David Kleinfeld - US grants

DESCRIPTION (Adapted from applicant's abstract): This program will allow scientists committed to quantitative approaches to problems in neuroscience to come to the Marine Biological Laboratory to work and interact among themselves and other members of the neuroscience community. The goals are to focus on experimental and theoretical issues related to the large-scale dynamics of nervous systems. The workgroup will focus on issues that arise in two complementary areas critical to the understanding of brain function. The first involves advanced signal processing methods relevant to neuroscience, particularly those appropriate for emerging multi-site recording techniques and noninvasive imaging techniques. The second involves the development of a calculus to study the dynamical behavior of nervous systems and the computations they perform. The workgroup will enable a close collaboration between experimentalists and theorists, particularly with regard to the analysis of data and the planning of experiments. The workgroup will meet annually for 3 weeks with 30-40 participants, split between senior and junior researchers, both experimentalists and theorists. The workgroup will have limited research lectures supplemented by tutorial lectures on the relevant mathematical and computational techniques. It is expected that researchers will spend the major fraction of their time collaboratively analyzing data, using state-of-the-art workstations and software, and discussing their results. This workgroup will provide a means to critically evaluate techniques for the processing of multi-channel data, of which imaging forms an important category. Such techniques are of critical importance for basic research and medical diagnostics. An attempt will be made to establish a repository of these techniques, along with benchmarks. A successful workgroup will insure the rapid dissemination of modern analytical techniques throughout the neuroscience community.

*** 9802469 This grant will support the development of a novel set of laboratory modules for undergraduate physics laboratories. The modules will emphasize several aspects of biophysics, showing how physical principles are realized in biological systems. They include: tracking of diffusing particles in solution; the forces on organelles; visual processing by the fly. Those interested in learning about these modules, for use in other schools, can attend a hands-on workshop at the host institution. ***

Multi-photon laser scanning microscopy is a powerful new tool to probe brain structure and function within the intact, living nervous system. For example, recent in vivo studies have shown that the anatomy and ionic behavior of neurons in the mammalian cortex can be probed with micrometer spatial resolution and millisecond temporal resolution. Yet formidable problems remain in the application of optical techniques to in vivo studies, particularly since light is strongly scattered by brain tissue so that it is difficult to resolve structures deep to the pial surface of cortex. Here we propose to advance multi-photon imaging techniques as a means to enable studies of brain homeostasis and neuronal and glial dynamics at the level of the middle and deep layers of mammalian cortex. Our technical program is focused on the optimization of pulsed lasers for deep imaging. We propose to construct a source that is tunable with center wavelengths from 525 nm to beyond the water absorption limit of approximately 1.3 mum, whose pulse shape is optimized for multi-photon absorption, and, most critically, whose trade-off between energy per pulse and repetition rate insures the greatest possible penetration depth into cortex while heating of the brain and possible photodynamic damage in minimized. The proposed improvements should extend the depth for diffraction limited imaging of brain structures from the current state-of-the-art of approximately 500 mum to over 1 mm. These improvements will be amenable to a broad range of extrinsic, intrinsic and genetically induced molecular indicators of brain function in vivo. Our research effort toward the study of cortical microcirculation provides a context for our technical development. Fine capillaries are ubiquitous throughout the depth of cortex and thus provide a test bed to readily explore issues of depth penetration and photodynamic damage. Our preliminary data shows that conventional two-photon laser scanning microscopy may be used to observe the flow of individual red blood cells in capillaries that lie in the superficial layers of cortex in rat. We will use our proposed advancements in penetration depth to map the spatial-, temporal-, and stimulus-dependence of changes in cerebral microcirculation throughout the entire depth of cortex as a means to address the microscopic connection between neuronal activity and local blood flow. The proposed advancement in the depth penetration of multi-photon methods will provide an essential tool for understanding the optical properties of normal and diseased tissues, and thus may substantially improve upon the effectiveness of two-photon diagnostic procedures and photodynamic therapies.

9987614
Terry Sejnowski - University of California, San Diego
IGERT: Graduate Training Program in Computational Neurobiology

This Integrative Graduate Education and Research Training (IGERT) award supports the establishment of a multidisciplinary graduate training program of education and research in computational neurobiology. The goal is to train a new generation of scientists and engineers with a broad range of scientific and technical skills who are equally at home measuring large-scale brain activity, analyzing the data with advanced computational techniques, and developing new models for brain development and function. This integrative training program is centered in the Department of Biology at UCSD and the Salk Institute, but includes faculty members from physics, chemistry, psychology, cognitive science, electrical engineering, computer science, and mathematics, as well as from biology and neuroscience. The training program will give all students hands-on experience in a wide range of advanced experimental and computational techniques through collaborative research between laboratories, industrial internships, and the opportunity to pursue research abroad. The faculty will participate in outreach programs to encourage and prepare underrepresented minorities for a career in computational neurobiology. Research areas in the training program include: (1) synaptic growth and plasticity; (2) neural dynamics; (3) neural population coding; (4) visual perception and memory; (5) stochastic learning algorithms; and (6) functional brain imaging.

IGERT is an NSF-wide program intended to meet the challenges of educating Ph.D. scientists and engineers with the multidisciplinary backgrounds and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing new, innovative models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In the third year of the program, awards are being made to nineteen institutions for programs that collectively span all areas of science and engineering supported by NSF. The intellectual foci of this specific award reside in the Directorates for Biological Sciences; Computer and Information Science and Engineering; Social, Behavioral, and Economic Sciences; Mathematical and Physical Sciences; Engineering; and Education and Human Resources.

DESCRIPTION: (Adapted from the Investigator's Abstract) Spatial navigation requires active sensory and motor processes. Animals must extract meaning from the sensory information they amass through their receptors as they search and locomote. A fundamental question in studies of sensory perception is how the blur of sensory input is converted by the nervous system into a stable perception. The proposed studies aim to elucidate the algorithm used by animals to extract a stable image of the world from input with actively mobile sensors. The experimental program addresses the question of active sensation in the context of tactile localization of objects accomplished by the exploratory whisking movements of vibrissae in the rat. The experiments involve trained animals and recording and stimulation techniques with implanted electrodes. The proposal has two major foci: First, what are the neurological reference signals in vibrissa sensory and motor cortex for whisker position? What parameters of whisking are controlled at this level? The second focus is on the sensorimotor loop linking sensory and motor vibrissa cortices. How does a fast spike train signal in sensory cortex get transformed into a slow motor control signal in motor cortex?

