Fig. 7

EphB2 deficiency inhibits NMDAR-dependent mitochondrial Ca²⁺ responses and mitochondrial membrane depolarization in neurons. WT and Ephb2^−/− forebrain neurons were obtained from P0 mice. a, b Mitochondrial calcium imaging using the FRET-based indicator 4mt.D3cpv was performed with drugs in the bath to inhibit voltage-dependent calcium channels, AMPA receptors, and voltage-dependent sodium channels. This should prevent APs and associated voltage-dependent calcium signals, leaving the “pure” NMDA signal, which was evoked by a brief (30 s) application of 20 μM NMDA. a Representative data from one coverslip each of WT and Ephb2^−/− cells showing the baseline 4mt.D3cpv FRET ratio in the presence of inhibitors and the response to NMDA (mean ± SEM). b Quantification of the baseline 4mt.D3cpv FRET ratio and peak amplitude of the response to NMDA (mean ± SD; n = 15/19 coverslips from 4 independent preparations; Student’s t-test). c, d Mitochondrial membrane potential imaging using the fluorescent dye Rh123. Under basal conditions, Rh123 accumulates within the mitochondrial matrix, where its high concentration leads to quenching. Mitochondrial membrane depolarization induces leakage of Rh123 from the mitochondria into the cytoplasm, where its fluorescence is dequenched resulting in an increase in fluorescence intensity. Rh123 fluorescence levels were measured in the nucleus to avoid possible contamination by fluorescence signals emerging from mitochondria. c Representative data from one coverslip each of WT and Ephb2^−/− cells during stimulation with 20 μM NMDA over ~ 10 min (mean ± SEM). d Quantification of the area under the curve during the first 9 min of NMDA treatment for 5 and 20 μM NMDA (mean ± SD; n = 10 coverslips from 4 independent preparations (5 μM), n = 16/21 coverslips from 4 independent preparations (20 μM); Student’s t-test). e Quantification of cytoplasmic Ca²⁺ levels under baseline conditions and the peak amplitude of the cytoplasmic Ca²⁺ response as measured using the ratiometric small molecule indicator fura-2 triggered by brief (30 s) stimulation with 20 μM NMDA (mean ± SD; n = 15/16 coverslips from 4 independent preparations; Student’s t-test). * p < 0.05