Spencer L. Smith, PhD - US grants

DESCRIPTION (provided by applicant): In the developing nervous system, initially promiscuous synaptic connections are remodeled by activity-dependent mechanisms, with axons strengthening their connections with some targets while completely disconnecting from others. In the neocortex, this process has been most thoroughly studied in the developing primary visual cortex, where decorrelating the activity of the two eyes dramatically reduces cortical binocularity. These functional changes in cortical physiology are widely accepted to be anchored in long-lasting changes in the efficacy of synaptic connections. However, the specific mechanisms by which synaptic plasticities lead to changes in the function of cortical circuitry remain elusive. The major objective of the work proposed here is to determine where in the cortical circuit experience-dependent changes in binocular responses are first observed, track the progression over time, and test the hypothesis that these changes are anchored in anatomical changes in synaptic connectivity. To address these questions, techniques such as 2-photon laser scanning microscopy, intrinsic signal optical imaging, and single-unit recordings, will be used to map circuit plasticity and follow changes in synaptic connectivity in vivo.

This award is made by the Instrument Development for Biological Research program (IDBR)in the Division of Biological Infrastructure (DBI; BIO Directorate).

The cerebral cortex, the outer layer of brain, has greatly expanded in surface are during mammalian evolution. This cortical region is parcellized into discrete functional areas including visual cortex, motor cortex, and language areas. These brain areas act in concert to support behavior. Although we have learned much about how to ascribe function to particular brain areas, we know little about the cellular mechanisms by which this concert is conducted. Model systems, including mice have discrete functional areas in their brains. However, the current tools that neuroscientists have for investigating activity in brains are limited to either a sparse sampling of neurons distributed over large areas, or a large density of neurons in a single area just 500-700 microns across. Thus, it is tremendously difficult to make progress in understanding how cortical areas act in concert to support behavior. The proposed research project will develop a new type of microscope which will be able to detect single neuron spiking across a field of view of several millimeters. This area can encompass five or more cortical areas in a mouse. In addition, this microscope will contain high speed spotlights for simultaneously imaging neuronal activity in multiple cortical areas. This time resolution is crucial for understanding the information neurons encode, their dynamics during behavior, and their connectivity. A community of scientists across the US and the globe will be cultivated to disseminate the research, aid in its implementation, and accelerate collaborative progress in neuroscience. Workshops will also be held to train scientists in advanced optics and neuroscience. Ultimately, this project will provide new technology that is crucial for the BRAIN Initiative, and will foster a broader scientific community for further progress in the field of two-photon imaging.

The research team will develop a two photon (2p) imaging system with a wide field-of-view (FOV) (~ 3 mm) and cellular resolution across the full FOV. To ensure high temporal resolution of recorded activity, they will also develop multiplexed beams that image brain regions within the FOV at high speed. These multiplexed beams can be dynamically reconfigured to target different areas within the full FOV, like spotlights. The approach is to model the full system and create optimized optical subassemblies, including a custom objective. The team will make calculated engineering tradeoffs to preserve cellular resolution while still achieving a wide FOV. High speed scanning will be developed using resonant scanners and photon counting electronics. This system is scalable, as the beam multiplexing can be modularized, and multiple modules can be stacked to increase the number of beams, so long as the fluorescence lifetime is shorter than the interval between laser pulses. Thus, the Trepan2p (Twin-Region, Panoramic 2p), will enable direct measurements of cross-correlations and moment-to-moment, dynamics in extended brain networks. This technology will enable previously impossible experiments, imaging neuronal activity with single cell resolution across extended neuronal circuitry in an array of model systems including mice and primates.

? DESCRIPTION (provided by applicant): Higher visual functions, including object recognition and global motion detection, are carried out by extrastriate visual areas, or higher visual areas (HVAs), downstream from primary visual cortex. These higher processing functions often fail to completely recover following congenital blindness or amblyopia. These observations suggest that early visual experience sculpts HVA circuitry, but this has not been determined. To address this gap in knowledge, we propose an integrative approach using genetically engineered mice. Our goal is to determine how experience sculpts the normal development of HVA circuitry and how circuit deficits are caused by visual deprivation. Our preliminary data indicates that a subset of HVAs is slow to develop after eye opening, and fails to completely recover visual responses following dark rearing. By contrast, a complementary set of HVAs is visually responsive at eye opening, and fully recovers following dark rearing. We will map the development of HVAs at multiple time points, starting at eye opening, using intrinsic signal optical imaging and two photon calcium imaging. We will use a paradigm we developed for high resolution receptive field mapping of local populations of neurons in parallel. We will use technology we have recently developed to examine activity correlations between visual cortical areas and map how these change in development. We will dark rear mice to determine the effect of visual deprivation on HVA circuitry. The results from this project will reveal the role of visual experience in sculpting HVA selectivity and circuit development.