DESCRIPTION (Adapted from the applicant's abstract): One approach for understanding the nature of neuronal computations involves the study of coherent spatio-temporal patterns that encompass many or all of the neurons in the intact brain. A powerful tool that facilitates this approach is the use of molecular indicators of the cell membrane potential to report the electrical dynamics of populations of neurons. When applied to invertebrates and lower vertebrates, studies that utilized this technical approach have shown that nervous systems represent sensory information and intended motor output on the scale of hundreds of neurons. This representation takes the form of traveling waves and other patterns whose form cannot be predicted on the basis of anatomy or conventional electrical recordings. Technology for the extension of such studies to the mammalian brain, on a routine basis, is the major goal of this work. The investigators propose to develop and build a multi-channel optical detector whose novel features, design flexibility and cost effectiveness should significantly advance the use of molecular indicators to report neuronal function. Numerical techniques for the removal of physiological noise contributions to the dynamic imaging data will also be further developed. There is a need for improved detection in the area of voltage-sensitive dye signals. These signals appear as small changes in light levels (11 part in 10(-5) to 10(-3). Further, they are modulated by both rhythmic and fluctuating physiological changes in the intrinsic optical properties of cortex. The proposed multi-site sensor will have the sensitivity, dynamic range, resolution and bandwidth to resolve the electrical contributions to the dye signal on top of these fluctuations. There is a general need for a modular, robust, and readily available multi- site detector in the cell biology and neurobiology communities. The proposed device can be used for the study of ions as well as electrical activity in a variety of cellular and tissue systems, both in vitro and in vivo, in which large dynamic range and quantum limited noise is essential. Present systems are mainly limited to the measurement of signals whose amplitude is well above theoretical noise limitations. There is a need for robust algorithms that will aid in the real-time analysis of single-trial dynamic brain images. These include techniques to estimate the contributions from vasomotion, respiration, and cardiac pulsation from the data and techniques to extract the weakly correlated components of functional activity. The proposed device is a research tool for the study of brain function in both the normal state and pathological states. These proposed studies of stimulus representation in the rat vibrissa sensorimotor pathway will serve as a scientific test-bed for the proposed technology.

A high-resolution, real-time, microscope that is based on third-harmonic generation and provides unique possibilities to visualize interfaces in refractive index or third-order nonlinear susceptibility in optically transparent media, and biological systems is being developed. The thin optical sections produced in the third harmonic microscope can be used in conjunction with standard surface rendering techniques to produce three-dimensional images. No fluorophore is necessary to label the specimen as is used in traditional laser fluoresence microscopy, as the signal is generated by endogenous interfaces. Thus, in third harmonic microscopy the images do not fade due to bleaching, and can be viewed for extended periods without loss of intensity or clarity. This imaging technique is therefore particular useful for imaging three-dimensional dynamics in microscopic systems. For dynamic measurements, imaging volumes must be repeatedly addressed in order to develop a reliable time series of transient phenomena. In the past, the effectiveness over which dynamic systems could be measured was limited by the bleaching characteristics of an exogenous label. Third harmonic imaging relies on naturally occurring boundaries within the sample to generate image contrast, and therefore does not fade, and is highly suitable for visualizing dynamic three-dimensional systems.
A diode-pumped, femtosecond Nd:glass oscillator optimized for microscopy will be constructed. The laser will produce third harmonic signals that are 16-21 times greater than can be achieved with commercially available lasers. The laser will also be beneficial for 2-photon imaging. Operating at a wavelength of 1.06 micro meter, this light penetrates tissue ~20% deeper than present femtosecond lasers that commonly operate at 800 nm. A novel microscope design will be constructed that can simultaneously capture fluoresence images and third harmonic images with perfect registration. This is important for fully quantifying the image contrast mechanisms in third harmonic microscopy, and enables a quantitative analysis of this new imaging process.
The third harmonic microscope will be used in conjunction with an image correlation spectroscopy technique developed specifically to allow rapid measurements of macromolecular aggregation within an intact cellular environment. By combining temporal and spatial autocorrelation analysis of an image time series, it is possible to obtain information on the molecular dynamics and transport properties as well as measuring the state of aggregation of the molecules as a function of time. The combined information can provide insight into the molecular mechanisms that govern many biochemical reactions in living cells. Measurement of molecular interactions and dynamics is integral for a full understanding of how cells build functional macromolecular assemblies as well as how they transduce signals across the plasma membrane following binding of external ligands to receptors localized to the cell surface.

The advent of ultrashort laser light pulses as a laboratory tool has provided the opportunity to probe and manipulate structure and function in biological systems. This award supports development of an instrument that will use nonlinear absorption to create micrometer-sized ablations in nervous tissue, with minimal collateral damage. The instrument can be integrated with existing nonlinear imaging devices, principally the multi-photon microscope. Prior work has shown that ultrashort laser pulses can be used for an all-optical approach to histology in which anatomical structure with micrometer resolution is obtained without the use of mechanical sectioning devices. Pilot data has also demonstrated that such pulses can block flow in specific, targeted blood vessels, thus enabling studies of the biofluid mechanics of the vessels. The proposed instrument development program will design and realize ultrashort pulsed laser technology capable of a mixture of optical ablation and nonlinear optical imaging. The effort will result in a near turn-key system that uses commercially available laser oscillators and regenerative amplifiers, together with a mixture of commercial and custom opto-mechanical and electronic components and software. The expected applications include the automatic anatomical mapping of neuronal and non-neuronal nuclei, vasculature, and subcellular structures within the rodent brain, and the optical induction of axonomy in which axons in the leech ganglion are cut to study circuit dynamics in the swim command network. Additional potential applications include submicrometer surgery to ablate organelles, and transient disruption of the blood-brain barrier. The design and realization of the instrument will expose students to use of state-of the art optical instrumentation for the pursuit of biological questions. The proposed work will specifically train two postdoctoral researchers in instrumentation and experimentation at the optics/biology interface.

DESCRIPTION (provided by applicant): The advent of ultrashort (femtosecond) laser light pulses as laboratory tool opens up new opportunities to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon microscopy. More recently, high-fluence ultrashort pulses have been exploited to reliably create micron-sized ablations in brain tissue with minimal collateral damage. These ablations are the driving technology in an all-optical histology, which allows anatomy to be imaged with micrometer resolution throughout the brain. These ablations can also be used to perturb neocortical blood flow as a means to probe normal and diseased tissues. Yet much additional effort is required to use and advance the mixture of multiphoton ablation and imaging techniques as a means to enable studies of neuronal and vascular architectonics. Our proposed instrumentation concerns the use of ultrashort laser pulses for focal ablation at high fluence (energy per area), and imaging, at lower fluence, to address open issues in neuroanatomy and neurovascular coupling. Two synergistic applications will serve as test beds for our technical goals: Establish all-optical based histology as a standard anatomical tool. This includes the optimization of parameters and the correction of spherical aberration for both ablation and imaging. We proposed to reconstruct neuronal and non-neuronal soma and vasculature positions throughout rat vibrissa sensory cortex and to reconstruct mitochondrial density and vasculature throughout vibrissa sensory cortex. Advance the optical induction and monitoring of targeted vascular blocks. We will perturb blood flow in connective arteriol networks as well as deep capillary networks to study flow dynamics in different angioarchitectures. The proposed advancement in the breadth of nonlinear optical methods will provide a novel tool for manipulating and probing tissues that is simultaneously imaged with two-photon microscopy. We will make these tools reliable and readily available to the biomedical community. The proposed model systems may substantially improve upon our understanding of brain architectonics and stroke formation. That may lead to improvements in preclinical models to assay therapeutics for stroke.

