Peter Saggau - US grants

There is compelling evidence for the involvement of presynaptic inhibition in controlling normal synaptic transmission and preventing excessive neurotransmitter release at mammalian central synapses. The presynaptic site is an effective target for modulation of synaptic transmission and presynaptic voltage-dependent calcium channels play a significant role in controlling transmitter release. To address our long-term objective of understanding the basic presynaptic mechanisms underlying modulation of synaptic transmission in mammalian central synapses, we propose to investigate the direct and indirect role of presynaptic calcium during inhibition of synaptic transmission at hippocampal CA3/CA1, MF/CA3 and PP/GC excitatory synapses. This in vitro study will employ hippocampal brain slices and optical imaging techniques. We will selectively load presynaptic terminals with ion-sensitive indicators to investigate resting levels and transients of presynaptic calcium and presynaptic potassium. In addition, we will use voltage- sensitive dyes to measure presynaptic action potentials. Specific blockers will be utilized to identify and quantify the types of presynaptic voltage-dependent calcium channels involved in synaptic transmission. We will study presynaptic calcium during the application of neuromodulators with presumed inhibitory presynaptic action and identify the types of calcium channels involved and their quantitative inhibition. Through the use of advanced optical techniques, the proposed studies will provide new and important insight into the presynaptic modulation of mammalian synaptic transmission. This insight will contribute to the understanding of normal and pathological synaptic transmission. Excessive release of excitatory neurotransmitter has been observed during episodes of epilepsy and after brain damage. Control of this release by presynaptically acting endogenous neuromodulators could be the basis of future therapeutic interventions. We will address the following specific aims: 1) To discriminate the types of presynaptic VDCCs at principal hippocampal excitatory synapses. 2) To investigate resting levels and influx of presynaptic Ca2+ during presynaptic inhibition of evoked synaptic transmission.

This proposal documents an instrumental improvement important in the dissertation research of Andrew Bullen. This research examines the conduction and integration of electrophysiological signals in the dendrites of cultured mamrr~ CNS neurons using fast optical recording techniques. Andrew is a student at the University of Pennsylvania School of Medicine who is undertaking his doctoral research at Baylor College of Medicine in order to exploit the unique instrumental resources available in the laboratory of Dr. Peter Saggau. The primary instrumental resource used in these investigations is a novel High-Speed Laser Scanning Fluorescence Microscope previously developed in Dr Saggau's lab. The bandwidth of this system (200,000 points/second) is considerably greater than existing scanning microscopes. This bandwidth is achieved through the incorporation of acoustic-optic deflectors as its only scanning mechanism. This instrument was previously used to record from brain slices (Saggau et al., 1990). This microscope has been further developed for simultaneous multisite recording from two fluorescent indicators in the dendrites of mammalian CNS neurons. These modifications have allowed studies of neuronal integration within a single cell and investigations of signaling between cultured neurons (Bullen & Saggau, 1994). These studies employed voltage-sensitive dyes and calcium indicators, but other probes are planned for future experiments. Notwithstanding its fast scanning rate and large bandwidth, the current system has several limitations that have been highlighted by these recent experiments. These limitations include modest spatial resolution, small scanning field size and poor signal-to-noise ratio in fine neuronal processes. A simple improvement is proposed to overcome these limitations and further enhance the performance of this instrument. By replacing the existing acousto-optic deflectors with equivalent devices able to produce more spots (70 vs 10 spots) over a larger initial deflection angle (40 mrad. vs 5 mrad.), the scanning field size will be significantly enlarged and the relative spatial resolution improved. The absolute spatial resolution will also be increased because the net spot size will be smaller (2 ~m vs 10 llm). Furthermore, the smaller spot size will enhance the signal- to-noise ratio and allow events of smaller magnitude to be resolved. These irnproved deflectors will also provide homogenous illumination and thereby facilitate a uniform signal-to-noise ratio throughout the scanning field. The proposed change can be implemented without compromising the existing bandwidth of the system or any major structural alterations. In summary, a minor change to an existing High-Speed Laser Scanning Fluorescence Microscope is proposed. This modification will make this instrument more sensitive and able to sample a larger area with greater spatial resolution. The impact of these features will allow simultaneous mulitsite optical recordings of small events from fine neuronal structures. Measurements of this nature were not previously possible and should significantly advance the understanding of information processing in the dendrites of mammalian CNS neurons. NSF FORM 1358 (1/94)

