Huizhong W. Tao, Ph.D. - US grants

DESCRIPTION (provided by applicant): The main goal of the proposed research is to examine the role of spike-timing-dependent plasticity in the functional modification of the visual cortex. Recent studies performed in the sponsor's laboratory have provided evidence that cortical space representation of cat V1 neurons can be modified by the relative timing of visual stimuli applied to adjacent retinal areas. I will further examine stimulus-timing-dependent cortical modifications with the following specific aims: Aim 1. To determine whether stimulus-timing-dependent modification of space representation is cortical in origin. I will examine whether the modification effect can undergo interocular transfer, which is believed to be intracortical. Aim 2. To determine the spatial specificity and time course of stimulu-timing-dependent modification of cortical receptive fields. I will use asynchronous local two-point stimuli to induce modification of cortical space representation. By varying the position of the conditioning stimuli, the spatial specificity of the modification effect can be determined. Further experiments will be carried out to determine the time course for induction and the persistence of the effect, with an aim to understand the potential roles of spontaneous or ongoing activity in reversing the stimulus-induced modifications. Aim 3. To examine the acute effect of moving stimuli in the induction of cortical modifications, I will investigate the acute effect of moving stimuli in shaping adult cortical receptive fields and its dependence on the velocity of the stimuli, thus providing further evidence for the involvement of spike-timing-dependent plasticity.

[unreadable] Description: (provided by applicant): The functional properties of sensory cortical neurons, as reflected in their response selectivity to stimulus attributes, are primarily determined by the spatiotemporal integration of sensory-evoked excitatory and inhibitory synaptic inputs to the cell. The objective of this project is to provide an understanding of excitatory and inhibitory synaptic mechanisms underlying the cortical cells' functional properties. The role of synaptic inhibition in shaping visual cortical processing has remained controversial. In addition, due to the difficulties in identifying and targeting cortical inhibitory neurons in vivo, the receptive field (RF) properties of these neurons, which are crucial to the function of synaptic inhibition, remain largely elusive. We propose to combine the in vivo whole-cell recording and two-photon imaging techniques, and exploit mouse genetic models, to determine the response properties of excitatory and inhibitory inputs in visual cortical neurons. In Aim 1, using "blind" whole-cell voltage-clamp recording coupled with histology, we will dissect the excitatory and inhibitory synaptic conductances of cortical excitatory neurons evoked by sparse flash stimuli. We will determine how simple and complex receptive field structures are determined by the spatial distribution of synaptic inputs. By reconstituting the membrane potential changes that result from these synaptic inputs, we will test the hypothesis that inhibitory inputs play a crucial role in sharpening the spatial discreteness of spike On and Off receptive fields. In Aim 2, we will perform two-photon imaging guided loose patch recording in a transgenic mouse line where inhibitory neurons are labeled with green fluorescence protein. We will examine visually evoked spike responses of both fluorescent inhibitory neurons and non-fluorescent excitatory neurons, and test the hypothesis that there are functional differences between these two groups of neurons, i.e. inhibitory neurons are less selective to stimulus attributes such as spatial phase and orientation. In Aim 3, by applying imaging guided whole-cell current-clamp and voltage-clamp recordings, we will test the hypothesis that the functional differences between excitatory and inhibitory neurons can be attributed to the difference in the strength of synaptic inputs they receive, rather than in the structure of synaptic input circuitry. These studies will provide novel insights into functional cortical circuitry. PUBLIC HEALTH RELEVANCE In the central nervous system inhibitory synaptic inputs control the gain of network activity and play a critical role in information processing. Abnormality in synaptic inhibition has been implicated in several cognitive disorders and age-related reduction in perceptual functions. The proposed project will advance our understanding of the role of inhibitory circuits in visual processing, and may provide important insights into how functional changes of inhibitory neurons can lead to deterioration of cognitive functions. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): To understand cortical functions, it is essential to determine how information contained in sensory input is represented and processed in individual cortical neurons that are morphologically and neurochemically diverse. To understand mechanisms underlying the representational and processing properties of individual cortical neurons, a blind, in vivo, whole-cell patch-recording technique has been applied to examine sensory-driven excitatory and inhibitory synaptic inputs onto cortical neurons. Results from such studies have provided new insights into synaptic circuit mechanism underlying cortical neurons'response properties. Although this technique can be combined with post hoc histological methods to reconstruct the morphology of recorded cells, its blind nature largely limits its potential in examining various cell types in the cortex, since it normally results in a biased sampling of excitatory pyramidal neurons in the cortex. In this exploratory project, we will study a new technique for revealing functional properties and synaptic inputs of inhibitory cortical neurons in vivo, two-photon imaging guided patch recording (TPGP) in which fluorescently labeled neurons are visualized by two-photon imaging and specifically targeted for patch recording. This technique has benefited from recent development of mouse genetics in labeling cells of specific types with fluorescence proteins such as green fluorescence protein (GFP), with their expression controlled by cell-type specific promoters. At an initial step, we will apply this recording technique to GFP-labeled GABAergic interneurons in the supragranular layers (L1-3) of mouse primary visual cortex (V1), to address the receptive field properties of these neurons and how these properties are determined by the integration of visually activated excitatory and inhibitory synaptic inputs.

