Thomas Stephen Otis, PhD - US grants

DESCRIPTION: (Applicant's Abstract) Glutamate transporters perform an essential function at excitatory synapses in the brain by helping to terminate excitatory signals. Based on their molecular diversity, distinct patterns of expression and propensity for modulation, it seems likely that glutamate transporters play additional, more active roles in excitatory synaptic function. However, their potential contributions to excitatory signaling remain unexplored. The broad objectives of our research are to understand glutamate transporter function at intact synapses and to define roles for transporters in excitatory signaling. This proposal uses whole-cell electrophysiological techniques applied to brain slices to focus on glutamate transporters at the parallel fiber (PF) to Purkinje neuron (PN) synapse in the cerebellum. PF synapses are regulated by a form of activity-dependent plasticity which is triggered by activation of a G- protein coupled glutamate receptor (mGluR1a). This receptor type is colocalized with a postsynaptic glutamate transporter present on PN dendrites. The close arrangement of these two molecules raises the possibility that glutamate transport controls mGluRla activity by limiting extracellular glutamate concentration at PF synapses. Each of the aims in the proposal specifically addresses a component of this hypothesis. The first aim examines the degree to which postsynaptic glutamate transporters limit glutamate concentration during PF synaptic activity. The second aim examines the effects of blocking glutamate transport and of manipulating glutamate concentration on the synaptic activation of mGluRla. The third aim tests if mGluRla modulates glutamate transport, a hypothesized negative feedback loop expected to regulate synaptic plasticity at PF synapses. These experiments illuminate a novel mechanism for controlling experience-dependent changes in excitatory circuitry in the brain. The findings should also lead to a better understanding of glutamate synapses and provide insight into diseases associated with malfunctions in excitatory transmission such as epilepsy and Amyotrophic Lateral Sclerosis.

The broad goals of this proposal test two hypotheses, (i) that extrasynaptic inhibition of cerebellar granule cells is mediated by receptors made up of the GABAA receptor (GABAR) subunits alpha6, beta3, and delta, and (ii) that receptors of this subunit composition are primary targets for certain general anesthetics, neuroactive steroids, and ethanol. The proposal relies on a combination of molecular approaches and electrophysiological analysis. This project carefully examines granule cell GABAR function in mice deficient in the beta3, and delta subunit genes and in a newly generated "knock-in" rat carrying a point mutation (R100Q) in the granule-cell specific GABAR gene alpha6.The R100Q mutation is hypothesized to enhance the sensitivity to various GABAR modulators including ethanol and benzodiazepines. Three specific aims will be undertaken: 1) Electrophysiological analysis of synaptic and extrasynaptic GABAR signals will test whether extrasynaptic inhibition is specifically disrupted in mice lacking the genes for the beta3 and delta subunits. 2) Measurement of synaptic and extrasynaptic signals in wild type and in the alpha6R100Q "knock-in" rat will be used to evaluate whether extrasynaptic GABARs are key determinants of behaviorally relevant concentrations of ethanol, general anesthetic, and neuroactive steroid action in cerebellum. 3) Mutant, fluorescently-tagged GABAR subunits designed to enhance or impair sensitivity to ethanol or general anesthetics will be introduced into wild type and knockout granule cells and extrasynaptic GABAR signals will be measured. These multidisciplinary experiments rigorously evaluate the molecular basis for extrasynaptic inhibition and test identify molecular determinants of ethanol, anesthetic, and neuroactive steroid action. Advancement in understanding the mechanisms responsible for anesthesia and ethanol intoxication will yield far-reaching clinical and societal benefits including improved treatments for psychiatric disorders and better therapies for severe alcohol abuse.

[unreadable] DESCRIPTION (provided by applicant): This grant proposes to generate protein-based, genetically-encodable sensors for the primary excitatory neurotransmitter glutamate and to express these fusion proteins in situ to study spatiotemporal aspects of excitatory synaptic signaling. A series of constructs based on fusion of a neuronal glutamate transporter (EAAT3 or EAAT4) and ion-sensitive variants of GFP will be generated. Sensor constructs will be evaluated in three stages. First, each will be characterized in HEK cells to establish whether glutamate elicits optical signals, and if so to determine the pH and chloride sensitivity and the kinetic properties of those signals (Aim 1). Next, viable constructs will be inserted into viral vectors suitable for expression in rat brain. The ability of constructs to generate optical signals in response to excitatory synaptic activity will be tested (Aim 2). Combining optical and electrophysiological measurements, constructs will be used to map the spatial pattern of active synaptic inputs to neurons under various conditions (Aim 3). Together these experiments will develop and establish the practical usefulness of a class of genetically-encodable indicators of excitatory synaptic transmission. Once developed, such indicators are expected to have an important impact on the study of information processing within neural circuits, allowing minimally invasive measurement of subthreshold synaptic signals. [unreadable] [unreadable] [unreadable]

