Rachel Wong - US grants

The processing of light information by retinal circuits depends critically on the interplay between excitatory and inhibitory neurotransmission. The output of retinal ganglion cells (RGCs) to higher visual centers is shaped by a balance of excitation and inhibition provided by their presynaptic partners, bipolar cells and amacrine cells. Direct inhibition onto RGCs and feedback inhibition onto presynaptic bipolar cells contribute to the final output profile of the RGCs. Previously, we examined how excitatory synapses are established at appropriate densities across the dendritic arbor of RGCs during development and discovered that perturbation of excitatory transmission from bipolar cells results in a reduction in glutamatergic synapses on RGC dendrites. Here, we propose to test the hypothesis that neurotransmission during development regulates the development of inhibitory inputs onto RGCs, and coordinates the maturation of the balance of excitation and inhibition onto these cells. We will also test the hypothesis that transmission influences the development of feedback inhibition onto the axon terminals of the bipolar cells. In Aim 1, we will determine the mature organization of inhibitory synapses onto RGC dendrites and examine how these patterns arise during development. This will be achieved using confocal and multiphoton imaging of RGCs with fluorescently tagged inhibitory postsynaptic sites. These experiments are needed to reveal the spatial and temporal relationships of the development of inhibitory and excitatory synapses on the dendrites of individual RGCs. In Aim 2, we will directly test the hypothesis that neurotransmission regulates inhibitory synapse development on RGCs using transgenic mice in which either glutamatergic or GABAergic transmission is markedly suppressed. In Aim 3, we will determine how feedback inhibition onto rod bipolar cells develop and ascertain the role of neurotransmission in the assembly of this synapse using the transmission-perturbed mice. Taken together, the results from these aims will advance our knowledge of how inhibitory circuits in the inner retina develop, and help define the role(s) of neurotransmission in attaining the proper wiring of retinal circuits or their miswiring in injury and disease.

Our broad goal is to understand how precise patterns of neuronal connections are established during development of the central nervous system. This proposal focuses on the role of presynaptic signaling in organizing in organizing the structure of the postsynaptic substrate, the dendritic arbor, during synapse formation and refinement of the retina. The dendrites of immature retinal ganglion cells (RGCs) undergo structural and functional remodeling as they contact and receive input from presynaptic cells. The role of activity-dependent and activity- independent signaling from two classes of retinal interneurons (cholinergic amacrine cells and bipolar cells) in the remodeling of RGC arbors will be investigated. Aim 1 will characterize the structural and potential functional relationship between the terminal processes of the retinal interneurons and the dendrites of RGCs before and during RGC dendritic remodeling. RGC arbors will be labeled with multicolors by a novel method of delivering carbocyanine dyes to retinal explants (collaboration with Jeff Lichtman). Cholinergic amacrines and a subset of bipolar cells will be labeled by introducing yellow fluorescent protein downstream of promoters specific to these cells, using knock-in technology. Aims 2 and 3 will assess the in vivo roles of presynaptic signaling by ablating these interneurons or blocking their release of neurotransmitter. Transmission will be blocked by knocking out the choline acetyltransferase gene, or by introducing the tetanus toxoid (TeTx) gene downstream of the cell-specific promoters. Ablation of cells will be carried out by replacing the TeTx gene with the diptheria toxin gene. Cre-lox technology will e used to ensure that gene or cell ablation and neurotransmission blockade occurs only when induced at an appropriate developmental stage. RGC arbors from wildtype and mutant mice will be reconstructed and compared. All transgenic mice will be generated in collaboration with Josh Sanes.

[unreadable] DESCRIPTION (provided by applicant): Synaptic connections in the nervous system are highly specific. In some regions of the central nervous system, synapse specificity arises from the reorganization of more diffuse, early patterns of connectivity whereas in other regions, precise patterns may be present from the very beginning. Thus, one of the most challenging and important issues in neurobiology concerns how neuronal circuits are established with precision during development. We are interested in understanding how neural circuits are formed and organized in the vertebrate retina, and in particular how interactions between potential pre- and postsynaptic cells guide this process during development. Although much knowledge has been gained from in vitro work, it is evident that examining this process in vivo will provide insight into the dynamic interactions that take place to establish synaptic specificity. The zebrafish is an ideal model for studying the in vivo development of retinal circuits. This is because synapse formation is completed within a few days after fertilization and the zebrafish embryo can be maintained transparent, making it suitable for in vivo imaging throughout the period of synapse formation and maturation. In this application, we propose to focus on the development of networks in the inner retina. We will determine how outgrowth and elaboration of the postsynaptic dendrites of retinal ganglion cells and the presynaptic terminals of amacrine cells, that together form the first retinal network, contact and form the synaptic region, the inner plexiform layer (IPL) during development. We will combine time-lapse in vivo imaging techniques with molecular approaches to elucidate the normal pattern of IPL development, and then apply these techniques together with the use of retinal mutants to ascertain the role of cell-cell interactions in organizing this synaptic layer. Together, the results of the proposed studies will further our understanding of the mechanisms underlying the structural and functional development of the inner retinal circuitry.

