Kristen M. Harris - US grants

Spiny neurons throughout the nervous system have thornlike protuberances arising from their dendrites. These dendritic spines are a site of asymmetric excitatory inputs to the neurons. Spine shape is thought to affect the efficacy of the synaptic input. The focus of this proposal will be to study the ultrastructure of dendritic spines on three different types of spiny neurons. The major emphasis will be on the pyramidal cells of area CA1 in the rat hippocampus. Comparison measurements will be made on granule cells of area dentata of the same hippocampus, and on cerebellar Purkinje cells of the same brain. I will use computer-assisted methods to reconstruct dendritic spines from serial electron micrographs. Then measurements of spine length, neck diameter, surface area and volume will be made. Synaptic surface area, the volume of spine cisternae and the diameter of the parent dendrite will also be measured. Then these measures will be used in computer programs that model how relationships among these structural features might affect synaptic efficacy. Ultrastructural studies of dendritic spines suggest that their necks shorten and widen following high frequency afferent activation. This structural change should increase the efficacy of synapses terminating on the spine by decreasing the resistance to synaptic current flow through the spine neck to the dendrite. In the hippocampus, high frequency stimulation of afferent input results in a long-lasting incrase in the physiological response of the postsynaptic cell. This change in response is referred to as long-term potentiation (LTP) and represents a candidate mechanism for information storage. In area CA1 of the rat hippocampus, the magnitude of LTP produced is about three times greater at postnatal day 15 than in the adult (Harris and Teyler, In Press). Spines will be studied to determine whether changes in there ultrastructure during development would account for this age related difference in LTP magnitude. In addition, LTP will be induced in the apical dendritic field of area CA1 in hippocampal slices at days 15 and 60. Spines in the vicinity of stimulation will be reconstructed and measured. Comparison measurements will be made on spines in the basillar dendritic field of the same slice, and on apical spines of non-potentiated slices. In order to unambiguously identify spines that contact stimulated axons, these axons will be filled with horse-radish peroxidase following the induction of LTP.

The Neuroscience Division of Children's Hospital works at the leading edge of developmental and genetic neuroscience, with particular strength in studies combining histochemistry and electron microscopy with computer-assisted reconstruction methods, to obtain three-dimensional views of neurons, their synaptic connections, chemistry, and developmental interrelationships. The pace of the research is severely constrained at present because of the progressively more limited reliability and stability of the JEOL transmission electron microscope that is at the heart of the instrumentation system. This grant application requests funds to replace the present electron microscope, after more than a decade of intensive use, with a new JEOL 100CX transmission electron microscope. The accessories required include: 1) 120KV accelerating voltage and minimum dose focusing to reduce speciman heating and subsequent destruction during exposure to the electron beam, 2) a goniometer with rotational holders for orienting ribbons of sections on sequential grids, 3) additional film canisters and cassettes to facilitate efficient photographing of serial sections from a single subject, or many different subjects at one photographic session, and 4) a free lens control system to allow continual as well as stepwise setting of the magnification. We expect the upgraded JEOL 100CX to serve several departmental investigators currently involved in biomedical research.

More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy (EM). Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Numerous anatomical studies have searched for anatomical correlates of LTP. These studies have lacked sufficient resolution to determine accurately the magnitude of the reported changes in the number of morphology of synapses and dendritic spines. A complete morphometric study is proposed to delineate the anatomical alterations at synapses and dendritic spines, to define their duration, and to relate these to various stages of LTP. The specific aims are: 1) To delineate the changes in synaptic and dendritic spine morphology that are associated with early and late phases of LTP in the hippocampus of postnatal day 15 (P15). This age is chosen because LTP is robust and enduring, even though the spines, synapses and dendrites have not attained their mature numbers or morphology. Because vigorous synaptogenesis is occurring at this age, multiple candidates exist for morphological plasticity that could subserve LTP. 2) To test whether mature synapses express morphological correlates of LTP that are similar to or different from those delineated at P15. It is essential for the understanding of mature memory mechanisms to establish whether mature synapses are similar to, differ only in degree from, or are categorically different from P15 synapses in their morphological plasticity. Methods: Standard procedures will be used to maintain in vitro hippocampal slices and induce and measure LTP extracellularly. Control hippocampal slices will receive the same stimulation paradigms as the LTP slices, But in the presence of DL-2-amino-phosphono-valeric acid (APV), a known blocker of LTP. Rapid microwave-enhanced fixation and routine processing will be used to prepare slices for electron microscopy. The irregularity in shape of dendritic spines and their synapses makes it impossible to extrapolate from partial measurements to their complete dimensions. Thus, the technically demanding approach of three-dimensional reconstruction from serial EM is required. Historically, the main drawback of serial EM has been the small sample sizes utilized because of the labor-intensive nature of the work. A new unbiased approach for sampling large areas of neutrophil in combination with serial EM is proposed to obtain accurate characterization of synaptic, spine, and dendritic morphologies and to allow for complete quantification of their ultrastructure in three dimensions. Health Relatedness: Improved understanding about the cell biology of learning and memory will help to clarify the cellular mechanisms underlying mental retardation or cognitive deterioration that occurs with many disorders of the central nervous system.

