光照条件变化对果蝇睡眠—觉醒节律的影响及刺五加的干预
摘要
目的：
以果蝇为模式生物,探讨光照条件变化和刺五加干预对果蝇睡眠时间和睡眠—觉醒节律的影响。
方法：
1、光照条件变化对2-7日龄野生型CS品系黑腹果蝇睡眠时间和睡眠—觉醒节律的影响：选择2日龄、3日龄、4日龄、5日龄、6日龄、7日龄果蝇作为观察对象,雌雄分组,每组32只。以果蝇睡眠时间和睡眠—觉醒节律为考察指标,利用果蝇活动监测系统(DAMS)进行连续7天24h监测,分别考察不同日龄果蝇在相同光照强度下不同光照环境(12/12h明暗交替环境、24h持续光照环境、24h持续黑暗环境和12/12h明暗颠倒环境)里的睡眠时间和睡眠—觉醒节律的变化；
2、刺五加对光照条件变化所致7日龄野生型CS品系黑腹果蝇睡眠时间和睡眠—觉醒节律的干预作用研究：选择7日龄果蝇作为观察对象,分为空白组、模型组、模型给药组,模型给药组分别给予浓度为0.25%、1%、4%的刺五加,连续给药4天,以果蝇睡眠时间和睡眠—觉醒节律为考察指标,利用果蝇活动监测系统(DAMS)对7日龄果蝇进行24h连续监测,分别考察不同光照环境下(12/12h明暗交替环境、24h持续光照环境、24h持续黑暗环境和12/12h明暗颠倒环境)刺五加对果蝇睡眠时间影响的量效关系以及对果蝇睡眠—觉醒节律的影响；
3、24h持续黑暗环境对7日龄野生型CS品系黑腹雌果蝇不同时间点的脑部5-HT和DA变化的影响：选择7日龄雌果蝇作为观察对象,以5-HT和DA为考察指标,考察12/12h明暗交替和24h持续黑暗环境下,7日龄雌果蝇在不同时间点(16：00、19：00、22：00、1：00、4：00和7：00)脑部5-HT和DA的含量变化；
4、刺五加对24h持续黑暗环境下7日龄野生型CS品系黑腹雌果蝇19：00脑部5-HT和DA含量的影响：选择7日龄雌果蝇作为观察对象,以5-HT和DA为考察指标,考察给予刺五加后7日龄雌果蝇19：00脑部5-HT和DA的含量变化；
5、刺五加干预对24h持续黑暗环境下7日龄野生型CS品系黑腹雌果蝇脑部睡眠相关基因表达的影响：选择7日龄雌果蝇作为观察对象,运用RT-PCR技术,以果蝇脑部per、tim、Obp99a、Sug、Hsc70-3基因为考察指标,考察给予刺五加前后24h持续黑暗环境下7日龄雌果蝇19：00脑部睡眠相关基因相对表达量的变化。
6、刺五加对12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除型雌果蝇睡眠时间和睡眠—觉醒节律的影响：选择7日龄cry基因敲除型雌果蝇和7日龄野生型Canton S品系黑腹雌果蝇作为观察对象,利用果蝇活动监测系统(DAMS),考察cry基因敲除型雌果蝇在12/12h明暗交替环境和给予刺五加后其睡眠时间和睡眠—觉醒节律的变化。
结果：
1、光照条件变化对2-7日龄果蝇睡眠时间和睡眠—觉醒节律的影响：
1.1与处于12/12h明暗交替环境下同日龄同性别果蝇比较：
①处于12/12h明暗颠倒环境的雌雄果蝇,2-7日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),24h总睡眠时间均无显著差异(p>0.05)。
②处于24h持续光照环境下,雌果蝇2-7日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),2日龄24h总睡眠时间延长、有显著性差异(*p<0.05),4-6日龄24h总睡眠时间均缩短、有极显著性差异(**P<0.01)；雄果蝇2-7日龄白天睡眠时间均无显著差异(p>0.05),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),2、3日龄24h总睡眠时间均缩短、有显著性差异(*p<0.05),5-7日龄24h总睡眠时间均缩短、有极显著性差异(**p<0.01)。
