| Objective:Using drosophila as model organism, the dissertation studies the effect of different light conditions and the intervention of CWJ on the sleep duration and sleep-wake rhythm of drosophila.Methods:1. Study the effect of different light conditions on the sleep duration and sleep-wake rhythm of2to7-day-old wild-type Canton-S strain of D. melanogaster. Drosophilas ranging in days from two to seven are chosen as research objects and are divided into groups sexually with32samples in each. DAMS (Drosophila Activity Monitor System) is made use of during the24hours to record the change of sleep duration and sleep-wake rhythm of drosophila of differen days under different light conditions (12/12h alternating light and dark environment,24h constant light environment,24h constant darkness environment and12/12h light and dark upside down environment) in the same light intensity.2. Study the effect of CWJ on the sleep duration and sleep-wake rhythm of7-day-old wild-type Canton-S strain of D. melanogaster under different light conditions.7-day-old drosophilas are picked out and divided into blank control group, model group and medication administration group with three doses in each. Medication administration group are given concentration of0.25,1and4percent of CWJ, which lasts for four days. DAMS is adopted to explore the dose-effect relationship of CWJ on the sleep duration and sleep-wake rhythm under different light conditions (12/12h alternating light and dark environment,24h constant light environment,24h constant darkness environment and12/12h light and dark upside down environment)after24h continuous monitoring in the same light intensity.3. Study the effect of24h constant darkness environment on the content change of5-HT and DA in7-day-old female wild-type Canton-S strain of D. melanogaster's brain. Choosing7-day-old female drosophila as the object of observation, the dissertation observes the content change of5-HT and DA in drosophila's brain in different time(16:00,19:00,22:00,1:00,4:00and7:00) in12/12h alternating light and dark and24h constant darkness environment and investigates its effect.4. Study the effect of CWJ on the content of5-HT and DA in7-day-old female wild-type Canton-S strain of D. melanogaster's brain.7-day-old female drosophilas are chosen to investigate the CWJ intervention on the content change of5-HT and DA in24h constant darkness environment from19:00on..5. Study the effect of24h constant darkness environment and CWJ intervention on the7-day-old female wild-type Canton-S strain of D. melanogaster's sleep behavior related gene expression. With the support of RT-PCR technique,7-day-old female drosophilas are chosen to explore the impact of24h constant darkness environment and CWJ intervention on their sleep behavior related gene expression from19:00on.6. Study the effect of CWJ on the sleep duration and sleep-wake rhythm of7-old-day female FBst0025859strain cry gene knockout drosophilas in24h constant darkness environment. With the7-old-day cry gene knockout drosophila and Canton S Drosophila as the object of observation, the changes of sleep duration and sleep-wake rhythm of7-old-day cry gene knockout drosophilas are recorded by DAMS after the intervention of CWJ in12/12h light and dark environment.Consequence:1. The effect of different light conditions on the sleep duration and sleep-wake rhythm of2to7-day-old drosophila.1.1Compared with the drosophilas of the same day and same sex in12/12h alternating light and dark environment.①There was a remarkably significant difference to male and female drosophilas that is2to7days old in12/12h light and dark upside down environment. Their sleep duration during the day is extended (**p<0.01) while night sleep time is shortened.(**p<0.01)But there was no significant difference in24h total sleep time.(p>0.05)②There is a remarkably significant difference to female2-7-day-old drosophila,whose sleep time during the day is extended(**p<0.01)while night sleep time is shortened(**p<0.01)in24h constant light environment. There is a difference (*p<0.05)to2-day-old female drosophila whose24h total sleep time prolongs.(*p<0.05),while there is a remarkably significant difference to4-6-day-old drosophila whose24hours total sleep time is shortened.(**p<0.01) To male2-7-day-old drosophila there is no marked difference in its sleep time during the day,but its sleep time at night is shortened,which differs greatly.(**p<0.01) There is difference to2-3-day-old drosophila whose24h total sleep time is shortened (*p<0.05) while there is remarkably significant difference to5-7-day-old whose total sleep time is also shortened.(**p<0.01)③In24h constant darkness environment female2-5-day-old drosophila's sleep time during the day prolongs(**p<0.01),while its sleep time at night is shortened,which differs significantly.