Maureen A. McCall - US grants

The central nervous system (CNS) is comprised of numerous pathways and nuclei. Most anatomically definable structures in the CNS are composed of many different cell classes, each of which is presumed to make a specific contribution to the function of the entire structure. A fundamental problem in understanding the role of structures with multiple, interconnected cell populations is that it has seldom been possible, using traditional neurobiological methods, to unequivocally define the role of single populations of neurons in a complex structure. In this application, we propose to use a recently developed technique, genetic ablation, to achieve the selection elimination of a class of neurons from the CNS and subsequently, to study the role of that cell class in CNS structure and function. In particular, we propose to eliminate an identified cell class from the retina. The goals of the proposed experiments are: 1) to identify retinal cell-class specific genes from subtracted retinal cDNA libraries, 2) to isolate the promoter regions of the identified genes, 3) to construct fusion genes by fusing the promoter of the identified gene to an attenuated diphtheria toxin gene, 4) to use this fusion gene to create transgenic mice and 5) to use these transgenic mice in a series of anatomical and neurophysiological assessments designed to characterize the effects of the ablation on the structure and function of the retina. In our preliminary experiments, we have successfully eliminated the rod photoreceptors from the retina, thereby demonstrating that this is a viable approach for the selective ablation of a specific cell population from the CNS. We have chosen the retina as our model system for the application of genetic ablation for a number of reasons. first, it is a laminar structure, with the somata of major, recognized cell classes, and their processes, lying in specific laminae. Second, retinal ganglion cells represent the sole output neurons of the retina and their responses reflect the integration of the activity of the more peripheral retinal elements. These cells, therefore, offer an accessible in vivo assay for the effects of an ablation. Finally, our laboratory has considerable expertise in the analysis of the retina, both anatomically and physiologically. While the experiments described in this application will be performed on neurons in the retina, we fell it is important to emphasize that the approach that we have chosen will be equally powerful in defining the function of any population of cells for which unique genetic material can be identified.

The retina is a layered structure and each layer is populated by different cell classes. Most cell classes are further subdivided on the basis of their shape and on the excitatory and inhibitory inputs they receive. In the retina, as well as in the rest of the brain, inhibition plays a central role in neuronal function by modulating excitatory signals. Most inhibitory neurotransmission is mediated by the release of either glycine or gamma amino butyric acid (GABA) from the pre-synaptic cell. GABA is detected by the post-synaptic cell by one of three receptors: GABAA, GABAB or GABAC. Each GABA receptor can be identified by its structure, sensitivity to GABA and selectivity for different agonists and antagonists. These receptors also differ in their pattern of expression in the brain. These structural and functional differences lead to the hypothesis that each GABA receptor mediates a different type of inhibition in the nervous system. In the rodent retina, the GABAC receptor is located only on the axon terminals of bipolar cells. Thus, the GABAC receptor should be able to modulate the excitatory output of the bipolar cells and, thus, ultimately modulate to the overall visual response of the retina.

The proposed experiments will use a mouse in which the GABAC receptor was eliminated (GABAC null mouse) via molecular genetic manipulations. The anatomy of the retina of the GABAC null mice will be examined and compared to controls to determine if the absence of the GABAC receptor alters retinal structure. The visual responses of all of the cells in the retina will be assessed in the GABAC null mice and compared to controls using a non-invasive technique, the electroretinogram. These data will determine how the absence of GABAC mediated inhibition affects the visual response at the level of the retina. These experiments represent the first attempt to define the function of an inhibitory input in the retina, using molecular genetic manipulations

