Дисертації з теми "Visual photoreceptors"

Abstract When one asks about the properties of visual signals stimulating nervous systems, the ultimate interest lies in determining how the signal is encoded and transferred across the receptor barrier, how much and what kind of information is passed further to the higher visual centers and what is lost. The research presented in this dissertation attempts to explore quantitatively some detailed aspects of information processing by microvillar photoreceptors. Until recently, three methods were used to measure or estimate information transfer from the visual scene to the photoreceptor: Shannon’s information capacity, the closely related linear coherence rate and the compression entropy rate. In the first research article of this dissertation, a novel information calculation method based on principal component analysis, the mutual information rate, was developed. In the second publication, the influence of a physiological delay in the photoreceptor response on the information rate estimates by the Shannon method-related coherence rate algorithm was explored and a technique to compensate the associated error was proposed. The third study addresses the question of whether photoreceptors can more efficiently transfer information arriving from natural sources than from common artificial visual stimuli. Natural stimuli have interesting statistical properties in the form of higher order correlations (HOC), arising from the presence of features representing surfaces, textures, and object boundaries. This problem was investigated in the most extensive study to date, using blowfly Calliphora vicina photoreceptors as a model. The individual photoreceptors encode input information by a form of Weber’s law, with the HOC in natural sequences reducing information transfer by decreasing the number of local contrast events that exceed the noise-imposed threshold.

I examine whether it is possible for the temporal resolution and reliability of a photoreceptor to vary across the eye. This possibility has not been addressed before in any animal and yet it is important for our understanding of how visual systems may be designed to register patterns of optic flow or to track moving targets. In the male blowfly, Calliphora vicina, I show that both spatial and temporal acuity are higher at the front of the eye and fall off with retinal eccentricity. The particular pattern of tuning observed emphasises the importance of tracking to the male fly, a behaviour commonly observed in flies prior to mating. To investigate this possibility further, in Chapter 3 I compare the responses of male and female photoreceptors to simulated target stimuli. For this purpose I use the housefly, Musca domestica, a species whose anatomical and behavioural sex-differences are well documented. The male photoreceptor responds much more powerfully to small moving targets than the female and response amplitude greatly exceeds that predicted from conventional models of photoreceptors dynamics. In particular, the male photoreceptor boosts the signals generated by targets moving within the behavioural regime of distances and speeds. These results allow the limits of male pursuit vision to be determined and demonstrate the impact of behaviour on retinal function. Adaptation of photoreceptor sensitivity and temporal resolution to ambient illumination is a well-known phenomenon. This process is usually considered to be complete within seconds. However, I show in M. domestica that significant improvements in temporal resolution and reliability can occur over a much longer period. These improvements are derived from a more consistent registration of the timing of photon absorption events and appear to be associated with a reduction in rhabdomeral surface area.

Un défi majeur dans le traitement des maladies dégénératives de la rétine par transplantation de photorécepteurs de remplacement est la difficulté d'induire les cellules greffées à croître et à maintenir des segments externes sensibles à la lumière dans la rétine hôte, qui dépend d'une interaction adéquate avec l'épithélium pigmentaire rétinien (EPR) sous-jacent. Pour une approche de traitement indépendante de l'EPR, nous avons introduit une opsine microbienne hyperpolarisante dans les précurseurs de photorécepteurs provenant de souris nouveau-nées et les avons transplantés dans des souris aveugles dépourvues de la couche photoréceptrice. Ces photorécepteurs transformés optogénétiquement ont réagi à la lumière et leur transplantation a permis de rétablir la fonction visuelle, comme en témoignent les enregistrements des cellules ganglionnaires et les tests comportementaux. Par la suite, nous avons généré des photorécepteurs à cônes à partir de cellules souches humaines pluripotentes induites (hiPSCs), exprimant Jaws, un autre opsine hyperpolarisante. Après la transplantation chez des souris aveugles, nous avons observé des réponses à la lumière au niveau des photorécepteurs et des cellules ganglionnaires. Ces résultats démontrent que la réparation structurale et fonctionnelle de la rétine est possible en combinant la thérapie par cellules souches et l'optogénétique
A major challenge in treatment of retinal degenerative diseases with transplantation of replacement photoreceptors is the difficulty in inducing the grafted cells to grow and maintain light-sensitive outer segments in the host retina, which depends on proper interaction with the underlying retinal pigment epithelium (RPE). For an RPE-independent treatment approach, we introduced a hyperpolarizing microbial opsin into photoreceptor precursors from newborn mice, and transplanted them into blind mice lacking the photoreceptor layer. These optogenetically transformed photoreceptors were light responsive and their transplantation lead to the recovery of visual function, as shown by ganglion cell recordings and behavioral tests. Subsequently, we generated cone photoreceptors from human induced pluripotent stem cells (hiPSCs), expressing the chloride pump Jaws. After transplantation into blind mice, we observed light-driven responses at the photoreceptor and ganglion cell level. These results demonstrate that structural and functional retinal repair is possible by combining stem cell therapy and optogenetics

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

Understanding how individual photoreceptor cells factor in the spectral sensitivity of a visual system is essential to explain how they contribute to the visual ecology of the animal in question. Existing methods that model the absorption of visual pigments use templates which correspond closely to data from thin cross-sections of photoreceptor cells. However, few modeling approaches use a single framework to incorporate physical parameters of real photoreceptors, which can be fused, and can form vertical tiers. Akaike’s information criterion (AIC c ) was used here to select absorptance models of multiple classes of photoreceptor cells that maximize information, given visual system spectral sensitivity data obtained using extracellular electroretinograms and structural parameters obtained by histological methods. This framework was first used to select among alternative hypotheses of photoreceptor number. It identified spectral classes from a range of dark-adapted visual systems which have between one and four spectral photoreceptor classes. These were the velvet worm, Principapillatus hitoyensis , the branchiopod water flea, Daphnia magna , normal humans, and humans with enhanced S-cone syndrome, a condition in which S-cone frequency is increased due to mutations in a transcription factor that controls photoreceptor expression. Data from the Asian swallowtail, Papilio xuthus , which has at least five main spectral photoreceptor classes in its compound eyes, were included to illustrate potential effects of model over-simplification on multi-model inference. The multi-model framework was then used with parameters of spectral photoreceptor classes and the structural photoreceptor array kept constant. The goal was to map relative opsin expression to visual pigment concentration. It identified relative opsin expression differences for two populations of the bluefin killifish, Lucania goodei . The modeling approach presented here will be useful in selecting the most likely alternative hypotheses of opsin-based spectral photoreceptor classes, using relative opsin expression and extracellular electroretinography.

