R Clay Reid - US grants

We will study the mechanisms by which visual information is transformed between the lateral geniculate nucleus of the thalamus (LGN) and layer 4 of primary visual cortex in cat. The functional transformations between receptive fields in the LGN and cortex are well known, but little is known of the mechanisms by which these transformations are achieved. We will study the connectivity between pairs of single LGN and cortical neurons and relate the degree of connectivity and its type (excitatory or inhibitory) to the visual functions of these pairs of neurons. Synaptic connections can be inferred by a lawful relation between the firing of the LGN neurons followed, on a short time scale (several msec), by the firing of the cortical neuron. The visual function of these neurons will be assessed with a method of automated receptive-field mapping (spatiotemporal white-noise analysis). This two-pronged study of both connectivity and function will allow us to test specific models of information processing by cortical cells. We will study the cortical mechanisms responsible for the selectivity for orientation and direction of motion in simple cells. The interpretation of our results concerning the connectivity between pairs of LGN and cortical neurons requires a second piece of information: what are the range of LGN afferents available locally for processing by single postsynaptic cortical cells? In additional experiments, we will therefore study the full range of thalamic inputs to any given point in the cortex. The number of afferents recorded and the density with which they are sampled will be far greater than in the more difficult LGN- cortex experiment. Thalamic afferents will be recorded directly in the cortex by silencing the cortical cells pharmacologically. In addition to multi-unit recording and automated receptive field mapping, this study will make integral use of optical imaging, a powerful new technique for mapping the function of neurons across the cortical surface in vivo. The type and scatter of afferents recorded in the cortex will be correlated with the local receptive field (e.g. their ocular dominance, preferred orientation, or color specificity) measured with imaging. This basic research on information processing in the visual cortex should further our knowledge of general mechanisms of cortical function.

nervous system; eye; Primates; Mammalia;

We will study the mechanisms by which visual information is transformed in the pathway from retina, to the lateral geniculate nucleus of the thalamus (LGN), and finally to layer 4 of primary visual cortex. The physiology of these three populations of neurons in the visual system have been extensively studied over the past forty years. Individual connections between these neurons have also been studied. Until recently, however, there has not been an explicit exploration of the interactions between multiple convergent inputs to single cells, or of the divergent projection from single cells to their multiple targets. The proposal is divided into three broad sections whose overarching goal is to understand the integration of multiple inputs to visual cortical neurons. In the first (A), we will study this problem directly by recording simultaneously in the LGN and layer 4 of visual cortex. In particular, we will concentrate on exploring synergistic interactions between near- synchronous inputs from convergent thalamic afferents. In the second section (B), we will study how the correlational structure of the thalamic inputs to visual cortex are determined by their retinal inputs. Preliminary results indicate that there is strong synchrony between groups of neurons in the thalamus. By recording simultaneously from neurons in the retina and LGN, we will study how synchrony in the LGN is caused by divergent input from the retina. Finally, in the third section (C), we will record simultaneously from all three levels in the pathway: retina, LGN, and visual cortex. This study will help us assess quantitatively the importance of the effects studied in (A) and (B) on the transmission of visual information from retina to cortex: single retinal ganglion cells diverge to synchronize to small pools of thalamic neurons, these synchronized neurons re-converge in a synergistic manner onto single cortical neurons. This basic research on information processing in the visual cortex should further our knowledge of general mechanisms of cortical function. Only by exploring the detailed interplay of multiple thalamic inputs to cortical neurons can we begin to understand functional disorders of this pathway, such as in certain forms of epilepsy.

vision; computer program /software; computer center; computer system design /evaluation; biomedical facility; image processing; vision tests; computer assisted diagnosis;

