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Cellular transplantation for photoreceptor replacement in retinal disease is limited by poor distribution, survival and integration of cells in vivo after standard delivery in saline vehicle. We were interested in addressing each of these barriers in order to improve transplant efficacy. To this end, we designed the first injectable biomaterial-based cell delivery vehicle to transplant adult stem cell progeny into the subretinal space of adult retina. A minimally-invasive and bio-resorbable blend of hyaluronan and methylcellulose (HAMC) was found to overcome cellular aggregation and non-contiguous distribution. The ability to direct stem cell differentiation toward a particular retinal lineage is another challenge facing clinical application. We showed that prospectively and clonally isolated multipotent mouse and human retinal stem cells (RSCs) could be directed toward a mature rod photoreceptor fate with the highest efficiency reported to date (>90%). Combinations of taurine and retinoic acid directed rod differentiation similar to rod development in vivo. RSC-derived rods exhibited morphology, protein and gene expression consistent with primary cultures of rods in vitro. When combined with the HAMC delivery vehicle, greater cell survival and improved integration of post-mitotic RSC-derived rods was observed in vivo compared to saline delivery. Improved donor rod survival was ascribed to the CD44 receptor – HAMC interaction in vitro and in vivo. Combined with HAMC delivery, disruption of the glial limiting membrane improved cell integration and resulted in the highest levels of integration of adult stem cell-derived rod photoreceptors relative to previous reports in the literature. Mature rod-lineage committed cells demonstrated higher integration potential compared to immature rods in this context. The integrated cells expressed Rhodopsin and elaborated outer segments. In the absence of the glial limiting membrane, rod integration depended on pro-survival signals from the environment. This work demonstrates that adult RSCs show promise for regenerative medicine strategies in the adult retina.

Syngnathidae (seahorse, pipefish, pipehorses & seadragons) are highly visual feeders with different species feeding on specific types of prey, a behaviour that has been related to snout length. Worldwide, many species have become threatened by habitat destruction, collection for the aquarium trade and exploitation for traditional medicine, as well as recreational and commercial bycatch. Attempts to establish aquaculture programs have been of limited success. Little is known about their visual capabilities in detail. The visual systems of fishes are known to have evolved specific adaptations that can be related to the colour of water in which they live and specific visual tasks such as predator detection and acquisition of food. This study examined the ocular and retinal morphology, photoreceptor structure and spectral sensitivity of adult individuals of a local pipefish (S. argus), local seahorse (Hippocampus subelongatus) which both inhabit green water seagrass beds, and a tropical species of seahorse (Hippocampus barbouri) from blue water coral reefs. Some juveniles were also investigated. Accordingly, we developed an understanding of the features that are common to all syngnathids and those that have evolved for specific environments. Cryosections of the eyes were taken to determine morphological distinctions of this group. Lens characteristics measured using a spectrophotometer determined 50% cut-off wavelengths below 408nm for all 3 species, hence no transmission of UV light to the retina. Histological examination determined a cone dominated fovea in the ventro-temporal retina and very large rods concentrated in the peripheral retina and adjacent to the optic nerve. Microspectrophotometry measured the absorption characteristics of the visual pigments within the photoreceptors showing the presence and maximum sensitivity (λmax) of rods, SWS single cones, and a broad, complex array of LWS double/twin cones. The results are discussed in relation to the light environment inhabited by each species and their feeding requirements. The implications for the design of suitable light environments for aquarium and aquaculture programs for the Syngnathidae are also discussed. Rearing success of this family of fish, for both the aquarium trade and re-stocking programs, would be advised to take lighting regimes and specifics of the animals’ vision into account

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains vi, 96 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 2011. / Printout. Includes bibliographical references (leaves 71-79). Also available on the World Wide Web.

