xi, 56 p. : ill. (some col.) A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / The development of a functional nervous system is paramount for the ability of animals to interact with their environments. Minor defects in nervous system function compromise the effectiveness of sensing and responding to stimuli. Severe defects in nervous system function often lead to extreme sensory, cognitive and motor skill impairment. The nervous system is a complex network of connections, with each neuron making functional contacts with several other neurons. Any single animal species generally exhibits a stereotyped pattern of neuronal connectivity, but the specific intrinsic and extrinsic signals that impart to a neuron its unique connective properties have only recently begun to be identified. In this study, we use the Drosophila visual system to examine neuronal connectivity. Our screen for genes involved in R7 photoreceptor connectivity led to the identification of the RhoGAP domain-containing protein dsyd-1 and the transcriptional repressor tramtrack . Flies harboring homozygous mutant dsyd-1 R7s fail to phototax towards UV light, an innate behavior mediated by the R7s. Subsequent analysis of axons of dsyd-1 R7s showed abnormal morphology in the region of presynaptic sites, suggesting that similar to its role in C. elegans , dsyd-1 is involved in presynaptic assembly. Further analysis demonstrated a requirement for dsyd-1 function in docking presynaptic components to terminal sites of contact. R7 axons are restricted to non-overlapping columns in the optic neuropil, thereby preserving spatial visual information in the retintopic map. The axon terminals of tramtrack mutant R7s exhibit overgrowth, similar to that observed in R7s that have loss of function mutations in genes involved in the activin signaling pathway. Previous studies have shown that activin signaling is involved in restricting R7 axons to their appropriate columns, and our results demonstrate that tramtrack may be functioning in the same pathway. One of two tramtrack isoforms, ttk69 , is expressed in photoreceptors after they have differentiated, and expression of ttk69 is specifically required for R7 axon targeting. / Committee in charge: Eric Johnson, Chairperson, Biology; Victoria Herman, Advisor, Biology; Bruce Bowerman, Member, Biology; Christopher Doe, Member, Biology; Tom Stevens, Outside Member, Chemistry

Abstract Vision is capable of providing an animal with a wealth of information very fast. Visually guided behaviours are numerous, ranging from foraging to navigation. Vision can be quite reliable in bright light, but the signals produced by the photoreceptors become progressively more unreliable with falling light intensities. In this thesis the usefulness of a novel virtual reality-based environment for insect vision research is reviewed, and the low-light vision of the American cockroach, Periplaneta americana, is assessed using the optomotor behavioural paradigm and intracellular photoreceptor recordings. The optomotor reaction is visual behaviour where an animal responds to a rotation of its environment by following the movement of its surroundings with its eyes or - like insects - by rotating its body in the direction of the movement. Placing the cockroach on a trackball in the middle of the virtual reality apparatus and projecting a rotating pattern of vertical stripes around it invariably causes an optomotor reaction if the cockroach is able to see the moving pattern. Presenting the cockroaches with the stimulus pattern at different low light levels and observing their abilities to follow the movement reveal the lowest light intensity at which they are able to use vision in guiding their behaviour. The compound eye photoreceptor signals at this behavioural threshold consist of singlephoton absorption events called ‘bumps’ at the extremely low rate of one bump every ten seconds. Furthermore, the role of the simple eyes or ocelli in the low-light vision of the cockroach is studied in the virtual reality by covering the compound eyes, the ocelli, or both. The ocelli seem to measure the light intensity and communicate this information to the compound eyes, and also have a direct effect on the general activity level of the cockroach.

