Rodney L. Parsons - US grants

Neurons in the vertebrate central nervous system receive and integrate information from numerous afferent synaptic inputs. Their action potential discharge is a function both of their basic membrane characteristics and the interaction at any given time of specific neurochemical transmitters which alter the membrane conductance properties. The complex organization of the vertebrate CNS and small size of most central neurons limits the use of many contemporary electrophysiological and pharmacological techniques needed to analyze basic membrane properties and mechanisms of transmitter action in vivo. We have developed a model system for the study of the biophysical properties and mechanisms of synaptic integration controlling the activity of defined vertebrate peptidergic neurosecretory cells in the caudal neurosecretory nucleus of fishes. Two electrophysiological approaches are planned. The first will utilize conventional intracellular recording techniques combined with pharmacological treatment to characterize basic membrane properties and to investigate the synaptic response following stimulation of specific afferent inputs. The second aspect will utilize voltage clamp techniques to analyze directly voltage-dependent and transmitter-induced alterations in membrane conductance. The specific aims will be: 1. To establish the basic electrophysiological and pharmacological characteristics of the caudal neurosecretory cells. 2. To analyze the synaptic response produced by stimulation of specific descending inputs and to determine the influence of different antagonists on the synaptic potentials. 3. To characterize the somal membrane ionic conductance systems, and determine the ionic basis of the different synaptic responses using voltage-clamp techiques. 4. To analyze the influence of putative transmitters on the biophysical properties of the somal membrane and compare their response with the physiological response produced by stimulation of specific presynaptic inputs. The results obtained should provide new information about the general membrane properties of neurosecretory cells as well as the mechanisms of synaptic intergration regulating the activity of this neuron type.

The parasympathetic cardiac ganglion of Necturus maculosus (mudpuppy) will be used to investigate the histochemical organization and synaptic integration occurring in a vertebrate cardiac ganglion. The mudpuppy cardiac ganglion contains two neuron types: postganglionic cells and intrinsic SIF cells. These two cell types can be identified in living preparations providing a unique opportunity to analyze the role of SIF cells within autonomic ganglia. The morphological and physiological basis for the effects of acetylcholine, catecholamine, serotonin (5-HT) and substance P on postganglionic neurons will be studied. The following specific questions will be addressed: 1) Identify the chemical nature of the synaptic profiles on postganglionic neurons. In preliminary studies, fibers and cells were found containing serotonin, catecholamines, and a substance P-like peptide in the mudpuppy cardiac ganglion. The synaptology in the ganglion of terminals immunolabeled for these substances will be determined using electron microscopy. 2) Determine the influence of substance P, 5-HT, and catecholamines on the membrane properties of postganglionic neurons and on synaptic transmission between the preganglionic fibers and postganglionic cells. Intracellular recording techniques and voltage-clamp procedures will be used to investigate the action of these transmitter substances on synaptic transmission between the preganglionic and postganglionic cells as well as on the conductance properties of individual postganglionic neurons. 3) Correlate electrophysiological response to applied neuroactive substances with synaptic terminal type and distribution to individual ganglion neurons. Following intracellular recording, neurons will be injected with HRP or lucifer yellow in order to understand the relationship of structure and function in synaptic integration. 4) Determine the effect(s) of SIF cell activity on the properties of adjacent postganglionic neurons. Individual SIF cells will be stimulated intracellularly to determine the influence of SIF cell activation on adjacent parasympathetic neurons and/or on transmission between preganglionic fibers and postganglionic cells. The results of this project should provide significant information about: a) the mechanism(s) of interaction of neurotransmitters on an identified postsynaptic cell, b) the role(s) of SIF cells in autonomic ganglia, and c) the type(s) of integration occurring in vertebrate cardiac ganglia for local reflex control of cadiac function.

