Japan Science and Technology Agency, International Cooperative Research Project and Solution-Oriented Research for Science and Technology, Calcium Oscillation Project, Wako-shi Saitama, Japan.
Front Cell Neurosci. 2011 Sep 29;5:18. doi: 10.3389/fncel.2011.00018. eCollection 2011.
Genetically encoded Ca(2+) indicators (GECIs) are promising tools for cell type-specific and chronic recording of neuronal activity. In the mammalian central nervous system, however, GECIs have been tested almost exclusively in cortical and hippocampal pyramidal cells, and the usefulness of recently developed GECIs has not been systematically examined in other cell types. Here we expressed the latest series of GECIs, yellow cameleon (YC) 2.60, YC3.60, YC-Nano15, and GCaMP3, in mouse cortical pyramidal cells as well as cerebellar Purkinje cells using in utero injection of recombinant adenoviral vectors. We characterized the performance of the GECIs by simultaneous two-photon imaging and whole-cell patch-clamp recording in acute brain slices at 33 ± 2°C. The fluorescent responses of GECIs to action potentials (APs) evoked by somatic current injection or to synaptic stimulation were examined using rapid dendritic imaging. In cortical pyramidal cells, YC2.60 showed the largest responses to single APs, but its decay kinetics were slower than YC3.60 and GCaMP3, while GCaMP3 showed the largest responses to 20 APs evoked at 20 Hz. In cerebellar Purkinje cells, only YC2.60 and YC-Nano15 could reliably report single complex spikes (CSs), and neither showed signal saturation over the entire stimulus range tested (1-10 CSs at 10 Hz). The expression and response of YC2.60 in Purkinje cells remained detectable and comparable for at least over 100 days. These results provide useful information for selecting an optimal GECI depending on the experimental requirements: in cortical pyramidal cells, YC2.60 is suitable for detecting sparse firing of APs, whereas GCaMP3 is suitable for detecting burst firing of APs; in cerebellar Purkinje cells, YC2.60 as well as YC-Nano15 is suitable for detecting CSs.
基因编码钙指示剂 (GECIs) 是一种有前途的工具，可用于细胞类型特异性和慢性神经元活动记录。然而，在哺乳动物中枢神经系统中，GECIs 几乎仅在皮质和海马锥体神经元中进行了测试，并且最近开发的 GECIs 的有用性尚未在其他细胞类型中系统地进行检查。在这里，我们使用重组腺病毒载体的子宫内注射，在小鼠皮质锥体神经元和小脑浦肯野细胞中表达了最新系列的 GECIs，包括黄色钙敏蛋白（YC）2.60、YC3.60、YC-Nano15 和 GCaMP3。我们通过在 33±2°C 的急性脑切片中同时进行双光子成像和全细胞膜片钳记录来表征 GECIs 的性能。我们使用快速树突成像检查 GECIs 对体细胞电流注入或突触刺激诱发的动作电位 (APs) 的荧光反应。在皮质锥体神经元中，YC2.60 对单个 APs 的反应最大，但衰减动力学比 YC3.60 和 GCaMP3 慢，而 GCaMP3 对 20Hz 下 20 个 APs 的刺激反应最大。在小脑浦肯野细胞中，只有 YC2.60 和 YC-Nano15 能够可靠地报告单个复杂峰 (CSs)，并且在测试的整个刺激范围内都没有信号饱和（1-10Hz 下 1-10 个 CSs）。在浦肯野细胞中，YC2.60 的表达和反应至少在 100 天以上仍可检测且具有可比性。这些结果为根据实验要求选择最佳 GECI 提供了有用的信息：在皮质锥体神经元中，YC2.60 适合检测 APs 的稀疏发射，而 GCaMP3 适合检测 APs 的爆发发射；在小脑浦肯野细胞中，YC2.60 和 YC-Nano15 都适合检测 CSs。
