Jason D. Shepherd, Ph.D - US grants

DESCRIPTION (provided by applicant): A major challenge in neuroscience is to understand how neuronal networks are modified through experience and how proteins/genes contribute to circuit modification. Neural circuits are refined during development through activity-dependent gene and protein expression. Similar macromolecular synthesis is essential for long-term forms of synaptic plasticity such as long-term potentiation (LTP) and depression (LTD). Efforts to identify molecules that underlie these forms of plasticity have revealed a set of genes that target to excitatory synapses. Among these, Arc is the most tightly coupled to behavioral encoding of information in neuronal circuits. Arc homeostatically regulates surface AMPA type glutamate receptors (AMPARs) by directly interacting with the endocytic machinery. However, very little is known about Arc's function at the level of neuronal circuits or its precise in vivo role in mediating information storage. The visual cortex is an ideal preparation to probe these questions as visual experience can be modulated to induce gross changes in neuronal activity. The overall goal of this proposal is to investigate the mechanisms that underlie Arc's role in modifying neural circuits in response to visual experience and how these processes are disrupted in neurological disorders. In previous experiments, we find that Arc plays a fundamental role in experience- dependent plasticity in mouse visual cortex (V1). Arc knocks out mice exhibit deficits in ocular dominance plasticity and in a newly discovered form of experience-dependent plasticity, stimulus-specific response potentiation. We also uncover an experience and Arc-dependent component to establishing the contralateral to ipsilateral ratio. How does Arc regulate experience-dependent plasticity in the visual cortex? The goal of aim 1 is to investigate the mechanisms underlying these phenotypes by utilizing slice electrophysiology in V1 cortical slices and investigating the role of Arc in 3 different types of synaptic plasticity;LTD, LTP and synaptic scaling. In vivo electrophysiology provides a powerful tool to assess experience-dependent plasticity, but it is difficult to identify specific networks of individual cells. The goal of aim 2 is to investigate the role of Arc in experience- dependent plasticity at the single cell level using 2-photon calcium imaging in vivo, which can measure neuronal activity in many cells with spatial precision. Aim 3 will directly test whether Arc mediates plasticity through its role in AMPAR trafficking. Finally, aim 4 intends to test the idea that Arc levels are critical for normal synaptic homeostasis and that abnormal Arc levels contribute to the synaptic dysfunction observed in neurological disorders, including Alzheimer's disease, Fragile X and Angelman Syndromes. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to understand, at the molecular and cellular level, how experience shapes and modifies the brain. We will investigate the role of the activity-dependent gene Arc in synaptic and experience-dependent information storage in mouse visual cortex. We will also test the hypothesis that normal Arc levels are critical for brain function and that abnormal regulation of Arc expression contributes to the synaptic dysfunction observed in neurological disorders.

Proposal # 1533611
Institute: University of Utah
Title: NCS-FO: Imaging synaptic activity deep in the brain using super-resolution cannula microscopy

Objective: This project will develop a tool for high-resolution (<100-nm) imaging of synapses in freely moving animals for neuronal studies. It will accomplish this goal by the development and integration of compact and lightweight cannula microscopy with in vitro fluorescence imaging with accompanying technology and methodologies for imaging synapses.

Non-Technical
The long-term vision of this project is to image with high resolution deep inside the brain of freely moving mice using inexpensive technologies so as to elucidate the fundamental basis of information processing and memory. Changes in synaptic strength at specific synapses are thought to underlie memory encoding and storage, yet there is very little experimental evidence for this theory in the intact brain due to technical limitations of visualizing the specific synaptic pattern involved in experience-dependent learning. This project aims to overcome this limitation by transforming a simple, inexpensive cannula into a super-resolution fluorescence microscope. Commercialization of this technology will be pursued after the fundamental science and engineering has been demonstrated for widespread dissemination.