The broad goals of this proposal test two hypotheses, (i) that extrasynaptic inhibition of cerebellar granule cells is mediated by receptors made up of the GABAA receptor (GABAR) subunits alpha6, beta3, and delta, and (ii) that receptors of this subunit composition are primary targets for certain general anesthetics, neuroactive steroids, and ethanol. The proposal relies on a combination of molecular approaches and electrophysiological analysis. This project carefully examines granule cell GABAR function in mice deficient in the beta3, and delta subunit genes and in a newly generated "knock-in" rat carrying a point mutation (R100Q) in the granule-cell specific GABAR gene alpha6.The R100Q mutation is hypothesized to enhance the sensitivity to various GABAR modulators including ethanol and benzodiazepines. Three specific aims will be undertaken: 1) Electrophysiological analysis of synaptic and extrasynaptic GABAR signals will test whether extrasynaptic inhibition is specifically disrupted in mice lacking the genes for the beta3 and delta subunits. 2) Measurement of synaptic and extrasynaptic signals in wild type and in the alpha6R100Q "knock-in" rat will be used to evaluate whether extrasynaptic GABARs are key determinants of behaviorally relevant concentrations of ethanol, general anesthetic, and neuroactive steroid action in cerebellum. 3) Mutant, fluorescently-tagged GABAR subunits designed to enhance or impair sensitivity to ethanol or general anesthetics will be introduced into wild type and knockout granule cells and extrasynaptic GABAR signals will be measured. These multidisciplinary experiments rigorously evaluate the molecular basis for extrasynaptic inhibition and test identify molecular determinants of ethanol, anesthetic, and neuroactive steroid action. Advancement in understanding the mechanisms responsible for anesthesia and ethanol intoxication will yield far-reaching clinical and societal benefits including improved treatments for psychiatric disorders and better therapies for severe alcohol abuse.

DESCRIPTION (provided by applicant): Two major impediments in the study of neural plasticity are the lack of tools for monitoring electrical signals in very small compartments such as dendritic spines, and the limited methods for studying electrical activity in groups of interacting neurons. For these reasons many neuroscientists agree on the need for optical sensors of membrane potential that are easy to use, have limited photoxicity, and the speed and sensitivity required for detection of individual action potentials (APs) in single neurons. In this application we propose to develop and apply a novel two-component optical approach for sensing membrane potential, based on Fvrster resonance energy transfer (FRET). A critical feature of our method is that it relies on the widely used neuronal tracer dye, DiO, as a donor in the FRET reaction while dipicrylamine (DPA), a molecule whose membrane partitioning is voltage sensitive serves as the acceptor. Preliminary data show that large and rapid fractional fluorescence changes (56 % per 100 mV, D ~ 0.1 ms) are observed in response to membrane depolarization of cultured cells. In cultured neurons and in neurons in brain slices, AP-induced optical signals are nearly 3-fold larger than with any other reporter, making it possible to detect subthreshold activity and APs in single trials from membrane areas less than a square micron. The application is organized into three aims. Aim 1 proposes to further characterize the system, establishing the effectiveness of two-photon sources, and carefully measuring the effects of DPA on electrical excitability. Using a stationary laser spot approach, Aim 2 proposes to measure membrane potential in single dendritic spines, a subcellular compartment regarded as a key site of neural plasticity. Aim 3 utilizes "diolistics" to label groups of functionally similar neurons to demonstrate that the DiO/DPA system can be used to monitor activity in small neural circuits. The proposal seeks to establish a robust and flexible new method for non-invasive monitoring of electrical signal flow within single neurons and anatomically-defined neural circuits. We expect that this new experimental approach will enable rapid progress in the study of neural plasticity both in preparations that are genetically-amenable and those that are not. PUBLIC HEALTH RELEVANCE: Current technologies for measurement of neural activity at the level of single neurons within circuits are severely limited and this prevents progress in understanding many brain diseases in which neural signaling is dysfunctional. This proposal presents a noninvasive optical method for measuring neural circuit activity;this novel strategy should enable advancements in our understanding of many of the underlying mechanisms of neurological diseases.