More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy. Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Several recent studies have shown that elevated calcium concentration in the dendritic spine, which is crucial for the induction of LTP, is regulated independently of that in the parent dendrite. A likely candidate for this independent calcium regulation, is the spine apparatus and smooth endoplasmic reticulum of dendritic spines. This FIRCA application extends the studies of the Parent Grant to delineate alterations in the dendritic spine apparatus and other organelles, with LTP. The aims are: 1) To determine in the hippocampal area CA1 of adult rats whether the spine apparatuses, tubes of smooth endoplasmic reticulum (SER), and/or their association with other organelles, including polyribosomes, mitochondria, microtubules, or PSDs are altered by LTP. Five groups of hippocampal slices will be evaluated to ascertain transient and enduring changes that are specific to LTP, including; 1 and 2) experimental slices showing potentiation when they are fixed at 4 minutes and 4 hours posttetanus by our new microwave-enhanced procedure (Jensen and Harris, 1989, appendix), 3 and 4) experimental slices that are tetanized in the presence of APV to block LTP and fixed at 4 minutes and 4 hours posttetanus, and 3) control slices that are fixed alter receiving an equal number of stimuli spaced so as not to induce LTP. First, a series sample analysis (Sec Harris et al., 1992, appendix), will be conducted to determine the relative frequencies of dendritic spines containing spine apparatuses and other organelles. Then a randomly-selected subpopulation of these dendritic spines will be photographed and reconstructed through serial sections at the highest magnification possible. Complete reconstructions will be made at both the host and the foreign laboratories. 2) At postnatal day 15 to determine whether the "pre-spine" apparatuses described in Harris et al., 1992, are induced by LTP to form the mature spine apparatus. To determine also whether the associations of these immature spine apparatuses with other organelles in the spines are altered by LTP in the immature hippocampus. Health Relatedness: The foreign laboratory is the first to have described the spine apparatus in the normal adult human cortex, and found that its structure is similar to that in the rodent brain. In addition the spine apparatus is grossly distorted in the hypertrophied spines of epitumorous human cortex. Understanding changes in the spine apparatus and other organelles in dendritic spines with LTP will be important for elucidating their role in the human brain.

More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy (EM). Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Numerous anatomical studies have searched for anatomical correlates of LTP. These studies have lacked sufficient resolution to determine accurately the magnitude of the reported changes in the number of morphology of synapses and dendritic spines. A complete morphometric study is proposed to delineate the anatomical alterations at synapses and dendritic spines, to define their duration, and to relate these to various stages of LTP. The specific aims are: 1) To delineate the changes in synaptic and dendritic spine morphology that are associated with early and late phases of LTP in the hippocampus of postnatal day 15 (P15). This age is chosen because LTP is robust and enduring, even though the spines, synapses and dendrites have not attained their mature numbers or morphology. Because vigorous synaptogenesis is occurring at this age, multiple candidates exist for morphological plasticity that could subserve LTP. 2) To test whether mature synapses express morphological correlates of LTP that are similar to or different from those delineated at P15. It is essential for the understanding of mature memory mechanisms to establish whether mature synapses are similar to, differ only in degree from, or are categorically different from P15 synapses in their morphological plasticity. Methods: Standard procedures will be used to maintain in vitro hippocampal slices and induce and measure LTP extracellularly. Control hippocampal slices will receive the same stimulation paradigms as the LTP slices, But in the presence of DL-2-amino-phosphono-valeric acid (APV), a known blocker of LTP. Rapid microwave-enhanced fixation and routine processing will be used to prepare slices for electron microscopy. The irregularity in shape of dendritic spines and their synapses makes it impossible to extrapolate from partial measurements to their complete dimensions. Thus, the technically demanding approach of three-dimensional reconstruction from serial EM is required. Historically, the main drawback of serial EM has been the small sample sizes utilized because of the labor-intensive nature of the work. A new unbiased approach for sampling large areas of neutrophil in combination with serial EM is proposed to obtain accurate characterization of synaptic, spine, and dendritic morphologies and to allow for complete quantification of their ultrastructure in three dimensions. Health Relatedness: Improved understanding about the cell biology of learning and memory will help to clarify the cellular mechanisms underlying mental retardation or cognitive deterioration that occurs with many disorders of the central nervous system.

