1997 — 2001 |
Pieribone, Vincent A |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Physiological Studies of Nerve Terminal Phosphorylation
The computational and cognitive abilities of the brain arise from the sheer number and variety of its internal communication links. The structural and functional unit of these communication links is the synapse. Modulation of synaptic strength is the basic property of all neuronal circuits and is believed to be a fundamental mechanism underlying the plasticity of the CNS. In recent years fundamental progress in the isolation and characterization of a variety of nerve terminal-enriched proteins has been made. The present study will examine the biochemical machinery that orchestrates synaptic vesicle docking and fusion. We will directly compare the effects on neurotransmitter release of the post-translational modification (protein phosphorylation) of different components of the release machinery. The first part of these studies will determine which of the selected proteins are substrates for a set of protein kinases implicated in nerve terminal function. The nerve terminal proteins VAMP, synaptotagmin, synaptophysin and SNAP-25 will be characterized as physiological substrates for Ca2+/ calmodulin-dependent protein kinase II, protein kinase C, cAMP-dependent protein kinase and casein kinase II using in vitro and in vivo assays. In each case, the sites of phosphorylation will be identified. We will then use direct injections of these proteins into the presynaptic element as a way to dissect their specific roles in the regulation of synaptic vesicles trafficking and neurotransmitter release. Using electron microscopy techniques we will compare ultrastructural changes in the nerve terminal resulting from these injections. Effects of phosphorylation of each of these proteins on specific protein-protein interactions that have been shown as physiologically relevant, will be assessed using quantitative in vitro binding assays. Phosphorylation state-specific antibodies will also be used for ultrastructural studies to localize these phosphorylation events within the synapse. Finally, fluorescently-labeled phosphorylation state-specific antibodies will be employed in injection experiments into living axons to monitor phosphorylation driven regulation in a functioning synapse. These studies will reveal the physiological role for phosphorylation of selected nerve terminal proteins in neurotransmitter release and release modulation.
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0.943 |
1998 — 2002 |
Pieribone, Vincent A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Synapsins and Animal Models of Synaptogenesis
They synapsins are a family of brain-specific synaptic vesicle-enriched phosphoproteins that regulate neurotransmitter release by tethering synaptic vesicle to active zones. Recently a variety of in vitro studies have indicated that these molecules also have trophic effects on the elongation on axons and the formation of synapses. The presence of synapsins accelerates the elongation of axons and the formation of synapses, while cultures of neurons lacking the synapses have a retarded rate of axon elongation and synapses. To determine if the synapsins act as trophic agents in vivo, we will examine mice lacking the various synapsins. We will compare the rate and degree of synapse formation in wild-type and synapsin-deficient mice. We will also examine changes in the degree of synapse loss with aging in synapsin-deficient mice. Hippocampal CA1 pyramidal neurons undergo cyclic dendritic spine sprouting and pruning during oestrus and following estrogen priming. The present studies will seek to determine if female mice lacking synapsins undergo similar degrees of synaptogenesis. Experiments will examine the granule cell mossy fiber sprouting that accompanies kindling, an experimental model of epilepsy, in mice lacking synapsin. The degree of sprouting will be quantified and compared between wild-type and synapsin-deficient mice. Studies will examine the rate and degree of sertonergic fiber regeneration following chemical lesions in wild-type and synapsin-deficient mice. Studies indicate that at least some of the actions of certain neurotrophic agents (e.g. NGF and BDNF) may be mediated through the synapsins, the response of regenerating serotonin axons to growth factor stimulation will also be assessed in synapsin-deficient mice. Following lesioning of the entorhinal cortex, axons from the contralateral perforant path collateralize and innervate the deafferented dentate. This regeneration is sensitive to trophic factors and has behavioral correlates. We will examine the histological regrowth and behavioral recovery of wild-type and synapsin- deficient mice following lesions of the entorhinal cortex. Finally, age- dependent behavioral deficits will be compared in wild-type and synapsin- deficient mice. These studies should establish the trophic role of the synapsins in adult animals and should lay the groundwork for future studies aimed at elucidating the mechanisms of synapsin actions and at harnessing this trophic action in Alzheimer's disease.
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0.943 |
2000 — 2002 |
Pieribone, Vincent A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Physiological Role of Actin in Synaptic Transmission @ John B. Pierce Laboratory, Inc.
