Fig. 3. Functional imaging of sensory coding in LEC LRIPs in SLM of CA1.

A. Diagram of in vivo imaging experiment. GCaMP6f was expressed in dorsal LEC, by injecting cre-dependent rAAV in Gad2-Cre/Ai 14 mice that also expressed tdTomato in all GABAergic neurons. A 40× water immersion objective was used for two-photon imaging through a cranial window over CA1 in head-fixed awake mice during multimodal sensory and behavioral stimuli presentation. B. Four examples of time averaged images of GCaMP6f fluorescence in LEC LRIP axons in SLM (green) with tdTomato labeling CA1 interneurons (magenta). C. Experimental design of single stimulus protocol. Imaging was performed in blocks of 4 trials, each 40 s in duration. After a 10 ± 3 s baseline one of four types of stimuli—aversive airpuff (A), water drop (W), tone (T) and light (L)—were presented in random order for 200 ms, except the water drop was limited to 50 ms to prevent satiation. Each block was repeated to obtain at least 5 trials per stimulus. The animal's behavioral response (running and licking) was monitored. ΔF/F traces showing increased Ca²⁺ signal in a single bouton on an LRIP axon in response to airpuff. D. Mean (± SEM) ΔF/F Ca²⁺ signal (PSTH) from responsive regions-of-interest (ROI) to indicated stimuli. E. Percentage of responsive boutons to the stimuli (air = 22.92%, water = 11.96%, tone = 13.64% and light = 5.65%). F. Scatter and mean (± SEM) plots of ΔF/F signals from individual responsive boutons (air = 0.55 ± 0.05, n = 68; water = 0.58 ± 0.07, n = 35; tone = 0.37 ± 0.03, n = 37; light = 0.23 ± 0.02, n = 18) G. Experimental protocol: Imaging was performed as described above, but in response to pairs of stimuli, presented in blocks of 10 trials, each 40 s long. Stimuli were randomized and paired stimuli were interleaved with single stimulus presentations. H. Mean (± SEM) ΔF/F Ca²⁺ signal (PSTH) from responsive ROIs to paired stimuli. I. Percentage of responsive boutons for paired stimuli (A+T = 32.8%; A+L = 45.3%; A+W = 25.4%; W+T = 13.3%; W+L = 15.6%; T+L = 14.1%). J. Scatter and mean (± SEM) plots of ΔF/F signals to paired stimuli from individual responsive boutons (A+T = 0.76 ± 0.07, n = 44; A+L = 0.74 ± 0.05, n = 58; A+W = 0.34 ± 0.03, n = 31; W+T = 0.48 ± 0.09, n = 17; W+L = 0.49 ± 0.04; T+L = 0.41 ± 0.045, n = 18).