Neuronal activity biases axon selection for myelination in vivo

Abstract

An essential feature of vertebrate neural development is ensheathment of axons with myelin, an insulating membrane formed by oligodendrocytes. Not all axons are myelinated, but mechanisms directing myelination of specific axons are unknown. Using zebrafish, we found that activity-dependent secretion stabilized myelin sheath formation on select axons. When VAMP2-dependent exocytosis was silenced in single axons, oligodendrocytes preferentially ensheathed neighboring axons. Nascent sheaths formed on silenced axons were shorter in length, but when activity of neighboring axons was also suppressed, inhibition of sheath growth was relieved. Using in vivo time-lapse microscopy, we found that only 25% of oligodendrocyte processes that initiated axon wrapping were stabilized during normal development and that initiation did not require activity. Instead, oligodendrocyte processes wrapping silenced axons retracted more frequently. We propose that axon selection for myelination results from excessive and indiscriminate initiation of wrapping followed by refinement that is biased by activity-dependent secretion from axons.

Figure 1. Axon selection is biased by electrical activity

(a) Confocal images show axon wrapping in Tg(phox2B:GFP) larvae. In control larvae (left), phox2B⁺ axons are frequently wrapped by nascent myelin sheaths marked by sox10:mRFP. TTX treatment (right) reduces the proportion of nascent sheaths wrapping phox2B⁺ axons. For each condition, the upper panels are lateral spinal cord views and the lower panels show orthogonal projections, which were generated at the dashed lines. Arrowheads point to axons (white) and nascent sheaths (magenta). Scale bars, 1 µm. (b) Quantification of the proportion of sox10:mRFP sheaths wrapping phox2B⁺ axons. P = 0.0005, t-test. (c) Quantification of the overall number of nascent sheaths per spinal cord hemisegment. P = 0.3426, t-test. For (b–c), n =22 control larvae (283 sheaths) and 23 TTX-treated larvae (283 sheaths). (d) Quantification of nascent sheath length; n =16 control larvae (406 sheaths) and 12 TTX-treated larvae (296 sheaths). P = 0.7468 (phox2B:GFP⁺) and P = 0.1540 (phox2B⁻), t-test. For all panels, error bars show s.e.m.; ***P < 0.001; n.s., not significant.

Figure 2. Veratridine reduces nascent sheath length but not sheath number or axon selection

(a) Quantification of touch response assays on control and veratridine-treated larvae. Data shown represent the proportion of larvae exhibiting either wild-type or prolonged touch-response phenotypes (see Methods). Scoring was performed three hours after treatment and again immediately prior to confocal imaging (24 hr post-treatment). For both control and treated groups, n = 80 (3 h post-treatment) and 76 (24 h post-treatment). (b) Representative confocal images show reporter expression in control and veratridine-treated Tg(phox2B:GFP); Tg(sox10:mRFP) larvae. M indicates the position of the Mauthner axon and arrowheads point to sites of phox2B⁺ axon wrapping. Scale bar, 5 µm. (c–e) Summary of axon selection, sheath number, and sheath length measurements from (b). Error bars show s.e.m.; t-test; *P < 0.05, **P < 0.01; n.s., not significant. For (c–e) n = 30 control and 28 veratridine-treated larvae. P = 0.2147 (c), P = 0.2173 (d), P = 0.0011 (e, left), and P = 0.0342 (e, right); t-test.