The role of the retinal pigment epithelial cells on the viability and renewal of photoreceptors has been well demonstrated in the Royal College of Surgeons (RCS) strain of rat. These rats are characterized by an inherited time-dependent degeneration of their photoreceptors. This degeneration is apparently due to the inability of the retinal pigment epithelial cells to adequately ingest fragments of photoreceptor membrane that are shed during the course of photoreceptor membrane renewal. The buildup of photoreceptor material in the interphotoreceptor space ultimately leads to the degeneration of photoreceptors in these animals. With regard to the pigment epithelial cells, neither the mechanism mediating the ingestion process in normal rats nor the nature of the defect of this process in RCS rats is understood.It is the goal of this proposed research to assay for the presence of phospholipase C in retinal pigment epithelial (RPE) cells and to determine possible modulators of the enzyme in an attempt to associate this with the process of phagocytosis.
Department of Biology

Abstract Sensory systems are considered to be optimized for their ecological niche. In vision this means highly organised regular structure and function, where nearly identical photoreceptors have graded light responses in order to be able to handle as much information as possible. Instead, cockroach compound eyes show large amounts of irregularities in their optics and structure, and unusually long axons. In this thesis photoreceptors of the cockroach were studied with intracellular recordings of their light responses, biophysical systems analysis, and modelling of the relations between the light stimuli and responses. Cockroaches prefer living in dark or extremely dim environments. However, they have large and complex compound eyes. The aim of this study was to find out the functional properties by which the visual system and especially photoreceptors have adapted to cope with, i.e. to see in, dim light conditions. The function of photoreceptors was found to vary randomly in many respects, and the long axons seemed to utilise action potential coding of visual signals. Through model simulations it was shown that signals of a group of these functionally variable and spiking photoreceptors, when pooled, could provide more reliable coding than signals of identical cells of any experimentally characterised type. This naturally sacrifices spatial resolution. The filtering dynamics of the photoreceptors is matched to low light intensities and their temporal resolution does not markedly improve with increasing light adaptation. Adaptation processes in the photoreceptors saturated near an intensity of about 1000 effective photons/s. These are all both unexpected and novel features of photoreceptor function. Spatial summation of functionally different photoreceptors and reduced temporal resolution and contrast coding abilities can be considered to be permanent optimizations to a dim environment.

Introdução: O tratamento do buraco macular idiopático ainda é acompanhado de incertezas em relação ao seu prognóstico. Ao longo dos últimos anos, têm sido utilizados o tempo de história, o tamanho e a acuidade visual pré-operatória dos buracos como critérios de orientação quanto ao resultado visual final. O aspecto final da parte mais externa da retina foveal tem merecido especial atenção em relatos recentes. Neste trabalho, avaliou-se, por meio da tomografia de coerência óptica, a integridade da camada de fotorreceptores, correlacionando o seu aspecto final com a acuidade visual obtida em casos de buraco macular idiopático, fechados cirurgicamente. Métodos: Pacientes operados com sucesso e com seguimento mínimo de 12 meses foram submetidos à avaliação oftalmológica completa incluindo a medida da acuidade visual final em escala logMar, biomicroscopia de mácula, oftalmoscopia indireta e angiofluoresceinografia. Colheram-se dados sobre idade, sexo, raça, tempo de história e tamanho do buraco além da acuidade visual pré-operatória. Para a classificação das lesões, utilizaram-se os critérios definidos por Gass. Imagens tomográficas da camada correspondente aos fotorreceptores da retina na região foveal foram criteriosamente obtidas através da tomografia de coerência óptica. Uma avaliação qualitativa desta camada foi realizada por examinador experiente, de forma mascarada, com os pacientes então classificados em três grupos (1, 2 e 3), em ordem crescente de integridade. Resultados: Foram incluídos no estudo 39 olhos de 37 pacientes, sendo eles 22 mulheres (59,5%) e 15 homens (40,5%). A idade média foi de 68,2 anos variando entre 51 e 83 anos. O tempo de seguimento pós-operatório médio foi de 38,5 meses. Entre todos os olhos estudados, 12 foram incluídos no grupo 1 (menor integridade); sete, no grupo 2 e vinte, no grupo 3. Na comparação entre os grupos 1 e 3, a acuidade visual final melhorou em ambos; porém, com maior intensidade no grupo 3 (p<0,001). O tamanho pré-operatório do buraco e o tempo de história da doença não impactaram significativamente o resultado visual final (p=0,223 e p=0,559, respectivamente). A acuidade visual pré-operatória média foi maior no grupo 3 que no grupo 1 (p=0,012). Conclusões: Os dados obtidos através deste estudo sugerem correlação entre uma maior integridade da camada dos fotorreceptores e um melhor resultado visual de longo prazo, em pacientes com buraco macular idiopático, operados com sucesso. Embora a visão tenha melhorado em todos os grupos, a melhora foi significativamente maior no grupo 3 que no grupo 1. Em relação ao tamanho pré-operatório da lesão e o tempo de história da doença, não se observou impacto significativo no resultado. Uma melhor acuidade visual inicial pré-operatória pode influenciar positivamente a acuidade visual final.
Purpose: To evaluate the integrity of the retina photoreceptor layer as determined by optical coherence tomography and its relationship with final visual outcome in successfully closed idiopathic macular holes. Methods: Successfully operated patients with a minimal follow-up of 12 months, underwent complete ophthalmic exam involving post-operative best corrected visual acuity using logMar scale, macula biomicroscopy and indirect ophthalmoscopy and fluorescein angiography. Complete epidemiologic data was collected as well as time duration, pre-op visual acuity, and size and Gass classification of the holes. Optical coherence tomography images of the layer corresponding to the transition between inner and outer segments of the photoreceptors were meticulously generated using commercially available Stratus OCT (OCT3). Qualitative analysis of this layer was carried out, in a masked fashion, by a certified and well experienced examiner. Based on increasing integrity showed in the images, patients were then distributed in 3 groups: 1, 2 and 3. Group 1 integrity from 1 to 33%, group 2 from 34 to 66%, and group 3 from 67 to 100%. Results: A total of 39 eyes of 37 patients were included in the study. Among all patients, 12 were included in group 1, 7 in group 3 and 20 in group 3. There were 22 female (59,5%) and 15 male (40,5%). Mean age was 68.2 years varying from 51 to 83 years of age. Mean post-op follow-up time was 38.5 months. When comparing groups 1 and 3, best corrected final visual acuity improved in both groups but dramatically in group 3 (better integrity) (p<0.001). Pre-operative size and time duration of the hole did not correlate with better visual acuity (p=0.223 and p=0.559 respectively). Mean pre-op visual acuity was better in group 3 than in group 1 (p=0.012). Conclusion: Data obtained in our study indicate that there is a strong correlation between better integrity of the photoreceptor layer, as determined by optical coherence tomography, and a more successful final, long term, visual acuity in patients with idiopathic macular hole successfully operated. Although final visual acuity has improved in all groups, there was statistical difference between groups 1 and 3. Pre-operative size and time duration of the hole did not have impact on final visual acuity. Better pre-op visual acuity may have an effect on final outcome.