DESCRIPTION (provided by applicant): Continued support is sought for the Core Grant for Vision Research at Harvard Medical School. The core faculty is made up of ten members of the Department of Neurobiology and six scientists from other departments at Harvard University with whom we share common interests. All of us study the visual system, which we approach at many different levels. Current research areas include: the cell biology of the cornea and lens; the physiology and pharmacology of retinal function; visual processing in the lateral geniculate nucleus (LGN), striate cortex and extrastriate cortex; synaptic physiology of the LGN; the molecular biology of circadian rhythms; and the development of the mammalian retina and central visual pathways. The Core Grant has one resource module and three service modules. The resource module consists of multiple facilities for Neural Imaging: (1) histological imaging with conventional and confocal microscopes, (2) physiological imaging with macroscopes, microscopes and a two-photon confocal, and (3) functional and anatomical MRI. The service modules include a Machine Shop with expertise in developing equipment for physiological experiments, a Laboratory Computer Module with expertise in hardware and software for data acquisition and visual stimulus generation, and an Electronics Module with expertise in electrophysiological hardware. The modules are staffed by personnel with extensive training in the appropriate fields and are supervised by established investigators. The vision community at Harvard is large and cohesive; many collaborations already existed five years ago when the Core Grant was established. The Core Grant has been a strong force in promoting further collaborations and has helped facilitate ongoing vision research at Harvard in an efficient and cost-effective manner. Within the Department of Neurobiology alone, there are four new members of the Core Grant since the last competitive renewal. With plans for continued recruitment, this community is expected to grow.

We will study the transmission of visual information through the lateral geniculate nucleus of the thalamus (LGN). Geniculate neurons have been studied extensively for the past 30 to 40 years with simple stimuli and with fixed gaze, but we still have only a dim understanding of geniculate activity during natural vision: when an alert animal scans a complex scene. Studies in both the anesthetized and the alert animal will provide a two pronged attack on this problem. Basic problems in geniculate physiology and information processing will be addressed first in the anesthetized animal, because visual stimuli are easier to control than in the alert animal and histology can be used to assign neurons in the magnocellular parvocellular or intercalated layers. In the alert animal, will be able to study how eye movements affect visual responses in each of the three geniculate divisions. In both sets of experiments, we will characterize receptive fields with the reduced stimuli typically employed in visual physiology, as well as with more natural stimuli. In later projects, multi-e4lectrode recording will be used to analyze the activity of the ensemble, rather than of individual neurons. Beyond this grant period, the long-term goal of this research program is to use our studies in the LGN as a foundation for the study of visual cortical physiology during perception. Much of visual physiology, in striate and especially extra striate cortex-is now being performed in the alert primate. We nevertheless have an imperfect understanding of the thalamic inputs to visual cortex when the eyes move. A more complete analysis of the interplay of vision and eye movements in the LGN will help put this emerging field on a more secure footing. This basic research on information processing in the visual thalamus-and on the relations between thalamus and cortex-should further our knowledge of general mechanisms of thalamocortical function. Only by exploring the normal activity of multiple thalamic inputs to cortical neurons can we begin to understand functional disorders of this pathway, such as in certain forms of epilepsy.

DESCRIPTION (provided by applicant): In the adult, synaptic connections from retina to thalamus and from thalamus to primary visual cortex are distinguished by their great specificity. Past work has shown that these connections are found only if the receptive field of the presynaptic neuron is well matched to the receptive field of its target. In the pathway from retina to thalamus (the lateral geniculate nucleus, or LGN), this relationship is straightforward. An LGN neuron receives only one or two strong inputs and the receptive-field centers of pre- and postsynaptic neurons are very similar. In the thalamocortical pathway, the relationship is more complicated. There is a large pool of potential thalamic inputs to any layer 4 cortical neuron, but only 1/3 of these afferents are functionally appropriate; it is this subset that makes functional connections. We will study animals in the weeks after eye opening, during which the specific connections seen in the adult are formed. Retinothalamic connections at eye opening are highly convergent. Over the next few weeks of development, the connections are pared down to the one-to-one (or several-to-one) connections seen in the adult. Much less is known of the fine-scale refinement in the thalamolocortical system. It is clear that refinement is proceeding simultaneously at two levels: between retina and LGN, and between LGN and visual cortex. Surprisingly little is known of the relative rates of this two-level process, or of how the process of maturation at one level affects the next. We propose to study three aspects of visual development in the weeks after eye opening. (A) We will compare receptive fields between levels (retina and LGN, or LGN and cortex) to assess directly the relative rates of functional maturation. (B) We will study correlations among nearby neurons, both in the retina and in the LGN. It is widely accepted that correlated activity among presynaptic neurons drives the refinement of their connections onto specific targets, but very little is known about these correlations in the weeks after eye opening. (C) Finally, we will examine how correlations in the retina caused by visual stimuli can instructively affect retinogeniculate connections: specifically how visual experience can modify geniculate receptive fields over the time-course of minutes to hours. This basic research will further our knowledge of normal development in the visual system and potentially help us understand disorders of neural development.

