Andrew D. Huberman - US grants

Project Summary/Abstract The overall goal of this research program is to understand how parallel retinofugal pathways are established during development. This proposal focuses on the question of how retinal ganglion cell (RGC) axons select their correct targets and target layers in the brain during development. RGC axon-target and axon-layer matching are both critical aspects of visual circuit organization and yet, very little is known about how they develop in mammals. We propose to study the development of these connections in transgenic mice where specific RGC subtypes express fluorescent proteins and in which specific genes are absent or misexpressed in the retinofugal pathway. The specific aims of this proposal are to 1) characterize the cellular events that enable RGCs carrying different qualities of visual information to recognize their appropriate targets in the brain, 2) test the hypothesis that the adhesion molecule cadherin-6 controls RGC axon-target matching 3) test the hypothesis that lamina specific targeting of axons from direction selective RGCs to the superior colliculus is mediated by the adhesion molecule cadherin-7. Results from these experiments should lead to new understanding of how mammalian visual circuits are established and inform strategies for maintaining and replenishing visual circuits in response to injury or disease.

? DESCRIPTION (provided by applicant): Visual neuroscience is finally beginning to achieve a vertically integrated understanding of the retina, bridging all levels from molecules to microcircuits to behavior. Success could be achieved for all retinal microcircuits in just a decade, if progress were sped up drastically. Such acceleration will be attained by generating the following foundational data and disseminating it to the community. (1) We will use genetic control of ganglion cell types to pinpoint their specific roles in a suite of ethologically relevan, visually guided behaviors. Our functional explorations will be guided by the tracing of downstream pathways into subcortical and cortical regions using genetic techniques. (2) We will apply two-photon calcium imaging and serial electron microscopy to a single patch of retina to yield complete information about its activity and connectivity, i.e. the functional connectome. This will be complemented by genetically targeted studies that correlate light and electron microscopy in separate retinas. (3) We will physiologically characterize inner retinal pathways through projective field measurements, i.e., massively parallel recording of population responses to direct stimulation of single bipolar and amacrine cells, employing both electrical and optical techniques. All these investigations will be linked by shared standards for typing neurons based on molecular markers, 3D structure, and functional signatures, as well as standardized visual stimuli and behavioral paradigms. We will initially develop the standards to facilitate collaboration between the members of this team. Disseminating them will enable the entire community to collectively pursue a vertically integrated approach to the retina. The ultimate impact will be transformative: a new generation of efficient coding theories and network models of retinal function solidly based on empirical data. Integrative neuroscience seeks to bridge the gap between the microscopic level of cells and the macroscopic level of behavior and mind. Such vertical integration is often professed as a goal, but has mostly been achievable only in invertebrate nervous systems. The retina now offers an unprecedented opportunity for vertically integrated neuroscience in the mammalian CNS. This approach will eventually extend to encompass the entire visual system.

Abstract Although mature retinal ganglion cells (RGCs) are normally unable to regenerate axons following optic nerve damage, studies from several labs, including those participating in this collaboration, have identified cellular, molecular, and physiological manipulations that enable some RGCs to regenerate injured axons from the eye to the brain. In spite of these efforts, however, the number of RGCs that survive after optic nerve injury and successfully regenerate axons into the brain remains small, thereby limiting meaningful visual recovery. The proposed research will combine a strong pro-regenerative therapy with novel transcriptomic and proteomic approaches and cutting-edge bioinformatic methods to identify new transcripts and proteins associated with the initiation and execution of a successful regenerative program. We will investigate the temporal sequence of changes in gene expression, protein translation, and protein transport down the regenerating optic nerve as mature RGCs undergo a transition from a normal intact state into a robust growth state, identify transcripts and proteins selectively expressed in the subset of RGCs that successfully extends axons into the nerve, and characterize the RGC subtypes with the highest potential to regenerate axons. 100-150 of the top candidate genes identified in the discovery phase will be tested for their ability to promote axon outgrowth in immunopurified RGCs in culture, and lead candidates from the intermediate screen will be tested for their ability to substantially augment levels of optic nerve regeneration in vivo, either in isolation or in combination with established pro-regenerative therapies. These latter studies will investigate the targeting of axons to appropriate central visual nuclei and tests of visual recovery. The integrated approach proposed here directly addresses the goal of identifying novel molecular targets to re- establish visual circuitry after injury to the visual system.

