来源:Koemans, TS 等人 果蝇求偶条件反射作为学习和记忆的衡量标准。J. Vis. Exp.(2017 年)。
该视频介绍了一种经典的条件反射测定,用于测试果蝇的学习和记忆,称为求偶条件反射。该测定基于在被不接受的预配对雌性拒绝后雄性求偶的减少。示例协议显示了可用于评估短期和长期记忆的程序设置。
此协议摘自 Koemans 等人,果蝇求偶条件反射作为学习和记忆的衡量标准,J. Vis. Exp. (2017)。
注意:在下面概述的协议中,描述了收集、训练和测试的一个重复。为了测试结果的可重复性,这些步骤应平行重复,多天,并与不同的果蝇组一起重复(表1)。该方案基于从鸡蛋到成虫的 10 天生命周期,这在 25 °C、70% 湿度和 12 小时光照/黑暗循环的恒定条件下饲养果蝇时是正常的。该方案的所有方面都假设条件在整个测定中保持不变。时间表示为培养箱中灯亮起前 (BLO) 或灯亮起后 (ALO) 的小时数,因为可以根据研究人员喜欢的一天中的时间方便地进行设置。仅使用 CO2 气体进行初始收集幼稚雄性果蝇和收集预先交配的雌性果蝇。该求偶条件反射协议由以下步骤组成:
1. 建立预先交配的雌性收藏培养物
2. 为男性测试对象的集合建立文化
3. 住宅区的准备 (图 2A)
4. 建立用于生产标准化预配母猪的交配瓶
5. 男性测试对象的集合
6. 训练
7. 测试
8. 视频数据分析与统计
<em>P{KK101437}VIE-260B</em> | VDRC | 101437 | Dhat-at-RNAi in 60100 background |
<em>P{KK108109}VIE-260B</em> | – | – | Control-RNAi in 60100 background (gift from K. Keleman) |
<em>w+, UAS-dcr2/yhh;;elav-Gal4 (III)</em> | – | – | panneuronal driver line |
Containers for plant tissue culture | VWR | 960177 | 175 mL plastic vials |
Folded filters | Whatman | 10311643 | Filter paper to enlarge area flies can pupate on |
Flat-bottom blocks (96-wells) | Qiagen | 19579 | Used for housing blocks |
MicroAmp Clear Adhesive Film | Applied Biosystems | 4306311 | PCR adhesive film as lid on flat-bottom blocks |
Razor blade | – | – | Any sharp will do |
Needle | – | – | 0.8 mm diameter |
Aspirator | – | – | Cut a 1mL pipet tip with scissors in order to have two pieces. The narrow tip of the pipettip is placed as fly entrance in a ~80 cm flexible hose. To prevent a fly from getting in the hose, a normal piece of cotton or small mesh gaze is placed in between the tip and the hose. The other half of the pipettip can be used as mouth piece at the end of the hose. |
Courtship chambers | – | – | file S1 can be opened with indicated CAD software |
Camcorder | Sony | – | camera specification: >4M pixels, full HD. |
For manual quantification, any simple video recording device has the potential to produce a video of sufficient quality to observe courtship behavior accurately. For automated quantification, there will likely be different requirements depending on the software to be used, and users should investigate this thoroughly if automated quantification is desired. | |||
<strong>Name</strong> | <strong>Company</strong> | <strong>Catalog Number</strong> | <strong>Comments</strong> |
power food | |||
Agar | Sigma | A7002 | |
Yeast | Bruggeman | – | |
Yeast extract | MP biomedicals | 0210330391 | |
Peptone | Sigma | P6838 | |
Sucrose | Sigma | S9378 | |
Glucose | Sigma | G7021 | |
MgSO4 | Sigma | M2643 | |
CaCl2 | Merck | 1023780500 | |
Methylparabene (CAUTION) | Sigma | H5501 | |
Propionic acid (CAUTION) | Sigma | P1386 | |
Demineralized water | – | ||
Yeast paste | – | yeast grains and water mixture in a 1:1 ratio | |
Name | Company | Catalog Number | Comments |
normal food | |||
Agar | MP biomedicals | 215017890 | |
Yeast | bruggeman | – | |
Corn flour | de Molen | – | |
Sugar | de Molen | – | |
Methylparabene (CAUTION) | Sigma | H5501 | |
Propionic acid (CAUTION) | Sigma | P1386 | |
Demineralized water | – |