Erik Jorgensen - US grants

Jorgensen 9733685 Abstract Exocytosis in neurons has become specialized in order to allow for the rapid secretion of neurotransmitter. A number of proteins involved in synaptic vesicle trafficking have been identified by biochemical purification from synaptosomes. Nevertheless, current models for neurosecretion are greatly limited because there is only an incomplete list of the proteins that are required for these processes. Genetic studies in the nematode, Caenorhabditis elegans, could potentially identify the complete complement of genes required for synaptic function. C. elegans is particularly advantageous for such studies because it is possible to select for mutants defective in secretion. Using such screens, over 38 genes have been identified which are required for normal neurotransmission. The efficiency of these screens has generated more mutants than can be easily characterized using standard genetic techniques. Techniques to rapidly map and clone these genes lags far behind the ability to obtain mutants. Mutagenesis strategies using transposons modified to contain foreign plasmid sequences could allow the immediate cloning of mutant genes without the need to map the mutations. The aims of this proposal are to: 1) Develop methods to mobilize recombinant Tc3 transposons in the germ line, 2) Develop techniques to mobilize a heterologous mariner element in C. elegans, 3) Train undergraduates to screen for neurotransmission mutants in C. elegans using a recombinant mariner element and to directly sequence the mutant genes in a single semester course. The tools developed by this work will benefit the larger scientific community and accelerate experiments designed to analyze neurotransmission. The techniques developed will greatly increase the efficiency of forward genetic methods. Additionally, the development of these tools and reagents will provide students with a more complete laboratory experience.

Neurotransmitters are released into the synaptic cleft using a common set of proteins involved in synaptic vesicle exocytosis and endocytosis. By contrast, certain synaptic proteins must be tailored to the needs of each specific neurotransmitter. These neurotransmitter-specific functions include biosynthetic enzymes, transporters for translocation across vesicular and plasma membranes, and specialized receptors on the postsynaptic surface. My laboratory is engaged in a longterm project to identify the gene products required by specific neurotransmitters. We are taking a genetic approach in the nematode Caenorhabditis elegans to identify mutants defective for the functions of a specific neurotransmitter, we then clone the cognate genes and characterize the proteins using biochemical assays. We propose to identify and characterize three proteins which function in neurotransmission: (1) the vesicular glutamate transporter, which as yet, remains uncharacterized, (2) a potential ancillary protein to the vesicular GABA transporter encoded by the unc-46 gene, (3) a novel excitatory GABA receptor encoded by the exp-1 gene. These studies are likely to result directly in the identification of vertebrate homologs of these proteins. Eventually, these experiments will lead to a better understanding of how the molecular structures of these proteins confer their functional properties. Ultimately, these studies might help explain the molecular behavior of neuroactive drugs and lead to the development of new and more specific pharmaceuticals which act on individual neurotransmitter systems.

This award provides partial support for purchase of a device used for rapid freezing of samples for electron microscopy. Following fixation at low temperature, cells preserved through use of high pressure freezing have improved ultrastructure and are suitable for immunocytochemistry. Use of high pressure permits the freezing of thicker samples than alternative procedures, and is thus useful for reliable freezing of whole organisms with cuticles, such as nematodes and flies, and for freezing of yeast and plant cells that have thick cell walls. The instrument will be used for study of synaptic ultrastructure in nematode mutants, protein localization at membranes of muscles and neurons, ultrastructure of yeast mitochondria, and developmental defects in plant mutants. The equipment will be housed in an imaging facility at the University of Utah where it will be available for other research and training use by students and faculty from the University and from throughout the Great Basin area.

C. elegans worms sense and respond to the presence of other individuals in a gender-specific manner: males are attracted to hermaphrodites, but hermaphrodites avoid one another. The C. elegans equivalent of a female is a self-fertilizing hermaphrodite that can also mate with a male. Male-attraction and hermaphrodite-avoidance are elicited by factors released into the environment by hermaphrodites. Both sexes are exposed to the same social signals, and the nervous systems of males and hermaphrodites are highly similar, yet their responses are diametrical. The goal of this project is to understand how molecules and nerve cells (neurons) generate these gender-specific behaviors. C. elegans is an animal model well suited for studies of behavior because genetic techniques are well-developed, the nervous system is well-characterized (the number and type of neurons do not vary significantly from individual to individual), and their wiring-how they are connected into circuits-is known. This project will use genetic techniques to identify and characterize molecules important for gender-specific behaviors, and laser microsurgery to identify neurons important for gender-specific behaviors. One of the eventual goals of this project is to test these models for how neural activity generates behavior. The C. elegans nervous system is simple enough that it should be possible to reprogram behavior. For example, hermaphrodites could be made to express the key behavioral genes of males, making the hermaphrodites attracted to other hermaphrodites. The project will train undergraduate students in genetics and neurobiology, and give them a practical basis for understanding fundamental principles. Additionally, this research will allow a postdoctoral fellow to establish a new area of scientific investigation and train him to become and independent investigator. The results from these studies will be made available by publication in open-access journals and presented to the public by the PI through community and university presentations.

