1985 — 2011 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Extracellular Matrix and Neuromuscular Development
DESCRIPTION (from applicant's abstract) Neurons and their targets exchange information of many sorts as synapses are formed, maintained, and modified. The investigators are using the skeletal neuromuscular junction, as the best studied of all synapses, to identify and characterize some of the target-derived cues that tell axons where, when, and how to form nerve terminals. Previous studies from this laboratory demonstrated that some of these cues are contained within the basal lamina (BL) that traverses the synaptic cleft, and suggest that others are associated with the muscle fiber membrane and the perisynaptic interstitial matrix. Subsequently, immunochemical methods were used to identify several candidate cues in the BL, membrane, and matrix. Recently, the investigators obtained evidence that one synaptic BL molecule, s-laminin/laminin beta-2 (a homologue of the laminin B1/beta 1 chain) is recognized by motoneurons in vitro and serves as a muscle-derived promoter of presynaptic deafferentation in vivo. The investigators will now extend this work to obtain a more complete picture of how beta-2 works, and how it interact with other signaling molecules to guide the transformation of a growing motor axon into a nerve terminal. The specific aims are to : (1) Elucidate the functions of the laminins and collagens of a synaptic BL , including laminin beta-2, a synapse-specific laminin alpha chain, and the collagen alpha 3-5(IV) chains, which we have found to be concentrated in synaptic BL. (2) Learn how the laminins and collagens of synaptic BL assemble into cleft material and are targeted to synaptic sites. (3) Isolate cellular receptors for synaptic BL components, so that we can learn how they convey information to the pre- and postsynaptic cells. (4) Identify components of the muscle cell membrane and perisynaptic matrix that, together with synaptic BL components, influence the behavior of motor axons. A combination of histological, biochemical, and molecular biological techniques will be applied to normal and mutant mice, chick embryos, and cultured cells to achieve these aims. Through this work, we hope to gain insight into the retrograde signals that target cells use to determine patterns of innervation.
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1 |
1991 — 1994 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Lineage Migration and Phenotypic Choice in Brain
The goal of this work is to learn where and when neuroepithelial cells in the brain make the decisions that determine the type of neuron or glial cell they eventually become. In particular, we want to know the extent to which intrinsic (lineage-derived) and extrinsic (environmental) factors influence phenotypic choice. Our experimental preparation for this study is the chick optic tectum, chosen because it is experimentally accessible and well-studied, and because it shares features of radial, laminar, and topographic organization with the less accessible mammalian cerebral cortex. Our major experimental method will be a technique of retrovirus- mediated gene transfer that we developed a few years ago. In this method, we infect a progenitor cell with a retrovirus in vivo, then use a histochemical stain for the retroviral gene product to identify the descendants of the infected cell. Using this method, we will first document the migratory paths of clonally-related cells. Our preliminary studies show that clonal cohorts begin to ascend from the ventricular surface along a restricted radial path, but then diverge into three streams: most of the cells continue radially, but small subsets follow two distinct tangential paths and acquire specific phenotypes. This pattern suggests a relationship between migratory path and cell fate. Accordingly, we will combine retroviral labeling with other methods to map the three streams, identify structures that act as migratory guides, and seek adhesive molecules that might influence migratory choices. Then, in a third set of experiments, we will construct and use retroviral vectors that transfer two genes--the marker plus a second, putatively neuroactive gene or its antisense copy. By producing and analyzing small cones of "transgenic" cells in an otherwise unperturbed environment, we hope to uncover some mechanisms that regulate neural differentiation and migration. For example, we will be able to ask what roles particular adhesive molecules play in migratory choices, and whether altering a cell's migratory path alters the phenotype it adopts. Finally, as time permits, we will apply these approaches to other areas--e.g., forebrain and cerebellum--that differ from each other and from tectum in their organization. These comparisons will provide insight into the variety of genealogical and migratory strategies that the nervous system uses to generate diversity.
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0.957 |
1993 — 1995 |
Sanes, Joshua R |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Research Training in Cellular &Molecular Neurobiology |
0.957 |
1995 — 1999 |
Sanes, Joshua R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Genetics of the Mammalian Neuromuscular System |
0.957 |
1995 — 2003 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Phenotypic and Synaptic Choices in the Optic Tectum
DESCRIPTION (Adapted from applicant's abstract): The long-term goal is to find how axons in the vertebrate brain determine when and where to form synapses. The current focus is on a fundamental determinant of connectivity throughout the brain, whereby axonal populations confine their terminal arbors and synapses to specific laminae within a target area. The experimental preparation is retinotectal projection in the chick, because it is experimentally-accessible and exquisitely laminated. Moreover, all retinal axons terminate in just 3 of 16 tectal laminae, the retinorecipient laminae (RRL), and each retinal axon terminates in just one of the RLL. Initial studies identified a set of five adhesive macromolecules that are selectively expressed in the RRL and showed that three of them, N-cadherin, SC1/DM-GRASP and a glycoprotein recognized by the Vicia villosa B4 agglutinin lectin (VVA-B4), are essential for lamina-specific arborization of retinal axons. To extend this work, aim one will identify the glycoprotein that binds VVA-B4 and elucidate the mechanisms that localize N-cadherin and SC1 to the RRL. Aim 2 will assess the roles of these three molecules in synapse formation per se, using both tissue slices and cultures of dissociated retinal and tectal cells. Aim 3 will test the hypothesis that, just as N-cadherin promotes the laminar selectivity of retinal axons, other cadherins promote lamina-specific behaviors of other tectal inputs. To this end, preliminary results have identified partial sequences of 24 distinct cadherins expressed in tecta. Aim 4 will test the hypothesis that one set of molecules (including N-cadherin and SC1) is involved in targeting retinal axons to RRL in general, whereas a second set promotes arborization of axonal subsets in individual laminae. To this end, this aim will examine selective innervation of individual retinorecipient laminae by defined subsets of retinal ganglion cells. Aim 5 will begin to extend analysis from the experimentally-accessible chick retinotectal synapse to the genetically accessible mouse retinocollicular synapse by completing the development of a method for selectively expressing neuronal genes along with an axonal marker in retinal ganglion cells of transgenic mice. The hope is that this strategy will eventually elucidate mechanisms that promote laminar-specific synapse formation in mammals.
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0.957 |
1995 — 1999 |
Sanes, Joshua R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Genetic Analysis of the Synaptic Cleft
DESCRIPTION (from the abstract): The central idea that underlies this project is that synaptic partners, either nerve and nerve or nerve and muscle exchange information during development and that the information that is exchanged regulates the development of both synaptic partners. In vivo studies by this investigator and others have shown that important mediators of both retrograde and anterograde information transfer are contained within the basal lamina that fills the synaptic cleft. It is clear that at the neuromuscular junction the basal lamina directs the development of both the postsynaptic and presynaptic components. Additionally, the basal lamina serves as a binding site for other molecules that might effect both pre- or postsynaptic elements. Several synaptic basal lamina constituents have been identified and some have been shown to effect either the motoneuron and/or the muscle in vitro. The goal of this project is to find out what roles these molecules play in the development and regeneration of the neuromuscular junction in vivo. The investigator, in collaboration with Dr. John Merlie, will achieve this goal through the generation and analysis of mutant mice that lack genes encoding each of five chosen synaptic basal lamina proteins. These proteins are s-laminin (the b2 subunit of laminin), agrin, collagen a3 (IV), collagen a5 (IV) and the collagenous subunit of acetylcholinesterase. There are good reasons to believe that each of these molecules plays an important role in neuromuscular development and in some instances such as s-laminin and agrin, more mechanistic knowledge of this role is available. In cases in which the mutant mice live postnatally, the investigator will transplant muscles to immunodeficient mice to analyze their innervation or re-innervation in a wild-type host. Finally, in collaboration with Dr. Snider, the investigator will access neuronal development and axonal trajectories in the spinal cords of mutant mice seeking affects of these genes on CNS development.