The advent of ultrashort laser light pulses as a laboratory tool provides an opportunity to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon scanning microscopy. More recently, nonlinear absorption has been exploited as a means to reliably and reproducibly create micrometer-sized ablations in brain tissue with minimal collateral damage. These ablations drive all-optical histology, which allows anatomy to be imaged with micrometer resolution and mapped throughout an entire brain. They can also be used to perturb neocortical blood flow as a means to probe normal and diseased vascular function. Yet much additional effort is required to advance this new technology as a means to enable studies of neuronal and vascular architectonics. Our technical program is focused on advancing the mixture of ablation and imaging. ? Optimization of pulsed lasers for nonlinear ablation. ? Optimization of objectives for high efficiency, in vivo ablation and imaging. ? Design and optimization of algorithms to reconstruct and analyze vascular and large-scale cellular anatomy. Our scientific effort involves the study of structural and functional anatomy in the brain. ? Automatic mapping of the relation among neuronal and nonneuronal nuclei, vasculature, and mitochondria or other subcellular structures within the rodent brain. This includes stroke-induced changes in angioarchitecture. ? Optical induction of localized, thrombotic and hemorrhagic strokes as a means to study the redistribution of blood flow and blood 02 in response to perturbations in brain homeostasis. The program will be carried by a partnership of three research groups with a history of collaboration: Kleinfeld with expertise in nonlinear imaging and cellular/systems neuroscience;Squier with expertise in laser and microscope design and biological imaging;and Ifarraguerri with expertise in algorithm design and biomedical imaging. The proposed advancement in nonlinear optical methods will yield novel tools for manipulating and probing tissues. We will make these tools reliable and readily available to the biomedical community. The proposed model systems may lead to improvements in preclinical models to assay therapeutics for stroke.

DESCRIPTION (provided by applicant): The advent of ultrashort laser light pulses as laboratory tool has opened up new opportunities to probe and manipulate anatomy and function in nervous systems. Ultrashort pulses are the essential means to drive the nonlinear absorption of light by biomolecules, which leads to a localized region of excitation and forms the basis of two-photon scanning microscopy. More recently, nonlinear optical absorption has been exploited as a means to reliably and reproducibly create micron-sized ablations in brain tissue with a minimum of collateral and thermal damage. These ablations can be use as the driving technology in an all-optical histology, which allows anatomy to be imaged with micrometer resolution throughout the entire brain. These ablations may also be used to perturb neocortical blood flow as a means to probe normal and diseased tissues. Yet much additional effort is required to use and advance the mixture of multi-photon ablation and imaging techniques as a means to enable studies of neuronal and vascular architectonics. Our proposed research concerns the confluence of nonlinear optics and anatomy. . Advance the mixture of multi-photon ablation and imaging to establish all-optical based histology as a standard anatomical tool. This includes the optimization of parameters and the advancement of software for combined ablation and imaging. . Reconstruct cell soma and vasculature positions throughout the vibrissa sensory areas in rat cortex. This information will be used to evaluate essential architectonic parameters, including correlations among cell densities in lateral as well as radial directions, as well as essential metabolic parameters, such as the interconnectivity within the vasculature and the distribution of somata relative to capillaries. These two goals, one technical and the other scientific, are intrinsically linked and will proceed in parallel. The proposed advancements will provide a novel tool for the automation of histology, which underlies an understanding of brain function. We will make this tool readily available to the biomedical community. The proposed model system may substantially improve upon our understanding of the large-scale structure of brain architectonics.

[unreadable] DESCRIPTION (provided by applicant): Our understanding of substance addiction may be considerably increased if we know how neurotransmitter levels vary in select regions of cerebral cortex. In particular, it is believed that using addictive drug alters attentional processing mediated by acetylcholine (ACh). However, further progress in ACh research in the normal and addicted brain is hampered by the shortcomings of current techniques to measure levels of ACh, as these methods have limited time resolution and spatial accuracy. To overcome these problems, we are developing a novel cell-based optical method to detect ACh in vivo. "Sentinel Cells" are engineered cells that express cloned ACh receptors together with a genetically encoded fluorescent probe that reports the activity of the receptors. Sentinel Cells are implanted in rat cerebral cortex and their optical signal detected by two-photon laser scanning microscopy, allowing these cells to be imaged several hundred of micrometers inside the brain. A preliminary realization with HEK293 cells as a substrate validates this approach. We utilize the Gq family of G-protein metabotropic receptors, which couples to the IP3 pathway, and express the M1 G-coupled receptor for ACh together with cytoplasmic expression of a Troponin-C-based calcium sensor. These cells are engineered to respond to heightened extracellular Ach levels via IP3-mediated release of Ca2+ from the endoplasmic reticulum into the cytoplasm, which leads to a change in the extent of fluorescent resonant energy transfer between cyan and yellow fluorescent proteins. In vitro data suggest an effective Kd of ~ 30 nM and a response time of ~ 1 second for our preliminary realization. Complementary in vivo data shows that cells injected into cortex can respond to Ach release from intrinsic cholinergic fibers. Our plan is, first, to continue the development and optimization of Sentinel Cells for the detection of Ach in acute in vivo preparations. Second, to extend the realization of Sentinel cells to fibriblasts and chronic in vivo measurements. Third, to extend the concept of Sentinel Cells with metabotopic receptors to other cell signaling molecules common to cortex. This includes the neurotransmitter gamma-amino butyric acid (GABA) and the neurovascular signaling molecules Neuropeptide Y (NPY), vasointestinal peptide (VIP), and somatostatin (SOM). In toto, Sentinel Cells provide a novel means for (near) real-time monitoring in drug abuse research and basic research in cortical physiology. We propose to develop a novel optical bioassay to detect extracellular signaling molecules in the live brain. This technique combines engineered cells expressing a transmembrane receptor and a fluorescent reporter with in vivo two-photon laser scanning microscopy. The assay has wide ranging applications, including, but not limited to research on abused substances. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): We address active sensation in the context of the tactile localization of objects, with an ultimate goal to decode how a perception is converted into a motor act. Our approach is to map the transformation of sensory input within the closed loops of the vibrissa sensorimotor system, in which animals sweep long hairs or vibrissae rhythmically through the space that surrounds their head,. The focus of the proposed new experiments are to delineate the control of brainstem nuclei, both sensory nuclei that transform input sensations and motor nuclei that direct the sensors, by descending cortical projections. Such control is of general relevance to all mammalian sensorimotor systems. The nested, closed loop structure of the rodent vibrissa system is a model for sensorimotor control in mammals up through primates. As a focus for scientific investigation and progress in both computational and health related issues, the vibrissa system enjoys a significant data base of anatomical and electrophysiological data, is compatible with standard molecular and physiological tools, and is amenable to a studies with awake behaving animals, albeit with a reduced behavioral repertoire compared to primates. The proposed experiments involve the cortical activation of brainstem nuclei electrical and optical based physiological and anatomical studies with awake, trained rodents. We begin at the sensory end. 1) Does feedback from primary sensory cortex to brainstem nuclei alter the sensory response? Centrifugal fibers from sensory cortex project to integrative sensory neurons in the brainstem. We will determine if and how this feedback path modulates the gain of the reafferent signal of sensor motion, a basic aspect of proprioception. This may be used, based on our past work, to code touch in terms of coordinates that are normalized to the region of interest. More generally, this work addresses gain of function through high-level control. We next consider an essential missing module in the vibrissa sensorimotor circuit: Where are the brainstem nuclei that drive rhythmic whisking? We ask: 2) Does the rhythm pattern generator of breathing control whisking as well? We ask if the rhythmic generator for breathing acts as a central clock, or if breathing and whisking have independent rhythmic generators that phase-lock under different conditions. This query bears on the general coordination of orofacial behaviors. We then build on this effort and ask: 3) Does feedback from motor cortex control the region of interest of rhythmic whisking? The interpretation of touch in neural pathways depends on how the region of interest that shape vibrissa movements are regulated. We will determine how the slowly varying signals observed in motor cortex are used to guide the range of motion set by brainstem motor nuclei. This query bears on the general issue of coordinating motor output on multiple time-scales. Our data may lead to fundamental concepts in signaling and circuitry in the normal state as well as dysfunction states.

