Chronic Ca2+ imaging of cortical neurons with long-term expression of GCaMP-X

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.