PDF(Pubmed)
Abstract：
Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2-3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.
摘要：
动态Ca2+信号反映了膜兴奋性的急性变化，也介导慢性过程中的信号级联。在这两种情况下，经常需要慢性Ca2+成像，但是受到基于钙调蛋白（CaM）的GCaMP固有的细胞毒性的挑战，一系列已被广泛应用的基因编码的Ca2+指标。这里,我们展示了GCaMP-X在皮质神经元慢性Ca2+成像中的表现，其中GCaMP-X通过设计是为了消除常规GCaMP和内源性(apo)CaM结合蛋白之间不需要的相互作用。通过在成年小鼠中在延长的时间范围内以高水平表达，GCaMP-X在感觉（晶须偏转）响应或自发Ca2波动的双光子成像中显示出较少的损伤和改善的性能，与GCaMP相比。对培养的表达GCaMP-X的皮质神经元进行1个月或更长时间的慢性Ca2+成像，揭示了自发/局部Ca2瞬态逐渐发展为自主/全局Ca2振荡。随着神经突长度和体细胞大小的形态学指标，振荡Ca2+的主要指标，包括费率，还检查了振幅和同步性。在病毒注射或药物诱导后2-3周左右，在新生或成熟神经元中表达GCaMP，GCaMP的更强或延长表达会加剧这种情况。相比之下,表达GCaMP-X的神经元损伤或扰动明显较少，完全突出了振荡Ca2+对神经发育和神经元健康的独特重要性。总之,GCaMP-X为涉及Ca2+探针的长时间和/或高水平表达的Ca2+成像应用提供了可行的解决方案。