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0.957 |
1996 — 1997 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transgenic Analysis of Synaptic Protein Function
The formation and maintenance of the neuromuscular synapse requires the coordinated expression and assembly of a large set of synaptic macromolecules. Over the past 3 years we have begun to study the role of a number of these synaptic proteins by creating knock-out mutations in their respective genes in mice. We have the following gene knock- out mutations in various stages of completion: S-laminin, a component of the synaptic based lamina, made by muscle, and predicted to be an adhesive molecule that may signal the nerve terminal to differential; 43k/rapsyn, a cytoplasmic extrinsic membrane protein that is hypothesized to be a structural component of the acetylcholine receptor (AChR) clustering mechanism; Agrin, a nerve derived component of the extracellular matrix that is hypothesized to be the signal that induces AChR clustering; AChR-epsilon subunit, the adult specific subunit of the AChR; DRP/utrophin, a novel homologue of dystrophin that is highly enriched at the synapse, and hypothesized to play a role in the regulation of AChR clustering. The synapses of these mutant animals will be analyzed throughout development by electrophysiological, immunocytochemical, and biochemical animals.
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0.957 |
1997 — 2002 |
Sanes, Joshua R |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Systems and Molecular Neurobiology |
0.957 |
2000 — 2002 |
Sanes, Joshua R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Core--Transgenic Facility
biomedical facility; genetically modified animals; animal colony; gene targeting; cryopreservation; developmental neurobiology; gene mutation; sperm; laboratory mouse;
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0.957 |
2000 — 2004 |
Sanes, Joshua R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Molecular Genetics of Mammalian Synaptogenesis
The most remarkable structural characteristic of the nervous system is the high degree of order with which its cells interconnect at sites of contact called synapses. Alterations in synapses underlie learning and memory; disruption of their development is likely to underlie numerous neurological and psychiatric diseases. The four participants in the program project propose to continue and enhance their collaborative, interdisciplinary studies on the intercellular interactions that regulate the formation and maturation of synapses. Their studies rely on a combination of recently developed methods for gene transfer and imaging which make it possible to gain fundamental insights into the cellular and molecular bases of these interactions. The focus for the next five years is on ways that activity-dependent and-independent regulatory programs cooperate to generate patterns of synaptic connectivity in mice. Two of the four investigators, Lichtman and Sanes, will combine their expertise in imaging and transgenic technology, respectively, to study the skeletal neuromuscular junction, whose relative simplicity and accessibility have made it the best understood of all synapses, In one set of studies, Sanes will use transgenic methods to block the electrical activity of some or all motor axons or muscle fibers during embryogenesis or postnatal life. Lichtman will image synapses in these lines over minutes to weeks, to learn how activity affects synaptic maturation and competition. Our studies will use genetically engineered mice in which only a few motor axons that supply a muscle are indelibly marked with a fluorescent tag; these will be imaged to elucidate position- and activity-dependent aspects of motor unit maturation. The other two investigators, Craig and Wong, will use many of the same lines and markers but extend the investigation to synapses in the brain. Wong will ask how presynaptic cells in the retina, and the activity they generate, shape the dendritic arbors of their postsynaptic cells in the retina, and the activity they generate., shape the dendritic arbors of their postsynaptic partners. Craig will use dissociated cell cultures to ask detailed developmental questions about how central (hippocampal) synapses. Her work will illuminate mechanisms that can not be studied in muscle, such as how individual neurons segregate synapses of different types to distinct domains.
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1 |
2000 — 2002 |
Sanes, Joshua R |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Genetic Manipulation of Neuromuscular Synaptogenesis
Pre- and postsynaptic cells generate signals that organize each other's differentiation. Considerable progress has been made in identifying some of these signals at the neuromuscular junction, and in elucidating their signal transduction mechanisms. Less is known about three other critical factors: electrical activity, which modulates numerous aspects of synaptic maturation; Schwann (glial) cells, which influence both nerve and muscle; and differences among muscles that bias synapse formation in favor of appropriate partners. This application proposes to apply transgenic technology to these issues. The first aim is to generate and analyze mice in which activity is blocked in three different ways: depletion of neurotransmitter, blockade of vesicle fusion, or deletion of the postsynaptic receptor. Comparison of these strains will allow us to assess which aspects of activity are important for each stage of synaptic development. In addition, each transgene will be conditional, allowing us to block activity at various stages of development (so we can bypass early lethality to study late steps in development) or in subsets of axons (so we can assay competitive interactions). The second aim applies the same methods to mice in which all or some Schwann cells are deleted. IN the third aim, we will use new transgenic lines in which a few of the motor axons that innervate a muscle are indelibly marked with a fluorescent label. By visualizing entire motor units in these mice, we will learn how they are arranged, how they develop, and whether their component synapses resemble each other. Finally, we will focus on one aspect of motor unit arrangement: the orderly mapping of a motor pool into a muscle's rostrocaudal axis. We recently found that mapping is degraded in transgenic mice over-expressing or lacking ephrins, protein previous implicated in retinotectal mapping. By analyzing the arrangement and development of motor units in ephrin mutant mice, we will learn how factors that promote selective synapse formation interact with the "nuts and bolts" that are shared by and play major roles in formation of all synapses of a class.
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0.957 |
2000 — 2001 |
Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Microarray Analysis of Single Retinal Ganglion Cells
DESCRIPTION (Applicant's abstract): Retinal ganglion cells (RGCs) process visual information in the eye and transmit it to the brain. All RGCs share key features, but distinct subtypes have been identified that differ in morphology, biophysical properties, visual responsiveness, synaptic inputs and synaptic targets. These RGC subtypes have been studied intensively with anatomical and physiological methods, both because they are critical determinants of visual perception and because they provide an accessible system for addressing general issues of neuronal diversification and categorization. Unfortunately, however, few if any molecular markers are known that distinguish functionally relevant mammalian RGC subclasses, so it has been impossible to answer basic questions about how many subtypes really exist, when they arise, how they acquire their definitive identities, or what molecules underlie their subtype-specific properties. Here, we propose to identify molecular markers for two pairs of RGC subsets that have already been defined structurally and physiologically, and are of clear functional importance: alpha- vs. beta-like (which differ in soma size, dendritic spread and briskness of response) and ON vs. OFF (which differ in inputs, outputs, dendritic stratification, and responsiveness to onset vs. termination of light). To this end, we have already generated and characterized transgenic mice in which the Green Fluorescent Protein (GFP) labels RGCs of multiple types in their entirely, allowing us to identify and isolate them. We will prepare and characterize cDNA from individual transgene-positive RGCs, using methods we have recently used successfully to characterize chick RGCs. The probes will then be hybridized to commercially available microarrays (Affymetrix GeneChips), allowing us to assess expression of about 27,000 genes in cells of each of four classes (alpha-ON, alpha-OFF, beta-like-ON, beta-like-OFF). The output will be analyzed to identify genes expressed by defined RGC subsets or that define novel subsets. Finally, candidate markers will be validated by in situ hybridization. We believe these experiments will (a) provide markers for developmental studies, (b) uncover genes that themselves play roles in subtype diversification or function, and (c) contribute to development of generally applicable methods for gene expression profiling from single cells.
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0.957 |
2004 — 2019 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Synaptic Choices in the Retinotectal System
DESCRIPTION (provided by applicant): Our long-term goal is to find out how axons and dendrites in the vertebrate brain determine when and where to form synapses. We focus on laminar specificity, a fundamental determinant of connectivity throughout the brain, whereby neuronal processes confine their arbors and synapses to specific laminae within a target area. Our object of study is the retinal ganglion cell (RGC), because it is relatively accessible, has a well-defined function, and displays exquisite laminar specificity: axons of distinct RGC subsets synapse in specific retinorecipient sublaminae of the superior colliculus, and their dendrites arborize in specific sublaminae of the inner plexiform layer (IPL), where they receive inputs from lamina-specified subsets of retinal interneurons. In work supported by this grant, we have obtained evidence that four related immunoglobulin superfamily (IgSF) adhesion molecules -Sidekick- 1, Sidekick-2, Dscam and DscamL- are critical determinants of an IgSF code that underlies some aspects of sublaminar specificity in the IPL. We will now use genetic methods to map the circuits that express these IgSF genes in mice, assess consequences of deleting them singly and in pairs, and determine whether they act cell-autonomously and/or homophilically. We will also test the possibility that close relatives of Sidekicks and Dscams, the Contactins, are additional components of the IgSF code. Then, we will use electrophysiological methods to relate circuit assembly to circuit function. We will map the receptive fields of IgSF-expressing RGC subsets, identify the interneurons that innervate then, and assess the effects of IgSF gene deletion on their properties. As an exacting test of our hypothesis, we will attempt to rewire a retinal circuit by replacing one IgSF gene with another, assessing the structural and functional effects of this swap. Finally, we will extend our analysis to the laminar targeting of RGC axons in the superior colliculus. We recently generated a map of projections that RGC subsets form in collicular retinorecipient sublaminae, and will now classify the target cells on which axons of these subsets form synapses. With this foundation, we will test our hypothesis, based on results from chick optic tectum, that members of the cadherin superfamily (particularly Type II cadherins) are involved in the targeting of RGCs to sublaminae in the superior colliculus. Together these studies will contribute to elucidation of mechanisms that promote lamina-specific synapse formation and, by extension, synaptic specificity generally.