More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy. Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Several recent studies have shown that elevated calcium concentration in the dendritic spine, which is crucial for the induction of LTP, is regulated independently of that in the parent dendrite. A likely candidate for this independent calcium regulation, is the spine apparatus and smooth endoplasmic reticulum of dendritic spines. This FIRCA application extends the studies of the Parent Grant to delineate alterations in the dendritic spine apparatus and other organelles, with LTP. The aims are: 1) To determine in the hippocampal area CA1 of adult rats whether the spine apparatuses, tubes of smooth endoplasmic reticulum (SER), and/or their association with other organelles, including polyribosomes, mitochondria, microtubules, or PSDs are altered by LTP. Five groups of hippocampal slices will be evaluated to ascertain transient and enduring changes that are specific to LTP, including; 1 and 2) experimental slices showing potentiation when they are fixed at 4 minutes and 4 hours posttetanus by our new microwave-enhanced procedure (Jensen and Harris, 1989, appendix), 3 and 4) experimental slices that are tetanized in the presence of APV to block LTP and fixed at 4 minutes and 4 hours posttetanus, and 3) control slices that are fixed alter receiving an equal number of stimuli spaced so as not to induce LTP. First, a series sample analysis (Sec Harris et al., 1992, appendix), will be conducted to determine the relative frequencies of dendritic spines containing spine apparatuses and other organelles. Then a randomly-selected subpopulation of these dendritic spines will be photographed and reconstructed through serial sections at the highest magnification possible. Complete reconstructions will be made at both the host and the foreign laboratories. 2) At postnatal day 15 to determine whether the "pre-spine" apparatuses described in Harris et al., 1992, are induced by LTP to form the mature spine apparatus. To determine also whether the associations of these immature spine apparatuses with other organelles in the spines are altered by LTP in the immature hippocampus. Health Relatedness: The foreign laboratory is the first to have described the spine apparatus in the normal adult human cortex, and found that its structure is similar to that in the rodent brain. In addition the spine apparatus is grossly distorted in the hypertrophied spines of epitumorous human cortex. Understanding changes in the spine apparatus and other organelles in dendritic spines with LTP will be important for elucidating their role in the human brain.

DESCRIPTION (Investigator's Abstract): The long-range goal of our research is to understand the structural basis of synaptic plasticity, especially in relationship to learning and memory. The current proposal will test whether conditions that do or do not induce long-term potentiation (LTP) result in structural changes in the rat hippocampus. LTP is an activity-dependent enhancement of synaptic transmission which involves activation of glutamatergic synapses on dendritic spines and is considered to be a good model of some forms of learning. Due to its long endurance (hours to weeks), LTP is thought to involve the formation of new synapses and/or the remodeling of existing synapses. This hypothesis is being tested directly by comparing synaptic structure in the in vivo hippocampal neuropil with hippocampal slices that obtain LTP; have LTP blocked by APV, an antagonist to the NMDA class of glutamate receptors; or receive only non-tetanic control stimulation either in normal media or media with APV. So far the slices with LTP show an increase in one type of dendritic spine (stubby), in the number of synapses per presynaptic bouton, and in glial processes surrounding the synapses. In contrast, the only difference between in vivo hippocampus and control slices is a substantial increase in spines having a large head (mushroom) with a parallel decrease in thin spines. Based on these results, the proposed experiments provide a comprehensive strategy to distinguish among activity-dependent structural changes that share or do not share the same mechanisms as LTP. The specific aims are: 1) determine whether activation of non-NMDA or metabotropic glutamate receptors is required for the structural changes; 2) ascertain when the structural changes first occur post-tetanus and whether they are altered during later stages of LTP; 3) determine when mushroom dendritic spines first increase in vitro and whether low frequency stimulation retains the high ratio of thin to mushroom spines that occur in vivo and in the tetanized slices. Physiological responses and LTP will be measured in hippocampal slices that will then be rapidly fixed by a microwave-enhanced protocol. The unbiased series sampling method and 3-dimensional reconstructions from serial electron microscopy will be used to determine the underlying frequencies and structure of different types of synapses and glial processes. Given the involvement of glutamatergic synapses in numerous neurological disorders, it is increasingly important to understand their role in normal brain function at the most basic levels.

More than 90% of the excitatory synapses in the central nervous system occur on dendritic spines. Changes in the structure of these tiny protrusions have long been implicated in learning and memory, though a clear delineation of such morphological changes has awaited the magnification and resolution provided by serial electron microscopy. Long term potentiation (LTP) is an enduring change in synaptic efficacy that is widely studied as a cellular memory mechanism. Several recent studies have shown that elevated calcium concentration in the dendritic spine, which is crucial for the induction of LTP, is regulated independently of that in the parent dendrite. A likely candidate for this independent calcium regulation, is the spine apparatus and smooth endoplasmic reticulum of dendritic spines. This FIRCA application extends the studies of the Parent Grant to delineate alterations in the dendritic spine apparatus and other organelles, with LTP. The aims are: 1) To determine in the hippocampal area CA1 of adult rats whether the spine apparatuses, tubes of smooth endoplasmic reticulum (SER), and/or their association with other organelles, including polyribosomes, mitochondria, microtubules, or PSDs are altered by LTP. Five groups of hippocampal slices will be evaluated to ascertain transient and enduring changes that are specific to LTP, including; 1 and 2) experimental slices showing potentiation when they are fixed at 4 minutes and 4 hours posttetanus by our new microwave-enhanced procedure (Jensen and Harris, 1989, appendix), 3 and 4) experimental slices that are tetanized in the presence of APV to block LTP and fixed at 4 minutes and 4 hours posttetanus, and 3) control slices that are fixed alter receiving an equal number of stimuli spaced so as not to induce LTP. First, a series sample analysis (Sec Harris et al., 1992, appendix), will be conducted to determine the relative frequencies of dendritic spines containing spine apparatuses and other organelles. Then a randomly-selected subpopulation of these dendritic spines will be photographed and reconstructed through serial sections at the highest magnification possible. Complete reconstructions will be made at both the host and the foreign laboratories. 2) At postnatal day 15 to determine whether the "pre-spine" apparatuses described in Harris et al., 1992, are induced by LTP to form the mature spine apparatus. To determine also whether the associations of these immature spine apparatuses with other organelles in the spines are altered by LTP in the immature hippocampus. Health Relatedness: The foreign laboratory is the first to have described the spine apparatus in the normal adult human cortex, and found that its structure is similar to that in the rodent brain. In addition the spine apparatus is grossly distorted in the hypertrophied spines of epitumorous human cortex. Understanding changes in the spine apparatus and other organelles in dendritic spines with LTP will be important for elucidating their role in the human brain.