Abstract for NSF proposal: IBN - 9723871 A significant problem for biomedical research of nerve tissue is the local stimulation of dendrites with bioactive substances such as neurotransmitters. The methods for electrical stimulation that are commonly used can only reach electrically excitable membranes and cannot directly activate the receptors of neurotransmitters. There exist chemically inactivated and photolabile substances that allow for adequate chemical stimulation. These so called caged compounds can be activated by disruption of their molecular cage with a flash of ultra-violet light. However, current photolysis systems used for this purpose lack speed and/or spatial accuracy. Multisite photolysis with high spatio temporal resolution of caged compounds would permit experiments in isolated nerve tissue that closely resemble the situation in the intact nervous system. This capability would add a new dimension to neuroscience and related biomedical fields. It is proposed to develop an optical stimulation system to locally release caged bioactive compounds by means of a pulsed, scanning UV laser beam. This novel system will make use of a scanning principle that allows for ultrafast, accurate, and computer-controlled random-positioning of laser beams. The proposed solution will exceed the capability of previous photolysis systems in its flexibility and spatio-temporal resolution by using acousto-optical effects to control position and intensity of the laser beam. With such devices, positioning and modulation times are in the microsecond range, permitting simultaneous multi-site release of caged compounds and thus multi-site stimulation.

9871994
Brownell
This is theoretical and experimental research on electro-osmosis in membranes in the outer hair cell lateral wall in the mammalian inner ear. The investigators claim that the outer hair cell can be regarded as a naturally occurring microelectromechanical (MEM) system, in which voltages induce fluid transport through the 33 nm wide extracisternal space. A set of differential equations has been developed to model the coupling between electrostatic potentials and fluid flow in the system. They will use this model to predict the effects of applied voltages on fluid transport under a variety of conditions. These predictions will be tested experimentally through optical measurements of the displacement of fluorescent molecules within the extracisternal space. The goal is to derive general principles about the interaction between voltages and fluid flow in nanostructures, which can be exploited in artificial MEM systems.
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There is compelling evidence for the involvement of presynaptic inhibition in controlling normal synaptic transmission and preventing excessive neurotransmitter release at mammalian central synapses. The presynaptic site is an effective target for modulation of synaptic transmission and presynaptic voltage-dependent calcium channels play a significant role in controlling transmitter release. To address our long-term objective of understanding the basic presynaptic mechanisms underlying modulation of synaptic transmission in mammalian central synapses, we propose to investigate the direct and indirect role of presynaptic calcium during inhibition of synaptic transmission at hippocampal CA3/CA1, MF/CA3 and PP/GC excitatory synapses. This in vitro study will employ hippocampal brain slices and optical imaging techniques. We will selectively load presynaptic terminals with ion-sensitive indicators to investigate resting levels and transients of presynaptic calcium and presynaptic potassium. In addition, we will use voltage- sensitive dyes to measure presynaptic action potentials. Specific blockers will be utilized to identify and quantify the types of presynaptic voltage-dependent calcium channels involved in synaptic transmission. We will study presynaptic calcium during the application of neuromodulators with presumed inhibitory presynaptic action and identify the types of calcium channels involved and their quantitative inhibition. Through the use of advanced optical techniques, the proposed studies will provide new and important insight into the presynaptic modulation of mammalian synaptic transmission. This insight will contribute to the understanding of normal and pathological synaptic transmission. Excessive release of excitatory neurotransmitter has been observed during episodes of epilepsy and after brain damage. Control of this release by presynaptically acting endogenous neuromodulators could be the basis of future therapeutic interventions. We will address the following specific aims: 1) To discriminate the types of presynaptic VDCCs at principal hippocampal excitatory synapses. 2) To investigate resting levels and influx of presynaptic Ca2+ during presynaptic inhibition of evoked synaptic transmission.

