Meyer B. Jackson - US grants

The patch clamp will be used to record single channel currents coupled to the GABA receptor of mouse spinal cord cell cultures. Data produced by GABA and by the GABA agonists muscimol and pentobarbital will be analyzed by a number of procedures designed to characterize the channel gating mechanism. This basic molecular information will then be used as a framework for the study of the mechanism by which barbiturates and benzodiazepines potentiate the GABA response. Different physiological responses to GABA have previously been identified. An attempt will be made to reduce the observed cellular responses to differences at the level of channel currents, and thereby obtain a biophysical basis for multiple GABA responses. Diversity in the properties of the GABA receptor channel will then be explored in cultures grown under conditions where spontaneous electrical activity is blocked, or where electrical activity is paroxysmal. In response to conditioning by pharmacological treatments which produce altered patterns of electrical activity, neurons may produce GABA receptor channels with different unitary properties and different physiological functions.

Patch clamp and molecular genetic techniques will be combined to study structure-function relationships in the Drosophila GABA-A receptor. The recent cloning of the GABA-A receptor of Drosophila provides an opportunity to carry out molecular genetic modifications of the receptor. The functional consequences of these structural alterations will be examined in expression systems such as Xenopus oocytes and transfected cells, and in a native cellular environment provided by cell culture of the Drosophila nervous system. In addition, Drosophila provides a powerful method of isolating mutants based on selection in terms of function, rather than site-directed mutagenesis. These advantages will be exploited to investigate the following basic problems relevant to the function of ligand-gated channels. We will use site-directed mutagenesis to determine the residues responsible for the anion selectivity of this channel. This information will then be exploited to elucidate the number of subunits in the native receptor and the number of copies of each cloned subunit in the native receptor. Ultimately, this work will lead to an understanding of the subunit stoichiometry of the GABA-A receptor of Drosophila. There is already some evidence to suggest that this receptor is a homomultimer, and if this is actually the case it would have considerable evolutionary and mechanistic significance. Proposed experiments will provide a clear test for this hypothesis. Along a different line, experiments will exploit mutants with reduced sensitivity to the classical GA BAA receptor antagonist picrotoxin. These mutants were isolated in fields sprayed with cyclodiene insecticides. Experiments proposed with these and other mutants will determine whether picrotoxin blocks by an allosteric mechanism, or by blockade of the open channel, or by both of these mechanisms. These experiments will also identify the residues of the GABA-A receptor that form the picrotoxin binding site. The information about the GABA-A receptor obtained in these studies will help researchers understand the nature of inhibition throughout the nervous system. Researchers will be able to develop better drugs in the treatment of epilepsy and many other neural disorders. The insight that will result from this study will be of general value in the development of drugs that act at other receptors in addition to the GABA-A receptor.

DESCRIPTION: The posterior pituitary is formed by nerve terminals emanating from the hypothalamus. The posterior pituitary releases two peptide hormones, vasopressin, which regulates blood circulation and renal function, and oxytocin, which regulates various reproductive functions. The nerve terminals of the posterior pituitary are unusually large, making them ideal for experimentation with patch clamp and imaging techniques. This provides a unique opportunity to investigate basic mechanisms underlying the regulation of neurosecretion. The present plan continues an investigation of membrane excitability in the posterior pituitary, emphasizing two recently discovered aspects of ion channel modulation. The first of these involves the labile gaseous signaling molecule nitric oxide (NO), which modulates ion channels in the posterior pituitary. Experiments will explore how NO and the NO signaling cascade modulate ion channels. The second involves sigma receptors, which modulate posterior pituitary ion channels in response to a number of ligands, including antipsychotic and psychotomimetic drugs. NO and sigma receptors employ novel mechanisms in the modulation of ion channels, and represent important additions to the repertoire of signaling pathways that affect electrical excitability. These mechanisms of ion channel modulation take on added significance in the context of the posterior pituitary, because they contribute to the regulation of neurosecretion. This study will examine how alterations in channel function influence action potential shape, calcium entry, and the propagation of electrical impulses through the complex terminal arborizations of the posterior pituitary. These factors influence release in profoundly different ways. Thus, this project will test basic hypotheses about how chemical signaling controls neurosecretion. Since axons generally extend over considerable distances, exhibit complex geometries, and have very large numbers of secretory specializations, these studies of the relationship between axonal geometry and ion channel modulation will have broad implications for the role of axon terminals in neural circuit function.

