Joseph R. Fetcho - US grants

DESCRIPTION: Much of the ability to move depends upon networks of neurons in the spinal cord and hindbrain. Recent evidence indicates that there is a relatively simple structural and functional organization in spinal cord that may extend into the hindbrain. This proposal explores whether there is a basic template to the organization of neurons and their wiring in hindbrain that is established during development and that underlies the control of movement. Such a template would provide a conceptual organization that would enhance in a major way the understanding of the organization of a part of the brain that is critical for normal movement and whose proper function is disrupted in disease and after spinal injury. The proposed work stems from the discovery that neurons in hindbrain are clustered into stripes based on neurotransmitter. The proposal takes advantage of the ability to see into the brain and spinal cord of intact larval zebrafish to explore 1) whether the stripes correspond to transcription factors and how the stripes develop, 2) whether neurons within a stripe are similar to one another and project in a regular way to neurons in other stripes, 3) whether the electrical properties of neurons within a stripe vary systematically with their position and age in a way that might lead to their orderly activation during normal movements and 4) whether the position of a neuron in a stripe reflects the speed of the movement in which it is activated. The proposed work will reveal basic principles that link development with the later structure and function of neurons in the hindbrain. These will inform us about brain organization in vertebrates, including humans, and should help us to interpret and eventually treat movement disorders. PUBLIC HEALTH RELEVANCE The ability to move depends upon nerve cells located in the back part of the brain, the hindbrain. This proposal outlines experiments to examine how the hindbrain is organized to control movements. The work is important because the control of movement by the brain is disrupted in spinal injury as well as in genetic diseases that affect movement.

Movements are produced by activity in populations of neurons, but little is known about the patterns of recruitment in neuronal populations and their relationships to behavior. This proposal outlines studies of how different forms of a simple vertebrate behavior-the escape behavior in zebrafish-are produced by activity in pools of hindbrain reticulospinal neurons and spinal interneurons. The work uses powerful new imaging techniques that take advantage of the transparency of larval zebrafish and allow the imaging of activity in cells in the intact animal, as well as the photoablation of cells to study the resulting behavior changes. The aims are: 1) to use photoabalations to test the hypothesis that the hindbrain contains serially repeated sets of functionally related reticulospinal neurons that act as a population to produce escape turns of different magnitudes; 2) To use photoablations to provide the first direct test of the functional role of at a class of spinal interneuron in a behavior; 3) To use calcium imaging to study how the spinal interneurons in a population are recruited during movements (escapes) of different strength and; 4) To use ablations, combined with imaging of activity, to study the links between reticulospinal activity and spinal interneuron recruitment. The work is of general importance because many of the descending commands for movement from the brain are channeled through the reticulospinal system and spinal interneurons, but we know little about the patterns of activity in these neuronal populations and their contributions to behavior. Each of the proposed aims not only provides a piece of the information needed for better understanding of one behavior, they also address fundamental issues in the control of movement that will apply broadly among vertebrates. These include the functional significance of hindbrain segmentation, the behavioral role of spinal interneurons, patterns of interneuron recruitment, and the links between reticulospinal neurons, premotor interneurons and behavior. The work is basic research dealing with principals of motor organization. The establishment of the principals by which normal movements are produced provides the foundation for understanding the disruptions of movement that occur in various diseases. The work is of additional significance because of the increasing importance of zebrafish as at a model system in development and genetics. The power of the eventual combination of development, genetics, functional imaging and behavioral studies make the further development of this model system especially important.

Abstract: Sleep and sleep states are fundamental not only to human life, but to every animal with a nervous system. Surprisingly, it is still not clear why they are so important. One compelling idea is that there are global shifts in the strengths of synaptic connections and excitability during sleep that act to keep synaptic function and neuronal excitability in a range where synapses and excitability of neurons can change relative to one another to allow for learning. If this does not happen, network function and behavior, whether in a worm or a human, degrade, leading ultimately to death. Such thinking about an important role of homeostatic mechanisms is moving to the fore in neuroscience, but what is needed to test hypotheses about global patterns of change in synapses and excitability is a model system and tools that allow us to monitor single synapses and neurons broadly in the living brain. We propose to develop and apply optical tools that allow us to examine patterns of scaling of synapses and excitability in the transparent zebrafish model where we can monitor these regularly and non-invasively over time during sleep and wakefulness. We will use these to directly test whether global resetting occurs during sleep. If sleep really involves such rescaling, the implications would be major, not only for a basic understanding of sleep, something that we should understand by now, but also for trying to restore functional states when sleep is impaired as a result of sleep disorders.