DESCRIPTION (Taken from application abstract): This project is directed at understanding the structure and function of synapses in the developing and mature brain, by quantitative and three-dimensional analysis of the structure, connectivity, and plasticity of presynaptic, postsynaptic, and glial elements. Such knowledge subserves understanding of the role of synaptic abnormalities in mental retardation. The informatics objective is to improve the methods of quantitative analysis and availability of anatomical data at the ultrastructural level by: 1. Developing an internet-accessible repository of synaptic and perisynaptic anatomy that will serve as a community resource for connecting the molecular and neuron levels of analyses. 2. Developing software for the public-domain that is internet accessible and third-party extensible to support and facilitate input and output of the database. 3. Improving the speed and accuracy of acquisition, alignment, calibration, segmentation, and 3D reconstruction of serial electron microscope sections from brain. 4. Developing software tools for quantitative comparison of different pre- and postsynaptic geometries and patterns of connectivity. The brain research objective is to test how synaptic structures can regulate synaptic function during development and plasticity by: 1. Investigating how synaptic and perisynaptic anatomy, change during development, during maintenance of slices in vitro, and during long-term potentiation (LTP) and long-term depression (LTD). 2. Examining how synaptic and perisynaptic anatomy differs during different levels of brain activity (hibernation, arousal, and wakeful activity). 3. Investigating the quantitative changes in spine and dendritic calcium due to activation of receptors coupled to the phosphoinositide second messenger system and determining the role this calcium signal plays in different types of synaptic plasticity (LTP, LTD, hibernation) using anatomical models from the database.

DESCRIPTION (provided by applicant): The long-term goal of this research is to specify changes in synapse structure in the brain that subserve learning and memory. Changes in synapse number or size have long been thought to underpin memory, but this hypothesis has not been proven because structural changes are difficult to measure, the altered synapses are difficult to identify, and the relevant circuits are not easily specified in mammals. To simplify this task the model system hippocampal long-term potentiation (LTP) is used to investigate these synaptic mechanisms. LTP is a protein synthesis-dependent enhancement in synaptic efficacy that can persist for months and there is abundant evidence that it plays an important role in learning and memory. Polyribosomes (PR) are structures where new proteins are synthesized. A discrete population of dendritic spines acquires PR and their synapses enlarge during LTP in hippocampal slices from immature rats. Missing from the slice experiments is information about whether the synaptic changes are sustained beyond several hours, whether the changes are strictly developmental, and whether similar changes occur in whole animals. The present experiments are designed to investigate synapses in the hippocampal dentate gyrus from mature rats that have undergone LTP after high-frequency stimulation in the medial perforant path. Quantitative serial electron microscopy and immunogold labeling will be used to distinguish changes in synapse structure and composition during different phases of LTP from 30 minutes to 3 months after its induction. Comparisons will be made between the potentiated medial perforant path synapses, the contralateral control medial perforant path synapses and the neighboring lateral perforant path and proximal associational synapses that become heterosynaptically depressed. Specific aims include: 1) Test for synapse enlargement at spines undergoing protein synthesis during LTP. 2) Investigate roles for synapse perforation, spinule formation, and cell adhesions in synapse enlargement and molecular components of synaptic remodeling during LTP. 3) Determine whether new dendritic protrusions give rise to enhanced connectivity during LTP. 4) Test NMDA receptor-dependence of structural and molecular changes to ensure they are related to synaptic plasticity, and not simply driven by neural activity. Understanding structural plasticity during LTP will elucidate mechanisms underlying normal changes as a basis for understanding brain pathology.