③处于24h持续黑暗环境下,雌果蝇2-5日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有显著性差异(*p<0.05),2、3日龄24h总睡眠时间均延长、有显著性差异(*p<0.05),4日龄24h总睡眠时间延长、有极显著性差异(**p<0.01),5-7日龄24h总睡眠时间均无著性差异(p>0.05)；雄果蝇2、3日龄表现为白天睡眠时间延长,具有极显著差异(*p<0.05,**p<0.01),夜晚睡眠时间缩短,具有极显著差异(**p<0.01),4-7日龄白天睡眠时间缩短具有极显著差异(*p<0.05,**p<0.01),夜晚睡眠时间无显著性差异(p>0.05),6、7日龄24h总睡眠时间缩短,具有显著性差异(*p<0.05,**p<0.01)。
1.2与处于相同光照环境下同性别果蝇的前一日龄组比较：
①处于12/12h明暗交替环境下,雌果蝇2-7日龄24h总睡眠时间均无显著性差异(p>0.05)；雄果蝇3、4日龄24h总睡眠时间均缩短、有显著性差异((?)p<0.05),6日龄24h总睡眠时间延长有显著性差异((?)p<0.05)。
②处于12/12h明暗颠倒环境下,雌果蝇2-7日龄24h总睡眠时间均无显著性差异(p>0.05)；雄果蝇3日龄24h总睡眠时间缩短、有极显著性差异(##p<0.01),其余日龄24h总睡眠时间均无显著性差异(p>0.05)。
③处于24h持续光照环境下,雌果蝇3、4日龄24h总睡眠时间均缩短,有显著性差异(#p<0.05,##p<0.01),7日龄24h总睡眠时间延长,有极显著性差异(##p<0.01)；雄果蝇3日龄24h总睡眠时间缩短,有显著性差异(#p<0.05),7日龄24h总睡眠时间延长,有显著性差异(#p<0.05)。
④处于24h持续黑暗环境下的雌雄果蝇3、4日龄24h总睡眠时间均缩短,有显著性差异(#p<0.05,##p<0.01),5-7日龄24h总睡眠时间无著性差异(p>0.05)。
1.3处于12/12h明暗交替环境下雌雄果蝇的睡眠—觉醒节律均呈周期性变化,即6：00—14：00和17：00—20：00活动明显增加,14：00—17：00和20：00—次日6：00睡眠明显增加,在7：00和19：00前后均出现活动高峰。与处于12/12h明暗交替环境下的果蝇比较：
①处于12/12h明暗颠倒环境下雌雄果蝇的睡眠—觉醒节律均呈现与比较组相反的周期性变化,即8：00—19：00和21：00—24：00睡眠明显增加,19：00—21：00和24：00—次日8：00活动明显增加,但在7：00和19：00前后同样出现活动高峰；
②处于24h持续光照环境下雌雄果蝇的睡眠—觉醒节律周期性基本消失,即7：00—次日7：00睡眠和活动交替且杂乱分布,随日龄的增加活动次数逐渐增加,同时7：00和19：00前后的活动高峰消失；
③处于24h持续黑暗环境下雌雄果蝇的睡眠—觉醒节律呈周期性变化,其变化与比较组的周期变化类似,在4：00—18：00活动明显增加,18：00—次日4：00睡眠明显增加,同样在7：00前后出现活动高峰,但19：00前后的活动高峰消失。
同时,处于以上三种光照环境下的果蝇的睡眠—觉醒节律均从4日龄开始显现,7日龄趋于稳定。
2、刺五加对光照条件变化所致7日龄果蝇睡眠时间和睡眠—觉醒节律的干预作用研究：
2.1与同一光照环境下7日龄同性别果蝇比较：
①0.25%、1%、4%的刺五加给药4天后,处于12/12h明暗颠倒环境的雌雄果蝇睡眠时间均无显著性差异(p>0.05)。