My thesis has accomplished 3 significant contributions to neuroscience. Firstly, I have discovered a novel example of vertebrate deep-brain photoreception. Spontaneously generated fictive locomotion from the isolated nervous system of pro-metamorphic Xenopus tadpoles is sensitive to the ambient light conditions, despite input from the classical photoreceptive tissues of the retina and pineal complex being absent. The photosensitivity is found to be tuned to short wavelength UV light and is localised to a small region of the caudal diencephalon. Within this region, I have discovered a population of neurons immuno-positive for a UV-specific opsin protein, suggesting they are the means of phototransduction. This may be a hitherto overlooked mechanism linking environmental luminance to motor behaviour. Secondly, I have advanced the collective knowledge of how both nitric oxide and dopamine contribute to neuromodulation within motor control systems. Nitric oxide is shown to have an excitatory effect on the occurrence of spontaneous locomotor activity, representing a switch in its role from earlier in Xenopus development. Moreover, this excitatory effect is found to be mediated in the brainstem despite nitric oxide being shown to depolarise spinal neurons. Thirdly, I have developed a new preparation for patch-clamp recording in pro-metamorphic Xenopus tadpoles. My data suggest there are several changes to the cellular properties of neurons in the older animals compared with the embryonic tadpole; there appears to be an addition of Ih and K[sub](Ca) channels and the presence of tonically active and intrinsically rhythmogenic neurons. In addition, I have shown that at low doses dopamine acts via D2-like to hyperpolarise the membrane potential of spinal neurons, while at higher doses dopamine depolarises spinal neurons. These initial data corroborate previously reported evidence that dopamine has opposing effects on motor output via differential activation of dopamine receptor subtypes in Xenopus tadpoles.

Vision plays a key role in life of many vertebrates, and the performance of visual system is often adapted to specific environments inhabited by individual species. Fish colonized a wide range of habitats and adjusted their visual abilities to maximize their success rates in hunting, reproduction and predator avoidance. This thesis is focused on molecular mechanism of visual system, namely on genes for photoreceptor proteins, opsins, of two major groups of teleost fishes: African riverine cichlids (family Cichlidae, order Cichliformes, part of larger taxa Percomorpha), and European freshwater cyprinids (family Cyprinidae, order Cypriniformes, part of larger taxa Ostariophysi). Two types of photoreceptor cells are present on retina: the cones and the rods. Actinopterygian fishes in general have four cone opsin types (SWS1, SWS2, RH2 and LWS) used for colour (photopic) vision, and one rod opsin type (rhodopsins) for vision in deteriorated light conditions (scotopic vision). In the present thesis, I focus on 1) DNA sequence and amino acid substitutions of the opsin genes, and on 2) gene expression levels of opsins sensitive to various wavelengths of light spectrum. The results of my work show that both cichlids (family Cichlidae) and cyprinids (family Cyprinidae) have a complete set of opsin genes in...

A retina é composta por células neuronais especializadas, que quando danificadas são incapazes de se regenerar. CTIPD são uma promissória fonte para substituir o tecido lesado pois são capazes de se diferenciar em quase todos os tipos celulares. O objetivo do estudo é avaliar o potencial de diferenciação das CTIPD em neurônios retinais. As CTIPD foram submetidas a formação de neuroesfera, a qual foi avaliada pela expressão de nestin, <font face=\"Symbol\">b-III-tubulin e Pax-6. A partir da neuroesfera foi realizada a indução da diferenciação em neurônios retinais, a qual foi avaliada pela expressão dos marcadores específicos da diferenciação retinal. Observou-se a reação positiva com nestin, <font face=\"Symbol\">b-III-tubulin e Pax-6 na esfera, sugerindo um comprometimento com a linhagem neural/retinal. Nas células diferenciadas observamos uma maior expressão de Chx-10 e Crx e menor de Nrl, Calbindin, Recoverin e Rhodopsin. Este estudo sugere que CTIPD apresentam potencial de desenvolvimento em neurônios retinais. Entretanto, estudos adicionais são necessários para elucidar a formação da retina neural. / Retina is composed of specialized neuronal cells, which in case of damage are unable to regenerate. CTIPD is a promissory source to replace the injured tissue because they can differentiate into almost all cell types. The aim of the study was to evaluate the differentiation potential of CTIPD in retinal neurons. CTIPD were submitted to neurosphere formation and evaluated by the expression of nestin, <font face=\"Symbol\">b-III-tubulin and Pax-6. From the neurosphere was performed the induction of differentiation into retinal neurons, which was assessed by the expression of specific markers of retinal differentiation. We observed a positive reaction with nestin, <font face=\"Symbol\">b-III-tubulin and Pax-6 in the sphere, suggesting a commitment to the neural/retinal lineage. In differentiated cells we found a higher expression of Chx-10 and Crx and a lower expression of Nrl, Calbindin, Recoverin and Rhodopsin. This study suggests that CTIPD show potential development into retinal neurons. However, additional studies are needed to elucidate the formation of the neural retina.