[unreadable] DESCRIPTION (provided by applicant): The dynamics response of individual neuronal vessels to sensory-stimuli is crucial to form a mechanistic understanding of functional imaging technologies, such as functional MRI (fMRI), as well as for understanding neurovascular dysfunction, as occurs in stroke and dementia. Toward this goal, we propose to characterize the stimulus-evoked cerebral hemodynamic response on the level of single arterioles and capillaries throughout a significant three-dimensional volume. Further, we will relate this characterization to the underlying neuronal electrical activity, the angioarchitecture, and the mitochondria density. Vibrissa sensory cortex of rat serves as our model system. Two-photon laser scanning microscopy (TPLSM), in conjunction with dyes that label the blood lumen, and all-optical histology, a related nonlinear optics technique, serve as our primary technology. As a prerequisite to the proposed measurements, we will improve the capability of TPLSM to allow rapid assessment of multiple blood vessels. This will allow us to characterize blood flow and blood vessel diameter at micrometer resolution throughout a 2 - 3 mm3 volume, along with correlations along flow in different vessels. [unreadable] [unreadable] Our analysis consists of three directions. [unreadable] Dynamical characterization of the diameter and flow dynamics of three classes of vessels, i.e., surface communicating arterioles, penetrating arterioles, and subsurface microvessels, in response to tactile single vibrissa stimulation. [unreadable] Ex vivo reconstruction of the exact angioarchitecture throughout the region of study by the in vivo vascular measurements, followed by three-dimensional mapping of the mitochondria density relative to the microvasculature. [unreadable] [unreadable] Our results will reveal, at a minimum: [unreadable] The characteristics, e.g., biphasic versus monophasic, of the temporal dynamics of the vessel diameter and blood flow changes of individual vessels. [unreadable] The dependence of the responses of a vessel on its distance from the center of the neuronal activity, its connectivity to major surface feeding arteries or penetrating arterioles, and its position relative to the local metabolic need as revealed by the mitochondria density. [unreadable] [unreadable] This work will bridge the critical gap between macroscopic functional imaging technologies such as fMRI and the microscopic understanding of single vessel responses to the neuronal activation. Stroke, vascular disease, and dementia are all dysfunctional states that relate to compromised cerebral blood flow. Our work will define the normal state of flow and bears on disruption to the normal state. It will help define optical- and MRI-based diagnostics for the detection of dysfunction and clinically appropriate interventionist therapies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This EUREKA proposal addresses two interlocked questions on neurovascular coupling: 1 - What are the design principles that link cortical blood flow dynamics to the underlying angioachitecture? In particular, is the microvasculature arranged in modules or does it form a continuum? 2 - What are the signaling mechanisms that neurons use to transform electrical activity into changes - both increases and decreases - in blood flow? In particular, is the relation between metabolism and blood flow mediated by vasoactive signaling molecules under the control of inhibitory interneurons? These are central questions in brain science, with many innovative hypotheses on the regulation and control of arterial transport. However, a dearth of experimental approaches has limited the formation of a central dogma on a vascular unit and the control of blood in the brain. Future progress will depend on the strong interplay of concepts from physics, neurophysiology, molecular biology, and experimental neurology. The confluence of this interplay, an essential feature of the proposed studies, should delineate emergent properties of vascular networks. Our approach makes use of four unique and in some cases developing tools: (1) "All Optical Histology" and associated computational procedures to reconstruct the angioarchitecture and cytoarchitecture of the mouse brain. These studies address the structure of the microvasculature. (2) Perturbation techniques, mediated by linear and nonlinear optical interactions, to modulate blood flow in targeted vessels. These studies probe the redundancy of flow, test models of experimental microstroke, and provide one means to study the consequence of changes in flow on neuronal activity. (3) Sentinel cells, formed by transforming HEK cells to respond to exogenous transmitters with an optical read-out, to directly measure patterns of vasoactive molecules. (4) In vivo two-photon guided patch to record from and stimulate inhibitory interneurons that release vasoactive substances. In combination with two photon measurements of blood flow, we can determine quantitative relations between neuronal activity and blood flow. These proposed questions bear on fundamental issues of blood flow in the normal brain including homeostasis and the underpinnings of blood-based imaging techniques such as fMRI - and issues in dysfunctional states - such as microstroke and microvascular diseases. PUBLIC HEALTH RELEVANCE: Uninterrupted blood flow in the brain is essential to maintain all aspects of cognition as well as homeostasis of bodily functions. The proposed work may significantly increase our understanding of how critical flow is regulated in both normal and dysfunctional states. Such understanding bears directly on the interpretation of diagnostics to monitor basal flow and the changes in flow that are induced by mental activity. Further, our work may suggest pathways for the potential treatment of microstroke and microvascular diseases.

Abstract Blood is a vital and limited resource in the brain: neuronal activity requires supplies of oxygen and glucose, and deprivation of flow to even restricted regions leads inevitably to cell death. How is the distribution of blood controlled relative to the changing needs of cortex? The answers bear directly on cortical function. Developmentally, they yield design rules for vascular architecture. From the perspective of physiology, the answers define state variables that relate neuronal activity to changes in blood flow. This has implications for neuroimaging, as the extraction of oxygen from blood is the basis of contrast in BOLD fMRI. Lastly, for neurology, the answers determine the resistance of cognitive processes to vascular trauma and disease. The current literature on molecular signaling between neurons and arterioles, based mainly on slice preparations, points to a competition between vasoactive signaling molecules that dilate arteriole smooth muscle and those that cause constriction. Mixtures of such signaling molecules are released by inhibitory interneurons, which directly contact muscle, and by excitatory neurons, which act via the excitation of astrocytes that encapsulate vessels. This picture is compelling yet incomplete, as natural stimuli change flow on the subsecond level while the literature points to changes on tens of seconds. The main challenge is to assay both signaling molecules and flow in vivo. We will meet this challenge by combining our strengths in in vivo electrophysiology and imaging with our methods for precise quantification of blood flow and newly engineered indicators for signaling molecules. We envision a model of neurovascular control that maps the activity of different neuronal subtypes to changes in vascular tone. The dynamics of the underlying signaling molecules form the state variables, much as channel dynamics form the state variables in single neuron dynamics. Such a model will clarify neurovascular disease models and neuroimaging studies.

DESCRIPTION (provided by applicant): We propose to advance a technology that, for the first time, will permit in vivo detection of any neurotransmitter that binds to a G-protein coupled receptor. This includes the monoamines, which play a fundamental role in neuromodulation and a biomedical role in addiction and mental disorders, and peptide transmitters, which are important for the neuroendocrine system and control of vascular tone and blood flow. At present, only limited in vivo assays are available to detect monoamines and peptide neurotransmission. This deficit is a major impediment to understanding normal signaling in the brain. To fill this gap of missing, we introduce Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for the optical measurement of exogenous receptor activity in vivo. A CNiFER is a clonal cell-line that is engineered to express a specific metabotropic receptor that couples to G proteins and a genetically encoded FRET-based Ca2+ sensor that detects changes in intracellular [Ca2+]. Stimulation of the metabotropic receptor leads to elevations in cytosolic [Ca2+], providing a direct and rapid readout of local neurotransmitter activity. CNiFERs are acutely or chronically implanted and fluorescence measured with in vivo two-photon microscopy. This new technology will make it possible to map the spatial patterns of in vivo signaling with up to ~ 100 5M spatial precision and ~ 1 s temporal resolution.

