Imaging of presynaptic activity in cultured cerebellar granule neurons

为了了解小脑神经元回路，测量动物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，我利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，而激发光有时会损伤细胞或使组织和细胞产生自身荧光。因此，采用发光技术可以获得更好的信噪比。我用的是对pH值敏感的高斯荧光素酶。我进行了随机突变来获得明亮的发光。为了可视化突触前活性，我们使用了VAMP2突触前蛋白。结果，我获得了突变体Gluc (mGluc)，其亮度是野生型Gluc的10倍左右。转染VAMP2 mGluc的神经元在去极化后表现出强烈的发光，表明该VAMP2-mGluc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.