Susumu Tonegawa - US grants

We propose to study diversity, somatic reorganization, and expression of mouse Ig genes. Using the recombinant DNA technique, we will clone a variety of DNA fragments containing the Ig genes and their flanking sequences and characterize these DNA clones by various methods including heteroduplex analysis, R loop analysis, restriction enzyme mapping and DNA sequencing. As the sources of cells we will use myelomas, hybridomas representing various stages of B cell differentiation and natural B lymphocytes fractionated by a fluorescence-activated cell sorter. We will also identify and characterize enzymes responsible for somatic recombinations. Our goal is to decipher at the molecular level the genetic mechanisms that are responsible for the vast antibody diversity and the expression of Ig genes including allelic exclusion, heavy chain switch and simultaneous synthesis of two heavy chain classes.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The objective of this research proposal is to further our understanding of T cell development and T cell function in health and disease. To this end we will use methods of molecular biology (cloning, polymerase chain reaction aided analytical methods, chromosome walking, site directed mutagenesis, gene transfection for transient and stable expression, gel retardation assays, construction of transgenic mice and of mutant mice using the ES cell homologous integration technique) as well as cell and thymus organ culture techniques, cell fusion techniques, flow cytometry, immunohistology and various in vivo assay for T cell functions (skin grafting, ear assay for delayed type hypersensitivity). We will study the molecular mechanism of the initial steps of alpha/beta and gamma/delta T cell differentiation, gamma/delta TCR repertoire generation, positive and negative selection alpha/beta and gamma/delta T cells in the thymus and the homing of gamma/delta T cell subsets to different epithelia. We will analyse TL and CD1 genes and the putative role of their products as antigen presenting proteins for gamma/delta T cells and investigate the molecular details and the putative biological significance of heat shock protein recognition by gamma/delta T cells. A major effort will be made to elucidate the in vivo function of gamma/delta T cells in lymphocyte development, in various immune responses (antibody responses, DTH responses, graft rejection) as well as in infectious diseases, auto immune diseases and tumor immunity.

The long term objective of this research proposal is to understand at the molecular and cellular levels how the immune recognition is controlled during the organism's development. Specific aims are: 1) to understand the molecular mechanism of the tissue-specific enhancer associated with the immunoglobulin gene locus, 2) to identify the cis- and trans-acting elements involved in the developmentally controlled expression of the T cell receptor Alpha and Beta genes and the T cell-specific gene Gamma, 3) to identify, isolate, and characterize the genes and gene products for the putative receptor on the immature T cells involved in the intrathymic selection of self MHC-restricted T cells. For these purposes we will use a variety of recombinant DNA, DNA sequencing, transfection, cell-free transcription, hybridoma, and transgenic mice techniques as well as more conventional biochemical techniques for proteins and nucleic acids. Since the B and T cell receptors play the pivotal roles in the functioning of the immune system, the vertebrate's major body defense device, through understanding of the developmentally controlled expression of the genes is the basis on which a variety of diagnostic and therapeutic methods can be developed. In addition, the information on the structure and function of anti MHC receptor will be useful in the clinical control of the allograft rejection.

The long-term objectives of this research proposal is to dissect the molecular and cellular mechanisms underlying the synaptic plasticity, long term potentiation (LTP) and long-term depression (LTD), in the hippocampus and/or cerebellum of the mammalian brain, and to examine the role of such plasticity as mechanisms for learning and memory, and for kindling, an animal model of epilepsy. To this end, a new genetic approach based on gene targeting in embryonic stem (ES) cells has been introduced. Four distinct strains of mutant mice with a non revertible mutation in one of four different enzymes whose functions are thought to be involved in at least some forms of synaptic plasticity will be analyzed or constructed and analyzed by a variety of methods including immunocytochemistry, in vitro and in vivo, electrophysiology, and behavioral paradigms such as Morris water maze fear conditioning, eyeblink conditioning, and epileptic behavior. These enzymes are the alpha isoform of Ca2+/calmodulin-dependent protein kinase type II, gamma and Beta isoforms of protein kinase C, and heme-oxygenase II required for the synthesis of carbon monoxide (CO) which may have a retrograde messenger activity in LTP. In addition, this application proposes to develop new methods with which a genetic deletion in a specific protein of interest can be induced in grown up animals or restricted to certain subregions of the brain. These methods are expected to expand substantially the power of the genetic approach in the analysis of brain functions. This proposal will help understand the fundamental molecular and cellular mechanisms for learning and memory as well as for epilepsy in mice. Information obtained is expected to be useful for the development of diagnostic and therapeutic methods for neurological diseases such as Alzheimer's disease and epilepsy.

