Fig. 4: Decreased ACh release in mPFC underlies defective temporal order recognition in Ngfr^−/− mice.

a Schematic diagram depicting the measurement of relative ACh levels by a fluorescent ACh3.0 sensor. Fiber photometry was used for recording the fluorescence response of ACh sensor during sample 1, sample 2, and test phase in temporal order recognition test. b Fluorescence of AAV-ACh3.0 expressed in the mPFC. Scale bar, 1 mm. c Pseudocolored fluorescence responses of ACh3.0 in mPFC during object encounters (time 0) in each phase of the temporal order recognition test. Phasic ACh release was absent in Ngfr^−/− mice. Color scale indicates ΔF/F₀. d Averaged ACh release upon object encounters (time 0). Thick lines indicate the averaged values between trials and the shaded areas represent SEM. e Group summary of peak ACh signals indicates no significant difference in ACh release when the mice explored the two identical objects in sample phases. ACh signals were significantly higher when the mice explored the earlier object from sample phase 1 (green circle) than that when they explored the later object from sample phase 2 (purple square). Sample phase 1, p = 0.54; sample phase 2, p = 0.97; test phase, p = 0.044. Ngfr^+/+, n = 3; Ngfr^−/−, n = 3. f Group summary of peak ACh signals during object interaction indicate significantly reduced ACh release in each phase of the temporal order recognition in Ngfr^−/− mice. Sample phase 1, p = 0.013; sample phase 2, p = 0.023; test phase, p = 0.0063. Ngfr^+/+, n = 3; Ngfr^−/−, n = 3. Numerical data are means ± SEM. Statistical analyses were performed by unpaired two-sided t-test in (e) and (f). *p < 0.05; **p < 0.01; ns not significant. Source data are provided as a Source Data file.