[unreadable] DESCRIPTION (provided by applicant): The Vision Training Program at Harvard Medical School seeks to train excellent students in molecular biology and related disciplines while preparing them to enter the field of vision at some later point in their careers. Students enter the Vision Training Program through two interdepartmental umbrella programs at HMS, the Biological and Biomedical Sciences (BBS) Program and the Neuroscience Program. Students take the curriculum required of their respective programs and learn about the visual system in two ways: (1 ) They are required to take an introductory course, Genetics 214: Biology of the Visual System, taught by several faculty of this training grant (and others) whose primary research interest is vision. As part of this course, the students design a research proposal aimed at understanding or treating a disease of the visual system; (2) The students organize and attend a monthly seminar series, "Molecular Approaches to Vision", in which an outside speaker visits HMS and gives a seminar to the HMS community. The students invite the speakers to have lunch and dinner with them. [unreadable] The students are supported for five years on this grant in order to maximize exposure to vision. By the end of their period of support, most students will have attended approximately 50 seminars on vision at HMS, met with and discussed the research of these 50 speakers, read approximately 25 papers on vision in Genetics 214, and researched a disease of the visual system and written a proposal aimed at furthering our understanding of such a disease. The students trained within this program will have the tools to make progress in our understanding of vision system disease and treatments [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of the proposed studies is to understand the functional organization of layers 2/3 in visual cortex of cats at the level of single neurons. Functional architecture of visual cortex has been studied with two approaches: (1) electrophysiology of single neurons, and (2) optical imaging, which provides functional maps at low resolution: >100 mu/m. The new technique of calcium imaging with two-photon laser-scanning microscopy (resolution <1 mu/m) bridges the gap between single-unit electrophysiology and conventional optical imaging. Using this technique, we will create the first maps of visual cortical function at the scale of single neurons. We call cortical organization at this level functional micro-architecture. We will examine functional micro-architecture with a three-pronged strategy: intrinsic-signal imaging, two-photon imaging, and electrophysiology. We will first obtain maps of visual cortex with conventional optical imaging, then zoom in with two-photon microscopy to examine smaller regions at single-cell resolution. In Aim 1, we will examine the fine-scale clustering of neurons according to receptive-field attributes such as orientation selectivity, direction selectivity, ocular dominance and retinotopy. In Aim 2, we will combine single-cell calcium imaging with electrophysiology: to calibrate the calcium signal, to examine co-active ensembles of neurons and (with intracellular stimulation) to determine the influence of one neuron on its potential targets in the circuit.

