Fig. 5. Inhibition of ACh release in the SI/nBM-mPFC circuit impairs temporal order recognition.

a Schematic diagram illustrating chemogenetic inhibition of SI/nBM to mPFC cholinergic projection by injection of Cre-dependent hM4D-mCherry AAV-Retro or control virus in ChAT-Cre mice. b Discrimination ratio of temporal order recognition decreased in the hM4D group. p = 0.012. mCherry, n = 4; hM4D, n = 5. c Pseudocolored fluorescence responses of ACh3.0 in mPFC during object encounters (time 0) in each phase of the temporal order recognition test for the mCherry and hM4D group. d Reduction in averaged ACh release upon object encounters in hM4D group in each phase of the temporal order recognition test. Thick lines indicate the averaged values of trials and the shaded areas represent SEM. e Group summary of peak ACh signals indicates reduced ACh release in hM4D group during object encounters. Sample phase 1, p = 0.035; sample phase 2, p = 0.025; test phase, p = 0.028. mCherry, n = 3; hM4D, n = 3. f Diagram of optogenetic inhibition of cholinergic projection from SI/nBM to mPFC. AAV9-DIO-eNpHR3.0, or control virus were injected into SI/nBM of ChAT-Cre mice. After 4 weeks, 589 nm light was applied through an optical fiber planted in mPFC to inhibit cholinergic terminals. g Decreased discrimination ratio in temporal order recognition by optogenetic inhibition of SI/nBM to mPFC cholinergic innervation. p = 0.047. mCherry, n = 5; eNpHR3.0, n = 8. h Schematic diagram depicting specific inhibition of MS to HP cholinergic projection by injection of Cre-dependent hM4D-mCherry AAV-Retro or control virus in ChAT-Cre mice. i Intact discrimination ratio in temporal order recognition after chemogenetic inhibition of MS to HP cholinergic projection. p = 0.58. mCherry, n = 4; hM4D, n = 4. Numerical data are means ± SEM. Statistical analyses were performed by unpaired two-sided t-test in (b), (e), (g), and (i). *p < 0.05; ns not significant. Source data are provided as a Source Data file.