(*p<0.05) There is a difference to2-3-day-old drosophila whose total sleep time is extended (*p<0.05)while it differ remarkably to4-day-old drosophila whose total sleep time is extended (**p<0.01).There is no difference to5-70day-old drosophila's total sleep time(p>0.05).To male2-3-day-old drosophila there is a remarkably significant difference whose sleep time during the day is extended(*p<0.05,**p<0.01),while its sleep time at night is shortened,which differs significantly(**p<0.01).Male4-7-day-old drosophila's sleep time during the day is shortened (*p<0.05,**p<0.01),which differs greatly；but its sleep time at night is no significant difference(p>0.05).Male6-7-day-old drosophila's24h total sleep time is shortened,which differs greatly (*p<0.05,**p<0.01)1.2Compared with the previous-day-old drosophila of the same sex in the same light condition:①There is no difference to2-7-day-old female drosophila in its total sleep time in12/12h alternating light and dark environment. But there is a significant difference to male3-4-day-old drosophila whose24h total sleep time is shortened while6-day-old drosophila's total sleep time is extended.②In12/12h light and dark upside down environment, there is no difference to female2-7-day-old drosophila in its total sleep time. But it differs greatly to male3-day-old drosophila because its total sleep time is shortened while there is no difference to drosophila of other days in its total sleep time.③Female3-4-day-old drosophila's24h total sleep time is shortened in24h constant light environment, which makes difference (#p<0.05,##p<0.01), while7-day-old drosophila's24h total sleep time is extended, which makes greater difference.(#p<0.05,##p<0.01,##p<0.01). There is a significant difference to male3-day-old drosophila whose24h total sleep time is shortened (#p<0.05) while7-day-old drosophila's24h total sleep time is extended (#p<0.05)④It differs greatly when the male and female3-4-day-old drosophila's24h total sleep time is shortened in24h constant darkness environment (#p<0.05,##p<0.01),but there is no different to5-7-day-old drosophila.1.3The male and female drosophila's sleep-wake rhythm shows cyclical changes in12/12h alternating light and dark environment. That is they move about between6:00-14:00and17:00-20:00and sleep between14:00and17:00,20:00and6:00the next day. And they move most frequently (Peak Activity) at about7:00and19:00.Compared with the drosophila in12/12h alternating light and dark environment:①Compared with sample group, the sleep-wake rhythm of male and female drosophila in12/12h light and dark upside down environment shows an opposite cyclical change. They sleep between8:00and19:00,21:00and24:00and fly between19:00and21:00,24:00and8:00the next day. But there is same peak activity at7:00and19:00.②Compared with sample group, the cyclical changes of male and female drosophila's sleep-wake rhythm in24h constant light environment almost disappear. Sleep and activity alternates chaotically between7:00and7:00the next day. With the increase of age, the number of activity also increases. At the same time the peak activity at7:00and19:00disappears.③Compared with sample group, the sleep-wake rhythm of male and female drosophila in24h constant darkness environment shows similar cyclical changes. They fly between4:00and18:00and sleep between18:00and4:00the next day. Similarly there is peak activity at7o'clock, but it disappears at19:00.Meantime, drosophila's sleep-wake rhythm in the above metioned three light environment begin to show on the four-day-old drosophila and become stable on the7-day-old drosophila.2. The effect of CWJ on the7-day-old drosophila's sleep time and sleep-wake rhythm under different light conditions.2.1Compared with the7-day-old drosophila of the same sex under the same light conditions:①There is no difference to the male and female drosophila's sleep time in12/12h light and dark upside down environment after0.25,1and4percent of CWJ is given to drosophila.②There is a significnat difference to drosophilas who are given differnet concentrations of CWJ in24h constant light conditions.1and4pecent of CWJ will shorten female drosophila's24h total sleep time (*p<0.05);1percent of CWJ will shorten female drosophila's sleep time at night (*p<0.05);4percent of CWJ will shorten female drosophila's sleep time both during the day and at night (*p<0.05);4percent of CWJ will shorten male drosophila's24h total sleep time (*p<0.05)③There is a remarkably significnat difference to drosophilas who are given differnet concentrations of CWJ in24h constant darkness conditions. 