DESCRIPTION: Funding is requested for a Gordon Conference on the Synapse which will be chaired by Drs. Jackson and Trussel. The meeting will be held in Augus of 1998 and will endeavor to look at the synapse in molecular terms (function of vesicle proteins, SNAREs and transporters), biophysical terms (the microphysiology of Ca in the terminal, determinants of the time course of synaptic responses, rates of vesicle recycling) and in cellular, integrative terms (dendritic integration, synapses in networks). The meeting will be held at Plymouth State College.

DESCRIPTION (Adapted from the Applicant's Abstract): The synchronous electrical discharges in the brain that occur during epilepsy can be mimicked very effectively in brain slices from the rat piriform cortex. The study of epileptogenic "hot spots" deep within this brain region holds great promise in revealing the cellular processes and circuitry responsible for epilepsy. This application proposes the use of voltage imaging and single-cell patch clamp recording to study epileptiform activity in piriform cortex. Voltage imaging produces a detailed picture of the time course of epileptiform activity over a substantial area. This allows us to follow the highly characteristic pattern of development of an epileptiform discharge, which is forecast by weak activity in one site, is initiated in another site, and then propagates through a slice along a specific trajectory. With the ability to use imaging techniques to visualize each of these phases in an epileptiform event, patch electrodes can be positioned to record from single cells in targeted regions and thereby obtain precise information about what individual neurons are doing as an epileptiform event progresses. Using voltage imaging and patch clamp techniques in combination we will test a number of hypotheses regarding the generation of epileptiform discharges, including the role of excitatory synaptic activity, the role of specific excitatory connections, the role of electrical activity in specific sites, the contributions of specific types of neurons, and the role of intracellular calcium. We will make parallel studies of interictal-like and ictal-like activity to weigh the relative contributions of cellular processes and circuitry to different kinds of epileptiform activity. These experiments will thus reveal basic mechanisms responsible for epileptiform activity and thus provide a better conceptual framework for investigating human epilepsy, and for developing pharmacological treatments.

DESCRIPTION (provided by applicant): Neurotransmitters and hormones are released by Ca2+-triggered exocytosis. This tightly regulated process controls the delivery of chemical signals throughout the nervous and endocrine systems. At present, we have a very poor understanding of the molecular mechanism of Ca2+-triggered exocytosis, and this limits our ability to investigate fundamental processes such as synaptic transmission and endocrine regulation. Exocytosis entails the fusion of a vesicle with a cell's plasma membrane. A pivotal step in exocytosis is the formation of a fusion pore, which initiates membrane fusion. The fusion pore can be detected in biophysical measurements, but its molecular properties are poorly understood and its chemical composition is unknown. The experiments proposed here will investigate 1) the mechanism by which released substances permeate the fusion pore, 2) the mechanism by which fusion pores open and close, and 3) the mechanism by which the protein synaptotagmin regulates the dynamics of the fusion pore. 4) Additional experiments will test the hypothesis that membrane spanning segments of the proteins synaptotagmin, syntaxin, and synaptobrevin form the fusion pore. These experiments employ electrical measurements of flux and current through single fusion pores. The experiments will be performed in PC12 cells, a clonal cell line that exhibits Ca2+-triggered secretion, and in which molecular manipulations are easily carried out. The three specific aims of this proposal approach the question of the molecular properties of the fusion pore from different directions. The improved understanding of fusion pore permeation, dynamics, regulation, and structure emerging from this work will lead to a better understanding of how nerve and endocrine cells release substances. This in turn will improve our understanding of the many physiological functions and systems under neural and endocrine control.