DESCRIPTION(Adapted from applicant's abstract): The synapse is the elemental unit of information processing and storage in the nervous system. Modulation of synaptic strength is widely believed to be the mechanism by which information is encoded in the brain. To understand synaptic plasticity, the specialized form of vesicle secretion that underlies neurotransmission must first be understood. The present studies use an experimental preparation of an isolated lamprey spinal cord in vitro. This preparation allows unparalleled access to a single pre-synaptic element for microinjection and the ability to record from neurons which receive monosynaptic inputs from such an element. Upon injection of various active compounds, the effects of these injections can be monitored as changes in the secretion of neurotransmitter and by alterations in synapse ultrastructure. Preliminary results indicate that synaptic vesicle endocytosis appears to be highly dependent on F-actin and that F-actin may serve as a scaffold by which synaptic vesicles return to the vesicle cluster following endocytosis. The studies proposed will involve injecting a variety of actin modifying agents directly into the pre-synaptic element of a living synapse and measuring the effects such injections have on neurotransmitter release and synaptic ultrastructure. The type of morphological alterations that occur in response to these injections will enable estimation of where in the synaptic vesicle cycle the reagent is having an effect. F-actin binding, disrupting, and capping agents will be injected. In addition, experiments will seek to determine if a major actin binding protein family (myosins), recently localized to nerve terminals, have actions in vesicle exocytosis or endocytosis. These studies will involve cloning lamprey homologues of myosin II and V, production of specific antibodies that disrupt myosin/actin interactions and injections of these antibodies into pre-synaptic regions. The effect such injections have in synapse ultrastructure and physiology will be assessed.
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0.909 |
2002 — 2003 |
Pieribone, Vincent A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Synuclein Function in the Synaptic Vesicle Cycle @ John B. Pierce Laboratory, Inc.
Several independent lines of evidence have linked the nerve terminal- enriched synuclein family of proteins to Parkinson's disease. Genetic studies have linked mutations in the alpha-synuclein gene to the disease in several families. Synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins are a major constituent of Lewy bodies, a hallmark histopathology of Parkinson's disease. Over-expression of synucleins in mice causes Lewy body-like neuropathology and motor deficits. While there is a wealth of information on the genetic and histological features of synucleins, very little information exists on the function of the proteins in normally functioning nerve terminals. We propose to study the function of these proteins in nerve terminals using classic neurophysiologic experiments in an isolated nerve terminals using classic neurophysiologic experiments in an isolated nerve terminal. Pre-synaptic injection of agents that will modify synuclein function will be made into the giant pre-terminal of a primitive vertebrate (lamprey). The effects of pre-terminal injections of several synuclein affecting agents will be analyzed with electrophysiologic and ultrastructural methods. Inj3ected agents will include: full length and fragments of lamprey synuclein, recombinant human alpha-synuclein containing Parkinson's disease associated mutations (A53T and A30P). Mock casein kinase I and src kinase phosphorylated (S129E/S87E) human alpha-synuclein and antibodies against lamprey synuclein. Using paired intracellular recordings between the pre- and post-synaptic elements we will examine the effects of injections on the ESPS generated by intraoxonal stimulation. Many parameters of synaptic vesicle release kinetics will be examined. In addition, giant pre-synaptic elements receiving injections of agents followed by varying levels of stimulation will be examined under the electron microscope. Effects of injections on the synaptic vesicle cycle can be established by characteristic changes in nerve terminal morphology. Establishing the function of the synucleins in normal neurotransmitter release will shed light on the function and dysfunction of the protein in Parkinson's disease.
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0.909 |
2003 — 2004 |
Pieribone, Vincent A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
A Protein Based Optical Probe of Membrane Potential @ John B. Pierce Laboratory, Inc.
DESCRIPTION (provided by applicant): Understanding nervous system function will require the simultaneous monitoring of the electrical activity of a large number of functioning neurons. It will be important to understand the temporal interactions between large groups in networked neurons to fully understand how the central system processes information. Optical recording methods offer advantages in that they provide the spatial and temporal resolution necessary to record salient neuronal events with limited perturbations of the system. Currently available voltage-sensitive dyes are very difficult to apply experimentally, produce small changes in fluorescence for a given potential change and cannot be targeted to a specific subset of neurons, thereby reducing the complexity of the signal obtained. The present application seeks to produce a protein-based, voltage-sensitive, optical probe for use in neurons. We have developed a probe (termed SPARC), which is based on the fusion of the rat mu1 skeletal muscle sodium channel with a fluorescent protein (i.e., GFP). This construct embodies many of the features we are seeking in an optical probe of membrane potential. Xenopus laevis oocytes, injected with the cRNA for SPARC, exhibit a fluorescent signal that undergoes a rapid (<1 ms), reproducible (>1000 pulses/hour tested) and reversible change (0.1-0.5% delta F/F/100mV) in intensity during depolarization of the cell membrane. This change can be easily recorded during action potential sized depolarization pulses (<2 ms). The present application seeks to increase the delta F/F and signal-to-noise ratio of the fluorescent signal arising from this probe and validate the use of this probe in neurons. This will entail the following studies: (I) fully characterize the presently developed SPARC probe. (II) Modify the nature and location of the fluorescent protein insertion site. (III) Alter the type and number of fluorescent proteins inserted at these sites. (IV) Finally, we will attempt to develop a FRET version of the probe to enhance the fluorescence change seen with voltage steps.