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1 |
2008 — 2013 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Imaging Core
The core we envision will be centralized but otherwise perform as extensions of individual labs: places where scientists come to do their experiments with the only one major difference being that the equipment will be shared and technical expertise is immediately available. This model is standard in such fields as high energy physics, astronomy, oceanography where the accelerators, telescopes and ships are always shared. As biomedical neuroscience moves into similarly sophisticated and expensive technologies the same transition must take place. MRI and PET are two fields where from the beginning scientists shared equipment. It is now time for microscopy to make the same transition. Accordingly, the Advanced Imaging Core will provide users with access to state-of-the art tools, as well as the training needed to use them effectively. /'. Tools for optical imaging: Optical imaging devices will be housed in a multiuser optical imaging laboratory under the aegis of the CBS. The facility will be housed in over 1500 square feet in the new Northwest Building on the Cambridge campus of Harvard University, which will open in the spring of 2008. The optical imaging facility will be in vertically adjacent space on the two floors occupied by CBS faculty. A stairway connecting the two areas will assure good both flow between the two large rooms while the two floor facility assure researchers on both floors have easy access. Geographically the facility will be located at the nexus of the departments of life, physical, and engineering sciences on the Cambridge campus. The facility will have online sign-up for all major equipment. Neuroscientists throughout the university will be welcome to use the equipment. Equipment for this facility will be provided by Harvard University, through the Center for Brain Science. Equipment will include laser scanning confocals (Zeiss 510, Zeiss Pascal, Olympus FV1000), a Zeiss Axioplan for conventional epifluorescence microscopy, a total internal reflection fluorescence (TIRF) microscope with a sophisticated incubation housing for long lasting cell culture experiments, and a structured illumination scope (Apotome, Zeiss). For automated microscopy we will include motorized the stages on the three main laser scanning scopes and data acquisition software that allows automatic, large-scale, continuous laser scan PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 60 Continuation Format Page Principal Investigator/Program Director (Last, first, middle): Sanes, Joshua R. montages. We expect to implement more sophisticated microscopes that surpass the diffraction limit of light in the near future and make them available. //. Tools for electron microscopy Through an initiative dubbed the Connectome Project, which has been funded by a foundation gift and Harvard, we have been able to purchase both transmission and scanning electron microscopes, and, as described above, design and construct new ancillary devices. This equipment will be the foundation of the high-throughput microscopy core. It will be housed in more than 1,200 square feet of laboratory space in the lowest basement level of the Northwest Building in Cambridge, beginning in the spring of 2008. This basement level will be shared with the Neuro-engineering facility, the magnetic imaging facility, and possible expansion space for advanced optical imaging. ///'. Training: Hands on training in optical techniques will be available from the technicians we hire if this grant is funded. Another aspect of training is also critical: modern microscope technique depends heavily on an understanding of the concepts of optical microscopy, photochemistry, and digital image processing. Most neurobiologists have little formal education in any of these areas. We believe that these tools cannot be used to full advantage if the user is not familiar with the underlying principles. For example many users of laser scanning microscopes have no appreciation that fluorescence emission saturates due to the relatively long lifetime of the excited state. Thus they excite with far more laser intensity than the in-focus sample region can accommodate causing a disproportionate amount of out of focus light in the final image. To help users we have begun to offer a course in optical imaging that provides a clear (no calculus required) conceptual framework for understanding the behavior of light, microscope optics, fluorescence, confocal, multiphoton, image restoration, and nanoscopy. This one semester course (3 hrs per week) is open to all students, fellows, and faculty. Beginning last fall it has been presented by way of a live video feed to the medical campus to minimize the difficulty of commutes between the campuses. Training in conventional and high-throughput electron microscopy will also be provided. Our sense is that 3D electron microscopy in particular is such a powerful analytic tool that once a facility is up and running many scientists will consider using this approach for their studies. At present we know from discussions with neuroscientists here that there is widespread interest in obtaining ultrastructure within a three dimensional context but most neuroscientists imagine that this will be too burdensome to carry out. Thus, one of our goals will be to educate the community about the potential of using these approaches. As already set up for the optical imaging component, we think that presentations advertising the capabilities of this equipment will be useful. Lichtman¿who already teaches a highly regarded graduate course on microscopy¿will use his syllabus as the basis of a mini-course on the three main tools we are developing that we will present to the user community each fall.
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1 |
2008 — 2013 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Administrative Core
3. Administrative Core The administration of the Center Core will be integrated with that of CBS. This is important for three reasons. First, it makes sense intellectually, since the cores are the key to implementing the CBS vision. Second, CBS has already established an administrative structure, so the Cores can be up and running with minimum delay. Third, it is financially efficient to avoid the creation of a separate bureaucracy. At present, three people share administrative duties in CBS: i) The Director of CBS and the P.I. of this Core Grant is Joshua R. Sanes, Professor of Molecular and Cellular Biology. Sanes was on the faculty at Washington University in St. Louis for over 20 years before moving to Harvard in 2004 to direct CBS. Sanes has studied molecules and mechanisms that regulate the differentiation of synapses and the specificity with which they form. In addition, he has pioneered transgenic methods for marking neurons so that their lineage can be followed and their synaptic connections monitored. He is a member of the National Academy of Sciences and the American Academy of Arts and Sciences and has served on the Board of Scientific Counselors and the National Advisory Council for National Institute of Neurological Disorders and Stroke of the National Institutes of Health. ii) The Executive Director of CBS is Kenneth Blum. Blum earned a PhD in Physics, and then did postdoctoral work in Neuroscience at Brandeis and MIT before becoming a senior editor at Neuron in 1999 and then its deputy editor in 2002. He moved to Harvard in 2004 to help lead CBS. Blum has conducted research in condensed matter physics, computational neuroscience, and systems neuroscience. In the latter category are studies on the role of hippocampal place cells in learning, memory, and navigation. His background is especially useful in building the ties between physical and life scientists necessary for the success of Cores B andC. iii) The CBS Coordinator is Ray Kohno. Kohno earned a BA in Cognitive Science from UC Berkeley. He handles the bulk of scheduling for faculty recruiting, seminars and other meetings. Together with Blum, he manages CBS finances and HR, and will oversee the financial aspects of the Cores. These three will devote considerable time to administration of the cores, but we do not request financial support for them here. With funds provided by the Administrative Core of this Grant, we will recruit a half-time staff assistant who will be entirely devoted to the day-to-day management of the grant. Most likely, this would be a full-time employee whose other half time will be used to assist one or more faculty members, who neither need nor could afford a full-time office manager. This Core staff assistant will report directly to Blum. His or her duties will include: ¿ Maintaining the Core web site (which will include information, protocols, and sign-up sheets) ¿ Scheduling meetings of the Core Executive Committee ¿ Maintaining record of core usage and expenditures ¿ Preparing scientific reports ¿ Ordering supplies at the request of Core personnel ¿ Providing limited administrative support to Core managers
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1 |
2008 — 2012 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Maturation and Maintenance of the Postsynaptic Apparatus
DESCRIPTION (provided by applicant): Pre- and postsynaptic elements exchange developmentally important signals as synapses form. The simplicity, size and accessibility of the skeletal neuromuscular junction (NMJ) make it an excellent preparation for studying these signals and the intracellular signal transduction mechanisms they use. We and others previously defined a rudimentary pathway for initial steps of postsynaptic differentiation at the NMJ in which the proteoglycan z-agrin is a nerve-derived signal, MuSK is its main receptor, and rapsyn is a critical intracellular effector leading to aggregation of acetylcholine receptors. We will now build on this foundation to analyze the dramatic alterations that transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches; alterations in molecular architecture; subdivision of the membrane into distinct domains; and changes in biophysical properties and metabolic stability. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze ways in which two main nerve-derived signals, agrin and acetylcholine, shape the postsynaptic membrane. Using newly generated conditional mutant mice, we will determine whether agrin is required not only for the formation of NMJs but also for their maturation or maintenance. Using targeted mutagenesis in vivo and time-lapse imaging of cultured myotubes, we will analyze roles of lpha-dystrobrevin and LL5_, two synapse-associated proteins that we have already implicated in postsynaptic maturation. Together, these studies on key transsynaptic signals (agrin and neurotransmitter) and intracellular mediators (alpha-dystrobrevin and LL5_) will provide a framework for understanding how postsynaptic maturation occurs and how it is regulated. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable. PUBLIC HEALTH RELEVANCE: Nerve cells communicate with each other at junctions called synapses. Synapses are the sites of information processing and plasticity in the normal nervous system, and the sites of defects believed to underlie many neurological and behavioral disorders. Many of the early steps in synapse formation have been described over the past several years, but less is known about how the initially-formed synapse is remodeled during postnatal maturation. We propose to study these processes at the skeletal neuromuscular junction (NMJ). The simplicity, size and accessibility of this synapse make it an excellent preparation for studying signals that regulate synaptic maturation and the intracellular signal transduction mechanisms they use. Moreover, dramatic alterations transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches; alterations in molecular architecture; subdivision of the membrane into distinct domains; and changes in biophysical properties and metabolic stability. Specifically, we will use the NMJ to analyze roles of two nerve-derived signals, agrin and acetylcholine, and two intramuscular signaling molecules, alpha-dystrobrevin and LL5_, all of which have already been implicated in postsynaptic development. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze how these molecules shape the postsynaptic membrane. Hypotheses derived from these studies will be tested using genetically engineered mutant mice. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable.