Las enfermedades neurodegenerativas cursan con la degeneración y muerte de las neuronas las cuales son incapaces de suplir su propia muerte. Debido a esta característica enfermedades como el Alzheimer, el Parkinson o el Glaucoma no tienen cura en la actualidad. La retina es una proyección del sistema nervioso central (SNC) encapsulada en el globo ocular y aislada del resto del SNC, lo que la hace fácilmente accesible a la manipulación experimental. En este trabajo estudiamos las alteraciones funcionales de la retina con el electrorretinograma de campo completo (ERG), técnica basada en el registro de la respuesta eléctrica retiniana tras la presentación de un estímulo de luz homogéneo. Así hemos estudiado los efectos de la sección del nervio, del aumento de presión intraocular y de la fototoxicidad, dichas lesiones afectan selectivamente a determinadas poblaciones neuronales retinianas e imitan enfermedades neurodegenerativas como el Glaucoma o la Degeneración Macular Asociada a la Edad.
Neurodegenerative diseases are characterized by degeneration and death of neurons which are unable to recover after a given insult, thus impairing functional recuperation. Because of this, there is no cure for diseases such as Alzheimer, Parkinson or Glaucoma. The retina is a projection of the central nervous system (CNS) located in the eye and therefore, isolated from the rest of the CNS. This makes the retina a very good model for experimental manipulation. In this work we have studied the functional changes in the retina by full field electroretinograme (ERG). This technique records the electric response of the retina after presentation of homogeneous light stimuli. We have studied the effects that optic nerve sections, increase of the intraocular pressure and phototoxicity have on ERG recordings. These lesions impair selectively certain retinal neuronal populations and imitate neurodegenerative diseases as Glaucoma or Age-related Macular Degeneration.

Photoreceptor replacement therapy is a promising strategy for treating retinal degenerative diseases leading to blindness. Previous work by our lab has demonstrated proof-of-concept that mouse embryonic stem cells (ESCs) can be differentiated, in a 3D culture system, into post-mitotic rod photoreceptor precursors for transplantation. However, rescue of visual function following transplantation of ESC-derived rod precursors has yet to be demonstrated. This project therefore sought to optimise the transplantation of mouse ESC-derived rod photoreceptor precursors into mouse models of retinal disease, define the optimal developmental stage for transplantation in an endogenous photoreceptor reporter ESC line, and thereafter, assess the extent to which visual function is restored in recipient animals post-transplantation. We assessed different aspects of optimising the transplantation protocol, such as dissociation method, AAV2 virus pseudotype used for labelling photoreceptors, and host immune suppression, and also characterised photoreceptor differentiation in a Crx.GFP ESC line. Additionally, we developed a multi-electrode array (MEA) set up and stimulus protocol that allowed us to detect visual responses from retinal explants. These studies demonstrated the generation of large numbers of healthy, developmentally-homogenous mouse ESC-derived rod photoreceptors for transplantation, and resulted in a significantly increased number of donor-reporter labelled cells observed in the host outer nuclear layer (ONL) following transplantation. Most importantly, using the MEA, we were able to demonstrate for the first time that mouse ESC-derived photoreceptor precursors were capable of transmitting light-evoked responses following transplantation into a mouse model of severe retinal degeneration. Taken together, these are encouraging first steps towards the use of stem cells for photoreceptor replacement therapy in retinal degenerative diseases.

Serpentes habitam grande diversidade de habitats na maior parte do planeta. Tamanha variedade ambiental implica o desempenho de distintos nichos ecológicos e padrões comportamentais, muitas vezes relacionados a diferentes adaptações de seus sistemas visuais. Não apenas a dispersão destes animais oferece oportunidades privilegiadas de investigação, os diversos e particulares históricos evolutivos neste grupo demarcam transições ambientais convenientes para esclarecer a influência da cena visual sobre a organização de sistemas visuais, comportamentos e a filogenia. A análise comparativa da retina destes animais traz informações a respeito de adaptações comportamentais e ecológicas relativas ao ambiente e padrões circadianos de atividade. Neste trabalho foi realizada a análise dos genes de fotopigmentos visuais e da morfologia dos fotorreceptores de duas espécies de serpentes da família Viperidae, Bothrops jararaca e Crotalus durissus terrificus. Três indivíduos de cada espécie foram obtidos junto ao laboratório de Herpetologia do Instituto Butantan. O RNA total foi extraído a partir de retinas homogeneizadas e convertido em cDNA por meio da reação de transcriptase reversa. Os genes de interesse foram amplificados com uso de primers específicos por meio de reação em cadeia de polimerase (PCR). Após purificação dos produtos de PCR foi realizado o sequenciamento dos genes de opsinas visuais expressos nas retinas das duas espécies, lws, rh1 e sws1. Cada opsina, maximamente sensível a uma banda espectral específica e presente em diferentes populações de fotorreceptores da retina, teve o seu pico de absorção estimado com base na estrutura proteica revelada. Adicionalmente, foi conduzida análise dos tipos celulares de células fotorreceptoras da retina das duas espécies de viperídeos, por meio da técnica de imunohistoquímica, visando caracterização morfológica dos fotorreceptores em que estão compreendidas cada classe de opsina. Os resultados obtidos apontam para os mesmos grupos morfológicos de fotorreceptores e a mesma sensibilidade espectral dos respectivos pigmentos visuais, das duas espécies analisadas: cones simples e cones duplos com o fotopigmento LWS, e pico de sensibilidade espectral (max) estimado em ~555nm; cones simples com o fotopigmento SWS1, e max estimado em ~360nm; e bastonetes, com o fotopigmento RH1, com max de ~500nm. Desta forma conclui-se similaridade do nicho ecológico e do histórico natural das duas espécies, que apontam para adaptações ao habito noturno. Isto demonstra o sucesso evolutivo e a versatilidade proporcionada pela disposição de retina duplex dominada por bastonetes. Em B. jararaca e C. d. terrificus, serpentes da subfamília Crotalinae, estas características de organização do sistema visual são somadas à capacidade de detecção de comprimentos de onda infravermelhos, o que aponta para similaridades em relação a serpentes do grupo Henophidia, consideradas evolutivamente mais primitivas, e consagra novamente a vantagens obtidas na manutenção deste padrão sensorial
Serpents inhabit a great diversity of habitats around the planet. Such environmental variability implies the performance of distinct ecological niches and behavior patterns that are related to different visual system adaptations. The diversity of environments inhabited by snakes and their evolutionary history provides a privileged investigative opportunity on the adaptive organization of the visual systems, specific behaviors and phylogeny. The comparative analysis of the retina of those animals provide many information concerning behavior and ecological adaptations related to their respective environment and circadian rhythm patterns. In this study, we performed genetic analysis of the opsin genes and morphological analysis of the photoreceptors of two snakes from the Viperidae family, Bothrops jararaca and Crotalus durissus terrificus. Three subjects of each species were collected at the Butantan Institute. Total RNA was extracted from homogenized retinas, and mRNA was converted to cDNA by reverse transcriptase reaction. The opsin genes lws, rh1 and sws1 were amplified by polymerase chain reactions (PCR), using specific primers. Each opsin is expressed in a different photoreceptor population and is maximally responsive to a determined spectral absorption peak (max) that was inferred according to the protein structure. Additionally, photorreceptor cell populations were analyzed using immunohistochemistry technique. Results point out to the same morphological cell populations and the same absorption peak in their respective opsins in the two species: double and single cones with the LWS photopigment and estimated max at ~555nm; single cones with the SWS1 photopigment and max at ~360nm; and rods with the rhodopsin RH1 photopigment and max at ~500nm. In this way, great similarity of ecological niche and natural history was concluded for both species, which present adaptations to the nocturnal habit. This should demonstrate the great evolutionary success and versatility attained by the rod-domminated duplex retina. In B. jararaca and C. d. terrificus, snakes from the Crotalinae subfamily, those retinal features are summed to the capability of infra-red detection, which point out to similarity with snakes from the basal Henophidia group