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0.909 |
2009 — 2015 |
Culurciello, Eugenio (co-PI) [⬀] Pieribone, Vincent A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
High-Speed, Wide Field Fluorescent Imaging of Cortex in Freely Moving Animals @ John B. Pierce Laboratory, Inc.
DESCRIPTION (provided by applicant): The study of neuronal activity in awake, freely moving animals is the most representative view of 'normal' neuronal function. Anesthetics and restraint have profound effects on neuronal physiology and the correlate animal behavior. The study of real time neuronal event processing requires recording methods with high temporal bandwidth (>500 hz) and the ability to detect physiologically relevant events (i.e. voltage changes). Currently only microelectrode recording of single neuronal units, multiunit activity and field potentials have sufficient temporal resolution and portability to allow recording of neuronal activity in freely moving animals. The proposed project will produce three miniature microscope/imaging systems which can be head-mounted and will allow the optical recording of changes in membrane voltage of neurons in freely moving rats. During the funded two-year ARRA project, two prototype devices and two image sensors were developed that collect wide field image sequences of fluorescent voltage dye signals. The current application will complete the fabrication and operationalize two different style, head-mountable, high-speed, fluorescence microscopes. This includes the design and fabrication of two different finalized, imaging sensors, custom optics for each, a stabilized illumination source and form fit microscope bodies with skull mounts. The studies proposed herein will also build and test a novel fluorescence microscope design that will reduce the height of the traditional microscope by eliminating the 45? dichroic mirror. We will also miniaturize our current camera control and recording system to be fully contained in a back pack on the rat. This back pack will contain a field programmable gate array, heat exchanger/coolant pump, SD memory and a battery to run the microscope fully autonomously. Finally, the instruments will be tested in imaging studies of the rat somatosensory cortex (barrel cortex). Cortical responses to object discrimination will be studied in freely moving rats. These studies will systematically engineer each component to maximize excitation light delivery, fluorescent light collection and recording speed while minimizing weight volume, energy usage and heat generation. The device will be capable of recording rapid (1000 fps) fluorescent image sequences of a >2 mm2 area of cortex and detect small changes in ?F/F (0.1%). The device will be small (<1 cm2 head mounted) and light weight enough (5 g) to be mounted on the head of a freely moving rat. The device is intended as a precursor to devices that can translate neuronal activity in humans into actions in real time, optical brain machine prosthetic.
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0.909 |
2013 — 2016 |
Hughes, Thomas E (co-PI) [⬀] Nitabach, Michael Pieribone, Vincent A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Genetically-Encoded Voltage Probe Development @ John B. Pierce Laboratory, Inc.
DESCRIPTION (provided by applicant): Neuronal electrical activity is the central underpinning of nervous system function. While understood as essential for over a century, the tools to study circuit level neurophysiology have remained largely unchanged in 50 years. The advent of molecular biology has dramatically advanced neurobiology by allowing molecular characterization of the nervous system but has not translated into significant gains in neural electrophysiology. Opto-molecular methods have revolutionized our study of neuronal connectivity, development, gene distribution, calcium signaling and recently, targeted neuronal activation (i.e. optogenetics). A glaring exception to this light-based revolution is the use of optical methods to monitor electrical activity. Intracellular calcium levels and metabolic signals are often used as a surrogate marker of electrical activity, however they are temporally delayed, do not detect subthreshold events and more often than not fail to capture the relevant suprathreshold activity. The PIs laboratories, as part of a multi laboratory collaboration have been developing genetically encoded voltage sensors based on fusions of green fluorescent protein orthologs and voltage sensing domains. Our grant members have published most of the significant advances in genetically-encoded voltage sensors in recent years. Our most recent probes, Arclight and ElectricPK significantly improved the signal size and response speed of fluorescent voltage probes. The current application will continue this successful collaborative search for voltage probes. We are seeking probes which combine large F/ V signal sizes, a range of useful response speeds and red-shifted fluorescence spectra. During this previous funded period time, we discovered that by altering the voltage sensor domain, the linker length, the fluorescent protein and by introducing point mutations in the fluorescent protein, we could develop probes with vastly superior signal size and response kinetics. We also confirmed, however, that a purely empirical step (i.e. large scale screening of single, incrementally-modified constructs) is required to make dramatic improvements in response properties. We will employ a staged evolution approach involving successive rounds of directed and random sequence modification followed by direct testing in mammalian cells. The current experiments will be an advance over all previous studies in two important ways: i) we will create vastly greater numbers (20x) of potential probe (thousands) using domain swapping and site directed / random mutagenesis and ii) the larger numbers of constructs will be prescreened by an automated, robotic microfluorimetry method which evaluates the fluorescence signal size and speed in electrically-active mammalian cells. Finally, all successful candidates will be validated for in vio functionality in Drosophila circadian neurons and rodent somatosensory/barrel cortex.