DESCRIPTION (provided by applicant): A two-photon scanning confocal microscope is requested for integration with a patch clamp setup. This integrated system will be used for experiments that combine imaging with simultaneous electrophysiological recording from living cells. Six investigators, Ed Chapman, Roberto Coronado, Robert Fettiplace, Meyer Jackson, Jeff Walker, and Lea Ziskind-Conhaim form a core of major users of this system. All of these investigators have expertise in both electrophysiology and imaging, and all have similar needs for a system that can combine laser scanning microscopy and patch clamping. They will investigate various aspects of signaling in cardiac myocytes, hair cells, presynaptic terminals, and neuroendocrine terminals. Calcium plays vital roles in the physiology of each of these systems. Most of the experiments to be conducted primarily with calcium imaging to obtain high resolution information about calcium signals in real time as cells are subject to electrical stimulation. Each of the six investigators has NIH funding for research that can be dramatically enhanced by this instrument. Among other advantages, this system will enable Chapman to investigate the regulation of fusion pores by synaptotagmin, Coronado to investigate Ca 2+dynamics at individual Ca 2+release sites, Fettiplace to study Ca 2+ in individual stereocilia, Jackson to investigate spatial variations in Ca 2+dynamics in nerve terminals, Walker to apply caged signaling molecules to T-tubules and Ziskind-Conhaim to investigate the role of specific cell types in spinal rhythmicity. The shared instrument grant will enhance all of these research programs by providing better signal-to-noise in Ca 2+ imaging, better tissue penetration in imaging experiments in slices, and better localization in caged compound photolysis.

Neurons and endocrine cells release signaling molecules by Ca2+?triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein on the vesicle membrane, and triggers the fusion of the vesicle membrane with the plasma membrane to expel some or all of the vesicle content into the extracellular space. To explore the mechanisms of this process our research focuses on the fusion pore, the initial aqueous passage between the vesicle interior and the outside of a cell. Studies of the fusion pore have given us valuable insights into the roles of specific molecules in the regulation of membrane fusion. The previous funding cycle saw progress in the identification of the vesicle protein synaptobrevin 2 as a fusion pore?forming protein in endocrine cells. This work revealed new unanticipated details in the structure of the fusion pore. We also showed that the transmembrane domain of this protein alters fusion pore stability by perturbing lipid bilayers. These findings raise new questions which we will attempt to answer by performing amperometry and capacitance recordings along with recordings of unitary synaptic current to obtain sensitive measures of fusion pore permeability and stability. We will build on insights from studies of endocrine fusion pores to test the role of the transmembrane domains of syntaxin and synaptobrevin in synaptic vesicle fusion pores. We will test the role of the vesicle protein synaptophysin in endocrine fusion pores by assaying flux and lipid perturbation. We will test the role of contacts between linkers and transmembrane domains of syntaxin and synaptobrevin in providing an energetic driving force for fusion pore expansion. These experiments will establish the specific functions of proteins associated with exocytosis. We will determine how these proteins contribute to the formation of specific structural intermediates of fusion, and how these proteins deform and remodel lipid bilayers to carry out these functions.