Technical abstract: This Major Research Instrumentation award supports a team of investigators at the University of California San-Diego to develop a state of the art infrared (IR) nano-spectrosopy system for ultra-fast (10's of fs) spectroscopic pump-probe measurements in the setting of a near field nano-scope enabling imaging at ultra-small length scales down to 5-10 nm. This one-of-a-kind system is a hybrid of an amplified ultra-fast laser beamline and an atomic force microscope. The IR beamline is based on a Ti:Sapphire amplifier driving an optical parametric amplifier, difference frequency and optical rectification unit. This versatile system enables intense pump pulses and highly stable probe pulses over a broad frequency range from visible to far-IR frequencies. The tunable repetition rate of the proposed Ti:S amplifier will allow the investigators to optimize data acquisition conditions of near-field spectra and images. The apparatus will allow a broad group of researchers to address some of the problems at the forefront of contemporary physics, chemistry, materials science and biological physics. These issues include among others: a spectroscopic characterization of ultrafast electronic and plasmonic dynamics in graphene, photo-induced phase transitions in correlated electronic systems, phase separation and competition between many body effects in high-Tc superconductors and dynamics of confined water and lipid bilayers. A common aspect of these projects is that they all require ultra-fast pump-probe measurements with nanoscale spatial resolution.
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Non-technical abstract: Infrared (IR) light provides a universal means for probing the vibrational and electronic properties of matter. Near-field IR optics enable spatial resolution beyond the so-called diffraction limit, thus opening access to the world of the ultra-small (down to ~5-10 nm) and overcoming the limitations of commonly employed ultrafast experiments using conventional diffraction-limited optics. This Major Research Instrumentation award supports a multi-disciplinary team of researchers comprised of a physicist, a chemist and a neuroscientist to develop a state-of-the-art facility for IR nano-imaging and spectroscopy analysis of materials and life science specimens from across the sciences. Once built and commissioned, the nano-spectrometer will meet the research and pedagogical needs of the sciences at the University of California San Diego and facilitate greater exposure of advanced scientific instrumentation to faculty, students, partners in industry and government laboratories. The commercialization of the instrumentation will help to maintain US leadership in manufacturing state-of-the-art tools for probing matter at the nanoscale.

? DESCRIPTION (provided by applicant): This proposal aims to develop new molecular techniques to map activities of neurons, manipulate the strength of communication between neurons and disrupt intracellular signaling. These 'optogenetic' approaches will be used to further our understandings of brain function on behavior and have important implications in our understandings of neurological conditions and neurodegenerative diseases. The first goal is to develop a technique where the researchers can use optical approach to identify synaptic connections that were active during the performance of a behavior task. This reporter system can be turned on with light, which defines the window of activity reporting, and fluorescence signal can be detected if there is significant activity between two defined cell groups. Many existing approaches can only be used to map excitatory connections, whereas the proposed approach can be used to identify activities between synapses utilizing any neurotransmitters. The approach will utilize a split fluorescent protein approach where its complementation and the generation of fluorescent signal is activity dependent. This approach will test whether a defined synaptic connection is involved in the performance of a behavior. The second goal is to develop a technique where the researchers can use light to modulate the strength of synaptic communication between neurons. Increasing synaptic strength is believed to underlie memory and learning, and its disruption has been implicated in drug addiction and many neurological conditions. Having the ability to modulate the synaptic strength experimentally can be used to interrogate how changes in synaptic strength alter learning and memory, leading to the observed adaptive behavior in the animals in both normal and pathological conditions. Many small protein fragments can alter synaptic strengths between neurons. A light-responsive protein can be used to functionally mask these protein fragments in the dark and light can be used to functionally release these protein fragments. This will permit rapid experimental control of synaptic strength and their functional effects can be studied in the behaving animals. This tool can be used to understand how alteration in synaptic strength changes during learning and adaption. The third goal of the project is to develop a technique where G-protein coupled receptor mediated second messenger pathway is inhibited by light. G-protein coupled receptors mediate the effects of neuromodulator and neuropeptides in the nervous system and they have great importance in modulating and/or mediating behaviors. Using a similar approach as described above, competitive binding peptides that disrupt G-protein coupled receptor-G protein interactions or peptides that directly inhibit the effectors of G- protein pathways can be masked and unmasked with light-responsive protein and light illumination. With this approach, light will turn off G protein activation or effectors of G-protein pathway rapidly to interrogate the behavioral effects of neuromodulators or neuropeptides in specific cells with defined temporal resolution.

? DESCRIPTION (provided by applicant): Neuronal circuits in the brainstem control life-sustaining functions, in addition to driving and gating active sensation through taste, smell, and touch. We propose to exploit the advent of molecular and genetic tools to undertake cell lineage marking, cell phenotyping, molecular connectomics, and methods from machine learning and image processing to construct an integrated anatomical and functional atlas of the brainstem. This will enable us to generate anatomical wiring diagrams for the brainstem circuits that control or facial actions. There are three phases to this work. (1) Reveal the identity and organization of brainstem nuclei. Motivated by striking similarities between the developmental plan for the spinal cord and brainstem, we will embrace and extend these efforts to interrogate the molecular composition of neurons that define individual nuclei with sensorimotor circuits in the murine brainstem. (2) Reveal brainstem neuronal circuits and their interactions. We will utilize Tran synaptic viral labeling to delimit pathways from specific muscles that are innervated by facial, trigeminal, hypoglossal, and laryngeal motor nuclei. This will reveal hitherto unknown brainstem circuits, including sites of modulation by higher brain areas. (3) Control the behavior of identified feedback circuits. We will manipulate specific populations of brainstem neurons using a battery of genetic tools to delineate or facial motor actions and motor synergies. The results from the above efforts will be a quantitative map of the functional organization of neurons in the brainstem that enable studies on computations that underlie or facial behavior. An understanding of these fundamental behaviors bears directly on the more general issue of how nervous systems deal with computations that can be performed autonomously, yet must interact synergistically. Thus our proposed program on brainstem circuitry and dynamics will yield general lessons about the nature of neuronal computation. The work performed under this proposal will serve as the basis for a larger national effort in brainstem neuronal computation.

Neuronal circuits in the brainstem are at the "front-end" of sensorimotor loops that underlie behavior and cognitive processing. They control life-sustaining functions that include breathing, movement and balance, sniffing, chewing, suckling in neonates, and, in rodents, whisking. These circuits further drive active sensation through taste, smell, balance, and touch. In this project the PI will delimit the circuits that control different motor actions and show how behaviors emerge as an assembly of these actions.