Vision requires the precise organization and function of neuronal circuits in the retina. Our  overall goal here is to advance the basic understanding of the cellular mechanisms that  regulate the formation and the maintenance of synaptic connections in the mammalian  retina. Like elsewhere in the nervous system, signals not only converge onto individual  neurons  from  multiple  input  types,  but  signals  from  an  individual  neuron  are  also  distributed  across  multiple  targets.  Together,  these  two  basic  motifs  of  synaptic  connectivity,  convergence  and  divergence,  underlie  the  complex  but  highly  organized  processing  of  neuronal  information.  Our  knowledge  of  the  mechanisms  that  sculpt  stereotypic patterns of convergence is expanding. In contrast, our understanding of how  divergence is shaped during development and disrupted in neurodegenerative conditions  is scarce. To fill this gap in knowledge, we will focus on the AII amacrine cell circuitry that  is  integral  to  the  rod  pathway,  which  is  responsible  for  scotopic  vision.  This  circuit  will  enable us to gain insight into the developmental mechanisms that organize an exquisite  arrangement of synaptic divergence at a single synapse (Aim 1), as well as mechanisms  that  distribute  synapses  from  a  single  cell  in  a  biased  but  consistent  manner  across  distinct  targets  (Aim  2).  We  will  use  a  combination  of  novel  imaging  approaches,  electrophysiology and transgenic animals to: (Aim 1) Determine the cellular mechanisms  that organize synaptic divergence at the rod bipolar cell -­ AII/A17 amacrine cell dyad, and  ascertain the factors that lead to disruption of this synapse after neurodegeneration due  to a rise in intraocular pressure (IOP), and: (Aim 2) Determine the cellular processes that  shape  output  connections  of  AII  amacrine  cells  onto  bipolar  cells  and  ganglion  cells  during  normal  development,  and  identify  the  processes  that  disrupt  these  connections  upon  IOP  elevation.  Together,  our  findings  will  greatly  advance  knowledge  of  the  mechanisms responsible for precision in circuit assembly, as well as offer new knowledge  of how this precision becomes altered in conditions of neuronal degeneration. 

DESCRIPTION (provided by applicant): Vision requires proper information transfer from photoreceptors to retinal ganglion cells (RGCs), the output neurons of the retina. This information is distributed by retinal bipolar cells (BCs) along many anatomically and functionally distinct channels. Because BC channels carry different chromatic and temporal information, the light response of a RGC is shaped by the unique combination of BC input it receives. To date, the circuitry of only a few functionally defined RGC types is known, largely because reconstructions by serial electron microscopy are technically challenging and time consuming. Using state-of-the art imaging approaches and molecular tools to visualize synapses and connectivity, we are able to readily reconstruct these circuits by light microcopy. We will establish, and compare, the connectivity patterns of three functionally distinct BC-RGC circuits in the mouse retina, in order to learn new principles or uncover distinct strategies by which BC input can shape RGC output (Aim 1). We will investigate how loss of neurotransmission (Aim 2), or death of retinal neurons (Aim 3) alters circuitry in the inner retina. Because the effects of activity blockade can vary according to how neurotransmission is perturbed in development or in disease, we will determine how disruption of input and/or output of BCs influence their connectivity with RGCs. We will use novel transgenic mice in which transmission is perturbed in distinct ways, and also mice in which activity is altered in either a few or entire populations of BCs. In Aim 3, we will determine the potential of mature BCs and RGCs to re-connect when cells from one or the other population are ablated. We will do this by using transgenic mice in which the magnitude and timing of cell death can be controlled. These findings will be particularly significant for designing cell-based therapies to restore vision. Together, the discoveries of this project will significantly increase our understanding of the cellular mechanisms that regulate the function, assembly and repair of retinal channels necessary for conveying information from photoreceptors to RGCs.