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1 |
2008 — 2013 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Genome Modification Core
c. Description The Biomedical Research Infrastructure (BRI) is an underground two floor, 120,000 square foot, 27,000 cage animal facility. This state-of-the-art automated facility is in the courtyard of the Biological Laboratories, which places it adjacent to the Northwest Building. The BRI houses a Genome Modification Facility (GMF) in an 800 square feet procedural room in the BRI with additional animal holding space for 1,000 cages in adjacent rooms. This room is equipped with 4 vibration free microinjection stations, micro-needle fabrication equipment, 4 banks of tissue culture incubators for embryo/ES cell culture, 3 tissue culture hoods, 3 laminar flow hoods for pathogen free minor surgeries, 12 stereomicroscopes of which 2 will be equipped with GFP optics and teaching video, automatic liquid nitrogen storage facilities, and computers. The GMF as a whole is governed by faculty committee of four individuals: Andrew McMahon (Molecular and Cellular Biology, Committee Chair), Joshua Sanes (CBS), and Kevin Eggan (HSCI). The committee oversees effective running of the GMF. The GMF staff consists of the following. ¿ The director, Manfred Baetscher, PhD, has broad experimental experience of genetic modification of mice and many years of managerial experience. He participates directly in production of genetically modified strains, remains current on emerging genetic approaches, and advises investigators on appropriate genetic strategies for their experiments. ¿ Transgenic research assistants: Two assistants, both with proven experience in either or both pronuclear injection of DMA and blastocyst injection and small animal surgery. ¿ Tissue culture technicians: A full time technician, soon to be hired will perform all aspects of ES cell culture, ES cell electroporation and clonal selection and DMA preparation forgenotyping. ¿ Animal husbandry: Two animal care technicians conduct all support functions for embryo generation and biopsy preparation. ¿ Administrative assistant: This individual is responsible for all ordering, billing and coordination between investigators and GMF. Finally, it is worth noting that the BRI will also contain behavioral testing suites equipped for phenotypic analysis of genetically modified mice, including models of neurological and psychiatric diseases. These will be supported from other sources, so are not described here, but it is our intention to integrate them with the GMF. The GMF provides the following services: ¿ Production of transgenic mice by pronuclear injection of DMA constructs in plasmids, BACs, or YACs ¿ ES cell targeting and injection to generate knock-out and knock-in mice ¿ Cryopreservation and storage of embryos and sperm for valuable strains not in current use ¿ Mouse strain resuscitation by in vitro fertilization and intracytoplasmic sperm injection (ICSI) ¿ Re-derivation of mouse strains by embryo transfer to render them pathogen-free ¿ The development of lentiviral vector based transgenes ¿ Consultation on genetic model creation ¿ Plans to offer gene targeting in embryonic stem cells and validation of DNA vectors and feeder lines We also hope the GMF will provide new functionality that we will add if this grant is funded, circumventing a bottleneck that many users of genetically modified mice encounter. There are a dozen or so strains that are highly useful for a broad variety of neuroscience applications. Most are publicly available but the waiting time to obtain them collaborators or stock centers (e.g., at Jackson Laboratories) is often 6 months or more, and the expense is considerable. Yet, few laboratories can afford to maintain stocks permanently of lines that may need only once every year or two. Lines in this category include: (a) Mice in which specific neuronal populations are marked with GFP or one of its spectral variants (e.g., motoneurons in YFP-16, layer 5 pyramids in YFP-H, very small numbers of forebrain neurons in GFP-M, or inhibitory interneurons in GAD65- GFP). (b) Mice in which expression of a reporter is conditional on expression of ere recombinase (e.g., Z/EG, Z/AP and thy1-stop-YFP). (c) Mice in which ere or flp recombinase is expressed in the neural tube generally (e.g., Nestin-Cre) or in specific populations of neurons (e.g., CaM kinase-Cre) or only following activation by tamoxifen (e.g., CAGS-CreER). So, we propose to maintain small colonies (approximately 4 cages of 4 males each) of each of about a dozen lines. The lines will be chosen by the Steering Committee. In most cases, the mice are already available in at least one user laboratory. Then, when any Harvard neuroscientist requires one of the lines, we will be able to provide two males within a week, and then replenish our own supply by breeding. We believe this repository will greatly decrease the barrier to use of valuable strains, thereby making possible risky or pilot experiments that would otherwise be prohibitively expensive or unduly slow.
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1 |
2008 — 2013 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Neuro Engineering Core
c. Description The Neuro-engineering core will consist of an integrated laboratory for designing, building, and testing devices. It will include electronics and machining equipment, an optical bench, and computers for design, testing, and software and hardware development. Harvard has committed ample space to support this core facility. It will be housed in 600 sq. ft. of basement laboratory space in the new Northwest Building. The technical staff will have office space with the faculty and students in the Center for Brain Science above ground, where they can answer questions, trouble-shoot, and brainstorm about technical solutions to problems faced by Harvard neuroscientists. 1) Machining: this will consist of the essential machines for cutting and shaping apparatus from metal and plastics. These are a lathe, a numerically controlled vertical milling machine, a drill press, and a band saw. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 65 Continuation Format Page Principal Investigator/Program Director (Last, first, middle): Sanes. Joshua R. 2) Electronics: this will consist of two long benches for electronic design, circuit assembly, trouble-shooting, and testing. Essential equipment includes multichannel digital oscilloscopes and multimeters, and a range of commonly used electronic supplies, including power supplies and common integrated circuits. 3) Optics: this will consist of an optical table with optical devices. Essential equipment will include lasers, highintensity LEDs, optical elements, optical fibers, cameras and photomultiplier tubes, and a microscope. 4) Computers: this will consist of desktop computers for developing data acquisition hardware and software.
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1 |
2008 — 2017 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Core Facilities For Analysis of Neural Circuit Structure and Function
DESCRIPTION (provided by applicant): The aim of this grant proposal is to support a set of core facilities that spreads understanding and use of new technologies to the wide range of neuroscientists in our community. The scientific cores are: (1) Light Microscopy, which enables neuroscientists to visualize neural circuit structure and function with great specificity and resolution using the newest super-resolution fluorescence microscopy, multiphoton, and confocal instruments; (2) Electron Microscopy, with TEM and a new high-throughput, serial scanning EM; and (3) Neuroengineering, which provides design and fabrication expertise to allow individual users to adapt the latest electronic, imaging, and computer technologies for their experiments. Two additional facilities, devoted to Neuroimaging (including human brain and small animal scanners) and Genome Modification (providing transgenic and targeted mutant mice for structural or functional analysis), are supported from other sources, but will be integrated with the three Cores supported here, providing neuroscientists with access to the full range of imaging modalities currently driving the field forward. An Administrative Core tracks the usage and finances of the scientific cores, oversees coordination among all cores, and facilitates interactions between the core users and core technical staff. Individuals will find equipment and services in these Cores that are difficult or impossible to support in individual labs, which lack sufficient technical expertise, money, and space. The users, members of Harvard's Cambridge-based neuroscience community, will be drawn closer together, through shared space, equipment, and techniques. We also understand that many of these newer technologies are difficult to understand and implement, and so they are not utilized as broadly as might be wished. Thus we propose not only to establish these Cores but also to adopt multiple strategies aimed at reducing barriers to their widespread utilization. These include formal and informal education, making most core services available without user fees, and providing ample technical support. Our ambitious model has begun to shift the paradigm for neuroscience research from complete reliance on individual laboratory-centered facilities to the more cost-effective and productive use of extraordinary shared facilities.
