Li I. Zhang - US grants

DESCRIPTION (provided by applicant): Most physiological studies in the primary auditory cortex (Al) have focused on neural spike output. However, to understand the processing and computation performed by auditory cortical neurons, it is necessary to examine the synaptic mechanisms underlying cortical response properties, i.e. to correlate the synaptic inputs of cortical cells with their outputs under various sound stimuli. In our pilot studies, we have developed techniques of in vivo whole-cell recording from auditory cortical neurons, as to establish a fundamental understanding of the synaptic connection basis for cortical responses. Here, I propose to systematically characterize the synaptic inputs, in terms of both excitatory and inhibitory inputs, underlying the frequency tuning and the supra-/sub-threshold structure of the frequency-intensity tonal receptive fields (TRFs) of single A1 neurons. In Aim 1, I will address how the tonal inputs are represented by a single Al neuron. I will determine the spatial relationship between supra- and sub-threshold TRFs of single A1 neurons, by recording tone-evoked membrane potential responses with in vivo whole-cell current-clamp method, and also characterize the change of subthreshold TRFs with the characteristic frequencies (CFs) of A1 neurons. In Aim 2, I will determine the role of spectrotemporal interaction between excitatory and inhibitory synaptic inputs in shaping the frequency tuning and TRFs of Al neurons. TRFs of pure excitatory and inhibitory synaptic inputs will be derived by using in vivo-whole-cell voltage-clamp recording. In particular, I will determine the role of the cortical inhibition in shaping the frequency tuning and TRFs. In Aim 3, I will characterize the contributions of thalamocortical and intracortical components to the excitatory synaptic TRFs of single A1 neurons by exploiting pharmacological approaches to silent the intracortical connections. With the understanding of the origins of the excitatory inputs, a basic model of synaptic input circuits underlying the TRFs of A1 neurons will be constructed. As a starting point, this project will specifically target the histologically determined excitatory pyramidal neurons in the input layers (layer 3-4) of adult rat A1. This study will be a direct extension of our pilot studies, and will generate information essential for understanding the cortical mechanisms underlying sound processing and representation in the auditory cortex. Taken together, the application of whole-cell recording technique in these studies will provide unique opportunities to address the fundamental issues concerning the mechanisms underlying auditory cortical responses, and are also likely to yield new level of information to the understanding of physiology and pathology of the auditory cortex.

An essential step to achieving an understanding of auditory cortical function is determining how information contained in sensory inputs is represented and processed in different individual cortical neurons. Recently, several laboratories have successfully applied a "blind" in vivo whole-cell voltage-clamp recording technique to cortical neurons. This technique can resolve sensory-driven excitatory and inhibitory synaptic inputs onto the recorded neuron, making it possible to construct a synaptic connectivity model to predict the neuron's response under arbitrary sensory stimulation. 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, especially those that are small in size or spatially sparse; the "blind" patch-clamp recording technique will normally result in a biased sampling of pyramidal neurons in the cortex. In this exploratory project, we will study a new technique for revealing functional synaptic inputs made onto different types of individual cortical neurons [unreadable] two-photon imaging guided whole-cell (TPGWC) recording [unreadable] in which fluorescence-labeled neurons are visualized by two-photon imaging and specifically targeted for patch recording. This technique has largely benefited from recent developments in mouse genetics in the labeling of specific cell types with fluorescence proteins, such as green fluorescence protein (GFP), whose expression is controlled by cell-type specific promoters. Initially, we plan to apply this recording technique to GFP-labeled GABAergic interneurons in the input layers (L3/4) of the mouse primary auditory cortex (A1), and aim at addressing two fundamental questions: a) What subthreshold/spike TRF properties are possessed by Al inhibitory neurons? b) How do cortical excitatory and inhibitory synaptic inputs determine subthreshold/spike TRF structure of A1 inhibitory neurons?