The long-term objectives of this research proposal is to dissect the molecular and cellular mechanisms underlying the synaptic plasticity, long term potentiation (LTP) and long-term depression (LTD), in the hippocampus and/or cerebellum of the mammalian brain, and to examine the role of such plasticity as mechanisms for learning and memory, and for kindling, an animal model of epilepsy. To this end, a new genetic approach based on gene targeting in embryonic stem (ES) cells has been introduced. Four distinct strains of mutant mice with a non revertible mutation in one of four different enzymes whose functions are thought to be involved in at least some forms of synaptic plasticity will be analyzed or constructed and analyzed by a variety of methods including immunocytochemistry, in vitro and in vivo, electrophysiology, and behavioral paradigms such as Morris water maze fear conditioning, eyeblink conditioning, and epileptic behavior. These enzymes are the alpha isoform of Ca2+/calmodulin-dependent protein kinase type II, gamma and Beta isoforms of protein kinase C, and heme-oxygenase II required for the synthesis of carbon monoxide (CO) which may have a retrograde messenger activity in LTP. In addition, this application proposes to develop new methods with which a genetic deletion in a specific protein of interest can be induced in grown up animals or restricted to certain subregions of the brain. These methods are expected to expand substantially the power of the genetic approach in the analysis of brain functions. This proposal will help understand the fundamental molecular and cellular mechanisms for learning and memory as well as for epilepsy in mice. Information obtained is expected to be useful for the development of diagnostic and therapeutic methods for neurological diseases such as Alzheimer's disease and epilepsy.

A major component of this Center is to use genetic manipulation of the mouse brain to unravel the molecular basis of learning and memory. One of the approaches here is to generate strains of mice in which a gene of interest has been eliminated in specific neuronal populations by use of the Cre recombinase. A prerequisite for this strategy is the availability of promoters that target Cre expression to specific populations of neurons in brain regions of interest. The purpose of this core is to identify appropriate genes and generate the targeted Cre mice. The PI proposes to isolate brain region specific transcripts using a combined subtractive hybridization-array screening strategy followed by in situ hybridization. This approach is based on existing commercial methods which require modification in most cases. The applicant is aware of this and has made substantial progress in optimizing the subtraction methodology. An alternative strategy, which the PI may well have considered, is to use in situ hybridization as the secondary screen (rather than using array technology). However, the proximity of the MIT Genome Center, which has extensive expertise in robotic screening methods, also favors the array approach. Following the isolation of transcripts, BACS will be identified for the clones having the requisite neuronal specificity and these used to generate BAC-Cre constructs. The use of BACs to construct transgenic mice has gained popularity and is probably the best approach available (assuming the candidate gene is represented in the commercial BAC libraries). The BAC fusion constructs will be used to make lines of transgenic mice that express Cre recombinase in specific populations of neocortical neurons. Subsequently, these mice will be crossed with lines of mice that harbor a gene of interest flanked by loxP sites.

This core consists of a central breeding and typing facility to coordinate the maintenance and distribution of mice used by investigators of the Center. The mice to be housed in this facility are explicitly defined. The maintenance of transgenic mice is critical to this Center, and the need for such a core is well justified with 5 of the 6 projects utilizing genetically modified mice. Therefore, it is appropriate to maintain a transgenic mouse Core within the Center. The level of mouse activity described in the application justifies the hiring of someone to manage the colonies and type offspring (particularly once new lines of mice are being generated). Unfortunately, with the information provided, it is difficult to estimate how many mice need to be genotyped over a given period, what services are already provided by the Comparative Medicine staff as part of their per diem charges (e.g. is colony management part of that charge?), or to what extent the hiring of such a position reduces per diem charges. Nevertheless, given that the Core is maintaining both single and double transgenic mice, the centralization of screening is a prudent move. This will ensure quality (validity of genotypes) and availability of mice to the members of the Center.