The receptive parts of most neurons (nerve cells) in the brain are the branches called dendrites. These dendrites are covered with spiny structures, which are part of the functional contacts between nerve cells called synapses. These spines receive signals from the synaptic terminals of other neurons. Neurotransmitters are the biochemical compounds that carry a signal across the small gap between the pre- and the post-synaptic sides, and generate the electrochemical membrane response in the post-synaptic cell. Dendritic size, complexity, and accessibility, together with the hundreds or thousands of synaptic contacts on a single cell, make it technically difficult to study the local dynamic mechanisms underlying this synaptic signaling at this microscopic scale. It is very difficult to elicit a physiological multi-site stimulation pattern and to perform multi-site recordings of the membrane potential, especially to measure local synaptic potentials concurrently at different sites in the same cell. This project uses novel optical methods in addition to the powerful 'patch-clamp' electronic technique for whole-cell physiological recordings. An optical workstation has been developed that uses a pulsed ultraviolet (UV) laser beam with a novel acoustico-optic control for microsecond timing of the position of a spot in the micrometer size range, in connection with a biochemical compound that acts as a 'cage' around neurotransmitter molecules to keep them 'invisible' for their receptors. When caged molecules are hit with the laser beam, the light energy immediately breaks the cage (photolysis) and free transmitter can act on the synapse. High-resolution differential-interference-contrast (DIC) microscopy of neurons in culture, with the pulsed random-access laser-scanning photolysis, and a new technique of laser-scanning fluorescence microscopy of voltage-sensitive dye bound to the cell membranes are combined to allow a new level of computer-controlled, non-invasive, high-resolution stimulation and recording. This powerful technology is used to examine the functional role of dendritic structural details, to characterize spatio-temporal interactions among dendritic synapses, the computational functions that can be assigned to dendrites in integrating signals, and the effects of prior activity on dendritic behavior.
Results will be important for understanding the fundamental role of dendrites for cellular information processing, and for refining computational compartmental models of nerve cells. The impact of the technology also will extend beyond cellular neuroscience to cellular biology. In addition, exceptional cross-disciplinary opportunities will be provided for postdoctoral and graduate training.

This award provides support for development of a type of confocal microscope that will be able to scan samples much more rapidly than current commercial instruments. Optical imaging techniques play an important role in studying structure and function of nerve cells, a role that has been facilitated by advanced techniques such as multiphoton microscopy. This type of microscopy allows collection of high-quality structural images as well as high-fidelity transient signals from light-scattering preparations such as living tissue. While the spatial resolution of existing instruments of this type is remarkable, their temporal resolution has been limited by use of mechanical devices to scan the specimen. As a result, drastic sacrifices of spatial resolution are usually necessary when monitoring fast cellular signaling. The instrument to be developed will combine the use of Multiphoton Microscopy with Acousto-Optic Laser Scanning. For structural Imaging, the instrument will permit significantly higher spatial resolution at fast frame rates. For observation of transient signals (functional imaging), it will allow the user to select points of interest from previously imaged structures and perform multi-site measurements at frame rates of more than 1000 per second. The instrument will be of great advantage for experimental work in neuroscience where it will allow study the computational properties of individual neurons in brain slices by imaging dendritic structures and their function, and to study processing of sensory information in intact brain. Other applications are likely to include imaging of structure and function in excitable non-neuronal tissue, such as heart, smooth and skeletal muscle, and imaging of other light-scattering biological preparations.