PURPOSE. Recessive mutations in CLN7/MFSD8 usually cause variant late-infantile onset neuronal ceroid lipofuscinosis (vLINCL), a poorly understood neurodegenerative condition, though mutations may also cause nonsyndromic maculopathy. A series of 12 patients with nonsyndromic retinopathy due to novel CLN7/MFSD8 mutation combinations were investigated in this study. METHODS. Affected patients and their family members were recruited in ophthalmic clinics at each center where they were examined by retinal imaging and detailed electrophysiology. Whole exome or genome next generation sequencing was performed on genomic DNA from at least one affected family member. Immunofluorescence confocal microscopy of murine retina cross-sections were used to localize the protein. RESULTS. Compound heterozygous alleles were identified in six cases, one of which was always p.Glu336Gln. Such combinations resulted in isolated macular disease. Six further cases were homozygous for the variant p.Met454Thr, identified as a founder mutation of South Asian origin. Those patients had widespread generalized retinal disease, characterized by electroretinography as a rod-cone dystrophy with severe macular involvement. In addition, the photopic single flash electroretinograms demonstrated a reduced b- to a-wave amplitude ratio, suggesting dysfunction occurring after phototransduction. Immunohistology identified MFSD8 in the outer plexiform layer of the retina, a site rich in photoreceptor synapses. CONCLUSIONS. This study highlights a hierarchy of MFSD8 variant severity, predicting three consequences of mutation: (1) nonsyndromic localized maculopathy, (2) nonsyndromic widespread retinopathy, or (3) syndromic neurological disease. The data also shed light on the underlying pathogenesis by implicating the photoreceptor synaptic terminals as the major site of retinal disease.

No description available.

Zebrafish have been used as a model to study the vertebrate retina due to its functional and structural similarities to the human retina. Photoreceptor cells (PRCs) are highly specialised type of neurons present in the retina. In zebrafish, PRCs can be divided into 5 different subtypes, rods and green, red, blue and UV sensitive cones. Mature PRCs are composed of different morphological compartments (basal domain, inner segment and outer segment), which are essential for their phototransduction ability. During development, these cells are known to arise from columnar neuroepithelium precursor cells and undergo a maturation process to become compartmentalised10. However, a detailed characterisation of this process is lacking in zebrafish. In this project, I aimed to establish and characterise in detail the stages of PRC maturation in zebrafish. Next, I aimed to investigate the role of candidate genes in this PRC maturation process. To label the plasma membrane of all cells, a zebrafish transgenic line was utilised. Furthermore, a novel zebrafish transgenic line that labels the outer segments of red sensitive PRCs was generated. This transgenic line enabled visualisation and volume quantification of outer segments of red sensitive cones. The use of both transgenic lines in combination with antibody stainings indicated that, from 72 hours post fertilisation (hpf) onwards, subtypes of PRCs exhibit differences in growth rate and morphology of their cell compartments. Additionally, differences in mitochondrial clusters and nuclei positioning were observed during the maturation process. From 72 hpf to 120 hpf, rough endoplasmic reticulum accumulation emerged specifically in rod like PRCs. Changes in chromatin organisation were observed in UV sensitive cones like PRCs from 120 hpf onwards. This showed that a high degree of complexity was observed even within the cone PRC subtypes. Lastly, the role of a candidate gene, crb2b, was examined by comparing PRC maturation process in WT and crb2b mutants. My results indicate that loss of Crb2b does not show obvious defects in PRC maturation. Results obtained in this dissertation provided a comprehensive characterisation of six independent PRC maturation stages using the criteria of cell compartmentalisation and growth, organellar distribution and localisation of cell polarity related protein complexes. This defined developmental timeline provides a platform to further study PRC maturation and function.

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 <i>in vivo</i> drug screening. The zebrafish RP model expresses a human rhodopsin transgene which contains a premature stop codon at position 344 (<i>Tg</i>(<i>rho:Hsa.RH1_Q344X</i>)). 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<i> (Tg</i>(<i>rho:Hsa.RH1_P23H</i>)). 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.

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.

