Edward M. Callaway, Ph.D. - US grants

The precise organization of connections within and between the cortical layers is crucial for the computations that underlie the extraordinary capabilities of the cerebral cortex. Nowhere is this more apparent than in the primary visual cortex, where the physiological response properties of neurons and the circuits they form have been described in considerable detail. One of the principal organizing features of the visual system is the presence of parallel processing streams that each carry and extract information about different features of the visual scene. The proposed studies are aimed at determining the normal sequence of events and the importance of activity in the generation of stream specific local circuits in macaque primary visual cortex. The anatomical organization of local circuits in newborn and embryonic animals will be assessed by reconstruction anterogradely-labeled axons following extracellular injections of biocytin made into coronal and tangential slices of primary visual cortex. Slices will be sectioned and double-stained for cytochrome-oxidase as well as biocytin allowing laminar and blob/interblob boundaries to be identified. Analyses of the relationships between axonal projections and laminar and blob/interblob compartments in developing animals will reveal when various functional stream-specific circuits arise and whether they emerge by the specific formation of collateral branches exclusively in correct regions or by the reorganization of initially exuberant projections. The organizational of functional local connections in developing animals will be studied using a combination of whole-cell recording and a novel caged-glutamate- based photostimulation method (Callaway and Katz 1993). This method allows the sources and strengths of connections to individual neurons to be determined. Experiments will focus on: 1) the development of connections to blob versus interblob regions of layer 2/3 originating from layers 4A, 4B, 4Calpha, and 4Cbeta, and from layer 2/3 itself, and 2) the development of layer specific projections from individual layer 6 pyramidal neurons to layers 3/4A, 4Calpha, and 4Cbeta. The influences of prenatal retinal activity and postnatal patterned visual experience on the development of these connections will be tested by depriving animals of normal activity patterns by either prenatal binocular enucleation or postnatal binocular lid suture. Results from the work proposed here will yield important insights into the mechanisms involved in the development of parallel visual processing circuits. These insights are expected to be useful for understanding the central effects of visual disorders such as strabismus and amblyopia and to aid in the development of strategies for their treatment. Dyslexia has been linked to specific anatomical and physiological deficits in the motion processing stream (Livingstone et al., 1991) and thus understand the normal development of functional stream-specific circuits and its dependence on activity may aid in understanding the cause(s) of dyslexia.

DESCRIPTION: (Adapted From The Applicant's Abstract) Visual perception is mediated by complex interaction amongst urons in the retina, visual cortex and subcortical brain structured. The importance of vision to humans and other primates in reflected in the enormous percentage of cerebral cortex devoted to processing visual information. Thus deficits in visual processing are particularly debilitating and arise from abnormalities not only in the eye, but also in cortical circuitry, For example, strabismus or amblyopia during childhood can have long-lasting effects on the cortical circuits that process visual information. There is also evidence that some forms of dyslexia result from central visual system abnormalities. The proposed studies are aimed at understanding the organization of neural circuits within and between the visual cortical areas V1 and V2, with the broader objective of contributing to understanding how neural circuits mediate the computations that underlie visual perception. In particular, these studies aim to identify: (1) how local circuits in V1 mediate integration and/or segregation of information arising from "parallel M and P retino-geniculate pathways; 2) whether V1 neurons receiving different combinations of M and P input project in turn to different functional compartments in V2; (3) the sources of local functional input to individual neurons in the deep layers of V1 and whether they correlate with the outputs of each deep layer cell type; (4) the organization of local circuits that mediate computations in V2. These goals will be achieved by using a combination of anatomical and physiological methods in living in vitro brain slice preparations. Neurons in area V1 and V2 will be intracellularly labeled and their axonal and dendritic arbors reconstructed to determine their contributions to local cortical circuits. The V1 neurons will also be subject to EM analyses to identify ultrastructural properties of the synapses formed by the various cell types in this area. Scanning laser photo-stimulation will be used to reveal the source of local functional input to individual superficial and deep layer neurons in V1. Some of the superficial V1 neurons will previously have been retrogradely-labeled allowing identification of the extra-striate cortical areas that they target, while deep layer neurons with the various output patterns will be identified by intracellular labeling of their axonal arbors. These combined photo-stimulation and anatomical approaches therefore allow the identification of functional input sources to cells whose outputs are anatomically identified. These analyses reveal the flow of visual information across multiple synapses, allowing an unprecedented view of visual cortical circuits.