DESCRIPTION (provided by applicant): We propose to use novel optical methods to study extrasynaptic GABAA receptors, a subset of inhibitory neurotransmitter receptors implicated in both acute alcohol action and in adaptation to chronic alcohol exposure. These new methods will allow us to examine pools of these receptors that, for technical reasons, have been experimentally inaccessible. Specific aim one proposes to use laser-based photolysis techniques to determine the properties of non- synaptic GABARs in various locations on cerebellar neurons. The second specific aim makes use of a novel optical voltage sensing method to examine GABAR function and pharmacology on axons and presynaptic terminals. The results will facilitate understanding of the molecular determinants of ethanol and sedative action in the brain and should speed the development of new therapies to address alcohol addiction and the chronic changes in brain function that occur during alcoholism. PUBLIC HEALTH RELEVANCE: We propose to use novel optical methods to study extrasynaptic GABAA receptors, a subset of inhibitory neurotransmitter receptors implicated in both acute alcohol action and in adaptation to chronic alcohol exposure. These new methods will allow us to examine pools of these receptors that, for technical reasons, have been experimentally inaccessible. The results will facilitate understanding of the molecular determinants of ethanol action in the brain and should speed the development of new therapies to address alcohol addiction and the chronic changes in brain function that occur during alcoholism.

DESCRIPTION (provided by applicant): This application describes a multifunctional microscope system capable of patterned excitation of various optically sensitive targets. This custom-designed instrument will be used to explore cutting edge questions in neural cell biology, single neuron physiology, and microcircuit function. It will incorporate a pulsed-IR source for PMT-based two-photon imaging;in addition it will have another optical path allowing for patterned, single-photon illumination at various wavelengths (405, 473 and 594 nm). Patterned illumination will be accomplished with spatial light modulator (SLM) technology which permits user defined patterns of illumination to be delivered to the specimen plane (Lutz et al., 2008;Nikolenko et al., 2008). A high speed CCD camera will be used as an alternative detector for experiments in which high time resolution (i.e.>1 kHz) optical measurements are necessary. This will allow optical measurements of electrical activity from many single neurons labeled with a novel voltage sensor developed by one of the group members. A survey of existing microscopy capabilities at UCLA demonstrates that no equipment with these capabilities is available;indeed two of the users have collaborations with overseas laboratories to enable the work. This instrument should allow experimental manipulation of several cutting edge optical tools with unparalleled spatial and temporal precision. These include: 1) photoactivatable or photoconvertible proteins (PA-GFP, Dendra2, DRONPA) useful for cell biological studies of nervous system function, 2) caged neurotransmitters that can be used to photostimulate individual neurons or circuits, 3) photosensitive channels (channel- and halorhodopsin) or reversible photo switches (PALs) to activate or inactivate specific circuit elements, and 4) a novel, ultra rapid optical reporter of membrane potential that enables simultaneous measurements of neural activity from many individual neurons. The application details how the instrument system will directly benefit more than 10 currently funded NIH projects on which the five major users serve as Principal Investigators. Research in these laboratories is directed at understanding fundamental issues in neurophysiology and neural cell biology including the molecular mechanisms involved in excitation-contraction coupling (Vergara), exocytosis (Schweizer), and neuronal plasticity (Martin) as well as aspects of microcircuit function in brainstem respiratory centers (Feldman), vestibular epithelium (Schweizer), and in cerebellar cortex (Otis). Various UCLA Departments and the UCLA School of Medicine will provide $92,922 in funding to enable the purchase of the system;they will also contribute more than $46,000 per year in ongoing funding to cover the service contract for the equipment. In addition, the user group will provide approximately $90,000 of electrophysiological, micro perfusion, and temperature control equipment so that neurophysiological experiments can be done in parallel with optical measurements under controlled physiological conditions. These pledges totaling well over $250,000 in institutional support are evidence of the scientific enthusiasm and intense need for this type of instrumentation at UCLA. PUBLIC HEALTH RELEVANCE: This grant would provide funding for a state-of-the-art microscope system incorporating advanced optical technology that enables parallel manipulation and/or measurement of neuronal activity within various brain microcircuits. The system will also permit sophisticated tracking and manipulation of optically-tagged signaling proteins with unprecedented precision in cell biological experiments. UCLA has a large and highly collaborative neuroscience research community and this equipment will accelerate progress in the laboratories of the NIH-funded major users whose research on basic mechanisms underlying disorders of breathing, movement, balance, and learning is currently funded by more than 15 PHS grants.

