Dissertations / Theses on the topic 'Inner retina'

The retina is able to adjust its signaling over a wide range of light levels. A functional result of this is increased visual acuity at brighter luminance levels, such as during the day, due to changes in the organization of retinal receptive fields. This process is commonly referred to as light adaptation. These organizational changes have been shown to occur at the level of the ganglion cells, the output neurons of the retina, which have shifts in their excitatory center-inhibitory surround receptive fields that increase their sensitivity to small stimuli. Recent work supports the idea that light-adapted changes in ganglion cell spatial sensitivity are due in part to inner retinal signaling changes, possibly including changes to inhibition onto bipolar cells, the interneurons at the center of retinal signal processing. However, it is unknown how inhibition to the bipolar cells changes with light adaptation, how any changes affect the light signal or what mediates the changes to the bipolar cells that have been suggested by previous reports. To determine how light adaptation affects bipolar cell inhibition, the inhibitory inputs to OFF bipolar cells were measured. OFF bipolar cells, which respond to the offset of light, in particular may be involved in retinal adaptation as they bridge dim- and bright-light retinal pathways. Their inputs were compared between dark- and light-adapted conditions to determine how any inhibitory changes affects their output onto downstream ganglion cells. We found that there was a compensatory switch from primarily glycinergic-mediated inhibition to OFF bipolar cells in the dark to primarily GABAergic-mediated inhibition in the light. Since glycinergic and GABAergic inhibition perform very different roles and are mediated by morphologically different cells, it is likely this switch underlies a change in the spatial distribution of inhibition to these cells. We found that the spatial inhibitory input to OFF bipolar cells became significantly smaller and narrower with light adaptation, translating to smaller inhibitory surrounds of the OFF bipolar cell receptive fields. Through a model, our data suggested that the OFF bipolar cell output to downstream ganglion cells was stronger in the light, due to the narrowing and reduction in the spatial input, to small light stimuli. This would effectively be one way the retina could use to increase visual acuity. Additionally, we found that the inhibitory changes to OFF bipolar cells with light-adaptation are partially mediated by dopamine D1 receptor signaling. Dopamine is released in the light and has been shown to be an important modulator of retinal light-adaptation. However, there are likely other factors involved in mediating inhibitory changes to OFF bipolar cells. Through these studies, we show that light adaptation heavily influences inner retina inhibition and likely plays a prominent role in determining and shaping light signals under different ambient light conditions which may ultimately be one mechanism for increasing visual sensitivity and acuity.

Purpose: To investigate species differences in the distribution and localization of alpha B-crystallin (ABC) in the normal retina and optic nerve head region, and to describe changes during outer and inner retina degeneration. Material and methods: Animals studied included mice, rats, cats, pigs, cows, and monkeys. Sections of the optic nerve and central retina were labeled with antibodies against ABC and glial fibrillary acidic protein (GFAP). Results: ABC was located in astrocytes and Muller cells with different intensities. During outer retina degeneration (dystrophic rat and Abyssinian cat), only late stages showed an increase in ABC in the retina and optic nerve head. Inner retina degeneration in the glaucoma mouse model showed no increase of ABC. In the monkey glaucoma model, only the innermost layer of the optic nerve head showed increased labeling for ABC. Conclusions: The distribution of ABC is species dependent and is (excluding the mouse) present in the nerve fiber layer of the retina and in the optic nerve head (localization of astrocytes). Chronic retinal degeneration does not necessarily lead to an over-expression of ABC. While in outer retinal degeneration induction was predominantly present in late stages, pressure-induced glaucoma led to a specific increase in ABC already in early stages indicating a local stress-response in this region.

The retina adjusts its signaling gain over a wide range of light levels. A functional result of this is increased visual acuity at brighter luminance levels (light adaptation) due to shifts in the excitatory center-inhibitory surround receptive field parameters of ganglion cells that increases their sensitivity to smaller light stimuli. Recent work supports the idea that changes in ganglion cell spatial sensitivity with background luminance are due in part to inner retinal mechanisms, possibly including modulation of inhibition onto bipolar cells. To determine how the receptive fields of OFF cone bipolar cells may contribute to changes in ganglion cell resolution, the spatial extent and magnitude of inhibitory and excitatory inputs were measured from OFF bipolar cells under dark- and light-adapted conditions. There was no change in the OFF bipolar cell excitatory input with light adaptation; however, the spatial distributions of inhibitory inputs, including both glycinergic and GABAergic sources, became significantly narrower, smaller, and more transient. The magnitude and size of the OFF bipolar cell center-surround receptive fields as well as light-adapted changes in resting membrane potential were incorporated into a spatial model of OFF bipolar cell output to the downstream ganglion cells, which predicted an increase in signal output strength with light adaptation. We show a prominent role for inner retinal spatial signals in modulating the modeled strength of bipolar cell output to potentially play a role in ganglion cell visual sensitivity and acuity.