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1 |
2008 — 2012 |
Lichtman, Jeff W [⬀] Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular and Molecular Analysis of Defects At Aging Neuromuscular Synapses
DESCRIPTION (provided by applicant): The nervous system undergoes disturbing changes with age. To elucidate key mechanisms underlying age-related decline in neural function, we will examine the neuromuscular system, which is very accessible, relatively simple, and the site of clinically significant age-related functional decline. Our initial analysis has led to four sets of results: 1) Neuromuscular junctions (NMJs) undergo many structural and molecular alterations as they age. 2) Preterminal portions of motor axons exhibit regions of abnormal thinning, distension and sprouting. 3) Atrophy and synaptic changes in aged muscles are correlated on a fiber-by-fiber basis. 4) Although NMJs in most muscles are ravaged by age, those in a few are spared. Here, we propose studies designed to explore the relationships among these changes, identify molecular defects that underlie them, and test one way to reverse them. First, we will follow up preliminary observation of a dramatic and specific decline in levels of three known synaptic organizing molecules at aging NMJs -laminins 4 and 2 and agrin. We will correlate changes in the levels and distribution of these proteins with structural alterations, and ask whether targeted null or conditional mutants with decreased levels of these proteins show premature synaptic aging. Second, we will seek transport defects that underlie axonal dystrophy by correlative light and electron microscopy, along with use of new transgenic mice in which mitochondria and synaptic vesicles are labeled. Third, we will determine the relationship between the synaptic abnormalities and sarcopenia, the clinically significant age-related decline in muscle mass and strength. Using transgenic mice in which single motor axons and muscle fibers of specific types are selectively labeled we will assess myogenic and neurogenic determinants of sarcopenia. Fourth, we will follow up our observation that extraocular muscles are spared from age-related neuromuscular decline. This result is intriguing because extraoculars are also spared in the invariably fatal disease, amyotrophic lateral sclerosis (ALS), suggesting parallel mechanisms underlying age-related and neurodegenerative defects. Finally, we will use transgenic rescue techniques to ask whether reintroduction of laminins or agrin attenuates age- related synaptic disorganization. Together, these studies will provide insights into age- related neural defects that may not only provide ways to ameliorate sarcopenia but also be generally applicable to the nervous system PUBLIC HEALTH RELEVANCE: The nervous system undergoes disturbing changes with age. The investigators propose to use the neuromuscular system to elucidate key mechanisms underlying age-related decline in neural function. This system is very accessible, relatively simple, and the site of clinically significant age-related functional decline. First, they will follow up preliminary observation of a dramatic and specific decline in levels of known developmentally important molecules (laminins and agrin) at aging neuromuscular junction (NMJs), the synapses made by motoneurons on muscle fibers. They will correlate changes in the levels and distribution of these proteins with structural alterations, and ask whether mutant mice with decreased levels of these proteins show premature synaptic aging. Second, they will seek defects in the transport of materials along nerve fibers to the NMJ. Third, they will determine the relationship between the synaptic abnormalities and sarcopenia, the clinically significant age-related decline in muscle mass and strength. Fourth, they will explore intriguing similarities in symptoms and muscle-specific susceptibility between neuromuscular changes in aged mice and those in the invariably fatal disease, amyotrophic lateral sclerosis (ALS). Finally, they will ask whether reintroduction of laminins or agrin attenuates age-related synaptic disorganization. Together, these studies will provide insights into age-related neural defects that may not only provide ways to ameliorate sarcopenia but also be generally applicable to the nervous system.
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1 |
2009 — 2012 |
Sanes, Joshua R |
U24Activity Code Description: To support research projects contributing to improvement of the capability of resources to serve biomedical research. |
Next Generation Brainbow Transgenes For Neural Circuit Analysis |
1 |
2009 — 2010 |
Meister, Markus [⬀] Sanes, Joshua R |
RC1Activity Code Description: NIH Challenge Grants in Health and Science Research |
Transgenic Strategy to Map Structure and Function of Neural Circuits in Retina
DESCRIPTION (provided by applicant): This application addresses broad Challenge Area 06, Enabling technologies, specific Challenge Topic 06-NS- 106: "Validating new methods to study brain connectivity." The long-term goal of this research is a fundamental understanding of how the eye communicates with the brain. More immediately, the research serves to validate and improve a set of genetic tools for the study of neural circuits. The retina is a complex network of neurons in the back of the eye that converts a visual image into streams of action potentials that travel through the fibers of the optic nerve to the brain. The circuits of the retina begin with the photoreceptor cells that sense the light, pass through bipolar cells and other interneurons, and end with the ganglion cells that form the optic nerve. In all, the retina uses over 50 different types of neurons;the ganglion cells alone comprise about 20 different types. Each of these ganglion cell types extracts and reports a different aspect of the visual scene. A long-term goal of visual neuroscience is to understand what kind of visual processing occurs in each of these streams, and how those processes are implemented by the elaborate neural circuitry of the retina. New genetic methods are beginning to accelerate our understanding of neural systems. In particular, there has been great interest in finding genetic markers that distinguish the brain's many different neuron types. Experience has shown that availability of such a marker, in combination with new molecular and physiological approaches, can dramatically accelerate scientific progress on the structure and function of the corresponding neural circuits. Here we propose to apply these methods to assemble a complete catalog of the ganglion cell types in the mouse retina and to analyze their visual functions. The specific research goals are: (1) to find genes that are expressed specifically in one type of retinal ganglion cell;(2) to construct transgenic mice based on these genes in which all neurons of a given type are marked;(3) to exploit these lines for targeted studies of the structure and function of retinal pathways. For each type of retinal ganglion cell, we will determine the distribution of the neurons across the retina, how their dendritic fields cover visual space, and where in the brain their axons project. At the single-cell level we will examine the shape and location of the ganglion cell's dendritic tree within the retina to deduce its likely synaptic partners among retinal interneurons. To analyze visual function, we will determine what image features each ganglion cell type extracts from the visual scene. In addition, we will assess its involvement in ecologically important computations, such as the processing of image movement, and adaptation to the visual environment. This research will lead to a qualitatively new understanding of retinal function. It will inform our understanding of higher visual areas that draw all their input from the retina. Furthermore, the work will validate and gather experience with a set of genetic tools that can generalize to all brain circuits. PUBLIC HEALTH RELEVANCE: This project concerns basic research into the function of brain circuits. It will develop and test new genetic methods for visualizing types of nerve cells, and exploit these markers to understand how the circuits process information. In the long run, this will enhance our understanding of how the brain works, and how it fails in certain disorders.
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1 |
2011 — 2015 |
Lichtman, Jeff W [⬀] Meister, Markus (co-PI) [⬀] Sanes, Joshua R Seung, Hyunjune Sebastian |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Resolution Connectomics of Mammalian Neural Circuits
DESCRIPTION (provided by applicant): The overall goal of this work is to develop and validate a new suite of technologies that can rapidly and routinely generate circuit diagrams of nervous system tissue, sometimes called connectomes. The small size of neuronal processes and the synapses that connect them require that reconstruction be done at nanometer resolution;the distributed nature of neuronal connectivity requires reconstruction of large volumes, extending over a millimeter or more. Our method meets these two seemingly incompatible challenges by combining novel sectioning, electron microscopic imaging and reconstruction technologies. Together, these advances will allow us to acquire data and map circuits at least 1000-fold faster than has previously been possible. As a first test, we will reconstruct the retinal circuit of a mouse in its entirety. Enough is known about retinal structure and function to make this an appropriate tissue to validate the method. At the same time, this background will allow us to pose and solve important problems about neural circuits that will be directly applicable to the brain. We will then use the method to compare neural circuits in young adult and aged retina, providing insight into the structural basis of age-related neural decline. Finally, we will test the application of this connectomic method to human tissue. The new methods introduced here will transform neuroscience in several ways. First, it will allow elucidation of the structural underpinnings of brain function. It will also provide insight into how neural circuits are refined in early life and altered in old age. Second, applied to the ever increasing number of animal models of human behavioral disorders, it will help researchers delve into pathologies of cognition, behavior, and affect, some of which likely arise from miswiring of neural circuits. Finally, the method can be applied to any biological tissue where three-dimensional reconstruction of multiple large-volume specimens would be informative. PUBLIC HEALTH RELEVANCE: The connectome of each individual is probably unique, containing the traces of experiences and learned behaviors. Until we can acquire and decipher connectomic patterns, we will not know how acquired information is organized and encoded in the brain, or whether, as many suspect, miswiring underlies a variety of developmental, aging, and behavioral disorders. As the human genome project demonstrated, biomedical science can be transformed by scaling up and speeding up technologies to the point where they can provide comprehensive information about essential biology;our brain circuit reconstruction will be fast enough to compare entire circuits from multiple individuals at nanometer resolution.