IDBR: Development of a streak microscope for measurement of fast multineuronal signals.

One of the greatest challenges in neuroscience is to understand how patterns of electrical activity in networks of neurons underlie brain function. Although the fundamental electrical signals (action potentials) and the basic hardware elements that generate these signals (neurons) are well known, the temporal (~ 1 kHz) and spatial (~10 micrometer) scales in which this signaling occurs prohibit parallel measurements from more than a small number of neurons. In recent years, new optical methods for tracking neural activity have been developed and offer great promise for overcoming some of these technical barriers. However, available microscopy methods and instrumentation are incapable of recording ensemble neural activity with sufficient spatial and temporal resolution. This proposal aims to address this issue directly by constructing a novel microscope optimized to record ensemble neuronal activity with temporal precision (> 8KHz) appropriate to resolve action potential activity in single neurons. The microscope¡¦s design is inspired by star trails observed in long exposure images of the night sky and is thus termed a fluorescent trails microscope (FTM). The FTM will be optimized to perform prolonged optical measurements of spatially distributed signals with submillisecond resolution. Rather than scanning a laser beam, the microscope will utilize a computer addressable diffractive element (a spatial light modulator or SLM) to generate continuous, patterned illumination of user-selected regions of interest. Images will be swept across a CCD or CMOS sensor in synch with the frame rate, allowing for the increased temporal resolution. This flexible design should be adaptable for use in a variety of experimental preparations including in vivo brain imaging and other biomedical applications involving time resolved fluorescence photometry. The proposed activity will foster specific cross-disciplinary, interactions and pre-college educational activities. Once fully developed, this technology will be used by a wide range of scientists at UCLA and beyond who are interested in measuring neuronal network activity as well as studying other biological phenomenon with collective properties. Construction of the instrument will involve recruitment of students in Physics from a nascent "Neurophysics Program" at UCLA who will be engaged in the interpretation of the resulting large quantity of experimental data requiring expertise in systems neurobiology and statistical physics. The project will provide a platform for students in multiple graduate disciplines (i.e., Physics, Neurobiology, Mathematics) to expand their knowledge base beyond the traditional boundaries of their respective fields as well as motivate campus wide interaction and collaboration. Lastly, there will be an opportunity for high school and undergraduate students to tour the laboratory and learn about the science behind the project, as well directly engage in the research, with a particular emphasis on recruiting those from underserved communities.

DESCRIPTION (provided by applicant): Effective coordination requires that the motor system predict proper movements. To make these predictions, the cerebellum integrates sensorimotor information and motor errors and, through a process of error-driven learning, build up feed-forward models of movement. Decades of cerebellar research have clarified a highly stereotyped circuit, identified roles for particular circuit elements, and suggested cellular mechanisms that might account for associative learning. However, fundamental questions remain unanswered. How do particular activity patterns in Purkinje neurons influence movement? What are the functional ramifications of the neurochemically-defined divisions in the motor map? Where within the cerebellar circuit do changes occur during cerebellum-dependent forms of motor learning? And finally, how do circuit changes alter cerebellum-dependent behavior? The following specific aims will be addressed in the project. In Specific Aim 1, we will interrogate the organization of the motor map in the simplex lobe of the mouse cerebellum using optogenetic stimuli. Preliminary data show that Purkinje neuron inhibition triggers rapid, highly stereotyped movements. Using high speed videography and motion tracking we will measure movement trajectories and speeds in response to activation or inhibition in various cerebellar neurons with patterned illumination. We will also make electrophysiological recordings from cerebellar neurons in awake mice to examine the effects of manipulating PN excitability on the circuit. In Specific Aim 2 we will test whether associative motor learning can be driven by pairing sensory stimuli with optogenetically-elicited reductions or increases in PN firing. In vivo electrophysiology will be used to determine how error signals contribute to this learning. In Specific Aim 3 we will test the hypothesis that manipulation of PN firing alters a prediction signa giving rise to feed-forward error signals. These interrelated aims make use of a novel behavioral preparation applying sophisticated optical patterning, optogenetic, electrophysiological, and behavioral methods to awake mice in order to answer fundamental questions about cerebellar physiology. Together, the proposed experiments are designed to resolve issues that have been debated for decades within the cerebellar field. We expect that our results will yield a much improved understanding of basic cerebellar physiology and resolve some long-standing mysteries regarding cerebellum-dependent learning. In addition, these findings are likely to provide conceptual insights into cerebellar dysfunction caused by inherited and sporadic forms of ataxia.

