Samuel S.-H. Wang - US grants

Basic mechanisms of synaptic integration will be explored at the parallel fiber synapse onto the rat cerebellar Purkinje neuron. Purkinje neurons were chosen for their dendrites, which have the most complex known arborization in the animal kingdom and receive about 100,000 inputs per Purkinje neuron. Methods to be used include whole-cell patch clamp recording, focal photolysis of caged compounds, video-rate confocal fluorescence microscopy of calcium-sensitive dyes, and advanced image processing. The hypothesis to be tested is: synaptic activity produces calcium and other second messenger signals that are localized in space and time, and this signal localization sets parameters of dendritic integration and synaptic plasticity. These studies will provide a greater understanding of how synaptic integration and plasticity contribute to thought, learning, and memory.

Synaptic learning rules describe the relationship between neural activity patterns and the resulting changes in synaptic strength. Such persistent modifications of synaptic strength are widely believed to underlie functional changes such as learning in the central nervous system. Increases and decreases in synaptic strength - long-term potentiation (LTP) and long-term depression (LTD) - are present in a wide variety of excitatory synapses and have been the focus of thirty years of intensive research. In recent years, whether an activity pattern induces LTP or LTD has been shown to depend on the exact timing of single presynaptic and postsynaptic action potentials. But in the most general case, the rules for how activity is mapped to plasticity are not well understood. A key issue in the study of LTP and LTD is the lack of a unifying model that explains synaptic plasticity in terms of individual plasticity events. Work in this proposal will identify separable components of bidirectional plasticity. The investigators will then measure the activity dependent properties of these components, and use these properties to account for the original rules observed in the entire synaptic ensemble. The model system to be used is the mammalian hippocampal Schaffer collateral-CA1 synapse, which has NMDA-type and metabotropic glutamate receptors that evoke postsynaptic dendritic calcium signals. Calcium is an intermediate bottleneck in synaptic plasticity, and this fact will be used to divide the project into separate questions: How is activity mapped to calcium? How is calcium mapped to plasticity? These questions will be pursued in three stages. First, bidirectional plasticity will be separated into component mechanisms of potentiation and depression/depotentiation. Second, rules will be measured by which activity is transformed into synaptic plasticity and to dendritic calcium signals, which are known to be necessary and sufficient to induce both potentiation and depression. Third, calcium signals will be identified that are optimally tuned to evoke potentiation or depression, and used to create a general model predicting plasticity for arbitrary patterns of synaptic activity. Taken together, these experiments will test the idea that calcium signals can be used to predict the type and amount of plasticity resulting from an arbitrary pattern of synaptic neural activity. Experimental tools include patch clamp recording, multiphoton laser scanning microscopy, and focal photolysis of caged neurotransmitters and second messengers. Particular use will be made of an instrument for uncaging at many sites at once (up to 100,000 locations per second). The proposed experiments may help provide a general framework for learning rules not only at hippocampal synapses, but in the rest of the vertebrate central nervous system.

This work will support the continued development of the institutions program in neurobiology through course development and graduate and postdoctoral training. Further, the work should result in continued development of experimental tools useful to the scientific community.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, 06-NS-106: Validating new methods to study brain connectivity. We propose to test a new method that provides substantial improvement over previous Cre-conditional viral tracers. The technology combines the Brainbow multicolor cell marking technology with the retrograde, circuit tracing properties of pseudorabies virus (PRV), a neuroinvasive alpha herpesvirus. We have constructed a prototype PRV Brainbow virus called PRV263 that we propose will enable simultaneous identification of distinct chains of neurons projecting to a phenotypically defined population of neurons, and promises to provide predictive data on the strength of different connections among those neurons. Importantly, these PRV Brainbow tracers will have distinct advantages over present tracers. Our concept takes advantage of conditional, site-specific recombination of the genome of a DNA virus to produce multiple reporters so that neurons upstream (presynaptic) of a Cre recombinase (Cre) expressing neuron will be a different color from the Cre-expressing neuron. This novel concept will be expanded to produce second generation prototypes of PRV Brainbow tracers that do not rely on Cre-transgenic mice and can be used in the many mammalian species susceptible to PRV infection. A third generation prototype will be constructed that not only marks circuits, but also reports on neuronal activity. In this latter concept, the PRV Brainbow virus also will include a genetically encoded calcium indicator that fluoresces when calcium is bound. As viral tracing of neural circuitry has become an essential tool in the neuroscience community, our new tracers will be immediately applicable for many ongoing fundamental research projects in neuroscience in a variety of animals. These new tools have promise to reveal detailed functional insights into neural circuit organization that have not been possible to achieve in the past. Viral tracing of neural circuitry has become an essential tool in the neuroscience community. The new, robust viral tracers that will result from our work have promise to reveal detailed functional insights into trans-neuronal spread of herpesviruses, as well as neural circuit organization that have not been possible to achieve in the past. These neural tracers would be powerful tools to elucidate brain micro-circuitry, providing a better understanding of nervous system functions.

