Extended Data Fig. 2: Rule shift task description and control experiments using weak light delivery or weak viral expression.
From: Long-range inhibition synchronizes and updates prefrontal task activity
a, Trial timeline. A mouse begins each trial when it is placed in the home cage, then makes a decision, indicated by digging in one bowl. If the mouse is correct, the food reward is consumed. The mouse is then transferred to the holding cage until the next trial. The intertrial interval (ITI) is longer after errors. b, For optogenetic inhibition behavior experiments in Fig. 2, Day 1: no light delivery during the initial association (IA), nor during the rule shift (RS); Day 2: no light is delivered while mice learn the IA, but once mice meet the criterion for learning (8/10 consecutive trials correct), we begin delivering continuous 532 nm light and test the animal for 3 additional IA trials, before switching to the RS portion of the task. c, For optogenetic inhibition dual-site voltage indicator experiments in Fig. 3, Day 1: no light delivery during the IA, nor during the RS; Day 2: no light delivery during the IA, but continuous light delivery of 638 nm begins during 3 additional IA trials, followed by the RS; Day 3: no light delivery during the IA, nor during the RS. d, For optogenetic inhibition + microendoscopic calcium imaging experiments in Fig. 4, Day 1: no light delivery during the IA, nor during the RS; Day 2: no light delivery during the IA, but continuous light delivery of 600 nm begins during 3 additional IA trials, followed by the RS; Day 3: no light delivery during the IA, nor during the RS. e, Control experiments to verify that weak light delivery does not affect RS learning. Experimental design: Day 1, no light delivery; Day 2, continuous 0.1 mW 532 nm light is delivered during the RS; Day 3, continuous light at 0.1 mW 638 nm for inhibition during the RS. f, Representative image showing AAV-DIO-eNpHR-mCherry (DIO-eNpHR) expression and a fiber-optic cannula in the same mPFC hemisphere in a PV-Cre mouse. Scale bar, 100 μm. g, h, Performance in the rule shift task did not vary across days in either controls (n = 4), or DIO-eNpHR-injected mice (n = 5; two-way ANOVA (task day × virus); interaction: F(2,14) = 0.01721, P = 0.983). i, Experimental design: Day 1, no light delivery; Day 2, continuous 2.5 mW 532 nm light is delivered during the RS. j, Representative image showing diluted (200 nL in 800 nL saline) DIO-eNpHR expression and a fiber-optic cannula in the same mPFC hemisphere in a PV-Cre mouse. Scale bar, 250 μm. k, Light stimulation of PV cells infected with lower virus titer did not alter RS performance across days (n = 2 mice; two-tailed paired t-test, P = 0.5000). l, Experimental design: Day 1, no light delivery; Day 2, light delivery during the inter-trial interval (ITI) during the RS for optogenetic inhibition of callosal PV terminals; Day 3, no light was delivered; Day 4, continuous light during the RS. m, Representative image showing DIO-eNpHR-mCherry (DIO-eNpHR) expression in one mPFC and a fiber-optic cannula implanted in the contralateral mPFC. Scale bar, 100 μm. n, o, Optogenetic inhibition of mPFC callosal PV terminals impairs rule shift performance in DIO-eNpHR-expressing mice only when delivered during both the trial and inter-trial intervals of the RS (n = 6) compared to controls (n = 3; two-way ANOVA (task day × virus); interaction: F(3,28) = 23.31, P < 0.0001). n, Performance of controls was not different from eNpHR-expressing mice on Day 1 (post hoc t(28) = 0.3489, P > 0.9999), Day 2 (post hoc t(28) = 0.6978, P > 0.9999), nor Day 3 (post hoc t(28) = 0.4652, P > 0.9999). o, Inhibition disrupts rule shift performance in DIO-eNpHR-expressing mice compared to controls when light is delivered continuously throughout the RS on Day 4 (post hoc t(28) = 9.886, P < 0.0001). Two-way ANOVA followed by Bonferroni post hoc comparisons was used. ****P < 0.0001.