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<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<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.

The most robust entrainment cue to the circadian clock is light. In response to this, organisms have evolved light detection mechanisms which allow them to measure and transduce irradiance information to specific centres in the brain. To perceive light, these receptors use photosensitive opsins that are remarkably divergent from those involved in image-forming photoreception. The main focus of this work was to understand if the photopigment function of the inner-retinal opsins can be inferred from their sequence and predicted structure. To this end we have isolated and characterised melanopsin and VA opsin from vertebrate species that have evolved under different environments.

Teacher attrition has serious consequences in hard to staff schools. Mostly poor and ethnic minority students are deprived of being taught by stable, experienced teachers. The purpose of this study was to explore the strategies used to effectively retain teachers in such schools through the perspective of teachers at a high school that comprises poor and ethnic minority students in southwest Georgia. The conceptual framework that guided this study was Chen's theory about race and social class which postulated that a high percentage of poor ethnic minority students results in low teacher morale. This study explored the reasons why teachers stay at a school where there is a high proportion of poor and ethnic minority students. In this research, the case study strategy of inquiry was employed and data were collected from interviews with 10 teachers (using a 16-question interview guide) to solicit their perspectives on the working conditions at their school. The data were then examined for patterns and themes in the text. The findings produced 4 consolidated themes that revealed (a) aspects of a successful environment created by the principal; (b) an effective mentoring program that was aimed at assisting, developing, and supporting new and inexperienced teachers; (c) good parental involvement where parents were enthusiastic about supporting the school and their child's educational progress; and (d) stable and charismatic leadership that promoted retention. If implemented at hard to staff schools, these best practices can lead to improved teacher morale, better prepared teachers, and higher student achievement.