DESCRIPTION (provided by applicant): Techniques for imaging of electrical activity in intact nervous systems hold great promise for the study of how networks of neurons work and how collections of neurons generate behavior as an emergent process through their complex synaptic interactions. Voltage imaging depends critically on the use of optical probes that convert changes in membrane potential into a detectable optical signal. Probes currently in wide use have serious limitations that impede progress in the investigation of neural networks with voltage imaging. This proposal will develop a new and novel approach to voltage imaging in living cells in brain slices from transgenic mice. This plan will extend a method termed hybrid voltage sensing (hVOS), which employs a genetically encoded fluorescent protein expressed in cells and targeted to the cell surface. Cells expressing this protein are treated with the synthetic compound, dipycrylamine (DPA), which absorbs light emitted by the fluorescent protein. DPA enters the hydrophobic interior of the plasma membrane lipid bilayer, and through resonant energy transfer, DPA can absorb light emitted by the membrane targeted protein, and thus quench its fluorescence. Because DPA carries a negative charge, it moves within the lipid bilayer in response to membrane potential. When the distance between DPA and the membrane targeted fluorescent protein changes, the amount of quenching changes so that detected fluorescence yields information about membrane potential. The first Specific Aim of this project is to perform critical tests of various fluorescent proteins to find one with optimal performance in hVOS imaging. The second Specific Aim will develop a method of hVOS that employs two fluorophores targeted to opposite faces of the plasma membrane. This will enable investigators to perform ratiometric calculations of membrane potential from two hVOS signals. These probes will enable investigators to target specific types of cells, either by the production of transgenic animals or by a variety of other molecular targeting techniques. In this way high-performance hVOS probes will serve as valuable tools for investigating network behavior, and thus contribute broadly to advances in many basic and applied areas of neuroscience. PUBLIC HEALTH RELEVANCE: This project will advance understanding of mental illness and neurological diseases by giving investigators general tools for the study of electrical activity in the brain. By studying electrical activity in specific kinds of neurons, researchers will learn how different kinds of cells contribute to brain function. This will allow clinical scientists to decide which types of neurons to target with drugs to correct abnormalities in neural function.

DESCRIPTION (provided by applicant): A new Assistant Professor will be recruited to the Department of Physiology of the University of Wisconsin School of Medicine and Public Health. The Department will recruit an outstanding researcher in the field of Synaptic Function, and this new faculty member will join a core of 5 existing Physiology Faculty working in this field. The new faculty member will develop an independent research program, and bring strength and new blood to an outstanding synergistic group of independent investigators already established at this institution. As an integral member of the Synaptic Function core group, the new faculty member will enhance the research activities of this strong team of investigators.

DESCRIPTION (provided by applicant): This proposal requests funding for renewal of an NIH Training Program in Molecular Biophysics (the MBTP) at the University of Wisconsin-Madison. Molecular biophysics takes a quantitative, physical, non- phenomenological approach to biology that is firmly rooted in the principles of condensed-phase physics and Physical chemistry. Biophysicists are driven primarily by their curiosity about how biological systems work at the molecular level. They are often responsible for development of state-of-the-art measurements that eventually enhance the capabilities of all biological scientists, including medical researchers. The MBTP Trainees are a highly sought cohort of graduate students combining strength in mathematics and physical science with a genuine interest in complex biological systems. Our current Trainees have a mean GRE Quantitative score of 760 on a scale of 800. The key elements in the Training Program are core coursework that ensures that all Trainees have a firm grounding in physical and biological principles;strong, interdisciplinary research at the forefront of molecular biophysics;coursework and informal training in proper conduct of scientific research;and close interaction with a thesis/mentoring committee that is formed in the second year and maintains a strong connection with the Trainee throughout the Ph.D. program. The UW- Madison offers a tremendous range of seminars, poster sessions, and auxiliary coursework that greatly enhance the scientific breadth of our Trainees. In the past two years, we have substantially altered the administrative structure and the content of the MBTP and the closely related Biophysics Graduate Degree Program (BGDP). A single Biophysics Steering Committee now administers both the MBTP and the BGDP. We are working hard to create a more coherent community of biophysics students on the UW-Madison campus. New requirements ensure uniformity of the training experiences of all biophysics students on campus, both MBTP Trainees (regardless of home department) and BGDP students. Through a common, weekly Biophysics Seminar class, poster sessions, Biophysics Evenings, all biophysics students share many opportunities to interact with faculty Trainers and with each other. By leveraging existing connections with outreach programs already existing on campus, the MBTP Trainers will enhance ongoing efforts to increase participation of underrepresented minority students in both the MBTP and the BGDP. To enforce accountability, any Trainer who accepts a new Trainee will be required to participate in one meaningful outreach activity each year. In the coming grant period, we also plan to improve self-evaluation of the effectiveness of the MBTP via exit interviews and a periodic survey of former Trainees at least five years after their Ph.D.

