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Fig. S1. Control showing lack of effect of pressure ejection delivery method on Ca2+ dynamics in cultured neurons. (A) Position of delivery microelectrode, containing modified HEPES buffer with additional 50 mmol/L NaCl. (B) Plot showing intracellular Fura-2 ratios from four neurons selected from the field shown in (A), and lack of response to 500 ms NaCl ejection pulse (delivered at 10 min). (C) Same field, after switching delivery electrode to one containing 50 mmol/L β-NAD+ in HEPES, placed very close to the position in (A). Identical 500-ms pressure pulses generated robust Fura-2 transients in the four neurons, and this was followed by sustained Ca2+ elevations after depolarization generated by bath application of KCl (40 mmol/L).

Fig. S2. Control showing lack of effect of localized application of acidic solutions on Ca2+ dynamics in cultured neurons. The plot shows intracellular Fura-2 ratios from four neurons, with lack of response to a pair of 500-ms pH 3.0 ejection pulses. These same neurons responded robustly after switching delivery electrode to one containing 50 mmol/L β-NAD+, and this was followed by sustained Ca2+ elevations after depolarization generated by bath application of KCl (40 mmol/L).

Movie S1. This set of four image sequences corresponds to data presented in Fig.  8. The top two panels show intracellular Ca2+ increases during repetitive NAD challenges, separated by 2 min (data from six neurons plotted in Fig.  8A). The bottom two panels show the lack of Ca2+ elevations when the same challenges are applied in Ca2+-free buffer. The full-scale color map corresponds to ratio changes from 0.4 to 1.2. Frame interval was 333 ms, and the full duration of each image sequence is 16.6 s.

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