Cellular Biology Service and Shared Instrumentation Component ? Project Summary The objective of the Vision Research Core is to enhance the productivity and efficiency of the research programs of the UW vision scientists, with special priority given to investigators holding NEI R01 grants. The core achieves this objective by: (1) giving investigators and their laboratory personnel access to resources that are outside the resources of individual R01 grants, (2) giving investigators and their laboratory personnel access to technical expertise that is outside the scope of individual labs, (3) providing training of laboratory personnel to enhance the capacity of individual labs, and (4) providing a culture that promotes collaboration. The Cellular Biology Services and Shared Instrumentation Component achieves this goal ? by providing access to state-of-the-art confocal and electron microscopes, ? by providing dedicated technical assistance in the use of the component?s microscopes, ? by providing assistance and training in preparation of tissue for microscopy, ? by providing assistance and training in image acquisition and analysis, and ? by maintaining component equipment in good working order through service contracts.

? DESCRIPTION (provided by applicant): The Graduate Program in Neuroscience, established at the University of Washington in 1996, is large and strong, with 48 students and 141 faculty members from 27 departments and 4 partner institutions across the city of Seattle. Our goal is to train the best neuroscientists possible, fostered by inclusion of students from diverse and underrepresented backgrounds. We have exceptional breadth and depth of research interests, including neurodevelopment, neurodegeneration, addiction, ion channel physiology and pathology, systems neuroscience, and computational neuroscience. The breadth of our faculty allows us to provide interdisciplinary training drawing from a variety of techniques and approaches, including neuroanatomy, biochemistry, molecular biology, physiology, biophysics, pharmacology, in vivo brain imaging, computational modeling and behavior. In addition to a solid core of required and elective courses, students also receive instruction in other key areas of professional development on topics including grant writing, public speaking and bioethics. Faculty mentors and the Graduate Training Committee closely monitor student progress to ensure that each student receives the guidance he or she needs to succeed. Graduates emerge from the program prepared to conduct independent research and equipped to pursue a variety of career paths. The intent of this proposal is to partially replace an Institutional Training Gran for Neurobiology (T32 GM007108) that the University of Washington has held for 40 years. T32 GM0007108 has been a crucial underpinning of our interdisciplinary Neuroscience PhD program, but is being retired because of administrative changes in the Institute of General Medical Sciences. The retiring award supported 12 students; here we seek support for 6. One of the primary attractions of our program is that it accommodates students with diverse academic backgrounds, and offers a wide selection of faculty with whom to work. This diversity of academic backgrounds, however, makes training grant support essential. It would be inappropriate to ask a faculty member to provide research support for a student whose background will not allow a fast track to productivity within that lab and on the specific research grant that will support the work. There are currently no other predoctoral training grants that are available to support early-stage students. Because the Graduate School funds our students in their first year, funds from this T32 would be used primarily to support students in their second year, when they are just beginning their dissertation research, and are still completing their elective coursework and teaching requirements. By supporting early-stage students while they remain substantially engaged in important components of their training outside their dissertation labs, this training grant will give our students greater independence and control at a critical stage of their graduate careers, and make a significant contribution to the continuing success of graduate training in neuroscience at the University of Washington.

This request seeks reimbursement for attending the Meeting of the Jointly Sponsored Institutional Predoctoral Training in the Neurosciences T32 Program Directors in Baltimore, MD on 2/19/19. I am the University of Washington JSPTPN T32 PD. The presentations and informal discussions in which I participated at the meeting will improve quantitative training in our Neuroscience Graduate Program throughout students? time in graduate school, beyond required coursework and electives in the first few years.

Microscopy and Histology Core Module ? Project Summary The objective of the Vision Research Core is to enhance the productivity and efficiency of the research programs of the UW vision scientists, with special priority given to investigators holding NEI R01 grants, and to promote collaborations among investigators. The Microscopy and Histology Core Module achieves these objectives by (i) Providing access to state of the art confocal and electron microscopes, including serial block facing scanning electron microscopes (ii) Providing dedicated technical assistance in the use of specialized confocal and electron microscopes (iii) Providing assistance and training in the preparation of tissue for microscopy (iv) Providing assistance and training in acquisition and analysis of images from core microscopes (v) Maintaining module equipment in good working order through service/maintenance contracts and support of biomedical engineer with expertise in the maintenance and repair of core equipment and computers