DESCRIPTION (provided by applicant): Brain function is based on the computation that occurs concurrently within and among nerve cells. At the level of the single nerve cell, information from thousands of synaptic inputs is received at the dendritic spines, where it is processed by the dendrites' specific morphology and distribution of voltage-gated ion channels. Two of our participating labs have been investigating various aspects of this Neuronal Computation, including nonlinear summation of synaptic potentials and signaling functions of dendritic action potentials. To understand these dendritic functions, it is absolutely necessary to analyze the complex interplay of structure and function. Such analysis requires computational models of the neuron incorporating both information about the neuron's structure and its distribution of ion channels. Building such models has been difficult because technical considerations have dictated that structure and function be acquired separately. Recent advances in optical imaging techniques, now allow us to acquire the structure of living nerve cells and perform multi-site recording of neuronal function during a single experiment. However, choices as of the sites for functional imaging must still be made. We propose to choose these optimal recording sites based on an on-line simulation of the nerve cell under study. Such a simulation requires that structural information be acquired, a morphological reconstruction be performed, and a compartmental model of the neuron be constructed, during the short time frame of an acute experiment. The output of the simulation will guide the functional imaging by identifying sites that will yield the most information about the process under study. Finally, the acquired functional imaging data would be incorporated into the computational model for further experiments. The goal of this project is the development of a computational and experimental framework to allow real-time mapping or functional imaging data (e.g., spatio-temporal patterns of dendritic voltages or intracellular ion) to neuronal structure, during the very limited duration of an acute experiment. In order to accomplish this goal, the research objectives of this proposal are the following: . To develop the theoretical framework and computational techniques for on-line, robust, and accurate morphological reconstruction of a fluorescently labeled live nerve cell from stack of optical sections obtained using non-invasive structural imaging. . To predict on-line a nerve cell's behavior using the reconstructed morphology and a priori knowledge regarding the distribution of ion channels embodied in a compartmental model. . To guide functional imaging based on predictions of the model and the reconstructed morphology. . To optimize the computational model of the neuron by minimizing error between the predictions and the data acquired during functional imaging. The impact of the proposed project is in its enhancement of the data acquisition process, particularly in optimizing the value of multi-site optical recordings, and in the focused and directed incorporation of data into quantitative computational models of nerve cells. Our computational and experimental framework will guide the efficient design of experiments and the generation of new hypotheses that can help reveal functional mechanisms underlying both normal and diseased states of the nervous system, both for us and for other researchers. The successful completion of the proposed research requires input from neuroscience, bio-imaging, biophysics, and computer science. Thus, our project demands collaboration and complementary expertise for its success - our team is uniquely suited to accomplish this challenge and also to attract and train excellent students.

DESCRIPTION (provided by applicant): Brain function is based on the concurrent computation within and among nerve cells. At the level of the single nerve cell, information from thousands of synaptic inputs is received at dendritic spines, where it is processed by the dendrites' specific morphology and its distribution of voltage-gated ion channels. It is well established that the distributions of spines, dendrites and channels change with age -- both during normal aging and as a result of neurodegenerative diseases associated with aging such as Alzheimer's disease. For most neurons in the central nervous system, the details of how the dendrites transform information in normal and pathological states remain poorly understood. In large part this is because of the massively parallel nature of the connectivity between neurons -- it has not been possible to experimentally stimulate neuronal synapses in large numbers with well-defined spatial and temporal patterns that mimic the physiological input to the neuron. Here, we propose a new approach that will employ a novel imaging workstation allowing hundreds of individual synapses to be activated across the spatial extent of the dendrites by multi-site photolysis of caged neurotransmitter. This project will build on and make extensive use of our existing collaborative infrastructure that allows acquisition of the structure of a neuron in a brain slice, reconstruction of the neuron's morphology, construction of a compartmental model, and optical high-speed multi-site functional recording. We will use the morphological reconstruction to identify the exact sites for stimulation and recording. Computer simulations of the neuron will be used online to optimize experiments by identifying regions of the neuron meeting experiment-dependent specifications, and offline to aid in understanding the underlying ionic mechanisms of nonlinearities in the neuron's response to stimulation. - The goal of this project is to determine the input/output function of pyramidal neurons in the hippocampal area CA1. In order to accomplish this goal, the research objectives of this proposal are to: - Implement a workstation for concurrent optical multi-site stimulation and recording. - Refine structural imaging and morphological reconstruction to identify dendritic spines. - Improve neuronal simulations by iterative fitting of experimental data in small branches. Generate optimal multi-synapse activation patterns and investigate the neuron's activity generated by these patterns.

This award supports development of a novel microscope that overcomes present limitations of temporal resolution during fast functional imaging of living tissues. The instrument will employ an inertia-free three dimensional scanning scheme, recently invented in the principal investigator's lab, that builds on two established techniques - Fast Acousto-Optic Laser Scanning and Multiphoton Microscopy. Critical to this has been the PI's development of a strategy to compensate for the dispersion of ultra-fast laser pulses when steered with acousto-optic deflectors. The instrument will allow researchers to interactively select sites-of-interest to perform multi-site functional imaging at frame rates of more than 1,000 per second. The microscope is expected to make possible the study of sensory information at cellular and subcellular level in intact brain and in excitable, non-neuronal tissue, such as heart, smooth and skeletal muscle, and imaging of other light-scattering biological preparations. The project will provide research training for number of graduate and undergraduate students in Engineering and Biology who will participate in design and construction of the instrument. In addition, portions of the theoretical work that provides the basis for the instrument will be incorporated in an imaging course taught by the investigator.