DESCRIPTION (provided by applicant): Synaptotagmin 1, a protein found on synaptic vesicles, serves as a Ca2+ sensor in neurotransmitter release. This protein has 16 homologues, and while these other proteins are widely thought to serve related functions essentially nothing is known about where they are or what they do. Preliminary data presented in this application shows that several synaptotagmin isoforms are abundant in the posterior and/or anterior pituitary, glands that release peptide hormones that control essentially all vital aspects of mammalian physiology. Prior to these new preliminary results, the pituitary had been established as a preparation amenable to quantitative biophysical study of how peptide-containing vesicles fuse with the plasma membrane to release their content into the circulation. Thus, the finding of novel synaptotagmin isoforms in the pituitary offers a unique opportunity to test their function in detail and elucidate their precise roles in excitation-secretion coupling. This project will study the pituitary synaptotagmin isoforms with complementary approaches, investigating their binding properties in vitro, and their localization and physiological function in the pituitary. The functional studies employ a strategy of using transgenic mice for gene ablation of specific synaptotagmin isoforms, and gene targeting to identify specific cell populations that express those isoforms for electrical recording. By performing capacitance recording in identified cells and nerve terminals we will determine how syt isoforms regulate exocytosis in terms of Ca2+ sensitivity and fusion pores. The experiments proposed here will test the functions of synaptotagmins 4, 7, 9, 10, 11, and 12 in the release of hormones that control growth, metabolism, stress, fluid balance, and reproduction. PUBLIC HEALTH RELEVANCE: These experiments will shed light on the function of a class of proteins with broad roles in neurological, mental, and endocrine function and thus contribute to improving treatment of neurological disorders and mental illness. By illuminating the mechanisms of control of the pituitary hormones oxytocin, vasopressin, growth hormone, adrenocorticotropin, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, and prolactin, these studies will improve our understanding of growth defects, abnormal stress responses, metabolic disorders, and reproductive health.

DESCRIPTION (provided by applicant): The hybrid voltage sensor (hVOS) technique enables researchers to use green fluorescent protein and other fluorescent proteins to image membrane potential. Because hVOS probes are encoded by DNA, genetic techniques can be used to target them to specific types of cells. This project will develop mice with hVOS probes targeted to specific types of neurons in the mouse brain. Animals will be developed with hVOS probes expressed in a wide variety of brain regions and cell types. Animals will also be developed that can be used to target very specific populations of cells. Slices of brain prepared from these animals will be very useful in imaging experiments. Essentially any brain region and any nerve cell type will be accessible to studies using the strategy advanced in this work. With these animals researchers will be able to study the electrical activity of large numbers of cells simultaneously. A hVOS probe developed in this laboratory was found to preferentially target axons. Animals expressing this hVOS probe will be useful in general studies of axonal excitability.

DESCRIPTION (provided by applicant): The Integrated Biological Sciences Summer Research Program (IBS-SRP) at the University of Wisconsin (UW) has an impressive record in providing authentic summer research experiences to undergraduates, most of whom are from groups underrepresented in the biological sciences research community. This application seeks support to expand this program by increasing the number of IBS-SRP students in UW neuroscience laboratories. The seven students supported by this grant will spend their summer in an outstanding neuroscience laboratory and conduct research under the supervision of scientists with experience in and commitment to the mentoring of undergraduates in research. The students will enter a highly structured program with weekly meetings and with many enrichment activities designed to help them connect their research with major concepts in the biological sciences. Additional enrichment activities provide students with guidance in preparing for graduate school and for developing the skills they will need as neuroscience researchers. This program will increase the diversity of neuroscience researchers and help students from diverse backgrounds in their preparation for careers in neuroscience.

