Berni Jimena, Muldal Alistair M, Pulver Stefan R
Department of Zoology, University of Cambridge, Cambridge, UK CBQ 3EJ.
J Undergrad Neurosci Educ. 2010 Fall;9(1):A5-A14. Epub 2010 Oct 15.
Here we describe a set of straightforward laboratory exercises that integrate the study of genetics, neuroanatomy, cellular physiology and animal behavior. We use genetic tools in Drosophila for visualizing and remotely activating ensembles of neurons with heat pulses. First, we show how to examine the anatomy of several neuronal populations using genetically encoded green fluorescent protein. Next we demonstrate how to use the warmth gated Drosophila TRPA1 (dTRPA1) cation channel to remotely activate neural circuits in flies. To demonstrate the cellular effects of dTRPA1 activation, we expressed dTRPA1 panneurally and recorded excitatory junctional potentials in muscles in response to warmed (29°C) saline. Finally, we present inexpensive techniques for delivering heat pulses to activate dTRPA1 in the neuronal groups we observed previously while flies are freely behaving. We suggest how to film and quantify resulting behavioral phenotypes with limited resources. Activating all neurons with dTRPA1 caused tetanic paralysis in larvae, while in adults it led to paralysis in males and continuous uncoordinated leg and wing movements in females. Activation of cholinergic neurons produced spasms and writhing in larvae while causing paralysis in adults. When a single class of nociceptive sensory neurons was activated, it caused lateral rolling in larvae, but no discernable effects in adults. Overall, these exercises illustrate principles of modern genetics, neuroanatomy, the ionic basis of neuronal excitability, and quantitative methods in neuroethology. Relatively few research studies have used dTRPA1 to activate neural circuits, so these exercises give students opportunities to test novel hypotheses and make actual contributions to the scientific record.
在此，我们描述了一系列简单直接的实验练习，这些练习整合了遗传学、神经解剖学、细胞生理学和动物行为学的研究。我们利用果蝇中的遗传工具，通过热脉冲来可视化并远程激活神经元集群。首先，我们展示如何使用基因编码的绿色荧光蛋白来检查几个神经元群体的解剖结构。接下来，我们演示如何利用温度门控的果蝇瞬时受体电位阳离子通道A1（dTRPA1）远程激活果蝇的神经回路。为了证明dTRPA1激活的细胞效应，我们在全神经元中表达dTRPA1，并记录肌肉中对温热（29°C）生理盐水的兴奋性连接电位。最后，我们介绍了一些低成本技术，用于在果蝇自由活动时向我们之前观察到的神经元群体传递热脉冲以激活dTRPA1。我们还建议如何在资源有限的情况下拍摄并量化由此产生的行为表型。用dTRPA1激活所有神经元会导致幼虫强直性麻痹，而在成虫中，会导致雄性麻痹，雌性则出现腿部和翅膀持续不协调运动。激活胆碱能神经元会使幼虫产生痉挛和扭动，而成虫则会麻痹。当激活一类伤害性感觉神经元时，会导致幼虫侧向翻滚，但对成虫没有明显影响。总体而言，这些练习阐释了现代遗传学、神经解剖学、神经元兴奋性的离子基础以及神经行为学中的定量方法的原理。相对较少的研究使用dTRPA1来激活神经回路，所以这些练习为学生提供了检验新假设并对科学记录做出实际贡献的机会。