②给予24h持续光照环境下果蝇不同浓度刺五加后,1%、4%的刺五加均能缩短雌果蝇24h总睡眠时间、有显著性差异(*p<0.05),1%的刺五加能缩短夜晚雌果蝇睡眠时间、有显著性差异(*p<0.05),4%的刺五加均能缩短雌果蝇白天和夜晚睡眠时间、有显著性差异(*p<0.05)；4%的刺五加能缩短雄果蝇24h总睡眠时间、有显著性差异(*p<0.05)；
③给予24h持续黑暗环境下果蝇不同浓度刺五加后,0.25%、1%、4%的刺五加均能减少雌果蝇24h总睡眠时间、白天睡眠时间和夜晚睡眠时间、均有极显著性差异(**p<0.01),1%的刺五加均可缩短雄果蝇24h总睡眠时间和夜晚睡眠时间、有显著性差异(*p<0.05),4%的刺五加均可缩短雄果蝇24h总睡眠时间和夜晚睡眠时间、有极显著性差异(**p<0.01)。
2.2经分析果蝇昼夜活动节律图与睡眠-觉醒图知,刺五加能部分增加12/12h明暗颠倒环境下雌雄果蝇的24h活动次数,但对活动节律和睡眠-觉醒节律影响不明显。刺五加能增加24h持续光照环境下雌雄果蝇24h活动次数和连续活动时间,减少果蝇片段化睡眠,影响睡眠时间,浓度为4%的刺五加时作用明显,但此浓度药对果蝇的睡眠-觉醒节律影响不明显。对于处于24h持续黑暗环境下的果蝇,刺五加通过增加雌果蝇白天活动次数和连续活动时间,减少其白天片段化睡眠,影响其睡眠-觉醒节律,其中0.25%的刺五加对此作用明显；刺五加通过增加雄果蝇24h活动次数和连续活动时间,减少其片段化睡眠,影响果蝇的睡眠-觉醒节律,1%的刺五加对此作用明显。
3、24h持续黑暗环境对不同时间点的7日龄雌果蝇脑部5-HT和DA变化的影响：随着睡眠的深入,处于12/12h明暗交替环境下雌果蝇脑部5-HT和DA含量均呈现先降低、后升高、再降低的趋势；处于24h持续黑暗环境下的雌果蝇脑部5-HT含量呈现先升高,后降低的趋势,且在16：005-HT含量较处于同时间点12/12h明暗交替环境下的雌果蝇脑部5-HT含量降低,有显著性差异(*P<0.05)；DA含量呈现逐渐升高的趋势,16：00、19：00、22：00的雌果蝇脑部DA含量比同时间点的处于12/12h明暗交替环境下的雌果蝇脑部DA含量降低,有显著性差异(*p<0.05),但二者间的含量差距随时间变化逐渐降低,1：00时二者含量相比较,无显著性差异(p>0.05),4：00、7：00时处于24h持续黑暗环境下雌果蝇脑部DA含量逐渐比处于12/12h明暗交替环境下雌果蝇脑部DA含量增高,有显著性差异(*p<0.05)。
4、刺五加对24h持续黑暗环境下7日龄雌果蝇19：00脑部5-HT和DA含量的影响：0.25%的刺五加给药4天对处于24h持续黑暗环境下7日龄雌果蝇19：00脑部5-HT含量的增加无显著性影响(p>0.05),但能显著增加DA的含量(*P<0.05)。
5、刺五加干预对24h持续黑暗环境下7日龄雌果蝇脑部睡眠相关基因表达的影响：与处于12/12h明暗交替环境下7日龄雌果蝇19：00脑部各基因的表达量比较,处于24h持续黑暗环境7日龄雌果蝇19：00脑部基因per是其7.32倍,tim是其3.99倍,Hsc70-3是其4倍,Obp99a是其3.81倍,Sug是其16倍。给予0.25%的刺五加4天后,24h持续黑暗环境下7日龄雌果蝇在19：00脑部各基因相对表达量较处于12/12h明暗交替环境7日龄雌果蝇19：00脑部各基因相对表达量均出现不同程度的降低,per降至2.2倍,tim降至0.29倍,Hsc70-3降至0.35倍,Obp99a降至0.96倍,Sug降至0.54倍。
6、刺五加对12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除型雌果蝇睡眠时间和睡眠—觉醒节律的影响：