[unreadable] DESCRIPTION (provided by applicant): Project Summary: Cortical circuits are composed of a complex network of many neuron types. Their function is dependent on how neurons are interconnected, how the connections function, and how the information delivered to individual neurons is integrated within the postsynaptic dendritic arbor. Despite extensive knowledge of the basic blueprint of cortical circuits, detailed knowledge about precisely which cell types are connected and how the multitude of connections onto a single neuron interacts is limited. The studies proposed here will reveal the laminar and fine-scale specificities of functional excitatory and inhibitory inputs to specific types of inhibitory neurons in the cerebral cortex. A novel laser scanning photostimulation method will be used to stimulate neurons that might make connections to neurons of interest, while recording electrical responses in those neurons to determine whether connections are present. With photostimulation it is possible to stimulate hundreds of sites to "map" the sources of functional input to a single neuron. Combining this method with paired intracellular recordings and cross-correlation analyses of postsynaptic currents allows identification of shared inputs on a fine scale. Results from these studies will reveal similarities and differences in the input to different types of inhibitory neurons. Interactions between excitatory and inhibitory inputs will be tested by combining photostimulation based mapping of input patterns with stimulation of a single inhibitory neuron that is connected to an excitatory neuron of interest. This will directly test the ability of inhibition to shape the sources of excitation to a single neuron. Relevance: Understanding the detailed organization of cortical circuits involving specific inhibitory neuron types is necessary to obtain a mechanistic understanding of the function of the cerebral cortex. Understanding the specific roles of inhibitory neurons in cortical function has important implications for human health, as these cell types and their activities are implicated in the cortical mechanisms that regulate attention and their disruption is implicated in schizophrenia. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Visual perception is mediated by complex interactions amongst neurons in the retina, visual cortex and subcortical brain structures. The importance of vision to humans and other primates is reflected in the enormous percentage of cerebral cortex devoted to processing visual information. Thus, deficits in visual processing are particularly debilitating and arise from abnormalities not only in the eye, but also in cortical circuitry. For example, strabismus or amblyopia during childhood can have long-lasting effects on the cortical circuits that process visual information. There is also evidence that some forms of dyslexia result from central visual system abnormalities. The proposed studies are aimed at understanding the organization and function of neural circuits to and within the primary visual cortex (Vl), with the broader objective of contributing to understanding how neural circuits mediate the computations that underlie visual perception. In particular, these studies aim to identify: 1) the detailed functional organization of afferent input from the LGN to V1; 2) the functional connectivity between excitatory neurons and distinct types of inhibitory neurons in V1 and how these circuits relate to the functional organization of V1; 3) how the in vivo visual response properties of individual, identified neurons correlate with the connectivity of these same cell types, as revealed by our previous and ongoing in vitro studies. These goals will be achieved by: 1) directly recording visual responses from the terminal arbors of LGN afferents in V1, 2) using a combination of anatomical and physiological methods in living in vitro brain slice preparations; 3) recording visual responses of V1 neurons and labeling them with dye to correlate anatomically distinct cell types with function. In vitro brain slice studies will use scanning laser photostimulation to reveal the sources of local functional input to individual inhibitory neurons in V1. The same cells will be intracellularly labeled, stained with antibodies, and their intrinsic membrane properties assessed; the combined physiological and anatomical approach allows the identification of functional input sources to cells whose outputs and physiology are also characterized. The proposed studies will allow an unprecedented view of visual cortical circuits - they will reveal the detailed functional connectivity of neurons in visual cortex and how these circuits relate to the functional properties of the component neurons.