Retinitis pigmentosa (RP) is a genetically and phenotypically heterogeneous condition that affects approximately 1 in 4000 individuals worldwide. The most common presentation of RP is a rod-cone dystrophy, where the degeneration of cone photoreceptors occurs secondary to advanced rod loss, leading to a significant decline in central vision and a corresponding reduction in patient quality of life. The mechanisms underlying secondary cone loss are poorly understood, particularly in disorders where the gene defect is unknown or manifest only in rod photoreceptors. Consequently, the thesis presented herein proceeds on several fronts. First, in the long term a greater understanding of the causes underlying cone loss in RP is likely to be beneficial, and so in chapter one a dominant cone degeneration is characterized using intrinsically fluorescent cone photoreceptors to track the degenerative process. Second, as we develop a greater understanding of the genetic etiology underlying RP it is likely that the number of large genes identified as being causative will increase. As currently there is no efficient way to deliver large genes to photoreceptors, chapter two explores the use of alternate viral vectors that might be used to deliver a large therapeutic transgene. Lastly, whilst our understanding of cone loss in RP remains incomplete, it is necessary to develop a broadly applicable therapy to slow or attenuate further cone loss in RP patients regardless of the underlying cause. In chapters three and four we examine the use of low molecular weight "growth factors‟, such as ciliary neurotrophic factor (CNTF), to preserve cone photoreceptors long-term using a rhodopsin knockout model of RP.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