①处于12/12h明暗交替环境下7日龄cry雌果蝇,与处于24h持续黑暗环境下7日龄CS雌果蝇比较,白天睡眠时间延长,有显著性差异(**p<0.01)；与处于12/12h明暗交替环境下7日龄CS雌果蝇相比较,白天睡眠时间延长,有显著性差异((?)p<0.01)；0.25%刺五加给药4天后,7日龄cry基因敲除型雌果蝇白天睡眠时间减少,有显著性差异(**p<0.01)。
②处于12/12h明暗交替环境下的7日龄cry基因敲除型雌果蝇的睡眠—觉醒节律呈周期性变化,其变化情况在处于12/12h明暗交替环境和处于24h持续黑暗环境下的7日龄CS雌果蝇的睡眠—觉醒节律之间,表现为4：00—19：00活动与睡眠交替进行,19：00—次日4：00睡眠明显增加,且7：00和19：00前后均出现活动高峰。
③给予0.25%刺五加4天后,处于12/12h明暗交替环境下7日龄cry基因敲除型雌果蝇的白天活动次数和连续活动时间增加,片段化睡眠减少,睡眠—觉醒节律部分恢复到12/12h明暗交替环境下7日龄cs雌果蝇的睡眠—觉醒节律。
结论：
1、同等光照强度下不同光照环境对果蝇的睡眠时间和睡眠—觉醒节律影响不同。
2、刺五加改善24h持续黑暗环境下7日龄CS雌雄果蝇睡眠时间和睡眠—觉醒节律的最佳给药剂量分别为0.25%刺五加给药4天和1%刺五加给药4天。但对12/12h明暗颠倒环境和24h持续光照环境下7日龄果蝇,刺五加改善其睡眠时间和睡眠—觉醒节律作用不明显。
3、处于12/12h明暗交替环境和24h持续黑暗环境下7日龄CS品系雌果蝇脑部5-HT和DA含量随时间的变化而变化。
4、刺五加改善果蝇睡眠—觉醒节律的机制可能与果蝇脑部神经递质5-HT和DA以及per、tim、Obp99a等基因变化有关。
5、刺五加能部分恢复12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除雌果蝇的睡眠—觉醒节律。
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.
以果蝇为模式生物,探讨光照条件变化和刺五加干预对果蝇睡眠时间和睡眠—觉醒节律的影响。
方法：
1、光照条件变化对2-7日龄野生型CS品系黑腹果蝇睡眠时间和睡眠—觉醒节律的影响：选择2日龄、3日龄、4日龄、5日龄、6日龄、7日龄果蝇作为观察对象,雌雄分组,每组32只。以果蝇睡眠时间和睡眠—觉醒节律为考察指标,利用果蝇活动监测系统(DAMS)进行连续7天24h监测,分别考察不同日龄果蝇在相同光照强度下不同光照环境(12/12h明暗交替环境、24h持续光照环境、24h持续黑暗环境和12/12h明暗颠倒环境)里的睡眠时间和睡眠—觉醒节律的变化；
2、刺五加对光照条件变化所致7日龄野生型CS品系黑腹果蝇睡眠时间和睡眠—觉醒节律的干预作用研究：选择7日龄果蝇作为观察对象,分为空白组、模型组、模型给药组,模型给药组分别给予浓度为0.25%、1%、4%的刺五加,连续给药4天,以果蝇睡眠时间和睡眠—觉醒节律为考察指标,利用果蝇活动监测系统(DAMS)对7日龄果蝇进行24h连续监测,分别考察不同光照环境下(12/12h明暗交替环境、24h持续光照环境、24h持续黑暗环境和12/12h明暗颠倒环境)刺五加对果蝇睡眠时间影响的量效关系以及对果蝇睡眠—觉醒节律的影响；
3、24h持续黑暗环境对7日龄野生型CS品系黑腹雌果蝇不同时间点的脑部5-HT和DA变化的影响：选择7日龄雌果蝇作为观察对象,以5-HT和DA为考察指标,考察12/12h明暗交替和24h持续黑暗环境下,7日龄雌果蝇在不同时间点(16：00、19：00、22：00、1：00、4：00和7：00)脑部5-HT和DA的含量变化；
4、刺五加对24h持续黑暗环境下7日龄野生型CS品系黑腹雌果蝇19：00脑部5-HT和DA含量的影响：选择7日龄雌果蝇作为观察对象,以5-HT和DA为考察指标,考察给予刺五加后7日龄雌果蝇19：00脑部5-HT和DA的含量变化；