Nervous systems evolved to solve many of the same problems in species as diverse as worms, fishes, and humans. Using collections of neurons, from 100 or fewer in small invertebrates to hundreds of millions in humans, animals behave in ways that allow survival and reproduction in demanding and often hostile environments. A major hurdle to revealing the principles by which diverse species achieve these goals is being able to monitor the structure and function with high resolution throughout the brain - a necessity because behavior emerges from broad interactions of neurons across brains, even in the simplest organisms. A team of investigators will push optical imaging to this goal through the development of a NeuroNex Neurotechnology Hub at Cornell University. Experts from physics, engineering and biology will work together to develop, demonstrate the utility of, and disseminate to other neuroscientists a suite of imaging tools that will overcome current technology barriers in studying how brains work. Furthermore, the Hub will provide a unique opportunity to educate the next generation of neuroscience researchers to work in interdisciplinary teams that combine the neuroscience, technology development, and big data analysis expertise required to make progress in understanding the brain. Graduate student trainees will also receive instruction in science communication and mentoring in career planning. Opportunities for undergraduate researchers will be provided and the PIs and other project members will become actively engaged in outreach to local-area schools, the broad public, and policymakers.

Large scale, noninvasive recording of neural activity in awake and behaving animals is essential to understand the function of the nervous system. This NeuroNex Neurotechnology Hub will develop and disseminate innovative neurotechnologies for noninvasive recording of neural activity across a large depth and volume, at multiple places in the central and peripheral nervous system, and with high spatial and temporal resolution. The newly developed optical imaging technologies will be employed in behaving animal models across multiple species in different phyla, including mammals, teleost fish, flies, and birds, and will be demonstrated by attacking important neuroscience questions in fruit fly, zebrafish, and mice. We will create a new lab, the Laboratory for Innovative Neurotechnology at Cornell (LINC), which will be shared by the Hub PIs and serve as the physical embodiment of the hub, and close the loop between technology development and biological questions. LINC will serve as a hub for dissemination by hosting, connecting, and training researchers and developers across multiple disciplines from both academia and industry. The dissemination of these new technologies will catalyze studies of how brains produce behavior in species across a broad range of sizes throughout animal phylogeny. This NeuroNex Neurotechnology Hub award is co-funded by the Division of Emerging Frontiers within the Directorate for Biological Sciences and the Division of Chemical, Bioengineering, Environmental, and Transport Systems within the Directorate of Engineering as part of the BRAIN Initiative and NSF's Understanding the Brain activities.

Vertebrate behaviors emerge from interactions of neurons across the brain, but the tools for revealing neuronal structure and function at the cellular level in living animals access only small portions of the brain. We must move toward access to structure and function anywhere in the brain of individual adult, behaving animals. In vivo three photon (3P) microscopy, a recent, but proven, technology allows optical access to deeper structures than ever before in intact mammalian brains, but much optimization remains to catalyze its wider adoption. The plan of this project is to extend the reach of 3P microscopy both within brains and through the scientific community by developing new technology and proving its worth for imaging structure and function anywhere in the brain of adult zebrafish ? a powerful vertebrate model. This project will extend the depth, speed and regional extent of imaging with 2P and 3P through a combination of technological improvements. Imaging depth will be enhanced by the development of a novel dual adaptive optics approach to correct optical aberrations that combines conjugate and standard adaptive optics to allow deep imaging through the skull with near diffraction limited resolution and improved signal. To enhance the breadth and speed of imaging, a novel approach will be developed with a light-weight, small, tandem piezo-fiber scan engine and a large field of view with the ability to raster scan any 2 to 4 sub-regions in the field. By determining and then applying optimum laser repetition rates and the best order of the nonlinear excitation as a function of depth, the number of neurons will be increased that can be imaged and reduce light exposure to improve longer term, repeated imaging through life. While the innovations will be useful for many animal models, they will be tested by imaging newly generated transgenic zebrafish lines made with CRISPR technology, as well as other established lines. The lines label neurons of different transmitter phenotype with membrane targeted fluorophores for structural imaging, or genetically encoded calcium indicators (gCaMPf or s) for functional imaging. The goal on the biological front is to provide the tools to allow the unique ability to image neuronal structure and function anywhere in the brain of an intact individual vertebrate at any time during its life, from embryo into adulthood.