(provided by candidate): All of brain function, from sensory perception to behavior, is derived from the pattern and properties of the synaptic connections among billions (in humans) of individual neurons. The long-term goal of this project is to understand molecular pathways that regulate synapse formation in vivo using a vertebrate model with a focus on the underappreciated electrical synapse. Electrical synapses are sites of direct communication between neurons that allow the passage of ions and small molecules. They are formed in a regulated manner between only a subset of potentially available partners and are composed of neuronal gap junction channels. Electrical synapses contribute extensively to neural circuits during development as well as to adult circuits from sensory perception to processing to motor output. However, the molecular mechanisms underlying the formation of the gap junction channels that form the electrical synapse are unknown. This proposal utilizes the Zebrafish Mauthner (M) circuit to investigate the genetics of electrical synapse formation. The M neurons are individually identifiable and their pre and postsynaptic partners, synapses, and function are exquisitely visualized in a living, vertebrate embryo. A forward genetic screen for mutations causing defects in the stereotyped M electrical synapses was performed that identified two distinct classes of mutations: 1) the Disconnect (Dis) class, which disrupts synapse formation, and 2) the Amped (Amp) class, which causes ectopic synapses to form along the M axon. Using an RNA-seq-based approach all three Dis mutations were positionally mapped, and one of the Dis mutants was found to be due to the loss of the autism- associated gene neurobeachin (nbea). This proposal will investigate Nbea's role in electrical synapse formation (Aim1), will clone the other Dis and Amp mutations identified in the pilot screen (Aim2), will examine the effect of the mutations on synapse function and behavior (Aim3), and will expand the pilot screen to elucidate further genes and pathways required for synaptogenesis (Aim4). During the two year mentored phase I will develop the model system by characterizing how the genes regulate electrical synapse formation in several ways: What are the temporal and spatial properties of synaptic cargo localization during in vivo synaptogenesis? How do the mutants affect the function of the synapse? How do the mutants affect neural network function and behavior? In Cecilia Moens' lab at the Fred Hutchinson Cancer Research Center (main mentor), I will learn to perform live cell imaging of fluorescently-tagged, synaptic proteins using spinning disc confocal microscopy. This technique will be applied to all mutants and will be the first live investigation of electrical synapse formation in vivo. To investigate M synapse and circuit function I will visit Joe Fetcho's lab at Cornell University to learn to perform electrophysiology n the M neural circuit and I will visit Michael Granato's lab at the University of Pennsylvania Perelman School of Medicine to learn behavioral analysis of the M-mediated escape behavior. The skills acquired will be brought back to Seattle where I will perform experiments on the mutants. For electrophysiology I will work with Rachel Wong at the University of Washington (main co-mentor) where I will receive ongoing training in electrophysiology and will have access to equipment for experiments. For behavior I will work in the Moens lab where we have the high- speed camera necessary to capture the M-mediated escape response. The electrophysiological and behavioral analysis will be applied to all mutants and will be essential for linking the cell-biological defects to functional deficits in the circuit. The training in the Fetcho and Granato las will be short and intensive, but both mentors will be available to me on an ongoing basis for technical expertise and guidance. The mentoring in the Moens and Wong labs will be ongoing, with extensive interaction and support. With this training I will have the necessary experience and a powerful set of tools and techniques to establish my own independent research group. During the independent phase of the project I will utilize the acquired skills to illuminate the molecular mechanisms that build gap junctions at the electrical synapse. The proposed studies will provide a detailed molecular, cellular, and functional view of how neural circuits form in a vertebrate in vivo. Disorders that cause neural circuit miswiring or synaptic imbalance are the basis of many neurological diseases including autism and epilepsy. In the case of autism, several molecular pathways (including Nbea examined here in Aim1) have been associated with the disorder. However a unifying theory explaining how these genes fit together to explain the syndrome remains elusive. Investigating the genetic pathways required for neural circuit wiring and synapse formation will lend insight into disease states that will ultimately allow for the identification of targets for therapy.

Public Health Relevance

The central nervous system contains billions and billions of neurons organized into connected circuits allowing for the transfer and processing of information, leading to perception, thought, and behavior. The brain is not just a jumble of wires randomly linked to one another, but instead very specific and reproducible circuits are created, and defects in the genes underlying their development lead to a number of diseases such as autism. This study proposes to investigate how genes regulate the creation of neuronal circuits, giving insight into this fundamental process, knowledge that is necessary to guide future therapeutic work that attempts to fix diseased brains.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Transition Award (R00)
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Special Emphasis Panel (NSS)
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Riddle, Robert D
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University of Oregon
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Miller, Adam C; Pereda, Alberto E (2017) The electrical synapse: Molecular complexities at the gap and beyond. Dev Neurobiol 77:562-574
Miller, Adam C; Whitebirch, Alex C; Shah, Arish N et al. (2017) A genetic basis for molecular asymmetry at vertebrate electrical synapses. Elife 6:
Marsh, Audrey J; Michel, Jennifer Carlisle; Adke, Anisha P et al. (2017) Asymmetry of an Intracellular Scaffold at Vertebrate Electrical Synapses. Curr Biol 27:3561-3567.e4
Shah, Arish N; Davey, Crystal F; Whitebirch, Alex C et al. (2016) Rapid Reverse Genetic Screening Using CRISPR in Zebrafish. Zebrafish 13:152-3
Shah, A N; Moens, C B; Miller, A C (2016) Targeted candidate gene screens using CRISPR/Cas9 technology. Methods Cell Biol 135:89-106
Miller, Adam C; Voelker, Lisa H; Shah, Arish N et al. (2015) Neurobeachin is required postsynaptically for electrical and chemical synapse formation. Curr Biol 25:16-28
Shah, Arish N; Davey, Crystal F; Whitebirch, Alex C et al. (2015) Rapid reverse genetic screening using CRISPR in zebrafish. Nat Methods 12:535-40