The "front-end" circuits of orofacial processing control life-sustaining functions that include breathing, movement and balance, sniffing, chewing, suckling in neonates, whisking in rodents, and vocalization. The functions, some of which share common muscles, must occur without compromising the patency of the airway. The control structure is not understood and nontrivial and it will serve as a model for the control and coordination of concurrent neuronal processes at any level in the nervous system. The application of control theory to nervous systems was proposed by the Cyberneticists of the 1940s to 1960s. These prescient notions were stymied by a lack of experimental tools to identify and control the circuits that underlie behavior. We now have sufficient tools to map brainstem circuits and the PI will exploit the tools of engineering and physics to gain insight into fundamental sensorimotor processes. The technical aspect of the the PI's approach involves the introduction of molecular tools based on the expression of lineage factors and constitutive proteins to identify brainstem neurons and track tracing tools based on trans-synaptic viruses to reveal connectivity The project will contribute to the education and the training of future multidisciplinary scientists through research-based education of undergraduate and graduate students. Technical aspects of work will be broadly disseminated through the involvement of the PIs and colleagues in graduate and post-graduate summer schools.

This is a development project to construct a scanning two- and three-photon microscope for deep imaging in the brain in support of activities related to neuronal circuit analysis and neurovascular coupling. The ability to image ever deeper in the brain with optical methods is a key enabling technology in our ability to decipher neuronal anatomy and circuit function as well as neurovascular function. Optical tools, together with labels of specific brain structures, are the only means to probe the geometry and state variables of single cells, e.g., voltage and second messengers, and the dynamics of brain vasculature in a noninvasive or partially invasive manner in vivo. The current method of choice for in vivo imaging makes use of two-photon microscopy with a 100-femtosecond pulsed laser sources to observe structure and dynamics throughout the upper ~ 500 micrometers of cortex of mice. Yet there is a clear need to image throughout the full depth of cortex, 1.0 to 1.2 micrometers in mice, to determine the complete flow of information in cortical processing. There is also a need to image deeper still into hippocampus and other subcortical structures without excavated overlying tissue, as well as to determine the loci of vascular control throughout gray and while matter. The initial proposed experiments, all of which depend on the proposed instrument, address topics in fundamental brain science as well biomedicine. Fundamental issues revolve around neuronal plasticity and memory formation and include: the formation of motor memories, where the learning of a behavioral task is believed to follow from the formation of patterns of correlated neuronal output in motor cortex; the transformation of sensory signals in cortex into memory traces, such as learned fear via the amygdala and induction of depression via the habenula; the role of specific gene products, known as inducible transcription factors, in synaptic plasticity; and understanding how the prodigious adult neurogenesis in the olfactory bulb is integrated into ongoing olfactory function. More applied issues concern the role of exposure to nicotine alone in changing the basis for memory formation, as well as issues in vasodynamics, including the locus for neuronal control of its own nutriment supply through the cortical vasculature and the impact of microinfarctions on cell death within the white matter, where myelinated fibers traffic information from sensory to motor areas that span the cortical mantle. Realization of this system will permit training of graduate students and postdoctoral fellows in state of the art in vivo optical imaging. UC San Diego, along with the greater La Jolla scientific community, supports a large and highly collaborative neuroscience community with graduate students and fellows who will pursue careers at institutes throughout the county, even the world. They will be inspired to think of new experiments based on the capabilities of imaging new vistas in the brain, as well as new associated technologies, particularly in the design of optical probes of yet unmeasured variables. Lastly, the high density of potential users within this community will facilitate unanticipated refinements of deep imaging and perhaps transform the proposed development project into a turn-key design for the benefit of the global neurosciences communities.


The PI proposes to build an instrument, whose design is motivated by three threads of work, that enables two- and three-photon imaging throughout the full depth of cortex and into deeper structures. First is the use of 100-fs pulsed laser light at wavelengths of 1.3 or 1.7 micrometers, where scattering is minimized but absorption by water is still weak; second is the use of an optical amplifier to increase the energy per pulse and drive fluorescence at greater depths, and third is the use of aberration corrective optics to counteract distortion of the incident beam with increasing depth into brain tissue.

? DESCRIPTION (provided by applicant): Neuronal circuits in the brainstem control life-sustaining functions, in addition to driving and gating active sensation through taste, smell, and touch. We propose to exploit the advent of molecular and genetic tools to undertake cell lineage marking, cell phenotyping, molecular connectomics, and methods from machine learning and image processing to construct an integrated anatomical and functional atlas of the brainstem. This will enable us to generate anatomical wiring diagrams for the brainstem circuits that control or facial actions. There are three phases to this work. (1) Reveal the identity and organization of brainstem nuclei. Motivated by striking similarities between the developmental plan for the spinal cord and brainstem, we will embrace and extend these efforts to interrogate the molecular composition of neurons that define individual nuclei with sensorimotor circuits in the murine brainstem. (2) Reveal brainstem neuronal circuits and their interactions. We will utilize Tran synaptic viral labeling to delimit pathways from specific muscles that are innervated by facial, trigeminal, hypoglossal, and laryngeal motor nuclei. This will reveal hitherto unknown brainstem circuits, including sites of modulation by higher brain areas. (3) Control the behavior of identified feedback circuits. We will manipulate specific populations of brainstem neurons using a battery of genetic tools to delineate or facial motor actions and motor synergies. The results from the above efforts will be a quantitative map of the functional organization of neurons in the brainstem that enable studies on computations that underlie or facial behavior. An understanding of these fundamental behaviors bears directly on the more general issue of how nervous systems deal with computations that can be performed autonomously, yet must interact synergistically. Thus our proposed program on brainstem circuitry and dynamics will yield general lessons about the nature of neuronal computation. The work performed under this proposal will serve as the basis for a larger national effort in brainstem neuronal computation.

DESCRIPTION (provided by applicant): The PI introduces novel instrumentation to image and then analyze and quantify the whole- cortex electrical dynamics of neurons and the analogous vasomotor dynamics of brain vasculature. The spatial-scales encompassed by this instrument span from cell resolution - at the one-micrometer scale - to all of mouse cortex - at the then-millimeter scale. Large-scale brain activity encompasses the neurovascular issue of the redistribution of blood flow to areas of heightened metabolic need as well as patterns of blood flow secondary to vasomotor activity. It further concerns neurological issues of correlated neuronal activity in attention and decision making, distant signaling in multisensory processing and, at a more abstract level, issues such as the patterning of electrical waves in the brain. Further, large- scale Thus the proposed instrument gains broad intellectual merit as a means to diagnose neuronal and neurovascular processing that extends across different cortical areas. Our approach involves the design and realization of a novel, large-field in vivo two-photon microscope with an unprecedented ten-millimeter field of view with one micrometer resolution across the field. This is sufficient to image the entire cortical mantle in mouse with subcellular resolution. Our design and realization must meet the complex yet not insurmountable challenges of maintaining an aberration-free beam across large scan-angles, which are traditionally associated with chromatic and spatial aberrations. While there is limited guidance in the published literature on correcting such problems, we are able to successfully address these issues in a systematic way. Our novel microscopy will be combined with the use of transgenic animals in which individual specific cell types express a functional reporter. This will allow us t record from specific cell types - deep to the pial surface - and thus provide an anatomical basis for neuronal and neurovascular dynamics. Our approach provides a qualitative improvement of past, whole-field imaging techniques in which uniformly stained neuronal tissue was studied with neither depth nor cell-specific information. Beyond our scientific interests in neuronal processing and neurovascular coupling, our unique instrument will be of service to investigations of many other topics in the physiology of whole systems, including for example cell migration in development and regeneration as well as the flow of cells and tissue in tumor genesis and metastasis. Technical aspects of work will be broadly disseminated through the involvement of the PI and colleagues in graduate and post-graduate summer schools.

