Article Figures & Data
- Figure 1.
The pulse-spread experimental paradigm. A, Neurofilaments switch between kinetically distinct on-track and off-track pausing states. When on track, the filaments switch between the on-track pausing state and anterograde and retrograde transported states moving at rates of va and vr, respectively. The motors linking the moving neurofilaments to the microtubule track are meant to represent kinesin-1 (anterograde) and dynein (retrograde). B, Diagram of a pulse-spread experiment in which photoactivatable neurofilaments are activated within an axon segment (dashed magenta box), after which the fluorescence (Fd and Fp) is measured within the flanking windows (dashed blue boxes) and neurofilament flux (ja and jr) is calculated (see Materials and Methods). C, Graph of the theoretical pulse-spread kinetics, showing the rise and fall of the fluorescence in the distal and proximal flanking windows (Fd and Fp, respectively) with time, normalized to the fluorescence in the central window immediately after photoactivation (Fc(0)). The rate of increase in the fluorescence at short times is given by the initial slopes Sp and Sd. The time of fluorescence activation is marked by the vertical dashed magenta line. D, Bright-field transmitted light microscopy images of the tibial nerve within the perfusion chamber at 10× (top; scale bar, 100 μm) and 100× (bottom; scale bar, 10μm) magnification. Myelinated axons can be distinguished by their myelin sheaths. E, Time-lapse fluorescence images from a pulse-spread experiment in the tibial nerve of an hThy1-paGFP-NFM heterozygous male mouse, showing three axons spaced apart by their myelin sheaths. The dashed magenta box marks the activated region, and the dashed blue boxes mark the proximal and distal flanking regions for one axon at t = 0 min. Note that there is some loss of fluorescence because of photobleaching in these raw images. Our quantitative analysis was confined to the first 4 min after activation, and the data were corrected for photobleaching (see Materials and Methods). Proximal is left; distal is right. Scale bar, 10 μm.
- Figure 2.
Determination of flanking window size. A, Example of a single axon imaged immediately after photoactivation, showing the four different lengths of flanking windows (2, 5, 10, and 15 μm) used in our analysis. B, Graph of the fluorescence in distal flanking windows of varying size generated by computational simulation (see Materials and Methods), showing the predicted rise and fall of the fluorescence. For our analyses, we measured the increase in the fluorescence at short times (marked by the dashed magenta box). C, Fluorescence increases measured in proximal and distal flanking windows of varying sizes from pulse-spread experiments conducted in the tibial nerves of 8-week-old male mice (n = 7 nerves, n = 92 axons). D, Approximation of the rate of fluorescence increase by linear regression (dashed black line) of the first 4 min of data using 15 μm flanking windows (solid green line, reproduced from C). E, An example of one axon at 0, 4, and 10 min after activation shown in grayscale (left) and pseudocolor (right). Proximal is left, and distal is right. We measured the rate of increase of the fluorescence in the proximal and distal flanking regions (dashed blue boxes) during the first 4 min after activation. This was a very small but measurable fraction of the activated fluorescence.
- Figure 3.
Neurofilament transport is bidirectional with an anterograde bias. A, B, Frames from representative time-lapse image series of pulse-spread experiments in the tibial nerve of an 8-week-old female (A) and an 8-week male (B) hThy1-paGFP-NFM mouse. C, A representative time-lapse image of a pulse-spread experiment in a nerve from an 8-week-old male hThy1-paGFP-NFM mouse perfused with saline containing the glycolytic inhibitors 2-deoxy-d-glucose and sodium iodoacetate. The activated regions (40 μm in length) are marked by dashed magenta boxes: proximal is left, distal is right throughout. The dashed blue boxes mark the proximal and distal flanking regions for one axon at t = 0 min. Note that there is some loss of fluorescence because of photobleaching in these raw images. Our quantitative analysis was confined to the first 4 min after activation, and the data were corrected for photobleaching (see Materials and Methods). Scale bar, 10 μm. D, Quantification of relative flux of the fluorescence in the proximal (P) and distal (D) flanking regions (each 15 μm in length) in the first 4 min following photoactivation (n = 107 axons in six nerves for the female group; n = 111 axons in eight nerves for the male group; n = 17 axons in five nerves for the male group treated with inhibitors). E, Neurofilament population transport velocity (positive is anterograde, negative is retrograde). F, Percentage of neurofilament transport in the anterograde direction, calculated as the ratio of the distal relative flux to the sum of the distal and proximal relative flux. Significance in F is tested against a theoretical population with a mean of 50% and an equivalent SD. Each data point in D, E, and F represents the measurement for a different axon. The boxes show the median, upper, and lower quartiles, and the whiskers show the minimum and maximum. The magenta points on either side of the boxes show the sample mean. The mean velocity and mean percentage anterograde were calculated using the average relative proximal and distal fluxes. n.s. p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
- Figure 4.