DESCRIPTION (provided by applicant): Brain function is based on the concurrent computation within and among nerve cells. At the level of the single nerve cell, information from thousands of synaptic inputs is received at dendritic spines, where it is processed by the dendrites' specific morphology and its distribution of voltage-gated ion channels. It is well established that the distributions of spines, dendrites and channels change with age -- both during normal aging and as a result of neurodegenerative diseases associated with aging such as Alzheimer's disease. For most neurons in the central nervous system, the details of how the dendrites transform information in normal and pathological states remain poorly understood. In large part this is because of the massively parallel nature of the connectivity between neurons -- it has not been possible to experimentally stimulate neuronal synapses in large numbers with well-defined spatial and temporal patterns that mimic the physiological input to the neuron. Here, we propose a new approach that will employ a novel imaging workstation allowing hundreds of individual synapses to be activated across the spatial extent of the dendrites by multi-site photolysis of caged neurotransmitter. This project will build on and make extensive use of our existing collaborative infrastructure that allows acquisition of the structure of a neuron in a brain slice, reconstruction of the neuron's morphology, construction of a compartmental model, and optical high-speed multi-site functional recording. We will use the morphological reconstruction to identify the exact sites for stimulation and recording. Computer simulations of the neuron will be used online to optimize experiments by identifying regions of the neuron meeting experiment-dependent specifications, and offline to aid in understanding the underlying ionic mechanisms of nonlinearities in the neuron's response to stimulation. - The goal of this project is to determine the input/output function of pyramidal neurons in the hippocampal area CA1. In order to accomplish this goal, the research objectives of this proposal are to: - Implement a workstation for concurrent optical multi-site stimulation and recording. - Refine structural imaging and morphological reconstruction to identify dendritic spines. - Improve neuronal simulations by iterative fitting of experimental data in small branches. Generate optimal multi-synapse activation patterns and investigate the neuron's activity generated by these patterns.

DESCRIPTION (provided by applicant): Brain function is based on the concurrent computation within and among nerve cells. At the level of the single nerve cell, information from thousands of synaptic inputs is received at dendritic spines, where it is processed by the dendrites' specific morphology and its distribution of voltage-gated ion channels. It is well established that the distributions of spines, dendrites and channels change with age -- both during normal aging and as a result of neurodegenerative diseases associated with aging such as Alzheimer's disease. For most neurons in the central nervous system, the details of how the dendrites transform information in normal and pathological states remain poorly understood. In large part this is because of the massively parallel nature of the connectivity between neurons -- it has not been possible to experimentally stimulate neuronal synapses in large numbers with well-defined spatial and temporal patterns that mimic the physiological input to the neuron. Here, we propose a new approach that will employ a novel imaging workstation allowing hundreds of individual synapses to be activated across the spatial extent of the dendrites by multi-site photolysis of caged neurotransmitter. This project will build on and make extensive use of our existing collaborative infrastructure that allows acquisition of the structure of a neuron in a brain slice, reconstruction of the neuron's morphology, construction of a compartmental model, and optical high-speed multi-site functional recording. We will use the morphological reconstruction to identify the exact sites for stimulation and recording. Computer simulations of the neuron will be used online to optimize experiments by identifying regions of the neuron meeting experiment-dependent specifications, and offline to aid in understanding the underlying ionic mechanisms of nonlinearities in the neuron's response to stimulation. - The goal of this project is to determine the input/output function of pyramidal neurons in the hippocampal area CA1. In order to accomplish this goal, the research objectives of this proposal are to: - Implement a workstation for concurrent optical multi-site stimulation and recording. - Refine structural imaging and morphological reconstruction to identify dendritic spines. - Improve neuronal simulations by iterative fitting of experimental data in small branches. Generate optimal multi-synapse activation patterns and investigate the neuron's activity generated by these patterns.