A key function of the nervous system is to sample data from the external world, generate internal signals, and transform them into meaningful information that can be used to trigger behaviour. In order to gain insight into the underlying mechanism for signal transformation, the visual system has been extensively studied: partly owing to the stimulus being reliably presentable, and the anatomy being well described. The Drosophila visual system is one such system, with the added advantage of genetic tractability. In this thesis, I studied the filtering property of visual neurons at two levels, biophysical and circuit levels. The first study looks at signal transformation at the biophysical level, at the input of the visual system, in photoreceptors. Voltage-gated potassium channels counteract the depolarization caused by opening of light sensitive channels, and the heterogeneous properties of their kinetics can fine-tune the photoreceptor’s frequency response to fulfill the animal’s ecological requirements. Shaker (Kv1) and Shab (Kv2) have been identified as fast and slow inactivating components of the photoreceptor’s outward currents, however a current with intermediate kinetics (IKf) has not been molecularly identified, but had been postulated to be Shal (Kv4). I focused on characterizing this current using whole-cell patch clamp in wild type and mutants, and using antibodies for Shal. My results from whole-cell patch clamp indicated that IKf in adult R1-6 cells are not Shal, from their voltage dependence and insensitivity to a Kv4 blocker. This calls for alternative molecular basis for IKf, which is likely to be a slow inactivating component of Shaker, or a combination of its many splice variants. The second study looks at signal transformation at the circuit level, at the output end, in the third optic neuropil, lobula. Visual projection neurons project from the lobula to the central brain, and have been proposed to carry behaviourally relevant visual features to higher brain regions. It was recently shown that optogenetic activation of individual visual projection neuron types could induce distinct behaviours such as takeoff and backward walking, linking these visual neurons to specific behavioural programs downstream. Using in vivo two-photon calcium imaging, I recorded visually evoked calcium responses from three of these cell types. Cell types that showed induced takeoff and backward walking preferentially responded to dark looming stimuli or fragmented expanding local features, suggesting their role in behaviours triggered by object approach. To explore how this visual information is transformed in the downstream circuit, we identified several candidate neurons that receive input from this cell type by anatomical overlap, and then validated their connections using optogenetic activation and calcium imaging. One downstream cell-type that projects bilaterally had very similar response properties to its upstream partner, whereas another cell-type that projects ipsilaterally seemed to filter out some information from its upstream partner. This is one of the first studies that functionally characterizes lobula visual projection neurons and their downstream partners in Drosophila, and their response properties agree with the general idea that visual information becomes increasingly selective as it is sent to higher brain regions.

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

High temporal resolution of vision relies on the rapid kinetics of the photoresponse in the light-sensing photoreceptor neurons. It is well known that the rapid recovery of photoreceptor membrane potential at the end of light stimulation depends on timely deactivation of the visual transduction cascade within photoreceptors. Whether any extrinsic factor contributes to the termination speed of the photoresponse is unknown. In this thesis, using Drosophilaas a model system, I show that a feedback circuit mediated by both neurons and glia in the visual neuropile lamina is required for rapid repolarization of the photoreceptor at the end of the light response. In the first part of my thesis work, I provide evidence that lamina epithelial glia, the major glia in the visual neuropile, is involved in a retrograde regulation that is critical for rapid repolarization of the photoreceptor at the end of light stimulation. I identified the gene affected in a slrp (slow receptor potential) mutant that is defective in photoreceptor response termination, and found it needs to be expressed in both neurons and epithelial glia to rescue the mutant phenotype. The gene product SLRP, an ADAM (a disintegrin and metalloprotease) protein, is localized in a special structure of epithelial glia, gnarl, and is required for gnarl formation. This glial function of SLRP is independent of the metalloprotease activity. In the second part of my thesis work, I demonstrate that glutamatergic transmission from lamina intrinsic interneurons, the amacrine cells, to the epithelial glia is required for the rapid repolarization of photoreceptors at the end of the light response. From an RNAi-based screen, I identified a vesicular glutamate transporter (vGluT) in amacrine cells as an indispensable factor for the rapid repolarization of the photoreceptor, suggesting a critical role of glutamatergic transmission from amacrine cells in this retrograde regulation. Further, I found that loss of a glutamate-gated chloride channel GluCl phenocopies vGluT downregulation. Cell specific knockdown indicates that GluCl functions in both neurons and glia. In the lamina, a FLAG-tagged GluCl colocalized with the SLRP protein in the gnarl-like structures, and this localization pattern of GluCl depends on SLRP, suggesting that lamina epithelial glia receive glutamatergic input from amacrine cells through GluCl at the site of gnarl. Since the amacrine cell itself is innervated by photoreceptors, these observations suggest that a photoreceptor — amacrine cell — epithelial glia — photoreceptor feedback loop facilitates rapid repolarization of photoreceptors at the end of the light response. In summary, my thesis research has revealed a feedback regulation mechanism that helps to achieve rapid kinetics of photoreceptor response. This visual regulation contributes to the temporal resolution of the visual system, and may be important for vision during movement and for motion detection. In addition, this work may also advance our understanding of glial function, and change our concept about the effect of glutamatergic transmission.

