Abstract
The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.
Highlights
Using the YTnC indicator, we identified spontaneous and drifting grating-evoked neuronal activity in the soma of neurons located in the mouse visual cortex (Fig. 5)
The extracted brains were further post-fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) overnight at 4 °C and stored in PBS with 0.1% sodium azide at 4 °C until sectioning
After rinses with PBS, the sections were incubated for blocking and permeabilization in PBS with 2% Triton X-100 and 5% goat serum at room temperature for 2 h
Summary
We aimed to compare the behavior of the YTnC and GCaMP6s indicators in C-terminal fusions with different neuron-specific motifs for subcellular targeting during their transient expression in cultured neurons
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