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

Abstract Dynamic Ca 2+ signals reflect acute changes in membrane excitability (e.g. sensory response), and also mediate intracellular signaling cascades normally of longer time scales (e.g., Ca 2+ -dependent neuritogenesis). In both cases, chronic Ca 2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca 2+ indicators. Here, we demonstrate that the recently developed GCaMP-X outperforms GCaMP in long-term probe expression and/or chronic Ca 2+ imaging. GCaMP-X shows much improved compatibility with neurons and thus more reliable than GCaMP as demonstrated in vivo by acute Ca 2+ responses to whisker deflection or spontaneous Ca 2+ fluctuations over an extended time frame. Chronic Ca 2+ imaging data ( ≥ 1 month) are acquired from the same set of cultured cortical neurons, unveiling that spontaneous/local Ca 2+ activities would progressively develop into autonomous/global Ca 2+ oscillations. Besides the morphological indices of neurite length or soma size, the major metrics of oscillatory Ca 2+ , including rate, amplitude, synchrony among different neurons or organelles have also been examined along with the developmental stages. Both neuritogenesis and Ca 2+ signals are dysregulated by GCaMP in virus-infected or transgenic neurons, in direct contrast to GCaMP-X without any noticeable side-effect. Such in vitro data altogether consolidate the unique importance of oscillatory Ca 2+ to activity-dependent neuritogenesis, as one major factor responsible for the distinctions between GCaMP vs GCaMP-X in vivo . For the first time with GCaMP-X of long-term expression in neurons, spontaneous and sensory-evoked Ca 2+ activities are imaged and evaluated both in vitro and in vivo , providing new opportunities to monitor neural development or other chronic processes concurrently with Ca 2+ dynamics.