Felice A. Dunn - US grants

DESCRIPTION (provided by applicant): To restore vision, we must understand how information is processed in the mature retina and how structural and functional organization are affected during degeneration. The divergence of signals at the first synapse in the visual system, where a single cone provides input to 10-12 types of cone bipolar cells, provides a unique opportunity to study the origin of parallel pathways. This synapse also exhibits convergence, where each type of cone bipolar cell receives inputs from a stereotyped number of cones. Our recent work demonstrates that three types of cone bipolar cells establish their unique patterns of structural contact with presynaptic cone photoreceptors according to different strategies and segregated timelines. However, we know little about how these differences translate into functional properties in the mature circuit. Moreover, how cone bipolar cell types respond to progressive loss of photoreceptors during disease is unclear. The long-term goal of the proposed work is to understand how visual information is parsed and processed in the retina at the cone-to-cone bipolar synapse, and to determine how this information is perturbed in disease. The objectives of the proposed work are to determine the functional properties of three morphologically characterized bipolar cells types, for which we already know structural connectivity patterns, and to determine these bipolars' structural and functional changes in a degenerating retina. In Aim 1, we will determine how cone convergence and divergence shapes the functional properties of three types of cone bipolar cells. We will make functional measures of the bipolar cells' spatial, temporal, and gain properties. In Aim 2, we will identify the effect of cone degeneration on bipolar cell structure, connectivity, and function. Many retinal diseases leading to blindness originate with death of photoreceptors. How disease progresses to affect postsynaptic neurons remains largely unknown. We will use laser ablation and transgenic approaches to control the extent and timing of cone death. Imaging and electrophysiology will allow us to determine the structural connectivity patterns, glutamate receptor distributions, and responses to light stimuli of bipolar cells following controlled cone death. The approach is innovative because we are separately determining the function and response of specific bipolar cell types to photoreceptor degeneration. The proposed work is significant because it will reveal how a bipolar cell's functional properties are determined by its anatomical connections with cones and will provide an understanding of how bipolar cells respond to photoreceptor degeneration as a model of potential circuit rearrangements in retinal disease.

There is a gap in knowledge of how loss of 50-80% of cone photoreceptors produces almost no change in visual acuity or sensitivity. While contributions from cortex have been examined, those from retina have been underappreciated. The long-term goal to understand how the retina functions robustly in the face of photoreceptor death will generate transformative insights into how neural plasticity compensates for cell death. Understanding this compensation is likely to lead to earlier diagnostics and more effective treatments. The overall objective of this proposal is to elucidate the fundamental synaptic and circuit-level mechanisms that allow the retina to function while compensating for photoreceptor death. This proposal focuses on the well-characterized circuit of the ON sustained alpha ganglion cell in mouse retina, a strong model circuit with identified cell types, maps of specific connections, accessibility to genetic manipulation, and quantifiable structure and function. Following genetic ablation of 50-75% of cones in adult retina with the diphtheria toxin receptor, these ganglion cells adjust receptive field structures and spike responses. The observations are congruent with adaptation, which adjusts integration and gain for stimulus statistics, e.g., greater integration and gain at lower light levels, or homeostatic plasticity, which involves remodeling circuitry or channel expression. The central hypothesis is that the retina can compensate for cone loss via mechanisms of adaptation and/or homeostatic plasticity that we will determine in two specific aims: (Aim 1) identify the extent and sites of compensation within the retinal circuit following partial cone loss in the adult and (Aim 2) determine the contributions of partial stimulation, mean adaptation and homeostatic plasticity to the retina's reaction to cone loss. The results of the first aim will identify the structural and functional consequences of cone loss on the direct excitatory pathway from cones to type 6 cone bipolar cells to ON alpha ganglion cells. The results of the second aim will determine how adaptation, changes in excitatory and inhibitory circuits, and intrinsic excitability contribute to changes in ganglion cell spatial and intensity encoding following partial cone loss. The approach is innovative for the genetic control over cone ablation in mature retina, the stage at which most human retinal diseases occur; functional and structural examination with cell-type specific resolution; and focus on synaptic and circuit mechanisms underlying a well known discrepancy between photoreceptor loss and visual function. The research is significant for (1) uncovering mechanisms that may mask visual deficits in early stages of photoreceptor loss; (2) suggesting diagnostics that could detect earlier onset of diseases causing cone loss; (3) establishing knowledge about the flexibility of a sensory circuit and how this flexibility pertains to a surviving circuit; (4) providing direct measures of how retinal function after partial cone loss is distinct from or similar to that in control retina?thus potentially influencing the design of treatments to restore retinal function following photoreceptor loss.

There is critical need to understand the causes, extent, and mechanisms of reactions to cell death so that effective treatments most appropriate for the state of the remaining circuit can be employed, and so that constructive compensation can be harnessed as a potential treatment in conditions where a portion of the circuit endures. Our long-term goal is to salvage neuronal circuits. Here, we will define the effects of controlled cell death on specific circuits, cell types, synapses, and proteins for the purpose of understanding the conditions that result in constructive (e.g., compensation through increasing synaptic gain) vs. destructive (e.g., aberrant spontaneous activity that corrupts signal) response. The mouse retina is an exceptional platform for this study because the primary sensory neurons, photoreceptors, can be manipulated under genetic control; cell types within specific circuits are identifiable and accessible; and the functional readout can be interpreted as visual sensitivity. We propose to ablate variable populations of rods in mature retina and determine the structural and functional effects on the primary rod bipolar cell pathway, the most sensitive retinal pathway: rods?rod bipolar cells?AII amacrine cells?ON cone bipolar cells?ON sustained alpha ganglion cells (abbr. ON alpha). ON alpha ganglion cells receive the greatest number of rod inputs, thus would be the most sensitive to rod loss. Our central hypothesis is that the retina has constructive reactions to input loss with the capacity to recover normal function up to an undefined threshold; beyond this threshold, destructive reactions begin. Unknown is this tipping point. Our preliminary data show that despite loss of half the rods, rod- mediated light responses in ON alpha ganglion cell spikes are comparable to control, suggesting compensation within the primary rod bipolar cell pathway. Thus, the premise is strong for constructive compensation within the retina following rod loss, and we will determine the induction parameters, sites, and contributions of this compensation to maintaining function in the following aims: (Aim 1) to determine the degree of input loss that induces constructive vs. destructive structural and functional changes, and (Aim 2) to locate the site(s) and mechanism(s) of compensation within a well-defined neural circuit. The approach is innovative for genetic control over the timing and degree of rod death; synaptic- and cell-type specific structural and functional investigation of a well-defined retinal circuit; and molecular tools to distinguish between cell ablation and synapse disassembly in triggering compensatory mechanisms. The results will be significant for (1) determining the degree of rod death that triggers the remaining circuit to undergo destructive or constructive responses, (2) identifying the sites and contributions of structural and functional compensation to maintaining retinal function, and (3) providing knowledge essential to the optimization and deployment of therapies to treat dysfunctional photoreceptors involving stem cells, genes, and prostheses, all of which rely on a stable retinal circuit and/or extensive knowledge of the state of the surviving retinal circuit.