Thesis (M.S.)--Miami University, Dept. of Zoology, 2008. / Title from first page of PDF document. Includes bibliographical references (p. 24-29).

No description available.

In contrast to mammals, zebrafish possess a high regenerative capacity even within the central nervous system including the retina. In the retina, lesions result in a loss of neurons and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate into all types of retinal neurons. Eventually the anatomical tissue architecture is restored within a few weeks or months depending on the degree of lesion. However, little is known about how retinal lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, quantitative behavioral assays were applied to assess visual function during homeostasis and regeneration in adult zebrafish. While the dark-light preference test has been identified to be too variable to assess visual function, a novel visiondependent social preference test as well as an automated two level OKR-based vision test have proved to be more suitable. Two different lesion paradigms have been compared in this study: light-induced lesions that target photoreceptor cells and neurotoxin-mediated lesions that act in a concentration-dependent manner either affecting only inner retinal neurons or all retinal cell types. It could be shown that visual function was significantly impaired after light lesion but fully though gradually recovered within 28 days. Vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7–10 days post lesion while vision under more demanding conditions - with low contrast and high level of detailwas regained only from 14 days post lesion onwards. The corresponding retinal morphology was monitored in vivo using optical coherence tomography and revealed a close correlation between structure and function. Injection of neurotoxins resulted in a multitude of morphological changes that impeded the analysis of functional recovery. In general, restoration of visual function after neurotoxin-mediated lesion was limited and highly dependent on the initial degree of damage. As suggested by the behavioral studies, visual functions are, at least partially, restored after lesion. However, no conclusions can be drawn on the restoration of the underlying retinal circuitry. Hence, large-scale electrophysiological recordings using multielectrode arrays have been established for the adult zebrafish retina allowing the recording of spontaneous and light-driven activity. This technique can be exploited to analyze the restoration of signal processing of the retina in future studies. In addition, the established tools can be applied to characterize visual mutants and models of retinal degenerative diseases both morphologically and functionally as exemplarily demonstrated here for the blind atoh7 mutant. It was shown that the adult atoh7mutant largely resembles the larval phenotype and displays distinct alterations in optic tectum structure as well as impairments of social behavior. Altogether, the established vision-dependent behavioral assays suggest that the degree and kinetics of functional recovery are highly dependent on the lesion paradigm as well as quality of morphological restoration. In combination with the established multielectrode array recordings, the field is provided with new readouts to study functional recovery that can be used in addition to existing criteria to assess regeneration of the adult zebrafish retina. The established toolbox can furthermore be exploited to analyze retinal function not only in a regeneration context but also in visual mutants, disease models or ageing. Eventually, a deeper understanding of the regenerative processes in zebrafish will provide new perspectives for the development of therapies for retinal degenerative diseases.

No description available.

[Truncated abstract] Microenvironmental changes associated with apoptotic neural degeneration may instruct a proportion of newly transplanted donor cells to differentiate towards the fate of the deteriorating host cellular phenotype. In the work described in this thesis, this hypothesis was tested by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult rats and mice, and then examining whether intravitreally grafted cells from a range of sources of donor neural tissue became incorporated into these selectively depleted retinae. Donor tissues were: a postnatal murine cerebellar-derived immortalised neural precursor cell line (C17.2); an adult rat hippocampal-derived clonal stem-like line (HCN/GFP); mouse embryonic day 14 (E14) primary dissociated retinal cells (Gt[ROSA]26); and adult mouse ciliary pigmented margin-derived primary neurospheres (Gt[ROSA]26). In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), and in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Some adult hosts received ON transection coupled with an autologous peripheral nerve (PN) graft. Donor cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0, 5, 7 or 14 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified, quantified and their differentiation fate within host retinae was assessed. Transplanted male C17.2 cells were identified in host retinae using a Y-chromosome marker and in situ hybridisation, or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal) using Xgal histochemistry or a beta-gal antibody. No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were, however, stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin (TUJ1), a protein highly iii expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased C17.2 cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.

In comparison to mammals, zebrafish are able to regenerate many organs and tissues, including the central nervous system (CNS). Within the CNS-derived neural retina, light lesions result in a loss of photoreceptors and the subsequent activation of Müller glia, the retinal stem cells. Müller glia-derived progenitors differentiate and eventually restore the anatomical tissue architecture within 4 weeks. However, little is known about how light lesions impair vision functionally, as well as how and to what extent visual function is restored during the course of regeneration, in particular in adult animals. Here, we applied quantitative behavioral assays to assess restoration of visual function during homeostasis and regeneration in adult zebrafish. We developed a novel vision-dependent social preference test, and show that vision is massively impaired early after lesion, but is restored to pre-lesion levels within 7 days after lesion. Furthermore, we employed a quantitative optokinetic response assay with different degrees of difficulty, similar to vision tests in humans. We found that vision for easy conditions with high contrast and low level of detail, as well as color vision, was restored around 7–10 days post lesion. Vision under more demanding conditions, with low contrast and high level of detail, was regained only later from 14 days post lesion onwards. Taken together, we conclude that vision based on contrast sensitivity, spatial resolution and the perception of colors is restored after light lesion in adult zebrafish in a gradual manner.