DESCRIPTION (provided by applicant): The mammalian central nervous system (CNS) is composed of a complex network of numerous distinct neuron types that are functionally and anatomically intertwined. The function of the circuits in which these neurons are embedded is dependent on the precise and cell type specific regulation of connectivity, synaptic function, and intrinsic integration of external signals. Thus, in order to conduct experiments to better understand the basic mechanisms of nervous system function or to develop and implement treatments for neurological dysfunction, it is highly desirable to have tools that can specifically target neuron types. Genetic methods therefore hold great promise for the study and therapeutic manipulation of CNS circuits because it is possible to harness the same mechanisms that regulate differential gene expression in neurons to drive the specific expression of transgenes. There are numerous transgenes which are being used or developed to allow studies or manipulation of neural circuits at the level of specificity of individual cell types. This genetic arsenal is likely to continue to grow in both size and sophistication within the next several years. The utility of all of these genetic methods, however, hinges on the ability to specifically drive gene expression in cell types of interest. This proposal aims to identify short promoter sequences from the genome of Fugu rubripes ("Fugu") which can be used to selectively drive gene expression in specific types of mammalian cortical neurons. The identification of such promoter sequences is expected to provide crucial tools for the study of cortical circuits. Short promoter sequences can be used not only in the generation of transgenic mice, but also can be used along with small capacity viruses for the control of gene expression in other species, or for the generation of non-murine transgenic animals or transgenic "knockdown" animals using lentivirus. Small capacity viruses are also among the most promising vectors presently available for human gene therapy in the brain, and their eventual use for this purpose will also require effective short promoters.

[unreadable] DESCRIPTION (provided by applicant): Understanding nervous system development and function requires elucidation of the relationships between the separate components of neural circuits and their function in an intact system. Revealing these relationships in intact systems has proven to be extremely difficult with conventional methods. The advent of multiphoton confocal microscopy, however, provides the opportunity for in vivo optical imaging deep within neuronal tissue with minimal photodamage. This method can be used to target electrode recordings to specific cell types or to optically probe the activity of identified cell types. Optical monitoring of neuronal activity can be done using measurements of fluorescent changes from calcium sensitive dyes or genetically expressed sensors of presynaptic vesicular release of neurotransmitter. In the present proposal, we plan to use multiphoton confocal microscopy, coupled with genetically encoded optical reporters and other small fluorescence molecules, to study the connectivity and the role of neural activity in the normal adult nervous system and in the formation of synapses during development and in disease states. These studies are only possible with the use of a multiphoton confocal microscopy system [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A new rabies virus-based method for mapping monosynaptic connections in neuronal circuits will be used to (1) label neurons and pre-synaptic terminals associated with those neurons and (2) determine if all pre-synaptic terminals impinging on the identified postsynaptic neuron are trans-synaptically marked by this approach. NCMIR developments in labeling chemistry for correlated light microscopy (LM) and electron microscopy (EM) will be extended to work with this system enabling high-resolution assessment of labeled terminals and associated structures in well-preserved specimens. The use of the new method being developed to produce [unreadable][unreadable][unreadable]multicolor EM[unreadable][unreadable][unreadable] images will also be explored to enable layering of these methods to address questions regarding detailed distribution of multiple molecular constituents in identified synaptic complexes.

DESCRIPTION (provided by applicant): The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neurons types. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate. The research proposed here is aimed at the development, refinement, and validation of a novel set of tools that will allow researchers to readily and systematically uncover neural circuits with cell type-specific resolution. These tools build on previous work in one of the PIs lab, developing and validating the potential for use of genetically modified rabies viruses, in combination with other genetic and viral technologies, to probe neural circuits. The new tools to be developed and tested include mouse lines and helper viruses which can be used to achieve cell type specific expression of genes that interface with the rabies tracing system. This will allow the modified rabies viruses to selectively infect specific cell types and to label the direct inputs to those cells. These new tools will be tested and protocols developed for their use in a broad range of nervous system structures, whose function is relevant to understanding disease states. New variants of rabies virus will also be generated in order to interface with the newly developed mouse lines. These variants will express genes to drive conditional expression of genes encoded in the genomes of the transgenic mice, such that inputs to specific cell types targeted for infection by the rabies virus can be identified. These new rabies viruses will also be tested and protocols developed for assaying neural circuits in a broad range of relevant structures. Overall, this project will result in the generation and validation of very valuable new tools which will then be available to the neuroscience research community. PUBLIC HEALTH RELEVANCE: Understanding neural circuits with increasingly sophisticated and higher resolution tools is crucial to understanding diseases that are caused by neural circuit disorders, including Parkinson's, neuromuscular disorders, paralysis, schizophrenia, depression, autism and attention disorders, among many others. The development of new tools for revealing circuits will therefore have a large impact on these diseases.

