Figure 1. PFC neurons show heterogeneous responses to reward-predictive cues.

a, Head fixation allowed two-photon microscopy in awake, behaving mice. b, Schematic of the Pavlovian conditioning paradigm. c, Example data showing anticipatory licking to the CS+ but not CS− after learning. d, Average change in lick rate during each cue for early and late conditioning sessions. e, Behavioral discrimination (licking during CS+ versus CS−; auROC-0.5) during early and late conditioning sessions wherein separate FOVs were examined (Early, n=30; Late, n=30; t(58)=43.0, p<0.001). f,g, Injections of AAVdj-CaMKii-GCaMP6s into PFC (f) and optical cannula implantation (g) allowed two-photon imaging of PFC neurons throughout conditioning (Early, n=1473 neurons; Late n=1571 neurons; n=8 mice). h,i, GCaMP6s expression across multiple FOVs in dorsomedial PFC (h) allowed recordings from hundreds of prefrontal neurons within individual mice (i). j,k, Perievent data showing example excitatory (j) or inhibitory (k) responses from example neurons during cue delivery after learning. l, Population data of all neurons showing few excitatory (red) or inhibitory (blue) cue responses (p<0.05 after correction; see Methods) to the CS+ and CS− during early sessions (CS+ versus CS−: χ²(2)=9.06, p=0.01). m, Population data of all neurons show many excitatory (red) or inhibitory (blue) cue responses to the CS+, but not CS−, during late sessions (CS+ versus CS−: χ²(2)=523.15, p<0.001). n,o, Heat plots from individual example neurons that showed excitatory (n) or inhibitory (o) responses during cue delivery. p,q, Population heat plots from all mice plots showing averaged cue responses after learning. Bar and line graphs represent the mean±SEM. Vertical dotted lines refer to timing of sucrose delivery. Scale bars=100µm; CS, conditioned stimulus; FOV, field of view; TI, trace interval.