Browsing by Subject "Retina"

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During development, cell-cell recognition events mediate crucial steps in the formation of organized cellular patterns critical for tissue function. In the nervous system, cell recognition cues guide migrating neurons during development to appropriate terminal locations and sculpt their characteristic sizes, shapes, and circuit connectivity. The retina contains a multitude of neuron types; however, neurons of the same cell type (homotypic) are patterned into evenly spaced arrangements known as “mosaics” across the retina surface. Disrupting mosaic formation impairs visual function, so it is important to understand the precise cellular and molecular mechanisms that allow homotypic neurons to recognize and adjust their proximity to neighbors. To understand this process, we studied two populations of interneurons, the OFF and ON starbursts amacrine cells (SACs), which require the cell-surface receptor MEGF10 to establish their mosaics. We find that SACs in Megf10 mutants still make lateral movements in the plane of the retina, but fail to recognize their proximity to homotypic neighbors. Using transgenic tools to visualize SACs early in development, we identify a transient developmental phase where SAC dendrite territories are bounded by homotypic somata, a relationship which is lost in SACs lacking MEGF10. Further, we determine that MEGF10 utilizes distinct signal transduction pathways in neurons from those identified in non-neuronal cells. Lastly, we demonstrate that specific amino acids within the intracellular domain of MEGF10 are required to recapitulate a cellular recognition-like event in a heterologous cell system. These findings support a model whereby MEGF10 signals in SACs by a distinct mechanism to mediate dendrite-soma interactions necessary to pattern the organization of retinal neurons.

Naturally-occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the central nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type – the astrocyte – has rarely been studied. It is presently unclear whether astrocytes are subject to significant amounts of developmental death, or how it occurs. Here we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia kill and engulf astrocytes to mediate their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally-occurring cell death, and demonstrate its importance for formation and integrity of the retinal gliovascular network.

Optical coherence tomography (OCT) is a non-invasive optical imaging modality that provides micron-scale resolution of tissue micro-structure over depth ranges of several millimeters. This imaging technique has had a profound effect on the field of ophthalmology, wherein it has become the standard of care for the diagnosis of many retinal pathologies. Applications of OCT in the anterior eye, as well as for imaging of coronary arteries and the gastro-intestinal tract, have also shown promise, but have not yet achieved widespread clinical use.

The usable imaging depth of OCT systems is most often limited by one of three factors: optical attenuation, inherent imaging range, or depth-of-focus. The first of these, optical attenuation, stems from the limitation that OCT only detects singly-scattered light. Thus, beyond a certain penetration depth into turbid media, essentially all of the incident light will have been multiply scattered, and can no longer be used for OCT imaging. For many applications (especially retinal imaging), optical attenuation is the most restrictive of the three imaging depth limitations. However, for some applications, especially anterior segment, cardiovascular (catheter-based) and GI (endoscopic) imaging, the usable imaging depth is often not limited by optical attenuation, but rather by the inherent imaging depth of the OCT systems. This inherent imaging depth, which is specific to only Fourier Domain OCT, arises due to two factors: sensitivity fall-off and the complex conjugate ambiguity. Finally, due to the trade-off between lateral resolution and axial depth-of-focus inherent in diffractive optical systems, additional depth limitations sometimes arises in either high lateral resolution or extended depth OCT imaging systems. The depth-of-focus limitation is most apparent in applications such as adaptive optics (AO-) OCT imaging of the retina, and extended depth imaging of the ocular anterior segment.

In this dissertation, techniques for extending the imaging range of OCT systems are developed. These techniques include the use of a high spectral purity swept source laser in a full-field OCT system, as well as the use of a peculiar phenomenon known as coherence revival to resolve the complex conjugate ambiguity in swept source OCT. In addition, a technique for extending the depth of focus of OCT systems by using a polarization-encoded, dual-focus sample arm is demonstrated. Along the way, other related advances are also presented, including the development of techniques to reduce crosstalk and speckle artifacts in full-field OCT, and the use of fast optical switches to increase the imaging speed of certain low-duty cycle swept source OCT systems. Finally, the clinical utility of these techniques is demonstrated by combining them to demonstrate high-speed, high resolution, extended-depth imaging of both the anterior and posterior eye simultaneously and in vivo.

Optical coherence tomography (OCT) is a non-invasive imaging modality that uses low coherence interferometry to generate three-dimensional datasets of a sample's structure. OCT has found tremendous clinical applications in imaging the retina and has demonstrated great utility in the diagnosis of various retinal diseases. However, such diagnoses rely upon the ability to observe abnormalities in the structure of the retina caused by pathology. By the time an ocular disease has progressed to the point of affecting the morphology of the retina, irreversible vision loss in the eye may already occur. Changes in the functionality of the tissue often precede changes to the structure. Thus, if imaging methods are developed to provide additional functional information about the behavior and response of the retinal tissue and vasculature, earlier treatment for disease may be prescribed, thus preserving vision for the patient.