DESCRIPTION (provided by applicant): The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neurons types. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate. The research proposed here is aimed at revealing the detailed connectivity of specific types of inhibitory cortical neurons. A novel Cre-dependent and rabies-based tracing system will be used to reveal the direct monosynaptic connections to neurons in primary somatosensory cortex of five different Cre-driver mouse lines. The intersection of Cre-expression in specific cell groups, with AAV infection and specific promoters, will define specific populations of inhibitory neurons that can be infected with the rabies virus. Transcomplementation and retrograde transsynaptic spread of the rabies virus will reveal the cells, across the entire brain, that make direct connections to the targeted cortical inhibitory neurons. To assess the specificity of local outputs from specific types of inhibitory cortical neurons, similar targeting methods will be used to selectively express channelrhodopsin (ChR2) in specific cell groups. Living brain slices will then be prepared and functional connections assayed by recording intracellularly from potential postsynaptic cell types of interest while optogenetically activating the ChR2 positive cells. The results of these experiments will shed light on the feasibility of various hypotheses about the functional roles of particular types of inhibitory cortical neurons and will guide future studies monitoring and manipulating the activity of specific cell types.

DESCRIPTION (provided by applicant): Our understanding of the neural mechanisms underlying action, perception and cognition has been limited by the lack of tools to modulate the activity of specific neural circuits in the awake, behaving animal. This technical hurdle has, in turn, limited our understanding of devastating disorders of the nervous system that result from dysfunction of neural circuits. The proposed research addresses this challenge by developing a new set of optogenetic techniques that will make it possible to selectively manipulate the activity of projection neurons with precise temporal control in the brain of the behaving non-human primate. We plan to inject viral vectors, specifically designed to be taken up by axon terminals, into the brain in order to get projection neurons to express the light-sensitive channels channelrhodopsin-2 and archaerhodopsin-3. We will then be able to selectively activate or inactivate projection neurons using light introduced into the brain with specially designed optitrodes. Our proposal focuses on projections from primary visual cortex (V1) to the superficial layers of the superior colliculus and V1 neurons projecting to the secondary visual area (V2), taking advantage of the well- defined retinotopic maps in these regions, and using physiological, behavioral, and histological methods to measure our ability to selectively manipulate the activity of projection neurons. The results of these studies are likely to have a large impact, because the tools and techniques developed in this research program will have wide applicability for those studying the relationships between functional neural networks in the CNS and primate behavior. They will also provide important tests of the feasibility of optical and virus-based methods as possible therapeutic approaches in CNS disorders. PUBLIC HEALTH RELEVANCE: Understanding the functional role of particular neural connections is crucial for unraveling the etiology of neuropsychological disorders such as neglect, Balint's syndrome, visual agnosia, schizophrenia, ADHD and autism. The goal of this project is to establish new techniques for selectively manipulating the activity of projection neurons in the behaving non-human primate under precise temporal control using combinations of physiological, molecular, and genetic methods. The results from our studies will demonstrate powerful new tools for studying the functional relationships between neural networks and primate behavior, and provide important tests of the feasibility of optical and virus-based methods as possible therapeutic approaches in CNS disorders.