DESCRIPTION (provided by applicant): This application requests funds to purchase a Prairie Instruments Ultima multiphoton microscope system. This new instrument will greatly extend the current capabilities for fluorescence microscopy at Princeton University. The Prairie system will allow researchers to perform multiphoton microscopy deep in thick specimens labeled with multiple fluorescent probes at high frame rates, while reducing photodamage and avoiding loss of signal from scattering of light entering and exiting the specimen. Existing microscopes at Princeton University are conventional confocal microscopes, limiting the depth of focus and the duration of observation that is possible. The Prairie system's multiphoton frame scanning capability allows researchers to acquire dynamic colocalization data with high spatial resolution in tissues where scattering prevents one-photon methods (e.g. confocal microscopy) from penetrating. Examples include fly embryos and ovaries, developing zebrafish, and mammalian tissues ranging from bone to brain. The system accomplishes fast frame scanning by using acousto-optical devices for beam steering, thus exceeding the scan speed limit imposed by using galvanometer-mounted mirrors. Substage detection will increase the signal collection by a factor of two or more compared with light collection through the excitation objective alone. In addition to these imaging features, a second laser and set of galvanometers allow photoactivation of fluorescent proteins and photolysis of synthetic "caged" signaling molecules. Overall, these capabilities will allow Princeton researchers to track rapidly moving intracellular particles, morphological change, and biochemical dynamics on a subcellular scale with movie-rate temporal resolution. Research currently undertaken at Princeton which will benefit greatly from the Prairie system include studies of mRNA transport and cell movement in Drosophila oocytes and embryos, herpes virus assembly and transport in neuronal cells, multicellular signaling by glia and neurons in intact brain tissue, and morphogenesis of normal and cancerous ducts in breast tissue. Study of each of these biological systems will provide valuable information pertaining to significant human health problems such as birth defects, viral infection, neurological disease, and cancer.

DESCRIPTION (provided by applicant): The cerebellum is important for the control of movement, sensory processing, and regulation of cognitive and emotional function. In adulthood, damage to this region in adulthood leads to debilitating problems with everyday life; in infancy, cerebellar damage dramatically increases the risk of autism, a neurodevelopmental disorder. The long-term goal of this laboratory is to understand how early damage to the cerebellum can lead to symptoms of autism. In particular, the proposed experiments will use new technologies to study the function of individual cerebellar neurons that are involved in learning to anticipate predictable events in both awake mice, both normal and in mice with genetic defects that cause autism in humans. The overall objective of this application is to understand the function of the cerebellum in awake, behaving animals and then to use that information to understand how this circuit malfunctions in mouse models of autism spectrum disorder. This contribution is significant because it will produce detailed and integrated knowledge of the function of an important neural circuit under realistic conditions and apply that knowledge to a common neurodevelopmental disorder. This approach is innovative because this laboratory has developed tools that allow the study of cells that previously could not be examined in awake animals. The work proposed in this application will therefore advance knowledge of how the genetic mutations that cause autism influence the function of neural circuits. In the long run, this information could lead to new approaches to diagnosis, treatment, and prevention of autism spectrum disorder.