DESCRIPTION (provided by applicant): Effective coordination requires that the motor system predict proper movements. To make these predictions, the cerebellum integrates sensorimotor information and motor errors and, through a process of error-driven learning, builds up feed-forward models of movement. Decades of cerebellar research have clarified a highly stereotyped circuit, identified roles for particular circuit elements, and suggested cellular mechanisms that might account for associative learning. However, fundamental questions remain unanswered. Where within the cerebellar circuit do changes occur during cerebellum-dependent forms of motor learning? How, at a cellular and circuit level, do motor errors drive these changes? And finally, how do circuit changes alter cerebellum-dependent behavior? The following specific aims will be addressed in the project. In Specific Aim 1, we will test the hypothesis that pauses in PN firing, evoked directly or indirectly by optogenetic stimuli, trigger rapid, highly stereotyped movements. Using high speed videography and motion tracking we will measure movement trajectories and speeds in response to activation or inhibition in various cerebellar neurons. We will also make electrophysiological recordings from cerebellar neurons in awake mice to examine the effects of manipulating PN excitability on the circuit. In Specific Aim 2 we will test whether associative motor learning can be driven by pairing sensory stimuli with optogenetically- elicited reductions or increases in PN firing. In Specific Aim 3 we will use n vivo electrophysiology to explore neural mechanisms of learning at key sites in the cerebellar circuit and to determine how error signals contribute to this learning. These interrelated aims make use of a novel behavioral preparation applying optogenetic, electrophysiological, and behavioral methods to awake mice in order to answer fundamental questions about cerebellar physiology. Together, the proposed experiments are designed to resolve issues that have been debated for decades within the cerebellar field. We expect that our results will yield a much improved understanding of basic cerebellar physiology and resolve some long-standing mysteries regarding cerebellum-dependent learning. In addition, these findings are likely to provide conceptual insights into cerebellar dysfunction caused by inherited and sporadic forms of ataxia and dystonia.

This NSF IDBR award is made to Prof. Katsushi Arisaka and collaborators at the University of California, Los Angeles, to develop a Bessel beam line confocal microscope. The goal of this project is to develop a system enabling the recording of three-dimensional cell structure in vivo, in real-time, with exceptional penetrating depth, and with minimal damage to the targeted sample. The proposed system will advance scientific understanding by facilitating cellular observation and systems level biologic analysis. Significantly, this system will enable the unprecedented high-speed recording of cell dynamics at super-resolution, in a temporal range permitting observation of developmental phenomena.

The broader impact is a cost-effective, easily configurable and user-friendly 3D imaging system for use by scientists towards the in vivo structural characterization of dynamic biological samples over a timespan of days. This collaboration will generate an available and reproducible microscope that significantly advances obtainable information concerning embryo development, dynamic neural network function, cellular differentiation and regulation, while enabling high-volume 3D cell imaging. This project directly integrates educational and research goals through incorporation of microscope development into a novel, interdisciplinary laboratory course developed by Dr. Arisaka at UCLA. Thus, development and construction of the system will serve as an educational platform directly fostering student learning. Moreover, the system will be housed in the Advanced Light Microscope Facility at the California NanoSystems Institute (CNSI), resulting in widespread availability to the extended scientific community.

This award is being made jointly by two Programs- (1) Instrument Development for Biological Research, in the Division of Biological Infrastructure (Biological Sciences Directorate), and (2) Biomedical Engineering, in the Division of Chemical, Bioengineering, Environmental and Transport Systems (Engineering Directorate).