Prakash Kara - US grants

[unreadable] DESCRIPTION (provided by applicant): The overall goal of this research is to understand how local networks of binocular cells in the visual cortex combine inputs from the left and right eyes. Understanding binocular vision at the local network level may contribute to the development of new forms of rehabilitation for restoring function to the compromised visual cortex in strabismus, amblyopia, or physical damage to the eye. The dominant approach for assaying the binocular properties of cortical neurons employs monocular tests of ocular dominance to infer binocular function. However, the relationship between ocular dominance and binocular disparity-a property that is critical for stereopsis and can only be assessed with simultaneous stimulation of both eyes-remains unclear. The functional micro-architecture of disparity tuning has never been described in any species. Furthermore, while great strides have been made in identifying the physiological and biochemical changes that result from monocular deprivation, there are clear inter-species differences (and variability amongst different studies in the same species) when ocular dominance is the only assay used to determine the binocular properties of cortical neurons. By using the new technique of two-photon calcium imaging in vivo, the specific goals of this proposal are to determine how these distinct binocular properties are mapped in cortical circuits, to assess whether there are systematic relationships between the mapping of ocular dominance and disparity tuning, and to evaluate how these properties are impacted by monocular deprivation. In Aim 1, we will resolve whether there is local mixing of neurons from different ocular dominance groups or whether these maps are as precise and smooth as those found for orientation selectivity. In Aim 2, we determine whether a micro-architecture for binocular disparity exists and whether ocular dominance can predict disparity tuning. In Aim 3, we examine whether monocular deprivation during the critical period disrupts the micro-architecture of binocular disparity. Clustering of cells that retain normal disparity tuning after monocular deprivation may provide important clues into the local circuit mechanisms that drive cortical neurons to become disparity selective. In Aim 4, we examine whether the single-cell fluorescence changes obtained with calcium imaging of neuronal cell bodies correlate with the single-cell firing rates obtained with electrophysiological methods. These results promise to shed new light on the functional organization of cortical circuits that mediate binocular vision, and the detrimental effects of altered visual experience. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): A single neuron in the visual cortex receives many thousands of synaptic inputs on its dendrites from other cortical neurons. The functions of these various synaptic inputs in sensory signal processing are unknown. We label single neurons in vivo such that the entire dendritic tree can be visualized and mapped functionally. We examine whether subsets of synapses on dendrites process different stimulus features in the environment. These synapses on cortical neurons may play an important role in experience dependent plasticity and the recovery of binocular vision after the initially detrimental effects of altered visual experience such as during amblyopia. PUBLIC HEALTH RELEVANCE: Amblyopia is a common cause of visual impairment in children where circuits within the brain favor the processing of visual stimuli from just one of the two eyes. This award will support research on structural and functional imaging of neurons in the visual cortex that may be affected by amblyopia.

DESCRIPTION (provided by applicant): The overall goal of this project is to determine the contributions from spiking, synaptic and astrocytic activity in shaping the feature selectivity of blood vessels in the sensory neocortex. This work is important to advance our understanding of brain function because vascular (hemodynamic) signals are now widely used to infer neural function in health and disease. Yet the mechanisms driving many of the spatial and temporal aspects of sensory-evoked hemodynamic signaling are poorly understood. We will perform two-photon functional imaging of neurons, astrocytes and blood vessels in the primary visual cortex of the cat. This animal model shares many sophisticated visual abilities and cortical circuit organizing principles with primates, including spatially precise cortical maps for encoding stimulus orientation. We will use the mapping of stimulus orientation as the probe to determine the contribution of synapses, spikes, and astrocytes in shaping sensory-evoked hemodynamic responses in individual blood vessels. We will combine sub-micron resolution imaging with cell-specific genetically encoded fluorescent sensors for detecting spiking activity (via gCaMP6 imaging) and synaptic activity (via iGluSnFr imaging). We also include artery-specific fluorescent labeling, intracellular recording from astrocytes, and pharmacological blockade of the astrocyte- specific glutamate transporter. In Aim 1, we test the hypothesis that the selectivit of sensory-evoked dilation of an individual blood vessel is predicted by the spatial pattern of synaptic activity (specifically, glutamate release) immediately surrounding the vessel, not the spatial integration of spiking activity. In Aim 2 we test the hypothesis that glutamate-driven astrocyte signaling is required for the rapid sensory-evoked activation of blood vessels. To our knowledge, this is the first study in any brain region that will extract sensory-evoked selectivity (tuning curves) from individual blood vessels and relate these single-vessel tuning curves to the local synaptic, spiking and astrocytic activity.

Non-technical description
This Research Infrastructure Improvement Track-2 Focused EPSCoR* Collaboration (RII Track-2 FEC) project involves the Medical University of South Carolina as the lead and the University of Alabama at Birmingham, Furman University, and University of South Carolina, Beaufort Campus as collaborative partners. Much of our knowledge of brain function comes from experiments using functional magnetic resonance imaging (fMRI), which directly measures blood flow to regions of the brain, but remains an indirect measure of neural activity. The precise relationship between neural events and hemodynamic response (increased blood flow) is unclear. This project will test the hypothesis that universal rules relate these two brain activities, using direct measurements in the brain and retina of mice and macaques.

Technical Description
The research will develop new instrumentation for in vivo imaging and cell subtype-specific stimulation in both the brain and the retina necessary for interpreting functional Magnetic Resonance Imaging (fMRI) measurements. These activities will help determine the extent of neurovascular coupling and provide a description of the micro-circuitry, which represents a critical and necessary step in understanding the full complexity of the brain. The consortium will track neural and vascular activity with micron-scale resolution, using two-photon microscopy and adaptive optics to measure the presence of synthetic dyes interacting with genetically encoded sensors. Cell subtype-specific stimulation will be monitored using optogenetic techniques. A computational team will analyze the data obtained to tease out hemodynamic signals. Experiments will be performed on macaques, selected because of the similarity in size and functional repertoire with the human brain. Studies in mice, where genetic and molecular tools are more readily available, will also be performed.

