Fig. 5
Ca2+ imaging of nodose (ND) neuron and Gq-DREADD-transfected GLUTag co-cultures. a, c Images of cells in our co-culture system: brightfield (top), fluorescence at 550 nm excitation (middle), and fluorescence at 488 nm excitation (bottom). Scale bars represent 50 µm. b, d Intracellular Ca2+ levels, represented as the ratio of Fura-2 fluorescence at 340 and 380 nm excitation, of the cells shown in a and c: mCherry-positive Gq-DREADD transfected GLUTag cell (top trace) and YFP-positive ND neuron (lower trace) identified using a NeuroD1-Cre/YFP reporter mouse. Drugs were applied as indicated above the traces. e Areas under the curve of the Fura-2 ratio change of ND neurons in response to CNO (10 µM) with and without PPADS (100 µM) pre-treatment. Values are expressed relative to the area of the response induced by ATP (100 µM) in each cell. Only ND neurons where a Ca2+ response could be evoked by CNO after wash out of the PPADS were included. Individual data points represent each ND neuron (n = 13). Statistical analysis performed using a paired t-test, *p < 0.05. f Percentage block of CNO responses in ND neurons by 100 µM PPADS, derived from the data shown in e. Individual data points represent each ND neuron and lines represent mean ± SEM (n = 13). Statistical analysis performed using one-sample t-test, ***p < 0.001. g Fura-2 imaging of ND neuron cultured with primary L-cells expressing Gq-DREADD under the control of the proglucagon promoter. Inset image to left is a brightfield image of the co-culture system. Drugs were applied as indicated above the traces. h Amplitude of Fura-2 ratio changes of ND neurons in response to CNO (10 µM) with and without PPADS (100 µM) pre-treatment. Values are expressed relative to the max response elicited by ATP (100 µM) in each cell. Individual data points represent each ND neuron (n = 7). Statistical analysis performed using a paired t-test, **p < 0.01. i Percentage block of CNO responses in ND neurons by 100 µM PPADs, derived from the data shown in h. Individual data points represent each ND neuron and lines represent mean ± SEM (n = 7). Statistical analysis performed using one-sample t-test, ***p < 0.001
