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

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