High visual sensitivity is a common but important characteristic of animal eyes. It is especially critical for night vision. In animal eyes, photoreceptors are the first to receive the incoming rays of light and they convert the light signals to electrical signals before passing the information to interneurons in the eye and finally to the brain. To function in dim light conditions, photoreceptors have developed high sensitivities to light. It is reported that both mammalian rod photoreceptors and Drosophilaphotoreceptors can detect single photons. The high sensitivities of photoreceptors largely depend on a high content of rhodopsin, a light-stimulated G protein-coupled receptor (GPCR), in light sensory organelles, outer segments in mammals and rhabdomeres in Drosophila. Two shared characteristics, the tightly packed photoreceptive membrane and the high concentration of rhodopsin in the membrane, work together to enable the photoreceptors to achieve the high content of rhodopsin in photosensory organelles in both mammals and Drosophila. In this thesis, I have used the Drosophilaeye as a model system to study the molecular mechanisms required for the maintenance of these two characteristics. In the second chapter, I present a new molecular mechanism of preventing Gq-mediated rhabdomeral degeneration. A new gene named tadr (for torn and diminished rhabdomeres), when mutated, leads to visual sensitivity reduction and photoreceptor degeneration. Degeneration in the tadr mutant is characterized by shrunken and disrupted rhabdomeres. The TADR protein interacts in vitro with the major light receptor Rh1 rhodopsin, and genetic reduction of the Rh1 level suppresses the tadr-induced degeneration, suggesting the degeneration is Rh1-dependent. Nonetheless, removal of phospholipase C (PLC), a key enzyme in phototransduction, and that of Arr2 fail to inhibit rhabdomeral degeneration in the tadr mutant background. Biochemical analyses reveal that, in the tadr mutant, the Gq protein of Rh1 is defective in dissociation from the membrane during light stimulation. Importantly, reduction of Gq level by introducing a hypomorphic allele of Gαq gene greatly inhibits the tadr degeneration phenotype. These results may suggest that loss of a potential TADR-Rh1 interaction leads to an abnormality in the Gqsignaling, which in turn triggers rhabdomeral degeneration independent of the PLC phototransduction cascade. We propose that TADR-like proteins may also protect photoreceptors from degeneration in mammals including humans. In the third chapter, I present a Drosophila CUB- and LDLa-domain transmembrane protein CULD that counteracts the visual arrestin Arr1-mediated endocytosis to retain rhodopsin in rhabdomeral membrane. CULD is mostly localized in rhabdomeres, but is also detected in scarce rhodopsin endocytic vesicles that contain Arr1. An intracellular region of CULD interacts with Arr1 in vitro. In both culdmutant and knockdown flies, a large amount of rhodopsin is mislocalized in the cell body of photoreceptors through lightdependent, Arr1-mediated endocytosis, leading to reduction of photoreceptor sensitivity. Expressing a wild-type CULD protein in photoreceptors, but not a mutant variant lacking the Arr1-interacting site, rescues both the rhodopsin mislocalization and the low sensitivity phenotypes. Once rhodopsin has been internalized in adult mutant flies, it is reversed only by expression of CULD but not by blocking endocytosis, suggesting that CULD promotes recycling of endocytosed rhodopsin to the rhabdomere. Our results demonstrate an important role of CULD in the maintenance of membrane rhodopsin density and photoreceptor sensitivity. We propose that a common cellular function of CUB- and LDLa-domain proteins, in both mammals and invertebrates, is to concentrate receptors including GPCRs in particular regions of cell membrane. In summary, the work addressed in this thesis has identified new molecular mechavii nisms underlying the maintenance of visual sensitivity in Drosophila, either through preventing Gq-mediated rhabdomeral degeneration or through antagonizing arrestin-mediated rhodopsin endocytosis. This work has advanced our understanding of visual biology and the general regulatory mechanisms of GPCR signaling, and may provide valuable clues to pathologic studies of human retinal degeneration disorders.

High temporal resolution of vision relies on the rapid kinetics of the photoresponse in the light-sensing photoreceptor neurons. It is well known that the rapid recovery of photoreceptor membrane potential at the end of light stimulation depends on timely deactivation of the visual transduction cascade within photoreceptors. Whether any extrinsic factor contributes to the termination speed of the photoresponse is unknown. In this thesis, using Drosophilaas a model system, I show that a feedback circuit mediated by both neurons and glia in the visual neuropile lamina is required for rapid repolarization of the photoreceptor at the end of the light response. In the first part of my thesis work, I provide evidence that lamina epithelial glia, the major glia in the visual neuropile, is involved in a retrograde regulation that is critical for rapid repolarization of the photoreceptor at the end of light stimulation. I identified the gene affected in a slrp (slow receptor potential) mutant that is defective in photoreceptor response termination, and found it needs to be expressed in both neurons and epithelial glia to rescue the mutant phenotype. The gene product SLRP, an ADAM (a disintegrin and metalloprotease) protein, is localized in a special structure of epithelial glia, gnarl, and is required for gnarl formation. This glial function of SLRP is independent of the metalloprotease activity. In the second part of my thesis work, I demonstrate that glutamatergic transmission from lamina intrinsic interneurons, the amacrine cells, to the epithelial glia is required for the rapid repolarization of photoreceptors at the end of the light response. From an RNAi-based screen, I identified a vesicular glutamate transporter (vGluT) in amacrine cells as an indispensable factor for the rapid repolarization of the photoreceptor, suggesting a critical role of glutamatergic transmission from amacrine cells in this retrograde regulation. Further, I found that loss of a glutamate-gated chloride channel GluCl phenocopies vGluT downregulation. Cell specific knockdown indicates that GluCl functions in both neurons and glia. In the lamina, a FLAG-tagged GluCl colocalized with the SLRP protein in the gnarl-like structures, and this localization pattern of GluCl depends on SLRP, suggesting that lamina epithelial glia receive glutamatergic input from amacrine cells through GluCl at the site of gnarl. Since the amacrine cell itself is innervated by photoreceptors, these observations suggest that a photoreceptor — amacrine cell — epithelial glia — photoreceptor feedback loop facilitates rapid repolarization of photoreceptors at the end of the light response. In summary, my thesis research has revealed a feedback regulation mechanism that helps to achieve rapid kinetics of photoreceptor response. This visual regulation contributes to the temporal resolution of the visual system, and may be important for vision during movement and for motion detection. In addition, this work may also advance our understanding of glial function, and change our concept about the effect of glutamatergic transmission.