The progressive desensitization which develops during closely repeated agonist applications or sustained agonist exposure is a common, but poorly understood consequence of almost all agonist- receptor interactions. The activation- desensitization sequence has been studied most extensively for the nicotinic AchR channel complex at the motor endplate of skeletal muscle or electric organs. In contrast, no comprehensive study of this important process has been completed for nicotinic AChR-channel complexes in vertebrate neurons. Therefore, the primary objective of the proposed study is to establish the kinetics of the activation- desensitization sequence occurring at nicotinic AChR-channel complexes in amphibian autonomic postganglionic neurons. These experiments will also test whether desensitization of the nicotinic receptor-channel complex occurs under physiological conditions and therefore is a mechanism by which the efficiency of ganglionic transmission can change during trains of repetitive preganglionic stimulation. The studies will be done using bullfrog sympathetic and parasympathetic ganglion cells. Individual ganglion cells in intact preparations and single enzyme dissociated cells will be utilized. Membrane currents will be measured in individual cells within intact ganglia using either a two microelectrode or a single electrode voltage clamp system. Whole cell patch clamp measurements of membrane currents will be obtained from enzymatically dissociated cells. The specific aims are to: 1. Establish the basic kinetic properties of desensitization of the nicotinic AChR in vertebrate ganglion cells. 2. Establish whether receptor channel activation kinetics determine desensitization kinetics in autonomic ganglion cells. 3. Establish whether AChR-channel desensitization is a physiological mechanism involved in integration within ganglia. The results of these experiments will provide important new information about the control of nicotinic-gated channels in vertebrate neurons.

The cardiac ganglion of the mudpuppy, Necturus maculosus, is an excellent model system for analysis of synaptic integration within a vertebrate cardiac ganglion. Two neuron types are present: parasympathetic postganglionic neurons and SIF cells. The SIF cells contain a number of neurotransmitters including: catecholamines, 5-HT, a substance P-like peptide and a galanin-like peptide. Further, there is an extensive substance P/GGRP immunoreactive afferent fiber input into the cardiac ganglion which make pericellular complexes around postganglionic neurons and SIF cells. We will establish the mechanism(s) by which the afferent fiber and SIF cell input influence the activity of the postganglionic neurons. The following specific questions will be addressed: a) Determine the function and source of the afferent transmission and on the conductance properties of the postganglionic neurons. Immunocytochemical and tracing studies will be done to establish the central origin (i.e., vagal versus spinal) and the peripheral distribution of the substance P/GGRP-immunoreactive fibers. b) Establish the role of SIf cells in the cardiac ganglion. Sif cells will be stimulated while recording from adjacent postganglionic neurons to test whether SIF cells influence postganglionic neuron activity. We will test whether the SIF cells respond to ACh, substance P and CGRP to establish whether these cells are activated by preganglionic and/or afferent transmitters. The action of other neurotransmitters, present in SIF cells such as 5-HT and dopamine, on the membrane properties of postganglionic neurons and ganglionic transmission will be tested. c) Establish the mechanism(s) by which galanin alters, excitability of the postganglionic neurons. The influence of galanin on membrane currents in isolated postganglionic neurons will be determined using cell attached patch and whole cell voltage clamp techniques. The kinetic properties of the galanin activated potassium conductance will be determined using cell attached patch and whole cell voltage clamp techniques. The kinetic properties of the galanin activated potassium conductance, will be determined from single channel activity. An analysis of galanin induced alteration of calcium and potassium conductances will be performed using whole cell voltage clamp procedures. This project will provide important new information in three areas: a) the mechanism(s) of action of specific neurotransmitters on an identified postsynaptic cell, b) the role(s) of SIF cells in autonomic ganglia, and c) the role of afferent fiber input and type (s) of integration which may occur in vertebrate cardiac ganglia that are important in local reflex control of cardiac function.