Purpose Intrinsically photosensitive retinal ganglion cells (ipRGCs) express a unique photopigment called melanopsin. Capable of direct phototransduction, the ipRGCs are also influenced by rods and cones via synaptic inputs.  Thus, the photoinput that mediates the pupil light reflex derives from both outer (rods and cones) and inner (melanopsin-mediated) retinal photoreception. This thesis has aimed to develop a pupillometric test that provides quantitative information about the functional status of outer and inner retinal photoreception in healthy eyes and in eyes with retinal degeneration. In addition to regulating the pupil light reflex, the ipRGCs signal light information for the circadian rhythm, thus, these two non-visual physiologic responses to inner retinal photoreception were examined simultaneously. Methods Pupil responses to a long and short wavelength light over a range of intensities (under conditions of light, mesopic and dark adaptation) were recorded using a customized infrared computerized pupillometer. Results were compared for two groups: patients with retinitis pigmentosa and controls. The response function threshold intensity and a half-max intensity was determined from the rod-weighted and cone-weighted pupil responses and correlated to extent of visual loss. The pupil response to light offset was assessed as a measure of direct melanopsin activation. Lastly, pupil responses to red and blue light at equal photo flux were recorded hourly during a 24-hour period and correlated to salivary melatonin concentrations in healthy subjects. Results In normal eyes, the blue light evoked greater pupil responses compared to equiluminant red light. With increasing intensity, pupil contraction became more sustained which was most apparent with the brightest blue light. In patients with retinitis pigmentosa, the pupil responses mediated predominantly by rod and cone activation were significantly reduced compared to controls, (p&lt;0.001) and the relative decrease in their contribution resulted in a greater influence of melanopsin on the post-stimulus response. Even at endstage retinal degeneration, pupil responses that derived predominantly from residual cone activity were detectable. The threshold intensity of the rod-mediated, but not cone-mediated, pupil response was also significantly reduced (p=0.006) in patients and the half-maximal intensity of rods correlated with severity of visual loss (r2=0.7 and p=0.02). In healthy controls, the melanopsin-mediated pupil response demonstrated a circadian modulation whereas the cone-mediated pupil response did not. Conclusion Early and progressive loss of rod function in mild-moderate stages of retinitis pigmentosa is detectable and quantifiable as a progressive loss of pupillary sensitivity to extremely dim blue lights obtained under conditions of dark adaptation. In advanced stages of retinal degeneration, chromatic pupillometry is more sensitive than standard electroretinography for detecting residual levels of rod and especially cone activity. In addition, selective wavelength pupillometry can assess non-visual light-dependent functions. The timing of the post-stimulus pupil response to blue light is in phase with melatonin secretion, suggesting a circadian regulation of this pupil parameter. / Bakgrund Jätteganglieceller (intrinsically photosensitive retinal ganglion cells, ipRGCs) är en klass av fotoreceptorer som utnyttjar ett unikt vitamin-A-baserat fotopigment som kallas melanopsin. Utöver deras direkta ljuskänslighet, mottar ipRGCs stimulerande och hämmande synaptiska signaler från andra fotoreceptorer (tappar och stavar) som därigenom kan modulera aktiviteten hos ipRGCs. Ögats pupillreflex medieras alltså av ljus både via yttre (stavar och tappar) och inre (melanopsin-medierad) retinal fotoreception, och den gemensamma afferenta pupillomotor-signalen leds till den pretectala nucleus olivarius via axoner från ipRGCs. Arbetet i denna avhandling syftar till att utveckla ett kliniskt pupilltest som ger kvantitativ information om yttre och inre retinala fotoreceptorers funktionella status hos friska försökspersoner och patienter med retinal degeneration. Förutom att styra pupillreflexen, skickar ipRGCs även impulser som påverkar kroppens dygnsrytm. Därför ingår även en delstudie i vilken ipRGCs aktivitet studeras genom att avläsa icke-visuella fysiologiska reaktioner på inre retinal fotoreception. Metoder Ljus av lång (röd) respektive kort (blå) våglängd presenterades med stegvis ökad ljusstyrka för att selektivt stimulera stavar, tappar eller melanopsin. Pupillreaktionerna registrerades med en infraröd datoriserad pupillometer och jämfördes mellan friska kontroller och patienter med retinitis pigmentosa. I uppföljande experiment gjordes mer noggranna tester i syfte att isolera aktiveringen av varje ljusmottagande element. Tröskelintensiteten för stav- eller tapp-medierad pupillreaktion bestämdes med linjär regressionsanalys. Reaktionskurvan för stavmedierad pupillreflex kvantifierades (halv-maximal intensitet) och jämfördes med svårighetsgraden av sjukdomen i två familjer med samma sjukdomsframkallande mutation för retinitis pigmentosa. För att undersöka icke-visuella reaktioner på inre fotoreception från ipRGCs, undersöktes pupillreaktion på rött och blått ljus varje timme under en 24-timmarsperiod och korrelerades till melatoninkoncentration i saliv hos friska personer med normal syn. Resultat I normala ögon, gav blått ljus en kraftigare pupillreaktion jämfört med rött ljus av samma ljusstyrka. Med ökande intensitet, blev pupillkontraktionen mer ihållande, vilket var tydligast med starkt blått ljus. Hos patienter med retinitis pigmentosa, var både tapp- och stav-medierad pupillreaktion signifikant reducerad jämfört med kontroller, (p&lt;0,001). Patienter med avancerad sjukdom och icke-reaktivt elektro-retinogram hade fortfarande mätbar pupillreflex, huvudsakligen härrörande från kvarvarande stavaktivitet. I två familjer med retinitis pigmentosa beroende på en enda missense-mutation av NR2E3 genen, var tröskelvärdet för stavmedierad pupillreflex signifikant reducerat (p= 0,006) och korrelerade till sjukdomens svårighetsgrad. Tappmedierad pupillreflex hos dessa patienter skilde sig dock inte signifikant från kontroller, trots att fotopiskt (tapp) elektroretinogram var klart avvikande. Hos friska kontroller visade melanopsinmedierat pupillsvar en dygnsvariation medan tapp-medierat pupillsvar inte gjorde det. Slutsatser Som tillägg till standardundersökningar kan selektiv våglängds-pupillometri (kromatisk pupillometri) vara användbart för utvärdering av funktionen hos stavar och tappar. Denna avhandling visar att tidig och gradvis förlust av stav-funktion i milt-måttligt stadium av retinitis pigmentosa är detekterbar och mätbar som en progressiv förlust av pupillens känslighet för mycket svagt blått ljus, efter mörkeradaptation. I avancerade stadier av retinal degeneration är kromatisk pupillometri känsligare än standardelektroretinografi för att detektera kvarvarande nivåer av stav- och speciellt tapp-aktivitet. Hos unga patienter, där elektroretinografi kan vara tekniskt svårt, är pupillometri en lovande teknik för att värdera yttre retinal fotoreception relaterad till synfunktion. Dessutom kan selektiv våglängdspupillometri ge information om icke-visuella ljusberoende funktioner. Pupillreaktionen på blått ljus varierar med melatoninsekretionen, vilket tyder på en cirkadisk reglering. Ytterligare studier krävs för att undersöka om selektiv våglängds-pupillometri även kan användas i samband med sjukdomar relaterade till störd dygnsrytm, som sömnlöshet och årstidsbunden depression.