Douglas G. McMahon - US grants

The research proposed here will examine the molecular basis of synaptic plasticity at electrotonic and glutamatergic synapses in the retina. SpecificaUy, the biophysical properties and molecular structures of the gap junction channels and glutamate receptors of teleost retinal horizontal cells will be characterized with particular attention to the mechanisms by which these synaptic membrane channels are modulated by the dopaminergic interplexiform system. This research will provide information on the molecular events during the first steps of visual information processing in the eye, and has broad implications for understanding the molecular basis of synaptic modulation in the central nervous system. In this regard it is potentiahy useful in ameliorating dysfunction of other brain dopaminergic systems, such as occurs in Parkinsonism and schizophrenia, and in understanding the role of glutamate receptors in neurodegenerative cell death and in learning and memory. Experiments will be conducted using retinal neurons isolated from the zebrafish (Brachydanio rerio). The biophysical properties of horizontal cell gap junction and glutamate-receptor channels will by described using patch and whole cell voltage clamp recordings from dissociated cells. The modulatory effects of dopamine on these channels will also be characterized. In addition, the action of other potential modulatory transmitters such as melatonin and VIP, and other intracellular messengers such as cGMP and PKC will be examined. The molecular structure of these ion channels will be elucidated by cloning of their respective cDNAs from a zebrafish cDNA library. Fragments of these genes will be obtained by PCR amplification of zebrafish DNA with primers designed by analysis of homologous mammalian genes. The retinally expressed fragments will be identified by Northern blotting, and then used to obtain full-length cDNAs. The resulting clones will be characterized as to their cell-specific expression by in situ hybridization, subcellular localization of their gene products by immunocytochemistry, and functional expression as channel proteins by expression in oocytes. Structure-function models of these channels reflecting these biophysical and molecular studies win then be produced.

Modulation of synaptic communication between neurons is a fundamental mechanism by which the nervous system responds to changing levels of stimuli, reflects previous experience and shapes functional connections during development. The broad goal of the research proposed here is to gain an understanding of the mechanisms of electrical synaptic plasticity at a molecular level using synaptic modulation in the outer plexiform layer of the retina as an experimental system. The proposed experiments focus on three specific aims; (1) extending successful studies of synaptic modulation at the single channel level to include modulation by nitric oxide, cGMP and pH, in addition to dopamine, (2) accelerating efforts in progress directed at cloning cDNAs for teleost retinal gap junction channel genes, and (3) expanding the scope of research to examine the regulation of assembly and maintenance of electrical synapses by dopamine, cell calcium and cell-cell adhesion. Successful completion of these experiments will contribute in a fundamental way to our understanding of the modulation of electrical synaptic transmission in the nervous system. This basic knowledge should be useful in understanding the pathophysiology of epilepsy, Parkinsonism, schizophrenia and senescence, where these processes are apparently deranged.

The physiology and behavior of virtually all eukaryotes, including humans, exhibits a pervasive daily rhythmicity. Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a freerunning period of about a day (i.e. circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the biological clock in mammals. The functional integrity of the SCN and the overt circadian rhythms it drives decline with age. In the aged human, this has a negative impact on health through fragmentation of sleep and endocrine rhythms. In rodents, age-related changes in circadian rhythms are correlated with losses in the SCN neuropeptide VIP and loss in SCN sensitivity to the neurotransmitter serotonin. The long-term objective of the research proposed here is to elucidate critical mechanisms of circadian pacemaker and to identify changes in these mechanisms related to the decline in circadian function during aging. We will address three questions regarding the mechanisms of SCN pacemaking and its aging: (1) What are the neurophysiological changes exhibited by SCN neurons as they oscillate? (2) What are the effects on SCN neurons and synapses of VIP and serotonIn? (3) Does compromise of neurotransmission through VIP, serotonin or GABA play a role in SCN aging? We will approach these objectives by combining patch- clamp and neural network array electrophysiology to study rodent SCN neurons in vitro. Successful completion of these experiments will reveal new and significant information regarding the fundamental mechanisms of circadian rhythmicity and the cellular basis of aging of a specific, localizable brain function. This will provide an expanded basis for understanding the underlying mechanisms of age-related disruption of biological timing where these processes are apparently deranged. The multidisciplinary approach to SCN aging afforded by coordination with our IRPG collaborators, Drs. Phyllis Wise and Marilyn Duncan, will enhance the impact of these independent research proposals.