5、刺五加干预对24h持续黑暗环境下7日龄野生型CS品系黑腹雌果蝇脑部睡眠相关基因表达的影响：选择7日龄雌果蝇作为观察对象,运用RT-PCR技术,以果蝇脑部per、tim、Obp99a、Sug、Hsc70-3基因为考察指标,考察给予刺五加前后24h持续黑暗环境下7日龄雌果蝇19：00脑部睡眠相关基因相对表达量的变化。
6、刺五加对12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除型雌果蝇睡眠时间和睡眠—觉醒节律的影响：选择7日龄cry基因敲除型雌果蝇和7日龄野生型Canton S品系黑腹雌果蝇作为观察对象,利用果蝇活动监测系统(DAMS),考察cry基因敲除型雌果蝇在12/12h明暗交替环境和给予刺五加后其睡眠时间和睡眠—觉醒节律的变化。
结果：
1、光照条件变化对2-7日龄果蝇睡眠时间和睡眠—觉醒节律的影响：
1.1与处于12/12h明暗交替环境下同日龄同性别果蝇比较：
①处于12/12h明暗颠倒环境的雌雄果蝇,2-7日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),24h总睡眠时间均无显著差异(p>0.05)。
②处于24h持续光照环境下,雌果蝇2-7日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),2日龄24h总睡眠时间延长、有显著性差异(*p<0.05),4-6日龄24h总睡眠时间均缩短、有极显著性差异(**P<0.01)；雄果蝇2-7日龄白天睡眠时间均无显著差异(p>0.05),夜晚睡眠时间均缩短、有极显著性差异(**p<0.01),2、3日龄24h总睡眠时间均缩短、有显著性差异(*p<0.05),5-7日龄24h总睡眠时间均缩短、有极显著性差异(**p<0.01)。
③处于24h持续黑暗环境下,雌果蝇2-5日龄白天睡眠时间均延长、有极显著性差异(**p<0.01),夜晚睡眠时间均缩短、有显著性差异(*p<0.05),2、3日龄24h总睡眠时间均延长、有显著性差异(*p<0.05),4日龄24h总睡眠时间延长、有极显著性差异(**p<0.01),5-7日龄24h总睡眠时间均无著性差异(p>0.05)；雄果蝇2、3日龄表现为白天睡眠时间延长,具有极显著差异(*p<0.05,**p<0.01),夜晚睡眠时间缩短,具有极显著差异(**p<0.01),4-7日龄白天睡眠时间缩短具有极显著差异(*p<0.05,**p<0.01),夜晚睡眠时间无显著性差异(p>0.05),6、7日龄24h总睡眠时间缩短,具有显著性差异(*p<0.05,**p<0.01)。
1.2与处于相同光照环境下同性别果蝇的前一日龄组比较：
①处于12/12h明暗交替环境下,雌果蝇2-7日龄24h总睡眠时间均无显著性差异(p>0.05)；雄果蝇3、4日龄24h总睡眠时间均缩短、有显著性差异((?)p<0.05),6日龄24h总睡眠时间延长有显著性差异((?)p<0.05)。
②处于12/12h明暗颠倒环境下,雌果蝇2-7日龄24h总睡眠时间均无显著性差异(p>0.05)；雄果蝇3日龄24h总睡眠时间缩短、有极显著性差异(##p<0.01),其余日龄24h总睡眠时间均无显著性差异(p>0.05)。
③处于24h持续光照环境下,雌果蝇3、4日龄24h总睡眠时间均缩短,有显著性差异(#p<0.05,##p<0.01),7日龄24h总睡眠时间延长,有极显著性差异(##p<0.01)；雄果蝇3日龄24h总睡眠时间缩短,有显著性差异(#p<0.05),7日龄24h总睡眠时间延长,有显著性差异(#p<0.05)。
