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NPJ Regen Med
2017 Jan 01;2:8. doi: 10.1038/s41536-017-0012-5.
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Serotonergic stimulation induces nerve growth and promotes visual learning via posterioreye grafts in a vertebrate model of induced sensory plasticity.
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The major goal of regenerative medicine is to repair damaged tissues and organ systems, thereby restoring their native functions in the host. Control of innervation by re-grown or implanted structures, and integration of the nascent nerves into behavioral/cognitive programs of the host, remains a critical barrier. In the case of sensory organs, this is particularly true, as afferent neurons must form connections with the host to communicate auditory, visual, and tactile information. Xenopus embryos and tadpoles are powerful models for such studies, as grafting techniques allow for the creation of eyes and other sensory structures along the body axis, and the behavior of the resulting organism can be quantitatively analyzed. Previous work has demonstrated that ectopic eyes could be grafted in blinded tadpoles, allowing some of the animals to learn in a simple light-preference assay. Here, we show that it is possible to improve the efficiency of the process in the context of a novel image-forming vision assay, using a drug already approved for human use. Innervation of the host by ectopic eyes can be increased by targeting a serotonergic signaling mechanism: grafts treated with a 5-HT1B/D agonist strongly innervate the recipient compared with untreated grafts, without large-scale disruption of the host nervous system. Blind animals possessing eye grafts with the augmented innervation demonstrate increased performance over untreated siblings in wavelength-based learning assays. Furthermore, treated animals also exhibit enhanced visual pattern recognition, suggesting that the increased innervation in response to 5-HT1B/D activation leads to enhanced functional integration of the ectopic organ with the host central nervous system and behavioral programs. These data establish a model system and reveal a new roadmap using small molecule neurotransmitter drugs to augment innervation, integration, and function of transplanted heterologous organs in regenerative medicine.
Fig. 1. Serotonin receptor 1B/D activation increases afferent innervation of eye grafts in Xenopus tadpoles. a Wild-type animals possess fully developed eyes by stage 47. b Microsurgery performed on stage 34 animals to remove developing eyetissue leads to blind animals that are otherwise healthy. c Grafts of eyeprimordia transferred from stage 24 donors to recipients result in ectopic eyes forming at the site of the graft. d Eyes forming from grafts display proper proximal-distal orientation with a clearly visible lens (arrow) facing outward from the body. e Grafts between tdTomato labeled donors and non-labeled recipients allow donor tissue to be visualized at later stages. f Higher magnification of the region posterior to the graft (white arrow) reveals little innervation of the host, with few if any neurites present in the fin or trunk region of the animal. g Treatment of grafts with the 5-HT1B/D activator Zolmitriptan does not alter the morphology of the ectopic eye. h Higher magnification of the region posterior to the graft (white arrow) reveals extensive afferent innervation in response to the treatment, with neurites present throughout the fin and trunk of the tadpole
Fig. 2. 5-HT1B/D-induced innervation is location-specific. a When developing eyetissue is removed in stage 24 embryos and replaced with labeled donor tissue of the same type, optic nerves are visualized (white arrow) crossing to the contralateral side of the brain and penetrating at the optic tecta. b Grafts at correct developmental locations do not change their innervation in response to 5-HT1B/D activation. c Animals with posterior grafts were visualized under fluorescence to determine if neurites reached the host brain. d Signal was never observed in the host brain (red arrows), e even when innervation was observed throughout the trunk and fin region of the animal (n = 20). Yellow boxes in (c) indicate location imaged at high magnification in (d) and (e)
Fig. 3. Time-lapse imaging of 5-HT1B/D-activated eye grafts reveals continued innervation and branching following treatment. a tdTomato labeled donor tissue was first observed innervating the host 4 days post-transplant. By 5 and 6 days post-transplant (b, c), neurites began to arborize and cover the ventralfin of the host. Arborization continued through days 7 and 8 (d, e), at which time high-branched neurites were present throughout the posterior of the recipient
Fig. 4. 5-HT1B/D activation does not alter host innervation. ai–iii Immunohistochemistry against acetylated-tubulin allowed visualization of lateral line components, nostrils, and motor axons. When comparing wild-type animals to 5-HT1B/D-activated (bi–iii) or 5-HT1B/D-activated blind animals (ci–iii), no differences were observed between treatments. a. aortic lateral line, a.l.lat anterior lower lateral line, c. caudal lateral line, m.a. motor axon, max. maxillary lateral line, mid.lat. middle lateral line, o.c. occipital lateral line, no. nostril, p. parietal lateral line, p.o. post-orbital lateral line, s.o. supra-orbital lateral line, u.lat. upper lateral line
Fig. 5. Eye grafts result in better performance in a visual assay when exposed to a 5-HT1B/D activator. a The training regime used with stage 48–49 tadpoles consisted of an innate preference test, a training phase, a rest period, and finally a learning probe. b Training and testing was executed using an automated training device. Motion tracking cameras under each arena recorded tadpole position/behavior and software issued punishments according to the training parameters. c Wild-type animals learn with high frequency in the device, while few blind animals show learning. Animals with untreated eye grafts do not learn at a greater frequency than blind individuals, while those with innervated grafts in response to 5-HT1B/D activation demonstrate significantly higher learning frequency than do blind animals. d When comparing basic behaviors between 5-HT1B/D innervated learners and non-learners, the only significant difference detected was that animals that learned in the device explored more of the arena than those which did not. e Comparing the time punished during training for 5-HT1B/D innervated animals reveals that learners receive significantly less punishments than individuals who do not learn in the device. n = 50, 38, 32, 43, and 24 for wild-type, wt + 5-HT1B/D, blind, ectopic eye, and ectopic eye + 5-HT1B/D, respectively. n = 8 for learners and 16 for non-learners. Asterisk indicates p ≤ 0.05. Values represent mean ± 1 standard deviation
Fig. 6.
Xenopus tadpoles follow a rotating pattern presented from below. a Schematic of rotation trial in which dishes containing 3–5 tadpoles were placed above a monitor that displayed a rotating patterns of triangles. Animals were given 15 min of the pattern rotating in the clockwise direction followed by 15 min in the counterclockwise direction. Behavior was scored and averaged across both phases of the trial. b Wild-type animals perform well in the assay, with 80% of individuals correctly following the direction of rotation and wild-type animals raised in a 5-HT1B/D agonist perform similarly. Blind animals and individuals with untreated eye grafts perform poorly, while animals with innervated grafts perform at intermediate levels. χ
2 = 18.76, p < 0.001. n = 34, 28, 25, 28, 23, for wild-type, wt + 5-HT1B/D, blind, ectopic eye, and 5-HT1B/D-activated, respectively. Values represent mean ± 1 standard deviation
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