DESCRIPTION (provided by applicant): Understanding the structure of cortical synaptic circuits is key to comprehending information representation and processing in the auditory cortex. However, due to technical limitations, the general structure of cortical synaptic circuits, and how this structure determines cortical function, remains largely unknown. As a first step to addressing this issue, in this project, we will investigate the patterns of excitatory and inhibitory synaptic inputs underlying the functional responses of individual cortical neurons and reveal the synaptic mechanisms determining or shaping these response properties. In the auditory cortex, patterns of synaptic inputs can be largely reflected by their frequency-intensity tonal receptive fields (TRFs). These patterns represent basic structural properties of synaptic input circuitry underlying the functioning of individual cortical neurons. Using an in vivo whole-cell recording technique, we will determine the spectrotemporal pattern of synaptic inputs for both excitatory and inhibitory neurons in the input layers of the adult rat auditory cortex. We will dissect the thalamocortical components of excitatory inputs by pharmacologically silencing the cortex. The cell type of recorded neurons will be determined by their spiking and morphological properties. We will determine excitatory and inhibitory synaptic mechanisms for the frequency/ intensity tuning of cortical pyramidal neurons by revealing the patterns of excitatory and inhibitory synaptic inputs with in vivo whole-cell voltage-clamp recording techniques. We will explicate the contribution of thalamocortical excitaotry inputs to the response properties of cortical neurons by developing a novel pharmacological approach to effectively and specifically silence the cortex. Finally, by distinguishing cortical inhibitory neurons according to histology and physiology, we will determine response properties of cortical GABAergic interneurons, and their underlying synaptic mechanisms.

DESCRIPTION (provided by applicant): Inhibitory synaptic circuits play important roles in shaping cortical processing. Our understanding of the functional engagement of inhibitory circuits composed of different inhibitory cell types however remains poor. The recent development of molecular and genetic tools in the mouse, in combination with the innovative techniques of in vivo electrophysiology, has now made it possible to systematically dissect synaptic circuitry underlying specific cortical functions. In this project, we will integrate multile approaches to investigate the synaptic, in particular inhibitory circuitry mechanisms underlying auditory processing in the mouse primary auditory cortex (A1). In the first part, we will apply in vivo cell-attached and whole-cell recordings to investigate synaptic mechanisms for specific laminar processing in A1, a direct extension of the previously funded project. Second, we will combine in vivo two-photon imaging and patch-clamp recordings and utilize optogenetic methods to dissect functional roles of different types of inhibitory neuron. Finally, with high-quality whole-cell recordings in awake behaving mice, we will investigate cortical synaptic circuitry mechanisms for auditory processing functions in awake cortex, and their modulation by different behavioral states.

PROJECT SUMMARY/ABSTRACT Identifying the diversity of cell types in the nervous system will allow for their selective manipulation and reveal their functional contributions in health and disease. However, this is not a trivial undertaking and is hindered by the lack of consensus on which properties to use for classification. Characteristics like anatomical location, connectivity, morphology, molecular profile, and electrophysiological properties have been used as classification systems, but singly, none provide a combined view of all these characteristics. To address this, we propose a multidisciplinary approach that will provide all of this information for each cell type of the mouse hippocampus and subiculum (HPF/SUB). We recently identified all HPF/SUB molecular domains and assembled their connectivity networks using tracing data from the Mouse Connectome Project (www.MouseConnectome.org). Our multiple retrograde tracer injections revealed that the HPF/SUB contain multiple intermixed populations of cells with unique projection targets, suggesting different cell types that could be defined based on their connectional start and end points with anatomic specificity. Therefore, here, we propose to use a quadruple retrograde tracing method to initially classify these neurons based on these connections. Subsequently, a two- step cre-dependent AAV tracing method will determine all outputs of each cell type. To determine their molecular identities, seqFISH, with preselected hippocampal marker genes, will be performed on the tissue from the quadruple retrograde tracing data. Importantly, seqFISH preserves spatial information so that the precise anatomic locations of the tracer-labeled cells and the genes will be retained. Next, rabies injections placed in targets of each HPF/SUB cell type will reveal their morphology. CLARITY and two-photon microscopy will enable morphological assessment in 3D and neuronal reconstructions for further analysis. To examine electrophysiologcial properties, each cell type will be labeled with retrograde tracers for identification purposes and ex vivo cell patch clamping will be performed on the labeled cells. Finally, cre-dependent viral tracing (TRIO) will determine inputs to the different HPF/SUB cells types. With the aid of Expansion Microscopy and two-photon imaging, a combined anterograde/rabies tracing strategy will show precise locations of select inputs to cell types. If successful, this project can be applied to characterize neuronal cell types of the entire brain. All data will be publicly shared. Images from the quadruple retrograde, two-step cre-AAV, and TRIO tracing experiments will be available in the iConnectome Cell Type Viewer. Graphic reconstructions of labeling from these experiments will be compiled and presented within a common neuroanatomic frame through a Cell Type Connectivity Map. The iConnectome Cell Type Morphology Viewer will showcase labeling from the double rabies experiments and provide details like 3D reconstructions and their morphological and electrophysiological properties. Cell type connections will be visualized in an interactive Web Connectivity Matrix. Our in-house informatics pipelines and algorithms will be further developed and optimized to support the proposed features of all viewers. 

