Byungkook Lim, Ph.D. - US grants

? DESCRIPTION (provided by applicant): Progressive stages of drug addiction are well defined by clinical observations and animal behavior experiments. Recognizing these stages of progression comes largely from observing outward signs of addicted behaviors. What is missing from understanding stages of drug addiction is the knowledge of neural circuits responsible for stage-specific symptoms. Many studies suggested that distinct cell types in nucleus accumbens (NAc) plays different roles in the drug addiction. However, the detailed circuit organization of different cell types in NAc and associated brain areas has not been fully elucidated. Moreover, how other brain areas engaged to modulate different subpopulations of neurons in NAc during the progression into drug addiction has not been systemically analyzed. This is partly because we lack the efficient methods to monitor the neural adaptation in circuit-specific manners in different brain areas at the same time as stages progress towards addiction. Using cocaine addiction in mice as a model system, I propose a new paradigm for studying drug addiction by monitoring the neural adaptation in brain circuitries related to different cell types in NAc during each stage of addiction progression. To achieve this, we use a monosynaptic rabies virus system expressing fluorescent marker or synaptic markers to generate brain-wide maps of neurons that form synapses with different cell types in NAc. First, we will elucidate afferent connections to different cell types in NAc in whole brain. Second, we will examine the dynamics of structural plasticity and synapses in different brain areas projecting to each cell type of NAc t the different stages of cocaine addiction at the same time in the same brain. By this means we will fill in some of the most important information missing in cocaine addiction research: which neural circuits engage, and what modifications do they undergo from stage to stage as addiction progresses? Understanding drug-induced circuit level modifications at different stages of addiction will provide a valuable framework for guiding future studies on drug addiction as well as the roles of reward circuitry, which eventually lead to develop better therapeutic strategy for this mental disorder. Furthermore, the approach used here for the anatomical and functional characterization of neural circuitry can be used to investigate models of any neuropsychiatric or neurodegenerative disease that progresses in stages.

? DESCRIPTION (provided by applicant): The anatomical substrates and cellular mechanisms underlying reward-dependent learning have been studied for decades, but the specific circuit and network interactions between the cortex, striatum, and midbrain that mediate action selection have not been systematically investigated. Here, we bring together three different investigators with specialized expertise in each of these three brain regions. As a team, the investigators are well positioned to perform cutting-edge cell-type-, region-, and projection-specific imaging and ontogenetic manipulations of neuronal ensembles during complex behavioral tasks designed to uncover the network-level representation of decision-related variables for action selection. The computational frameworks for analyzing these data are provided by reinforcement learning models. In Aim 1, the focus is on the striatum, which arguably lies at the center of reward-dependent learning. It is the point of intersection for sensorimotor and contextual information from cortex, and reward- and motivation-related information from the midbrain. Using microendoscopy with calcium imaging from genetically-specified neuronal subtypes in different striatal subregions during behavior, we will identify the key decision-related variables represented in the striatum during action selection tasks. In Aim 2, we will image from specific populations of dopamine neurons in the midbrain that project to different striatal subregions, in order to decipher their role in reward-related signaling. In Aim , we will image large ensembles of neurons in the motor cortex (M1 and M2) using two-photon microscopy, with a focus on neurons projecting to distinct cell types and subregions of striatum. All three aims will use the same behavioral tasks, and the same analysis techniques, in order to facilitate the integration of data from all three brain regions into a single coherent model for vertebrate action selection.