④处于24h持续黑暗环境下的雌雄果蝇3、4日龄24h总睡眠时间均缩短,有显著性差异(#p<0.05,##p<0.01),5-7日龄24h总睡眠时间无著性差异(p>0.05)。
1.3处于12/12h明暗交替环境下雌雄果蝇的睡眠—觉醒节律均呈周期性变化,即6：00—14：00和17：00—20：00活动明显增加,14：00—17：00和20：00—次日6：00睡眠明显增加,在7：00和19：00前后均出现活动高峰。与处于12/12h明暗交替环境下的果蝇比较：
①处于12/12h明暗颠倒环境下雌雄果蝇的睡眠—觉醒节律均呈现与比较组相反的周期性变化,即8：00—19：00和21：00—24：00睡眠明显增加,19：00—21：00和24：00—次日8：00活动明显增加,但在7：00和19：00前后同样出现活动高峰；
②处于24h持续光照环境下雌雄果蝇的睡眠—觉醒节律周期性基本消失,即7：00—次日7：00睡眠和活动交替且杂乱分布,随日龄的增加活动次数逐渐增加,同时7：00和19：00前后的活动高峰消失；
③处于24h持续黑暗环境下雌雄果蝇的睡眠—觉醒节律呈周期性变化,其变化与比较组的周期变化类似,在4：00—18：00活动明显增加,18：00—次日4：00睡眠明显增加,同样在7：00前后出现活动高峰,但19：00前后的活动高峰消失。
同时,处于以上三种光照环境下的果蝇的睡眠—觉醒节律均从4日龄开始显现,7日龄趋于稳定。
2、刺五加对光照条件变化所致7日龄果蝇睡眠时间和睡眠—觉醒节律的干预作用研究：
2.1与同一光照环境下7日龄同性别果蝇比较：
①0.25%、1%、4%的刺五加给药4天后,处于12/12h明暗颠倒环境的雌雄果蝇睡眠时间均无显著性差异(p>0.05)。
②给予24h持续光照环境下果蝇不同浓度刺五加后,1%、4%的刺五加均能缩短雌果蝇24h总睡眠时间、有显著性差异(*p<0.05),1%的刺五加能缩短夜晚雌果蝇睡眠时间、有显著性差异(*p<0.05),4%的刺五加均能缩短雌果蝇白天和夜晚睡眠时间、有显著性差异(*p<0.05)；4%的刺五加能缩短雄果蝇24h总睡眠时间、有显著性差异(*p<0.05)；
③给予24h持续黑暗环境下果蝇不同浓度刺五加后,0.25%、1%、4%的刺五加均能减少雌果蝇24h总睡眠时间、白天睡眠时间和夜晚睡眠时间、均有极显著性差异(**p<0.01),1%的刺五加均可缩短雄果蝇24h总睡眠时间和夜晚睡眠时间、有显著性差异(*p<0.05),4%的刺五加均可缩短雄果蝇24h总睡眠时间和夜晚睡眠时间、有极显著性差异(**p<0.01)。
2.2经分析果蝇昼夜活动节律图与睡眠-觉醒图知,刺五加能部分增加12/12h明暗颠倒环境下雌雄果蝇的24h活动次数,但对活动节律和睡眠-觉醒节律影响不明显。刺五加能增加24h持续光照环境下雌雄果蝇24h活动次数和连续活动时间,减少果蝇片段化睡眠,影响睡眠时间,浓度为4%的刺五加时作用明显,但此浓度药对果蝇的睡眠-觉醒节律影响不明显。对于处于24h持续黑暗环境下的果蝇,刺五加通过增加雌果蝇白天活动次数和连续活动时间,减少其白天片段化睡眠,影响其睡眠-觉醒节律,其中0.25%的刺五加对此作用明显；刺五加通过增加雄果蝇24h活动次数和连续活动时间,减少其片段化睡眠,影响果蝇的睡眠-觉醒节律,1%的刺五加对此作用明显。
3、24h持续黑暗环境对不同时间点的7日龄雌果蝇脑部5-HT和DA变化的影响：随着睡眠的深入,处于12/12h明暗交替环境下雌果蝇脑部5-HT和DA含量均呈现先降低、后升高、再降低的趋势；处于24h持续黑暗环境下的雌果蝇脑部5-HT含量呈现先升高,后降低的趋势,且在16：005-HT含量较处于同时间点12/12h明暗交替环境下的雌果蝇脑部5-HT含量降低,有显著性差异(*P<0.05)；DA含量呈现逐渐升高的趋势,16：00、19：00、22：00的雌果蝇脑部DA含量比同时间点的处于12/12h明暗交替环境下的雌果蝇脑部DA含量降低,有显著性差异(*p<0.05),但二者间的含量差距随时间变化逐渐降低,1：00时二者含量相比较,无显著性差异(p>0.05),4：00、7：00时处于24h持续黑暗环境下雌果蝇脑部DA含量逐渐比处于12/12h明暗交替环境下雌果蝇脑部DA含量增高,有显著性差异(*p<0.05)。