Le développement de l’uvéite auto-immune expérimentale (UAE) se fait en plusieurs étapes, commençant par l’activation en périphérie de lymphocytes T auxiliaires auto-réactifs spécifiques d’antigènes rétiniens, leur migration vers l’œil, où ils sont réactivés de façon antigène-spécifique et CMH II (complexe majeur d’histocompatibilité de classe II)-dépendante pour enfin induire la rupture de la barrière hémato-rétinienne (BHR), permettant le recrutement aspécifique de cellules inflammatoires responsables des dommages tissulaires. Tous ces processus représentent des cibles potentielles pour des thérapies biologiques ciblées. Dans cette perspective, notre travail a pour but d’approfondir la compréhension des mécanismes impliqués dans le recrutement de cellules inflammatoires, la présentation locale d’antigènes rétiniens et la rupture de la BHR interne lors de l’induction de l’UAE par transfert adoptif.L’expression de molécules d’adhésion par les cellules de la BHR joue un rôle central dans l’infiltration de cellules inflammatoires dans l’œil. Dans ce contexte, nous avons d’abord montré qu’à l’instar de ce que nous avions démontré pour VCAM-1, l’expression d’ICAM-1 est fortement induite dans la rétine durant l’UAE, avec une intensité et une extension corrélées à la sévérité de la maladie. Cependant, alors que VCAM-1 est uniquement inductible, une expression basale d’ICAM-1 est détectée dans la rétine naïve. Le ligand d’ICAM-1, LFA-1, est exprimé de façon ubiquitaire par les cellules immunes circulantes, contrairement au ligand de VCAM-1, VLA-4, qui n’est exprimé que par une minorité de cellules. Par ailleurs, nous avons observé une répartition tissulaire différente de ces deux molécules d’adhésion dans la rétine. En effet, si ICAM-1 prédomine dans l’épithélium pigmentaire rétinien, VCAM-1 est fortement exprimé au niveau des lésions de vasculite, à la fois sur les cellules endothéliales et gliales péri- vasculaires. Ces 2 sites correspondent respectivement à la BHR externe et interne. Ces différences majeures en termes de distribution rétinienne des molécules d’adhésion pourraient refléter des voies d’entrée distinctes pour les cellules inflammatoires lors de leur pénétration dans l’œil.Comme les lymphocytes T auto-réactifs n’induisent la maladie qu’après avoir localement reconnu leur antigène, nous nous sommes ensuite intéressés à identifier les cellules présentatrices d’antigène (CPA) potentielles exprimant du CMH II dans la rétine lors de l’UAE. Nous avons tout d’abord observé une forte induction de l’expression de molécules du CMH II dans la rétine lors de l’inflammation intraoculaire, corrélée avec la sévérité de la maladie. Celle-ci est associée avec l’induction de l’expression de molécules de co-stimulation, particulièrement sur les cellules exprimant fortement le CMH II. L’expression la plus forte de CMH II se retrouve dans la rétine interne, au niveau des vaisseaux enflammés et s’étend vers les couches externes de la rétine et l’espace sous-rétinien dans les uvéites sévères. Nous avons identifié 3 populations de CPA potentielles exprimant le CMH II dans la rétine :des cellules CD45-CD11b- non-hématopoïétiques exprimant faiblement le CMH II et des cellules CD45+CD11b+ hématopoïétiques exprimant plus fortement le CMH II, pouvant être subdivisées en cellules Ly6C+ et Ly6C-. L’analyse bio-informatique à l’aveugle du transcriptome de ces 3 populations mène à une ségrégation claire des échantillons, avec un enrichissement en marqueurs de macrophages et de microglie dans les cellules Ly6C+ et Ly6C-, respectivement. Cependant, l’expression de Ly6C ne permet pas une ségrégation absolue entre macrophages infiltrants et microglie résidente. L’analyse fonctionnelle à l’aide de DAVID (Database for Annotation, Visualization and Integrated Discovery) révèle que les 2 populations de cellules hématopoïétiques sont plus compétentes dans la présentation d’antigène associée au CMH II et l’activation des lymphocytes T que les cellules non-hématopoïétiques.Paradoxalement, nos données n’ont pas mis en évidence d’expression de CMH II par les principales cellules de la BHR que sont les cellules endothéliales et les cellules de l’épithélium pigmentaire rétinien. Cependant, il est bien établi que les cellules endothéliales rétiniennes subissent un changement majeur de phénotype lors du développement d’une UAE. Afin d’investiguer de façon globale les mécanismes sous-jacents à la rupture de la BHR interne, nous avons étudié la régulation de l’expression génique des cellules endothéliales rétiniennes lors de l’uvéite non-infectieuse. En accord avec les données de nos travaux précédents, l’analyse du transcriptome des cellules endothéliales rétiniennes n’a pas mis en évidence d’expression de CMH II lors de l’UAE. En revanche, cette approche nous a permis d’identifier 65 gènes modulés dans les cellules endothéliales rétiniennes lors du développement d’une UAE, confirmant non seulement l’implication de certaines molécules dont le rôle pathogénique est déjà connu, mais procurant également une liste de nouveaux gènes candidats et de voies fonctionnelles potentiellement associées à la rupture de la BHR lors d’une uvéite non- infectieuse.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