? DESCRIPTION (provided by applicant): Despite the increased prevalence of major depressive disorder (MDD) and the continued investment into identifying effective cures, most treatments merely alleviate symptoms rather than addressing causes. Long-term treatments are generally required for this disease and often include many side-effects. The onset of depression is often precipitated by stressful and/or aversive stimuli. Animals, too, are susceptible to the effects of stress. For example, repeated social defeat stress in rodents induces pervasive and long-lasting behavioral changes similar to the symptoms seen in patients with MDD including impaired social interaction, lack of motivation, helplessness and anhedonia. While the behavioral changes associated with depression have been well-studied, there is relatively little knowledge about the accompanying changes in the neural circuitry. Thus, we will anatomically and functionally dissect the distinct neural circuits mediating various stress-induced behaviors in mice to better understand the mechanistic changes in the brain accompanying MDD in human patients, which will help to devise revolutionary circuit-specific and stage-specific diagnosis and treatments. To accomplish this, we will examine the neural circuit mechanism of ventral pallidum (VP), one of the major components of reward circuitry, underlying depressive behaviors elicited by repeated social defeat stress in combination with a variety techniques to address circuit-level mechanisms, including optogenetics, viral mediated tracing, electrophysiology, and real time in vivo fiber photometry. Our preliminary findings showed that different types of neurons in VP project to different target structures which may be involved in different aspects of depressive behaviors. First, we will define the projection specific roles of VP circuity in depressive behaviors induced by repeated social defeat stress using optogenetics and viral tracing methods. Second, using in vivo fiber photometry and newly developed viral tools, we will directly monitor the dynamics of neural activity of VP circuitry in cell-type and projection specific manner in vivo in real time during social interaction before and after the repeated social defeat stress, and their response to anti-depressant treatment. Third, using ex vivo electrophysiology analysis we will examine the circuit-specific electrophysiological and synaptic changes induced by the stress. The accomplishment of the proposed works will be greatly beneficial to both the research and treatment of MDD, and will also provide a fundamental framework for studying mental disorders in circuit-specific manner.

? DESCRIPTION (provided by applicant): Despite the increased prevalence of major depressive disorder (MDD) and the continued investment into identifying effective cures, most treatments merely alleviate symptoms rather than addressing causes. Long-term treatments are generally required for this disease and often include many side-effects. The onset of depression is often precipitated by stressful and/or aversive stimuli. Animals, too, are susceptible to the effects of stress. For example, repeated social defeat stress in rodents induces pervasive and long-lasting behavioral changes similar to the symptoms seen in patients with MDD including impaired social interaction, lack of motivation, helplessness and anhedonia. While the behavioral changes associated with depression have been well-studied, there is relatively little knowledge about the accompanying changes in the neural circuitry. Thus, we will anatomically and functionally dissect the distinct neural circuits mediating social stress-induced behaviors in mice to better understand the mechanistic changes in the brain accompanying MDD in human patients, which will help to devise revolutionary circuit-specific and stage-specific diagnosis and treatments. To accomplish this, we will examine the neural circuit mechanism of ventral pallidum (VP), one of the major components of reward circuitry, underlying depressive behaviors elicited by repeated social defeat stress in combination with a variety techniques to address circuit-level mechanisms, including optogenetics, viral mediated tracing, electrophysiology, and real time in vivo fiber photometry. Our preliminary findings showed that parvalbumin-positive (PV) neurons in VP project to different target structures which may be involved in different aspects of depressive behaviors. First, we will anatomically define the efferent connections of VP PV neurons. Second, we will define projection specific roles of VP PV neurons in depressive behaviors induced by repeated social defeat stress using optogenetics and viral tracing methods. Third, using ex vivo electrophysiology analysis we will examine the circuit-specific electrophysiological and synaptic changes induced by the stress. The accomplishment of the proposed works will be greatly beneficial to both the research and treatment of MDD, and will also provide a fundamental framework for studying mental disorders in circuit-specific manner.