horizontal cell; enzyme activity; nitric oxide; glutamate receptor; protein kinase; neural information processing; retina; receptor sensitivity; cell cell interaction; biological signal transduction; neural transmission; synapses; retinal adaptation; cyclic GMP; electrophysiology; voltage /patch clamp; fish; single cell analysis;

The physiology and behavior of virtually all eukaryotes, including humans, exhibits a pervasive daily rhythmicity. Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a free-running period of about a day (i.e., circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the master biological clock in mammals. Recent research has revealed that (i) the ability to generate neurophysiological circadian rhythms reside in single SCN neurons, and (ii) the fundamental mechanisms for rhythms resides in single SCN neurons, and (ii) the fundamental mechanisms for rhythms generation within these neurons involve transcriptional negative feedback loops among putative circadian clock genes. A critical for circadian neurobiology is to establish means to monitor real time circadian gene expression in single SCN neurons so that the precise roles of putative clock genes, and the linkage of the intracellular molecular clock with its neurophysiological output, can be investigated in detail. Toward these ends we have created three kinds three of transgenic mice incorporating the reporter constructs fosdGFP. Toward these ends we have created three kinds of transgenic mice incorporating the reporter constructs fosdGFP, (dGFP) appropriate for assaying dynamic gene expression. Our hypothesis is that SCN neurons from fosdGFP and perdGFP mice will display circadian gating of induction and circadian rhythms in dGFP fluorescence correlating with the previously documented endogenous rhythms in c-fos and mper1 RNAs. We propose three specific aims to test and expand our hypothesis. Aim 1-Intact Animal Induction Experiments-to validate the function of the transgene reporters. Aim 2 In Vitro Induction Experiments-to test for acute induction of transgene reporters. Aim 2- In Vitro Induction Experiments-to test for acute induction of transgene reporters in vitro. Aim 3-In Vitro Circadian Experiments-to test for circadian rhythms in fos or per-driven gene expression. Successful completion of these aims will aims will establish promoter driven dGFP transgene reporters as an approach for assessing the molecular circadian rhythms in individual SCN neurons. This will provide a methodological foundation for expanded future research on the molecular basis of the mammalian SCN clock which should increase our understanding of the mechanism of seasonal affective disorder, age-related disruption of biological timing, and jet lag.

DESCRIPTION (from applicant's abstract): The goal of this training program is to prepare promising graduate and medical students, postdoctoral and medical resident fellows for successful careers in Cellular and Molecular Neuroscience of Sensory Systems. This program arises from the perceived benefits of integrated training in the fundamental principles which shape the function of all of our senses and is guided by the following conceptual framework: (I) The sensory systems of the human body are fundamental contributors to the health and well-being of people and are critical substrates for disease processes. (II) These neural systems share many core mechanisms of organization and function, which are especially apparent at the cellular, molecular and genetic levels. (III) Common mechanisms are also reflected in sensory pathologies- specific disease processes often impact multiple sensory systems. (IV) Graduate and postdoctoral research training which integrates cellular, molecular and genetic aspects over a range of sensory systems will enhance the ability of trainees to conduct innovative health-related research. Core mechanisms span multiple sensory systems as do disease processes (e.g. Charcot-Marie-Tooth disease, Norrie disease, Retinitis pigmentosa, Usher's syndrome, Aging and sensory senescence). Thus, there is a need for research training, which integrates across the traditional boundaries of each special-sense tissue. The proposed training faculty consists of 12 members with expertise in the auditory, chemosensory, mechanosensory and visual systems. Faculty research approaches range from mutagenesis and molecular genetics through cell and molecular biology, to intact animal physiology, behavioral analysis and clinical research. The institutional resources, level of extramural support and training histories of the training faculty engender a superb training environment.