Project Summary The way a cortical neuron processes sensory information is determined by its functional synaptic input circuit. This circuit contains three elements: 1) input connectivity from excitatory and inhibitory presynaptic neurons; 2) the strength and dynamic properties of each input; 3) processing properties of each presynaptic neuron. Although separate deciphering of each of these elements helps to extract how excitatory and inhibitory inputs interact to generate of output response properties of the neuron, an integral study addressing all these components in an intact system is necessary but remains to be a tremendous challenge. In vivo whole-cell voltage-clamp recording provides a unique and valuable approach for us to directly isolate and reveal the summed functional excitatory and inhibitory synaptic inputs under specific sensory stimuli. As the spatiotemporal properties of excitation and inhibition are determined by the above circuit elements, it provides a means to bridging the gap between connectivity with function. In this project, we will continue to harness the strength of this approach to understand various visual processing functions, and extend its application to awake mouse primary visual cortex (V1). First, we will determine the tuning relationship between inhibition and excitation underling orientation selectivity and spatial receptive fields in excitatory neurons in different cortical layers. Using neuron modeling and dynamic clamp recording, we will examine the diverse roles of inhibition in shaping functional selectivity. Next, based on our recent discovery of an interesting correlation between the direction tuning of excitatory responses under moving stimuli and the spatial asymmetry of excitatory input strengths evoked by stationary stimuli, we will examine how direction selectivity can be generated de novo in the cortex by testing a novel hypothesis that the spatial asymmetry can be converted into differential temporal summation under stimuli of opposite directions. By optogenetic silencing of cortical excitatory neuron spiking, the origin of this spatial asymmetry will also be examined. Finally, through optogenetics assisted cell identification, we will apply the whole-cell voltage-clamp recording to PV inhibitory neurons, and determine the mechanisms for their generally weak selectivity. Together, the proposed experiments will generate important new insights into how functional cortical synaptic circuits are organized and how cortical processing and sensory perception may go awry under neurological disease conditions which result in disrupted excitation-inhibition balance.

DESCRIPTION (provided by applicant): Various functional properties of visual cortical neurons, as usually measured by their output responses, undergo progressive developmental maturations, the process of which can be susceptible to experience-dependent modifications during a critical period (CP). The functional changes of neuronal responses during development or induced by specific sensory experience can be attributed to changes in cortical synaptic circuitry, the nature of which has remained largely unknown. This is mainly due to a lack of measurements of stimulus-evoked excitatory and inhibitory synaptic inputs to neurons in the developing visual cortex, techniques for which remain very challenging. In this project, we will explore the possibility of probing into the functional synaptic circuits of the developing cortex b applying the-state-of-the-art in vivo whole-cell voltage-clamp recording and two-photon imaging guided patch-recording in young mice at various developmental stages. We will focus on orientation selectivity (OS) and ocular dominance (OD) properties in layer 4 of the primary visual cortex (V1). In Aim1, we will determine the progression of OS development by recording spike responses at different developmental stages. We will then carry out voltage-clamp recordings to determine excitatory and inhibitory inputs underlying OS during stages when significant sharpening of OS occurs. We will test the hypothesis that the developmental sharpening of OS can be mainly attributed to a broadening of inhibitory tuning, rather than a sharpening of excitatory tuning or a decrease in excitation/inhibition (E/I) ratio. We will also record from genetically labeled inhibitory neuron subtypes to test the hypothesis that the broadening of inhibitory tuning can be attributed to a weakening of OS of specific inhibitory neurons. In Aim2, we will compare eye-specific excitatory and inhibitory inputs to excitatory neurons between mice experiencing monocular deprivation (MD) during the CP and age-matched control mice. We will determine whether the MD-induced OD shift away from the deprived eye is mainly attributed to a weakening of synaptic excitation or a strengthening of synaptic inhibition driven by that eye. Finally, we will examine how specific inhibitory neurons shift their OD in response to MD. This line of research will greatly enhance our understanding of synaptic circuitry mechanisms underlying the normal cortical functional development as well as the plasticity induced by visual deprivation.