DESCRIPTION (provided by applicant): We plan to address two fundamental questions about cerebral cortical circuits: " What are the functional roles of different neuronal classes, pyramidal cells and subclasses of inhibitory interneurons, in cortical computations? " What are the properties of spontaneously active ensembles and how do these ensembles relate to the neurons'functional properties? We will use two-photon calcium imaging of the rodent visual cortex in vivo to measure the orientation selectivity of all neurons in a volume layer 2/3 (~500 5m on a side, ~10000 neurons). The unprecedented ability to record neural activity so completely will enhance our understanding of two aspects of cortical circuits: the diversity of cell classes and the richness of spontaneous activity. We will use anatomical and genetic techniques to identify pyramidal neurons and various classes of interneurons. Spontaneous activity will be assessed with two-photon calcium imaging in the absence of sensory input. Great advances have been made over the past several years on the molecular and cell- physiological classification of different neuronal subclasses. These advances have had little impact on in vivo studies of neural function, but now calcium imaging with single-cell resolution can show the functional correlates of classes of neurons that have been identified by genetic techniques. Spontaneous activity in the cerebral cortex has been well documented, but it is poorly understood. Simultaneous cellular-level monitoring of entire neural circuits is crucial for advancing the study of this correlated activity and its relation to visual function. A wide-field resonant-scanning system will be used so that we can image thousands of neurons in a three-dimensional volume simultaneously. This large-scale overview of the function of entire local circuits will enhance our understanding of cerebral cortical function in health and disease. This basic research on cortical sub-networks is directly applicable to clinical disorders of brain function. The neurological and psychiatric diseases with the largest impact on public health, Alzheimer's disease, stroke, epilepsy, depression and schizophrenia, are all disorders of cortical activity. The approaches outlined in this proposal will characterize cortical circuits with unprecedented completeness;they can be used to study both normal brains and models of neurological disease.

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. The wiring diagrams of neuronal circuits may be obtained from electron microscopy (EM) images of serially cut, ultrathin (<50 nm) sections through nervous tissue. In the mammalian brain, even local circuits may span hundreds to thousands of microns in space. Images spanning hundreds of microns at nanometer resolution consume many gigabytes of storage. We are working with people at PSC to develop a high throughput pipeline for processing and analysis of these very large data sets.

DESCRIPTION (provided by applicant): It has been known for over a century that small electrical currents applied to the brain can activate neurons, trigger movements, and change behaviors. Recent years have seen an explosion of applications of brain stimulation to the treatment of neurological disorders. Worldwide, more than 60,000 deaf patients have recovered hearing through cochlear implants. Similarly, deep brain stimulation has proven highly valuable in the treatment of intractable movement disorders including Parkinson's. Today, its use is being expanded to a variety of severe, otherwise untreatable disorders, including refractory depression, obsessive-compulsive disorders, and even to promote recovery of function in stroke patients. Although electrical stimulation is routinely used today in human brain implants, we do not know how electrical stimulation acts to change neuronal activity in the brain. There is considerable debate, for example, as to whether deep brain stimulation activates activity in the targeted area or whether it suppresses it. The fundamental issue to be addressed, therefore, is how populations of cells respond to electrical stimulation. Are all the cells in the targeted area activated? Are cells recruited one by one as current is increased, or is there a threshold at which a set of cells fire in response to stimulation? The new technique of in vivo two-photon imaging of calcium signals provides a unique opportunity to answer these questions. With this technology, we can image the activity of thousands of neurons simultaneously as electrical stimulation is applied. We can thus measure how virtually every neuron in a local volume is affected by stimulation, and provide basic answers to the questions posed above. We have two specific aims. First, we will use single electrodes to stimulate in the visual cortex and assess the spatial distribution of cells that are activated (or suppressed) by microstimulation with various currents and pulse trains. Second, we will stimulate through a multi-site electrode with closely spaced contacts (50 5m separation), and ask how stimulation effects vary as the location of the current injection is varied. Does stimulation at different sites activate different cell populations or do these populations overlap? Do responses from different sites sum linearly or are there nonlinear interactions between these responses? These simple protocols will help ground a 50- year literature on cortical stimulation. More generally, this work will establish protocols for the use of calcium imaging to calibrate the effects of stimulation in neural circuits. PUBLIC HEALTH RELEVANCE Electrical stimulation of the brain is used in cochlear implants to provide auditory sensation to the deaf, in experimental visual prostheses to provide visual sensation to the blind, and in treatments to relieve patients from the symptoms of Parkinson's disease. Our proposed studies will use a new imaging technique to visualize at high-resolution brain activity during stimulation, which has previously been impossible. Measuring how neurons respond to electrical stimulation will help devise better treatments for diseases of the brain.