? DESCRIPTION (provided by applicant): Processing of sound is a collective activity of neuronal circuits that begins with the transmission of acoustic input from the cochlea to the brain. Auditory nerve fibers activate neurons in the cochlear nuclei, which then signal synaptically to other neurons in the cochlear nuclei as well as to higher centers. The dorsal cochlear nucleus (DCN) has long been considered a primary auditory nucleus because it receives input directly from auditory nerve fibers, but T stellate cells of the ventral cochlear nucleus (VCN) also project with excitatory synapses to the DCN. What is the relationship between these two sources of acoustic input to the DCN? Are the same neurons targeted by auditory nerve fibers and T stellate cells? Do T stellate cells in the VCN that are activated by a particular sound target cells in the DCN that are activated by the same sound? More generally, does the connectivity within the auditory brain stem reflect a cell-type based code or a more subtle functional code? Although circuitry can be studied by existing anatomical and functional techniques at the level of connections between different cell types, classical techniques cannot readily address the interactions that occur within populations of neurons that cooperate during the performance of specific tasks or sensory responses. The present application introduces a novel experimental design that will target neurons linked by their participation in behaviors under defined experimental conditions. Newly developed mouse models will use immediate-early gene expression to target a genetically-encoded voltage sensor to electrically active neurons. The voltage probe, a hybrid voltage sensor (hVOS), can detect subthreshold synaptic potentials in a single cell in a brain slice in a single trial without averaging. Immediate-early genes will be activated by presenting animals with auditory stimulation in the form of pure tones or white noise. This will drive hVOS probe expression selectively in the neurons that respond to auditory stimulation. Slices from the cochlear nuclei will then be used in voltage imaging experiments to study the circuit relations within these unique sets of labeled neurons. This approach will thus reveal the synaptic connections between selected populations of neurons linked by participation in responses to particular sounds. Pure tones will drive hVOS probe expression in isofrequency laminae in the VCN and DCN. hVOS imaging will then reveal the interactions between functionally related, labeled T stellate cells within these isofrequency bands in the VCN, as well as between T stellate cells and their targets in the DCN. We will then turn to white noise sound to target neurons that respond to broadband stimuli and assess their functional connections. We will use both pure tones and noise to label cells and investigate interactions between T stellate cells and their targets to learn whether T stellate cells are major sources of acoustic input. These experiments will reveal new features of the circuitry engaged in specific forms of auditory processing. We will determine if neurons that respond to specific sounds form unique networks dedicated to the sensory inputs that activate them.

? DESCRIPTION (provided by applicant): The nervous system operates through the generation and transformation of patterns of electrical activity distributed sparsely through a vast array of interconnected neurons. These patterns encode information ultimately arising from sensory inputs. Within this framework the storage of information by the nervous system can be viewed as an imprinting process that enables a network to recapitulate patterns of activity stored by prior experience. The canonical operation of pattern completion is one of the most fundamental forms of network operation envisaged by neuroscientists, and this concept has had a major influence on efforts to understand cognitive function. Pattern completion results when a representation has been stored by selective strengthening of synapses between participating neurons. When a subsequent event activates only a subset of these neurons, representing a part of this pattern, this activity can then spread to the entire set of neurons through the newly strengthened synapses to reconstruct the original pattern. These ideas have been developed within two very different disciplines, mathematical/computer modeling of neural networks and experimental studies of behavior. Until recently these ideas had not been studied in an intact neural circuit, leaving important hypotheses about neural network function without direct experimental tests. The present study will use voltage imaging to test hypotheses about pattern completion in hippocampal slices. Voltage imaging generates maps of electrical activity distributed through a slice, and these maps represent 'patterns' of electrical activity. By quantification of image similarity using methods derived from digital image processing and information theory, comparisons of these maps provide a rigorous experimental test of pattern completion. Recent work from this laboratory laid the foundation for this experimental approach and demonstrated that a pattern of activity can be stored in the CA3 region of a hippocampal slice by long-term potentiation (LTP). Subsequent partial inputs then retrieved the complete pattern. The present project will develop this in vitro approach for the study of information storage and recall. Aim 1 will evaluate neuromodulators known to influence both LTP and behavior to determine whether acetylcholine and norepinephrine receptors can modify information storage and pattern completion in the hippocampal CA3 region. Aim 2 will address temporal aspects of information storage, first testing the role of input timing (spike-timing dependent plasticity), and then investigating the complementary issue of persistence of the information trace in relation to the decay of different forms of LTP. Aim 3 will use a genetically-encoded voltage sensor to evaluate information stored during an animal's experience. This Aim will also evaluate the hypothesis of pattern completion at the level of single cells and synapses. This work will provide novel experimental tests for computational models of neural network function, and advance our understanding of the mechanisms by which neural circuits store, recall, and process information.