? DESCRIPTION (provided by applicant): Various functional properties of visual cortical neurons, as usually measured by their output responses, undergo progressive developmental maturations, the process of which is often susceptible to experience-dependent modifications during a critical period (CP). The functional changes of neuronal responses during normal development or induced after specific sensory experience can be caused by changes in the functional cortical synaptic circuitry, the nature of which has remained largely unknown. This is mainly due to a shortage of direct measurements of sensory-evoked excitatory and inhibitory synaptic inputs to developing cortical neurons, techniques for which remain very challenging. In this project, we will probe into functional synaptic circuits in the developing mouse visual cortex by combining several cutting-edge approaches, including in vivo whole-cell voltage-clamp recording, two-photon imaging guided patch recording, Ca2+ imaging and optogenetic manipulations. We will focus on visual receptive field (RF) and orientation selectivity (OS) properties in layer 4 of the primary visual cortex (V1). In Aim1, we will determine the progression of RF development by recording spike responses of single neurons in pre-CP, during CP and post-CP stages. We will then carry out voltage-clamp recordings to elucidate excitatory and inhibitory synaptic inputs underlying the RF. We will also carry out imaging guided recording and Ca2+ imaging of genetically labeled inhibitory neuron subtypes, in particular, parvalbumin (PV) positive neurons, to elucidate how their RFs are developed. In Aim2, by optogenetically activating cortical inhibitory neurons, we will isolate the thalamocortical input (and derive the intracortical input) to an excitatory neuron by silencing spiking of cortical excitatory neurons. W will investigate how developmental changes of these two components of cortical excitation contribute to the maturation of OS. By mapping visual RFs of the thalamocortical input, we will also investigate how developmental changes in specific spatial arrangements of thalamic inputs lead to maturation of their orientation tuning. Finally, for both aims, we will test whether the observed developmental processes are shaped by visual experience by comparing animals reared in darkness with age-matched animals reared normally. This line of research should greatly enhance our understanding of synaptic circuitry mechanisms underlying normal cortical functional development as well as plasticity induced by visual deprivation.

PROJECT SUMMARY Although great efforts have been dedicated to characterizing neuronal cell types in the brain, systematic studies on the brain-spinal cord connectome and associated spinal neuronal types are lacking. In this project, a team of seven laboratories proposes to use a highly innovative and multidisciplinary approach to systematically characterize neuronal types in the spinal cord based on their anatomy, connectivity, neuronal morphologies, molecular identities, and electrophysiological properties. In Aim 1, we will use a newly developed AAV anterograde transsynaptic tagging method to label spinal cord neurons that receive descending inputs from different brain regions, and use a retrograde viral tracer, AAVretro, to label spinal neurons that project to defined brain regions. These tagged neurons will be imaged in the intact whole spinal cord with a newly developed fast 3D light sheet microscopy technique, and targeted for recording in slice preparations. The axonal collateral patterns, dendritic morphologies, and electrophysiological properties will be compared between different input/output-defined spinal neuron groups. In Aim 2, the gene expression patterns of the tagged neurons will be determined in situ by sequential bar-coded FISH (seqFISH), with candidate marker genes obtained from online resources, or from single-cell sorting and RNA sequencing (Dropseq). In Aim 3, all collected data on connectivity, anatomical cell type distribution map, neuronal morphologies, molecular identities, and electrophysiological properties will be used for classifying spinal neuron types connected with brain, and an open-source data portal will be established which will allow users to search, view, and analyze the multi-modal and integrative cell-type specific data. Together, we aim to construct a comprehensive cell- type atlas of the mouse brain-spinal cord connectome.