Figure 3. Activity-dependent competition during axon selection

(a) Confocal images show expression of UAS:GFP or UAS:TeNT-GFP in single phox2B⁺ axons and nascent sheaths marked by sox10:mRFP. Images on the left are lateral views and the right panel shows orthogonal projections, generated at the dashed lines. Arrowheads point to ensheathed axons. Scale bars, 1 µm. (b) Summary of the percentage of axons selected for myelination. For each condition, the number selected and overall number of axons analyzed is indicated. (c) Quantitative measurements show the wrapping efficiency of phox2B⁺ axons expressing from the indicated plasmids. Data are expressed as the percent of total axon length ensheathed at the time of imaging. P = 0.0096 (upper asterisk), P = 0.0294 (lower asterisk), P = 0.7257 (lower comparison, n.s.), Mann-Whitney test. (d) Quantification of nascent sheath length. Left bars show the average length of sheaths wrapping GFP⁺ axons, and the right bars show lengths of nascent sheaths wrapping neighboring, unmarked axons in the same larvae. P < 0.0001 (left comparison), P = 0.0002 (right comparison), Mann-Whitney test. For (c–d), n corresponds to the numbers of axons indicated in (b) derived from 17 (UAS:GFP), 17 (UAS:TeNT-GFP), 22 (UAS:TeNT-GFP + TTX), and 29 (UAS:Kir2.1; EGFP) larvae. Error bars show s.e.m.; *P < 0.05, ***P < 0.001; n.s., not significant.

Figure 4. Accumulation of Syn-GFP vesicles at myelin ensheathment sites

(a) Representative confocal images show Syn-GFP vesicle puncta (white) and nascent myelin sheaths marked by the sox10:mRFP reporter (magenta). (b) Representative time-lapse confocal images show stationary and motile Syn-GFP⁺ vesicle puncta at unmyelinated axon segments (left panels) and ensheathment sites (right panels). The upper panels show images acquired 30 s prior to the onset of time-lapse imaging, and the lower panels show time-lapse images acquired at 10 s intervals. For (a–b), Syn-GFP puncta at unmyelinated segments and ensheathment sites are indicated by blue and yellow arrowheads, respectively. Images are lateral views with dorsal up and anterior right. The Mauthner axon (M) is marked for reference. Scale bars, 2 µm. (c) Summary of vesicle motility measurements show the proportion of motile and stationary vesicles at unmyelinated segments and ensheathment sites. Error bars represent s.e.m.; n = 272 puncta at unmyelinated segments (12 larvae), 31 puncta at ensheathment sites (7 larvae); ***P = 0.0009, t-test.

Figure 5. Activity-dependent secretion is not required for initial axon wrapping

(a) Representative time-lapse confocal images show initiation of axon wrapping in the ventral spinal cord of sibling control (left panels) and Tg(neuroD:TeNT-GFP) larvae (right panels). Images are lateral views and time (min) relative to wrapping initiation is indicated at the left. Arrowheads point to prospective sheaths that fail to stabilize. Scale bar, 2 µm. (b) Summary of time-lapse measurements show the proportion of prospective sheaths that are stable for at least 90 min. n = 12 control (66 sheaths) and 8 TeNT-GFP larvae (57 sheaths); P = 0.5624, Mann-Whitney test. (c) Measurements of nascent sheath lifetime amongst transient ensheathments during time-lapse imaging in (a). n = 12 control (50 prospective sheaths) and 8 TeNT-GFP larvae (41 prospective sheaths); P = 0.0411, Mann-Whitney test. For (b–c), error bars show s.e.m., *P < 0.05; n.s., not significant.

Figure 6. Nascent myelin sheaths are stabilized by activity-dependent secretion

(a) Representative confocal images show the retraction of existing sheaths during 15-hr time-lapse imaging in sibling control and Tg(neuroD:TeNT-GFP) larvae. Images are lateral views of the dorsal spinal cord and the time (min) relative to the start of image acquisition is indicated for each image. For demonstrative purposes, sheaths stable for the entire time-lapse are shaded in blue and retracting sheaths are shaded red. Scale bar, 5 µm. (b) Summary of time-lapse measurements show the frequency of sheath retraction. n = 7 control (99 sheaths) and 9 Tg(neuroD:TeNT-GFP) larvae (129 sheaths). P = 0.0071, Mann-Whitney test. (c) Quantitative measurements show the relationship between sheath length and stability. Bars represent the mean ± s.e.m. n = 7 control larvae (72 stable sheaths, 6 retracting sheaths) and 9 Tg(neuroD:TeNT-GFP) larvae (106 stable sheaths, 19 retracting sheaths). P = 0.0034 (left, **), P = 0.0002 (right, ***), P = 0.3737 (upper n.s.), P = 0.6105 (lower n.s.), Mann-Whitney test; For (b–c), **P < 0.01, **P < 0.001.