? DESCRIPTION (provided by applicant): Working memory, the ability to temporarily hold multiple pieces of information for mental manipulation, is central to virtually all cognitive abiliies. Working memory has been closely associated with multiple kinds of neural activity dynamics, such as persistent neural activity, activity ramps, and activity sequences. The neural circuit mechanisms of these dynamics remain unclear. This proposal will apply advanced technologies such as virtual reality, automated monitoring of behavior, in vivo microscopy, ontogenetic, and neural circuit reconstruction to solve fundamental problems in the understanding of working memory. The accumulation of evidence over time scales of seconds, a type of working memory critical for decision-making, will be used as a test bed for studying working memory. The proposal will build upon a rodent evidence-accumulation paradigm that allows quantitative, temporally precise parameterization of working memory and decision-making. The paradigm will be implemented with head-fixed rodents behaving in a virtual reality system (Aim 1), providing mechanical stability that enables the use of two-photon calcium imaging to observe neural activity related to working memory in the neocortex, basal ganglia, and cerebellum (Aim 3). Brain activity will also be perturbed using ontogenetic to probe the roles of brain regions and specific cell types in the formation and stabilization of memory (Aim 2). Finally, we will develop methods for probing the roles of cell types and connectivity in working memory through correlative serial electron microscopy and light microscopy as well as imaging of population responses to ontogenetic stimulation of single cells or groups of cells (Aim 4). This three-year project will produce a catalog of the types of neural circuit dynamics that are related to working memory across many brain regions. In subsequent years, this catalog will be mechanistically investigated by the anatomical and physiological methods developed in Aim 4. The long-term goal of this project is to arrive at a complete, brain-wide understanding of the cellular and circut mechanisms of activity dynamics related to working memory. The understanding is expected to take the form of a new generation of models containing cognitive variables distributed across brain regions, as well as models that explicitly represent neural circuit dynamics. This achievement will be a crucial step towards a mechanistic understanding of the neural basis of cognition.

? DESCRIPTION (provided by applicant): A long-term goal of the BRAIN Initiative is to track activity in large numbers of neurons individually in behaving animals, thus capturing the information processing that is done by brain circuitry. A universal signal in neurons when they are active is the messenger ion calcium. By examining changes in calcium concentration following each action potential in an individual neuron, researchers can track that neuron's activity, but at present, the time resolution of this approach is limited. The overall objective of this application is to use calcium-sensitive probes in spike-counting mode to detect individual action potentials as discrete events in space and time. The proposed experiments will produce new technology that can follow activity with millisecond resolution in hundreds to thousands of neurons at once. This contribution is significant because it will allow researchers to image important neural circuits noninvasively with millisecond resolution in awake animals. This approach is innovative because the investigators will use a novel, ultrafast response mode of a highly popular calcium sensor protein to obtain time resolution that is not otherwise possible. The work proposed in this application therefore advances our ability to understand how the brain functions in health and disease. In the long run, this information could lead to new approaches to diagnosis, treatment, and prevention of a variety of brain disorders.

? DESCRIPTION (provided by applicant): Many pathways that carry signals from one neuron to another involve calcium as a messenger molecule. By examining changes in calcium concentration within individual neurons, researchers can learn how these neurons interact within circuits. A long-term goal of this laboratory is to produce indicators that can track the full rang of calcium changes in large numbers of neurons in behaving animals across days to weeks. In particular, the proposed experiments will produce new technology that can follow activity in neuronal cell types that are involved in important brain functions, from fine motor control to reward learning. The overall objective of this application is to design probes that can follow rapi neural activity in awake, behaving animals and to demonstrate their usefulness in studying cell types whose activity cannot be recorded using currently available techniques. This contribution is significant because it will allow researchers to investigate the function of important neural circuits under realistic conditions. This approach is innovative because this laboratory will develop tools that allow the study of cells that previously could not be examined in awake animals. The work proposed in this application will therefore advance our ability to understand how the brain functions in health and disease. In the long run, this information could lead to new approaches to diagnosis, treatment, and prevention of a variety of brain disorders.

