Extended Data Figure 7: Validations of NT fluorescent sensor.

a: Illustrations of fiber placements for the PVT-BLA NT calcium photometry experiment.

b-f: NT sensor validation in HEK cells. (b) Representative images of sensor expression (top) and response to 100 nM NT (bottom) of the GRAB_NTS1.0 sensor and EGFP-CAAX in HEK293T cells. (Scale, 20 μm). (c, d) Time courses (left) and group summary of peak response ΔF/F0 (right) of GRAB_NTS1.0 sensor and EGFP-CAAX towards 100 nM NT; SR: SR142948 NTSR1 antagonist; n = 101, 102 and 115 cells from 3 cultures for Saline, SR and GFP respectively. (Unpaired t-test with Welch’s correction was performed. Saline vs. SR: t_101.2=34.18, ***P<0.0001, effect size= −2.550 ± 0.07461, CI₉₅= −2.698 to −2.402. Saline vs. GFP: t₁₀₂=34.56, ***P<0.0001, effect size= −2.583 ± 0.07474, CI₉₅= −2.731 to −2.435.) (e) Representative traces showing the response to NT (top) and subsequent addition of SR (bottom). The traces were the average of 3 different regions of interest (ROIs) on the scanning line. Each trace was fitted with a single-exponential function to determine t_on and t_off, and their group summary. n = 20 and 21 cells from 4 cultures for t_on and t_off. (f) Excitation and emission spectra for the GRAB_NTS1.0 sensor in the absence and the presence of NT.

g-j: NT sensor validation ex vivo on BLA slices. (g) After mice performed reward and punishment learning task, we sliced the BLA and recorded fluorescent responses to different concentrations of NT perfusions using fiber photometry by placing the fiber on the top of the BLA. (h) Illustrations and microphotographs of fiber placements and BLA slices. (i) Example traces of NT fluorescence in responses to single perfusions of ACSF, 10 nM NT, and 300 nM NT. (j) Normalized dose-response curve for GRAB_NTS1.0 in response to different concentration of NT perfusions (9 slices from 5 separate mice).

k-l: Representative images and illustrations of fiber placements for the NT sensor photometry experiment.

n and N denote number of neurons or slices and mice in each group, respectively. Error bars and shaded regions around the mean indicate s.e.m.