Many critical daily rhythms are endogenous, driven by an internal master pacemaker or biological clock, with a free-running period of about a day (i.e. circadian). The suprachiasmatic nuclei (SCN) of the hypothalamus are the apparent site of the master biological clock in mammals. Recent research has revealed that (i) the ability to generate neurophysiological circadian rhythms resides in single SCN neurons, and (ii) the fundamental mechanisms for rhythms generation within these neurons involve transcriptional-translational negative feedback loops among circadian clock genes. A critical challenge for circadian neurobiology is to define the links between the intracellular molecular clock with its neurophysiological inputs and outputs. Toward this end we have created transgenic mice incorporating a circadian reporter construct which expresses a degradable form GFP under the control of the mPer1 promoter. We will combine our ability to visualize the cellular dynamics of per1 promoter activation in the living SCN with double label immunocytochemistry, patch clamp electrophysiology, gene expression profiling and multielectrode array recording to (i) identify light- entrainment-transducing SCN neurons and the physiological and molecular events which occur in them during the phase-resetting process, (ii) measure individual neuronal rhythms in SCN slices en masse to provide a novel view of pacemaker structure, (iii) identify specific populations of rhythmic SCN neurons and examine the mechanisms by which the molecular clock loop is output to the cell membrane. Successful completion of these aims will elucidate critical links between gene regulation and circadian function of the SCN. This will increase our understanding of the fundamental mechanisms underlying normal biological clock function and our understanding of disease processes in which the clock is deranged.

DESCRIPTION (provided by applicant): The vertebrate retina is pervasively influenced by circadian (daily) rhythmicity. Across vertebrate species there is a wide variety of retinal rhythms including - rod and cone disc shedding; dopamine and melatonin synthesis; ERG b-wave amplitude; and visual sensitivity. The long-term goal of our research is to elucidate the retinal cell types and mechanisms critical for circadian organization of retinal function. By mapping the temporal and spatial distribution of the circadian clock gene Per 1 in the mouse retina, using transgenic mice in which neurons transcribing Per 1 are marked with a dynamic green fluorescent protein (GFP) reporter, we have established that Per 1 clock gene rhythms are concentrated in neurons of the inner retina, in part, within dopaminergic amacrine cells. In addition, the McMahon lab has recently established transgenic mouse lines which express a red fluorescent protein (RFP) reporter of tyrosine hydroxylase (TH) gene transcription to mark dopaminergic neurons. Here we propose to use these unique mouse models for functional studies examining three critical aspects of the circadian organization of the retina: I. Targeted Electophysiology of Per 1 - Expressing Dopaminergic Amacrine Cells - we will examine if these neurons exhibit intrinsic circadian rhythms in spike frequency and ionic currents. II. The Roles of Dopaminergic Amacrine Cells and Photoreceptors in Retinal Rhythmicity - we will determine if these cell populations support circadian rhythms in overall retinal clock-gene expression. III. Characterization of Per 1 - Expressing Ganglion Cells and Ganglion Cell Rhythmicity - we will determine the functional types of Per 1 - expressing ganglion cells (i.e. ON, OFF, transient), and whether ganglion cell activity is rhythmic. Completion of these aims will produce important insights into the mechanisms by which the retinal circuits match their performance to ambient conditions and provide an expanded basis for understanding the underlying mechanisms of photoreceptor degeneration and myopia, pathological eye conditions affected by the retinal circadian clock and its melatonin/dopamine outputs.

