Zhao Y and Szaro BG (1995), The optic tract and tectal ablation influence t...

XB-ART-19693

J Neurosci 1995 Jun 01;156:4629-40.

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The optic tract and tectal ablation influence the composition of neurofilaments in regenerating optic axons of Xenopus laevis.

Zhao Y , Szaro BG .

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Neurofilaments have been proposed to regulate axonal stability and diameter through changes in number and subunit composition. We have found that pathway and target innervation directly influence the molecular composition of neurofilaments within regenerating optic axons of Xenopus laevis. Immunocytochemistry was used to examine neurofilaments within two abnormal visual pathways. The first was an aberrant, transient retinoretinal projection, which formed when some axons entered the contralateral optic nerve at the chiasm. The second was formed by regenerating axons deprived of their normal targets by surgical ablation of both optic tecta. Distal to an orbital nerve crush, the neurofilament proteins NF-L, NF-M, NF-H, and XNIF disappear from degenerating fibers. In normally regenerating axons, these neurofilament proteins emerge in a progression reminiscent of development. In the aberrant retinoretinal projection, levels of XNIF, NF-L, and -M remained lower than in normally regenerating axons, whereas NF-H and a phosphorylated form of NF-M were undetectable for at least 35 d after nerve crush. Normally, these two latter forms reappear between 15 and 21 d after surgery. Thus, this transient, incorrect axonal projection expressed neurofilaments in a very different pattern from correctly regenerating axons. In tecta-ablated frogs, staining of phosphorylation independent epitopes of XNIF, NF-L, and -M increased normally after axons entered the tract, but that of NF-H and phosphorylated NF-M remained low for at least 42 d after axotomy. Thus, separate parts of the visual pathway influence the complexity of neurofilaments.

???displayArticle.pubmedLink??? 7540681
???displayArticle.pmcLink??? PMC6577719
???displayArticle.link??? J Neurosci
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Species referenced: Xenopus laevis
Genes referenced: crot ctdspl neff1 nefm tecta.2 tfdp1
???displayArticle.antibodies??? Nif Ab1