DESCRIPTION: (Adapted from the applicant's abstract) This is an application to establish a Silvio O. Conte Center for Neuroscience Research (CCNR) on GENETIC, PHYSIOLOGICAL, AND BEHAVIORAL STUDIES OF MEMORY. The long-term objective of the Center is to elucidate the molecular, cellular, and neuronal ensemble mechanisms of mammalian learning and memory. The CCNR attacks the problem at three levels. First, it seeks to understand the elementary mechanisms of synaptic plasticity in the hippocampus and neocortex, and how these are developmentally and regionally regulated. Second, the Center seeks to understand how neuronal activity in the hippocampus and neocortex represents information about the outside world, how this information is compared with previous experience, and how these representations are consolidated into long-term memory. Third, the Center seeks to connect the synaptic plasticity and the neuronal correlates of learning with behavioral measures of memory. The elementary mechanisms of synaptic plasticity will be studied by applying electrophysiological techniques to brain slices derived from the hippocampus and the visual and inferotemporal cortices of mice with several specific spatially targeted genetic lesions (knockout mice). A novel electrophysiological technique that permits the analysis of single visualized synapses of cultured neurons will also be used for the analysis of these mice. In order to understand how neuronal activity represents learned information, the Center will seek to identify alterations in the activity of neuronal ensembles in the hippocampus, inferotemporal cortex, and prefrontal cortex by applying the novel multiple tetrode recording techniques to monkeys or rodents that are undertaking various memory tasks. Finally, in order to connect synaptic plasticity and neuronal correlates of learning with behavioral measures of memory, the Center takes advantage of the new methods of regionally and temporally selective genetic disruptions in mice, some of which have already been developed in the Center (hippocampus CAI-selective) while others (neocortex and neocortical subregion-specific) are proposed to be developed as a core project. The proposed research is truly interdisciplinary covering molecular genetics of mice, electrophysiology of brain slices and cultured neurons from rats and mice, and multiple electrode recordings of monkeys, rats, and genetically engineered mice performing specific memory tasks. Six investigators from three institutions and the seventh investigator as a subcontractor, each possessing complimentary and well-recognized expertise and state of the art experimental technologies will join forces and collaborate extensively in the CCNR framework. The Center's research is highly relevant to mental health and illness because mnemonic impairments are a hallmark of aging and major neurological diseases such schizophrenia, Alzheimer's disease and Parkinson's disease.