ABSTRACT NOT PROVIDED

laboratory mouse

The goal of the Conte Physiological and Behavioral Core is to enable Conte investigators to explore the physiological function of serotonergic neurons and circuits and the behavioral phenotypes of mouse models resulting from manipulation of 5-HT signaling and its development. Neuronal function will be assayed by electrophysiological experiments performed by core staff in consultation with investigators. The core will also provide equipment, personnel, facilities and expertise for a wide range of behavioral assays of investigator-generated mouse models. The Research Strategy of the Core is to provide infrastructure, technical assistance and services for i) visualized patch clamp electrophysiology of raphe 5-HT neurons and hippocampal neurons and ii) behavioral analyses of transgenic mice. These include the following: Sensorimotor and Somatosensory behavior using manual and semi-automated analyses in both home cage and novel testing environments and at distinct stages of circadian activity; Cognition, Anxiety and Social Interactions in which quantitative technologies are implemented to explore behaviors linked to anxiety, aggression, and social dynamics; Depression Models and SSRI Responsiveness/Reversal where the impact of changes in engineered or developmentally altered strains for response to 5-HT modulating antidepressants and reversal of behavioral deficits is assessed. The Physiological Unit of the Core consist of two dedicated electrophysiology rooms/rigs along with all necessary accessory tools for modern slice electrophysiology. The Behavioral Studies Unit uses space and resources affiliated with the Vanderbilt Laboratory for Neurobehavior, which occupies ~9000 sq ft of dedicated and modularly designed murine behavioral testing facilities. The Core Director is Douglas McMahon, PhD, who will also head the Physiological Studies Unit of the Core. Dr. McMahon is a highly experienced neurobiologist with more than 30 years experience in electrophysiology and imaging of the nervous system. The Core Co-Director is Gregg Stanwood, PhD, who will head the Behavioral Studies Unit of the Core. Dr. Stanwood is an experienced behavioral neuropharmacologist and developmental neurobiologist. Another key person in the Core is Dr. Hideki Iwamoto, an electrophysiologist with direct experience working with mouse 5-HT signaling models. Two leaders in the field, Drs. Irwin Lucki (Univ. of Pennsylvania) and Jacqueline Crawley (UC Davis), will serve as consultants to further aid our efforts. Each of the individual Projects makes extensive use of the Physiology and Behavioral Core to accomplish key aspects of their research plans.

DESCRIPTION (provided by applicant): We request funds for the purchase of a shared Multiphoton Imaging and Electrophysiology Workstation. This workstation will consist of a dedicated multiphoton fixed stage upright microscope (Olympus Fluoview 1000 MPE) equipped with optics appropriate for imaging living brain slices or retinal explants, as well as electronics micromanipulators and other support items (electrode puller, vibratome, perfusion chamber, etc.) necessary for experiments combining multiphoton imaging with patch-clamp electrophysiology. The user group is made up of cellular and molecular neuroscientists of both the College of Arts and Sciences and the Medical Center of Vanderbilt University who study a broad range of problems in neuroscience using advanced imaging and electrophysiological techniques, and all have a critical experimental needs for multiphoton imaging of neural tissue in terms of the increased depth of penetration, the spatially restricted imaging and stimulation, the decrease in phototoxicity and the absence of visible light stimulation afforded by deep IR multiphoton imaging. The Olympus Fluoview 1000 MPE system requested has been uniquely configured based on the needs of the user group with significant attention to the highest performing immersion objective lenses for the specific needs of electrophysiology experiments as well as a dual transmitted light path for delivering light and pattern stimuli to living retinal preparations. The instrument will be located in and maintained by the Vanderbilt Cell Imaging Shared Resource (CISR) on the 7th floor of the Biological Sciences Building/Medical Research Building III (BSB/MRBIII). This building houses many of the users, including those from CAS, and is conveniently accessible to all users. The expertise and institutional support for this MPE microscope are exceptional. Drs. Piston and Wells, Scientific Director and Managing Director of the CISR, respectively, are world-renowned experts in multiphoton imaging. The CISR has an 18-year history of assisting over 275 investigators with cutting-edge imaging expertise. Vanderbilt University College of Arts and Sciences and Medical Center have committed $300,000 in funds to enhance the purchase and operation of the instrument.

DESCRIPTION (provided by applicant): The proposal is for the renewal of support for a graduate training program at Vanderbilt University that is structured to support the early phases of neuroscience predoctoral education and training. In support of the overall NIH mission, the overarching objective of the program is to provide an exceptional training environment for the next generation of neuroscientists, and is built on the foundation of a strong training faculty wit exceptional records of scholarship, research support and graduate mentoring. The heart of this mission is expressed in the academic and research goals of the program, which are to provide our students with a strong didactic foundation in the neurosciences through our core curriculum offerings, and to provide them with the opportunity to carry out state-of-the-art neuroscience research in the laboratories of a group of highly successful and committed mentors. In addition, the program has strong emphases on professional development and diversity, with the objective of building the requisite skills needed for success in graduate school and beyond, and of training an inclusive cadre of future independent investigators in neuroscience research. The Neuroscience Graduate Program at Vanderbilt is an interdisciplinary program that encompasses four different colleges and schools and 18 departments. Students can enter the program either directly or via three umbrella feeder programs (IGP/MSTP/CPB). Traditional and emerging areas of research strength in the program include: attention, brain evolution, cell signaling, cognitive neuroscience, circadian function, CNS drug development, development and developmental disabilities, molecular genetics, neurodegeneration and neurotoxicity, neuroimaging, plasticity, psychiatric illness, sensory and multisensory systems, synaptic transmission, and vision. The program is currently home to 83 trainees and 64 training faculty. The proposal requests an increase in support from 7 to 10 slots and provides the rationale and justification for this request.