[unreadable] DESCRIPTION (provided by applicant): Vision requires the integration of many aspects of the visual field into a cohesive whole that is used by the organism to navigate its environment. The mammalian retina is a laminar structure and each layer is populated by several cell types that combine to define the response properties of the retinal ganglion cells (RGCs), which transmit the signal to the brain. The many aspects of the visual scene are encoded by two parallel pathways within the retina that either detect increases or decreases in light intensity. There are many types of RGCs and their responses are defined by the inputs they receive. One key element in shaping their response properties is inhibition, which like other regions of the brain is mediated by glycine and gamma amino butyric acid (GABA). GABA inhibition is mediated by three receptors, GABAA, GABAB and GABAC, all of which are present in the retina. One challenge then is to determine how each of these inhibitory and excitatory inputs shapes the responses of the RGCs. A new tool towards attaining this goal is the ability to manipulate the mouse genome and selectively remove specific inputs, the effect of which then can be assessed on the RGCs. However, unlike the extensive literature for cat, rabbit and primate, relatively little is known about the fundamental properties of murine RGCs. The goal of this proposal is to characterize the response properties of mouse RGCs both in vivo and in vitro, and in particular determine the role that GABACR-mediated input plays in shaping the responses. The specific aims are (1) to determine the effect of eliminating, genetically, GABACR-mediated inhibition on a subset of RGC response properties recorded in vivo, and (2) correlate structure and function of a diverse set of RGCs, and determine the impact of eliminating GABACR-mediated input on the response properties of each of these cell types. These data will enhance our understanding of the role of inhibition in shaping the response of the output cells of the retina, and therefore important aspects of integration of information in the visual field. [unreadable] [unreadable]

Program Director/Principal Investigator (Last, First, Middle): PROJECT SUMMARY/ABSTRACT The goals of this research are focused on understanding signaling mechanisms that regulate the development of visual function and spatial organization of retinal ganglion cells and how they are altered in retinal disease. Normal vision begins in the retina and is initiated by photoreceptor detection of light. Parallel pathways of information flow are initiated at the first synapse between photoreceptors and depolarizing and hyperpolarizing bipolar cells, which detect luminance increases and decreases and/or vision under dark- and light-adapted conditions. As a result, the pathways extend the dynamic range of information processing in the intensity domain, and increase specialization of information processing for salient environmental features, e.g., motion, direction and size. Signaling within these circuits is refined by inhibitory inputs in the outer and inner retina and these interactions culminate in and define the receptive field organization of the retinal ganglion cells. The receptive field is a basic property, shared across all sensory neurons in all species. It defines the types and range of environmental stimuli that each cell encodes. Thus, understanding how receptive field properties develop is key to understanding visual function in the retina and the ganglion cells are a vital part because they represent both the culmination of all retinal processing and the scaffold for the rest of visual processing. Because basic RF spatial organization is already present at the onset of visual responses in ganglion cells, the processes underlying their development have been relatively intractable to investigation. That normal vision requires signaling through the depolarizing and hyperpolarizing pathways is amply indicated by the visual defects that occur in patients with and mouse models of congenital stationary night blindness (CSNB), where depolarizing bipolar cell processing is eliminated. We have three unique mouse models of CSNB1 that we will continue to use to probe the synaptic circuitry underlying CSNB, in which normal photoreceptor function is retained. The nature of the changes in spontaneous and visually-evoked responses across GCs in the three mutants provides a unique opening to study the development of receptive field organization and ganglion cell signaling. The phylogenetic conservation of receptive field organization suggests that our findings in the mouse will be relevant to primate peripheral retinal processing. We suggest that the characterization of these mouse models represents a significant opportunity, not afforded by other mouse or vertebrate models and represents a critical step in our understanding both the disease mechanisms in CSNB1 and normal retinal development and function. PHS 398/2590 (Rev. 11/07) Page Continuation Format Page

Project Summary/Abstract A major cause of blindness in North America is hereditary retinal degeneration and Retinitis Pigmentosa (RP) is the leading cause. RP is a group of inherited diseases characterized by the onset of night blindness, the early loss of the peripheral visual field, and ultimately the loss of central vision. The most common form of autosomal dominant form of RP is caused by a Pro23His mutation in rhodopsin that results in retinal degeneration ? i.e. P23H retinopathy. We have created and characterized a miniature pig model of P23H retinopathy to investigate therapeutic options to prevent or delay the onset of visual loss. Multiple therapeutic approaches have been or are being considered to prevent/delay the progression of RP, including cell-based, gene and drug therapies. Gene transfer in Leber's Congenital Amaurosis (LCA), a small subset of RP patients, shows very strong therapeutic promise. That said, gene therapy for other forms of RP faces several challenges including the wide variety of mutations associated with the disease. Cell?based transplantation of rod photoreceptors derived from a number of sources seeks to reverse the progression of RP. Although the early stage of RP is marked by the onset of rod degeneration in mid- peripheral retina, only in the late stages of the disease when retinal degeneration approaches the macula and cone degeneration ensues do most patients find themselves with a severe visual handicap. It also is well established that in RP, cone survival depends on normal rods. We have preliminarily observed that transplanted rod photoreceptors derived from pig induced Pluripotent Stem Cells (iPSCs) injected beneath the retina in our pig model of P23H retinopathy will rescue cones morphologically and functionally in the area centralis (analogous to the macula in man). We will confirm this observation, explore the synergistic effect of neuroprotection with rod derived cone viability factor (RdCVF) and further investigate the mechanism of cone loss in our model. Our studies have the potential to prevent the onset of functional blindness in the majority of patients with RP, not only those with the P23H mutation.