Technical:
The objective of this proposal is to image neuronal activity, neuron structure and protein localization deep in the brain with sub-100nm resolution using computational cannula microscopy (CM) and novel molecular reporters of synaptic activity. CM will allow imaging of the brain in awake, freely moving animals at unprecedented spatial resolution. Current techniques in freely moving animals are limited to imaging the brain near the surface, include large and heavy head stages with moving parts, and cannot penetrate deep into the brain without significant damage to surrounding tissue. The ultimate goal of this proposal is to allow imaging of individual synapses in freely moving animals. We have already developed the framework for in vitro fluorescence imaging using CM. During this project, we will extend CM to enable: (1) super-resolution (< 100nm resolution) fluorescence microscopy and (2) deep-brain imaging (depth > 1mm) with the vision of imaging activity and protein localization in individual synapses in the deep brain of freely moving animals. Changes in the strength of individual synapses are thought to underlie learning and memory in the brain, yet this fundamental theory of brain function lacks tangible experimental evidence to support it in vivo. Our project will enable studies that address the causal role of molecular events at individual synapses in mediating behavior and information processing.

The overarching goal of this proposal is to characterize the protein machinery that mediates trafficking of postsynaptic membrane proteins critical for synaptic plasticity. Endo- and exocytosis of membrane proteins are critical trafficking steps in all cells, but are especially dynamic and finely tuned in neurons. Unlike presynaptic endocytosis and synaptic vesicle recycling, the precise molecular processes that underlie postsynaptic trafficking still remain poorly defined. Little is known about how specific receptors are removed from the protein scaffolding complex at the postsynaptic density (PSD) or the functional role of protein-protein interactions within the PSD during synaptic plasticity. Our previous work identified the immediate early gene Arc as a critical mediator of memory storage in the brain and showed that Arc regulates AMPA-type glutamate receptor trafficking. We conducted unbiased proteomic screens and discovered novel Arc interacting proteins. Based on these screens, we hypothesize that Arc acts as a novel postsynaptic clathrin adaptor protein. Based on this hypothesis, we will test whether Arc binds lipids, recruits cargo to endosomes (e.g. AMPARs) and interacts with PSD proteins to release receptors from their scaffolding within the PSD. To study protein trafficking within nanodomains of synapses we have developed a novel live super-resolution light microscopy approach. This study will provide mechanistic insight into how Arc mediates multiple forms of synaptic plasticity and also broadly elucidates the protein machinery that is involved in postsynaptic trafficking of membrane proteins at excitatory synapses. Arc lies at a critical nexus as a critical synaptic effector protein and has been implicated in neurological and psychiatric disorders in human patients. Thus, this work will also shed light on synaptic dysfunction associated with neurological diseases.

PROJECT SUMMARY Optical imaging methods are well-established in neuroscience, but high-speed, high- resolution volumetric imaging of neural activity in deep tissue remains a challenge. A number of techniques address limited aspects of this goal, and most are applicable primarily to acute preparations. We propose to develop and test a novel approach to achieve three-dimensional ?deep-tissue? imaging for high spatial and temporal resolution neural recording by combining aspects of embedded optical probes with computational imaging techniques. Rather than use a single micro-endoscopic probe, we propose to utilize an array of narrower probes, or optrodes, to reduce the volume of tissue displacement. Computational imaging through each probe can be performed to achieve a field of view (FOV) at a desired distance from the probe tip. Combining the fields of view from multiple probes arranged in an array then provides a composite image field that is much larger than achievable from a single micro-endoscope. In our approach, each ?0.1 mm diameter probe of the array acts as an independent micro- endoscope. In order to achieve full-field imaging across the array, the individual fields must intersect, and the computational method must be scaled to accommodate, and stitch, multiple fields. In pursuit of these goals, we propose three Aims: Optimizing the FOV of a single micro-endoscope - The purpose of this Aim is to characterize the FOV for an individual probe at multiple depths, and optimize the FOV to about 0.3mm through control over the shape of the probe tip and light collection numerical aperture. Accelerating calibration and reconstruction - In this Aim, we will pursue efficient computational approaches for calibration based upon ray-tracing simulations and image reconstruction based on deep learning. Scaling the FOV with an endoscope array - The computational image reconstruction method will be scaled to accommodate small micro-endoscope arrays (e.g. 4 element) arranged in a hexagonal lattice with FOV of 0.6mm at a 1.5mm depth.