Cilj ovog istraživanja je da se ispita uticaj centralne debljine makularne regije (CMT) i prisustva subretinalne tečnosti na vidnu oštrinu (VA) kod pacijenata obolelih od dijabetesnog makularnog edema, kao i uticaj očuvanosti kontinuiteta spoja spoljašnjeg i unutrašnjeg segmenta fotoreceptora (IS/OS – kompleks) i spoljašnje granične membrane (ELM) kao prognostičkih faktora u poboljšanju vidne oštrine nakon primenjene terapije kod pacijenata obolelih od dijabetesnog makularnog edema (DME). Materijal i metode: u ovu retrospektivno prospektivnu kliničku studiju nasumično je uključeno 100 pacijenata koji su podeljeni u dve grupe. Grupu A – prospektivni deo studije je činilo 50 pacijenata (50 očiju) kod kojih je dijagnostikovan dijabetesni makularni edem i kod kojih je inidikovana primena terapije, laserftotkogaulacije i/ili anti-VEGF terapije (bevacizumab). Grupu B – retrospektivnu grupu je činilo 50 pacijenata (58 očiju) koji su prethodno lečeni od dijabetesnog makularnog edema primenom laserfotokoagulacije i/ili anti-VEGF terapije (bevacizumab). Nakon kompletnog oftalmološkog pregleda koji se sastojao od određivanja vidne oštrine (optotipima po Snellenu), biomikroskopije, merenja intraokularnog pritiska i pregleda očnog dna – fundusa primenom panfundoskopa izvršena je optička koherentna tomografija u svih pacijenata (primenom aparata Stratus® OCT, Carl Zeiss, Meditec i Copercnicus® Optopol). Analiza OCT snimka, je obuhvatila određivanje centralne debljine makule (CMT), prisustva subretinalne tečnosti kao i procenu stanja očuvanosti kontinuiteta spoja spoljašenjeg i unutrašnjeg segmenta fotoreceptora (IS/OS kompleks) i očuvanost kontinuiteta spoljašnje granične membrane (ELM). CMT je izračunat primenom softvera OCT aparata i izražen kao srednja vrednost za svih 9 ETDRS polja. Prisutvno subretinalne tečnosti je klasifikovano kao pozitivno ukoliko je identifikovano makar u jednom preseku OCT tomograma .Očuvanost kontinuiteta IS/OS kompleksa i ELM je analizirana u svakom pojedinačnom snimku i podeljena u 3 kategorije. Prva – ukoliko je očuvano u svim presecima, druga – ukoliko je očuvano samo u pojedinim presecima i treća – ukoliko se IS/OS kompleks i ELM nisu mogli identifikovati na nalazu OCT tomograma. Rezultati ukazuju da prisustvo subretinalne tečnosti pre primenjene terapije nema statistički značajnog uticaja na poboljšanje vidne oštrine nakon primenjene terapije u pacijenata grupe A (pA=0,915), a statistička značajnost nije potvrđena ni kod pacijenata koji su prethodno tretirani od DME – grupa B (pB=0,772). Srednja vrednosti CMT i VA u pacijaneta grupe A iznosila je 474μm±140,67μm odnosno 0.25±0.20. Nakon primenjene terapije srednja vrednost vidnih oštrina iznosila je 0.41±0.25, dok su vrednosti srednje vrednosti CMT iznosile 343.68μm±99.03μm. Potvrđeno je statistički značajno poboljšanje vidne oštrine nakon primenjene terapije (pVA=0,0001) i statistički značajno smanjenje centralne debljine makule (pCMT=0,0001). Korelacija VA sa vrednostima CMT pre primenjene terapije pokazuje statističku značajnost sa negativnom korelacijom (r=-0,391; p=0,005) dok se nakon primenjene terapije ne uočava statistički značajna korelacija (r=-0,047; p=0,746). Analizom vrednosti CMT pre primenjene terapije sa vrednostima VA nakon terapije se uočava statistički značajna negativna korelacija, odnosno veće vrednosti CMT pre primenjene terapije ograničavaju poboljšanje vidne oštrine nakon primenjene terapije (r=-0,393; p=0,005). Evaluacija OCT tomograma, pre primenjene terapije, u pacijenata grupe A utvrđen je u potpunosti očuvan kontinuitet IS/OS kompleksa i ELM u svim presecima u 23 odnosno 27 očiju, u pojedinim presecima u 18 odnosno 16 očiju, i nije mogao biti identifikovan u 9 odnosno 7 očiju. U pacijenata grupe A nakon primenjene terapije uočava se statistički značajno poboljšanje vrednosti VA u zavisnosti od očuvanosti kontinuiteta IS/OS kompleksa (F=5,550, p=0,007) i ELM (F=5,428, p=0,008). Univarijantna odnosno multivarjiantna analiza podataka za granične vrednosti vidnih oštrina od 0,1 i koraka poboljšanja od 0,1 ukazuje na statističku značajnost prediktora IS/OS kompleksa (p=0,012 i p=0,032) i ELM (p=0,003 i p=0,018) u poboljšanju vrednosti vidnih oštrina nakon primenjene terapije. Pacijenti sa očuvanim kontinuitetom IS/OS kompelsa u svim presecima imaju 9,5 puta (OR=9,500 ) veću šansu za poboljšanje VA nakon primenjene terapije u odnosu na pacijente gde kontinuitet IS/OS kompleksa nije uočljiv. Pacijenti sa očuvanim kontinuitetom IS/OS kompleksa u pojedinim presecima imaju 7 puta veću šansu (OR=7,000) za poboljšanje vidne oštrine nakon terapije u poređenju sa onima kod kojih IS/OS nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u svim presecima imaju 34,5 puta (OR=34,500 ) veću šansu za poboljšanje vidne oštrine u odnosu na pacijente gde ELM nije uočljiv. Pacijenti sa očuvanim kontinuitetom ELM u pojedinim presecima imaju 18 puta veću šansu (OR=18,000) za poboljšanje VA nakon terapije u odnosu na one kod kojih ELM nije uočljiv. Pored statistički značajnog uticaja očuvanosti kontinuiteta IS/OS kompleksa i ELM na poboljšanje vrednosti vidnih oštrina nakon primenjene terapije, uočava se i pozitvna korelacija između vidnih oštrina pre i nakon terapije (r=0,869; p=0,0001). U pacijenata grupe B srednja vrednost CMT odnosno VA iznosila je 253,72μm±75,27μm odnosno 0,68±0,29. Postoji statistički značajna razlika u vrednostima VA u odnosu na očuvanost kontinuiteta IS/OS kompleksa (F=107,913, p=0,0001) i ELM (F=25,619, p=0,0001). Poređenjem vrednosti parametara za obe posmatrane grupe uočava se statistički značajna razlika u vrednostima CMT koje su bile manje u grupi B (t=5,355, p=0,0001) i srednjim vrednostima VA ( t=5,137, p=0,0001) koje su bile veće u grupi B. Analizom očuvanosti kontinuiteta IS/OS kompleksa (χ2=0,119, p=0,730) i ELM (χ2=2,957, p=0,085) ne uočava se statistički značajna razlika. Zaključak: Odnos vidnih oštrina sa centralnom debljinom makule prikazuje različite vrednosti vidnih oštrina za iste vrednosti centralne debljine makule. Značajan uticaj na vidnu oštrinu pacijenata obolelih od DME ima očuvanost integriteta spoljašnje granične membrane (ELM) i spoja unutrašnjeg i spoljašnjeg segmenta fotoreceptora (IS/OS kompleks) evaluiranih na osnovu OCT snimka – tomograma. Očuvanost integriteta ELM i IS/OS kompleksa u svim presecima na OCT tomogramu pre primenjene terapije u pacijenta sa DME se mogu smatrati pozitivnim prognostičkim faktorom u poboljšanju vidne oštrine nakon primenjene terapije. U pacijenata kod kojih je kontinuitet ELM i IS/OS kompleksa očuvan u svim pravcima vrednost CMT pre primenjene terapije nema uticaj na poboljšanje vidne funkcije nakon terapije. Integritet IS/OS kompleksa i ELM ima pozitivnu korelaciju sa vidnom oštrinom bez obzira na vrstu primenjene terapije, anti-VEGF odnosno laserfotokoagulacije. Prisustvo subretinalne tečnosti ne utiče na vidnu oštrinu pacijenata obolelih od DME. Vrednosti VA pre terapije utiču na poboljšanje vidne oštrine nakon terapije.
The aim of this study was to investigate the influence of the central macular thickness (CMT) and the presence of sub retinal fluid on visual acuity (VA) in patients with diabetic macular edema, as well as the impact of preservation and continuity of the photoreceptor inner/outer segment junction (IS / OS - complex ) and external limiting membrane (ELM) as a prognostic factor in improving visual acuity after the applied therapy in patients with diabetic macular edema (DME). Materials and Methods: this retrospective - prospective randomized clinical study included 100 patients who were divided into two groups. Group A - a prospective part of the study, consisted of 50 patients (50 eyes), with the diagnosis of diabetic macular edema in which laser photocoagulation and / or anti-VEGF therapy (bevacizumab) was indicated. Group B - retrospective group, consisted of 50 patients (58 eyes), who were previously treated for diabetic macular edema either with laser photocoagulation and / or anti-VEGF therapy (bevacizumab). After complete ophthalmologic examination, which consisted of the determination of visual acuity (measured with Snellen charts), biomicroscopy, intraocular pressure measurement and inspection of the fundus, optical coherence tomography was performed in all patients (using the Stratus® OCT, Carl Zeiss Meditec and Copercnicus® Optopol). Analysis of OCT image, included the determination of the central macular thickness (CMT), presence of sub retinal fluid, as well as an assessment of the preservation of the continuity of the photoreceptor inner/outer segment junction (IS/OS - complex) and external limiting membrane (ELM). CMT is calculated using software of the OCT apparatus and expressed as the mean value for all 9 ETDRS fields. Presence of sub retinal fluid is classified as positive if it is identified in at least one cross-section of OCT tomogram. Preserved continuity of IS / OS complex and ELM is analyzed in each individual OCT cross-section image and divided into 3 categories. First - if it is preserved in all cross sections images, the second - if it is preserved only in certain sections and the third - if the IS / OS complex and ELM were not able to identify in OCT tomograms. The results indicate that the presence of sub retinal fluid before the applied therapy has no statistically significant effect on improving visual acuity after the applied therapy in patients of group A (pA = 0.915), and statistical significance was not also confirmed in any of the patients who were previously treated by DME - Group B (pB = 0.772). Mean CMT and VA values of patients in group A was 474μm ± 140,67μm and 0.25 ± 0.20. After receiving therapy mean visual acuity was 0.41 ± 0.25, while the value of the mean CMT was 343.68μm± 99.03μm. Significant improvement in visual acuity was achieved after the treatment in group A (pVA = 0.0001) together with statistically significant reduction in central macular thickness (pCMT = 0.0001). Correlation of VA with the values of CMT before applied therapy shows statistically significant negative correlation (r = -0.391; p = 0.005), while after the applied therapy statistical significance was not observed (r = -0.047; p = 0.746). Analyzing the values of CMT before the applied therapy with the values of VA after the treatment statistically significant negative correlation was observed, higher values of CMT before the applied therapy restrict visual acuity improvement after the applied therapy (r = -0.393; p = 0.005). Analyzing OCT tomograms in the patients in group A, before the applied therapy, fully preserved continuity of IS/OS complex and ELM in all the sections was found in 23 and 27 of the eyes, in certain sections in 18 and 16 of the eyes, and could not be identified in 9 and 7 eyes. Statistically significant improvement in VA, after the applied therapy, in patients in group A is observed, depending on the preservation of continuity of IS/OS complex (F = 5.550, p = 0.007) and ELM (F = 5.428, p = 0.008). Univariate and multivariate analysis with cut off VA value of 0.1 and step improvements of 0.1 points to statistically significant predictor of IS/OS complex (p = 0.012 and p = 0.032) and ELM (p = 0.003 and p = 0.018) in improving the VA after the applied therapy. Patients with preserved continuity of IS/OS complex in all sections are 9.5 times (OR = 9.500) more likely to improve the VA after receiving therapy compared to patients where continuity of IS/OS complex is not noticeable. Patients with preserved continuity of IS/OS complex in the some sections are 7 times more likely (OR = 7.000) for the improvement of visual acuity after treatment compared to those in which the IS/OS is not detectable. Patients with preserved continuity of ELM in all sections are 34.5 times (OR = 34,500) a greater chance to improve visual acuity compared to patients where ELM is not apparent. Patients with preserved continuity of ELM in the some sections are 18 times more likely (OR = 18,000) to improve the VA after treatment compared to those in which the ELM is not apparent. In addition to statistically significant impact of preservation of continuity of IS/OS complex and ELM for VA improvement after the treatment, statistically significant positive correlation between visual acuity before and after treatment (r = 0.869; p = 0.0001) was observed. In Group B patients, the mean CMT and VA value was 253,72μm±75,268μm and 0.68 ± 0.29. There is a statistically significant difference in the VA values compared to the preservation of continuity of IS/OS complex (F = 107.913, p = 0.0001) and ELM (F = 25.619, p = 0.0001). Comparing the values of parameters for both groups, statistically significant difference in CMT values and mean VA was observed. CMT values were lower (t = 5.355, p = 0.0001) while VA values were higher (t = 5.137, p = 0.0001), in group B. The analysis of preservation of continuity of IS/OS complex (χ2 = 0.119, p = 0.730) and ELM (χ2 = 2.957, p = 0.085) did not show a statistically significant difference. Conclusion: The relationship of visual acuity with central macular thickness shows the different levels of visual acuity for the same value of the central macular thickness. A significant impact on VA in patients with DME has maintained integrity of the external limiting membrane (ELM) and the photoreceptors inner/outer segments junction (IS/OS complex) evaluated on the basis of OCT - tomograms. Preservation of the integrity of the ELM and IS/OS complex in all sections of the OCT tomogram before applied therapy in patients with DME can be considered a positive prognostic factor in improving visual acuity after receiving therapy. In patients with preserved continuity of ELM and IS/OS complex in all sections before applied therapy the CMT value has no effect on the improvement of visual function after treatment. Regardless of the type of applied therapy, anti-VEGF and/or laser photocoagulation preserved integrity of IS/OS complex and ELM has a positive correlation with visual acuity. The presence of sub retinal fluid does not affect the visual acuity in patients with DME. The values of VA before treatment influence the improvement of visual acuity after treatment.

