the dark period was very short: baseline, 0%; night 1, 0.07%; night 2, 0.34%; night 3, 0.03%; night 4, 0.01%; night 5, 0.06%; and night 6, 0%.