Extended Data Figure 2. GCaMP6s faithfully reports SFO^Nos1 neuron activity in acute slices.

a, Expression of GCaMP6s in SFO^Nos1 neurons from AAV5-EF1α-FLEX-GCaMP6s (scale bar, 100 µm). b, Representative fluorescence images of a neuron given a 30 pA current injection for 700 msec in acute slice (1 of 9 cells). c, Representative traces showing calcium responses in response to 30 pA current injections of increasing duration to produce increasing numbers of action potentials (1 of 9 cells). d, Relationship between number of action potentials and ΔF/F for the representative neuron in panel c (shaded area denotes 95% confidence interval). e, R² and P-value for linear relationship between number of action potentials and ΔF/F (n = 9 cells). Panels f–j demonstrate that SFO^Nos1 neurons are homogeneously responsive to both angiotensin and salt challenge. f, Representative fluorescence images showing SFO^Nos1 neuron activity before and during bath application of angiotensin (1 of 3 experiments; red circles, identified neurons). g, 24/27 (~90%) identified SFO^Nos1 neurons were activated by bath application of angiotensin (red line, mean; grey lines, individual activated neurons). h, Quantification (****P < 0.0001, two-way repeated-measures ANOVA, n = 24 activated neurons). i, Experimental design to test if a single population of SFO neurons is responsive to both angiotensin and salt challenge. j, Co-localization of Agtr1α::GFP and salt challenge-induced cFos indicates that SFO^Nos1 neurons are homogeneously responsive to both angiotensin and salt challenge (scale bars, 100 µm). k, Experimental design to test if a SFO neurons express the excitatory neuron marker CaMK2α. l, Co-localization of CaMK2α::mCherry and Nos1::GFP indicates that SFO^Nos1 neurons are excitatory (scale bar, 100 µm). Values are mean ± s.e.m. (error bars or shaded area).