Genom åren har intresset för och satsningar på underhållning i tredimensionellt format (3D) gått i vågor. Idag har de flesta biografer stöd för att visa 3D-filmer. Utöver detta intresse har det på senare år även börjat komma mer teknik för 3D i hemmet. Det har också introducerats många nya mer immersiva och intuitiva inmatnings-enheter som bidrar till under-hållning med element av virtual reality hemma. Företagen Sony, Nintendo och Microsoft har alla lanserat olika typer av avancerade tekniker för sådana inmatnings-enheter till sina spel-system. Dessa tekniker bidrar på olika sätt till mer immersiv underhållning. På de sätt som många av teknikerna används idag blir däremot interaktionen fortfarande hämmad av att dessa kräver att man står, respektive tittar, i en viss riktning.Rapporten återger en experimentell studie som tar sikte på att undersöka om det är möjligt att med kommersiellt tillgänliga medel skapa immersiv virtual reality som är portabel och buren – för underhållning i hemmet. Inledningsvis redogörs kortfattat för människans upp-fattning av djup samt hur olika 3D-displayer fungerar. Härefter kommer vi in på virtual reality och betydelsen av en hög nivå av immersion i detta sammanhang. Beträffande virtual reality kommer däremot utgångspunkten vara mer teoretisk, för att ge en bild av åt vilket håll utvecklingen går. Denna del går därmed längre än vad experimentet omfattar, eftersom denna teori inte ännu fullt ut omsatts i praktiken. Hypotesen för experimentet är att tekniken ”head tracking”, i form av en huvudburen rörelsedetektor som känner av huvudets orientering, till viss del kan vara en lösning på problemet med immersion – eftersom jag som användare då inte är hänvisad till en viss plats. Slutligen analyseras såväl teori som experiment och man kommer fram till att VRD är en möjlig lovande framtida teknik. Hypotesen bekräftas till viss del och rapporten mynnar ut i en slutreflektion där det konstateras att teknik för att skapa ökad immersion och VR hemma finns tillgänglig – även om en del ytterligare arbete för datahantering skulle krävas för att optimera denna.
Through the years, interest in and focus on entertainment in three dimensional form (3D) has gone in waves. Today, most cinemas have support for showing 3D-movies. In addition to this interest, an increasing amount of technology for 3D at home has become available in recent years. A number of different new, more immersive and intuitive input devices with elements of virtual reality for home use, have also been introduced. The companies Sony, Nintendo and Microsoft have all launched various types of such advanced input technology for their game consoles. These techniques contribute in various ways to more immersive entertain-ment. In the way many of these technical solutions are used today, they are still limited by the requirement of standing and looking in a specific direction. The report reflects an experimental study that aims to explore the feasibility of using commercially obtainable material to create immersive virtual reality for home entertainment, which is portable and wearable. Initially it explains the basics of human depth perception, and how different 3D displays work. Hereafter, we will look at virtual reality and the im-portance of a high level of immersion in this context. In regards to virtual reality however, the starting point will be of a more theoretical nature, to give an idea of in which direction the development is heading. This part thereby goes further than what the experiment covers, because of this theory not yet beeing fully applied in practice. The hypothesis for the experiment is that the technology "head tracking", in the form of a head-mounted motion-sensor that detects the orientation of the head, in part, may be a solution to the problem of immersion – as the user isn’t dependant on a specific location.Finally an analysis of both theory and experiment is made in which it is concluded that VRD might be a promissing future technology. The hypothesis is partially confirmed and the report culminates in a final reflection where it is found that technology for creating a higher level of immersion and VR at home is available – even though some additional work with data handling would be required.

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The inner retinal complex is a well-defined layer in spectral-domain OCT scans of the retina. The central edge of this layer at the fovea provides anatomical landmarks that can be observed in serial OCT scans of developing full-thickness macular holes (FTMH). Measurement of the movement of these points may clarify the mechanism of FTMH formation. This is a retrospective study of primary FTMH that had a sequence of two OCT scans showing progression of the hole. Measurements were made of the dimensions of the hole, including measurements using the central edge of the inner retinal complex (CEIRC) as markers. The inner retinal separation (distance between the CEIRC across the centre of the fovea) and the Height-IRS (average height of CEIRC above the retinal pigment epithelium) were measured. Eighteen cases were identified in 17 patients. The average increase in the base diameter (368 microns) and the average increase in minimum linear dimension (187 microns) were much larger than the average increase in the inner retinal separation (73 microns). The average increase in Height-IRS was 103 microns. The tangential separation of the outer retina to produce the macular hole is much larger than the tangential separation of the inner retinal layers. A model based on the histology of the Muller cells at the fovea is proposed to explain the findings of this study.