? DESCRIPTION (provided by applicant): A fundamental question in neuroscience is how changes in gene expression are translated into changes in neuronal physiology and ultimately into changes in behavior. The brain's 24-hour timing mechanism, or biological clock, is a neural system that is uniquely suited to the study of the genes-to-behavior problem. A key gap in our knowledge is how the cycling of the neurophysiological rhythms in clock neurons is interconnected with the molecular clockworks to ultimately drive behavior. We conceptualize the clockworks in neurons as being composed of 3 interconnected layers or levels - genes, membrane and messengers. Here, we hypothesize that the neurophysiological and TTFL levels of the clock are coordinated through mutual reinforcement of the membrane and gene level rhythms, as mediated by the messenger Ca++, and CREB. We will use transgenic circadian reporter mice combined with optogenetics to address the following questions - How does manipulation of SCN neuron spike frequency modify the phase and period of the TTFL? We have successfully instituted both stimulatory and inhibitory optogenetic control of SCN neuron spike frequency, and used this approach to modify the phase and period of the SCN TTFL in vitro. Here we will determine (i) the mechanisms by which altering SCN spike frequency resets the phase of the SCN clock by examining membrane Ca++ fluxes, CamKII or MAPK activation, CREB/CRE induction and (ii) whether the ongoing membrane spike frequency rhythm is necessary to maintain molecular TTFL rhythmicity. How does altering SCN neuron spike frequency affect organization of the SCN? We have found that optogenetic spike- frequency-mediated clock resetting requires intercellular communication via SCN neuron action potentials and network communication through VIP. Here we will determine the mechanisms underlying SCN network effects of optogenetic stimulation and whether additional network communication through GABA and AVP signaling may be critical. In addition, (ii) we will define how spike frequency induced resetting may reconfigure the SCN network to alter free-running period as well as phase. Can manipulation of in vivo SCN spike frequency per se encode circadian behavior? We have used optogenetic stimulation of the SCN in vivo to reorganize circadian behavior and will use this approach to test for rescue of behavioral rhythms in arrhythmic backgrounds and whether optogenetic manipulation of the daily SCN spike frequency profile can mimic photoperiod encoding. Successful completion of these aims will provide novel insight into mechanisms by which SCN neurons generate and sustain coherent 24-hour rhythms that drive behaviors. As the clock genes are widely expressed in the brain and contribute to neuronal excitability in learning and memory and addiction, this work will also point to general mechanisms of genetic control of neuronal function and behavior. In addition, successful definition of how SCN spike frequency may encode circadian behavior and photoperiod would open the way for manipulation of SCN activity as a future therapeutic approach for circadian disorders.

PROJECT SUMMARY A fundamental question in neuroscience is how environmental signals may have long-lasting effects on neural circuits and neural function. The circadian clock and circadian photoperiod are associated with mood disorders, but the neurobiological mechanisms are unknown. Dysregulation of serotonin neurotransmission is implicated in neurobehavioral disorders, such as depression and anxiety, and alterations in the serotonergic phenotype of raphe neurons has dramatic effects on affective behaviors in rodents. The serotonergic dorsal raphe nuclei receive light input from the circadian visual system, as well as polysynaptic input from the biological clock nuclei, and dorsal raphe serotonin neurons respond acutely to tonic illumination with increased spike rate and to changes in the circadian light cycle with gene activation. Our laboratory has demonstrated that seasonal circadian photoperiods (winter ?like ?short days? vs. summer-like ?long days?) can induce enduring changes in mouse dorsal raphe serotonin neurons - programming their spontaneous neural activity, and altering depression and anxiety-like behaviors. Here we seek to elucidate the mechanistic basis photoperiodic programming of serotonin neurons, focusing on electrophysiology, gene regulation and maternal-fetal vs neonatal developmental windows. We will examine neural mechanisms of photoperiodic programming of dorsal raphe serotonin neurons using both multi-electrode array and whole cell electrophysiology; altered gene regulation in serotonin neurons induced by photoperiodic programming using RT-PCR, RNA-seq gene expression analysis of FACS sorted serotonin neurons and RNA Scope in situ hybridization to determine the photoperiod programming transcriptome, and the gene network in serotonergic neurons driven by photoperiodic programming. We will test the hypothesis that the Pet-1 transcription factor is a critical node for photoperiodic programing and define critical periods for the enduring effects of photoperiod. Successful completion of these aims will reveal novel mechanisms by which a pervasive environmental signal ? the daily light cycle ? can influence the long-term function of brain serotonergic neurons and the behaviors they mediate.