Neuromodulators are essential signaling molecules that regulate many neural processes, including cognition, mood, memory, and sleep, through their influence on brain circuits. Monitoring the release and distribution of neuromodulators in behaving animals is critical for understanding the diverse functions of these molecules. A major impediment to developing this understanding is the lack of tools that can monitor these compounds at the temporal, spatial and concentration scales relevant to these brain processes. Filling this technological gap is one of the most pressing needs in neuroscience research. Our proposal directly bridges this gap by developing a platform of new tools for chronic, non- invasive monitoring of neuromodulators at millisecond, subcellular, and nanomolar resolution. Genetically-encoded fluorescent indicators for calcium and glutamate have transformed investigation of dynamic brain processes in the major model systems, including worms, flies, rodents, and increasingly primates. Building on our prior experience in developing these tools, we now propose to build a new suite of GPCR-activation-based (GRAB) genetically-encoded fluorescent indicators for neuromodulators. Our preliminary data shows we can generate GRABs with >500% fluorescence change and nanomolar affinity in mammalian cells. We propose to further develop and validate these prototypes in cultured neurons, flies, rodent brain slices, anesthetized and behaving mice to maximize their utility. In Aim 1, we will develop GRAB indicators for acetylcholine, serotonin, and norepinephrine by iteratively screening libraries that systematically vary in insertion site, linkers, cpGFP sequence, and FP-GPCR protein surface interface. The dimensions of optimization will be dF/F, membrane surface expression, affinity, and non-disruption of endogenous signaling. Our targeted performance levels are >10x dF/F, nanomolar range affinity and <10 millisecond on-rates in vitro. In Aim 2, performance of top candidate GRAB indicators from the in vitro screen will be validated following long-term expression in drosophila olfactory system, in brain slice, in anesthetized and behaving mouse cortex. Feedback from these experiments will guide iterative optimization in Aim 1. Successful completion of our Aims will yield a suite of powerful molecular constructs, cell-type specific viral tools and technical approaches that will be broadly disseminated to the neuroscience community. The GRAB indicators can be easily integrated with existing mouse models of human mental disorders. Since these probes for neuromodulators are well-suited for a wide range of preparations, and a large number of investigators, they will have a multiplicative impact on our understanding of neural circuit function and dysfunction when combined with other advances supported by the BRAIN Initiative.

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.

Project Summary In this RO1 renewal application, we will explore functional roles of a higher-order thalamic nucleus, the lateral posterior nucleus (LP), in auditory cortical processing. LP is a rodent homologue of the pulvinar nucleus. While previous studies have been mostly focused on involvements of pulvinar in visual functions, there are salient connections between LP/pulvinar and auditory cortex, suggesting potential involvements of LP/pulvinar in auditory information processing as well. However, our knowledge on how LP influences auditory processing in the cortex is lacking. Recently in our preliminary experiments we observed that LP activity could suppress auditory responses in the primary auditory cortex (A1) of awake mice. This has prompted us to propose an extensive investigation into the functional contribution of LP to auditory cortical processing in A1. In Aim 1, we will perform in vivo recordings from individual neurons in supragranular and granular layers of A1 in awake mice and examined their auditory response properties before and after optogenetically inactivation and activation of LP. We will test the hypothesis that LP plays a role in enhancing auditory processing in A1 through a surround-suppression mechanism. In Aim 2, we will specifically manipulate the activity of the LP to A1 axon terminals either optogenetically or chemogenetically, and examine A1 neuron cell types that are innervated by the LP-A1 projection. We will test the hypothesis that the LP modulation of A1 responses is primarily mediated by the LP projection to layer 1 inhibitory neurons. In Aim 3, by manipulating activity of superior colliculus (SC), we will test the hypothesis that a SC-LP-A1 pathway mediates the bottom-up suppressive modulation of A1 responses. In addition, it can also mediate a cross- modality modulation of A1 responses by visual signals. Together, these experiments will enhance our understanding of functional roles of non-primary sensory thalamic nuclei in general.