? DESCRIPTION (provided by applicant): Cortical activity is tightly regulated to support adaptive behavior, but the mechanisms underlying this regulation are unclear. In this project, we will investigate how cortical activity is regulated in vivo, directly at the site of synaptic input Dendrites actively process synaptic input using voltage-gated ion channels and NMDA receptors. We recently showed that these mechanisms support dendritic spiking in awake mice. These dendritic spikes propagate to the soma as depolarizations that can trigger conventional axonal spikes, and thus represent a layer of computational processing that contributes to neuronal selectivity. A recently elucidated circuit motif involving neuromodulation and dendrite-targeting interneurons could play a key role in regulating dendritic spiking during sensory processing and behavior. Here, we use dendritic patch clamp recordings, optogenetics, and new multiphoton imaging technology to interrogate this circuit motif, its effects on dendritic spiking, and its activity during sensory processing and behavior. Since dendritic spiking is an essential component of synaptic integration in cortical circuitry, and dysfunctional synaptic integration is implicated in complex psychiatric and neurological disorders, results from this project can eventually contribute to new therapeutic strategies.

Multiphoton neuroimaging is a powerful approach for measuring neural activity in the brain, offering subcellular resolution of thousands or more neurons at time. However, current technology has technical limitations that restrict what experiments are possible. Therefore, this project will create new technology through a NeuroNex neurotechnology hub, called Nemonic (NExt generation Multiphoton NeuroImaging Consortium). The Nemonic project has three parts. First, the development component will create new systems in a series of Case Studies to enable currently impossible neuroscience experiments. Second, the dissemination component will spread this technology broadly to other neuroscience labs through open source resources, workshops, and industry partnerships. Third, the advancement component will push the fundamental technology of multiphoton neuroimaging into the next frontier. Two specific technologies will be developed: miniaturized photonic systems for multiphoton neuroimaging; and super-resolution imaging to image submicron structures. Also, a series of meetings will foster novel collaborations to more rapidly advance engineering technologies that are relevant to multiphoton neuroimaging in the future. Technology developed in the Nemonic project will also be relevant to manufacturing, 3D printing, and photonics. This work will also increase partnerships between academia and industry, enhance STEM training, and recruit and support the scientific training and careers of women and URM scientists.

The Case Studies will develop new instrumentation for large field-of-view two- photon and three-photon imaging, scalable temporal multiplexing, and integrated behavior. This technology will be developed for compatibility with an array of model systems. The focus will be on calcium and glutamate imaging, in cell bodies and processes, and other fluorescent indicators can be employed. The workshops will cover optical design, fabrication, assembly, and use, for an audience of neuroscientists and engineers. One advancement project will develop high peak power ultrafast lasers with transform limited pulses, with integrated, beam conditioning, beam steering, focusing, and detection systems. The other advancement project will develop super-resolution multiphoton imaging using spatial frequency modulation, adaptive optics, and novel pulse conditioning. Together, this work will advance multiphoton neuroimaging and a suite of related technology, through research, enhanced training, and industry partnerships. This NeuroTechnology Hub award is co-funded by the Division of Emerging Frontiers within the Directorate for Biological Sciences as part of the BRAIN Initiative and NSF's Understanding the Brain activities.

Project summary Vision plays a key role in our ability to navigate through the environment, from identifying landmarks and obstacles to determining location and heading. While studies of visual cortex have provided an understanding of properties such as orientation selectivity and object recognition, much less is known about how cortical circuitry extracts and processes features from the visual scene to support navigation. In particular, there are two challenges. First, the nature of the visual stimulus is dramatically different in navigation, where the subject's movement through the world creates a complex and dynamic visual input, in contrast to standard synthetic stimuli presented to stationary subjects. Second, the types of visual features and computations that must be performed are different in navigation than in standard detection or discrimination paradigms. Our goal in this proposal is to determine how the brain extracts relevant visual features from the rich, dynamic visual input that typi?es active exploration, and investigate how the neural representation of these features can support visual navigation. We will investigate this through three parallel aims, that build up from the representation of the visual scene in V1 during freely moving navigation, to the computation of speci?c variables needed for navigation. In our ?rst aim, we will measure the visual input in freely moving mice using miniature head-mounted cameras, together with neural activity in V1, to determine how neural dynamics represent the visual scene during natural navigation. In our second aim, we will use large ?eld-of-view two-photon imaging of multiple cortical areas, while mice navigate in a naturalistic open-world virtual reality system, to determine how visual features are represented across visual cortical areas. In our third aim, we will use 2-photon imaging in mice in a rotational arena to determine how visual input is used to dynamically update a key navigational variable: heading direction. Together, this project bridges foundational measurements in freely moving animals with mechanistic circuit investigations, to provide insights into an important aspect of visual system function.