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. The principal goal of this study is to understand the functional organization of neurons in layers 2/3 of primary visual cortex (V1). We are examining the organization of cells with defined response properties, both in their relation to larger compartments like cytochrome-oxidase blobs and orientation maps, as well as in their fine-scale arrangement. These issues have been studied in the past with two approaches: electrophysiological recordings, which yield functional characterizations of single neurons, and intrinsic signal imaging, which provides an overview of functional maps in cortex but with very low resolution (less than 100 m). The key aspect of our present work is that we are using a new approach, calcium imaging with two-photon laser-scanning microscopy, which bridges the gap between single-unit electrophysiology and conventional optical imaging. The technique gives us single cell resolution from an essentially complete sample of cells in a given patch of cortex, and, to our knowledge, this is the first time it has been used to study cortical microarchitecture in primates.

DESCRIPTION (provided by applicant): Twenty-one neuroscientists within Harvard's Neuroscience Program request continued funding for six pre-doctoral positions within our. Training focuses on the study of visual pathways from retina to brain, and cellular, molecular and developmental neurobiology of the visual system. The faculty are distributed throughout the university. Eleven faculty members are in basic science departments at the Medical School, six are in hospital based laboratories, and four are in the Faculty of Arts and Sciences. Over the past 15 years, Harvard University has greatly expanded the number faculty members who study the molecular, developmental, and neural-systems approaches to visual science. Students can choose laboratories among a large community of vision researchers, most of whom are affiliated with the NEI Core Grant in Vision research. The new goal of the Visual Neuroscience Training Program is to build a larger, coherent group of students who have a sense of community based in this Harvard-wide vision community, and who are trained by its faculty. The grant will support three students each in their second and third years, after they have chosen a dissertation lab, but advanced students remain actively involved with the program. This creates a large cohort of affiliated students. We train and supervise these students with courses, thesis committees, seminars, symposia, a Training Grant retreat, and our "Systems-Vision" journal club. Thus throughout their graduate careers, trainees interact with the faculty and with each other. Many vision scientists visit Harvard every year to give seminars;trainees at all levels interact with them over lunch and in lab visits. Through these activities, we will help train a new generation of vision scientists whose scientific careers will help us understand all aspects of the visual system: development, information processing, and disease.

DESCRIPTION (provided by applicant): A fundamental but unsolved question in neuroscience is how specific connections between brain cells (neurons) underlie information processing in the neural circuits. Even the smallest local circuit in the cerebral cortex consists of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand the cerebral cortex is that we don't have an actual wiring diagram of any single cortical circuit. But even if we had a wiring diagram, we would need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to study neurons in the visual cortex whose responses to sensory stimuli have been characterized with new imaging techniques. This will allow us to see the function of literally every neuron in a cube 1/2 millimeter on a side. We will then use high-resolution and high-throughput anatomical techniques to uncover some of the connections between these functionally characterized neurons. Recent advances in functional imaging and serial-section electron microscopy allow us to study this difficult problem. We will address these questions in the mouse visual cortex, an emerging model of visual processing that is amenable to genetic manipulation and in vivo imaging techniques. We will use two- photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large- scale serial-section electron microscopy to trace circuits in the same piece of cortex. By combining these methods, we will collect data sets that provide a physiological and structural overview of a well-studied brain circuit. It is now possible to study these circuits on their own terms: in all of their complexity and with data sets that are in many senses complete. PUBLIC HEALTH RELEVANCE: Many of the neurological and psychiatric diseases with the largest impact on public health-Alzheimer's disease, stroke, epilepsy, and autism-are functional disorders that likely have correlates in disordered brain connections. The proposed studies will characterize the functional connectivity of brain circuits with unprecedented resolution and completeness. In mouse models of functional brain disorders, the approaches we develop will greatly improve our ability to study the relationship between altered connections and functional deficits.

