Figure 2: Analysis of peptide and urine-evoked Ca²⁺ responses in intact VSNs.

(a) Example of somatic Ca²⁺ recordings from a single VSN to different concentrations of SIINFEKL. A response is defined as a stimulus-dependent deviation in Ca²⁺ fluorescence that exceeds twice the standard deviation of the mean of the baseline fluorescence noise. (b) SIINFEKL dose–response curves (grey) each recorded from a single VSN (n=12 cells, 8 slices). Relative changes in peak fluorescence (ΔF/F) induced by a given stimulus were normalized to the maximum peak response measured in a given VSN to construct a dose–response curve. The smooth curves (b,d) are fits of the Hill equation in combination with an iterative Levenberg–Marquardt nonlinear, least-squares fitting routine (chi-square test: P=0.96–0.99), defining threshold, EC₅₀ and saturation. The black curve represents the mean dose response curve; EC₅₀ value and SD are indicated. (c) Comparison of Ca²⁺ responses in a single VSN to various concentrations of SIINFEKL and diluted urine (DU) obtained from B6/OVA⁺, B6/OVA⁺/β₂m^−/− and B6 mice. Repeated presentations indicate that Ca²⁺ peak responses were highly reproducible and did not change over time under these conditions. (d) Estimating the active concentration of SIINFEKL in B6/OVA⁺ urine (black triangle) from Ca²⁺ responses of a single VSN to known concentrations of SIINFEKL. Same cell as in c. (e) Summary of response profiles of VSNs stimulated successively with SIINFEKL and different urine types as indicated. The SIINFEKL concentration in B6/OVA⁺ DU estimated from the dose–response curve of each cell is indicated. Arrows indicate the time points at which stimulus application was turned on. Non-responders are not listed in the table.