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1 |
2011 — 2012 |
Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Roles of Sad Kinases in Formation and Maturation of Multiple Synaptic Types
DESCRIPTION (provided by applicant): Synapse formation and maturation require signaling between the synaptic partners and signal transduction within each of them. To learn how assembly of the presynaptic neurotransmitter release apparatus is assembled, we focused on two genes, SAD-A and SAD-B. They are the mammalian orthologues of SAD-1, a gene required for presynaptic differentiation in C. elegans. Because SADs are kinases, we hope they will provide a valuable starting point for elucidating regulatory mechanisms that govern assembly of nerve terminals. Unfortunately, initial genetic tests of this idea gave complex results because the two genes play redundant roles and are involved in multiple steps in neuronal development and because SAD-A/B double mutants die at birth, before most synapses have formed. We therefore developed two genetic strategies to circumvent these limitations of pleitropy and lethality. First, we generated a conditional allele to ablate expression in selected neuronal types. Second, we generated alleles sensitive to a specific inhibitor, which allows precise temporal control of SAD-A/B activity and facilitates substrate identification. Using these new reagents, we have obtained preliminary results indicating that SADs are indeed required for complete presynaptic differentiation of several and perhaps most synaptic types. Here we propose to confirm and extend these results, and to initiate tests of our hypothesis that SAD kinases are critical components of pathways that lead from target-derived synaptic organizing molecules to assembly of nerve terminals. First, we will use the conditional allele to bypass neonatal lethality and characterize presynaptic defects in four peripheral and central excitatory synaptic types. We will also ask whether SADs are also required for development of inhibitory synapses, and whether SADs regulate post- as well as presynaptic development. Second, we will assay synaptic development and function with SAD-A and -B mutant alleles that render the kinases selectively inhibitable by an ATP analog to which unmodified kinases are insensivitive. These alleles provide us with precise and reversible temporal control over SAD kinase activity, both in vivo and in cultures generated from the mutant mice. We can therefore ask when during development SADs are required, whether their activity is required for synaptic maintenance in adults, and whether acute inhibition of the kinases affects the function of synapses that have developed normally. Finally, we will initiate studies aimed at learning how synaptogenic signals activate SADs and how SADs, in turn, coordinate presynaptic differentiation.
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1 |
2012 — 2015 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cell Surface Molecules That Require Arrangement of Retinal Neurons and Arbors
DESCRIPTION (provided by applicant): Orderly and specific connections among neurons of multiple subtypes underlie the function of neural circuits. Our own work has used retina as a model to elucidate molecules and mechanisms that underlie specificity, focusing on the matching of pre- and postsynaptic partners in particular synaptic laminae. There is another sort of order, however, that has received less attention: the arrangement of cells and their neurites in the orthogonal (x-y) plane. Processes that contribute to this arrangement include mosaic spacing of neuronal somata, tiling of dendrites, and self-avoidance of processes within a single arbor. These processes are believed to ensure uniform coverage of the visual field, precise connectivity, and appropriate receptive field size. Their molecular bases remain unknown in vertebrates. Recently, we began analyzing two sets of cell surface proteins that we suspected to be involved in laminar specificity: MEGF10 and 11, and a cluster of 22 related gamma protocadherins (Pcdhgs). Unexpectedly, preliminary results suggest that both are involved in regulating the arrangement of specific neurons and their dendrites in the x-y plane. Moreover, both affect the same cell type, starburst amacrine cells (SACs), but in different ways: MEGF10/11 regulate the mosaic arrangement of SAC somata whereas Pcdhgs are required for self-avoidance of their dendrites. We will now use these results as starting points to obtain insights into the mechanisms that underlie these common but little-studied aspects of circuit assembly. First, we will use gain- and loss-of function methods in vivo to characterize the role of MEGF10/11 in mosaic formation. We will ask whether MEGF10/11 is effective only during development, whether its effects endure, whether it can disrupt mosaics after they form, and whether the two homologues have distinct effects. Second, we will ask whether defects in self-avoidance observed in conditional Pcdhg mutant mice are cell-autonomous and whether they reflect problems in dendrite formation or refinement. We will then use genetic methods to reduce the repertoire of Pcdhg isoforms that retinal cells express. We can thereby test the role of isoform diversity in the process and learn whether different isoforms play different roles. Third, we will ask whether retinal subtypes other than SACs use MEGF10/11 or Pcdhgs to pattern their somata or arbors. Finally, we will combine studies in vitro and in vivo to initiate analyses of the signaling mechanisms by which MEGF10/11 and Pcdhgs function. We will ask whether MEGF10/11 act as receptors, as ligands, or as both ligand and receptor (that is, homophilically). For Pcdhgs, we will ask how the initially adhesive interaction that Pcdhgs appear to promote is translated into the repellent one required for self-avoidance. Together, these results will further our understanding of two poorly understood gene families and of a poorly understood set of processes important for patterning neural circuits.
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1 |
2014 — 2017 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Light Microscope Core Core Facilities For Analysis of Neural Circuit Structure A
The Light Microscopy Core enables neuroscientists to visualize neural circuit structure and function with great specificity and resolution using both the newest super-resolution fluorescence microscopy technology and multiphoton and confocal instruments. By the cost-effective and productive. use of extraordinary shared microscopes, the Light Microscopy Core contributes to the generation of valuable neuroscience research and promotes a new model for interdisciplinary neuroscience, ultimately to address fundamental issues of human health.
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1 |
2014 — 2017 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Electron Microscope Core Core Facilities For Analysis of Neural Circuit Structu
The Electron Microscopy Core enables neuroscientists to visualize neural circuit structure with nanometer resolution using traditional transmission electron microscopy and the high-throughput electron microscopy developed at Harvard for Connectomics. Like the Light Microscopy Core, the Electron Microscopy Core enables valuable neuroscience research and promotes a new model for interdisciplinary neuroscience, ultimately to address fundamental issues of human health.
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1 |
2014 — 2017 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Neuroengineering Core Core Facilities For Analysis of Neural Circuit Structure A
This innovative core implements a unique concept. With support from this P30 grant, we have been able to offer the expertise of a brilliant engineer-neuroscientist (Edward Soucy, PhD; see Personnel below) to our entire community. Working with another engineer-neuroscientist (Joel Greenwood, PhD, supported from University funds), he routinely solves what appear to be insuperable engineering problems-including designing and fabricating instrumentation, electronics, software for data acquisition and analysis, and imaging. Soucy designs and builds novel systems for specialized projects. One benefit is tailor-made solutions that are cost-effective, because the expense of commercial devices often comes from the need to make them applicable to a wide range of experiments. This cost-effectiveness stretches NIH dollars. We estimate that the savings realized from building rather than buying equipment total more than $3,000,000 over that past 4 years. A second benefit is that solutions engineered for one group often are applied rapidly to other groups-many labs here have adopted inexpensive LED light sources and two-photon microscopes, for example. The final benefit is that essential education and training for young neuroscientists. In some cases, Soucy trains students and postdocs to use custom-built instrumentation. In other cases, he guides students and postdocs who are designing and fabricating devices. He assists young scientists programming in LabView and Matlab. The array of the problems he has solved, the equipment he has designed and built, and the publications that have resulted, are now documented in this proposal for renewed funding. The impact on NINDS grant-holders and other H/C neuroscientists is difficult to overstate and impossible to replace. The Neuroengineering Core enables neuroscientists to surmount their most challenging technological and engineering obstacles. By the cost-effective use of a talented staff engineer and diverse fabrication facilities for trained users, the Neuroengineering Core will contribute to the generation of valuable basic neuroscience research and promote a novel approach to surmounting technological obstacles to scientific discovery, ultimately to address fundamental issues of human health.
