| ObjectiveIntrinsically photosensitive retinal ganglion cells (ipRGCs) were able to synthesize a photosensitive protein- melanopsin, involved in regulation of circadian rhythms, papillary light reflex and other non-imaging visual functions. In this study, mice of different genotypes by constructing sleep model were used with brain stereotactic and multiple polysomnography technology to observe and record EEG and EMG in the normal 12 h:12 h light dark cycle and light pulse administered at 1 h after the turning off of light, and further to investigate the role of ipRGCs in the sleep activity.Methods1. Experimental animals 8-week-old male mice (SPF grade) weighing 25-28 g were used and divided into four groups according to different genotypes. These mice were provided by Xue Tian’s group of Key Laboratory of Brain Functions and Diseases, University of Science and Technology of China.2. Genotyping Toe tissues of 2 week aged mice were taken to PCR. These PCR products were taken to 1% DNA agarose gel electrophoresis for identification. In addition to WT newborn mice, the gene identification is required for all of other genotyping mice before all experiments. The transgenic mice of this experiment include melanopsin knock out (MKO), melanopsin only (MO) and coneless, rodless and melanopsin knock out (TKO).3. Recording method Mice were fixed on brain stereotactic adapter after intraperitoneal anesthesia by avertin (30 ml/kg), inserted EEG and EMG electrodes, sutured wounds, and finally placed in the recovery cages of shield boxes with lateral position. Operated mice were placed in recovery cages to recover for 7 days and adapted in the recording cages to 3 days before formal recording. The normal 12 h:12 h light dark cycle,1 h (21:00-22:00) and 3 h (21:00-24:00) light pulse administered at 1 h after the turning off of light (21:00) were used to detect the variation of sleep.4. Data analysis According to the polysomnography results, each 4 s was regarded as one epoch. The sleep-wake cycle was divided into wakefulness with low-amplitude fast wave in EEG and obvious activity in EMG; non-rapid eye movement sleep (NREM) with high-amplitude slow wave in EEG and subdued activity in EMG; rapid eye movement sleep (REM), with low-amplitude slow wave EEG and no activity in EMG. Total sleep time (TST) was composed of REM and NREM.5. Statistical analysis SPSS 17.0 software was used for statistical analyses. The experimental data were presented as mean ± SEM. Two samples were compared using t test, groups were compared using ANOVA and those at P< 0.05 were considered as the level of significance.Results1. In the normal 12 h:12 h light dark cycle, WT, MKO and MO mice had a regular day-night rhythm which could be granted by the optical signal and no significant difference in wakefulness, REM sleep and NREM sleep.2. Compared with the corresponding time of the normal 12 h:12 h light dark cycle, the amount of wakefulness in WT mice (n= 10) was decreased 67.31% (P< 0.01) along with an increase of REM sleep 3061.11% (P< 0.01), NREM sleep 929.95% (P< 0.01) and TST 997.36% (P< 0.01) during 1 h light pulse. The total amounts of wakefulness were decreased 50.15% (P< 0.01), REM sleep, NREM sleep and TST were increased 785.15% (P< 0.01),286.21% (P< 0.01),308.65% (P< 0.01), respectively, during 3 h light pulse. The numbers of transition of NREM sleep to wakefulness, wakefulness to NREM sleep, NREM sleep to REM sleep and REM sleep to wakefulness were all increased during the light pulse (P< 0.01).The short term of wakefulness and NREM sleep were all increased during the light pulse (P< 0.01). The short term of REM sleep were increased (P< 0.05) in 1 h light pulse while increased (P< 0.01) in 3 h light pulse.3. Compared with the corresponding time of the normal 12 h:12 h light dark cycle, the amount of wakefulness, REM sleep, NREM sleep and TST in MKO mice (n= 8) had no significant difference. The numbers of transition of NREM sleep to wakefulness, wakefulness to NREM sleep, NREM sleep to REM sleep and REM sleep to wakefulness were all had no significant difference during the light pulse. The short term of wakefulness, REM sleep and NREM sleep were had no significant difference in the light pulse.4. Compared with the corresponding time of the normal 12 h:12 h light dark cycle, only the amount of REM sleep in MO mice (n= 8) increased 106.75% (P< 0.05) during 1 h light pulse. The total amounts of wakefulness were decreased 18.92% (P < 0.01), REM sleep, NREM sleep and TST were increased 137.38% (P< 0.05), 33.63% (P< 0.05),40.65% (P< 0.01), respectively, during 3 h light pulse. The numbers of transition of NREM sleep to wakefulness, wakefulness to NREM sleep, NREM sleep to REM sleep and REM sleep to wakefulness were all had no significant difference during the light pulse. The long and short term of wakefulness, the short term of REM sleep and NREM sleep were had no significant difference in the light pulse.5. TKO mice (n= 8) could not be entrained according to the light-dark cycle and exhibited free-running rhythm. The amount of wakefulness, REM sleep, NREM sleep and TST had no significant changes neither 1 h nor 3 h light pulse.ConclusionIntrinsically photosensitive retinal ganglion cells play an important role in light induced sleep of mice. |
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