The dentate gyrus generates new neurons throughout life, long after the neurogenesis of early development has ended. Adult neurogenesis makes important contributions to neuroplasticity and learning, and its impairment has been linked to neurodegeneration, learning disability, and epilepsy. New adult-born neurons integrate into existing mature neural circuitry, but retain the physiological attributes of immature neurons. Compared to mature neurons, new adult-born neurons form synapses more readily, are more excitable, and are more plastic with respect to induction of long-term potentiation of synaptic transmission. The study of adult neurogenesis is a very active field, and dramatic advances are being made in understanding the molecular control mechanisms and cellular properties of new adult-born neurons. Although the circuitry and network properties of new adult-born neurons ultimately play a critical role in their functions, the lack of suitable experimental tools and methods has impeded progress in this direction. This proposal will investigate network activity of new adult-born neurons by using a genetically targetable fluorescent probe in the hybrid voltage sensor (hVOS) family. hVOS employs a fluorescent protein that can be targeted for expression in defined populations of cells. hVOS imaging can record action potentials and subthreshold synaptic potentials from single neurons in hippocampal slices in a single trial (without averaging). We propose to express hVOS probe in newborn neurons and image their electrical activity. We will express probe in newborn neurons with retrovirus, as well as with a new hVOS Cre reporter mouse that can target probe expression to genetically defined populations of cells by crossing with Cre drivers. We will use an inducible Tbr2-CreERT2 driver to target probe to new adult-born neurons with high temporal resolution. The two expression approaches will be compared to check for consistency. hVOS imaging in hippocampal slices will then allow us to monitor the electrical activity of multiple new adult-born neurons simultaneously. We will stimulate the different granule cell inputs, perforant path axons and mossy cells, and use hVOS to record subthreshold synaptic potentials and action potentials. Experiments will test hypotheses about the organization of newborn neuron circuits by evaluating the properties, correlations, and synchronization of evoked electrical responses recorded in many new adult-born neurons simultaneously. We will directly test the hypothesis that mature granule cells synapse with immature granule cells. We will use this imaging approach to determine whether new neurons integrate into existing neural circuitry as functional clusters with shared synaptic inputs, evaluate clones of newborn neurons descended from a common progenitor cell, and explore the evolution of circuits as neurons mature. This work will reveal the circuit relations of new adult-born neurons, and will introduce a novel technique for the study of neural networks to the field of adult neurogenesis.