The roundworm Caenorhabditis elegans is a model organism that senses and responds to sex pheromones in a gender-specific manner: males are attracted to female pheromones, but females avoid the pheromones. The goal of this project is to understand how molecules and nerve cells generate gender-specific behaviors. This species is very well-suited for studies of behavior since it is the only organism for which we know the complete wiring diagram of the nervous system. In this study the pheromones will be purified and their molecular structure determined. These pheromones bind to receptors on the surface of the olfactory cells and signal to the brain that females are present. In addition, the brain circuit required for male response to the pheromones will be determined. One of the eventual goals of this project is to test models for how brain activity generates behavior. The C. elegans nervous system is simple enough that it should be possible to reprogram behavior and thereby confirm models for nerve cell function and behavior. Part of this project will be carried out by undergraduates, thus the proposed research will train students in genetics and neurobiology, and give them a practical basis for understanding fundamental principles. The results of this proposal will be made available by publication in scientific journals and presented to the public by the PI in community and university presentations.

DESCRIPTION (provided by applicant): Genetic analysis relies on the ability to introduce, eliminate or modify genes at will. Such techniques are advanced in genetic model organisms such as yeast, flies and mice, but are limited in the worm C. elegans, which is one of the most commonly used model organisms. In worms such methods are somewhat crude: gene knockouts are by random mutagenesis and restoring gene function is via multicopy extrachromosomal arrays or random gene integrations. Despite these limitations C. elegans research has contributed to some of the most important discoveries in biology in the last 35 years: Ras - MAP kinase pathways, cell death pathways, RNA interference and posttranscriptional regulation by microRNAs are examples. We propose to develop techniques to insert, delete or modify genes in the C. elegans genome. The techniques rely on mobilizing transposons to engineer the genome; the methods will be a resource for the whole C. elegans research community. Aim 1. Improved single copy insertion. We will characterize insertion sites on each chromosome and increase the efficiency of transgene insertions. We will also devise transient selection reagents that can be used in a wild-type background. Aim 2. Universal insertion sites. We will generate universal insertion sites on all chromosomes that will be compatible with a single targeting plasmid. This will substantially increase the versatility of the technique. Aim 3. Gene targeting. We will develop a strategy to manipulate genes in their endogenous context. This technique will allow researchers to engineer mutations, including knock-outs, without any extraneous DNA changes in the gene except the intended mutation.

The objective of this proposal is to increase the resolving power and the image acquisition speed, of super-resolution fluorescence microscopy by about an order of magnitude (to 5-10nm). Resolution in conventional photoactivatable-localization microscopy is limited by the localization precision, which, in turn is proportional to 1/sqrt(N), where N is the number of collected photons. In the proposed approach, we can achieve localization precision that is proportional to 1/N. This is achieved by: (1) reduction in the effective size of the point-spread function (PSF) as a result of a novel grating system, (2) increase in the number of detected photons due to interference with a high intensity reference wave and (3) utilization of phase information in addition to the intensity of the signals using a novel optical correlator for optical filtering.

The intellectual merit arises from the potential acquisition of fluorescence images with macro-molecular (sub-5nm) resolution enabled by a relatively simple addition to conventional confocal microscopes. The transformative nature of the proposed technique is a result of the higher resolution and faster imaging, which will elucidate the fundamental interactions between proteins and sub-cellular nanostructures with unprecedented spatial and temporal resolution. Such mechanistic understanding will prove to be essential for future advancements in biology.

The broader impacts of this project include (1) training of the scientific workforce via an innovative, inter-disciplinary course, (2) recruitment of under-represented students via a hands-on demonstration module to be utilized at 3 large outreach events, and (3) widespread dissemination via commercialization of the subject technology.