PROJECT SUMMARY AND ABSTRACT A comprehensive understanding neuronal cell type diversity is an essential guide to selective manipulation and illuminating cell type specific functional contributions toward health and disease. Accordingly, the Brain Initiative Cell Census Network (BICCN) is unifying the efforts of laboratories with unique expertise in anatomy, genetics, electrophysiology, and function to classify neurons and create a common 3D atlas with integrated cell type data. To this end, our proposed collaboratory aims to anatomically characterize neuronal cell types of the mouse limbic system. Using mesoscale quadruple retrograde tracing, we will initially characterize cell types based on the anatomical location of their connectional start and end points [e.g., ACB(contralateral)?BLAa?ACB(ipsilateral)]. A two-step cre-dependent AAV tracing strategy using advanced viral tools will subsequently validate and refine specific axonal projections, collaterals, and projection fields [e.g., ACB/X/Y?BLAa?ACB/X/Y]. Injections of G-deleted rabies in CLARITY-processed tissue will label morphological features of cell types. Cre-dependent TRIO viral tracing will determine discrete inputs to each cell type, providing deeper characterization of connectivity. Novel TRIO using flp recombinase in cre- dependent mice will define projection patterns of genetically-defined cell types. Newly constructed AAV and rabies viruses tagged to spaghetti monster fluorescent proteins, applied in combination with Expansion Microscopy and multiphoton imaging, will determine the spatial organization of different synaptic inputs to the cell types. Collectively, experiments will reveal cell type anatomic location, morphology, and comprehensive connectivity. Initial efforts will focus on the limbic system, with the design extensible to neuronal characterization of the entire brain. A web-based visualization platform will be developed to enable viewing and analysis of cell type anatomy data in 2D and 3D. An online visualization tool similar in function to our iConnectome viewer will present quadruple retrograde and TRIO tracing images. Digitized, reconstructed quadruple retrograde, cre-AAV, and TRIO labeling will be placed atop the Allen Reference Atlas (ARA) to create an online 2D connectivity map, allowing easy comparison of cell type specific inputs and outputs. Common Coordinate Framework (CCF) registration and reconstruction of cre-AAV labeling experiments will provide the cell type specific 3D context of projections, with input and morphological information integrated into the viewer. An interactive, weighted and directed matrix will present an intuitive visualization of all connectivity data. 3D reconstructed neurons will also be hosted on Neuromorpho.org for interspecies comparison. Our current informatics pipelines will be extended and optimized to support the proposed viewer features. We expect our technologies to elucidate diverse cell type specific networks and provide foundations for the overarching goal of the BICCN of creating a comprehensive 3D cell type atlas.

Project Summary Neuron loss and the reorganization of neural circuits in the superficial layers of entorhinal cortex are hallmarks of Alzheimer?s disease and temporal lobe epilepsy, and memory impairments are among the troubling symptoms of these diseases. Despite the knowledge that superficial entorhinal cell layers are selectively vulnerable in these diseases, the local connectivity of entorhinal circuits, how different functional cell types within these layers emerge, and how each cell type contributes to memory and spatial processing is only beginning to be revealed. The entorhinal cortex has extensive recurrent connectivity between its layers, and harbors many functional cell types such as grid cells, head-direction cells, border cells, context-selective cells, and other types of spatial and nonspatial cells. However, this functional diversity neither maps directly onto particular cell layers nor onto anatomically defined cell classes within layers. Each cell?s functional identity may therefore predominantly be determined by local microcircuits. The objective of this proposal is to examine whether functional cell identities in mEC, including grid cell firing and context-selective firing, emerge from circuit computations. We focus on local connectivity within the superficial layers and hypothesize that layer II pyramidal cells selectively contribute to rate coding in layer II stellate cells and that layer III inputs selectively contribute to spatial coding, including grid firing, in layer II stellate cells. This hypothesis will be tested in two specific aims. First, we will use viral tracing and patch clamp recordings with optical stimulation in entorhinal slices to determine the connectivity of mEC layer II pyramidal cells and layer III pyramidal cells and, for comparison, layer II stellate cells. Second, we will record from mEC cells in behaving mice while optogenetically stimulating or inhibiting entorhinal cell populations. In two separate subaims, we will examine (1) the effects of layer II pyramidal cell manipulations on the spatial and context-selective firing patterns of layer II stellate cells and layer III pyramidal cells and (2) the effects of layer III pyramidal cell manipulations on spatial and context- selective coding by layer II cells. Results from our aims will identify how local entorhinal circuits contribute to the generation of specialized entorhinal cell types, including grid cells and context-selective cells. This will not only fill gaps between theoretical models and experimental data, but also develop methods to selectively manipulate different functional cell types in mEC. Our results will therefore advance our understanding of the contribution of entorhinal cell types and cell layers to spatial and memory processing and thereby suggest strategies for restoring entorhinal circuit function and ameliorating the progression of neurodegenerative diseases.