*Experimental Program to Stimulate Competitive Research

PROJECT SUMMARY Our knowledge of signal processing in various parts of the human brain has been heavily influenced by non- invasive functional magnetic resonance imaging (fMRI) experiments. FMRI infers the location and selectivity of neural activity from vascular signals. However, brain circuits are much more complex than regional differences in neuronal selectivity. Specifically, the largest part of the brain (neocortex) accounts for up to 80% of the brain volume and is divided into six distinct layers. Specific computations, e.g., local processing vs. feedforward inputs vs. vs. feedback inputs, are done in specific cortical laminae. Thus, if high-resolution layer-specific fMRI is shown to reflect the repertoire of neural computations performed across these cortical layers, it would be an invaluable refinement to non-invasive imaging. However, despite the widespread usage of low-resolution fMRI, a detailed understanding of how neural activity generates vascular responses remains unknown. The goal of this project is to elucidate the link between neural and vascular signals across laminae by combining two-photon imaging of neural and vascular responses with ultra-high-field (UHF) fMRI. Experiments will use sensory visual stimuli that induce layer-specific responses. In cat primary visual cortex (V1), which has a functional architecture (e.g., maps for stimulus orientation) similar to human V1, we will measure neural activity (synaptic and spiking) with single-cell resolution together with vascular signals (blood flow, blood volume, and oxygenation) in individual vessels across the entire cortical thickness. We will also perform UHF lamina-specific fMRI in cat (9.4 and16.4 T) and human (7 and 10.5 T) V1 to relate fMRI signals to the single- vessel responses. Lastly, we will develop a model to relate lamina-specific vascular signals to neural activity. In Aim 1, we test the hypothesis that vascular signals selective for stimulus orientation are present in cortical layers 2/3 (and 5/6) while untuned responses occur in layer 4 and pial vessels. Grating visual stimuli will be used, while varying orientation and eye preference (ocular dominance) systematically. Since binocular integration is stronger outside layer 4, eye preference vascular signals should be most prominent in layer 4. In Aim 2, we will test the hypothesis that in any given cortical lamina, glutamate release in regions around an individual blood vessel best accounts for the selectivity of vascular responses compared to spiking activity?in terms of the preferred stimulus orientation and tuning width. Aim 3 is to build a computational model to determine effective minimum voxel size for BOLD fMRI. The model will be tested against simultaneously measured vascular and neural activity to natural scene stimuli using two-photon imaging. If the source signals at the finest spatial scales have laminar specificity, we can correlate laminar-specific fMRI signals to differences in neural processing. To our knowledge, this is the first study that brings together such a wide repertoire of approaches into a single project to understand the neural and laminar basis of fMRI.

Project Summary Spinal dorsal horn interneurons (IN) integrate somatosensory inputs and control their access to spinal projection neurons (PrN) that transmit nociceptive information to supraspinal components of pain pathways. The heterogeneity of dorsal horn neurons, limited knowledge on their connectivity, and lack of in vivo neurophysiological analysis of identified IN currently preclude comprehensive mapping of circuits involved in pain processing. Our long-term goal is to define dorsal horn neuronal networks in vivo, in the context of somatic sensory stimuli with established behavioral outcomes. We propose to establish a platform for multidisciplinary analysis of dorsal horn circuits based on: 1) monosynaptic transfer of non-toxic self- inactivating modified rabies virus (SiR) for targeted expression of fluorescent reporter proteins and Ca2+ indicators; 2) in vivo three-photon Ca2+ imaging of synaptically connected neurons using genetically encoded Ca2+ indicators; 3) CLARITY-based structural analysis; 4) single-cell transcriptional profiling of functionally characterized synaptically connected neurons. For proof-of-concept of this network analysis, the proposed project is focused on spinal PrN and their presynaptic IN. Information on these IN is extremely limited. We will conduct structural and transcriptomic profiling of PrN neurons and their presynaptic IN and examine in vivo their activity in response to sensory stimuli. Our central hypothesis is that this complementary analysis will identify modality-selective subtypes in the population of IN that are presynaptic to PrN. In Specific Aim 1, we will establish the structural connectivity of PrN and their presynaptic IN by labeling them differentially through monosynaptic gene transfer. CLARITY-based immunohistochemistry and volumetric imaging will be used to establish the structural relationship between the labeled neurons and classify them based on previously established morphological and neurochemical criteria. In Specific Aim 2, we will define responses of PrN and their presynaptic IN to innocuous and noxious thermal and mechanical sensory stimuli using in vivo three- photon Ca2+ imaging. Following in vivo imaging, we will use CLARITY methodology for morphological and neurochemical analysis of the functionally characterized neurons. In Specific Aim 3, we will characterize PrN and their presynaptic IN based on single-cell transcriptional profiling by fluorescently labeling their nuclei through monosynaptic gene transfer and conducting single-nucleus transcriptomic analysis. In addition, we will carry out proof-of-concept experiments for in vivo genetic tagging of neurons following three-photon Ca2+ imaging to correlate single-cell transcriptomes with physiological response properties. Impact: The proposed project will establish a new approach for integrated structural, transcriptional and functional mapping of dorsal horn microcircuits that will address critical gaps in our understanding of spinal pain processing. Monosynaptic tracing of different subsets of dorsal horn neurons in future studies will allow us to systematically and comprehensively explore connectivity in dorsal horn networks.