GRANT=P50MH58880-01A1-0002 DESCRIPTION: (Adapted from the Application) This project contributes to the Center's overall objectives by examining the roles of the NMDA receptor and calcineurin (PP2B) functions in hippocampal CA1 pyramidal cells in hippocampus-dependent learning. How synaptic plasticity in the hippocampus contributes to hippocampus-dependent learning is one of the major goals of the Center. Activation of NMDA receptors in CA1 neurons is known to play a pivotal role in the induction of several forms of synaptic plasticity such as LTP and LTD at CA1 synapses. On the other hand, calcineurin is known to play a critical role in the induction of LTD. Our previous studies on CA1-specific NR1 KO (CA1 KO NR1) mice which were conducted collaboratively between our and Wilson's laboratories showed that NMDA- dependent synaptic plasticity at CA1 synapses plays a crucial role in spatial learning. These studies also suggested that the spatial learning impairment exhibited by the NR1 KO mice is probably due to the animal's inability to generate normal place fields in the CAI area which may be required for place-specific, coordinated firing of neurons in downstream brain areas. We will further test this hypothesis by recording the activities of neuron ensembles in the subiculum and the deep layers of the entorhinal cortex of CA1 KO NR1 mice. While NMDA receptor-mediated synaptic plasticity seems to play a crucial role in spatial learning, the relative contributions of LTP and LTD in this cognitive process remain unknown. We predict that a loss of calcineurin will result in a selective loss of LTD with preservation of LTP. We therefore plan to construct CA1-specific PP2B knockout mice (called CA 1 KO Crib 1) by the method already available in Tonegawa's laboratory. These mice will be analyzed by subjecting them to electrophysiological and behavioral studies. In addition to the procedures already utilized in our laboratories during the analysis of the CA1-specific N-R1 KO mice, expertise in Bear's laboratory on LTD will also be applied (Specific Aim #3). Another set of experiments revisits the unsettled issue of whether the hippocampus plays a crucial role not only in spatial learning but also in non-spatial learning. We will address this issue by subjecting the CAIKNR1 mice to a non-spatial learning paradigm, social transmission of food preference (Specific Aim #2). Individual Project #2 also contributes to the Center's overall objectives by examining the role of cAMP-dependent transcriptional factor CREB in the hippocampal CA1 area as well as in the neocortex in memory consolidation and maintenance (Specific Aim #4). Earlier studies by others with Drosophila and Aplysia implicated CREB-dependent protein synthesis in long term memory or long term facilitation. Although an effort was made by others to extend this notion to mammals using unrestricted CREB KO mice, the lack of regional and temporal restriction in the gene knockout and the incomplete inactivation of CREB activity prevented a firm conclusion. In addition, no information could be obtained as to where, for instance in hippocampus or parietal cortex, CREB is required in the consolidation and maintenance of hippocampus-dependent long term memory. We plan to address this central issue in memory research by creating and analyzing a new set of CREB KO mice in which gene knockout is complete and is restricted to the CA1 area or to the parietal and temporal cortices (see Core #1 for the construction of the latter mice). We also propose to produce and analyze CREB KO mice in which the gene knockout can be induced in a specific area of the brain after maturation in order to study adult function of CREB independent of its developmental function. Another CREB mouse to be made using a dominant negative form of CREB (dn CREB) will allow spatially-restricted inducible inhibition of endogenous CREB function. The inducible knockout or inhibition system will enable us to study the roles of a specific gene product (in this case CREB) in different phases of themnemonic process such as acquisition vs. consolidation (or maintenance) of memory. With all of these CREB gene manipulated mice, our or Bear's laboratory will analyze LTP and LTD (or depotentiation), particularly late LTP (L-LTP) at CA1 synapses of the hippocampus or at Te-2 synapses of the neocortex. Both our laboratory and Wilson's will subject the same mice to spatial learning and memory paradigms to assess possible impairments in the learning and/or memory phases of the behavior. In a closely related study as part of Individual Project #6, Bear's laboratory will extend the behavioral analysis to include visual recognition memory. All these studies may permit correlations of physiological and behavioral defects and also may identify brain site(s) and phases(s) of the mnemonic process in which CREB plays a crucial role. Finally, Wilson's laboratory will analyze some of these CREB mice by large-scale recordings in order to correlate defects in synaptic plasticity and behaviors to defects in the activity of neuronal ensembles.

DESCRIPTION: (Adapted from the application). Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by prominent impairments in memory, which have been attributed to synaptic loss and altered synaptic plasticity in neural circuits subserving memory. Elucidation of the cellular and molecular mechanisms underlying memory formation may therefore lead to novel therapeutic strategies. Manipulation of the mouse genome has been exploited to address the contribution of specific genes to neural phenomena associated with synaptic plasticity, including hippocampal long-term potentiation (LTP), spatial learning and memory. The applicant and colleagues have recently developed methodology that enables gene disruption or transgene expression in restricted regions of the postnatal murine brain. These techniques have permitted disruption of NMDA receptor function exclusively in pyramidal cells of hippocampal area CA1, demonstrating a requirement for NMDA receptor-dependent plasticity at CA1 synapses in hippocampal LTP, place cell formation, and spatial learning and memory. The proposal is to employ this approach to test the hypothesis that mitogen-activated protein kinase (MAPK) function is required for synaptic plasticity and associated phenomena in the mammalian brain. The Ras/MAPK cascade is a highly-conserved signalling pathway that mediates cellular responses to a variety of stimuli. The extracellular-signal regulated kinase (ERK) subfamily pathway is activated by Ras-dependent and -independent mechanisms in response to stimuli associated with synaptic plasticity in neurons, including neurotrophins, glutamate and calcium. However, the role of the ERK pathway in learning and memory in the mammalian brain remains to be established. Mice transgenic for a dominant-negative form of MAPK/ERK kinase (MKK1) will be generated in order to inhibit ERK function in subregions of the postnatal brain. The resulting mice will be analyzed for hippocampus-dependent and hippocampus-independent spatial learning and memory, hippocampal and neocortical LTP and LTD, hippocampal place-cell formation, and cAMP response element-binding protein (CREB)-dependent gene expression.