Vision begins when light is converted to an electrical signal in the photoreceptors. Increases in light decrease release of the neurotransmitter, glutamate, from cone and rod photoreceptor terminals, and decreases in light increase its release. These changes in synaptic glutamate concentration are detected by two classes of bipolar cells that then transmit the signal vertically through the retinal circuit to the ganglion cells. The neurotransmitter changes also are detected by horizontal cells that provide lateral transmission in the form of feedback and feedforward inhibition. There are two classes of bipolar cells, hyperpolarizing (HBCs) and depolarizing (DBCs). HBCs utilize ionotropic glutamate receptors and hyperpolarize in response to a light flash. DBCs utilize a metabotropic glutamate receptor, mGluR6, that signals to TRPM1, depolarizing in response to a light flash. Defects in transmission in DBCs results in complete congenital stationary night blindness (cCSNB). Mutations in GRM6, NYX, TRPM1, GPR179 and LRIT3 cause cCSNB. The mechanism by which mGluR6 signals TRPM1 is largely unknown. The long term goal of this project is study the molecular interactions between LRIT3 and known and known components DBC signal transduction components. The specific aims are: 1) determine the function of LRIT3 domains on rod and cone DBC mGluR6 signalplex assembly and signaling, and 2) Define protein interactions required for DBC assembly and synaptic function, and 3) determine the function of LRFN1 and 2 on cone synapse function. At the completion of this project, we will have characterized the function of LRIT3 and how it interacts with currently unknown presynaptic partners. Further, we will have identified new candidate genes for congenital stationary night blindness.

Retinal ganglion cells (GCs) integrate excitatory and inhibitory inputs and perform computations that encode diverse features of the visual scene. The output of these functionally and morphologically diverse GCs establish visual signaling streams, maintained throughout the visual pathway. Ultimately, they create our perception of the size, shape and color of objects and their relationships in space. They establish relationships of objects in time, so that we know when it is stationary or moving, and when moving, its direction and velocity. To this end, there is a general division of labor between the retina's inhibitory neurotransmitter systems. GABA, and its receptors, modulate spatial vision, whereas glycine, and its receptors, modulate temporal vision. There are five glycine receptor subunits (one ? and 4?s). The GlyR? subunits combine with a single ? subunit to make functional receptors with diverse biophysical properties. Our work shows that all GCs expresses one or more GlyR??s and the composition differs across GC type. We hypothesize that variety enhances the diversity of inhibitory functions across and within GC types and are used to encode the visual scene. However, the specific function of GlyR? subunits is almost completely unknown. We know that the responses (and transmitter release) of the GABA and glycinergic amacrine cells presynaptic to GCs are modulated by other glycinergic amacrine cell input. This means that until GlyRs can be selectively eliminated in GCs or ACs, we cannot disambiguate the role of direct GlyR? inhibition to GCs from GlyR? modulation in the upstream circuit that provides input to the GCs. We developed a novel AAV-shRNA knockdown (KD) approach that eliminates expression of individual G lyR? subunits in GCs, while leaving their upstream expression intact. We propose to use this approach to define the role of GlyR? direct inhibition in 7 identified GC types and by extension the role of isolated GABA inputs in those same GCs. In Aim 1 we ask if a single GlyR?, GlyR?1 expressed by the 4 functionally diverse ?GCs modulates the same or different aspects of their visual responses. In aim 2 we ask if two different GlyR? subunits, expressed in the same GC, increase the diversity of inhibition to modulate different aspects of the visual response in a single GC type. We use electrophysiological assays to characterize the spiking responses of the GCs, underlying currents and the relationships between the kinetics of excitatory and inhibitory input with the postsynaptic response. This proposal addresses the novel concept: that diverse glycine subunit specific inhibition interacts with presynaptic inputs to controls visual computation.