DESCRIPTION (provided by applicant): Visual perception is mediated by complex interactions amongst neurons in the retina, visual cortex, and subcortical brain structures. The importance of vision to humans and other primates is reflected in the enormous percentage of cerebral cortex devoted to processing visual information. Thus, deficits in visual processing are particularly debilitating and arise from abnormalities not only in the eye, but also in cortical circuitry. For example, strabismus or amblyopia during childhood can have long-lasting effects on the cortical circuits that process visual information. There is also evidence that some forms of dyslexia result from central visual system abnormalities. The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. The proposed studies are aimed at revealing the organization and function of mouse visual cortical areas. Such studies will provide a crucial framework for detailed functional investigations of visual cortical circuits using novel genetic, viral and transgenic approaches tha have been developed at the cutting edge of neuroscience over the last several years. Because these tools are most powerful in mice and many basic principles of the organization and function of cortical circuits are conserved from mice to humans, development of the mouse system represents in extremely important direction for future studies. The 3 aims will reveal: the visual receptive field properties of neurons in each of nine mouse extrastriate visual areas; the functional properties of V1 neurons that project to extrastriate visual areas; and the connections between extrastriate visual areas and to subcortical structures.

DESCRIPTION (provided by applicant): A hallmark of primary visual cortex is its organization into maps of visual space, orientation and ocular dominance. Despite remarkable advances in our ability to measure the structure of cortical maps and their mutual relationships, many important questions remain unanswered. How do these maps develop? Why are maps missing in some species? What role do maps play, if any, in cortical computation? The central goal of our research is to seek answers to these fundamental questions of cortical development, organization and function that have eluded us for decades. Our working hypothesis is that the blueprint for the formation of simple-cell receptive fields and orientation maps in primary visual cortex is encoded in the spatial layout of retinal ganglion cell (RGC) mosaics. To test the idea we will analyze the spatial statistics of RGC mosaics, reconstruct the retinal input to given orientation domains, and test the micro-organization of cortical maps. If the these ideas are confirmed, they can offer a definitive account for the origin of orientation maps in primary visua cortex and, more broadly, profoundly influence the way we conceive of cortical maps, their development and function.

Project Summary Neural circuits are composed of networks of specific cell types that are interconnected in precise patterns to give rise to normal brain function. Past studies have investigated the rates of connections that are formed by cultured neurons in vitro and these have revealed abnormalities both in a mouse model of autism (MeCP2 KO mice) and for human induced pluripotent stem cell (hiPSC)-derived neurons from schizophrenic patients (Brennand et al., 2011). But simple separation of cortical neurons into just excitatory versus inhibitory groups barely scratches the surface of the diversity of cortical cell types, the known specificity of connections between specific cell types in the abnormal or normal brain. In Project 5 we propose to develop and then implement novel tools that will allow assays of the cell-type specificity of connections that are formed in cultures of hiPSC- derived neurons. These assays are based on the use of glycoprotein deleted rabies viruses for trans-synaptic labeling of connected neurons, and conditional expression of EnvA/TVA targeting systems for directing the initial infection of rabies virus to specific cell types (Wickersham et al., 2007b; Marshel et al., 2010; Wall et al., 2010)

? DESCRIPTION (provided by applicant): Deciphering how neural circuits within the mammalian brain give rise to perception, cognition, and behavior is central to understanding how the nervous system functions. Neural circuits operate over a vast range of spatial and computational scales, from high-level circuits that integrate information across multiple brain regions, to microcircuits that perform simple input/output transformations within a specialized brain structure. Each level of analysis is important for formulating responses to environmental conditions. However, studying a specific neural circuit is extremely difficult, as most nervous system structures contain many types of neurons with inextricably intertwined axons and dendrites. To overcome this obstacle, the glycoprotein (G)-deleted rabies vector system was developed to identify direct synaptic inputs to a particular neuronal population. By pseudo typing the G-deleted rabies vector with a foreign envelope protein, such as EnvA, the vector selectively transduces target neurons genetically engineered to express the EnvA receptor. If these cells also express rabies glycoprotein the vector travels retrograde exactly one synaptic step and transduces direct presynaptic neurons. The rabies genome can be altered to encode any gene of interest, including fluorescent proteins to reveal the cytoarchitecture of presynaptic cells, or neuroscience tools (e.g., calcium indicators or light-gated ion channels) to monitor or manipulate circuit activity. Thus, the G-deleted rabies vector system allows the fine- scale manipulation of specific cell types within a circuit, allowing investigators to test hypotheses linking these circuts to behavior. This technology has revolutionized the study of neuronal circuits, creating high demand for these cutting-edge reagents. Laboratories that focus on understanding neural circuits, however, typically do not have the resources or expertise to produce high quality rabies vectors or associated helper vectors that are necessary to perform these experiments. Because of this, the Salk Institute's Gene Transfer, Targeting, and Therapeutics (GT3) Core, which currently generates the rabies vectors, is inundated with requests for ready-to- inject viral reagents and demand exceeds production capacity. This R24 application proposes to expand the GT3 Core's capacity for maintaining, propagating, and distributing all G-deleted rabies vector variants and helper vectors (Aim 1). The GT3 Core will also incorporate newly developed tools into the technology platform as they are innovated (Aim 2). Establishing this central rabies production facility will lower reagent costs (through economies of scale) and improve the reproducibility of study findings. Between-lab cost sharing mechanisms will enable the distribution of small aliquots, facilitating pilot experiments and removing the greatest barrier to technology uptake by new laboratories. Newly generated reagents will be immediately distributed to the neuroscience community without publication restrictions, thereby speeding the pace of discovery. These efforts will broaden the impact of this technology and ensure that neuroscientists studying circuits are equipped with the most modern analytic tools.

