Imaging of presynaptic activity in cultured cerebellar granule neurons

为了了解小脑神经元的回路，测量动物在运动或学习时颗粒细胞的突触前活动是很重要的。在我的研究中，我使用荧光素酶开发了突触前活动的报告蛋白。由于荧光素酶的发光是化学反应的产物，它不需要激发光，有时会伤害细胞或从组织和细胞中产生自发荧光。因此，使用发光应该提供更好的信噪比。我用的是对pH敏感的赤霉菌荧光素酶。我进行了随机诱变以获得明亮的发光。用VAMP2突触前蛋白观察突触前活动。结果，我得到了突变体Gluc(MGluc)，它的亮度大约是野生型Gluc的10倍。VAMP2 mGluc转基因神经元在神经元去极化后显示出较强的发光，提示该突触前报告蛋白可用于监测突触前活动。

To understand the cerebellar neuronal circuit, it is important to measure the presynaptic activities of granule cells when animals are moving or learning. In my research, I developed the reporter proteins for presynaptic activity using luciferase. Because luminescence of luciferase is product of chemical reaction, it does not need excitation light, which may sometimes hurt the cells or make autofluorescence from tissue and cell. Therefore using luminescence should give a better signal to noise ratio. I used Gaussia luciferase, which was pH sensitive. I performed the random mutagenesis to obtain bright luminescence. To visualize presynaptic activity, VAMP2 presynaptic protein was used. As a result, I obtained mutant Gluc (mGluc), which showed about 10 times brighter than wild type Gluc. The VAMP2 mGluc transfected neuron showed strong luminescence after neuronal depolarization, indicated this VAMP2-mGluc presynaptic reporter protein can be used to monitor presynaptic activity.