DESCRIPTION (provided by applicant): A fundamental but unsolved question in neuroscience is how specific connections between neurons underlie information processing in the cortical circuits. Local circuits in the cerebral cortex consist of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand these circuits is that we have never been able to reconstruct their actual wiring diagrams. But if we had a partial or even complete wiring diagram, we would also need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to develop and apply new approaches for large-scale electron microscopy, towards the goal of mapping wiring diagrams of cortical circuits in a functional context. We will use two-photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large-scale serial-section electron microscopy to trace circuits in the same piece of cortex. Recent advances in functional imaging and serial-section electron microscopy (EM) allow us to study this difficult problem, but before we can reap the benefits of these approaches, considerable technical work is necessary. Functional imaging with two-photon microscopy is a technically mature field, but approaches for large-scale serial-section EM are still in their infancy. We propose to apply the dual approach of functional imaging followed by high-resolution anatomical imaging-which we have already performed once in a large pilot project-with the goal of improving the technologies specifically for large-scale EM, correlated with functional studies. Our four-year goal is to create a high-throughput system for generating correlated structure/function data sets from the cortex. In particular, we will build a new EM imaging system, a second-generation Transmission EM Camera Array (TEMCA), that will allow us to capture very large three-dimensional data sets (300 to 500 micrometers on a side) in a week, rather than months. We propose to address one class of question: are there subnetworks within each local cortical circuit that process distinct information? But the approach is general and can be applied to a wide range of questions, including clinically relevant ones. Are neural connections disrupted near plaques in Alzheimer's disease? When stem cells incorporate into a circuit, do they form connections that play a functional role? For the first time, these questions should be within our reach. By developing high-throughput methods for large-scale imaging, we will begin to study neural circuits on their own terms: in all of their complexity and with data sets that are in many senses complete.

DESCRIPTION (provided by applicant): A fundamental but unsolved question in neuroscience is how specific connections between brain cells (neurons) underlie information processing in the neural circuits. Even the smallest local circuit in the cerebral cortex consists of tens of thousands of neurons, each making thousands of connections. Perhaps the biggest reason we don't understand the cerebral cortex is that we don't have an actual wiring diagram of any single cortical circuit. But even if we had a wiring diagram, we would need to know what each neuron in a circuit is doing: its physiology. In this proposal we plan to study neurons in the visual cortex whose responses to sensory stimuli have been characterized with new imaging techniques. This will allow us to see the function of literally every neuron in a cube 1/2 millimeter on a side. We will then use high-resolution and high-throughput anatomical techniques to uncover some of the connections between these functionally characterized neurons. Recent advances in functional imaging and serial-section electron microscopy allow us to study this difficult problem. We will address these questions in the mouse visual cortex, an emerging model of visual processing that is amenable to genetic manipulation and in vivo imaging techniques. We will use two- photon calcium imaging to see the activity of neurons in a functioning local circuit. We will then use large- scale serial-section electron microscopy to trace circuits in the same piece of cortex. By combining these methods, we will collect data sets that provide a physiological and structural overview of a well-studied brain circuit. It is now possible to study these circuits on their own terms: in all of their complexity and with data sets that are in many senses complete.

