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The Photoconvertible Fluorescent Probe, CaMPARI, Labels Active Neurons in Freely-Moving Intact Adult Fruit Flies.
Frontiers in Neural Circuits ( IF 3.5 ) Pub Date : 2020-04-14 , DOI: 10.3389/fncir.2020.00022
Katie A Edwards 1 , Michael B Hoppa 2 , Giovanni Bosco 1
Affiliation  

Linking neural circuitry to behavior by mapping active neurons in vivo is a challenge. Both genetically encoded calcium indicators (GECIs) and intermediate early genes (IEGs) have been used to pinpoint active neurons during a stimulus or behavior but have drawbacks such as limiting the movement of the organism, requiring a priori knowledge of the active region or having poor temporal resolution. Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) was engineered to overcome these spatial-temporal challenges. CaMPARI is a photoconvertible protein that only converts from green to red fluorescence in the presence of high calcium concentration and 405 nm light. This allows the experimenter to precisely mark active neurons within defined temporal windows. The photoconversion can then be quantified by taking the ratio of the red fluorescence to the green. CaMPARI promises the ability to trace active neurons during a specific stimulus; however, CaMPARI’s uses in adult Drosophila have been limited to photoconversion during fly immobilization. Here, we demonstrate a method that allows photoconversion of multiple freely-moving intact adult flies during a stimulus. Flies were placed in a dish with filter paper wet with acetic acid (pH = 2) or neutralized acetic acid (pH = 7) and exposed to photoconvertible light (60 mW) for 30 min (500 ms on, 200 ms off). Immediately following photoconversion, whole flies were fixed and imaged by confocal microscopy. The red:green ratio was quantified for the DC4 glomerulus, a bundle of neurons expressing Ir64a, an ionotropic receptor that senses acids in the Drosophila antennal lobe. Flies exposed to acetic acid showed 1.3-fold greater photoconversion than flies exposed to neutralized acetic acid. This finding was recapitulated using a more physiological stimulus of apple cider vinegar. These results indicate that CaMPARI can be used to label neurons in intact, freely-moving adult flies and will be useful for identifying the circuitry underlying complex behaviors.



中文翻译:

光转换荧光探针CaMPARI在自由移动的完整成年果蝇中标记活跃的神经元。

通过映射活动神经元将神经回路链接到行为 体内是一个挑战。遗传编码的钙指示剂(GECI)和中间早期基因(IEG)都已被用于在刺激或行为过程中查明活跃的神经元,但存在诸如限制生物体运动等缺点。先验了解活动区域或时间分辨率较差。钙调制的光活化比例积分器(CaMPARI)旨在克服这些时空挑战。CaMPARI是一种可光转换的蛋白质,仅在高钙浓度和405 nm光的存在下才能从绿色荧光转换为红色荧光。这使实验人员可以在定义的时间窗口内精确标记活动神经元。然后可以通过取红色荧光与绿色的比率来量化光转换。CaMPARI有望在特定刺激下追踪活动神经元。但是,CaMPARI在成人中的用途果蝇在飞蝇固定过程中仅限于光转化。在这里,我们演示了一种在刺激过程中允许多个自由移动的完整成年苍蝇进行光转换的方法。将苍蝇放在装有用乙酸(pH = 2)或中和的乙酸(pH = 7)润湿的滤纸的皿中,并暴露于光转换光(60 mW)中30分钟(打开500 ms,关闭200 ms)。光转换后,立即将所有果蝇固定并通过共聚焦显微镜成像。对DC4肾小球(一束表达神经元的神经元)的红绿比率进行了定量Ir64a,是一种离子型受体,可以感应 果蝇触角叶。暴露于乙酸的果蝇的光转化率比暴露于中和乙酸的果蝇高1.3倍。使用苹果醋的更生理性刺激可以概括这一发现。这些结果表明,CaMPARI可用于标记完整的,可自由移动的成年果蝇中的神经元,并将用于识别复杂行为的潜在电路。

更新日期:2020-04-14
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