Higashijima Shin-ichi, Masino Mark A, Mandel Gail, Fetcho Joseph R
Department of Neurobiology and Behavior, State University of New York, Stony Brook, New York 11794-5230, USA.
J Neurophysiol. 2003 Dec;90(6):3986-97. doi: 10.1152/jn.00576.2003. Epub 2003 Aug 20.
Genetically encoded calcium indicators, such as cameleon, have offered the promise of noninvasively monitoring activity of neurons, but no one has demonstrated whether these indicators can report calcium transients in neurons of behaving vertebrates. We show that cameleon can be expressed at high levels in sensory and spinal cord neurons in zebrafish by using neural-specific promoters in both transient expression experiments and in a stable transgenic line. Using standard confocal microscopy, calcium transients in identified motoneurons and spinal interneurons could be detected during escape behaviors produced by a touch on the head of the fish. Small movements of the restrained fish during the behavior did not represent a major problem for analyzing the calcium responses because of the ratiometric nature of cameleon. We conclude that cameleon can be used to noninvasively study the activity of neurons in an intact, behaving vertebrate. The ability to introduce an indicator genetically allows for studies of the functional roles of local interneurons that cannot easily be monitored with other approaches. Transgenic lines such as the one we generated can also be crossed into mutant lines of fish to study both structural and functional consequences of the mutations.
基因编码钙指示剂，如变色龙蛋白，为无创监测神经元活动带来了希望，但此前无人证明这些指示剂能否报告行为中的脊椎动物神经元的钙瞬变。我们发现，通过在瞬时表达实验和稳定转基因品系中使用神经特异性启动子，变色龙蛋白可在斑马鱼的感觉神经元和脊髓神经元中高水平表达。利用标准共聚焦显微镜，在触碰鱼头部引发的逃避行为期间，可检测到已识别的运动神经元和脊髓中间神经元中的钙瞬变。由于变色龙蛋白的比率特性，行为期间受约束鱼的微小移动对分析钙反应而言并非主要问题。我们得出结论，变色龙蛋白可用于无创研究完整行为中的脊椎动物的神经元活动。通过基因方式引入指示剂的能力使得对难以用其他方法监测的局部中间神经元的功能作用进行研究成为可能。我们所构建的这类转基因品系还可与鱼类突变体系杂交，以研究突变的结构和功能后果。
