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

Abstract

Dynamic Ca2+ signals reflect acute changes in membrane excitability (e.g. responses to stimuli), and also mediate intracellular signaling cascades that normally take longer time to manifest (e.g., regulations of transcription). In both cases, chronic Ca2+ imaging has been often desired, but largely hindered by unexpected cytotoxicity intrinsic to GCaMP, a popular series of genetically-encoded Ca2+ indicators. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging with long-term probe expression in cortical neurons, which has been designed to eliminate the unwanted interactions between conventional GCaMP indicators and endogenous (apo)calmodulin-binding proteins. By expressing in live adult mice at high levels over an extended time frame, GCaMP-X indicators showed less damage and improved performance in two-photon imaging of acute Ca2+ responses to whisker deflection or spontaneous Ca2+ fluctuations. Chronic Ca2+ imaging data (³1 month) were acquired from cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients would progressively develop into autonomous/global Ca2+ oscillations. Besides the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined along with the multiple stages (from neonatal to mature) during neural development. Dysregulations of both neuritogenesis and Ca2+ oscillations were observed typically in 2-3 weeks, which were exacerbated by stronger or prolonged expression of GCaMP. In comparison, neurons expressing GCaMP-X exhibited significantly less damage. By varying the timepoints of virus infection or drug induction, GCaMP-X outperformed GCaMP similarly in cultured mature neurons. These data altogether highlight the unique importance of oscillatory Ca2+ to morphology and health of neurons, presumably underlying the differential performance between GCaMP-X and GCaMP. In summary, GCaMP-X provides a viable option for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

Injectable Fluorescent Neural Interfaces for Cell-Specific Stimulating and Imaging

“…Efforts to refine delivery methods have also helped to reduce toxicity. GCaMP-XC, a recent innovation, shows promise with its reduced toxic effects and faster kinetics, offering strategies to balance the toxicity and efficacy of the calcium indicator in vivo . GCaMP’s intricate composition, targeted delivery with micron-level resolution, exceptional stability, versatile kinetics, and seamless wireless compatibility make it an optimal choice for imaging , when integrated with mesh electrodes in brain implants.…”

“…GCaMP-XC, a recent innovation, shows promise with its reduced toxic effects and faster kinetics, offering strategies to balance the toxicity and efficacy of the calcium indicator in vivo. 74 GCaMP's intricate composition, targeted delivery with micron-level resolution, exceptional stability, versatile kinetics, and seamless wireless compatibility make it an optimal choice for imaging 75,76 when integrated with mesh electrodes in brain implants. This integration holds the potential to enhance the wireless decoding and imaging of large neural populations, addressing challenges often faced with conventional indicators due to their toxicity and limited compatibility.…”

“…While GCaMP exhibits significant biocompatibility for in vivo applications, exploring its potential cellular toxicity is imperative for brain implants. Variants like GCaMP6m and jGCaMP7 can detect subtle changes in single action potentials but may affect neuronal morphology and calcium dynamics differently . Advances in GCaMP technology include genetic modifications and the introduction of peptides such as RS20, leading to the development of fast and sensitive sensors such as XCaMP and R-CaMP2.…”

“…Variants like GCaMP6m and jGCaMP7 can detect subtle changes in single action potentials but may affect neuronal morphology and calcium dynamics differently. 74 Advances in GCaMP technology include genetic modifications and the introduction of peptides such as RS20, leading to the development of fast and sensitive sensors such as XCaMP and R-CaMP2. Efforts to refine delivery methods have also helped to reduce toxicity.…”

Daily two-photon neuronal population imaging with targeted single-cell electrophysiology and subcellular imaging in auditory cortex of behaving mice

“…The GCaMP-XC virus was used to track cells because it reduces neuronal toxicity while improving calcium quality and reliability, thus allowing long-term, high expression (Geng et al, 2022). Chronic two-photon imaging was performed after the implantation of cranial window (Figure 2, see detail in the section "Materials and methods").…”