Many Individual Projects of the proposed Silvio O. Conte Center for Neuroscience Research (CCNR) at MIT require genetically engineered mice. Furthermore, many of these mice are proposed to be generated and/or analyzed in collaborative arrangements involving two, three or even four laboratories. Another characteristic feature of these mice is that many of them are conditionally engineered and, therefore, require crossing of two or three component strains that are modular (e.g., Cre, floxed, tTA and Otet mice). Many of these component mice are crossed in different combinations for different specific aims across multiple collaborative projects. Therefore, we propose to set up central breeding colonies of these component mice at MIT in collaboration with its Department of Comparative Medicine which supervises and services MIT's entire animal facility. The central colonies are meant to be for the maintenance of the various strains and occasional supplying of these strains to individual laboratories. The cost for the intercrossing of mice to be used in specific experiments described in the Individual Projects will be charged to the individual faculty's budget. The mutant and transgenic mice to be produced, maintained, and used in Heinemann's lab at the Salk Institute will be charged to his individual budget. The cost to generate the strains described in Core #1 and individual projects will be charged to the corresponding budgets. Only after the component mice are established will they be transferred to Core #2 for maintenance. The mouse holding space will be provided by MIT. The standard maintenance services, cage changes, bedding, food, water, and health monitoring will be provided by the staff of the Department of Comparative Medicine for which the Core will pay per diem charges. In Year 1 about 15 to 20 strains will be maintained and we expect that about 10 strains will be added in subsequent years as they are newly made. It is expected that some colonies such as fNR1, CA1-Cre, and CA3-Cre, will be relatively large (50 cages) because of the heavy demands from multiple projects while others such as global NR1 KO, global GluR6 KO, and CA3-NR1 KO mice to be used occasionally as a control will be kept small (10 cages). The service provided by this Core consists of (1) maintenance of the colonies, (2) genetic typing of individual mice, and (3) shipment of mice to the Salk Institute and other outside collaborators. Core #2 will permit supplies of identically maintained mouse strains to the collaborating labs within the Center and sometimes beyond the Center.

The central hypothesis of this project is that synaptic plasticity in each of multiple hippocampal excitatory circuits contributes to the overall role of the hippocampus in learning and memory by providing distinct and complementary functions. While such ideas have been modeled extensively, empirical studies have been scant due to technical difficulties. The recently invented cell type-restricted gene knockout technique and multidisciplinary analyses of the resulting mutants provide an effective approach. Following our previous studies on CA1 and CA3 circuits, this project focuses on dentate gyrus (DG). We propose to generate an NMDA receptor (NR) knockout mouse strain in which the gene ablation is selective in adult DG granule cells. By subjecting the mutant mice to specifically designed behavioral protocols, we will test the hypothesis that the plasticity at the perforant path (PP)-DG synapses plays a crucial role in pattern separation and other specific aspects of hippocampus-dependent learning and memory. Applying the multielectrode recording technique to the mutant mice undergoing a specifically designed spatial memory task ( wagon wheel maze"), we will seek, in collaboration with Matthew Wilson, hippocampal neuronal activity correlates of the putative behavioral impairments. We also propose to generate GluR6 (G6) knockout mouse strains in which the gene ablation is selective either in DG granule cells or CA3 pyramidal cells. In collaboration with Steve Heinemann, we will determine whether pre- or post-synaptic G6 gates plasticity at the mossy fiber (MF)-CA3 synapses. By subjecting these mutants to several behavioral tasks, we will examine whether the MF synaptic plasticity plays a crucial role in specific aspects of memory, pattern separation, pattern completion and rapid one-trial learning. We will also seek hippocampal neuronal activity correlates of putative behavioral impairments. We will extend these studies to cell type-restricted, reversibly inducible NR1 and G6 mutant mice when they become available from the Center's Core #1 project. These multidisciplinary and collaborative studies will advance our fundamental knowledge about the roles of hippocampal circuits in learning and memory and, thereby, contribute to mental health and illness because mnemonic impairments are a hallmark of aging and major neurodegenerative diseases such as Alzheimer's and Parkinsons's disease.