DESCRIPTION (Taken from application abstract): This project is directed at understanding the structure and function of synapses in the developing and mature brain, by quantitative and three-dimensional analysis of the structure, connectivity, and plasticity of presynaptic, postsynaptic, and glial elements. Such knowledge subserves understanding of the role of synaptic abnormalities in mental retardation. The informatics objective is to improve the methods of quantitative analysis and availability of anatomical data at the ultrastructural level by: 1. Developing an internet-accessible repository of synaptic and perisynaptic anatomy that will serve as a community resource for connecting the molecular and neuron levels of analyses. 2. Developing software for the public-domain that is internet accessible and third-party extensible to support and facilitate input and output of the database. 3. Improving the speed and accuracy of acquisition, alignment, calibration, segmentation, and 3D reconstruction of serial electron microscope sections from brain. 4. Developing software tools for quantitative comparison of different pre- and postsynaptic geometries and patterns of connectivity. The brain research objective is to test how synaptic structures can regulate synaptic function during development and plasticity by: 1. Investigating how synaptic and perisynaptic anatomy, change during development, during maintenance of slices in vitro, and during long-term potentiation (LTP) and long-term depression (LTD). 2. Examining how synaptic and perisynaptic anatomy differs during different levels of brain activity (hibernation, arousal, and wakeful activity). 3. Investigating the quantitative changes in spine and dendritic calcium due to activation of receptors coupled to the phosphoinositide second messenger system and determining the role this calcium signal plays in different types of synaptic plasticity (LTP, LTD, hibernation) using anatomical models from the database.

DESCRIPTION (provided by applicant): Neuronal dendrites and synapses are structurally distorted in individuals with mental retardation and other neurological disorders. Dendrites and synapses also differ greatly in their appearance in normal brains. Hence, variability in structure-function relationships must be understood in the normal brain to be able to draw conclusions about the distortions. A rigorous plan is proposed to identify structure- function relationships along dendrites in the mature hippocampus, a brain region long known to be involved in learning and memory. Six core subcellular structures will be investigated along dendrites and into dendritic spines that host excitory synapses, including microtubules for transport, polyribosomes for protein synthesis, Golgi apparatus for posttranslational modifications, endosomes for membrane recycling, and smooth endoplasmic reticulum (SER) and mitochondria for calcium regulation. Connectivity relationships among dendrites, axons and perisynaptic astroglia will also be discerned. Several recent findings from this laboratory reveal the power of serial section transmission electron microscopy (ssTEM) to investigate these relationships. Dendrites containing more microtubules had more synapses. Dendritic spines with polyribosomes or perisynaptic astroglia had larger synapses. Different spines contained endosomes from those containing SER. Only large, mature spines contained a spine apparatus, which is similar to the Golgi apparatus. A cellular memory mechanism, known as long-term potentiation (LTP), enhanced several of these relationships in both mature and immature hippocampus. New dendritic spines formed and existing spine synapses enlarged by 5-30 minutes after the induction of LTP. Spine membrane was supplied from local recycling endosomes. Only spines that acquired polyribosomes had enlarged synapses two hours later. Here it is proposed to determine whether this structural plasticity relates to recognized distance- dependent changes in dendritic function and caliber among dendrites in the mature hippocampal area CA1. We will assess whether or not dendrites of varying caliber have different levels of structural plasticity after the induction of LTP. We will ascertain whether dendrites with more or larger synapses connect with more of the axons surrounding them after the induction of LTP. We will determine distance-dependent differences in dendrite structure, composition, and connectivity throughout the CA1 apical and basilar dendritic arbors. We will enhance and develop new Neuroinformatics tools to collect and share these content-rich data. We predict greater understanding will emerge about distance dependent dendritic and synaptic structure and function. PUBLIC HEALTH RELEVANCE Neuronal dendrites and synapses appear structurally distorted in individuals with mental retardation and other neurological disorders. Dendrites and synapses are also structurally diverse in normal brains;hence the variability in structure-function relationships must be understood to draw meaningful conclusions about these distortions. A rigorous plan is proposed to use neuroinformatics tools and three-dimensional reconstruction to identify important structure-function relationships along the length of dendrites, at their synapses, and with their neighboring axons and astroglia during long-term potentiation, a well-studied cellular mechanism of learning and memory.

