McKeown CR et al. (2013), Neurogenesis is required for behavioral recover...

XB-ART-46333

J Comp Neurol 2013 Jul 01;52110:2262-78. doi: 10.1002/cne.23283.

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Neurogenesis is required for behavioral recovery after injury in the visual system of Xenopus laevis.

McKeown CR , Sharma P , Sharipov HE , Shen W , Cline HT .

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Nonmammalian vertebrates have a remarkable capacity to regenerate brain tissue in response to central nervous system (CNS) injury. Nevertheless, it is not clear whether animals recover lost function after injury or whether injury-induced cell proliferation mediates recovery. We address these questions using the visual system and visually-guided behavior in Xenopus laevis tadpoles. We established a reproducible means to produce a unilateral focal injury to optic tectal neurons without damaging retinotectal axons. We then assayed a tectally-mediated visual avoidance behavior to evaluate behavioral impairment and recovery. Focal ablation of part of the optic tectum prevents the visual avoidance response to moving stimuli. Animals recover the behavior over the week following injury. Injury induces a burst of proliferation of tectal progenitor cells based on phospho-histone H3 immunolabeling and experiments showing that Musashi-immunoreactive tectal progenitors incorporate the thymidine analog chlorodeoxyuridine after injury. Pulse chase experiments indicate that the newly-generated cells differentiate into N-β-tubulin-immunoreactive neurons. Furthermore, in vivo time-lapse imaging shows that Sox2-expressing neural progenitors divide in response to injury and generate neurons with elaborate dendritic arbors. These experiments indicate that new neurons are generated in response to injury. To test if neurogenesis is necessary for recovery from injury, we blocked cell proliferation in vivo and found that recovery of the visual avoidance behavior is inhibited by drugs that block cell proliferation. Moreover, behavioral recovery is facilitated by changes in visual experience that increase tectal progenitor cell proliferation. Our data indicate that neurogenesis in the optic tectum is critical for recovery of visually-guided behavior after injury.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 msi1 sox2 tecta.2 tubb2b
???displayArticle.antibodies??? H3f3a Ab9 Msi1 Ab2 Tubulin I+II Ab1

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	Fig. 1. Xenopus laevis tadpoles exhibit an avoidance response to moving stimuli. A: Visual avoidance behavior apparatus. Animals are placed in the chamber and a moving stimulus is presented from below. Video images are captured from above. B: Still frames of a video sequence showing stage 47 tadpoles' behavioral response to the upward moving stimulus (66.67 ms/frame, every other frame is shown). Tadpoles do not respond to stationary dots (B, i–iv, left panels) and swim in a straight trajectory, as shown in the drawing (B, v). In contrast, tadpoles exhibit an avoidance behavior in response to moving stimuli (B, vi–ix, right panels) and abruptly change their trajectory upon an encounter with a perpendicularly approaching dot, indicated by arrow in the drawing of the swim trajectory (B, x). Time stamps shown in panels Bi–iv also correspond to Bvi–ix. C: Both pigmented and albino tadpoles similarly display a strong avoidance behavior to a stimulus size of 0.4 cm in diameter (n = 12 animals each).
	Unilateral injury to the optic tectum. A: Schematic of the injury paradigm. Stage 47 tadpoles were surgically injured by aspirating a focal region of optic tectal cells (black) from the caudal right tectal lobe, caudomedial to the region of retinal axon innervation (gray). B,C: DIC images show the tectal lobe before surgery (B) and after surgery (C). Arrows in C indicate wound area. D: Quantification of the tectal volumes before and after surgery. Surgery decreases the volume of the right tectal lobe without affecting the volume of the left tectal lobe. n = 16 animals, P < 0.01. E,F: Single optical sections of fluorescent FM4-64 membrane dye labeling before (E) and after surgery (F). The tectal lobes are outlined in white. The injury site in the right lobe was between the arrows. G,H: DiI labeling of the RGC axons in intact (G) and injured (H) tecta. Injury does not alter gross morphology of the RGC axon arbors. Scale bars = 100 μm in B–F; 30 μm in G,H.
	Fig. 4. Phospho-histone 3 (PH3) immunoreactivity increases after injury. The right tectal lobe of stage 47 tadpoles was injured and changes in cell division were assessed in the right and left tectal lobes by immunolabeling for PH3 over the course of 5 days to identify dividing cells in M phase of the cell cycle. Data were analyzed as individual optical sections, but are presented as confocal Z-projections of the tectal lobes, which are outlined. A–J: PH3 labeling of dividing cells in the tectal lobes 24 hours (A,B), 48 hours (C,D), 3 days (E,F), 4 days (G,H), and 5 days (I,J) after injury in the intact left tectal lobe (A,C,E,G,I) and in the injured right tectal lobe (B,D,F,H,J) of the same animals. K: Total counts of PH3-labeled nuclei in the injured (black line) and intact (gray line) tecta over a 5-day period after injury. n = 25 animals total (5 for each timepoint). P < 0.05, *P < 0.01, n.s. = not significant. Data are average ± SEM. Scale bars = 100 μm.
	Figure 5. Cell Proliferation increases after injury. The right tectal lobe of stage 47 tadpoles was injured and changes in cell proliferation were assessed by exposing animals to IdU for 2 hours. Data were analyzed as individual optical sections, but are presented as confocal Z-projections of the entire tectum from whole-mount brains. A–C: IdU incorporation in the tectum 2 hours after injury (A), 24 hours after injury (B), and 48 hours after injury (C). D: Total cell counts of IdU-positive cells per tectal lobe in the injured vs. intact tectum over a 48-hour period after injury. n = 15 animals total (5 for each timepoint), **P < 0.01. Data are average ± SEM. Scale bar = 100 μm.
	Figure 6. Proliferating cells are musashi1-expressing neural progenitor cells. Stage 47 tadpoles were injured in the right tectum. Two days later animals were exposed to CldU for 2 hours and processed immediately for CldU immunolabeling. A–F: Representative single optical sections from confocal images of 30-μm sections through the optic tectum of tadpoles labeled with antibodies to musashi1 (Msi1, green) and CldU (magenta). D–F: enlargements of the boxed area marked in C. 93.3 ± 2.5% of CldU-positive cells in injured tectal lobes were double-labeled with musashi1 antibodies (n = 7 animals). Scale bars = 50 μm in C and 20 μm in F.
	Figure 7. Injury increases the generation of new neurons. The right tectal lobe of stage 47 tadpoles was injured. Two days later tadpoles were then exposed to CldU for 2 hours in rearing solution and were allowed to develop in the absence of CldU for 2 days. A–F: Representative images of single optical sections of N-β-tubulin (green; A,C,D,F) and CldU (magenta; B,C,E,F)-immunoreactivity in 30-μm vibratome sections. D–F: Enlargements of boxed area in C. Scale bars = 50 μm in C; 20 μm in F.
	Figure 8. Tectal cells generated after injury differentiate into neurons. In vivo time-lapse images of tectal cells labeled by expression of turboGFP in Sox2-expressing neural progenitors cells at the time of injury. Time-lapse images of the uninjured (A–D) and injured (E–H) tecta collected 1, 2, 4, and 7 days after injury are shown. Scale bar = 70 μm.

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Banerjee, A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin. 1988, Pubmed