The ability to detect and effectively escape from predators is critical to an animal's survival, and the ability to do so effectively depends on its sensory system. Vision is the dominant if not sole sensory system fiddler crabs use for predator avoidance. In this thesis I investigate the photoreceptor properties of two fiddler crab species, Uca vomeris and Uca dampieri, and their escape behaviour under field conditions with the aim to further our knowledge of how these crabs use visual information to organise their escape behaviour. In an attempt to elucidate the spectral sensitivities of the crabs' photoreceptors, intracellular electrophysiological recordings were performed and are presented in Chapter 2. Temporal resolution of their visual system was investigated using both intracellular recordings and electroretinograms (ERGs) and presented in Chapter 3. The following two chapters present the results of behavioural experiments into the effect of the predator's elevation in the crabs' visual field on two stages of the crabs' predator avoidance response: the run home stage (Chapter 4) and the burrow descent stage (Chapter 5). I found that both species of fiddler crabs have a UV-sensitive photopigment, plus one or two photopigments with a peak sensitivity in the blue part of the spectrum (400-500 nm). Their temporal resolution, measured as critical flicker fusion frequency (CFF), was similar to other non-flying animals at around 70 Hz. When measured as integration time and time-to-peak, however, their temporal resolution was very high, comparable even to flying animals. The behavioural experiments revealed that at the run home stage, the crabs perceive objects changing in elevation as most dangerous. Additionally, when far from the burrow they respond earlier to objects appearing low in the visual field, whereas when close to their burrow, such as at the stage of the burrow descent, they are more sensitive to objects that are seen high in the visual field. These investigations into the visual system and into predator avoidance behaviours of fiddler crabs move us closer to establishing them as a model system where we understand the first stage of sensory processing, which in turn will allow us to investigate how further neural processing leads to the functional behavioural output that can be observed in the field.