Project Summary / Abstract A fundamental but unsolved question in neuroscience is how specific connections between brain cells (neurons) underlie information processing. Circuits in the cerebral cortex?the part of the mammalian brain that underlies high-level sensory, motor, and cognitive function?consists of tens of thousands of neurons, each of which sends and receives thousands of connections. Perhaps the biggest reason we don't understand the cerebral cortex is that we don't have an actual wiring diagram of any single cortical circuit. But even if we had a wiring diagram, we would need to know what each neuron in a circuit is doing: its physiology. In this proposal, we plan study the functional logic of networks in the visual cortex by examining groups of connected neurons whose responses to visual stimuli have been characterized. We will combine two techniques to tackle this difficult problem. Connections between neurons will be determined with a modified virus that allows us to specifically labels ensembles of neurons, all of which connect with a single 'target' neuron in each experiment. Once the neurons are labeled, we will use an advanced form of scanning-laser imaging (calcium imaging with two-photon microscopy) that allows us to make movies of each neuron's activity in response to carefully chosen visual stimuli. Together, these tools will allow to probe the functional logic of cortical circuits: the relationship between neuronal function and the wiring between neurons. The cerebral cortex is a network of networks. There are many different cortical regions each of which has its distinct inputs and outputs and distinct physiological properties. For instance, roughly ten visual cortical areas process different aspects of visual stimuli. We will start by studying the functional logic of wiring within three visual cortical areas: V1, AL and PM. We will then examine the functional logic of connections between these areas. The viral strategy that we employ is currently the only method that allow for both local and inter- areal connection to be studied along with physiology. The data we collect will help us understand feedforward, lateral, and feedback connections in cortical circuits and will form the foundation of new, data-driven models of cortical function.

Project Summary/Abstract Connectomics is a new field, created with the goal of densely or completely mapping the connections in the brain. Because this goal is at present only achievable for small organisms, connectomics has taken on two forms in the study of larger brains. Macroscale connectomics is used to describe the connections between brain areas, which in experimental animals is achieved with tracers, while humans it is typically pursued at a very coarse scale with diffusion imaging, a form of MRI. Microscale connectomics aims to create dense wiring diagrams of local circuits, small volumes of the brain with neurons that form rich networks within a local neighborhood. Macroscale connectomics lacks cellular resolution, while microscale connectomics can only reconstruct small volumes, with little information about the source of inputs entering the volume, or the targets of axons exiting it. We propose to develop a technique to bridge the gap between the microscale and the macroscale by creating experimental and analytical methods for mapping individual axons over long distances, concentrating on the largest 50% of myelinated axons (long-distance ?wires?) in the brain. For the current RFA to develop ?Tools to Facilitate High-Throughput Microconnectivity Analysis? we are specifically targeting one of the recommended goals, to ?Develop a high-quality toolbox of methods for efficiently mapping and annotating projections? in the human brain. The method?based on high-resolution 3D imaging of antibody-stained axons?can be scaled to analyze entire brains, but here we propose to apply it to the posterior pole of a macaque brain, a large (~10 cm3) volume will contain >20 visual cortical areas. The goal is to trace most of the larger axons (>1 um) between these areas, thus creating a dense axonal connectomics data set from the cortical visual system, allowing us to examine not only the hierarchy of visual areas, but also the structure of computational maps. Ultimately, this approach will permit whole-brain analysis of axonal projections, with targeted examination of key circuits for microscale connectomics. While microscale connectomics of whole human brains will remain impossible for the foreseeable future, dense axonal mapping should be achievable in the next 5 to 10 years. This proposal offers a pathway towards this new type brain-wide anatomy: Axonal Connectomics.

Project Summary/Abstract Primary visual cortex (V1) is the best studied part of the brain in multiple different mammalian species. Macaque V1 has been particularly intensively studied because of the close homologies between the visual systems of macaques and humans. We understand a great deal about the visual response properties of V1 neurons but, aside from some rough ideas about parallel pathways, we have limited understanding of how the cortical circuit transforms the visual information it receives. The rough outline of the cortical network is known, such as the axonal pathways that distribute information between the layers, but little is known of the network structure of connections between individual neurons. In particular, almost nothing is known about how visual response properties of neurons relate to their interconnections, even for pairs of neurons let alone over the entire network. Over the past decade, we have developed a new approach to studying cortical circuits?functional connectomics?that promises to address this question. Microscale connectomics has been developed by multiple groups with the goal of densely or completely mapping individual synaptic connections in the neural circuits with serial-section electron microscopy. Functional connectomics seeks to relate network structure with the physiological properties of individual neurons within a circuit. So far, virtually all research in microscale connectomics has been performed on mice or non-mammalian species. With recent advances in the scale of volumetric EM reconstructions and machine segmentation, it is time to perform cortical connectomics in species whose physiology is far better understood. In the macaque, we will use two-photon calcium imaging to record the visual responses of tens of thousands of neurons in a single circuit. This same circuit will then be reconstructed with EM connectomics, yielding a data set with rich information about visual response properties, neuronal type (as classified through morphology), and connectivity. This data set will be used to explore competing models of information in the macaque visual system. The close homology between macaque and human V1 offers a unique opportunity to compare their detailed network structure, while the structure/function relationships learned for the macaque will build a bridge for understanding the human network in a functional context.