DESCRIPTION (provided by applicant): The goal of this application is to establish a Center of Biomedical Research Excellence (COBRE) in Neuroscience at the University of Vermont (UVM). The specific aims of this proposal are: Specific Aim 1: Establish the research and intellectual infrastructure to support a University-wide Center for Neuroscience Excellence at UVM. Goals of this aim include: 1) to establish a mentoring program to support junior neuroscience faculty; 2) to establish an imaging/physiology core to support the research projects of the junior faculty; 3) to establish a cellular/molecular core to support the research projects of the junior faculty; 4) to establish a University-wide Neuroscience Seminar Series and Annual Neuroscience Retreat; 5) to establish a mechanism of communication between basic scientists and clinicians that facilitates development of translational research and 6) to establish a University-wide mechanism for Neuroscience Graduate Education. Specific Aim 2: Support the research development of a core group of junior faculty who will be future leaders in the Center for Neuroscience Excellence. Faculty to be supported and their project titles are: 1) Dr. Rona Delay, Department of Biology: Chloride homeostasis in olfactory neurons; 2) Dr. William Falls, Department of Psychology:Molecular and genetic analysis of learned fear reduction in mice; 3) Dr. Anthony Morielli, Department of Pharmacology: Kinase and cytoskeletal regulation of potassium channels; 4) Dr. Matthew Rand, Department of Anatomy and Neurobiology: Proteolytic modulation of Notch signaling in neurogenesis and 5) Dr. George Wellman, Department of Pharmacology: Mechanisms of cerebral vasospasm in subarachnoid hemorrhage. UVM has established investigators in three areas of neuroscience: molecular/developmental, cellular/systems and clinical/behavioral neuroscience. Award of this application would provide a mechanism to significantly expand research strength in these existing areas of neuroscience emphasis, to integrate basic with clinical neuroscience, and to promote research collaborations university-wide. A level of excellence in research and training will be fostered that is not possible without external support. Productivity of both junior and senior investigators will be stimulated, and a long term mentoring framework will be created. UVM is a small institution in a rural state with limited resources; an award would substantially expand our research infrastructure and significantly increase faculty competitiveness.

DESCRIPTION (provided by applicant): Pituitary adenylate cyclase activating polypeptides (PACAP) are potent cardiovascular regulatory neuropeptides. We hypothesize that a critical site of PACAP action is within the peripheral parasympathetic cardiac ganglia that serve as local integrative centers. In guinea pig parasympathetic cardiac ganglia, all postganglionic neurons are innervated by PACAP-immunoreactive fibers, and PACAP peptides potently depolarize and increase excitability of the postganglionic cardiac neurons. The increase in membrane excitability of guinea pig cardiac parasympathetic neurons by PACAP is greater than that produced by any other neuropeptide studied to date, but mechanism(s) responsible for the PACAP-induced depolarization and increase in excitability are not known. The primary goal of the project is to establish the mechanisms underlying the PACAP-induced increase in excitability and depolarization of guinea pig cardiac neurons. The following specific aims are proposed. Aim 1: Establish the second messenger transduction cascades responsible for generation of the PACAP-induced increase in membrane excitability and depolarization. Aim 2: Establish the membrane ionic mechanisms underlying the PACAP-induced increase in membrane excitability. Aim 3: Establish the membrane ionic mechanisms generating the PACAP-induced inward current. Aim 4: Establish whether the PACAP-induced rise in intracellular calcium (Ca2+)i in cardiac neurons occurs from either Ca2+ influx or from Ca2+ release from IP3- or ryanodine-sensitive stores, and test whether the rise in (Ca2+)i is an initial step in the generation of the membrane depolarization or increased excitability. The results of these experiments will provide key, new insight into mechanisms determining PACAP-induced excitatory actions on neurons in the parasympathetic cardiac ganglia; ganglia that determine the extent of parasympathetic inhibitory drive on cardiac function.