Many animals use vision to guide their behaviour and to collect relevant information about their environment. The diversity of visual environments and of visually guided tasks has led to a large variety of specialisations of eyes and visual systems. Our knowledge, however, about how the anatomical and physiological properties of eyes and the behavioural strategies of animals relate to the visual signals that are important to them in their natural environment, is extremely limited. In this thesis, I make use of optical, physiological and behavioural analyses to reconstruct the flow of visual information that the fiddler crab Uca vomeris experiences during its daily life on the mudflat. I present a detailed analysis of the first stage of visual processing, the sampling by the ommatidial array of the crabs' compound eye and demonstrate how regional specialisations of optical and sampling resolution reflect the information content and behavioural relevance of different parts of the visual field. Having developed the first intracellular electrophysiological preparation in fiddler crabs, I then examine the spectral sensitivities of photoreceptors - the basis for colour vision. I show that the crabs possess an unusual trichromatic colour vision system featuring a UV-sensitive and a variety of short-wavelength receptor types based on the coexpression of two short-wavelength sensitive pigments. Finally, the natural visual signals that predatory and non-predatory birds present to fiddler crabs are described. The visual cues the crabs use when deciding whether and when to respond to these potential predators are analysed and compared to those used in dummy predator experiments. The crabs use a decision criterion that combines multiple visual cues - including retinal speed, elevation and visual flicker. Neither of these cues accurately predicts risk, but together they reflect the statistical properties of the natural signals the crabs experience. The complex interactions between the design of the crabs' visual system, the stimuli they experience in their natural context and their behaviour demonstrate that neither of them can be understood without knowledge of the other two.
Research School of Biological Sciences (RSBS, now RSB), and the Australian National University for providing funding through an ANU PhD scholarship; the Australian Department of Education, Employment and Workplace Relations for an International Postgraduate Research Scholarship; the German National Academic Foundation and the Zeiss Foundation for support through a Heinz-Dürr Scholarship; and the Australian Institute of Marine Sciences for providing accommodation and facilities during fieldwork in Queensland.

The overall objective of this thesis was to understand better the mechanisms that shape the diversity in colour vision of fish, and to explore the adaptive significance of this divergence. Among the vertebrates, teleost fish show the greatest diversity in colour vision systems. The cichlid model system illustrates that the visual system of fish may differ among species, sexes, individuals, and life stages of individuals. The large number of available cone opsin genes, which have resulted from multiple opsin gene duplications, facilitates this high degree of variation in the mechanisms of colour vision. In general, cichlids possessed complements of four to five cone pigments, and these complements varied across species, sexes, and individuals. Additionally, lens transmission, cone pigment expression, post-receptoral sensitivity, and retinal circuitry differed across life stages of individuals. My results suggest that the diversification of colour vision across species and across life stages of individuals contributes to sensory adaptations that enhance both the contrast of zooplanktonic prey, and the detection of optical signals from conspecifics. Therefore, both natural and sexual selection may have worked in concert to shape colour vision in fish. Since light is more complex under water than on land, fish required four to six cone classes to reconstruct the colour signals reflected from aquatic objects. This suggests that the large number of cone pigments in fish have likely evolved to enhance the reconstruction of the complex colour-signals in aquatic environments. Taken together, these findings improve our understanding of the variable nature of fish colour vision, and, more generally, help unravel the evolution of photoreceptors and colour vision.
Thesis (Ph.D, Biology) -- Queen's University, 2012-05-14 13:16:50.276

Retinitis Pigmentosa (RP) is an incurable inherited retinal degeneration affecting approximately 1 in 4,000 individuals globally. The aim of this dissertation was to identify drugs that can help patients suffering from the disease. To accomplish this goal, the zebrafish was utilized as a model for RP to perform in vivo drug screening. The zebrafish RP model expresses a human rhodopsin transgene which contains a premature stop codon at position 344 (Tg(rho:Hsa.RH1_Q344X)). This zebrafish model exhibits significant rod photoreceptor degeneration beginning at 7 days post fertilization (dpf). To assess the visual consequence of this rod degeneration the zebrafish behavior visual motor response (VMR) was assayed under scotopic conditions. The Q344X RP model larvae displayed a deficit in this VMR in response to a scotopic light offset. This deficit in behavior was utilized to perform a drug screen to identify compounds that could ameliorate the deficient Q344X VMR. The ENZO SCREEN-WELL® REDOX library was chosen to be screened since oxidative stress may increase RP progression in a non-specific manner. From this library, a β-blocker, carvedilol, was identified as a compound that improved the Q344X VMR behavior. This drug was also able to increase rod number in the Q344X retina. Carvedilol was shown to be capable of working directly on rods by demonstrating that the drug can signal through the adrenergic pathway in the rod-like human Y79 cell line. Since carvedilol is an FDA-approved drug, this screening paradigm was utilized to screen the Selleckchem FDA-approved library to identify more drugs that can potentially be repurposed to treat RP like carvedilol. Additionally, this scotopic VMR assay was used to demonstrate that it can identify behavioral deficits in the P23H RP model zebrafish (Tg(rho:Hsa.RH1_P23H)). This dissertation work provides a potential FDA-approved drug for RP treatment and sets the foundation for future drug screening to identify more drugs to treat different forms of RP.

abstract: Why do many animals possess multiple classes of photoreceptors that vary in the wavelengths of light to which they are sensitive? Multiple spectral photoreceptor classes are a requirement for true color vision. However, animals may have unconventional vision, in which multiple spectral channels broaden the range of wavelengths that can be detected, or in which they use only a subset of receptors for specific behaviors. Branchiopod crustaceans are of interest for the study of unconventional color vision because they express multiple visual pigments in their compound eyes, have a simple repertoire of visually guided behavior, inhabit unique and highly variable light environments, and possess secondary neural simplifications. I first tested the behavioral responses of two representative species of branchiopods from separate orders, Streptocephalus mackini Anostracans (fairy shrimp), and Triops longicaudatus Notostracans (tadpole shrimp). I found that they maintain vertical position in the water column over a broad range of intensities and wavelengths, and respond behaviorally even at intensities below those of starlight. Accordingly, light intensities of their habitats at shallow depths tend to be dimmer than terrestrial habitats under starlight. Using models of how their compound eyes and the first neuropil of their optic lobe process visual cues, I infer that both orders of branchiopods use spatial summation from multiple compound eye ommatidia to respond at low intensities. Then, to understand if branchiopods use unconventional vision to guide these behaviors, I took electroretinographic recordings (ERGs) from their compound eyes and used models of spectral absorptance for a multimodel selection approach to make inferences about the number of photoreceptor classes in their eyes. I infer that both species have four spectral classes of photoreceptors that contribute to their ERGs, suggesting unconventional vision guides the described behavior. I extended the same modeling approach to other organisms, finding that the model inferences align with the empirically determined number of photoreceptor classes for this diverse set of organisms. This dissertation expands the conceptual framework of color vision research, indicating unconventional vision is more widespread than previously considered, and explains why some organisms have more spectral classes than would be expected from their behavioral repertoire.
Dissertation/Thesis
Doctoral Dissertation Biology 2016