PROJECT SUMMARY Drug craving during prolonged periods of abstinence is a major factor driving repeated cycles of drug abuse. In light of the increasing prevalence of drug abuse, it is imperative that we obtain a clear understanding of the neural circuit plasticity and associated molecular mechanisms underlying drug craving specifically. The ventral pallidum (VP) is the major output structure of the mesolimbic reward circuitry and is suggested to be the final common pathway for reward and motivational processing by relaying information from the nucleus accumbens (NAc) and ventral tegmental area (VTA) to the lateral habenula (LHb), VTA, and subthalamic structures. However, little is known about the circuit level organization and function of VP neurons in drug addiction, especially in the context of drug seeking following prolonged withdrawal. Therefore, we will anatomically and functionally probe the neural adaptations of a molecularly-defined subset of VP output neurons using the cocaine self-administration paradigm in mice, in order to better understand the mechanisms underlying cocaine seeking after a prolonged period of withdrawal. To accomplish this, we propose to study withdrawal-induced neural adaptations in specific subcircuits originating in the VP by using multiple cutting-edge techniques including optogenetic manipulation, in vivo monitoring of neural activity, viral-mediated tracing, ex vivo electrophysiology, and molecular profiling methods in a mouse cocaine self-administration model of drug addiction. Our preliminary data indicate that dopamine receptor 3 (Drd3) signaling is selectively upregulated in the VP during withdrawal from cocaine self-administration, and that knockdown of Drd3 in the VP, but not in the NAc, inhibits cocaine seeking behavior after prolonged withdrawal, but not sucrose reward seeking, strongly suggesting that VP Drd3 signaling may play a major role specifically in cocaine-induced craving and drug seeking behavior. We will first define the afferent and efferent connections of Drd3-expressing VP neurons. Second, we will examine the circuit- specific neural adaptations of VP Drd3 neurons and their role in cocaine seeking. Third, we will examine how VP Drd3 neuronal activity regulates VTA dopaminergic neuronal activity and dopamine release in NAc and VP during prolonged withdrawal from cocaine self-administration using cutting-edge imaging techniques. The accomplishment of this project will be greatly beneficial in providing a framework for studying drug addiction in a circuit-specific manner, as well as in developing a strategy for the treatment of drug addiction.

A comprehensive understanding of brain cell diversity and cell-to-cell communication (connectivity via specialized junctions between cells called synapses) is essential for understanding the anatomical substrates and cellular mechanisms that underlie brain function. Elucidation of the detailed synaptic organization of specific neural circuits is a necessary component for achieving this goal. Progress in this area of neuroscience has been limited by a lack of tools that can simultaneously reveal multiple cell types with distinct molecular and physiological properties within the same brain areas, as well as highlighting in detail how these cells are connected to each other. This project utilizes a novel molecular-biological strategy for defining multiple cell types and their interconnections in mouse brains, enabling investigators to mark individual cells that express combinations of engineered genetic elements. This technique is combined with an existing method that inserts two incomplete parts of a fluorescent molecule onto the surfaces of different nerve cells. If those cells communicate by forming a connection (synapse) with each other, the fragments of the fluorescent molecule come into close enough contact to become functional again, and synapses are identifiable as colored dots of fluorescent light. Both graduate and undergraduate students will be involved in the development and optimization of these tools, and the genetic constructs will be distributed via a non-profit agency; all protocols and sequence information will be made widely available through the PI's website. These new tools will greatly enhance the speed and precision with which neuroscientists can study the detailed organization of brain circuitry, and the synaptic connections made onto specific neurons.

This project introduces novel molecular and viral strategies for defining multiple cell types and their synaptic organization. It is based on a novel viral strategy to achieve the intersectional expression of transgenes among specific neuronal populations in transgenic mouse lines that express Cre, flippase (FLP), and other recombinases. A novel site-specific recombinase system (phiC31 and a phiC31-dependent single inverted open reading frame [pSIO]) is used to acheive virus-mediated, specific-transgene expression without cross-reactivity to other recombinases, both in vitro and in vivo. This phiC31/pSIO recombinase system is then combined with "enhanced Green fluorescent protein Reconstitution Across Synaptic Partners" (eGRASP) to label synaptic contacts made by specific inputs. By providing a novel experimental framework for identifying the cellular constituents of local neural circuits and examining the synaptic organization of multiple presynaptic inputs onto specific neurons, and by optimizing these tools for general use and making them freely available to the scientific community, this project is expected to accelerate discovery about the organizational and functional properties of neural circuitry.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