DESCRIPTION (provided by applicant): An award of the Centers of Biomedical Research Excellence (COBRE) established a Center for Neuroscience Excellence at the UVSM that greatly enhanced the research infrastructure for neuroscience investigators across the UVSM campus. The specific aim of this supplemental application is to markedly increase the research sophistication and innovation of optical imaging for neuroscience investigators at UVSM by requesting equipment funds to purchase a Bio-Rad Radiance 2100 Multiphoton Microscopy System that will be housed in the COBRE-supported Multi-user Neuroscience Imaging and Physiology Core. Multiphoton imaging is a state-of-the-art technique that allows investigators a window on individual cellular function within the larger context of a tissue. Specifically, one can perform high resolution, time lapse optical analysis in living tissues or organisms over extended periods of time because photolytic damage is minimized. Moreover, one can image deep within tissues or in vivo at high resolution because the light scattering by tissues that occurs in traditional epifluorescence or confocal fluorescence microscopy is minimized by use of long-wavelength excitation in multiphoton microscopy. Sophisticated imaging is a requisite for satisfactory completion of many of the projects supported by the COBRE award and by National Institutes of Health (NIH)-funded projects to other neuroscience investigators at UVSM. The multiphoton system will allow investigators at UVSM to be at the forefront of optical imaging and thus significantly increase their potential for garnering future research support. Purchase of this instrument will have immediate impact on the research programs of 17 neuroscience faculty at UVSM distributed across five departments and three colleges. This level of innovation and sophistication in imaging is presently not available at UVSM.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This proposal is submitted in response to the Notice of Limited Competition (NOT-RR-03-001) forwarded from the National Center for Research Resources (NCRR) to Principal Investigators of Center of Biomedical Research Excellence (COBRE) grants. Award of COBRE grant 1 P20 RR16435 established a Center for Neuroscience Excellence at the University of Vermont (UVM) and established a coherent infrastructure for Neuroscience research across the UVM campus. [unreadable] [unreadable] The rationale for the present application stems from the outcome of an evaluation by the External Advisory Committee of the Neuroscience COBRE in December, 2002. The committee was generally impressed with the caliber of the neuroscience at UVM; however, one arena in which further development was suggested was to establish the capacity for proteomics based research. As a consequence, the specific aim of this supplemental application is to facilitate the ability of UVM researchers to incorporate proteomics into their studies by requesting: a Ciphergen ProteinChip AutoBiomarker System, a Leica Laser Microdissection System, and a Sorvall Discovery 100 SE Ultracentrifuge. The Leica Laser Microdissection System and the Ultracentrifuge complement the Ciphergen ProteinChip System because they improve upon existing equipment necessary for preparing samples. These equipment items will be installed in the COBRE Cellular/Molecular Core Facility, where their use will be overseen by the Core Director, Dr. Sheryl White, and operated by a full time research technician assigned to the Facility. [unreadable] [unreadable] Acquisition of this equipment will have an immediate impact on the research programs of fifteen neuroscience faculty at UVM distributed across six departments and three colleges. Thus, these instruments would markedly increase the research competitiveness of neuroscientists at UVM by making available state-of-the-art technology that does not currently exist on campus, which would allow them to ask highly sophisticated questions. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Funds are requested to purchase a Zeiss LSM MP multiphoton microscope with a Coherent Chameleon Ultra II Ti:Sapphire pulsed laser. This instrument will replace our current BioRad Radiance Multiphoton Microscope, which no longer meets the needs of our users. In addition, Carl Zeiss is phasing out their legacy BioRad systems and no longer stocks replacement hardware or provides software upgrades for the Radiance 2300 system. The dedicated multiphoton microscope will be housed in the Imaging/Physiology Core Facility supported by the NCRR Center of Biomedical Research Excellence (COBRE) in Neuroscience grant (5P20 RR16435, 07/01/06 - 06/30/11). The competitive renewal of the COBRE grant is expected to start 7/1/01/2011 and provide 5 additional years of Core support. The Neuroscience COBRE Imaging/Physiology Core is administered by a full-time, highly trained microscopist. Acquisition