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. 2022 Sep 9;15:942548. doi: 10.3389/fnmol.2022.942548

FIGURE 4.

FIGURE 4

G protein coupled receptor (GPCR) and CICR signaling are required for cold-evoked Ca2+ dynamics. (A–C) Cold-evoked in vivo GCaMP6 imaging of CIII md neuron cell bodies. (A) Schematic of cold stimulus delivery and imaging system. (B) Plot of ΔF/Fbaseline over time of CIII md neuron cell body (ddaF) expressing GCaMP6m and gene knockdown for GABAB-R2, IP3R, or RyR compared to control (LucRNAi). (C) Max ΔF/Fbaseline for cold-evoked GCaMP response in CIII md neuron cell body. NAverage = 15. Statistics: One-way ANOVA with Holm-Šídák’s for multiple comparisons. (D,E) In vivo analysis of CaMPARI2 responses to noxious cold in CIII md neurons expressing gene knockdowns for GABAB-R2, IP3R, or RyR compared to control (LucRNAi). (D) Graphical schematic of cold plate assay combined with photoconverting (PC) light and post hoc imaging. (E) Left: Representative images of CIII md neurons expressing CaMPARI2 and gene specific RNAi. Cell bodies are outline in dashed white line. Scale bar: 5μM. Right: CaMPARI2 responses of CIII md neurons (ddaA and ddaF) plotted as fluorescence ratios of Fred/Fgreen. NAverage = 62. Statistics: Kruskal-Wallis with Benjamini, Krieger and Yekutieli for multiple comparisons. Significant differences indicated via asterisks, where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Created with BioRender.com.