A visão de cores em vertebrados necessita de pelo menos duas classes de cones, (fotorreceptores presentes na retina) e a existência de um substrato neural para que os fótons de luz sejam comparados, processados e posteriormente resultar na sensação da cor. Primatas do Velho Mundo, incluindo humanos, apresentam visão de cor tricromata, enquanto que primatas do Novo Mundo apresentam um polimorfismo nos genes dos pigmentos visuais e, entre os primatas, são os únicos que podem apresentar indivíduos com visão dicromata ou tricromata. O polimorfismo encontrado em primatas do Novo Mundo ocorre devido à variabilidade dos genes que expressam as opsinas responsáveis por absorver comprimentos de onda médios ou longos. Os estudos genéticos das opsinas são essenciais para compreensão do processamento e da sensação de cores nesses animais, e podem ajudar a entender a evolução da visão de cores nos Primatas. O objetivo deste trabalho é caracterizar a diversidade dos pigmentos visuais (LWS/MWS e SWS1) das espécies de primatas do Novo Mundo através de análises genéticas e descrever a sequência de aminoácidos observados para estimar o pico de sensibilidade espectral das opsinas. Foram coletadas amostras de sangue, fezes e/ou pelo de seis gêneros de primatas provenientes de diferentes regiões do Brasil (Pará, Rio de Janeiro, Rio Grande do Norte e São Paulo) e pertencentes às espécies Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth e Brachyteles arachnoides e posteriormente foram analisados os genes que expressam as opsinas nesses indivíduos. As sequências de aminoácidos encontradas nas posições importantes do gene SWS1 (52, 86, 93, 114 e 118) foram diferentes para algumas espécies. No gene SWS1 as espécies C. apella, L. lagotricha, A. belzebuth e B. arachnoides apresentam a sequência de aminoácidos LLPAT e as espécies C. jacchus, A. caraya e A. clamitans apresentaram a sequência de aminoácidos LLPGT. Foi descoberto que variações de aminoácidos na posição 50 do gene SWS1 em primatas do Novo Mundo podem ser importantes na determinação do pico de absorção espectral dos pigmentos expressos por este gene. Os genes LWS e MWS de indivíduos da espécie C. jacchus foram estudados e os aminoácidos localizados nas posições 180, 277 e 285 das opsinas foram identificados. Os resultados dos alelos encontrados nesses grupos tiveram cinco combinações diferentes (SFT, SYA, SYT, AYA e AYT), os alelos AYA e SYA foram descritos pela primeira vez neste grupo e a partir do resultado genético foi inferido o pico de absorção espectral da opsina. Este trabalho preencheu algumas lacunas da bibliografia e trouxe novas informações a respeito da diversidade genética dos pigmentos visuais em primatas do Novo Mundo / Color vision in vertebrates requires the presence of at least two different classes of cones in the retina, and a neural substrate capable to compare the activation of the different photoreceptors, which ultimately leads to color perception. Old World Monkeys (OWM), including humans, have trichromatic color vision, whereas New World Monkeys (NWM) have visual pigment genes polymorphism and among primates, are the only group with dichromatic or trichromatic individuals in the same species. This polymorphism in NWM occurs due to the variability of genes that express the opsins responsible for absorbing medium or long wavelengths. The genetic studies of color vision are fundamental for the comprehension of color perception in these animals and it could help to understand the color vision evolution in Primates. The aim of this work is to characterize the visual pigment diversity (LWS/MWS and SWS1) in NWM species by genetic analysis and estimate the opsin spectral absorption peak, based on the amino acid sequence. Blood, feces and hair were collected from six primate genres from different regions of Brazil (Pará, Rio de Janeiro, Rio Grande do Norte and São Paulo): Cebus apella, Callithrix jacchus, Alouatta clamitans, Alouatta caraya, Lagothrix lagothricha, Ateles belzebuth and Brachyteles arachnoides. The amino acid sequences found in important positions of the SWS1 gene (52, 86, 93, 114 and 118) were different among some species. In C. apella, L. lagotricha, A. belzebuth and B. arachnoides was found the amino acid sequence LLPAT. In C. jacchus, A. caraya and A. clamitans the amino acid sequence was LLPGT. It was observed in previous studies that residue 50 of the SWS1 gene in the New World primates is important to determining the spectral absorption peak of the visual pigments expressed by this gene. The LWS and MWS genes of C. jacchus have been studied and the amino acids located at positions 180, 277 and 285 have been identified. Five different combinations were found among the individuals analyzed: SFT, SYA, SYT, AYA and AYT. Two alleles, AYA and SYA, were described for the first time in this species. The present study filled some gaps in the literature and brought new information on the genetic diversity of visual pigments in New World primates