? DESCRIPTION (provided by applicant): The activity and plasticity of dendritic spines and synapses underlie normal cognitive processes, such as learning and memory and are the basis for the complex circuitry found in the brain. Dendritic spines, which are actin-rich protrusions that emanate from the dendrite shaft, comprise most postsynaptic terminals of excitatory synapses. Not surprisingly, abnormalities in dendritic spines are associated with a number of neurological disorders, including Fragile-X syndrome, Down's syndrome, Alzheimer's disease, autism, schizophrenia, and epilepsy. Despite the importance of spines and synapses in the central nervous system, the molecular mechanisms that regulate the activity and plasticity of these structures are not well understood largely because of the current lack of available technologies for probing these structures at single spine/synapse levels. Furthermore, the capability to study synaptic activity and plasticity in individual spines and synapses would provide significant insight into the function and molecular mechanisms that regulate these structures. We are developing novel neuron-glia co-culture microfluidic devices with integrated graphene sensors and electrodes and combining them with scanning photocurrent microscopy to detect and stimulate spine plasticity at sub- synaptic resolution (Specific Aim I). We will use this technology to record electrical properties at individual dendritic spines and synapses and to examine the effects of different electrical stimuli on these structures. Since reorganization of te actin cytoskeleton is thought to underlie the activity, plasticity, and function of dendritic spine and synapses, we will explore the role of actin-binding protein VASP in regulating synaptic activity and plasticity (Specific Aim II). We will alter the expression of VASP and determine the effect on the electrical properties of individual dendritic spines and synapses with the graphene probes. Moreover, we will determine the contribution of this protein to synaptic plasticity. The development of the proposed microfluidic platforms will be of great interest and benefit to neurobiologists by providing a powerful technology for investigating the mechanisms that underlie the electrical activity and plasticity of dendritic spines and synapses at a single synaps level.

Project Summary/Abstract This proposal seeks to establish a T34 MARC undergraduate training program at Vanderbilt University that is structured to support the enhancement of achievement and retention in STEM majors of students from underrepresented groups. In support of the overall NIH mission, the overarching objective of the program is to provide an exceptional training environment for the next generation of diverse and inclusive biomedical scientists and faculty, and is built on the foundation of a strong training faculty with exceptional records of scholarship, research support and undergraduate mentoring, and an exceptional institutional environment with core emphases on undergraduate research Immersion, and institution-wide dedication to increasing diversity and Inclusive Excellence. The heart of this mission is expressed in the academic and research goals of the program, which are to provide our students with a strong didactic foundation in the STEM disciplines through core curriculum offerings, and to provide them with the opportunity to carry out state-of-the-art research in the laboratories of a group of highly successful and committed mentors, here at Vanderbilt, and for one summer at another institution. In addition, the program has strong emphases on professional and career development, and on quantitative literacy and rigorous science, with the objective of building the requisite skills needed for success in graduate school and beyond, and of training an inclusive cadre of future independent investigators in biomedical research. The proposed MARC Program at Vanderbilt is an interdisciplinary program that encompasses 40 faculty preceptors from 3 different colleges and schools and 7 departments, including the departments of Biological Sciences, Biomedical Engineering, Chemistry, Computer Science, Neuroscience, Physics and Astronomy, and Psychology. These departments currently host ca. 700 exceptionally qualified undergraduates that are MARC eligible, providing an outstanding opportunity for recruitment. The proposal requests support for 20 MARC students each year and provides the rationale and justification for this request.