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Neuronal activity biases axon selection for myelination in vivo.

Hines JH, Ravanelli AM, Schwindt R, Scott EK, Appel B - Nat. Neurosci. (2015)

Bottom Line: Using zebrafish, we found that activity-dependent secretion stabilized myelin sheath formation on select axons.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA.

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.

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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.
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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.

Mentions: To test whether activity-dependent signals that bias axon choice originate in neurons, we next inhibited synaptic vesicle exocytosis selectively in phox2B+ axons by targeted overexpression of tetanus neurotoxin (TeNT), a protease that cleaves select Vesicle-Associated Membrane Protein (VAMP) family proteins including Synaptobrevin/VAMP2. This is a potent inhibitor of synaptic vesicle exocytosis in many animal models including zebrafish16,17. We cloned a 2.1 kb genomic fragment of the zebrafish phox2B gene and generated the Tg(phox2B:GAL4) line. When crossed to Tg(UAS:mCherry-CaaX), mCherry was faithfully co-expressed in axons marked by phox2B:GFP, validating the specificity of this new transgenic line (Supplementary Fig. 1). We injected Tg(phox2B:GAL4) embryos at early cleavage stage with DNA plasmids encoding UAS:GFP or UAS:TeNT-GFP, resulting in mosaic expression in single phox2B+ axons of 4-day larvae. When phox2B+ axons expressed GFP as a control, 55.3% were myelinated at the time of imaging, whereas only 31.6% expressing TeNT-GFP were myelinated (Fig. 3a,b). Quantitatively, we found that single TeNT-GFP+ axons were less well wrapped, because the percent length of axons ensheathed by sox10:mRFP+ sheaths was reduced (Fig. 3c). Notably, when oligodendrocytes did wrap TeNT-GFP+ axons, nascent sheaths were shorter in length (Fig. 3d).


Neuronal activity biases axon selection for myelination in vivo.

Hines JH, Ravanelli AM, Schwindt R, Scott EK, Appel B - Nat. Neurosci. (2015)

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.
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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.
Mentions: To test whether activity-dependent signals that bias axon choice originate in neurons, we next inhibited synaptic vesicle exocytosis selectively in phox2B+ axons by targeted overexpression of tetanus neurotoxin (TeNT), a protease that cleaves select Vesicle-Associated Membrane Protein (VAMP) family proteins including Synaptobrevin/VAMP2. This is a potent inhibitor of synaptic vesicle exocytosis in many animal models including zebrafish16,17. We cloned a 2.1 kb genomic fragment of the zebrafish phox2B gene and generated the Tg(phox2B:GAL4) line. When crossed to Tg(UAS:mCherry-CaaX), mCherry was faithfully co-expressed in axons marked by phox2B:GFP, validating the specificity of this new transgenic line (Supplementary Fig. 1). We injected Tg(phox2B:GAL4) embryos at early cleavage stage with DNA plasmids encoding UAS:GFP or UAS:TeNT-GFP, resulting in mosaic expression in single phox2B+ axons of 4-day larvae. When phox2B+ axons expressed GFP as a control, 55.3% were myelinated at the time of imaging, whereas only 31.6% expressing TeNT-GFP were myelinated (Fig. 3a,b). Quantitatively, we found that single TeNT-GFP+ axons were less well wrapped, because the percent length of axons ensheathed by sox10:mRFP+ sheaths was reduced (Fig. 3c). Notably, when oligodendrocytes did wrap TeNT-GFP+ axons, nascent sheaths were shorter in length (Fig. 3d).

Bottom Line: Using zebrafish, we found that activity-dependent secretion stabilized myelin sheath formation on select axons.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA.

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.

Show MeSH