Actin Cytoskeletal Dynamics Do Not Impose an Energy Drain on Growth Cone Bioenergetics

The growth cone was defined as the distal 10 microns of the axon shaft. If a growth cone exhibited lamellipodia then the distal most extent of the lamellipodium was taken as the distal most point on the growth cone, if not the distal most extent of the axon shaft was used as the defining distal point. Quantitative analysis of images was performed using Zeiss AxioVision software. The region of interest encompassing the growth cone was outlined and measurements of mean pixel fluorescence intensity in all relevant channels was determined for generation of ratios. For each channel, the mean intensity value obtained from the background of the image not containing any cells was subtracted from the mean intensity in the growth cone region of interest prior to determination of the ratio. Acquisition settings were chosen to generate maximum pixel values between 20-30% of the dynamic range to allow for detection of both decreases and increases in intensity in response to experimental treatments.