Charles S. Zuker - US grants

The aim of the research proposed in this grant is to gain insight into the cellular and molecular basis of phototransduction in the neuronal photoreceptor cell. It is expected that the results obtained from these studies will help our understanding of the basis of sensory reception and information processing in biological systems. Over the past 22 months we have concentrated our efforts in the isolation and characterization of genes important for photoreceptor cell function. We have isolated the genes encoding four distinct Drosophila opsins. One is expressed in the six outer photoreceptor cells (R1-R6), the second in the central R8 photoreceptor cell, and the other two in the UV sensitive R7 photoreceptor cells. We have determined the structure and nucleotide sequence of each of these genes. We have used P element-mediated gene transfer to introduce the cloned structural gene for the R1-R6 opsin into the Drosophila germline and restored the ninaE mutant phenotype to wild-type. In an attempt to study the contribution of the various opsins to the specific functional properties of the different photoreceptor cell types, we have genetically engineered Drosophila lines that express R8 opsin in the R1-R6 photoreceptor cells. The experiments proposed in this grant are a continuation, and an extension, of these studies. Focusing on the Drosophila visual system, we will: (1) Attempt to isolate the additional genes encoding the different opsins expressed in each photoreceptor cell type of Drosophila. (2) Determine the primary sequence and gene structure of each of these opsin genes. (3) Determine the anatomical sites of expression of the different rhodopsins, and (4) attempt to determine the basis for the photoreceptor cell specificity of the different genes. We will use oligonucleotide site-directed mutagenesis to mutate selected amino acids and regions of the rhodopsin molecule and reintroduce the mutated genes into the germline of Drosophila melanogaster by P element-mediated gene transfer to: (5) Study the basis of the different spectral sensitivities of the various opsin proteins, and (6) analyze structure-function relationships in the rhodopsin molecule. The latter two aims will also be approached by constructing hybrid genes between the different opsins. These constructs will also allow the expression of the different opsins in photoreceptor cells in which they are not normally expressed so that we can study the physiological activities and specific properties of the different rhodopsins in relation to the various photoreceptor cell types. (7) Finally, we will isolate genes encoding other polypeptides involved in the phototransduction process.

Although much is known about the molecular basis of vision and olfaction, almost nothing is known about the molecular biology of taste. Humans and flies have four basic taste modalities, sweet, sour, bitter and salty. This grant focuses on a molecular genetic dissection of taste in Drosophila melanogaster, a model system particularly well suited for a genetic dissection of this process in vivo. It is expected that results obtained from these studies will be important in elucidating the molecular basis of taste, and will help our understanding of sensory signaling in general. We will: (1) Develop a comprehensive genetic screen to isolate mutations affecting taste cell function, including sweet, bitter and salty pathways. (2) We will screen for autosomal recessive mutations so as to generate an extensive genetic database for detailed analysis and (3) characterize the mutants genetically and physiologically. In particular, we will developed a preparation suitable for physiological analysis and use electrophysiological recording together with genetic epistasis to localize the site of action of the defective genes.(4) We will isolate the defective genes and determine their entire nucleotide sequence. Wild type and mutant copies will be introduced back into flies by P-element mediated germ line transformation and tested for rescuing and/or induction of taste cell dysfunction. We will study in detail those sequences which based on their patterns of expression and genetic and physiological criteria most warrant further investigation. We will define the normal function of the affected loci, and determine the molecular basis of the defect. Finally, (5) we will use the Drosophila genes to isolate the corresponding human homologs, and make those sequences available to the community working on vertebrate models.