DESCRIPTION (provided by applicant): This revised proposal requests continued funding of an established graduate training program in neuroscience at the Institute for Neuroscience (INS) at the University of Texas at Austin. This program has grown substantially, with addition of a new director and numerous faculty members in Neurobiology, Psychology, Pharmacology and Toxicology, Computer Science, Biomedical Engineering, Kinesiology, Communication Sciences &Disorders, and Nutrition. Faculty also have appointments in Centers of Excellence (Center) including: Center for Learning and Memory, Center for Perceptual Systems, Institute for Cell and Molecular Biology, Waggoner Center for Alcohol and Addiction Research, Imaging Research Center and Computational Visualization Ctr. Research interests span molecular, biochemical, physiological and electrophysiological, computational neuroscience, behavior, Neuro-ethology and evolution of the nervous system. All training faculty have substantial funding giving graduate student's broad options for training in cross-disciplinary research among collaborating laboratories. Funding is requested for three new pre-doctoral trainees per year, each are funded for two years (totaling 6 per year). Students are required to complete the following: laboratory research rotations and present seminars based on that work, two core neuroscience courses, a responsible conduct of science/ethics course, a course on experimental design and statistics, and four electives including a neurobiology of disease course. Students join a research lab by the beginning of year two and complete coursework and qualifying exams by the end of year two. Graduate students are required to participate in Neuroscience seminar series and specialized journal clubs. We plan to continue successful recruitments of minority students. In summary, this program has particular strengths in the neurobiology of perceptual systems, learning and memory, and addiction research, and also provides excellent opportunities in areas ranging from computational to cellular approaches. This broad interdisciplinary training provided will prepare our trainees for research in neuroscience at multiple levels which are of great importance to discovering causes and treatments for brain diseases. RELEVANCE (See instructions): The major health problems in the US are brain diseases, including Alzheimer's, Schizophrenia, Alcoholism, Drug Addiction, Depression and Bipolar Disorders, yet our incomplete understanding of the neurobiology of these conditions provides for few effective treatments. The goal of this proposal is to provide state-of-the-art training for the next generation of neuroscientists who will help to solve these health problems.

DESCRIPTION (provided by applicant): Dendritic spines host >90 percent of excitatory synapses; they are lost or have abnormal structure in many developmental disorders that disrupt central nervous system function. The overall goal is to understand the role of spine and synapse structure in the normal development of learning and memory. Long-term potentiation (LTP) is a synaptic model of learning and memory well-suited to investigate this process. Spines are thought to be important because they sequester core structures and molecules needed for the protein synthesis-dependent or late phase of LTP (L-LTP) lasting >3hr. A clear understanding requires the nanometer resolution of 3D reconstruction from serial section electron microscopy, an approach pioneered in this laboratory. Rigorous experiments are proposed to test whether formation of dendritic spines and structural synaptic plasticity provide general mechanisms for the developmental regulation of L-LTP in hippocampus, a brain region crucial for learning and memory. Aim 1 is to test the hypothesis that the abrupt onset of L-LTP at postnatal day (P)12 is associated with first occurrence of dendritic spines and capacity for structural synaptic plasticity. The experiments will determine what differentiates dendritic, axonal, spine, and synaptic structure and composition at P12, from P8 and P10 when L-LTP is not produced by one bout of TBS. They will test whether production of L-LTP at P12 results in a balanced elimination of small spines and enlargement of remaining synapses as occurs in mature hippocampus and whether pre- and postsynaptic structural remodeling are synchronized during development with the ability to express L-LTP. Aim 2 is to test the hypothesis that dendritic spines are induced by TBS and then serve to sustain L-LTP after a second bout of TBS at P10, but not at P8, when multiple TBS do not produce L-LTP. Aim 3 is to ascertain the developmental onset of L-LTP and its ultrastructural correlates in mouse hippocampus as a foundation for future work using genetic manipulations. The outcomes promise new insight into the synaptic basis of learning and memory, essential knowledge to design effective treatments for developmental brain disorders.

? DESCRIPTION (provided by applicant): The long-term goal is to understand how synapse structure in the adult central nervous system supports learning and memory. We study hippocampal long-term potentiation (LTP), a form of synaptic plasticity that is viewed as a cellular substrate for learning and memory. The focus is on dendritic spines, the tiny protrusions that host more than 90% of excitatory synapses in the brain, are modified during LTP, learning, and memory, and are severely distorted in a variety of neurological disorders. Recently, we have shown that initially saturated LTP can be subsequently augmented if more than 1.5 hours elapse between episodes of LTP induction, with 100% success achieved in adult mouse hippocampus after a 4 hour interval. These findings support the hypothesis that spacing episodes of LTP engages mechanisms that might underlie the advantage of spaced over massed learning. Using 3D reconstruction from serial section electron microscopy (3DEM), we have discovered several changes in synapse structure that manifest over time after the initial saturation of LTP. Both in vivo and in vitro, many synapses had nascent zones, dynamic edge regions that have a postsynaptic density but lack the presynaptic vesicles normally found at active zones. Nascent zones rapidly acquired presynaptic vesicles, thereby converting to active zones however by 30 min. By 2 hr., both nascent and active zones were enlarged, and the greatest synapse enlargement occurred on spines that contained smooth endoplasmic reticulum (SER) and polyribosomes. We will test the hypothesis that the capacity to modify synaptic structure is initially saturated by LTP, and time is required for synapses to recover or grow in preparation for later augmentation. We will verify successful LTP induction with physiology following various pharmacological and genetic manipulations in mature mouse hippocampus prior to performing the more time consuming 3DEM and immunolabeling. We aim to test the following hypotheses regarding mechanisms of augmentation: 1) That receptors and presynaptic docking sites must accumulate at synapses enlarged after the initial saturation of LTP. 2) That SER-dependent synapse growth and spine clustering serve the augmentation of LTP. 3) That protein synthesis-dependent growth of synapses is required for augmentation of LTP. 4) That absence of candidate molecules involved in building or stabilizing synapses disrupts saturation or augmentation of LTP. Upon completion of these aims we will know which elements of structural synaptic plasticity are integral to the augmentation of LTP. Outcomes will provide new understanding of the mechanisms of nascent zone conversion, synapse growth, and spine clustering, and whether these processes are coupled to SER expansion, local protein synthesis, and synapse adhesion in preparation for the subsequent augmentation of initially saturated LTP in the mature mouse brain. The results should ultimately inform the development of new strategies to repair dysfunctional synaptic circuits.