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	Figure 1. Camera lucida drawings of representative sections showing the optic pathway at progressively more caudal levels in sections cut transversely through the head. A, Section at the level of the proximal optic nerve (on) near its exit from the eye (e). B, Section illustrating the optic nerve (on) approximately midway between the eye and the chiasm. C, Section illustrating the location of the optic tract (or), which runs along the surface of the brain at the arrowhead. D. Section cut at the level of the rostral optic tectum (t).
	Figure 2. Regenerating optic axons that have followed an anomalous pathway into the contralateral optic nerve. The regenerating axons were labeled by injection of WGA-HRP into the eye from which they originated. Sections were cut transversely and are viewed here under Nomarski optics. In all panels, lateral is to the left; dorsal is up. A, Labeled retinoretinal axons coursing through the contralateral chiasmatic optic nerve (con) just past the optic chiasm. The optic chiasm is located to the right of the edge of the panel. The arrowhead at or indicates labeled optic axons projecting normally into the contralateral optic tract (of). B, Labeled regenerating optic axons within the contralateral optic nerve (on), halfway between the eye and the optic chiasm (see Fig. 1B for orientation). C, Labeled regenerating optic axons within the proximal region of the contralateral optic nerve (on) (see Fig. 1A for orientation). D, Labeled regenerating optic axons entering the contralateral eye. The labeled axons are seen within the optic nerve head at the papilla of the retina @r) and extending into the neighboring retina. The vitreous (v) of the eye is to the left. The choroidal pigment (cp) is naturally black and does not represent WGA-HRP labelling. Scale bar in C applies to all panels.
	Figure 3. Regenerating optic axons in a crushed optic nerve, 5 weeks after surgery. Axons were labeled by immunofluorescent staining with antibodies directed against NF-M/ind (XC10C6, A), NF-M/P (H053, B), and NF-H (DP-1, C). The sections were taken at approximately the same level as shown in Figure 1B. Scale bar in C is for all panels.
	Figure 4. Regenerating optic axons m&outed into the contralateral optic nerve, 5 weeks after surgery, labeled either by intraocular injection of WGA-HRP (A) or by immunofluorescent staining with antibodies directed against various forms of the neurofilament proteins (B-D). The aberrantly projecting axons are indicated by the arrowheads in all panels. A, WGA-HRP labeled misprojected regenerating optic axons within the contralateral optic nerve, visualized with Nomarski optics. B, The section adjacent to A, stained with the antibody (XC10C6) directed against NF-M/ind and visualized with the fluorescence microscope. The staining of the misprojecting axons was less intense than that of the normal uninjured axons (ua), which are projecting normally through their own optic nerve. C, The next section adjacent to B, stained with the antibody (DP-1) directed against nonphosphorylated NF-H. No immunoreactivity was detectable in the misprojecting axons. D, The next section adjacent to C, stained with the antibody (H053) directed against NF-M/P. Again, no immunoreactivity was detected in the misprojecting axons. The sections were taken at approximately the same level as shown in Figure lB, and are from the same sections as those shown in Figure 3. Scale bar in D is for all panels.
	Figure 5. Regenerating optic axons that have followed a normal pathway into the contralateral optic tract, 4 weeks after a nerve crush. The regenerating axons are indicated by the arrowheads in all panels. A, Regenerating axons labeled with intraocularly injected WGA-HRP and viewed under Nomarski optics. B-D, Sections adjacent to that in A, immunostained with antibodies directed against nonphosphorylated epitopes of NF-M (B), NF-H (C), and phosphorylated NF-M (D), viewed under fluorescence. The sections were taken at the level shown in Figure 1C. Scale bar in D is for all panels.
	Figure 6. Transversely cut cross sections through the middle of the optic tecta from a normal frog (A) and from one whose optic tecta were ablated (B). The sections were stained with cresyl violet. The magnification is the same for both A and B.
	Figure 7. Projection of the regenerating optic axons of a tecta-ablated animal (C, D, F, G) as compared to that of a sham-operated control animal (A, B, E), 3 weeks after surgery. The regenerating optic axons were labeled by intraocular injection of WGA-HRP Sections were cut transversely. Lateral is to the left in A, C, E, F and to the right in B, D, G. Dorsal is up in all panels. A, Labeled regenerating optic axons within the contralateral optic tract (cot) in a sham-operated control animal. B, Labeled regenerating optic axons within the ipsilateral optic tract (iot) on the same section as shown in A. C, Labeled regenerating optic axons within the contralateral optic tract (cot), the nucleus Bellonci (nb), and the corpus geniculatum thalamicum (c) in a tecta-ablated animal. D, Labeled regenerating optic axons within the ipsilateral optic tract (id) on the same section as shown in C. A-D are taken from approximately the level illustrated in Figure 1 C. E, Labeled regenerating optic axons within the contralateral optic tectum (t) in the sham-operated, control animal. This section was taken at approximately the level shown in Figure 1D. F and G, Labeled regenerating optic axons within the posterior thalamic/pre-tectal nuclei @z) on both the contralateral (F) and the ipsilateral (G) sides of the tecta-ablated animal. These views are from sections located slightly rostra1 to those shown in Figure 1D and panel E. Scale bar in E is for all panels.
	Figure 8. Anti-NF-M/ind (XClOC6) immunofluorescent staining of the uncrushed (A) and regenerating, crushed (B) optic nerves of a shamoperated, control animal, as compared to those of a tecta-ablated animal (C, uncrushed; D, regenerating), 4 weeks after surgery. The sections were taken from a level near that shown in Figure IB. Scale bar in C is for all panels.
	Figure 9. Sections taken from the same animals, and neighboring those shown in Figure 8, but stained with the monoclonal antibody (HO5 directed against NF-M/l? A and B show the uncrushed and regenerating crushed nerves, respectively, of the sham-operated control; and C and show the comparable sections of the tecta-ablated animal. Scale bar in D is for all panels.
	Figure 10. Sections stained with the antibody (DP-1) directed against non-phosphorylated NF-H, taken from the same animals and neighboring those shown in Figures 8 and 9. The uncrnshed and regenerating crnshed nerves of the sham operated control are shown in A and B, respectively; and comparable views from a tectal ablated animal are shown in C and D. The unlabeled arrowheads in D point to the outer circumference of the otherwise unstained optic nerve. The arrowheads labeled on point to the optic nerves in A-C, whereas the arrowheads labeled pn point to neighboring peripheral nerves in all panels. Scale bar in D is for all panels.
	Figure Il. Regenerating, crushed optic nerves (on) from a sham operated control (A and C) and a tectal ablated (B and D) frog, 6 weeks after surgery. The sections in A and B were stained with the antibody directed against NF-H (DP-1), whereas those in C and D were stained with the antibody to NF-M/P (H053). A and C, and B and D represent two pairs of serial sections, respectively. The arrowheads labeled pn point to adjacent peripheral nerves. Scale bar in D is for all panels.