Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama, Japan.
PLoS One. 2018 Apr 25;13(4):e0194707. doi: 10.1371/journal.pone.0194707. eCollection 2018.
Sensory processing is regulated by the coordinated excitation and inhibition of neurons in neuronal circuits. The analysis of neuronal activities has greatly benefited from the recent development of genetically encoded Ca2+ indicators (GECIs). These molecules change their fluorescence intensities or colours in response to changing levels of Ca2+ and can, therefore, be used to sensitively monitor intracellular Ca2+ concentration, which enables the detection of neuronal excitation, including action potentials. These GECIs were developed to monitor increases in Ca2+ concentration; therefore, neuronal inhibition cannot be sensitively detected by these GECIs. To overcome this difficulty, we hypothesised that an inverse-type of GECI, whose fluorescence intensity increases as Ca2+ levels decrease, could sensitively monitor reducing intracellular Ca2+ concentrations. We, therefore, developed a Ca2+ indicator named inverse-pericam 2.0 (IP2.0) whose fluorescent intensity decreases 25-fold upon Ca2+ binding in vitro. Using IP2.0, we successfully detected putative neuronal inhibition by monitoring the decrease in intracellular Ca2+ concentration in AWCON and ASEL neurons in Caenorhabditis elegans. Therefore, IP2.0 is a useful tool for studying neuronal inhibition and for the detailed analysis of neuronal activities in vivo.
感觉处理是由神经元回路中神经元的兴奋和抑制的协调调节的。神经元活动的分析极大地受益于最近开发的基因编码 Ca2+指示剂（GECIs）。这些分子的荧光强度或颜色会响应 Ca2+水平的变化而变化，因此可以用于灵敏地监测细胞内 Ca2+浓度，从而能够检测神经元兴奋，包括动作电位。这些 GECIs 是为了监测 Ca2+浓度的增加而开发的；因此，这些 GECIs 不能灵敏地检测神经元抑制。为了克服这个困难，我们假设一种反向 GECI，其荧光强度随着 Ca2+水平的降低而增加，可以灵敏地监测细胞内 Ca2+浓度的降低。因此，我们开发了一种名为反向-pericam 2.0（IP2.0）的 Ca2+指示剂，其在体外结合 Ca2+时荧光强度降低 25 倍。使用 IP2.0，我们成功地通过监测秀丽隐杆线虫 AWCON 和 ASEL 神经元细胞内 Ca2+浓度的降低来检测到潜在的神经元抑制。因此，IP2.0 是研究神经元抑制和体内神经元活动详细分析的有用工具。
