Chronic Ca 2+ imaging of cortical neurons with long-term expression of GCaMP-X.

Dynamic Ca 2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca 2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca 2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca 2+ 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 Ca 2+ fluctuations, in comparison with GCaMP. Chronic Ca 2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca 2+ transients progressively developed into autonomous/global Ca 2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca 2+ , including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca 2+ 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 Ca 2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca 2+ imaging applications involving long-time and/or high-level expression of Ca 2+ probes.