DESCRIPTION (provided by applicant): Visual perception is mediated by complex interactions amongst neurons in the retina, visual cortex, and subcortical brain structures. The importance of vision to humans and other primates is reflected in the enormous percentage of cerebral cortex devoted to processing visual information. Thus, deficits in visual processing are particularly debilitating and arise from abnormalities not only in the eye, but also in cortical circuitry. For example, strabismus or amblyopia during childhood can have long-lasting effects on the cortical circuits that process visual information. There is also evidence that some forms of dyslexia result from central visual system abnormalities. The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. The proposed studies are aimed at revealing the organization and function of mouse visual cortical areas. Such studies will provide a crucial framework for detailed functional investigations of visual cortical circuits using novel genetic, viral and transgenic approaches tha have been developed at the cutting edge of neuroscience over the last several years. Because these tools are most powerful in mice and many basic principles of the organization and function of cortical circuits are conserved from mice to humans, development of the mouse system represents in extremely important direction for future studies. The 3 aims will reveal: the visual receptive field properties of neurons in each of nine mouse extrastriate visual areas; the functional properties of V1 neurons that project to extrastriate visual areas; and the connections between extrastriate visual areas and to subcortical structures.

? DESCRIPTION (provided by applicant): The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neurons types and their functional interactions in the intact brain. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate and to selectively manipulate and measure the activity of specific cell types in the context of the normal functioning network. The research proposed here is aimed at revealing the detailed connectivity and function of specific types of inhibitory cortical neurons. Intersectional genetic methods and Cre- and Flp-driver mouse lines are used to target gene expression to specific subsets of VIP positive inhibitory cortical neurons. The direct synaptic inputs and outputs of these neurons are then determined by monosynaptic rabies tracing or by optogenetic activation and whole cell recordings in brain slices. These same cell types will be optogenetically activated in vivo to assess their impact on other cells in the intact network and in the generation of sensory receptive fields. These experiments are designed to test hypotheses about the interactions between specific neural elements and their contributions to perception, cognition, and behavior.

PROJECT SUMMARY The role of a neuronal cell type in the generation of perception and behavior depends on three major factors. These are: the sources of synaptic input, how synaptic inputs are integrated to give rise to spiking output, and the population of neurons that receive the output. This Research Segment (Segment 2 - Anatomy. Linking Cell Types Defined by Epigenetic Profiling to Neural Circuits) will link putative cell types defined by epigenetic profiling (Segment 1) to their inputs and outputs. This knowledge will link genetically targetable cell types to circuits, allowing future manipulative studies to assess their functional roles within intact neural circuits. Nearly all brain regions, including those that will be dissected and subject to epigenetic profiling in Segment 1, either contain multiple intermingled cell types or they are composed of nuclei too small to be selectively dissected. However, because individual nuclei or intermingled cell types typically project axons to unique distant brain structures, they can be identified based on their axonal outputs. Efforts here will generate epigenetic profiles of neurons with known projections, to link profiled populations to their outputs. Monosynaptic rabies tracing will then be used to link inputs across the entire brain to cell types defined based on their outputs. Finally, putative cell type specific enhancers, whose ability to drive cell type specific expression will be initially evaluated in Research Segment 4, will be further characterized. This includes further characterization of the cell types that drive expression from identified enhancers using anatomical analysis of their distributions within the brain, as well as the patterns of projections of their axonal arbors. The ability to use enhancers to target cell types for selective gene expression and anatomical characterization of the targeted cell types will make available the full arsenal of molecular and genetic tools for interrogating the functional roles of these cell types within intact neural circuits.