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0.909 |
2014 — 2016 |
Pieribone, Vincent A |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Development of Protein-Based Voltage Probes @ John B. Pierce Laboratory, Inc.
? DESCRIPTION (provided by applicant): The use of genetically encoded fluorescent activity probes represent the most advanced method to monitor the electrical activity of networks of neurons without using electrodes. While genetically encoded calcium indicators have been evolved to produce robust signals in a variety of different neuronal preparations, fluorescent probes of membrane potential have not been well evolved. Current voltage probes, while finally in expanded use, will need considerable improvement if the goal of recording the activity of a large number of neurons simultaneously in vivo is to be achieved. The goal of this project is to discover protein-based fluorescent voltage probes with signal to noise characteristics that allow routine optical recording of action potentials from single cortical neurons in vivo. We are seeking probes with significantly improved signal to noise characteristics, red-shifted fluorescence spectra, faster on and off rates and better plasma membrane expression. This project brings together leading genetic probe scientists to: design new probe scaffolds, robotically screen large incrementally modified libraries of probes, identify probes with improved response properties, then validate these new probes under standardized experimental conditions in a range of 'real world' neuronal preparations with increasing levels of complexity. Finally, we will make all probe reagents (i.e. plasmid DNA, AAV particles, transgenic flies, etc.) as well as supporting validation data readily available to the research community.
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0.909 |
2017 — 2019 |
Pieribone, Vincent A |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High Throughput of Protein-Based Voltage Probes @ John B. Pierce Laboratory, Inc.
A significant motivation of the BRAIN Initiative is the desire to understand information processing in neuronal tissues in situ. Towards this end a steadily growing number of neuroscience investigators are turning to optical methods to monitor neuronal activity as opposed to more traditional methods that rely on electrodes. The most commonly used method involves genetically encoded, fluorescent calcium indicators (i.e. GCaMPs) combined with multiphoton or wide field microscopy. These methods allow activity measurements in large numbers of identified neurons from intact animals. Advancement of these studies rely on the development of improved, genetically encoded, protein-based indicators. Fluorescent intracellular calcium indicators produce robust signals in response to neuronal activity, however they i) exhibit very slow kinetics relative to action potentials, ii) have poor individual action potential reporting fidelity and iii) cannot enable visualization of hyperpolarizations of membrane potential. Fluorescence voltage indicators provide signals which are richer in information, more temporally relevant and offer a more direct measure of neuronal electrical activity. A number of new and more practically useful fluorescent voltage indicators have been developed over the past decade with improved properties. During a previous funding period our laboratory developed a high throughput workflow to create and screen protein-based, voltage sensitive indicators. Using this platform we have discovered several novel indicator templates and new indicators. With the steady increase in throughput of our screening workflow we have seen, as expected, more rapid improvements in indicator properties which has translated to greater practical use. However, the protein design and modification space is enormous hence we propose further scaling up of the screening throughput and widening the template design space. We propose to screen upwards of 1150 novel constructs per day or ~8000 per week. We will include random mutagenesis in the process given the newly developed screening capacity. With this new platform we are seeking to develop : i) indicators with maximum total ?burst? photon output in response to action potentials, ii) fluorescence output increases from a weak resting level lasting of between 2-40 ms in response to action potential-type voltage transients, iii) positive voltage/fluorescence output slope relationship indicators, iv) indicators with green, red and near IR emission spectrum, v) indicators targeted to subcellular regions of the neuron, and vi) probes with reduced bleach rates.
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0.909 |