Project Summary Working memory, the ability to temporarily hold multiple pieces of information in mind for manipulation, is central to virtually all cognitive abilities. Recent technical advances have opened an unprecedented opportunity to comprehensively dissect the neural circuit mechanisms of this ability across multiple brain areas. The task to be studied is a common form of decision-making that is based on the gradual accumulation of sensory evidence and thus relies on working memory. A team of leading experts propose to investigate the neural basis of this behavior using the latest techniques, including virtual reality, high-throughput automated behavioral training, large-scale cellular-resolution imaging in behaving rodents, manipulation of neural activity in specific brain areas and cell types, and automated anatomical reconstruction. In particular, the researchers will identify key brain regions that are required for this decision task through systematic, temporally specific inactivations via optogenetics technology, across all of dorsal cortex and in key subcortical areas, and use quantitative model-fitting to evaluate the effects. They will use state-of-the-art two-photon calcium imaging methods and electrophysiology to characterize the information flow in many individual neurons within these brain areas during the task. In addition, they will use cutting-edge anatomical reconstructions and new functional connectivity methods, within and across brain regions, to evaluate the interactions of these physiologically characterized neurons. The long-term goal of this project is to arrive at a complete, brain-wide understanding of the cellular and circuit mechanisms of activity dynamics related to working memory. Finally, they will use sophisticated computational methods to incorporate this new understanding into a realistic circuit model that will support a tightly integrated process of model-guided experimental design, in which the model suggests the most informative experiments and their results are then fed back to improve the model?s fidelity. This process is expected to produce the most accurate and detailed multi-brain-region biophysical circuit model of a cognitive process in existence. In addition, the proposed research will enable researchers to generate and test a variety of hypotheses about the neural basis of evidence accumulation, working memory, and decision-making. Taken together, these achievements will represent a crucial step toward a mechanistic understanding of how the brain works with information.

Project  Summary    Flexible behavior is central to virtually all cognitive and social abilities. Recent technical advances have opened an  unprecedented opportunity to comprehensively dissect the neural circuit mechanisms of this ability across multiple  brain areas in freely behaving animals. This proposal focuses on the cerebellum, a structure that is a major site of  pathology in autism spectrum disorder. Damage to the cerebellum at birth leads to a 36­fold increase in the risk of  autism,  and  this  region  is  also  a  principal  site  for  co­expression  of  autism  risk  genes.  Thus  cerebellar  development  may  act  as  an  intermediate  mechanistic  step  in  transducing  inherited  autism  risk  into  neurodevelopmental  phenotypes.  In  this  project,  a  multidisciplinary  team  of  leading  experts  proposes  to  investigate  the  neural  basis  of  this  disorder  using  advanced  technologies,  including  unbiased  automatic  classification  of  behavior,  large­scale  cellular­resolution imaging in behaving rodents, mouse genetic models for  autism, and manipulation of neural activity in specific cerebellar areas and cell types. In genetic mouse models of  autism,  the  researchers  will  identify  modes  of  behavior  based  on  physical  poses,  and  relate  these  modes  to  classical  behavioral  tests,  such  as  eyeblink  conditioning,  and  to cerebellar circuit dysfunction. In adult wild­type  and autism model mice, the researchers will use optogenetic methods to perturb specific cerebellar lobules while  quantifying  the  effects  on  behavioral  dynamics  and  learning.  In  juvenile  model  mice,  the  researchers  will  use  chemogenetic methods to identify long­lasting patterns of behavioral disruption and relate these patterns across  behaviors to build a quantitative map of these perturbations. In addition, they will use in vivo dendritic imaging to  evaluate the influences of cerebellar perturbation on neocortical neuron structure. All of these results will inform  modeling  of  cerebellar­neocortical  interactions  to  better  understand how these differently wired regions interact  during learning and development. The long­term goal of this project is to arrive at a chain of explanation, centered  on  principles  of  convergent  neuroscience,  to  understand  causal mechanisms of neurodevelopmental disorders.  This  project  will  join  genetics  with  circuit  function,  local  cerebellar  anatomy  with  behavioral  outcomes,  and  classical  behavioral  tests  with  modern  unbiased  methods.  This project is expected to produce an accurate and  detailed  understanding  of  cerebellar  contributions  to  normal  and  aberrant  neurodevelopment.  In  addition,  the  proposed research will enable researchers to generate and test a variety of hypotheses about the neural basis of  flexible  behavior.  Taken  together,  these  achievements  will  represent  a  crucial  step  toward  a  mechanistic  understanding  of  how the brain develops its complex ability to respond flexibly to the environment, from birth to  adulthood.   