Project Summary Neuropeptides are essential neuromodulators in the brain. They are released into the extrasynaptic space, where they diffuse over long distances and signal through G protein coupled neuropeptide receptors. Neuropeptides control cognition, sensorimotor processing, and energetics through changes in vascular tone and blood flow in the nervous system. Pharmacological and molecular genetic studies have implicated alterations in neuropeptide signaling as a contributor to brain dysfunctions, including migraines, addiction, motivation and stress. Although widely expressed in the brain, remarkably little is known about when and where neuropeptides are released. Monitoring the release of neuropeptides in real-time in awake animals performing complex behaviors would be transformative, enabling the elucidation of the function of neuropeptides in regulating neural circuits in the brain. In response to RFA-MH-16-775, we propose to develop and validate an innovative neurotechnique for optically measuring release of neuropeptides in a cell-specific and circuit-specific processes in the brain. The new technology is based on cell-based neurotransmitter fluorescent engineered reporters, referred to as CNiFERs, which were original developed for detecting the release of classical, small molecule neurotransmitters. A CNiFER is a clonal HEK293 cell that is engineered to express a specific G-protein coupled receptor and a genetically encoded fluorescence-based intracellular calcium sensor. CNiFERs are implanted in the brain, where they produce minimal inflammation and remain viable for days, and have been used successfully to measure volume transmission of dopamine, norepinephrine and acetylcholine in vivo during learning. Three neuropeptide CNiFERs will be developed and used for test-bed validation projects within our own laboratories: Orexin, which is important in sleep regulation as well as drug seeking and reinstatement, Somatostatin which has been implicated in depression, motivation and learning, and Vasoactive Intestinal Peptide, which as been implicated in neuroplasticity and learning. For collaborative projects, we will further construct four additional neuropeptide CNiFERs for detecting release of Dynorphin, Corticotropin-Releasing Factor, Neuropeptide Y and Substance P. Each neuropeptide CNiFER will be subjected to rigorous in vitro testing prior to their use to study the dynamics and consequences of release of neuropeptides in vivo.

Principal Investigators(Last, first, middle):KLEINFELD, DAVID and ROSEN, BRUCE R. Functional magnetic resonant imaging (fMRI) is the only means to infer neuronal activity within the entire volume of the human brain. A powerful aspect of fMRI concerns coordinated fluctuations in the amplitude of blood oxygen level dependent (BOLD) signals across distant regions of the brain, which are interpreted as resting-state functional connections. Here we address the underlying biophysical mechanism that underlies resting-state functional connectivity. Our hypothesis is that the natural ultra-low frequency oscillations in the smooth muscle of arteriole walls, termed vasomotion, acts as an intermediate oscillator that links oscillations in neuronal activity with the blood oxygenation and thus fMRI signals. Rodent models permit us to test this hypothesis through detailed two-photon imaging, advanced fMRI measurements, and manipulations of cortical vascular dynamics and blood oxygenation under controlled conditions. We then advance the spatial resolution of ultra high field MR imaging in humans to image single intracortical vessels, with 100 micrometer or better resolution, to test whether vasomotion may be a unifying mechanism for resting and task-driven fMRI signals. The results of these studies have two consequences. One is to provide the underpinnings for interpreting resting state connectivity relative to neuronal projections. The second is a new model of mapping functional connections via changes in arteriole volume. In particular, the strong homologies between the physiology of rodents and primates suggest that these methods can be extended to map resting- state functional connections in the human brain with higher resolution and greater precision than previously achieved. This new mechanistic insight will advance our use of fMRI to study cognition and a variety of neuropsychiatric disorders.

Principal Investigator (Last, first, middle): KLEINFELD, DAVID We propose to advance our understanding of the key factors involved in the distribution of blood within the brain. Our focus begins with the relation of flow dynamics to the topology of the underlying angioachitecture. In cortex, a highly interconnected network of surface vessels and a network of subsurface vasculature were shown to be effective in distributing blood and in providing a robust immunity to occlusions. In contrast, the penetrating arterioles that shuttle blood from the cortical surface to the underlying microvessels were shown to be a bottleneck to the supply of blood within the brain and a locus for cognitive decline after a microstroke. We will determine if other brain areas follow the same vascular plan or rather have a different set of design rules. Our initial emphasis is on hippocampus, given the great sensitivity of hippocampal neurons to ischemia. While the topology of the vasculature is fixed, the diameter and thus the resistance to flow of individual can change. First, the diameter of brain blood vessels changes with vasomotion at a frequency of 0.1 Hz. It is not known if this slow signal phase-locks to vasomotion in a distant region or in the contralateral hemisphere that is also activated by a common stimulus. We will determine the effects of awake sensory stimulation on vasomotion along pathways over large regions of cortex. The finding of activity induced correlation of vasomotion should have implications as a basis for the functional connectivity derived by bold oxygenation level dependent (BOLD) functional magnetic resonant imaging (fMRI). A second aspect of neurovascular signaling concerns changes in diameter in arterioles and potentially microvascular capillaries in response to modulators released by stimulus-induced neural activity. The mechanism by which neural activity leads to both vasoconstriction and to vasodilation is a puzzle. We will determine the competitive nature of vasoactive signaling by concomitant measure of extrasynaptic modulator concentration and activation of specific neuronal subtypes in terms of their affects on changing blood flow through vasoconstriction or vasodilation. We note that all of the proposed experiments make use of a broad range of technologies, ranging from behavior to physiology to probes to imaging, and thus provide a fertile test bed for scientific discovery as well as a means to train the next generation of neuroscientists as generalists.

Overview - Abstract Brainstem function is necessary for life-sustaining functions such as breathing and for survival functions, such as foraging for food. Individual motor actions are activated by specific brainstem cranial motor nuclei. The specificity of individual motor actions reflects the participation of motor nuclei in circuits within closed loops between sensors and muscle actuators. However, these loops are also nested and connect to feedback and feedforward pathways, which underlie coordination between orofacial motor actions. A key question for this proposal is how different actions are coordinated to form a rich repertoire of behaviors, such as rhythmic motions linked to breathing, and the orchestrated displacements of the head, nose, tongue, and vibrissae during exploration. We postulate that the best candidate interface for orofacial motor coordination are premotor and pre2motor neuron populations in the brainstem reticular formation: these neurons project to cranial motor nuclei, receive descending inputs from outside of the brainstem, and interconnected to each other. Our approach exploits and expands upon a broad spectrum of innovative experimental tools. These include state-of-the-art behavioral methods to study motor actions and their coordination into behaviors. From an experimental perspective, the underlying neuronal circuitry for each orofacial motor action may be accessed via transsynaptic transport starting at the muscle activators or associated sensors in the periphery. These studies will make use of molecular, genetic, and functional labeling methods to enable cell phenotyping and circuit tracing. These data will establish the Components, i.e., brainstem nuclei connectivity for all Research Projects. These studies are complemented by in vivo electrophysiology and optogenetics in order measure and perturb the signal flow during exploration and decision-making: these studies will establish orofacial ?Wiring Diagrams?. The sum of these techniques will permit us to elucidate the functions of intrinsic brainstem circuits and their modulation by descending pathways. Our data will be integrated in two ways. First we will begin development of computational models of the dynamics of active sensing by the orofacial motor plant and brainstem circuits. These will initially focus on the vibrissa system, starting with characterizations of mechanics and mechano-neuronal transformations of vibrissa movement and extending to exploration of brainstem circuits that drive vibrissa set-point and rhythmic whisking. Finally, vibrissa feedforward pathways will be computationally modeled to explore how sensory input affects vibrissa dynamics. Second, to record connectivity data that arises from our experimental tracing studies, we will construct an Trainable Texture-based Digital Atlas that utilizes machine learning to automate anatomical annotation of brainstem nuclei. The Atlas is designed to allow accurate 3D alignment of labeled neurons, even when labeled neurons reside in small sub-regions outside of well-defined brainstem nuclei, based on triangulation to Atlas landmark structures. Further, digitization of serially sectioned brain data sets allows 3D reconstruction and alignment of small brainstem subregions as well as the collation of this data from different brains into the same Atlas. Our proposed program on brainstem circuitry and dynamics will yield general lessons about the nature of neuronal computation. The analytic and anatomical tools developed for these studies will be made available through our data science core to the larger neuroscience community.

