Figure 6.
Local glycolysis is required for axonal retraction. (A) On the left, a schematic representation of the PDMS-based microfluidic devices and experimental design used for local application of drugs at the distal compartment (in blue; LPI plus or minus 2DG) or at the proximal somatic compartment (in light red; plus or minus 2DG). The two compartments are connected by small channels in which axons extend from the proximal compartment to reach the distal chamber. On the right, bright-field images of the three compartments: (1) somatic chamber containing the cortical neuronal cell bodies; (2) channels for axonal extension; and (3) distal axonal chamber with axonal tips that have exited the channels. (B) Bright-field and fluorescence images after 15 min of fluorescein injection in the growth cone chamber. Due to a pressure gradient, fluorescein does not diffuse into the axonal channels. (C) Axonal retraction assay was performed when growth cones reached the distal growth cone chamber (DIV4–7). Video microscopy was performed in bright-field at a frequency of 1 frame/minute for 15 min before the induction of retraction by LPI injection at the distal chamber. For the local inhibition test, 2DG was injected with LPI in the axonal chamber (2DG distal) or the 2DG was injected in the somatic compartment simultaneously with LPI injection in the growth cone chamber (2DG proxi). Boxplot showing values of axonal retraction 20 min after LPI injection (control: n = 21, three independent experiments; 2DG distal n = 30, three independent experiments; 2DG proxi n = 16, two independent experiments). Statistics: one-way ANOVA followed by Tukey HSD post-test correction; ***P < 0.001. (D) Proposed model of axonal retraction fueling by energetic coupling between actomyosin and glycolysis. The ATP that fuels NMII contraction and axonal retraction is generated locally and on demand by the glycolytic enzymes associated with actomyosin.

Local glycolysis is required for axonal retraction. (A) On the left, a schematic representation of the PDMS-based microfluidic devices and experimental design used for local application of drugs at the distal compartment (in blue; LPI plus or minus 2DG) or at the proximal somatic compartment (in light red; plus or minus 2DG). The two compartments are connected by small channels in which axons extend from the proximal compartment to reach the distal chamber. On the right, bright-field images of the three compartments: (1) somatic chamber containing the cortical neuronal cell bodies; (2) channels for axonal extension; and (3) distal axonal chamber with axonal tips that have exited the channels. (B) Bright-field and fluorescence images after 15 min of fluorescein injection in the growth cone chamber. Due to a pressure gradient, fluorescein does not diffuse into the axonal channels. (C) Axonal retraction assay was performed when growth cones reached the distal growth cone chamber (DIV4–7). Video microscopy was performed in bright-field at a frequency of 1 frame/minute for 15 min before the induction of retraction by LPI injection at the distal chamber. For the local inhibition test, 2DG was injected with LPI in the axonal chamber (2DG distal) or the 2DG was injected in the somatic compartment simultaneously with LPI injection in the growth cone chamber (2DG proxi). Boxplot showing values of axonal retraction 20 min after LPI injection (control: n = 21, three independent experiments; 2DG distal n = 30, three independent experiments; 2DG proxi n = 16, two independent experiments). Statistics: one-way ANOVA followed by Tukey HSD post-test correction; ***P < 0.001. (D) Proposed model of axonal retraction fueling by energetic coupling between actomyosin and glycolysis. The ATP that fuels NMII contraction and axonal retraction is generated locally and on demand by the glycolytic enzymes associated with actomyosin.

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