Díez-García Javier, Matsushita Shinichi, Mutoh Hiroki, Nakai Junichi, Ohkura Masamichi, Yokoyama Jennifer, Dimitrov Dimitar, Knöpfel Thomas
Laboratory for Neuronal Circuit Dynamics, RIKEN Brain Science Institute, 2-1 Hirosawa, Saitama 351-0198, Japan.
Eur J Neurosci. 2005 Aug;22(3):627-35. doi: 10.1111/j.1460-9568.2005.04250.x.
Genetically encoded fluorescent Ca2+ indicator proteins (FCIPs) are promising tools to study Ca2+ signaling in large assemblies of nerve cells. Currently, there are few examples of stable transgenic mouse lines that functionally express such sensors in well-defined neuronal cell populations. Here we report the generation and characterization of transgenic mice expressing an FCIP under the 5' regulatory sequences of the Kv3.1 potassium channel promoter. In the cerebellar cortex, expression was restricted to granule cells. We first demonstrated reliable measurements of Ca2+ transients from beams of parallel fibers and compared the FCIP signals with intrinsic autofluorescence signals. We demonstrate that, in a transgenic line that exhibits a high expression level of the FCIP, autofluorescence signals are negligible and stimulation-induced fluorescence transients represent FCIP signals. Using frontal cerebellar slices we imaged antidromic activation of granule cells following electrical stimulation of parallel fibers and orthodromic activation of beams of parallel fibers following electrical stimulation of granule cells. We found that paired pulse-induced presynaptic Ca2+ transients of parallel fibers are not affected by blockade of N-methyl-D-aspartate receptors.
基因编码的荧光钙指示剂蛋白（FCIPs）是研究神经细胞大集合中钙信号传导的有前途的工具。目前，在明确定义的神经元细胞群体中功能性表达此类传感器的稳定转基因小鼠品系的例子很少。在这里，我们报告了在Kv3.1钾通道启动子的5'调控序列下表达FCIP的转基因小鼠的产生和表征。在小脑皮质中，表达仅限于颗粒细胞。我们首先证明了从平行纤维束可靠测量钙瞬变，并将FCIP信号与固有自发荧光信号进行了比较。我们证明，在一个表现出FCIP高表达水平的转基因系中，自发荧光信号可以忽略不计，刺激诱导的荧光瞬变代表FCIP信号。使用额叶小脑切片，我们对平行纤维电刺激后颗粒细胞的逆向激活以及颗粒细胞电刺激后平行纤维束的顺向激活进行了成像。我们发现，平行纤维的配对脉冲诱导的突触前钙瞬变不受N-甲基-D-天冬氨酸受体阻断的影响。
