運用光學紀錄與光基因轉殖激發觀察果蠅幼蟲中運動神經的信號傳遞

Observe the Signal Propagation of Motor Neuron in Drosophila Larvae with Optical Record and Opto-genetic Stimulation

林恒毅(Hen-Yi Lin)

指導教授 ： 朱士維

國立臺灣大學/理學院/物理研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.00605

摘要

果蠅的腦部結構與人腦十分相似。近年來，非活體的果蠅腦部幾何關聯研究已經逐漸建立起一套完整的架構，促使我們更全面地理解腦部的運作方式。然而，我們尚未藉由直接觀察果蠅腦神經間的信號傳遞，以證明果蠅在活體內的神經連結。既往的作法，是利用顯微電極來記錄細胞膜上的電位變化，以探測神經信號。然而，這種方式具有侵入性，且由於電極的尺寸大小，使得這種方法在空間上有其極限存在。相較之下，以光學影像觀察神經，提供我們一種非侵入的觀測方式，且能在小於微米影像解析度下，同時觀察大量的神經單位—在活體的神經研究中，這種方式更為理想。

實驗中，我們利用鈣離子染劑作為我們的光學指示劑。藉由捕捉鈣離子染劑在神經上的螢光變化，神經上的動作電位動態即可被觀察到。在初步的神經信號展示中，我們選了果蠅幼蟲的運動神經(MN)作為觀測的目標，因為它在結構上較為單純且容易擷取。樣本中的運動神經有接受綠色螢光蛋白(GFP)的基因轉殖，方便我們判斷這些神經在幼蟲中的位置。幼蟲的運動神經經染色後，我們使用共軛焦顯微鏡的影像觀察神經上的蛋白螢光及染劑螢光的重疊程度，來判斷神經染色是否成功。接著，我們使用氯化鉀溶液(KCl)或光敏感通道(ChR2)作為激發源來激發不同形式的神經信號。運動神經上的鈣離子染劑以約0.5秒一張的速率擷取，將動作電位的傳遞視覺化。

擁有較低侵入性、更廣的觀察視野、及更高的解析度等優勢，結合光學紀錄及光基因轉殖激發的方法提供我們一個比傳統方式更吸引人的選項。隨著光學及基因轉殖的發展，我們的目標是理解活體果蠅腦內的連結機制。

關鍵字

光敏感通道 ； 鈣離子影像 ； 光學 ； 果蠅幼蟲 ； 神經信號 ； 光學刺激

並列摘要

Drosophila brain is structurally similar to human. In recent years, Drosophila connectome in vitro has been geometrically established, meriting our understanding of brain functioning comprehensively. Nevertheless, the direct functional proof of neural connection should be verified in vivo, through witnessing the signal propagation between two adjacent neurons. To detect neural signal propagation, the conventional method is to use microelectrodes to stimulate and record electrical signals across neural membranes. However, this approach is invasive and spatially limited to one or few selected neurons with resolution limited to the size of electrodes. In contrast, observing the neural signal with optical imaging provides physically noninvasive observation with sub-micrometer spatial resolution on tens to hundreds of neurons simultaneously, which is more ideal for connectome study in vivo.
In our experiment, calcium dye is used as an optical indicator to detect neural signals. By capturing the variation of calcium dye fluorescence on neurons, the dynamics of action potential along neurons can be observed. As a preliminary demonstration of neural signal detection, the motor neuron (MN) of Drosophila larvae is selected as the site of observation, due to its structural simplicity and accessibility. The MN is genetically labeled with green fluorescence protein (GFP) to assist us in identifying its location. The larval MN were stained with calcium dye, and then imaged with a confocal microscope to confirm the spatial overlap of GFP and calcium dye, manifesting that the MN was successfully stained with calcium dye. Subsequently, we apply KCl solution or channelrhodopsin-2 (ChR2) as stimuli to generate neural activities with different patterns. Fluorescence of calcium dye on MN is recorded approximately 0.5 sec. each frame (256X256 pixels), to visualize the propagation of action potential.
Possessed with lower invasion, broader view, and higher resolution, the method combined with optical record and opto-genetic stimulation provides us an attractive alternative in contrast to conventional methods. With further development on optics and trans-genetics, our goal is to characterize neural connectome of Drosophila in vivo.

並列關鍵字

ChR2 ； Calcium imaging ； optics ； Drosophila larvae ； neural imaging ； calcium dye ； voltage dye

參考文獻

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