The sense of taste is a major form of chemosensory input in the animal kingdom. Taste perception is responsible not only for attraction and repulsion to various food sources, but also for providing important information about the chemical environment. Our long-term goal is to define the components required for orchestrating the response of a taste receptor cell to its cognate ligands, and to help elucidate the logic of taste coding. This grant focuses on the identification of taste signaling molecules. We will: (1) Generate single cell libraries, perform differential screens and use subtraction approaches to identify genes expressed in subsets of taste receptor cells. These studies will use genetically marked cells isolated from mice expressing fluorescent reporters in defined subsets of taste receptor cells. (2) Develop heterologous expression system to (a) assay taste receptor function, and (b) to functionally clone novel receptors and downstream signaling molecules. We will also isolate and characterize "accessory" proteins required for receptor trafficking and maturation. Finally, (3) we will attempt to identify the genes responsible for taste defects in the sac, soa, rua and qui mutant strains.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic, 06-NS-106: Validating new methods to study brain connectivity. Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. One challenge in mapping neural circuits is to unambiguously identify synaptic partners. Traditionally, synaptic connectivity has been studied using electrophysiology and electron microscopy - methods that provide critical detail but are impossible to apply in large scale. We aim to develop and validate a system to more easily identify synapses between selectively tagged neurons in the mouse. Recently, a system named GFP Reconstitution Across Synaptic Partners (GRASP), has been developed in invertebrates to study synaptic connectivity. It relies on genetic expression of two non-functional, complementary GFP fragments that are exposed on the extracellular sides of different cell populations. GFP reconstitution, and therefore fluorescence, occurs at the sites of close contact (e.g. synapses) between these cells. GRASP has many advantages: it can be genetically targeted to specific neuronal populations, it is a fluorescent system that can be readily visualized using traditional microscopy, and can be easily adapted to answer a wide variety of different questions about synaptic connectivity of neurons. To validate this technology for use in mammalian systems, we will initially test a battery of GRASP constructs in cell culture and an insect model. The most promising combinations will then be used to generate general-use transgenic lines that can be employed in concert with the Cre/LoxP and the tet-TTA systems to control expression of GRASP in time and space. In addition, we will develop viral carriers for GRASP as an alternate means of delivery and spatial restriction. We will use GRASP to help address questions of connectivity in the mammalian taste system, thereby providing validation of its utility to study mammalian neural circuits. Ultimately we anticipate that the genetically engineered GRASP mouse lines and viral vectors generated in this study will provide a toolbox that will be of considerable value for the entire neuroscience community. PUBLIC HEALTH RELEVANCE: Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. We aim to develop and validate a system to more easily identify synapses between selectively labeled neurons in the mouse. We anticipate that our genetically engineered GRASP mouse lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.

DESCRIPTION (provided by applicant): GRASP (GFP Reconstitution Across Synaptic Partners), originally developed in invertebrates, is a genetically controllable fluorescence-based system for identifying sites of contact between two cells (or cell populations). It relies on the expression of membrane-tethered split (i.e. non-functional) GFP modules that must come together to reconstitute GFP fluorescence (spGFP1-10 & spGFP11); since this functional complementation requires close apposition between membranes (<100 nm), GRASP is a powerful system to identify synapses between two cells. The objective of this application is to develop and validate GRASP methods for use in mammalian and Drosophila neural circuits. We will generate a battery of GRASP tools, including viral vectors for targeted expression of spGFPs, produce transgenic (and knock-in) lines expressing spGFPs under the control of the Cre or tetracycline inducible systems, and engineer Drosophila chromosomes harboring GRASP components under orthogonal expression control systems. Together, these reagents will afford temporal and spatial selectivity of GRASP expression, and provide a versatile platform for circuit mapping. We propose to test these components in two different models: a thermosensory circuit screen in flies and a Cre reporter screen in mice. We will also engineer a multicolor GRASP as a novel synaptic fingerprinting technique. We expect the results of these studies to significantly enhance the arsenal of tools available for circuit mapping, and be helpful in the characterization of normal and diseased nervous systems.

DESCRIPTION (provided by applicant): In this proposal, we make use of novel two-photon calcium imaging technology, optogenetic tools, circuit tracing, and genetic targeting techniques to dissect the role of selective cortical fields in driving rewarding/appetitive and aversive behaviors. At a more fundamental level, the work presented in this research proposal may help identify new substrates for understanding (and perhaps managing) eating and metabolic disorders such as obesity, anorexia, bulimia, and diabetes. Using two-photon calcium-imaging in live animals, our lab recently identified a gustotopic map in the insular cortex, where each quality is represented in a distinct and separate cortical field. In this proposal we examine how neural activity in these cortical fields drives appetitive and aversive behaviors:) In aim 1, we wll activate or inactivate activity in distinct cortical fields of awake behaving animals while monitoring their behavioral responses. In aim 2, we will perform in vivo calcium imaging studies and optogenetic stimulation in the insular cortex after changing the hedonic value of sensory stimuli, and examining potential changes in their representation in the cortex. In aim 3, we will trace the projection patterns of distinct insular cortex fields to identify their targets in the brin. The results obtained from this research proposal should contribute to our understanding of how animals use brain circuits mediating rewarding/appetitive and aversive responses to orchestrate behavior.