DESCRIPTION (provided by applicant): This interdisciplinary and multi-institutional training program seeks to support the education of future researchers who can apply the tools of Mathematics, Physics and Engineering to problems of Brain Research. Traditional Neuroscience uses reductionism to formulate hypotheses and tests them experimentally, while Theroretical and Computational Neuroscience builds on information Theory, Dynamical Systems Theory, and Computer Science to create theoretical models to be tested numerically. Collaborations of Neuroscientists, individually trained in experimental and computational approaches, are not unusual on the basis of experimental data. In extension of this, we advocate a synergistic use of both approaches to control the experiment itself, and propose to train pre- and postdoctoral students accordingly. Commensurate with our escalating knowledge of neural function, the complexity of experiments to analyze both healthy and diseased brain function is ever-increasing. In this situation, it is necessary to utilize the analytic and predictive nature of Theroretical and Computational Neuroscience not only between but rather during experiments. To meet this challenge, we will recruit both pre- post-doctoral students with previous training in Mathmatics, Physics and Engineering, and associate them with dual mentors of expertise in both theoretical and experimental Neuroscience. In addition to using theoretical tools, these students will be trained in state-of-the-art experimental methods, specifically those for complex multidimensional data acquisition, processing and visualization, as these are most prominent in Advanced Imaging Techniques. We have devised a curriculum to best educate these interdisciplinary students. For predoctoral students, training will be a well-balanced combination of classroom instruction and hands-on labs. For both pre- and postdoctoral students, there will be active journal clubs, mentor-guided research with an internship in the lab of the co-mentor, and conference presentations. Our training faculty of 23 is drawn from six institutions in and around the Texas Medical Center in Houston. All faculty members are also members of the Gulf Coast Consortium for Theoretical and Computational Neuroscience, which is part of the Gulf Coast Consortia for Interdisciplinary Bioscience Research and Training. Both our spectrum of represented disciplines and existing facilities makes this an ideal site for the proposed training program.

Neuroplasticity is central to fundamental processes in the brain, including learning, and long-lasting changes in neural circuits that result from employing substances of abuse. In contrast to significant advances at the cellular/molecular level, our understanding of the functional organization and reorganization of brain circuits remains limited. Increasing in vivo evidence suggests that single cells remain plastic into and during adulthood. However, our knowledge of the functional properties of single cells is primarily descriptive. This limits our understanding of the underlying mechanisms involved in assembling and modifying neuronal tuning functions during plasticity. In order to understand how the properties of cells change during plasticity, it is imperative to record from a population of interconnected neurons in vivo. More than half of all synaptic contacts in the cortex arise from neurons within a -200 j.lm radius from the target cell. Importantly, connections between cells in the cerebral cortex are predominantly along cortical depth. Therefore, we need to monitor simultaneously the activity of all adjacent neurons in a cortical volume, and thus record in three dimensions. To date, no experimental tool exists that would allows us to do this. This project seeks to establish novel in vivo methods that will allow us to analyze neural circuits in three dimensions. For this purpose, we will advance two technologies to record from and manipulate circuits in the mammalian cortex: (1) Ultra-fast three-dimensional two-photon imaging, and (2) Optical manipulation of neural activity with single-cell and single-spike resolution using optogenetic tools. The proposed developments have become possible because of recent technical advances in ultra-fast multi-photon microscopy and light-activated ion channels. In short: Inertia-free nearinfrared laser scanning technology allows for in vivo fast structural and functional imaging as well as for high resolution optical stimulation. The proposed project will combine these key technologies to generate the infrastructure and the experimental skills to study the function and plasticity of cortical microcircuits and thus will help researchers to understand mechanisms of neuroplasticity in the intact brain.