Proposal # 1533611
Institute: University of Utah
Title: NCS-FO: Imaging synaptic activity deep in the brain using super-resolution cannula microscopy

Objective: This project will develop a tool for high-resolution (<100-nm) imaging of synapses in freely moving animals for neuronal studies. It will accomplish this goal by the development and integration of compact and lightweight cannula microscopy with in vitro fluorescence imaging with accompanying technology and methodologies for imaging synapses.

Non-Technical
The long-term vision of this project is to image with high resolution deep inside the brain of freely moving mice using inexpensive technologies so as to elucidate the fundamental basis of information processing and memory. Changes in synaptic strength at specific synapses are thought to underlie memory encoding and storage, yet there is very little experimental evidence for this theory in the intact brain due to technical limitations of visualizing the specific synaptic pattern involved in experience-dependent learning. This project aims to overcome this limitation by transforming a simple, inexpensive cannula into a super-resolution fluorescence microscope. Commercialization of this technology will be pursued after the fundamental science and engineering has been demonstrated for widespread dissemination.

Technical:
The objective of this proposal is to image neuronal activity, neuron structure and protein localization deep in the brain with sub-100nm resolution using computational cannula microscopy (CM) and novel molecular reporters of synaptic activity. CM will allow imaging of the brain in awake, freely moving animals at unprecedented spatial resolution. Current techniques in freely moving animals are limited to imaging the brain near the surface, include large and heavy head stages with moving parts, and cannot penetrate deep into the brain without significant damage to surrounding tissue. The ultimate goal of this proposal is to allow imaging of individual synapses in freely moving animals. We have already developed the framework for in vitro fluorescence imaging using CM. During this project, we will extend CM to enable: (1) super-resolution (< 100nm resolution) fluorescence microscopy and (2) deep-brain imaging (depth > 1mm) with the vision of imaging activity and protein localization in individual synapses in the deep brain of freely moving animals. Changes in the strength of individual synapses are thought to underlie learning and memory in the brain, yet this fundamental theory of brain function lacks tangible experimental evidence to support it in vivo. Our project will enable studies that address the causal role of molecular events at individual synapses in mediating behavior and information processing.

PROJECT SUMMARY/ABSTRACT Hearing loss is the second most common health condition among adults. Untreated hearing loss is associated with an increased risk of depression, anxiety, social isolation, dementia, and all-cause mortality. Despite this, only 14% of people who would benefit from hearing aids use them. The most common reason adults give for not using hearing aids is that hearing aids do not help them in the very situations they struggle to hear in the most?complex listening environments (CLEs). CLEs are places with many different sound sources, like restaurants. Indeed, restaurants are the most difficult listening environments for the majority of adults with hearing loss. Hearing aid technologies that are designed to improve speech perception in noise, such as noise reduction and directional microphones, show some modest benefits when tested in a laboratory environment. In the real world, however, these technologies show no benefits. Hearing aids can therefore demonstrate efficacy (how well they can work in the best possible scenario, i.e., in a laboratory test), but fail to be effective (how well they work in the real world). We propose that listening environment complexity differences between the lab and the real world drives the hearing aid efficacy-effectiveness gap. We further propose that the effect of environment complexity on listening performance is moderated by cognitive ability, and the benefit of hearing aid features on listening performance is moderated by environment complexity. Our aims are designed to test these hypotheses in the laboratory using controlled experimental paradigms, as well as in the real world using field experiments. The proposed study is informed by information theory, which defines complexity as the amount of information in a communication system. Under this theory, complexity can be measured using entropy to quantify how much information is in the system. Our team will apply this framework to understanding communication in real world CLEs. In Aim 1, we characterize the relationship between complexity, cognitive ability, and hearing aid feature efficacy in the lab using a controlled experimental paradigm. In Aim 2, we characterize the relationship between complexity, cognitive ability, and hearing aid feature effectiveness in the real world using smartphones to record real world environments and deliver surveys to participants, as well as experimental hearing aids that record how features process signals in real-time. The findings from this study will: enhance our understanding of how hearing aid users perceive real world CLEs, extend information theory to quantify the complexity of real world CLEs and its effect on listening performance, provide important insight into the factors that underlie the hearing aid efficacy- effectiveness gap, and provide groundwork that may help guide developments to close the efficacy-effectiveness gap through clinical and engineering approaches. The proposed study directly addresses the NIH/NIDCD's strategic plan priorities to increase our understanding of the interactions among auditory and cognitive functions to help explain perception in real world listening environments and improve hearing aid performance in background noise and real environments.