Article Figures & Data
Figures
- Figure 1.
Doxycycline regulation of astrocyte-specific transgene expression to induce nodal remodeling. A, EGFP expression serves as a marker for dnVAMP2 expression in the optic nerve. EGFP expression (green) was absent in the optic nerves of [gfap]dnVAMP2 mice supplied DOX (DOX-ON) from gestation through adulthood (P70). In contrast, EGFP expression (green) was prevalent in astrocytes in the optic nerves of [gfap]dnVAMP2 mice on a diet that did not contain DOX (DOX-OFF) from gestation through adulthood (P70). EGFP expression in astrocytes was also elevated when [gfap]dnVAMP2 mice were raised on a DOX-ON diet through development, but DOX was removed from their diet from P40 to P70 (30 d DOX-OFF). The paranodal region is marked by Caspr1 labeling in red. B, Western blotting for EGFP expression confirmed the results of immunocytochemistry. Actin was used as a loading control. C, Box plots of average nodal gap length measured per animal. Individual points represent the average of 10 microscope fields per animal. Maximum, minimum, and median nodal gap lengths are represented within the box plot as whiskers (top and bottom) and mid-line (solid black). The mean nodal gap length of all animals within each condition is marked with a dashed red line. Optic nerve nodal gap length was larger in lifelong +dnVAMP2 mice than in lifelong –dnVAMP2 mice (mean nodal gap length ± SEM: 0.646 ± 0.026 vs 0.787 ± 0.036 μm; lifelong –dnVAMP2 12 h light/dark cycle vs lifelong +dnVAMP2 12 h light/dark cycle, respectively; t test, t(12) = −3.17, p = 0.008).
- Figure 2.
Astrocyte-regulated nodal lengthening proceeds normally during binocular visual deprivation. A, Experimental timeline used to test whether visual experience influences the astrocyte-mediated mechanism of node of Ranvier plasticity. Exocytosis from astrocytes was reduced throughout life by withholding DOX from [gfap]dnVAMP2 transgenic mice throughout gestation and adulthood to express the dnVAMP2 gene continuously. At P40, after myelination of optic nerve is complete, a subset of these animals underwent 30 d of binocular deprivation (24 h darkness), and nodes of Ranvier were compared with similar animals experiencing normal visual conditions in a 12 h light/dark cycle. The lengths of the nodal gaps were compared in both groups of animals at P70. B, Representative confocal microscope fields from +dnVAMP2 animals maintained under normal 12 h light/dark cycle (top) or under 24 h darkness (bottom) from P40 to P70. The paranodal region was labeled with Caspr1 (red), and the node was identified by sodium channel Nav1.6 immunocytochemistry (blue). C, Examples of typical individual nodes of Ranvier from +dnVAMP2 animals maintained from P40 to P70 under the normal 12 h light/dark cycle (top) or under 24 h darkness (bottom). D, Box plots of nodal gap length. Individual points represent the average of all nodes measured in each of 10 microscope fields per animal. Maximum, minimum, and median nodal gap lengths are represented within the box plot as whiskers (top and bottom) and mid-line (solid black). Mean nodal gap length of all animals within each condition is marked with a dashed red line. Nodal gap length was not significantly different in lifelong +dnVAMP2 animals following binocular visual deprivation compared with similar animals maintained under normal light conditions (mean nodal gap length ± SEM: 0.787 ± 0.036 vs 0.813 ± 0.017 μm; lifelong +dnVAMP2 12 h light/dark cycle versus lifelong +dnVAMP2 24 h darkness, respectively; t test, t(9) = −0.65, p = 0.530). n.s. = not significant.
- Figure 3.