DESCRIPTION (provided by applicant): Super-resolution Workstation for Imaging Live Biological Nanostructures. A novel research instrument will be developed for visualizing and measuring living biological structures that are below the resolution limit of conventional light microscopy. This super-resolution microscope does not require any mechano-optical adjustments during image acquisition and will thus allow for fast imaging of sub-resolution structures free of inherent mechanical artifacts. The instrument will combine two established techniques -- the spatial resolution enhancement of Standing Wave Microscopy with the temporal resolution enhancement of Acousto-Optic Laser Scanning. The proposed instrument will be unique in its performance and will be of particular advantage in applications where the dynamics of sub-resolution living biological structures are to be studied. The PI has previously conceived and constructed a series of advanced imaging instruments necessary for his long-term biological research goal to understand information processing in single neurons and small neuronal populations. All developed instruments utilized the PI's expertise with Diffractive Optical Elements, specifically Acousto-Optic Devices. The optical properties of these elements are rapidly adjustable, i.e. with electronically produced sound waves in the radio frequency range, making acousto-optic devices unique building blocks for advanced imaging instrumentation. The proposed imaging workstation will be developed in a two-step approach, resulting in improved spatial resolution in three dimensions. The inertia-free control of the necessary illumination patterns by acousto-optic devices will result in a highly versatile instrument with superior mechanical stability and imaging speed. The proposed workstation for fast super-resolution imaging would be of high importance in biomedical research. It would vastly improve the way important intracellular structures can be visualized and their function monitored, including mitochondria, endoplasmic reticulum, and microtubules. Specifically in experimental Neuroscience such an instrument would support the study of various aspect of synaptic transmission, including presynaptic vesicle clusters and postsynaptic dendritic spine necks. For example, the fragile sub-resolution structure of spine necks is susceptible to changes during development and plasticity, but also to a number of neurological diseases. In general, the availability of the proposed super-resolution imaging capability would be transformational and benefit large communities in the biomedical field. PUBLIC HEALTH RELEVANCE (provided by the applicant): Although the proposed imaging workstation was conceived for Biomedical Research, it has also great potential as a diagnostic tool. Changes in subcellular structure and function often coincide with various states of numerous diseases. The proposed instrument will allow microscopic inspection and functional testing of subcellular structures in live, non-fixed cellular specimen at unparalleled spatio- temporal resolution.

DESCRIPTION (provided by applicant): All-Optical High-Throughput Functional Connectivity Mapping using Advanced Microscopy and Optogenetic Tools We propose an innovative and translational approach to map functional connections between cells in neuronal populations. Connectivity maps are the fundamental step to analyze neuronal networks. Traditionally, such maps were established by electrically recording from pairs of neurons by means of micropipettes. More recently, multi-patch protocols have been used, requiring complicated equipment and highly skilled experimenters. High-resolution connectomes are presently established by advanced histological techniques, involving serial sectioning and electron microscopy, however, this approach primarily produces anatomical maps and identification of functional connections remains difficult. Optogenetic tools and two-photon microscopy have dramatically changed functional connectivity mapping. For example, neurons expressing light-activated ion channels can be optically depolarized above their spiking threshold, and postsynaptic signals can be monitored in connected cells. Initially, hybrid approaches were taken, activating presynaptic neurons optically and measuring postsynaptic signals by whole-cell recording. More recently, all-optical mapping methods have been explored; combining light-activated channels to optically evoke activity and optical indicators to monitor activity. However, when using an all-optical approach to generate functional connectivity maps, two main challenges arise: Firstly, activating individual presynaptic neurons will produce hard to detect optical signals in postsynaptic cells as these sub-threshold postsynaptic potentials do not generate spiking. Secondly, concurrent optical stimulation and recording usually requires two separate excitation wavelengths and thus two costly lasers. Fortunately, both challenges can be met. Building on our expertise in advanced optical imaging, we will utilize 3D laser scanning technology developed in our lab and successfully applied by many research groups. To reliably detect single synaptic connections, we will optically activate presumed postsynaptic cells just at firing threshold and presumed pre- synaptic cells well above threshold. Keeping postsynaptic cells at 50% firing probability will result in readily detectable optical signals, maximizing the sensitivity for both discriminating excitatory and inhibitory connections. For concurrent stimulation and recording, we will take advantage of the small two-photon excitation volume. While this effect was seen as an obstacle to recruit sufficient light- activated channels for supra threshold stimulation, we will utilize it to employ a single wavelength to independently stimulate by scanning illumination of cell bodies and record by single-point illumination. Overall, the proposed protocol for determining functional connections by pure optical means is ideally suited for high-throughput functional connectomics. The combined use of multi-photon excitation and 3D laser scanning makes the translation from brain slices to in vivo cortex straightforward.