Abstract Recent studies by our group have revealed that the neuronal gene Arc, a master regulator of synaptic plasticity and information storage in the brain, acts as a repurposed retroviral Gag protein that forms capsids with the capacity to transmit genetic information between cells. These findings lead to a paradigm shift in the way we view both mechanisms of cognition and more generally how cells can signal to each other. This transformative R01 application will address these questions using a synergistic team of neuroscientists and virologists who will apply their expertise to Arc, intercellular gene transmission, and neuronal development. We will determine what genetic messages are transferred between neurons in Arc particles, how these particles enter ?target? neurons to deliver their RNA cargo to cell cytoplasm, and how delivery of this cargo influences the neuronal and synaptic processes that underlie memory and cognition. The methodologies to address these questions, as well as the potential impact of the answers, make this application ideally suited to the transformative R01 mechanism.

  Abstract  Post-­mitotic  neurons  in  the  mammalian  brain  form  synapses  that  dynamically  remodel  throughout  an  individual?s  lifetime  to  encode  short-­  and  long-­term  memories.  Synaptic  plasticity  involves  spatiotemporal  fine-­ tuning  of  gene  expression  levels  in  response  to  environmental  stimuli,  including  rapid  transcription  of  immediate  early  genes  on  the  time  scale  of  minutes  and  longer-­term  global  chromatin  remodeling.  The  cis-­ acting  genetic  and  epigenetic  elements  that  govern  activity-­dependent  expression  are  of  outstanding  interest  toward understanding how experiences sculpt the brain. Here, we submit a proposal entitled ?Elucidating the 3-­ D epigenetic determinants of activity-­dependent gene expression in mammalian neurons?. We have assembled  an  interdisciplinary  team  with  critical  expertise  in  genome  folding,  epigenetics,  chromatin  engineering,  neurobiology,  synaptogenesis,  electrophysiology,  and  computational  biology.  We  aim  to  elucidate  the  causal  link among long-­range looping interactions, epigenetic modifications on the linear genome, expression of their  spatial  target  genes,  and  the  activity  of  mammalian  neurons.  We  hypothesize  that  immediate  early  genes  will  functionally engage in singular short-­range loops to rapidly activate expression on the time scale of seconds to  minutes in response to the environmental stimulus of neuronal activation. By contrast, we posit that secondary  response  genes  will  spatially  connect  via  architectural  proteins  into  complex,  long-­range,  pre-­existing  topological  configurations  to poise  the genome  for  a  second  wave  of  expression  on  the order  of  hours  to  days  in  response  to  neuronal  firing.  To  test  our  hypotheses,  we  will  create  high-­resolution  genome  folding  maps  using  the  Hi-­C  during  a  time  course  of  activation  in  mouse  hippocampal  neurons.  We  will  identify  activity-­ dependent  enhancers  and  gene  expression  genome-­wide  and  determine  their  temporal  profile  with  respect  pre-­formed and  activity-­dependent  loops.  We  will formulate  mathematical  models  to predict  activity-­dependent  expression  of  immediate  early  genes  and  secondary  response  genes  from  the  timing  of  enhancer  activation  and looping contacts. By integrating single nucleotide variants linked to autism, schizophrenia, bipolar disorder,  addiction,  and  attention-­deficit/hyperactivity  disorder  with  our  models,  we  will  predict  the  specific  target  genes  and  potential  pathways  involved  in  neurological  disease.  Finally,  we  will  dissect  the  functional  role  for  loops  and  enhancer  activity  in  regulating  the  activity-­dependent  transcription  of  Bdnf  and  c-­fos  using  CRISPR  genome  editing  of  architectural  protein  binding  motifs  and  CRISPRi  inhibition  of  specific  enhancers.  Our  work  will  uncover the genome?s  long-­range  interaction  landscape  in  mammalian neurons and  reveal  the causal  link  between  the  3-­D  Epigenome  and  the  kinetics  of  transcriptional  response  to  environmentally  stimulated  neuronal activation.