Reduction in astrocyte exocytosis in adult mice lengthened nodal gap length regardless of modulation of the visual system by dark exposure. A, Experimental timeline for adult-onset +dnVAMP2 binocular deprivation used to eliminate possible confounds of reducing exocytosis in astrocytes in gestation and during myelination. Astrocyte vesicular release was reduced in adulthood via +dnVAMP2 expression begun at P40 by removing DOX from the diet at that time. The mice were then subjected to 30 d of binocular deprivation (24 h darkness) and nodes of Ranvier were compared at P70 with similar animals with normal visual experience (12 h light/dark cycle). B, Representative confocal microscope fields from adult-onset +dnVAMP2 animals maintained under normal 12 h light/dark cycle (top) or under 24 h darkness (bottom) from P40 to P70. The paranodal region was labeled with Caspr1 (red), and the node was identified by sodium channel Nav1.6 (blue). C, Examples of typical individual nodes of Ranvier from adult-onset +dnVAMP2 animals maintained from P40 to P70 under normal 12 h light/dark cycle (top) or under 24 h darkness (bottom). D, Box plots of average nodal gap length measured per animal. Individual points represent the average of nodes of Ranvier in 10 microscope fields per animal. Maximum, minimum, and median nodal gap lengths a represented within the box plot as whiskers (top and bottom) and mid-line (solid black). Mean nodal gap length of all animals within each condition is marked with a dashed red line. Nodal gap length was not significantly different in adult-onset +dnVAMP2 animals following 30 d of binocular visual deprivation compared with similar animals maintained under normal light conditions (mean nodal gap length ± SEM: 0.912 ± 0.020 vs 0.882 ± 0.021 μm; adult-onset +dnVAMP2 12 h light/dark cycle vs adult-onset +dnVAMP2 24 h darkness, respectively; t test, t(7) = 1.03, p = 0.336). n.s. = not significant.
- Figure 4.
Long-term binocular deprivation does not alter length of nodes of Ranvier in adult optic nerves with normally functioning astrocyte vesicular release. A, Experimental timeline to test whether binocular deprivation affects node of Ranvier gap length when exocytosis from astrocytes is unimpaired. Animals with unimpaired vesicular release from astrocytes were raised by supplying DOX during gestation and throughout life. A control group of lifelong –dnVAMP2 mice was maintained under normal visual conditions of a 12 h light/dark cycle from birth until P70, while similar animals undergoing binocular visual deprivation were raised under normal visual conditions of 12 h light/dark cycle until P40 and then maintained under 24 h darkness from P40 to P70. B, Representative microscope fields from –dnVAMP2 animals maintained under normal 12 h light/dark cycle (top) or under 24 h darkness (bottom) from P40 to P70, the paranodal region, were labeled with Caspr1 (red), and the node is identified with Nav1.6 (blue). C, Examples of typical individual nodes of Ranvier from –dnVAMP2 animals maintained under normal 12 h light/dark cycle (top) or under 24 h darkness (bottom) from P40 to P70. The paranodal region was labeled with Caspr1 (red), and the node was identified with sodium channel Nav1.6 immunostaining (blue). D, Box plots of average nodal gap length measured per animal. Individual points represent the average of 10 microscope fields per animal. Maximum, minimum, and median nodal gap lengths are represented within the box plot as whiskers (top and bottom) and mid-line (solid black). Mean nodal gap length of all animals within each condition is marked with a dashed red line. Nodal gap length was not significantly different in lifelong –dnVAMP2 animals following binocular visual deprivation compared with similar animals maintained under normal light conditions (mean nodal gap length ± SEM: 0.646 ± 0.026 vs 0.675 ± 0.025 μm; lifelong –dnVAMP2 12 h light/dark cycle vs lifelong –dnVAMP2 24 h darkness, respectively; t test, t(14) = −0.80, p = 0.435). n.s. = not significant.
- Figure 5.
Two-way ANOVA of the effects of vesicle release from astrocytes and visual deprivation in all experimental conditions tested. The results show that vesicular release from astrocytes was the primary factor determining nodal gap length across all conditions (p < 0.001), and that sensory deprivation did not have a significant effect on nodal gap length (p = 0.476). There was no significant interaction between these two variables (p = 0.092; n.s. = not significant; *** p < 0.0001). Box plots of average nodal gap length measured per microscope field in all conditions. Outliers are represented with Xs. Maximum, minimum, and median nodal gap lengths are represented withing the box plots as whiskers (top and bottom) and midline (solid black). Mean nodal gap length of all animals within each condition is marked with a dashed red line.
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