As part of the NSF National Research Infrastructure for Neuroscience, this Neurotechnology Hub will develop new approaches to examine the brain in greater detail. Three-dimensional electron microscopy (3DEM) has helped reveal new insights into the role of tiny connections between cells in the brain. However, 3DEM has been limited in impact by the rate of data analysis. This Neurotechnology Hub will improve the 3DEM instrumentation to collect information in greater detail, develop better algorithms to process the information, and link the workflows with high performance computing to greatly increase the rate of knowledge discovery. These innovations and capabilities will be shared with the scientific community through active training on the approach, and through open access to the new software and data. By way of this Neurotechnology Hub, 3DEM will become part of the national infrastructure for neuroscience research. To help address the grand scientific challenge of understanding the brain, this project will apply the improved 3DEM approach across several different mammalian species including humans to identify similarities and differences, and their relationship to behavior, learning, and memory.

This Neurotechnology Hub is motivated by challenges in understanding synapses, the tiny points of inter-neuronal communication. The variance in synapse dimensions, connectivity, and subcellular content across species is simply not known, yet required to determine whether model systems represent human brain functions. Current approaches are limited by resolution, inefficient data collection, and analysis bottlenecks. Addressing these challenges, the project will: (1) Develop simultaneous multi-detector and tilt-tomography on the scanning electron microscope operating in the transmission mode. Add-on hardware and software will improve axial resolution from 45 to 10 nm (or less), while maintaining in-plane resolution of 1-2 nm. (2) Integrate automated and interactive tools that speed and improve analysis of synapses in large data volumes. The enhanced resolution will increase data volume but reduce major image processing difficulties by producing more isotropic images. (3) Integrate the enhanced electron microscopy (EM) with high performance computing to increase throughput; to disseminate images, metadata, analyses, and software in a way that facilitates uptake into existing cell type and brain databases; and to provide a venue to develop 3DEM communities. (4) Apply the new technology to image hippocampus and comparable parts of cortex in mice, rats, and humans. This NeuroTechnology Hub award is co-funded by the Division of Emerging Frontiers within the Directorate for Biological Sciences and the Office of Advanced Cyberinfrastructure within the Directorate for Computer and Information Sciences, as part of the BRAIN Initiative and NSF's Understanding the Brain activities.

The overall goal is to understand synaptic mechanisms of learning and memory. Long-term potentiation (LTP) is a model of learning and memory that is well-suited to investigate these processes. Dendritic spines host about ninety percent of excitatory synapses in the brain and are well known to show structural plasticity following induction of LTP. The developmental onset of dendritic spines coincides with an abrupt developmental onset for LTP lasting more than three hours (L-LTP) at postnatal day 12 (P12) in rat hippocampus. At P10 and P15, LTP enhances synaptogenesis and small spine formation. With maturation, the LTP-accelerated synaptogenesis shifts to a process that enlarges specific synapses and retains spine clusters locally but is balanced by reduction in spine numbers elsewhere on the dendrite. The spine clusters are locally delimited by the availability of smooth endoplasmic reticulum (SER), an organelle critical for regulating calcium, and the transport of lipids and proteins, and by the presence of polyribosomes, which mediate local protein synthesis. The LTP-produced synapse enlargement is greatest on spines that contain a spine apparatus, which is a structure derived from SER that provides synthesis and post-translational modification of transmembrane proteins. Structural changes in presynaptic axons are also developmentally regulated following LTP and mirror the spine changes with new boutons forming to accommodate the LTP-accelerated synaptogenesis at P15, and fewer boutons occurring with spine reduction at P60. Thus, LTP in developing hippocampus accelerates synaptogenesis, whereas resource-dependent synapse growth and spine clustering occur on mature dendrites. This homeostatic balance in synaptic plasticity is hypothesized to be disrupted with cognitive decline in the aging brain. A comprehensive analysis of structural synaptic plasticity during maturation and senescence is proposed as a foundation for understanding lifelong changes in cognitive capacity. Specifically, the aims are: 1) To determine whether shifts in synaptic plasticity underlie maturational milestones in learning. 2) Determine generality of resource-dependent synapse growth and spine clustering across circuits. 3) To determine whether LTP-related structural plasticity diminishes in the aging hippocampus as cognitive capacity declines. 4) To test whether removal of the spine apparatus impairs resource-dependent synapse growth and spine clustering following LTP. Outcomes promise essential insight into the synaptic basis of learning and memory across lifespan and will provide basic knowledge that could inform new therapies for developmental and age-related brain disorders.