Project Summary Visual perception is mediated by complex interactions amongst neurons in the retina, visual cortex, and subcortical brain structures. The importance of vision to humans and other primates is reflected in the enormous percentage of cerebral cortex devoted to processing visual information. Thus, deficits in visual processing are particularly debilitating and arise from abnormalities not only in the eye, but also in cortical circuitry. For example, strabismus or amblyopia during childhood can have long-lasting effects on the cortical circuits that process visual information. There is also evidence that some forms of dyslexia result from central visual system abnormalities. The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types across multiple cortical areas. The proposed studies are aimed at revealing the organization and functional impact of local and long-distance feedback connections within and between mouse visual cortical areas. These studies are conducted in mice to take advantage of the range of molecular, genetic, and viral tools that can be used to elucidate brain circuits and link them to function using optogenetic and imaging methods. The 2 aims will reveal: the fine-scale organization of V1 microcircuits linking cortical layers 2/3 and 5, and the functional impact of feedback from layer 5 to layer 2/3; and the organizational principles and functional impact of cortico-cortical feedback.

Abstract Understanding the exact cell-type composition in the different regions of the mouse brain is a fundamental step when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although major categories of cell-types present in the mouse brain have been defined through a handful of specific markers, the different subtypes within these categories, as well as their location and connectivity are far from understood. Epigenomic signatures such as DNA methylation (mC) and open chromatin are stable modifications that persist in post-mitotic cells throughout their lifetime, defining their cellular identity. Open chromatin as well as methylation patterns are cell type specific, differentiating the major types of cells i.e., neurons and glia, in the rodent and human cortex, as well as differentiating neuronal types in mouse brain. Research Segment 1 of this center proposes to produce a catalog of methylation and open chromatin patterns at the single-cell level throughout the entire mouse brain. Analysis of the combined data will permit the discovery of cell-type specific regulatory regions that will allow the production of transgenic mouse lines and viral tools that will become available to the community.

Project Summary Understanding the circuit mechanisms that give rise to perception and behavior requires linking neuronal activity to connectivity. This can be accomplished at multiple scales and ideally can be related to further studies using activity manipulations to demonstrate causality. Recent work in the mouse visual system has revealed the contributions of specific cell types to the generation of visual receptive field properties as well as state-dependent changes in the representation of visual information. But it is unknown whether the cortical circuit mechanisms and principles revealed in the mouse are preserved across species. This project aims to develop and refine molecular, genetic, viral and large scale optical and electrical recording tools for use in the non-rodent cortex. Paradigms will be established by which these tools can link visual function to cortical modules, cell types, and connectivity. Experiments that expand knowledge of the role of specific cortical cell types to be comparable to data collected in mice are required to evaluate what are common circuit mechanisms and principles of cell type specific computations versus circuits that are specialized to particular species or functions. Systematically controlling visual stimuli while conducting recordings of activity with these tools will create data sets that make it possible to test whether principles and functions of specific circuit motifs emerging from studies in the mouse cortex can be generalized to higher species. Specific aims are organized around levels of selectivity at which visually-evoked activity will be linked to circuits: 1) modules, 2) cell types, and 3) connectivity. These aims share two different basic approaches for recording dynamic activity from large neuronal populations ? two-photon calcium imaging and high-density (128 and 384 channel) laminar silicone electrode arrays. Aim 1 will link visually evoked neuronal activity to modular and laminar organization of primary visual cortex (V1). This knowledge can be combined with known relationships between connectivity and modular/laminar organization to link circuits to function. Aim 2 will link visually evoked neuronal activity to V1 cell types by: combining 2-photon calcium imaging with post mortem identification and antibody staining; and recording activity of single neurons with high-density laminar electrode arrays and then identifying cell types based on electrical images. Aim 3 will directly link visually evoked neuronal activity to connectivity using cross-correlation analysis of recordings from up to 150 neurons recorded simultaneously with high-density laminar electrode arrays.