Pulse-spread time-lapse image series of axons at different ages. A–D, Frames from representative time-lapse image series of pulse-spread experiments in the tibial nerves of male hThy1-paGFP-NFM mice that were 2 (A), 4 (B), 8 (C), and 16 weeks (D). The data for 8 weeks is reproduced from Figure 3 to allow side-by-side comparison with the other ages. The top frame represents the preactivation image. The dashed magenta box marks the activated region. The dashed blue boxes mark the proximal and distal flanking regions for one axon at t = 0 min. Note that there is some loss of fluorescence because of photobleaching in these raw images. Our quantitative analysis was confined to the first 4 min after activation, and the data were corrected for photobleaching (see Materials and Methods). Proximal is left; distal is right. Scale bar, 10μm.
- Figure 5.
Neurofilament transport slows with age. A, Fluorescence measurements from the proximal (magenta) and distal (gray) flanking windows of axons from each age group, showing mean (dot) and SEM (bars) of the full dataset (92 axons in seven nerves at 2 weeks; 84 axons in six nerves at 4 weeks; 111 axons in eight nerves at 8 weeks; 135 axons in seven nerves at 16 weeks), with respect to time after activation. Dashed black lines show linear regressions fit to the first 4 min of each dataset. B, Quantification of the relative flux of the fluorescence in the proximal (P) and distal (D) flanking regions in the first 4 min following photoactivation in nerves from male hThy1-paGFP-NFM mice 2, 4, 8, and 16 weeks of age. The 8 week data are the same data shown in Figure 3. C, Neurofilament population transport velocity variation with age (positive is anterograde; negative is retrograde). D, Percentage of neurofilament transport in the anterograde direction for each age group, calculated as the ratio of the distal relative flux to the sum of the distal and proximal relative flux. Significance in D is tested against a theoretical population with a mean of 50% and an equivalent SD. Each data point in B, C, and D represents the measurement for a different axon. The boxes show the median, upper, and lower quartiles, and the whiskers show the minimum and maximum. The magenta points on either side of the boxes show the sample mean. The mean velocity and the mean percentage anterograde were calculated using the average relative proximal and distal fluxes. n.s. p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
- Figure 6.
Neurofilament transport velocity does not correlate with axon diameter. A, Quantification of axon diameter in photoactivated axons at 2, 4, 8, and 16 weeks of age. B–E, Scatterplots of retrograde and anterograde neurofilament transport flux and neurofilament population velocity versus axon diameter at 2, 4, 8, and 16 weeks of age. Linear regression of scatterplots with 95% confidence intervals are shown in magenta, along with the p-values of each regression. The data in this figure come from the axons analyzed in Figure 5.
- Figure 7.