The inner plexiform layer (IPL) of the Vertebrate retina is a highly organized synaptic region amassed with a myriad of processes from different cell types, like ganglion cells (GCs), amacrine cells (ACs) and bipolar cells (BCs). Their dendrites stratify at different levels within this cell-free zone, e.g. so-called subbands. Further, Müller glial cells (MCs) span radially through all retinal layers. In this study, I have focused on cholinergic ACs and MCs, and their possible roles in the formation of the IPL in the embryonic chicken retina. In the first chapter I have analysed the in vivo development and differentiation of starburst amacrine cells (SACs) and their role in cholinergic stratification. Type-I and type-II SACs are mirror-symmetrically arranged ACs on both sides of the IPL, which send their processes into the latter, where they stratify as subbands ‘a’ and ‘d’. Acetylcholine is the predominant neurotransmitter of these cells, e.g. they are cholinergic. Using the cholinergic markers choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), I show here immunohistochemically that an early postmitotic pool of cells in the inner half retina expresses first AChE, and then also ChAT. SACs and GCs are derived from this pool. By embryonic stage E6, two narrow rows of ChAT+ cells appeared in the future IPL, separated by a narrow band of AChE. Thus, AChE might be playing a guiding role for ChAT+ processes. Type-I SACs retained their high ChAT+, but not AChE expression, which was less pronounced in type-II SACs. Remarkably, type-I SACs in the INL were neighbored by strongly AChE-reactive cells which were ChAT-, a finding whose functional implications needs further attention. In chapter II, I show that the earliest ChAT+ cells also co-expressed the ganglion cell-specific marker Brn3a and CRABP. By E6, one set of cells down-regulated both ChAT and CRABP, but up-regulated Brn3a, marking the specification of GCs, while another set of cells reacted to the contrary, marking the specification of the two future types of SACs. By E8, type-I cells at the IPL/INL border lost their expression of CRABP, while type-II SACs in the IPL retained this protein. A co-localization of the early GC marker Brn3a with ChAT+ cells revealed that early GCs transiently express ChAT. Throughout these developmental stages, both the GCs and the ACs expressed AChE. Now following, I have performed in vitro experiments to analyze the role of SACs and Müller glial cells during IPL development, using both 3D retinal spheroids and retinal explants. In chapter III, I describe the establishment of a novel explant culture system, presenting the development of all retinal layers of an in vivo retina, except the GCL. Although being used here for studies of the inner retina, it is worthwhile to mention that with these explants the initial formation of photoreceptor outer segments can be followed. In continuation to the above mentioned in vivo studies, in chapter IV, I have similarly analyzed IPL formation by using both in vitro systems. Since type-II SACs are closely associated with GCs, I postulated that GCs could influence their differentiation, and in turn the development of the IPL as well. To test this hypothesis, I investigated the development of the IPL in explants, in which GCs are completely lost due to the absence of an optic nerve. In addition, 3D retinospheroids were used, where cells are completely dissociated and allowed to form histotypical structures, but again mostly in absence of GCs. Interestingly, in both in vitro culture models the IPL still developed, including both cholinergic subbands and migration of type-II SACs. This is strong evidence that major parts of the IPL develop in absence of GCs. Besides SACs, early Müller glial cells (MCs) have been suggested to provide guiding cues to establish the neuritic plexus of the IPL. Therefore, in the last chapter V, I have used the specific gliotoxin DL-alpha aminoadipate (AAA) in retinal spheroids to target MCs and thereby analyze the role of MCs during IPL development. At a sub-toxic concentration of 0.4 mM AAA, though the IPL was developed, the cholinergic stratification was severely disturbed, indeed suggesting an organizing role of MCs during IPL development. Moreover, thereby the numbers of inner retinal cells increased, while photoreceptor differentiation was inhibited, indicating further roles of MCs in cell lineage regulation.

Studies demonstrate neuro-retinal damage in patients with diabetes and no clinically visible diabetic retinopathy. It is unknown which retinal regions are most vulnerable to diabetes. We hypothesized that the standard and slow-flash (sf-) multifocal electroretinogram (mfERG) and adaptive optics (AO) imaging will localize retinal regions of vulnerability. Fifty-five adolescents with diabetes and 54 controls underwent mfERG testing to isolate predominately retinal bipolar cell activity and sf-mfERG testing to isolate three oscillatory potentials (OPs) from intraretinal amacrine and interplexiform cells. Greatest mfERG delays were in the superior temporal quadrant and at 5°-10° eccentricity. Greatest sf-mfERG delays were found at different eccentricities for each OP. Twenty adolescents with diabetes and 14 controls underwent AO imaging. No significant differences in cone photoreceptor density were found; however, patients showed a trend towards reduced density in the superior nasal region. Inner retinal structures may be more susceptible to damage by diabetes than outer retinal structures.