Trillions of synapses connect billions of neurons in neural circuits that allow sensation, thought, action, learning, and memory. This NeuroNex Network involves the development of new approaches to determine the strength of connections between neurons?synaptic weight--in the brain. Understanding synaptic weight is crucial, yet even a clear definition remains elusive, despite more than a century of searching. This NeuroNex Network assembles world experts to study synapses from molecules to behavior, to answer this fundamental and ambitious question: What constitutes synaptic weight, and what role does it play in shaping neural circuits? Synaptic weight is hypothesized to involve the differential composition and co-occurrence of key proteins and subcellular resources. Multidisciplinary approaches are used to assess these features in well-defined states of neural circuits involving multiple cell types, brain regions, and diverse behaviors. Consistent predictors of synaptic state are mapped onto neural connectomes to enhance understanding of how synaptic weight influences circuit organization and function. New electron microscopy technologies developed and used in this project bridge gaps in image size and resolution needed to achieve deeper understanding of brain function and regulation from nanoscale to circuit levels. A long-lasting, far-reaching impact involves leveraging work from this NeuroNex Network with other BRAIN Initiative projects to enable acquisition and sharing of the new knowledge. Future applications, even beyond the brain, of the knowledge and tools developed here will give rise to data that address fundamental and novel principles of complex self-organizing systems. The NeuroNex Network also involves training the next generation, including through inter-laboratory and fellow exchanges.

What constitutes synaptic weight, what role does it play in shaping neural circuits, and how does it change during growth and plasticity? Answers require a shift away from thinking about synapses as isolated entities. Synapses are not simply on or off one-bit machines; instead the information content stored in synapse size, as a proxy for weight, is much higher. Synaptic weight is controlled over broad temporal and spatial scales dynamically regulated by neural activity. New evidence points to subcellular resources (endoplasmic reticulum, mitochondria, endosomes, ribosomes) as brokers that drive synaptic efficacy and plasticity. This project seeks to understand how synapse composition and structure predict synaptic weight and function at a scale that reveals biological mechanisms at the subcellular level. A new 3D electron microscopy (EM) approach is developed using conical tilt tomography on the scanning EM operating in the transmission mode (tomoSEM). TomoSEM fills the current resolution-to-volume gap between methods of structural biology (high resolution, small volumes) and connectomics (relatively low resolution, larger volumes). TomoSEM eliminates major artifacts of other EM methods while reducing human effort and cost. The investigators comprise world experts in protein chemistry, cell biology, connectomics, and behavior. Experts in EM implement, validate, and deploy tomoSEM. Experts in image analysis, geometry, statistics, machine learning, and multilevel modeling create platforms to search data for hidden order. These strategies share international resources to overcome limits of accumulating data locally one synapse at a time. This project is co-funded by Emerging Frontiers in the Directorate for Biological Sciences.

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.

Successful learning and long-term memory retention are central to a successful society, starting with early education in schools and extending throughout life. For over 100 years research has shown that spaced learning is much more effective than massed learning for long-term memories. The efficiency of focused learning falls after an hour, which is paralleled in lab experiments at the level of single synapse between neurons, whose strength is saturated by focused stimulation. This project seeks to understand the synaptic mechanisms that eventually lead to continued synaptic growth on the time scale of many hours. The project hypothesis is that over this time period, regions inside the synapse open up to make room for a larger and stronger synapse. This research is the first step toward helping those with learning disabilities and new ways to enhance learning in others.<br/><br/>The goal of this research is to build imaging, analytical, and computational tools to investigate the structure of nanodomains within the synapse. The nanodomains comprise nascent and active zones of synapses. The nascent zones have a fully defined postsynaptic region but lack presynaptic vesicles and hence are silent. New EM tomographic imaging combined with new computational analyses will refine understanding of nascent zones as they recruit presynaptic vesicles and are thus converted to active zones in support of synaptic plasticity that underlies the advantage of spaced learning. Existing and newly acquired large data sets will be analyzed at scale with artificial intelligence. This research will be transformative for Data-Intensive Neuroscience and Cognitive Science. The data sets and AI tools will be shared broadly with the neuroscience community through the NSF-funded 3Dem Portal (3dem.org) at the Texas Advanced Computing Center (TACC). The objectives of this project are: 1) Create computational tools to delineate nascent zones automatically by mapping presynaptic vesicle docking sites in serial sections of synapses in the hippocampal CA1 region and dentate gyrus at various times after induction of LTP or cLTD, compared to control stimulation. 2) Apply a new computational analysis based on information theory and overall synapse size to measure the storage capacity of synapses, refining the definition of synaptic weight as encompassing the enlarged active zones obtained during the conversion of nascent zones. 3) Perform realistic Monte Carlo reaction-diffusion simulations of synaptic nanodomain 3D structure and function using MCell to provide a functional estimate for the boundary between nascent and active zones and determine how changes in nascent and active zones alter efficacy at synapses during saturation and recovery of LTP and cLTD.<br/><br/>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.