Figure 2.
Cannabinoids and RHOA activation’s effect on energetic metabolism and local ATP levels at the growth cone and near axonal shaft. (A) Box plot showing OCR and ECAR measured in neuronal hippocampal cultures 5 min after injection of 10 µM oligomycin (oli), 30 mM 2DG, or DMSO (control) using a Seahorse analyzer. Values were normalized to the values measured before injection for each well (n = 4). On the right, the evolution of total ATP measured by luminescence after addition of metabolic drugs. Boxplot n = 4 (wells). Two-way ANOVA followed by a Bonferroni correction revealed statistical differences in the oli group as compared with control at 2 min P < 0.01, and the following times points P < 0.001, in the oli+2DG as compared with control 2 min P < 0.01, and the following times points P < 0.01, in the 2DG as compared with control at 2 min P < 0.01, while no differences were detected at the other time points. (B and E) Confocal images of hippocampal growth cones expressing the reporter Perceval before and after 2 min treatment with 10 µM oligomycin, 30 mM 2DG, or 0.2 U/ml calpeptin. The ATP channel, ADP channel, mask obtained from the ATP channel, and the ATP/ADP ratio calculated inside the mask are shown. LUT: look-up table from the maximum value (+) to the minimal value (−). Histograms with the distribution of ATP/ADP before (blue) and after metabolic inhibition (yellow or red) or calpeptin (green) treatment. (C) Boxplot showing OCR and ECAR measured in HEK293 cells stably expressing CB1-eGFP after 5 min of 100 nM WIN injection as in A. Values were normalized to the values measured before injection for each well. n = 6 (triplicates, two independent cultures). (D) Boxplot showing OCR and ECAR measured in hippocampal neurons after 5 min of 0.2 U/ml calpeptin injection. Values were normalized to the values measured before injection for each well. n = 4 (duplicates, two independent cultures). (F) Working model of how ATP can be provided to NMII for actomyosin contraction and axonal retraction: (1) by the cytosolic pool or (2) by energetic coupling. Statistics: Student’s t test (B and C) or one-way ANOVA followed by Tukey HSD post-test correction (A); *P < 0.05.**P < 0.01.***P < 0.001.

Cannabinoids and RHOA activation’s effect on energetic metabolism and local ATP levels at the growth cone and near axonal shaft. (A) Box plot showing OCR and ECAR measured in neuronal hippocampal cultures 5 min after injection of 10 µM oligomycin (oli), 30 mM 2DG, or DMSO (control) using a Seahorse analyzer. Values were normalized to the values measured before injection for each well (n = 4). On the right, the evolution of total ATP measured by luminescence after addition of metabolic drugs. Boxplot n = 4 (wells). Two-way ANOVA followed by a Bonferroni correction revealed statistical differences in the oli group as compared with control at 2 min P < 0.01, and the following times points P < 0.001, in the oli+2DG as compared with control 2 min P < 0.01, and the following times points P < 0.01, in the 2DG as compared with control at 2 min P < 0.01, while no differences were detected at the other time points. (B and E) Confocal images of hippocampal growth cones expressing the reporter Perceval before and after 2 min treatment with 10 µM oligomycin, 30 mM 2DG, or 0.2 U/ml calpeptin. The ATP channel, ADP channel, mask obtained from the ATP channel, and the ATP/ADP ratio calculated inside the mask are shown. LUT: look-up table from the maximum value (+) to the minimal value (−). Histograms with the distribution of ATP/ADP before (blue) and after metabolic inhibition (yellow or red) or calpeptin (green) treatment. (C) Boxplot showing OCR and ECAR measured in HEK293 cells stably expressing CB1-eGFP after 5 min of 100 nM WIN injection as in A. Values were normalized to the values measured before injection for each well. n = 6 (triplicates, two independent cultures). (D) Boxplot showing OCR and ECAR measured in hippocampal neurons after 5 min of 0.2 U/ml calpeptin injection. Values were normalized to the values measured before injection for each well. n = 4 (duplicates, two independent cultures). (F) Working model of how ATP can be provided to NMII for actomyosin contraction and axonal retraction: (1) by the cytosolic pool or (2) by energetic coupling. Statistics: Student’s t test (B and C) or one-way ANOVA followed by Tukey HSD post-test correction (A); *P < 0.05.**P < 0.01.***P < 0.001.

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