Les photorécepteurs sont des neurones très spécifiques dédiés à la phototransduction et reposant sur une machinerie cellulaire très complexe. La dépolarisation permanente dans le noir des photorécepteurs déclenche une transmission synaptique constante et extrêmement spécifique qui requièrent une quantité d'énergie considérable. Les photorécepteurs peuvent dégénérer lorsque la phototransduction ou l'apport énergétique sont altérés. Le syndrome d'Usher conduit à une surdité et une cécité. La recherche du rôle des protéines usher dans les photorécepteurs a été freinée par l'absence de phénotype rétinien dans les modèles. De la même façon, la compréhension des mécanismes moléculaires conduisant à l'atteinte des cônes dans la rétinopathie diabétique a été entravée par l'absence de symptômes vasculaires et neuronaux dans les modèles. Durant ma thèse, j'ai caractérisé deux modèles animaux des syndromes Usher et HANAC. Des atteintes neuronales ont été démontrées par électrorétinogramme et par l'observation de changements morphologiques des cellules. Dans les modèles Usher, j'ai également montré une neuroprotection des photorécepteurs par plusieurs stratégies. Dans le modèle HANAC, les atteintes neuronales étaient associées à une tortuosité vasculaire anormale une augmentation de la perméabilité vasculaire et l'expression accrue de VEGF. Les évaluations phénotypiques de ces trois modèles fournissent un nouvel aperçu de la physiopathologie des dégénérescences des cônes dans le syndrome d'Usher et dans les maladies vasculaires complexes. Ce travail ouvre surtout la voie au développement et à l'évaluation de nouvelles stratégies thérapeutiques pour ces maladies menant à la cécité. / Photoreceptors are very specific neurons dedicated to phototransduction, which relies on very complex machinery. The maintained depolarization in darkness triggers a constant and thus very specific type of synaptic transmission. These require high energy need. As a consequence, photoreceptors can degenerate in various hereditary retinal diseases when phototransduction or energy consumption are altered. The Usher syndrome is such a hereditary disease leading to both deafness and blindness. If Usher proteins are involved in the mechanotransduction in hair cells, investigating their role in photoreceptors has been hamperedby the lack of a retinal phenotype in murine models. Similarly, understanding themolecular mechanisms of cone dysfunction in diabetic retinopathy has beenhampered by the lack of vascular and neuronal symptoms and neuronal models. During my PhD, I have developed animal models of Usher and HANAC syndromes both leading to cone photoreceptor dysfunction and damage. Cone dysfunction was demonstrated by electroretinogram recording and by morphological changes, retinal gliosis and microglial activation. In the Usher models, I also demonstrated photoreceptor neuroprotection by different strategies. In the HANAC model, neuronal dysfunction was associated as in diabetic retinopathy to blood vessel tortuosity, blood vessel permeability and incresead VEGF expression levels. These phenotypic evaluations of mouse models provide new insights into the physiopathology of cone photoreceptor degeneration in Usher syndrome and in complex vascular diseases. It also open the way for the development and assessment of new therapeutic strategies for these diseases leading to blindness.