Anterograde neurofilament flux is higher in proximal nerve segments. A, Diagram showing the relative location of the sciatic and tibial nerve segments in relation to the spine and hindlimb. B, Frames from a representative time-lapse image series of a pulse-spread experiment in the proximal sciatic nerve. The activated region is marked by the dashed magenta box, and the proximal and distal flanking regions are marked by the dashed blue boxes for one axon at t = 0 min. Note that there is some loss of fluorescence because of photobleaching in these raw images. Our quantitative analysis was confined to the first 4 min after activation, and the data were corrected for photobleaching (see Materials and Methods). Proximal is left, and distal is right. Scale bar, 10 μm. C, Quantification of the relative fluorescence flux in the distal (D) and proximal (P) flanking windows in the first 4 min following photoactivation (111 axons in 8 nerves for the tibial; 95 axons in 10 nerves for the sciatic). The tibial nerve data used for comparison is the 8-week-old male cohort shown in Figures 3 and 5. D, Neurofilament population transport velocity in the sciatic and tibial nerves (positive is anterograde; negative is retrograde). E, Percentage of neurofilament transport in the anterograde direction in the tibial and sciatic nerves. Significance in E is tested against a theoretical population with a mean of 50% and an equivalent SD. The boxes show the median, upper, and lower quartiles, and the whiskers show the minimum and maximum. The magenta points on either side of the boxes show the sample mean. The mean velocity and mean percentage anterograde were calculated using the average relative proximal and distal fluxes. n.s. p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.
- Figure 8.
Extrapolation of relative flux to infinite flanking window size using mathematical modeling. A–E, Population averages of the relative fluorescence flux in the proximal and distal flanking regions measured over the first 4 min plotted against flanking window size for 2–16-week-old tibial nerve and 8-week-old sciatic nerve. Data from the experiments shown in Figures 3, 5, and 7. Dashed black lines show the optimal fits using our mathematical model, allowing extrapolation of the relative flux to window sizes beyond the physical limit imposed by the camera field of view.
Group Relative anterograde flux
(% ΔFd / Fc(0) /min)Relative retrograde flux
(% ΔFp / Fc(0) /min)Velocity (mm/d) Anterograde (%) Measured Model Difference Measured Model Difference Measured Model Difference Measured Model Difference Tibial nerve 2 weeks 0.299 0.415 +39% 0.195 0.231 +18% 0.060 0.106 +77% 60.5 64.2 +6% 4 weeks 0.178 0.218 +22% 0.135 0.184 +36% 0.025 0.020 −21% 56.9 54.2 −5% 8 weeks 0.154 0.152 −1% 0.122 0.113 −7% 0.018 0.022 +22% 55.8 57.3 +3% 16 weeks 0.096 0.091 −5% 0.068 0.060 −12% 0.016 0.018 +11% 58.7 60.5 +3% Sciatic nerve 8 weeks 0.202 0.202 0% 0.125 0.130 +4% 0.044 0.041 −7% 61.8 60.8 −2% Table compares the average measured neurofilament transport flux, velocity, and percentage anterograde for the data reported in Figures 5 and 7, with predicted values based on modeling an extrapolation to infinite window size. The average velocity and average percentage anterograde were calculated using the average relative proximal and distal fluxes (Eqs. 9, 10). The percentage difference (“difference”) between the experimentally derived (“measured”) and mathematically modeled (“model”) values are shown.
- Movie 1.
Pulse-spread experiment in 8-week-old male tibial nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
- Movie 2.
Pulse-spread experiment in 8-week-old female tibial nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
- Movie 3.
Pulse-spread experiment in 8-week-old male tibial nerve with glycolytic inhibition. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Note that the proximal and distal ends of the activated regions remain sharp, with no spread of fluorescence into the flanking regions. Proximal is left; distal is right.
- Movie 4.
Pulse-spread experiment in 2-week-old male tibial nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
- Movie 5.
Pulse-spread experiment in 4-week-old male tibial nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
- Movie 6.
Pulse-spread experiment in 16-week-old male tibial nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
- Movie 7:
Pulse-spread experiment in 8-week-old male sciatic nerve. Time zero corresponds to 1 min postactivation, after relaxation of the paGFP from the dark state (see Materials and Methods). The length of each activated axonal segment was 40 μm. The time-stamp format is minutes:seconds. There is noticeable photobleaching during the 30 min movie; our analyses were performed during the first 4 min, and our measurements of fluorescence intensity were corrected for photobleaching. Proximal is left; distal is right.
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