0.25,1and4percent of CWJ will largely reduce female drosophila's24h total sleep time, sleep time during the day and night sleep time (**p<0.01).1percent of CWJ will reduce male drosophila's24h total sleep time and night sleep time;4percent of CWJ will largely reduce male drosophila's24h total sleep time and night sleep time.2.2CWJ will partly increase the number of male and female drosophila's activity within24hours in12/12h light and dark upside down environment through the analysis of drosophila's sleep-wake activity and sleep-wake chart, but it has no significant effect on the rhythm of drosophila's sleep-wake activity and sleep-wake. CWJ can increase the number and the time of male and female drosophila's activity within24hours in24h constant light environment, reduce the fragments of drosophila's sleep and affect the sleep time, especially when4percent of CWJ is given. However, it will not influence the rhythm of drosophila's activity. To the drosophila in24h constant darkness environment,0.25percent of CWJ has such effect and1percent of CWJ affect male drosophila greatly.3. The effect of24h constant darkness environment on the content of5-HT and DA in7-day-old female drosophila's brain. With the deepening of sleep, the content of5-HT and DA in7-day-old female drosophila's brain first decreases, then increases and decreases again in12/12h alternating light and dark environment. While the content of5-HT and DA in female drosophila's brain firstly increases and then decreases in24h constant darkness environment, and the content of5-HT decreases significantly at16:00in12/12h alternating light and dark environment (*p<0.05). The content of DA increases gradually and the content of DA in female drosophila's brain at16:00,19:00,22:00greatly decreases compared with it in the12/12h alternating light and dark environment at the same time (*p<0.05). However, the gap between them shortens gradually. After1o'clock, the content of female drosophila's DA in24h constant darkness environment increases more than it in12/12h alternating light and dark environment (*p<0.05) 4. The effect of CWJ on the content of7-day-old female drosophila's5-HT and DA in24h constant darness environment.0.25percent of CWJ is given to female drosophila for4days at19:00in24h constant darkness environment. It makes no difference to the content of female drosophila's5-HT, but it can greatly increase the content of DA (*p<0.05)5. The effect of24h constant darkness environment and the intervention of CWJ on the7-day-old female drosophila's sleep behaviour related gene expression. In contrast with of7-day-old female drosophila's gene expression quantity at19:00in12/12h alternating light and dark environment, the gene per of7-day-old female drosophila at19:00in24h constant darkness environment is7.32times, tim is3.99times, Hsc70-3is4times and Obp99a is3.81times, and Sug is16times. After given0.25percent of CWJ for4days, the gene expreesion quantity of both groups both decrease to varying degrees, such as per decreases2.2times, tim decreases0.29times, Hsc70-3decreases0.35times, Obp99a is0.96times, and Sug is0.54times.6. The effect of CWJ on the7-day-old female cry gene knockout drosophilas's sleep time and sleep-wake rhythm in12/12h alternating light and dark environment:①Compared with the7-day-old CS female drosophila in24h constant darkness environment, the7-day-old cry knockout female drosophila's sleep time in12/12h alternating light and dark environment is longer. Compared with the7-day-old CS female drosophila in12/12h alternating light and dark environment, its sleep time is longer (▲▲p<0.01). It makes great difference when0.25percent of CWJ was given to7-day-old cry knockout female drosophila as a result of the reduction of its sleep time (**p<0.01)②There is a cyclical sleep-wake rhythm change of7-day-old CS female drosophila in12/12h alternating light and dark environment. The changes are just between the12/12h alternating light and dark environment and24h constant darkness environment of7-day-old CS female drosophila. cry knockout female drosophila's sleep and activity alternates chaotically between4:00and19:00. They usually sleep from19:00to4:00the next day and peak activity appears at about7:00and19:00.③The frequence and time of7-day-old cry knockout female drosophila's activity in12/12h alternating light and dark environment increases after they are given0.25percent of CWJ. At the same time, fragmental sleep also decreases and sleep-wake rhythm partly resumes to that of7-day-old CS female drosophila in12/12h alternating light and dark environment.Conclusion:1. Different light conditions have different effect on the drosophila's sleep duration and sleep-wake rhythm in the same light intensity.2. The best dose of CWJ to improve the7-day-old CS female drosophila's sleep duration and sleep-wake rhythm in24h constant darkness environment is the concentration of0.25percent of CWJ (lasting for4days) and the concentration of1percent of CWJ (lasting for4days). But it is not obvious for CWJ to improve its sleep duration and sleep-wake rhythm in12/12h alternating light and dark environment and24h constant light environment.3. The content of7-day-old CS female drosophila's5-HT and DA will change as time changes in12/12h alternating light and dark environment and24h constant light environment.4.5-HT, DA and the change of gene per, tim, Obp99a in drosophila may have some relationship with the mechanism of CWJ's improvement of sleep-wake rhythm.5. CWJ can recover in part the sleep-wake rhythm of7-day-old cry knockout female drosophila in12/12h alternating light and dark environment. |
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