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It was expected that calcium imaging could identify fluctuations of intracellular Ca2+ concentrations in Pre-I neurons without signal averaging. The calcium signals recorded from the rostral cut surface of the pFRGs in dye-injected preparations (Fig. 2a, b; Supplementary Video 1) closely corresponded to the membrane potential trajectory of single Pre-I neurons. In the membrane potential recordings, more than 70% of Pre-I neurons in this region showed only weak inspiratory inhibition (Fig. 2d). The group data from 14 experiments were: C4 burst rate 5.9 1.5/min, pre-inspiratory phase duration 897 590 ms, inspiratory phase 917 101 ms, and the post-inspiratory phase duration 3593 825 ms. This post-inspiratory phase duration was comparable to that of previous electrophysiological measurements [10]. In the present measurements, we could not clearly focus on individual stained cells because the dye injection method used here was effective in labeling cells located in regions slightly deeper than the cut surface (approximately 50 μm depth), but not for cells in the rostral cut surface plane due to insertion of dye-containing glass micropipettes into the tissue. Therefore, it is possible that calcium signals might originate from multiple cells.

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