Project Summary/Abstract The overall goal of this research program is to understand how to regenerate mammalian central visual pathways. This proposal specifically focuses on the question of how to regenerate projections from output neurons of the eye, retinal ganglion cells (RGCs) to their targets in the brain (collectively referred to `retinofugal pathway')- a circuit absolutely essential vision. Significant progress has recently been made in identifying molecular programs capable of triggering some RGC axon regeneration. The next crucial milestones for the field are to discover ways to increase the number RGC axons that regenerate and the distance they regro. It is also crucially important to determine whether regenerating RGCs can find and reconnect to the correct targets, such visual capacities return. The three major aims of this proposal are to: 1) test the hypothesis that specific forms of visual stimulation can enhance the number and distance of RGC regenerating axons 2) test the hypothesis that combining visual stimulation with molecular triggers of RGC axon growth can cause RGC axons to regrow long distances back into the brain and 3) determine whether regenerating RGCs are capable of pathfinding back to and re-connecting to their appropriate targets, as well as avoiding targets incorrect for their function. Results from these experiments should lead to new understanding of how mammalian visual circuits can be replenished in response to injury and pave the way for the cultivation of tools applicable to humans suffering from vision loss.

Project Summary/Abstract The overall goal of this proposal is to elucidate how to regrow and reconnect injured optic nerves and tracts to specific target neurons in the brain. Specifically, this proposal investigates mechanisms that promote the regeneration of connections made by direction selective retinal ganglion cells (DSGC) to their the accessory optic targets in the brainstem (collectively referred to as the ?Accessory Optic System,? or ?AOS?). The AOS serves a crucial role in vision by generating slip-compensating eye movements whenever the head or the eyes move at slow speeds. In the absence of proper AOS connectivity and function, images appear blurry and perceptual performance is severely degraded. From a practical standpoint, understanding how to regenerate the mammalian AOS, and defining the cellular and molecular underpinnings of that regeneration, represent an ideal model for parsing regeneration of other visual parallel pathways and also mammalian CNS circuits generally. The AOS is comprised of known retinal neurons and circuits, and the central targets and information carried in this pathway are rather well understood. Indeed, significant progress has been made by our and other groups in identifying genetic markers for the DSGCs that drive AOS function and also cellular and molecular pathways that wire them to their targets. Moreover, both of our laboratories have adopted and expanded state-of-the-art approaches to measure AOS function at the whole animal level with quantitative rigor. In parallel to our work, the field of CNS visual system regeneration has reached the crucial milestone of identifying molecular and activity-based manipulations that allow some retinal ganglion cell (RGC) axons to regenerate following axotomy. The next crucial milestone is to figure out how to ensure accurate reconnection of specific RGC types with their correct targets in the brain. Importantly, it remains unclear whether, after damage to the retina or optic nerve, RGCs and/or their targets re-express, or maintain expression of, the receptors or ligands that enabled them to correctly wire up with one another during development. It is also imperative to determine how the specificity of axon-target matching at the level of cell types and targets, impacts circuit function and behavior. Now that the molecular programs for these developmental steps have started to become clear, this essential issue relating to optic nerve regeneration can finally be approached with deep rigor, and we propose here do that in the context of the AOS. The four major aims of this proposal are to: 1) Test the hypothesis that AOS-projecting RGCs are among the cohort of RGC types capable of regenerating in response to increases in mTOR activation and/or RGC firing. 2) Test the hypothesis that damage to AOS-projecting RGCs and their axons triggers robust changes in axon guidance receptors and ligands in the relevant RGCs and targets. 3) . Test the hypothesis that re-introduction of specific guidance receptors and ligands to AOS-projecting RGCs can be used to steer their axons to desired brain areas. 4) Test the hypothesis that regeneration of a small fraction of total retinofugal connectivity is sufficient to replenish functional recovery of the optokinetic reflex and nystagmus necessary for image stabilization.

NEUROGENETICS OF VISION PROJECT SUMMARY Genetics and in particular, genetic-based approaches such as transgenes and viruses that enable researchers to express proteins of interest in desired sets of neurons in the retina and brain, are pivotal to make significant progress in modern basic visual neuroscience and toward the treatment of visual disorders. The last decade has brought forth a wide and powerful arsenal of genetic tools for identifying the neurons that comprise the visual system of flies and rodents. Because they enable delivery of a wide range of gene cargo, such tools also allow for selective manipulation of cells of interest. Indeed, in comparison to just a decade ago, nowadays it is straightforward to label a given cell type in vivo, and thereby visualize its unique morphology, and then compare it to other cells of different types, selectivity target them for electrophysiology, calcium- or voltage-dye imaging, and then reversibly silence or activate them. Last but not least, genetic tools can be leveraged to explore the signature pattern of RNA expression present in different cell types in order to probe their homology across species and/or relevance to mutations associated with human diseases.