Project Summary/Abstract The long-term goal of this proposal is to characterize the connectivity of specific circuits in the mouse visual system whose gross architecture is highly conserved across species from mouse to primate. The objective is to identify the neural circuits that transmit visual information from the retina, through different regions of the dorsal lateral geniculate nucleus (dLGN), to specific populations of neurons in the primary visual cortex (V1) that contribute to visual perception and behavior. The central hypotheses of this proposal are: (1) that neurons in the shell and core of the dLGN receive different types of visual information from the retina and, therefore, can transmit different types of visual information to V1 neurons; (2) The balance of core and shell input that V1 neurons receive influences their tuning properties. The experiments outlined in this proposal will test these hypotheses by pursuing three specific aims: (1) Identifying and characterizing the retinal ganglion cell (RGC) types that provide input to neurons in the core of the dLGN, as well as the neurons that those dLGN neurons contact in V1; (2) Determining whether genetically-identified neurons in V1 receive input from unique patterns of RGCs; and (3) Determining how input from neurons in the core of the dLGN influence tuning properties in genetically-identified populations of V1 neurons. This proposal is technologically innovative; it will use novel mouse lines as well as a combination of rabies circuit tracing, whole-cell recording, optogenetic stimulation, chemogenetic silencing, and two-photon calcium imaging to accomplish its aims. The proposed research will yield significant findings that will provide considerable insight into how information is encoded, processed, and ultimately transmitted throughout the mouse visual pathway. These findings are of utmost importance as the computations performed in these pathways generate a representation of the visual scene and ultimately make characteristic contributions to perception and behavior. The proposed research will also help determine the extent to which visual processing in the mouse visual system does, or does not, mimic visual processing in the primate visual system. Understanding the extent to which computations performed in the visual pathway overlap in mouse and primate visual systems is critical for determining how research done in in the mouse visual system translates to the primate ? and therefore human ? visual system. Indeed, to successfully develop strategies to restore sight across a wide range of afflictions of the visual system, it is critical to first understand how the visual system gives rise to our sense of the world around us. The work proposed here will unequivocally move us closer to this goal.

Project Summary The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neuron types and their functional interactions in the intact brain. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate and to selectively manipulate and measure the activity of specific cell types in the context of the normal functioning network. The research proposed here is aimed first at revealing the detailed connectivity of inhibitory cortical neurons by developing and using a novel monosynaptic rabies circuit tracing strategy in which input neuron types are classified using single cell sequencing. The work will also develop and use a novel high throughput strategy for identification of cell type specific enhancer elements. The approach used will identify enhancers that can drive transgene expression in specific types of inhibitory cortical neurons. Together these studies will allow the development and testing of new hypotheses about the functional contributions of specific inhibitory cortical neuron types to perception,cognition and behavior.

Project Summary The function of the nervous system is dependent on complex interactions between networks of neurons composed of multiple neuron types. Understanding how these networks function both in health and disease is dependent on understanding the precise connectivity between specific neuron types and their functional interactions in the intact brain. It is therefore apparent that, in order to have an adequate understanding of the nervous system, it is necessary to have detailed descriptions of neuronal connectivity with the same level of precision at which these systems operate and to selectively manipulate and measure the activity of specific cell types in the context of the normal functioning network. Such studies would be greatly facilitated by the ability to target gene expression to specific cell types in the context of AAV vectors. The research proposed here is aimed at developing and using a novel high throughput strategy and PCR-activated cell sorting for identification of cell type specific enhancer elements. The approach used will identify enhancers that can drive transgene expression in specific types of cortical neurons. Further studies will valdiate these enhancers in a real-use context for ability to facilitate imaging with genetically-encoded calcium indicators and to facilitate optogenetic inactivation. These reagents will allow future studies testing the functional contributions of specific inhibitory neuron types to perception,cognition and behavior.