4、刺五加对24h持续黑暗环境下7日龄雌果蝇19：00脑部5-HT和DA含量的影响：0.25%的刺五加给药4天对处于24h持续黑暗环境下7日龄雌果蝇19：00脑部5-HT含量的增加无显著性影响(p>0.05),但能显著增加DA的含量(*P<0.05)。
5、刺五加干预对24h持续黑暗环境下7日龄雌果蝇脑部睡眠相关基因表达的影响：与处于12/12h明暗交替环境下7日龄雌果蝇19：00脑部各基因的表达量比较,处于24h持续黑暗环境7日龄雌果蝇19：00脑部基因per是其7.32倍,tim是其3.99倍,Hsc70-3是其4倍,Obp99a是其3.81倍,Sug是其16倍。给予0.25%的刺五加4天后,24h持续黑暗环境下7日龄雌果蝇在19：00脑部各基因相对表达量较处于12/12h明暗交替环境7日龄雌果蝇19：00脑部各基因相对表达量均出现不同程度的降低,per降至2.2倍,tim降至0.29倍,Hsc70-3降至0.35倍,Obp99a降至0.96倍,Sug降至0.54倍。
6、刺五加对12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除型雌果蝇睡眠时间和睡眠—觉醒节律的影响：
①处于12/12h明暗交替环境下7日龄cry雌果蝇,与处于24h持续黑暗环境下7日龄CS雌果蝇比较,白天睡眠时间延长,有显著性差异(**p<0.01)；与处于12/12h明暗交替环境下7日龄CS雌果蝇相比较,白天睡眠时间延长,有显著性差异((?)p<0.01)；0.25%刺五加给药4天后,7日龄cry基因敲除型雌果蝇白天睡眠时间减少,有显著性差异(**p<0.01)。
②处于12/12h明暗交替环境下的7日龄cry基因敲除型雌果蝇的睡眠—觉醒节律呈周期性变化,其变化情况在处于12/12h明暗交替环境和处于24h持续黑暗环境下的7日龄CS雌果蝇的睡眠—觉醒节律之间,表现为4：00—19：00活动与睡眠交替进行,19：00—次日4：00睡眠明显增加,且7：00和19：00前后均出现活动高峰。
③给予0.25%刺五加4天后,处于12/12h明暗交替环境下7日龄cry基因敲除型雌果蝇的白天活动次数和连续活动时间增加,片段化睡眠减少,睡眠—觉醒节律部分恢复到12/12h明暗交替环境下7日龄cs雌果蝇的睡眠—觉醒节律。
结论：
1、同等光照强度下不同光照环境对果蝇的睡眠时间和睡眠—觉醒节律影响不同。
2、刺五加改善24h持续黑暗环境下7日龄CS雌雄果蝇睡眠时间和睡眠—觉醒节律的最佳给药剂量分别为0.25%刺五加给药4天和1%刺五加给药4天。但对12/12h明暗颠倒环境和24h持续光照环境下7日龄果蝇,刺五加改善其睡眠时间和睡眠—觉醒节律作用不明显。
3、处于12/12h明暗交替环境和24h持续黑暗环境下7日龄CS品系雌果蝇脑部5-HT和DA含量随时间的变化而变化。
4、刺五加改善果蝇睡眠—觉醒节律的机制可能与果蝇脑部神经递质5-HT和DA以及per、tim、Obp99a等基因变化有关。
5、刺五加能部分恢复12/12h明暗交替环境下7日龄FBst0025859品系cry基因敲除雌果蝇的睡眠—觉醒节律。
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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