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1 |
2014 — 2016 |
Regev, Aviv (co-PI) [⬀] Sanes, Joshua R Schier, Alexander F (co-PI) [⬀] Zhang, Yi |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Comprehensive Classification of Neuronal Subtypes by Single Cell Transcriptomics
? DESCRIPTION (provided by applicant): To understand the brain, we need a parts list of its cell types. The list will need to integrate molecular, functional and morphological data, but of these, molecular classification is best suited for comprehensive categorization and the only approach that can lead directly to genetically accessing the types; such access is essential in order to mark and manipulate neurons and to allow rigorous comparison of neurons from normal and diseased brains. We will apply the emerging method of single-cell transcriptional profiling (scRNA-seq) to this task. We will first rigorously compare and optimize cutting- edge methods for cell isolation, transcriptional profiling, and computational analysis to establish an efficientand effective pipeline for categorization. Then, we will apply our suite of methods to two brain regions - mouse retina and zebrafish habenula - that differ in several ways but share key features: they are accessible and compact and it is feasible to map their cell types comprehensively. In each case, we will perform unbiased and exhaustive profiling of 1,000's of neurons, to ensure that even rare classes of neurons are included in the survey. We will validate gene modules obtained from profiling by in situ hybridization, integrate them with structural and functional data, and provide standardized and comprehensive maps of cell type. Finally, we will apply what we have learned to a larger region, the mouse habenula. Profiling and classification in this structure will not only provide a stringent test of our ability to scale up our methods, bu also allow us to ask two important and interesting questions: to what extent cell types are conserved across species (zebrafish vs. mouse habenula) and to what extent cell types are conserved across regions (mouse retina vs. habenula). Together, insights, methods and reagents obtained in this work will provide an essential toolkit for tackling the whole brain.
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1 |
2014 — 2017 |
Sanes, Joshua R |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Admin Core Core Facilities For Analysis of Neural Circuit Structure and Functio
The Administrative Core integrates all of the scientific cores and tracks the usage and finances of these facilities.
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1 |
2015 — 2016 |
Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Screen For Determinants of Synaptic Specificity in Outer Retina.
? DESCRIPTION (provided by applicant): Neural processing of visual information begins at the first synapses of the retina, which are made by rod and cone photoreceptors with horizontal and bipolar cells (HCs, BCs) in a thin synaptic layer called the outer plexiform layer (OPL). These interneurons, along with amacrine cells, pass the information to retinal ganglion cells, which send it to the brain. Connectivity in the OPL is specific in at least three ways: rods and cones synapse almost entirely on rod BCs and cone BCs, respectively (cellular specificity); they synapse with axons and dendrites of HCs, respectively (subcellular specificity); and their synapses are confined to outer and inner strata of the OPL, respectively (laminar specificity). To date, few molecules have been found that mediate any of these aspects of synaptic recognition. The objective of this proposal is to identify such molecules. Our approach is to screen candidates in vivo in mice. Few such screens have been performed in any mammal, but the large size and accessibility of OPL synapses, along with recent technical advances in gene transfer and genome modification, now make it possible to analyze dozens of genes in a manageable period. To prepare for this screen, we have: (a) characterized molecular markers that label all synaptic partners in the OPL; (b) analyzed their expression during the period of synapse formation; (c) optimized gene transfer methods by electroporation in vivo; (d) shown that these methods can be used to effectively attenuate gene expression in rods and cones using shRNA and Cas9/CRISPRs for loss of function studies, and to ectopically express genes for gain-of-function studies; and (e) purified developing rods and cones by FACS sorting and used RNA-Seq to obtain transcriptome information from them. We will now use transcriptomic data to select ~50 genes that encode transmembrane or secreted molecules differentially expressed by developing rods and cones. We will attenuate their expression in developing retina, then use multi-label confocal microscopy to seek altered synaptic patterns in the OPL. Finally, for the most promising of these genes, we will conduct expression analysis as well as additional loss- and gain-of-function studies to elucidate their roles in synapse formation. In addition to initiating a deep analysis of synaptogenesis and synaptic selectivity at this clinicall important synapse, our results will be useful in two ways. First, they will provide reagents and insights for studies of less accessible synapses elsewhere in the brain. Second, they may guide optimization of methods to restore vision by photoreceptor replacement. Replacement methods have shown recent promise, but may fail if the new photoreceptors fail to make appropriate synapses. Molecules we identify could be useful in enhancing the efficacy of this strategy.
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2016 — 2020 |
Sanes, Joshua R |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Combinatorial Roles of Cadherins in Retinal Circuit Assembly.
? DESCRIPTION (provided by applicant): Orderly and specific connections among neurons of multiple types underlie the function of neural circuits. Our work has used mouse retina as a model to identiy molecules and mechanisms that underlie this specificity. The retina is not only important and fascinating in its own right, but is also a particularly accessible portion of the central nervous system. Moreover, we and others have generated a set of transgenic lines that allow specific retinal cell types to be marked and manipulated. We focus on the inner plexiform layer (IPL) in which dozens of types of interneurons (amacrine and bipolar cells) form stereotyped patterns of connectivity with ~30 types of retinal ganglion cells (RGCs), endowing each of them with sensitivity to visual features such as motion in a particular direction or color contrast. Wiring up these largely hard-wired circuits seems likely to require many cell type-specific molecules. Over the past several years he have identified several candidates, and tested them in vivo using loss- and gain-of-function strategies. During the past project period, we found two roles for members of the cadherin superfamily of recognition molecules in assembly of the IPL. First, a pair of classical cadherins (Cdh8 and Cdh9) play instructive roles in directing axonal arbors of two bipolar cell types (BC2, BC5) to appropriate sublaminae within the IPL. Second, a group of clustered gamma protocadherins (Pcdhg's) are required for dendritic patterning of starburst amacrine cells (SACs) in the lateral plane. Fortuitously, BC2, BC5 and SACs are all components of a direction-selective circuit that also includes ON-OFF direction-selective RGCs, which send information about motion in four cardinal directions to the rest of the brain. These results provide us with the opportunity to address a long- standing issue: how do members of multigene families work together in combinations to pattern neural circuits. Using new genome editing methods, we have generated double and triple mutants that lack (a) both Cdh8 and its closest relative Cdh11, (b) Cdh9 and its closest relatives, Cdh6 and Cdh10, and (c) Pcdhgs and their closest relatives, the alpha-Pcdhs. Importantly all of these genes are expressed by cells of the direction-selective circuit. Our aims now are to analyze mutants in which genes of the cadherin superfamily have been mutated singly and in combination. We will use molecular, histological and electrophysiological methods to analyze the consequences of these perturbations on the structure and function of the direction-selective circuit. Together, these studies will allow us to take a first step toward confronting the disturbing reality that analysis of single genes is insufficient to understand the assembly of complex neural circuits.
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2017 — 2018 |
Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Cell Type-Specific Vulnerability of Neurons to Axonal Injury: Comprehensive Mapping of Types and Gene Expression Analyzed by High Throughput Single Cell Rnaseq
Recovery is distressingly poor following acute (e.g., stroke or traumatic injury) or chronic (e.g., neurodegenerative disease) insults to the human central nervous system (CNS). Many neurons die and those that survive are generally incapable of growing new axons and re-establishing synaptic connections with their partners. Over the past few decades, however, some of the factors that limit neuronal survival and regeneration have been identified, and these discoveries have led to the development of interventions aimed at promoting recovery. Nonetheless, few if any are sufficient to restore useful function, so further advances are needed. In seeking them, a useful strategy has been to compare adult CNS neurons, which regenerate poorly, with neurons that grow or regenerate well ?for example, those in the developing mammalian CNS, the adult mammalian peripheral nervous system, or the CNS of lower vertebrates. Here, we propose a complementary approach: comparing vulnerable and resilient cell types within a single tissue. We will use the mouse optic nerve as a model. The optic nerve carries the axons of retinal ganglion cells (RGCs), the sole retinal output neurons, to the brain. There are ?40 RGC types in mice; all are similar in most ways, but they are tuned to distinct visual features and exhibit limited molecular differences. Following optic nerve crush (ONC) to sever all RGC axons, ~80% of the RGCs die within 2 weeks. Virtually none of the survivors regenerate axons spontaneously, but limited regeneration can be provoked in several ways. Recently, we used molecular markers to assess the fate of a small set of RGC types following ONC. We found dramatic differences in their ability to survive following ONC, and in the ability of survivors to regenerate following treatment. We also found a gene selectively expressed by a resilient type that can promote regeneration when overexpressed. These findings led us to believe that a deeper understanding of type-specific differences in response to injury could lead to new strategies for improving recovery. To identify these differences, we will use a high- throughput single cell RNA sequencing (scRNAseq) method called Drop-seq that we helped pioneer. We have now used Drop-seq to profile >80,000 cells from control retina, generating molecular profiles for over half of all retinal types and optimizing a scalable pipeline for further studies. Using Drop-seq and customized computational approaches, we will now profile RGCs in control retina and 2 weeks after ONC to comprehensively map the vulnerability of all types. We will then profile RGCs at 3, 12 and 48 hours after ONC, to find transcriptomic differences that correlate with, and could contribute to their differential vulnerability. Our results will provide a foundation for (a) identifying the cell types on which available interventions act, (b) elucidating their mechanisms of action, so they can be improved, and (c) discovering new ways to transform vulnerable populations into resilient ones.