Many Individual Projects of the proposed Silvio O. Conte Center for Neuroscience Research (CCNR) at MIT utilize genetically engineered mice as key experimental tools. Most of these mouse strains are conditionally engineered (i.e., spatially and/or temporally restricted) and, hence, require state-of-the-art technology. The role of Core #1 is twofold: to provide the technical knowhow regarding the transgenics and knockout mice to the Center's individual laboratories and to generate some of the strains that are proposed to be used by the Center's multiple laboratories as multidisciplinary collaborations. In Specific Aims #1 to #3, the Cre-loxP recombination system will be combined with the tetracycline-transactivator (tTA) system to accomplish cell type-restricted, reversibly regulatable expression of a gene. Specific Aim #1 will attempt to regulate the NMDA receptor (NR)-1 gene in the dentate gyrus (DG) granule cells, while Specific Aim #2 will seek to regulate the GluR6 (G6) gene in the DG granule cells or CA3 pyramidal cells. These mutant mice provide invaluable tools in the Center's collaborative attempt (Individual Project #2) to understand the roles of synaptic plasticity in distinct hippocampal excitatory synapses in specific aspects of learning and memory. Specific Aim #3 will attempt to regulate the NR1 gene in the superficial layers of the entorhinal cortex and will serve the Center's collaborative effort (Individual Project #1) to understand the role of these receptors in place field formation. The objective of Specific Aim #4 is to produce a dorsal forebrain-restricted tTA mouse which is an important component mouse for the generation of multiple transgenic strains needed in the Center's collaborative attempt to understand how synaptic plasticity in the visual cortex subserves the receptive field plasticity (Individual Project #6). Core #1 will also provide training and the facility to Individual Project #7 which proposes to generate transgenic lines of PSD-GFP fusion proteins to study the morphological changes accompanying synaptic plasticity (Individual Project #7). Finally, Core #1 will provide advice and instructions to the attempt to genetically manipulate cortical function of monkeys (Individual Project #3).

Many Individual Projects of the proposed Silvio O. Conte Center for Neuroscience Research (CCNR) at MIT require genetically engineered mice. Furthermore, many of these mice are proposed to be generated and/or analyzed in collaborative arrangements involving two, three or even four laboratories. Another characteristic feature of these mice is that many of them are conditionally engineered and, therefore, require crossing of two or three component strains that are modular (e.g., Cre, floxed, tTA and Otet mice). Many of these component mice are crossed in different combinations for different specific aims across multiple collaborative projects. Therefore, we propose to set up central breeding colonies of these component mice at MIT in collaboration with its Department of Comparative Medicine which supervises and services MIT's entire animal facility. The central colonies are meant to be for the maintenance of the various strains and occasional supplying of these strains to individual laboratories. The cost for the intercrossing of mice to be used in specific experiments described in the Individual Projects will be charged to the individual faculty's budget. The mutant and transgenic mice to be produced, maintained, and used in Heinemann's lab at the Salk Institute will be charged to his individual budget. The cost to generate the strains described in Core #1 and individual projects will be charged to the corresponding budgets. Only after the component mice are established will they be transferred to Core #2 for maintenance. The mouse holding space will be provided by MIT. The standard maintenance services, cage changes, bedding, food, water, and health monitoring will be provided by the staff of the Department of Comparative Medicine for which the Core will pay per diem charges. In Year 1 about 15 to 20 strains will be maintained and we expect that about 10 strains will be added in subsequent years as they are newly made. It is expected that some colonies such as fNR1, CA1-Cre, and CA3-Cre, will be relatively large (50 cages) because of the heavy demands from multiple projects while others such as global NR1 KO, global GluR6 KO, and CA3-NR1 KO mice to be used occasionally as a control will be kept small (10 cages). The service provided by this Core consists of (1) maintenance of the colonies, (2) genetic typing of individual mice, and (3) shipment of mice to the Salk Institute and other outside collaborators. Core #2 will permit supplies of identically maintained mouse strains to the collaborating labs within the Center and sometimes beyond the Center.