of the Zeiss LSM 7 MP dedicated multiphoton microscope, a modern, modular, and actively supported system, would greatly enhance the capabilities of the Core and would meet the emerging needs of the present Core investigators and provide the opportunity for additional funded investigators to significantly expand the scope of their research programs. The new Zeiss multiphoton microscope, model LSM 7MP, offers impressively rapid acquisition speed, about 4 times higher than the speed of our BioRad Radiance 2100 MPD two-photon microscope. The high acquisition speed combined with the superior sensitivity and lower noise levels makes LSM 7 MP perfect for studying the activity of living neurons and other cell types in the brain. In recent years, multiphoton photolysis of caged compounds (uncaging) is increasingly used as a tool to quickly deliver calcium and other substances intracellularly or in the extracellular space with great precision in time, location and volume. Furthermore, multiphoton uncaging allows deeper penetration into live tissues, with smaller volumes compared to single photon photolysis. Our highly innovative research projects that investigate the neuro-vascular coupling in the brain and cerebral arteriole smooth muscle and endothelium function absolutely require the use of photolysis of caged calcium, IP3, glutamate and ATP. The Zeiss LSM 7 MP is capable of two photon photolysis localized within multiple arbitrarily defined regions of interest without slowing down the acquisition speed. This will allow us to simultaneously uncage and image living cells or tissue with a speed that is adequate for monitoring and recording the changes in cellular activity that occur as a result of uncaging. Moreover, this will enable us to simultaneously or consecutively uncage compounds in more than one cell or location thus providing us with the unique opportunity to study the interactions and communications between different cells or structures in the brain. PUBLIC HEALTH RELEVANCE: This instrument would support research designed to understand the development and normal functioning of the brain and autonomic organs and their alteration in response to disease. Imaging of cellular responses within brain slices and organs will provide key insight into the regulation of neuronal, vascular, and connective tissue function and their interdependence. This insight will inform development of therapeutic approaches for diseases that strike these tissues.

DESCRIPTION (provided by applicant): Funds are requested to purchase a Yokogawa CSU-W1 spinning disk confocal system coupled to a Nikon Eclipse NI-E upright microscope with Andor EM CCD detectors. This instrument will replace a Noran/Prairie Technology Scanning confocal microscope, which was purchased in 1995. The new instrument will be housed in the Neuroscience Imaging/Physiology Core Facility supported by the NIGMS/NCRR Center of Biomedical Research Excellence (COBRE) in Neuroscience grant (P30 RR032135/P30 GM103498, 7/1/2011-6/30/2016). The Neuroscience COBRE Imaging/Physiology Core is administered by a full-time, highly trained microscopist. Acquisition of the Yokogawa CSU W-1, a modern, modular, and actively supported system, would greatly enhance the live imaging capabilities of the Core, expanding the high-speed, high-resolution microscopy resources available to investigators within the College of Medicine and in other Colleges on the UVM campus. These investigators routinely record optical measurements of fast, localized intracellular events, such as calcium transients from living cells and tissues. The Yokogawa spinning disk will significantly improve the capabilities of the Core to meet the emerging needs of the present Core investigators, while providing the opportunity for additional funded and new investigators to expand the scope of their research programs. The Yokogawa system will use an upright rather an inverted microscope platform, allowing better access to intact thin tissues like isolated vessels or gut, bladder or urothelial strips by employing high numerical aperture physiology dipping lenses. The Yokogawa system can simultaneously record two low light signals with dual 512x512 EM CCD cameras at 52 fps (frames per second), a rate well above our other instrumentation. Higher resolution (2560X2160) imaging for brighter samples can be achieved with the Zyla sCMOS camera at 100 fps. These cameras allow great flexibility for the multiple sample types encountered in our multi-user facility. In addition, the Yokogawa system will have multiple laser lines, greatly broadening its experimental capacity by allowing simultaneous measurements of multiple fluorescent signals from defined cells. The COBRE Imaging/Physiology Core has a long history of supporting live imaging research and publication, but its instrumentation for high speed acquisition is obsolete, and can no longer meet current research demands.