Project Summary/Abstract Over the past decade, serial-section electron microscopy has come into its own as a method to study the connectivity of neural circuits, from local circuits in mammals to entire invertebrate brains. Recently, the emphasis in the field has been to create increasingly large data sets, while comparatively little effort has been spent on making the tools of EM connectomics available to a large number of circuit neuroscientists. Obstacles exist at multiple levels. Manual approaches to serial sectioning are prohibitively difficult, while automated approaches require complex, expensive equipment that is difficult to deploy. High throughput scanning EM is limited to multi- beam approaches that are extremely expensive. Transmission EM is far less expensive, but automated approaches to sectioning remain challenging and require expensive substrates that are hard to manufacture and difficult to use. We propose to develop a new approach, already prototyped by our group and our industry partner, to establish a robust platform optimized to achieve the widest possible adoption. The system will center on an open source serial sectioning robot implementing a novel collection approach. The goal is to create a system that can be used at a variety of scales, from the current state of the art (1 mm3 or greater), to small volumes that can be sectioned and imaged routinely. Up to now, each published EM volume for connectomics has required a multi-year effort. Instead, our goal is to use volume reconstruction as an assay, rather than an end unto itself, in the context of other experiments. In the final year, we will create data sets that test the flexibility and robustness of the approach by creating EM volumes ranging from 50µm on a side to very large volumes encompassing >1 mm3.

Project Summary/Abstract Over the past decade, there have been transformative advances in three areas of mammalian neuroscience. First, our ability to record from large populations of neurons has dramatically increased with the advent of new electrode technologies and improved multiphoton imaging. Second, the study of brain connections in their entirety, connectomics, has come into its own as a field. Most recently, there has been a revolution in the classification of neuronal types, largely by probing which genes are translated into RNA in cells (transcriptomics). What is lacking is a way to bring these three fields together, particularly in the studies of non-human primates and humans. The goal of the current RFA is to create innovative tools for use in humans and non-human primates. In particular, two suggested topics are to develop: (1) ?novel methods for tagging individual neurons such that cellular components of a functional circuit can be explored? and (2) ?innovative approaches to bridge scales of experimental approach. Studies that can explore molecular and cellular mechanisms of neural activity in broader contexts are encouraged.? To achieve these goals, we present an innovative approach to characterize neuronal cell types in macaques and humans, combining transcriptomics, inter-areal connectivity and functional studies at multiple scales, from individual neurons to entire brains. We will build the necessary tools to create integrated atlases of individual brains that combine six modalities into a common reference frame: (1) functional MRI, to measure functional properties of brain areas at 0.5-1 mm resolution, (2) widefield optical imaging, to map bulk neuronal activity at the cortical surface with ~100 µm resolution, (3) multiphoton calcium imaging, to map neuronal activity in individual neurons across multiple cortical areas, (4) diffusion tensor imaging (DTI), to map axonal tracts in the white matter with 0.5-1 mm resolution, (5) ?axonal connectomics?, to map projections of individual myelinated axons from efferent cell bodies to their postsynaptic targets, and (6) multiplexed FISH, to assay transcriptomic identities of the same cells whose physiology and projection targets have been defined. Data from first three modalities will be collected, starting with in vivo studies of macaques and correlated with subsequent analysis of brain tissue with the last three modalities. Only the last three steps will be used in the study of human brain tissue, although functional MRI could, in principle, be obtained from research institutions with appropriate programs for prospective studies.