Within the last decade, significant technological advances in OCT systems have enabled high-speed and high sensitivity image acquisition using either spectral domain OCT (SDOCT) or swept-source OCT (SSOCT) configurations. Such systems use Fourier processing to extract structural information of a sample from interferometric principles. But such systems also have access to the optical phase information, which allows for functional analysis of sample dynamics. This dissertation details the development and application of methods using both intensity and phase information as a tool for studying interesting biological phenomena. The goal of this work is an extension of techniques to image the vasculature in the retina and enhance the clinical utility of OCT.

I first outline basic theory necessary for understanding the principles of OCT. I then describe OCT phase imaging in cellular applications as a demonstration of the ability of OCT to provide functional information on biological dynamics. Phase imaging methods suffer from an artifact known as phase wrapping, and I have developed a software technique to overcome this problem in OCT, thus extending its usefulness in providing quantitative information. I characterize the limitations in measuring moving scatterers with Doppler OCT in both SDOCT and SSOCT system. I also show the ability to image the vasculature in the retina using variance imaging with a high-speed retinal imaging system and software based methods to correct for patient motion and create a widefield mosaic in an automated manner. Finally, future directions for this work are discussed.

In the central nervous system, billions of neurons interconnect with precision to form morphologically complex and functionally diverse neural circuits. The stereotypical fashion by which neurons assemble suggests that cell-surface molecular cues can act as identity tags during development. These cell surface receptors allow neurons to distinguish between circuit partners, incorrect connections and homotypic neighbors. Multiple EGF-like domains 10 (Megf10) was previously identified to mediate homotypic recognition of certain retinal cell types. Genetic evidence suggests that MEGF10 acts as both ligand and receptor to initiate cell-cell repulsion. Although its significance in cell-cell recognition has been demonstrated, the exact mechanism of how MEGF10 mediates mosaic formation remains unclear. Specifically, the biochemical basis of MEGF10-MEGF10 interaction is largely unknown nor do we have knowledge on what molecules are involved in signaling transduction. Further, MEGF10 is also expressed in glia cells, but it has not been tested if this MEGF10 recognition event is neuron specific.To address these questions, we decided to first characterize the molecular components of the MEGF10 complex. We determined MEGF10 complex composition through co-immunoprecipitation (co-IP) and chemical crosslinking and discovered that MEGF10 forms a lateral complex. Truncation and co-IP studies reveal that the interacting motifs are located on the ectodomain of MEGF10. Such binding is not restricted to MEGF10 as we also discovered hetero-multimers between MEGF10 and MEGF12. Next, to identify other molecules in the MEGF10 signaling pathway, we performed IP to isolate native MEGF10 interacting complexes and conducted proteomic analysis. We found previously known MEGF10-interacting molecules such as Dynamin1 and Traf4, as well as novel MEGF10 associating candidates. Our identification of interacting molecules that facilitate cytoskeletal and membrane rearrangement suggests that MEGF10 activates these cellular processes. Finally, we characterized Müller glia organization through a genetic-based labeling method. With a MEGF10 mutant mouse, we determined that MEGF10 is not necessary for glial array formation. We conclude that MEGF10 has distinct functions in neurons vs. glia. This study sets the stage to describe the molecular mechanism by which MEGF10 mediates cell-cell recognition, potentially uncovered novel MEGF10 interactions, and distinguishes MEGF10 neuronal function from its role in glia.

One of the central problems in neuroscience is that there is a lack of understanding of the diversity and functions of cell types in the brain. Even in brain areas that have been studied extensively, such as the retina, much remains to be learned about the diversity and functions of cell types. Morphological, functional and genetic studies have yet to converge on a consistent picture of cell type diversity in the retina, because the field lacks a standardized approach to classify cell types. A systematic classification approach is essential to provide an unambiguous appreciation of cell type diversity, and a better understanding of the organization and function of the retina. In the first portion of this dissertation, we present a novel approach that classifies retinal ganglion cells (RGCs) in a quantitative, verifiable and reproducible manner. We utilize diverse visual stimuli and a multi-electrode array, to record simultaneously from multiple RGCs, and show that there are at least 13 RGC types with distinct functional properties. In the second portion of the dissertation, we present a quantitative determination and comparison of the spatiotemporal receptive field (RF) structures and neural coding properties across these RGC types. Determining the RF structure of RGC types is important, because it constrains the computations performed by retinal circuits and identifies the signals available to retinal recipient areas. We find that RGC types exhibit functional asymmetries in terms of their RF size, temporal integration, and response nonlinearities. We also show that no RGC types exhibited RFs that were strictly independent in space and time. These results provide several new insights into the computations performed in the rodent retina, and highlight the importance of understanding cell type diversity to further our understanding of how the retina works and the role it plays in visual processing.