Ca2+ triggers the release of transmitters from nerve terminals and hormones from endocrine cells. Ca2+ signals are initiated by Ca2+ entry through voltage-gated Ca2+ channels, and shaped by Ca2+ binding to cytosolic Ca2+ buffers. The channels have been extensively studied, but much less is known about the buffers. These proteins rapidly bind 97.5-99.5% of the Ca2+ upon entry, and together with the Ca2+ sources and sinks form a highly regulated but very dynamic system. The complex interplay between transport and binding presents a formidable challenge to the quantitative study of cellular Ca2+ signaling. Buffers limit the rise in Ca2+, set up steep gradients around sites of entry, control Ca2+ diffusion, limit the rate of Ca2+ extrusion and sequestration, and determine the availability of Ca2+ for downstream signaling targets. The molecular structures of cytosolic Ca2+ buffers are known and their Ca2+ binding properties have been well studied in vitro. However, their concentrations in cells are hard to measure, their binding properties can change in cytoplasm, and their anchoring within cells often restricts their mobility. This application proposes to use fluorescence imaging in posterior pituitary nerve terminals to explore cytosolic Ca2+ buffers in situ. Early Ca2+ imaging work provided measurements of the endogenous buffering capacity, denoted as ?e (the ratio of total to free Ca²+). However, the in situ binding properties are rarely characterized. It is difficult to go from ?e to concentration and Kd, but we need this information because buffer saturation can reduce ?e by one or two orders of magnitude. This application will use our innovative new method that combines patch clamping and Ca2+ fluorescence to follow the titration of Ca2+ binding sites in situ. This method goes well beyond measurements of ?e to characterize multiple endogenous Ca2+ binding species. In pituitary terminals this method identified two Ca2+ buffers, and determined their Kd and concentration. Western blots revealed the well-known cytosolic Ca2+ buffers calretinin and calbindin D28K, and their Kd?s are consistent with our measurements. We will improve our approach and use it to examine buffering in different nerve terminal compartments, characterize diffusion in situ, and investigate the mobility of each species to assess its influence on Ca2+ diffusion. Genetic ablation and computer simulation will test hypotheses about the biological functions of calretinin and calbindin D28K. We will explore the role of these proteins in secretion and determine how they control Ca2+ access to the exocytotic Ca2+ trigger. We will test the hypothesis that buffer saturation facilitates release, and that buffers contribute to differences in facilitation of the two pituitary hormones, oxytocin and vasopressin. We will explore the potential roles of buffers in reproductive functions of oxytocin by comparing sexes, and potential roles in fluid balance functions of vasopressin by evaluating water-deprived animals. This work will illuminate the role of cytosolic Ca2+ buffers in endocrine function and clarify longstanding issues in the field of excitation-secretion coupling.

Multi-PI: Xinyu Zhao, Meyer Jackson, University of Wisconsin-Madison. Title: Integration of Experience-Induced Gene Expression and Circuit Functions Understanding the complex relationships between cells, gene networks, neural circuits, and behavior requires techniques that can probe the molecular makeup of distinct types of neurons, evaluate their properties, and test their roles in higher level functions. Genes expressed within specific populations of neurons determine their electrical properties and these properties together with their synaptic connectivity collectively shape the electrical activity of neural circuits. This is especially well illustrated by a population of neurons defined by expression of the Ca2+ binding protein parvalbumin (PV). PV interneurons (PVIs) are sparsely distributed, fast-spiking cells that provide feedback and feedforward inhibition to principal neurons. One of the most well-defined network functions of PVIs is in the coordination of neuronal networks and their associated oscillations. PVIs entrain cortical networks to drive gamma oscillations (30-100 Hz) and control their frequency and strength. PVI-mediated gamma oscillations are known to have important roles in sensory processing, attention, working memory, and cognition. However, the gene networks that control PVI functions and their impact on gamma oscillations remain unclear. PVIs are readily modified by environmental conditions and experience. PV immunoreactivity increases after exploration of a novel environment, rearing under environmental enrichment (EE), and voluntary running (VR). These changes occur in brain regions associated with cognition, including hippocampus, prefrontal cortex, and amygdala. The molecular mechanisms underlying PVI changes during behavioral adaptation remain unknown. Although studies suggest that behavioral adaptions affect gamma oscillations, a role for PVIs in the link between behavioral adaption and gamma oscillations has not been established. This application takes a multidisciplinary approach to address the fundamental question of how PVIs contribute to behavioral adaptations. Our overarching hypothesis is that changes in gene expression that modify the cellular properties of PVIs will alter network oscillations, enabling PVIs to serve as a critical hub in behavioral adaptations. We will determine whether behavioral adaptation mobilizes networks of genes in PVIs, and assess the contributions of these networks to PVI physiology and gamma oscillations. This project combines the unique expertise of co-PIs Zhao (genetic regulation of neurodevelopment) and Jackson (neurophysiology and neural circuits) and co-Is Roy (system biology and machine learning) and Rosenberg (computational and system neuroscience). By integrating experimental data with gene network analysis and computational modeling of multicellular networks, this work will reveal how changes in molecular/cellular properties impact the emergent properties of neural circuits.