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2018 — 2019 |
Do, Michael Tri Hoang Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Defining Cell Types of Foveal and Peripheral Retina by High-Throughput, Single-Celltranscriptional Profiling
PROJECT SUMMARY Visual sensation and computation begin in the retina, and retinal defects are the leading causes of vision loss in the developed world. Accordingly, the retina has been studied in detail. Over the past decade, the mouse has emerged as a favored model for retinal analysis, thanks to its ready availability, the vast array of molecular and genetic tools available, and the central position of mouse models in biomedicine generally. Exploiting these strengths, several groups, including ours, have identified genes selectively expressed by many of the >100 retinal cell types (grouped into 5 neuronal classes and several non-neuronal cell types), and such studies have led to deep insights into the structure, function and development of retinal circuits. Unfortunately, however, the mouse has a serious limitation as a model of human retinal function and dysfunction. Most visual perception in humans and other primates relies on a central specialization called the fovea; other mammals, including mice, lack this structure. Thus, diseases that affect the fovea?such as age-related macular degeneration, which is a major cause of blindness?cannot be modeled in the mouse. Yet, research on the primate retina lags behind that of the mouse retina, and molecular studies of the primate retina lag further still. We propose to help bridge this gap in knowledge by classifying the cell types that comprise the foveal and peripheral retina of macaques, and determining the gene expression patterns that define each type. To this end, we will use high-throughput, single-cell RNA sequencing (scRNAseq) methods, which allow profiling of thousands of cells at moderate cost. We helped develop one such method, called Drop-seq, and combined it with computational clustering to analyze >80,000 cells from the mouse retina; this effort resulted in molecular profiles for most cell types and an optimized, scalable pipeline for future studies. This pipeline is well suited for tissue that is difficult to obtain and for small samples such as those from the fovea. As a first step, we will perform scRNAseq on 50,000 cells each from foveal and peripheral retina, then use our bioinformatic methods to divide their profiles into groups. We will then make a tentative identification of these groups with cell types, based on expression of orthologous genes obtained from a taxonomy of retinal cell types in mouse that is now nearing completion in our hands. We will then turn to a comprehensive classification of retinal ganglion cells, which we will purify from foveal and peripheral retina using a cell-sorting protocol that we developed for mouse and have now adapted to macaque. Finally, we will develop methods for relating gene expression patterns of macaque retinal neurons to their morphology, providing protocols that can be used for detailed characterization in the following years. Taken together, our proposed experiments constitute early steps toward a complete identification of cell types in the primate retina; this, in turn, will generate new insights into foveal specializations, and lay the foundation for analysis of normal and diseased human retina.
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2021 |
Sanes, Joshua R |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High Throughput Single Cell Transcriptomic Approach to Identify Susceptible Cell Types and Gene Expression Changes in Human Glaucoma
ABSTRACT Glaucoma, the leading cause of irreversible blindness worldwide, results from loss of retinal ganglion cells (RGCs), which carry visual information from the eye to the rest of the brain. Current treatment strategies center on lowering intraocular pressure (IOP), the only known modifiable risk factor for glaucoma. However, significant unmet need persists, and a neuroprotective strategy targeting glaucomatous RGCs directly would offer a powerful complementary approach. To date, however, no neuroprotective therapies have successfully entered the clinic. One major obstacle is that little is known about which of many human RGC types are most susceptible, and what molecular changes occur in RGCs prior to their demise. This project uses single cell transcriptomic profiling and integrative computational analysis to address these gaps in our knowledge. Over the past five years, we have helped to develop methods for high throughput single cell RNA sequencing (scRNA-seq) and applied them to retina ? first in mice, then in non-human primates, and subsequently in humans and in mouse models of neuronal injury. Most recently, we have implemented the related method of single nucleus RNA-seq (snRNA-seq) so that we can profile tissue obtained post-mortem, frozen and banked. We now propose to obtain and analyze single nucleus transcriptomes of RGCs from well-characterized glaucomatous and normal human retinas. The number of samples must be large, because glaucoma is a heterogeneous disease with diverse genetic and non-genetic risk factors. It is therefore important to study diverse groups, so we can determine whether they converge on common molecular patterns. Specifically, we will profile at least 2000 RGCs from each of 200 human donor eyes, 150 from individuals with verified glaucoma and 50 from age-, sex- and race-matched controls. From the data we obtain, we will (a) determine whether specific RGC types are selectively resilient or vulnerable in glaucoma and (b) identify genes differentially expressed between glaucomatous and normal RGCs of each type. Finally, we will perform similar analysis on a high-IOP mouse model of glaucoma, helping us understand the extent to which diseases processes in humans are accurately modeled in mice. Our results will provide a powerful resource of sufficient power to transform our view of this prevalent, complex and incompletely understood blinding disease.
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2021 — 2024 |
Sanes, Joshua Hanken, James [⬀] |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Creating a Novel Museum-Based Resource For Neuroscience: Mass Whole-Slide Imaging of the R. Glenn Northcutt Collection of Comparative Vertebrate Neuroanatomy and Embryology
This project will enable online access to a unique, irreplaceable resource for comparative studies of the evolution and development of the vertebrate brain. The R. Glenn Northcutt Collection of Comparative Vertebrate Neuroanatomy and Embryology, housed in Harvard’s Museum of Comparative Zoology (MCZ), is the world’s largest and most taxonomically diverse collection of histological preparations of developing and adult vertebrate brains mounted on glass microscope slides. It is of particular interest and relevance to the current generation of neuroscientists who use molecular and genetic approaches to elucidate mechanisms underlying evolutionary innovations, but the slides are fragile and access to them is difficult and time consuming. By utilizing a whole-slide-imaging workflow developed through a novel collaboration with Harvard’s Center for Brain Science, the project will allow professional scientists, educators and students to easily and routinely access the slides’ content via high-resolution digital images. Such access will facilitate use of the slides in research and education and complement novel technologies for studying brain structure, development and function. It will facilitate collaborations between the neuroscience and biodiversity communities and, together with other projects that seek mass digitization and sharing of biological collections, it will enhance the ability of natural history institutions to more fully serve both science and society beyond their traditional constituencies. Indeed, the project’s imaging workflow and associated training components offer an exemplar method for rapid and cost-effective digitization that can be used by other institutions, whose slide holdings number in the millions, most of which remain dark data.
The project will use a high-throughput, semi-automated slide scanner to make high-resolution digital images of the approximately 33,000 glass microscope slides in the Northcutt Collection. When completed, the project will provide online access to approximately 500,000 serial sections and whole-mount preparations of adult brains and embryos of more than 240 genera and 270 species of living vertebrates. A cost-effective whole-slide imaging workflow will be utilized to process as many as 360 slides/week while yielding excellent image resolution (0.2 and 0.4 μm/pixel at 40X and 20X magnification, respectively). A digital image of each slide will be served to potential users via two online portals: MCZbase, the Museum of Comparative Zoology’s permanent specimen database; and MorphoSource, an NSF-supported online repository for specimen digital imagery. Both portals will be configured with Girder and SlideAtlas, two open-source software tools for whole-slide image viewing, downloading and analysis. In addition, for as many as 20 species widely used to teach comparative vertebrate anatomy, digital images of a subset of histological sections labelled to point out principal brain regions will be uploaded to BrainMaps, an online resource for vertebrate neuroanatomy. Finally, the project will produce several resources for training both students and professionals in methods for mass digitization and computer-assisted visualization of slide collections regardless of subject matter. These resources include four “how-to” videos, two online tutorials, undergraduate and graduate student internships and a graduate-level course in museum studies.
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.
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