ABSTRACT Stroke is one of the leading cause of long-term disability. However, there is no fundamental treatment for this condition, and current therapies only offer limited benefits. It is necessary to identify and develop better therapeutic strategy. One approach is to understand how animals endogenously recover from experimental stroke models and to leverage these findings to clinical applications. In this vein, much effort has been devoted to the identification of recovery-related genes and proteins. However, to fully understand the role of newly identified molecules, we need to know how these molecules are involved in the dynamics of the neural circuit mechanisms in recovery from stroke. The interruption of blood flow to the brain rapidly induces a cascade of degeneration, inflammation, and reduced neuronal excitability. Therefore, recovery requires the re-normalization of neuronal excitability through the reorganization of surviving neural circuits. However, no clear information on the role of different neuronal types in this neural adaptation after stroke. Therefore, delineating the cell type-specific adaptations is an important first step toward understanding the mechanisms of stroke recovery. In this proposal, we adapted the photothrombotic stroke model aims to induce an ischemic damage in primary motor cortex (MOp), which impairs the forelimb movement. This impaired movement is recovered within 3-4 days after the stroke. Thus, we will study the role of the neural reorganization of nearby circuitry, especially in secondary motor cortex (MOs), in behavioral recovery after the stroke in primary motor cortex (MOp). Interestingly, our preliminary results showed that the inhibition of MOs activity after stroke inhibit the behavioral recovery, suggesting that the neural adaptation in MOs may mediate the recovery from MOp stroke. Using 2-photon microscopic imaging of excitatory and inhibitory MOs neurons before and after the stroke, we will examine the dynamics of different types of neurons in MOs and manipulate the activity of those neurons selectively to examine the cell type specific roles in recovery from stroke. Furthermore, we will also examine the role of individual synapses made by different inputs to different cell types in MOs in recovery from stroke. To achieve this, we adapt the newly developed tool, enhanced green fluorescent protein reconstitution across synaptic partners (eGRASP), to label and monitor synapses between defined pre- and postsynaptic partners using longitudinal 2-photon imaging. Understanding the cell type specific neural adaptation and the dynamics of synaptic inputs to different cell types in MOs after stroke will provide novel insight for the circuit mechanism of the recovery after stroke.

SUMMARY The basal ganglia are a group of subcortical nuclei that regulates motor and cognitive functions. Recent identification of neuronal heterogeneity in the basal ganglia suggests that functionally distinct neural circuits defined by their molecular identity and efferent projections exist even within the same nuclei. This distinction may account for a multitude of symptoms associated with basal ganglia disorders such as Parkinson's disease (PD). However, our incomplete understanding of the basal ganglia functional organization has hindered further investigation of individual circuits that may underlie distinct behavioral symptoms in different disease states. The external globus pallidus (GPe) is a central basal ganglia nucleus that can influence numerous downstream regions. While the prevailing circuit model assumes that the GPe is a homogeneous population of neurons transferring the signal in the indirect pathway of the basal ganglia, accumulating evidence suggests that neurons in the GPe are more heterogeneous than previously appreciated. Although GPe is known to be a nucleus with GABAergic neurons, we have identified novel cell types expressing VGLUT2, glutamatergic neuronal marker, at the outer layer of GPe. In our careful anatomical and molecular examination showed that VGLUT2GPe neurons project mainly to inner part of GPe, making synaptic contacts onto other neuronal populations. Recent evidence showed that the distinct cell types in GPe may have different roles in modulating basal ganglia circuitry and associated behaviors. Thus, elucidating the anatomical and functional organization of VGLUT2GPe neurons will provide novel cellular and circuit information to understand basal ganglia function. The progressive nature of behavioral deficits associated with PD is very well documented in human patients. However, what neural adaptations associated with behavioral deficits at different stages of PD are not fully understood. In this application, we try to address this with two different animal models. First, as in our preliminary results and recent reports, we will administer different doses of neurotoxin administration to induce different degrees of DA neuronal loss, which elicit the different behavioral deficits. Second, we will confirm the neurotoxin- induced PD-related behaviors in MitoPark mice which show the progressive loss of DA neurons. Examining the circuit adaptation in two animal models will provide an important information on the neural mechanisms underlying the progressive nature of PD. Therefore, using cutting-edge techniques including optogenetic, genetic and viral-mediated manipulation, in vivo multi-unit recording, and so on, we will decipher roles of VGLUT2GPe neurons in behavioral deficits in these two animal models for PD.