Genetically Encoded Calcium Indicators Can Impair Dendrite Growth of Cortical Neurons

Abstract

A battery of genetically encoded calcium indicators (GECIs) with different binding kinetics and calcium affinities was developed over the recent years to permit long-term calcium imaging. GECIs are calcium buffers and therefore, expression of GECIs may interfere with calcium homeostasis and signaling pathways important for neuronal differentiation and survival. Our objective was to investigate if the biolistically induced expression of five commonly used GECIs at two postnatal time points (days 14 and 22-25) could affect the morphological maturation of cortical neurons in organotypic slice cultures of rat visual cortex. Expression of GCaMP3 in both time windows, and of GCaMP5G and TN-XXL in the later time window impaired apical and /or basal dendrite growth of pyramidal neurons. With time, the proportion of GECI transfectants with nuclear filling increased, but an only prolonged expression of TN-XXL caused higher levels of neurodegeneration. In multipolar interneurons, only GCaMP3 evoked a transient growth delay during the early time window. GCaMP6m and GCaMP6m-X C were quite "neuron-friendly." Since growthimpaired neurons might not have the physiological responses typical of age-matched wildtype neurons the results obtained after prolonged developmental expression of certain GECIs might need to be interpreted with caution.

GABAergic Inhibition of Presynaptic Ca2+Transients in Respiratory PreBötzinger Neurons in Organotypic Slice Cultures

“…Thus, earlier variants of GECIs (GCaMP6 fused to synaptophysin) can report on presynaptic Ca 2+ in neurons in primary culture ( Brockhaus et al, 2019 ; Ferron et al, 2020 ), and in the calyx of Held synapse following viral injection of GCaMP6 variants three weeks prior ( Singh et al, 2018 ). Biolistic transfection, electroporation, or AAV transduction of hippocampal, hypothalamic, and cortical organotypic slice cultures with plasmids encoding GCaMP3/6 variants or RFP-based GECIs, result in strong somatodendritic labeling, but no apparent presynaptic labeling ( Podor et al, 2015 ; Shen et al, 2018 ; Gasterstädt et al, 2020 ; Kim et al, 2020 ). Interestingly, jGCaMP7 variants show labeling of presynaptic boutons of the Drosophila larval neuromuscular junction ( Dana et al, 2019 ).…”

Encoding, transmission, decoding, and specificity of calcium signals in plants

“…Single-wavelength indicators include Oregon Green 488 BAPTA-1, and Fluo-4 and Fluo-5 acetoxymethyl ester and pentapotassium, each containing a fluorophore and calcium chelator, with increased dynamic range for qualitative analysis ( Kanchiswamy et al , 2014 ; Lock et al , 2015 ). Limitations include variability in determining intracellular calcium levels as a consequence of dye extrusion, uneven loading, dye retention, photobleaching, and cell toxic side effects ( Gasterstädt et al , 2020 ; Li and Saha, 2021 ). Ratiometric or dual-wavelength dyes are UV-excitable, and quantitatively detect fluctuations in target ion concentrations.…”

Chemogenetic Silencing of Differentiating Cortical Neurons Impairs Dendritic and Axonal Growth

“…We investigated organotypic slice cultures of rat visual cortex at two postnatal time points, DIV 10 and DIV 20, with quantitative assessment of dendritic and axonal maturation of pyramidal cells and multipolar interneurons. Cultures were prepared from pigmented Long-Evans rats at P1 as described (Gasterstädt et al, 2020;Gonda et al, 2020). The visual cortex was extracted and cut sagittally into 350 µm thick slices (McIlwain tissue chopper, Ted Pella, Redding, CA, United States).…”