Project 3. Abstract Descending control of orofacial behavior (Kleinfeld lead; Mukamel, Svoboda, Wang) This Research Project will define the connectivity and neural mechanisms of descending control by motor cortex of orofacial behavior. Orofacial movements can be rhythmic and coordinated with each other. However, individual movements can also be specifically controlled to achieve behavioral goals, such as consuming a reward at a particular location and time. We will use a behavioral assay in which mice have to make a directional tongue movement at a particular time to receive a reward. This further serves as an example of set- point control. The brainstem level controls for licking are driven by the motor neurons in the hypoglossal nucleus whose activity in turn is modulated by premotor neurons in the intermediate nucleus of the reticular formation. Our studies will explore the anatomical connectivity, i.e. ?Components? and signal flow, i.e., ?Wiring Diagrams? that converge on this region of the reticular formation. We will further refine the description of hypoglossal premotor subregions, nominally referred to here as ?hIRt?, that are relevant to licking. Our preliminary data indicates that motor cortex can direct the timing of licking bouts and direction of licking, but not the timing of individual licks. We will trace these command signals from the motor cortex through the superior colliculus and into the hIRt. We will measure neural signals in another descending pathway from the basal ganglia that converges on the superior colliculus and the hIRt. We will measure how these descending inputs are synaptically coupled to defined neuron types in the hIRt. Together this project will provide a mechanistic account of how descending signals from multiple sources are integrated at the level of premotor neurons in the brainstem, complementing our studies on projections that control the set-point of whisking (Project 2). An additional focus will set the stage for the next generation dissection of the neural circuits in the brainstem. This requires molecular profiling of specific cell classes to provide transcriptomes that will facilitate future fine-grained analyses, including genetic labeling, manipulation, and transsynaptic tracing. Specific populations of premotor neurons will be isolated by transsynaptic labeling and mRNA will be isolated using Translating Ribosome Affinity Purification (TRAP), a technology that isolates mRNAs associated with translating ribosomes and is especially favorable in the densely myelinated brainstem. This is followed by deep sequencing and yields a new method, TRAP:Seq.

PROJECT SUMMARY/ABSTRACT ? PROJECT 1 We propose to leverage our state-of-the-art expertise in vivo optical imaging and data analysis, combined with behavioral training, electrophysiology, and modeling, to investigate fundamental aspects of the pial neurovascular circuit in mice. This circuit is composed of a fully connected albeit irregular lattice of pial arterioles that undergo rhythmic oscillations - in the ~ 0.1 Hz vasomotor band - in isolation. The pial circuit integrates neuronal activity from neighboring vessels, underlying neurons, and subcortical regions to produce dynamic patterns of coherent oscillations in arteriolar diameter across the cortical mantel. These patterns contain regions at slightly different frequencies, i.e., they parcellate, in a manner that partially reflects the underlying neuronal input. We seek to understand and model this parcellation, which is readily measured with optical and functional MR imaging, and quantify how it defines brain state. Aim 1 seeks to formulate an understanding of fundamental physiology of the pial neurovascular circuit. This includes testing if brain arterioles truly act as non-linear interacting oscillators, so that they entrain and phase lock rather than passively filter. In Aim 2 we explore the competitive conditions that break locking between oscillators so that parcellation can occur. These experiments gain from our ability to use sensory stimuli from different modalities - touch, vision and audition - each of which targets a different brain area. They also gain from our ability to drive subcortical inputs, particularly those involved in homeostatic brain function, and use direct optogenetic stimulation where needed. Lastly, these experiments gain from interaction with the neuromodulatory investigations of Project 2, as subcortical neuromodulation provides both regional and cortex-wide control of neuronal excitability. The experimental plan is motivated by the theory of phase-coupled oscillators that dates from Yoshiki Kuramoto's 1975 Lecture Notes. In this regard, progress on Aims 1 and 2 are strongly interwoven with the theory effort of Project 4. Aim 3 will connect the dynamics of the pial neurovascular circuit with the dynamics of the penetrating arterioles; these vessels source energy substrates to the parenchyma. These experiments, also in rodents, involve deep imaging of the cerebral mantel with CBV fMRI and adaptive optics two photon imaging. Together with direct measurements of oxygen transport in Project 2, these data provide input for calculations of oxygen tension throughout the cortical mantle. This, in turn, provides a means to couple BOLD fMRI and/or CBV fMRI to pial neurovascular dynamics. All told, the experimentation and analysis of Project 1 will provide a way forward to infer the state of the human mind from MR imaging (Project 3).

Principal Investigator (Last, first, middle):KLEINFELD, DAVID Project summary Two-photon laser scanning microscopy is indispensable for imaging the structure and function of the mammalian brain with subcellular resolution. However, the resolution and efficiency decreases with tissue depth as a result of scattering and optical aberrations. Adaptive optics can improve multi-photon imaging by synthesizing a distortion to the wavefront of the excitatory beam that compensates for aberrations in the wavefront that are created by the tissue. Our system utilized adaptive optics to enable investigators to probe subcellular dynamics in individual synapses along the full depth of cortex. This is a crucial advance, particularly as layer 5 and 6 cortical output neurons lie deep to the surface of the brain. Many contemporary studies within the neuroimaging community are limited by the current inability to record synaptic dynamics within output regions of cortex, e.g., layer 5, as opposed to within the dominant input layer, i.e., layer 4, and the intermediate levels, e.g., layers 2/3. We will remove this limit and thus open up a new subfield of in vivo studies on subcellular determinates of cortical output. Our proposed work incorporates good engineering practice in the design of our current adaptive optics two-photon microscope design. We will disseminate accurate plans and construction details to enable other laboratories to duplicate this system. We will further educate the neuroimaging community on the principles of adaptive optics and the design and utility of adaptive optics-based two-photon microscopes. This effort includes workshops at UC San Diego. Throughout the period of the proposed grant, we will continue to advance the adaptive optics two-photon system and update and expand our user base. Proposed new directions include a rapid shift in focus together with aberration correction for diffraction limited focus over planes separated by as much as 300 µm and the incorporation of a resonant scanner for fast cell-based imaging. Lastly, we will form a team effort among users and incorporate feedback from the team to extend adaptive optics into new areas of inquiry in neuroscience as they arise.