Figure 1. Mitochondrial Ca²⁺ extrusion is caffeine dependent in WT neurons but not in NCLX KO neurons.

(A) Representative fluorescence traces of mitochondrial Ca²⁺ [Ca²⁺]m changes monitored in WT and NCLX KO primary cultured hippocampal neurons. Neurons were pre-loaded with Rhod2-AM and initially superfused with Ringer’s solution. The [Ca²⁺]m signals were first triggered by caffeine containing (2 mM) Ringer’s solution and then neuronal depolarization trigged by Ringer’s solution containing high K⁺ (50 mM).

(B) Quantification of [Ca²⁺]m oscillations (per 300 s) shown in (A) for WT (n = 162) and NCLX KO neurons (n = 126).

(C) Quantification of [Ca²⁺]m influx rates shown in (A) for WT (n = 38) and NCLX KO neurons (n = 54).

(D) Quantification of [Ca²⁺]m efflux rates shown in (A) for WT (n = 164) and NCLX KO neurons (n = 106).

(E) Representative fluorescence traces of [Ca²⁺]c changes in WT and NCLX KO neurons pre-loaded with Fura 2-AM (1 μM) evoked by caffeine and high-K⁺ Ringer’s solution as in (A).

(F) Quantification of [Ca²⁺]c oscillations (per 300 s) shown in (E) for WT (n = 170) and NCLX KO (n = 166) neurons.

(G) Quantification of [Ca²⁺]c influx amplitude of the data shown in (E) for WT (n = 166) and NCLX KO neurons (n = 99).

(H) Representative fluorescence traces of [Ca²⁺]m changes monitored in WT and NLCX KO. [Ca²⁺]m signals were evoked only by high-K⁺ Ringer’s solution.

(I) Quantification of [Ca²⁺]m influx rates of data shown in (H) for WT (n = 160) and NCLX KO neurons (n = 114).

(J) Quantification of [Ca²⁺]m efflux rates shown in (H) for WT (n = 138) and NCLX KO neurons (n = 145).

(K) Representative fluorescence traces of mitochondrial Mg²⁺ [Mg²⁺]m tracked in WT primary cultured neurons (days in vitro [DIV] 10–15). To track the mitochondrial Mg²⁺ transport, neurons were pre-loaded with magnesium green (2 μM) and MitoTracker deep red (200 nM), and analysis focused on regions that showed MitoTracker deep red staining. Cells were initially superfused with Ringer’s solution at pH 7.4 and then caffeine containing (2 mM) Ringer’s solution.

(L) Quantification of basal and caffeine evoked [Mg²⁺]m rise shown in (K) for WT (n = 48).

All summary data represent mean ± SEM. ***p < 0.001, ****p < 0.0001, N.S., nonsignificant.