Project Summary: Core 4, Brain Registration and Pathway Tracing    Working memory, the ability to temporarily hold multiple pieces of information in mind for manipulation, is  central to virtually all cognitive abilities. This multi-component research project aims to comprehensively  dissect the neural circuit mechanisms of this ability across multiple brain areas. The behavior to be studied is  a type of decision-making task that is based on the gradual accumulation of sensory evidence and thus relies  on working memory. A full understanding of how this behavior relates to this brain function requires  explanation at multiple levels: from neural activity in particular regions to how those regions interact in  brain-wide networks via specific pathways. These levels of analysis require distinct technical approaches,  which are often difficult to relate to one another rigorously. This Core will promote rigor and reproducibility in  the proposed research by producing an anatomical framework to standardize and compare the various types  of data that will be collected. The facility will serve several essential functions in building a broad integrative  structure for the project. First, it will produce standardized functional maps that will be used to accurately  determine the boundaries of visual cortical regions before cellular-resolution imaging or inactivation studies.  Second, it will register all studied brain areas into an anatomical context that includes connectivity and  functional significance. Automated cell-recognition methods will be used to survey directly imaged regions  and indirectly connected regions, and to classify neurons and other objects of interest. Third, it will support  long-distance tracing across synapses to identify paths of connectivity between distant brain regions involved  in evidence accumulation. Fourth, it will organize this information in a relational database that links all the  experiments, in a format that can be shared with the neuroscience research community. As technologies for  functional mapping, registration, and tracing advance over time, this facility will evaluate new methods, adopt  those that will substantially improve the Core?s capabilities, and train project personnel in their use. Taken  together, these functions are essential for placing recorded and perturbed neural activity into a brain-wide  anatomical context, which will enable the integration of information produced by individual experiments and  techniques into a coherent theoretical framework. 

PROJECT SUMMARY/ABSTRACT The cerebellum integrates sensory, motor, and internal information to rapidly guide and fine-tune action. This process has been investigated most extensively for movement control, but the cerebellum is also involved in the updating of internal states such as reward and working memory. Previous work from this laboratory shows that the cerebellar region crus I is required for evidence accumulation and decision-making. These findings, along with preliminary data, led to the hypothesis that cerebellar processing of sensory and internal information evolves over the course of learning to exert moment-to-moment predictive influence and shape flexible behavior. The proposed experiments will determine, with quantitative rigor, how cognitive regions of the cerebellum contribute to neural coding, predictive learning, and forebrain target activity. Past studies of cerebellar contributions to cognition have been hampered by the coarseness with which neuronal activity could be monitored and perturbed, pathways traced, and behavior measured. This proposal will overcome these limitations by using advanced tools, including two-photon calcium imaging, whole-brain transsynaptic viral tracing, high-density silicon probe recording, and optogenetic perturbation. Aim 1 will determine how predictive information in cerebellar activity influences working memory. In an evidence-accumulation decision task that distinguishes neural activity related to evidence accumulation, information retention, and decisions, preliminary data show that optogenetic inactivation of crus I removes the dependence of decisions on previous evidence, indicating a necessary role in evidence integration. This aim will examine the main cerebellar pathway with optogenetics, two-photon imaging, and many-electrode recording to probe learned cerebellar contributions to sensory processing, working memory, decisions, and motor output with subsecond time resolution. Aim 2 will characterize learning and transfer of working memory-related neural dynamics. This aim will examine how task representations evolve during learning in Purkinje cells and deep-nuclear neurons to test the idea that intrinsic cerebellar signals involved in movement preparation provide a foundation for learning neural responses that accumulate sensory evidence over time. Aim 3 will evaluate how cerebellar areas involved in cognition shape activity in connected forebrain areas. This aim will use transsynaptic viral tracing to identify pathways from crus I through midbrain and thalamus to their targets in the neocortex, and then specifically perturb and monitor these pathways to identify their contribution to task performance. The long-term goal of this project is to build a quantitative explanatory framework for cerebellar function in complex behavior. The results are expected to inform computational models that predict and explain the impact of detailed cerebellum-forebrain interactions. Together, these studies will significantly advance basic neuroscience of the cerebellum and contribute to understanding of syndromes marked by cerebellar dysfunction, including attention-deficit hyperactivity disorder and autism spectrum disorder.