Barry Ache - US grants

Odors are complex mixtures of substances. It is difficult to predict behavioral or physiological responses to odor mixtures from the responses to the individual componentsof the mixtures. Mixtures are often significantly less stimulatory than predicted, a phenomenon called mixture suppression. This is at least partly due to poorly understood interactions that occur when multiple odorants activate the olfactory pathway simultaneously. This project addresses this problem by considering several possible mechanisms in mixture suppression: namely, mutually interfering mechanisms of transduction by the receptors and inhibitory synaptic connections in the olfactory part of the central nervous system. The research animal is the spiny lobster, in which it is known that receptor and central events both contribute to mixture suppression in olfaction. Electrophysiological methods are used to determine if different odor components activate opposing transduction processes inreceptor cells. Similar methods as well as cell staining and special techniques to localize the various substances by which nerve cells communicate with each other are used to identify inhibitory cells in the central nervous system and their connections in its olfactory areas. Mixture suppression provides a specific context in which to interpret the role of these inhibitions in recognizing odor quality. Since basic molecular and cellular processes tend to be conserved in evolution, the results can be expected to contribute to the general understanding of how complex odors are recognized in animals other than lobsters, including humans.

Experiments are proposed that investigate two novel and complementary approaches to labelling ensembles of physiologically active neurons in the olfactory system The primary technique is a molecular approach exploiting use of a proto-oncogene c-fos, which is expressed at low levels in most cells. It is based on the finding that diverse stimuli induce expression of Fos, the protein product of this gene, which can be detected histochemically. Experiments will determine if olfactory stimulation can induce c-fos expression in the somata of primary receptor cells, as well as in those of higher-order olfactory neurons. The second approach is based on the activity-induced endocytosis of fluorescent dyes into cellular processes at active synapses. Experiments will determine if perfusion of these dyes into the brain during olfactory stimulation selectively labels neuropil regions which are functionally active. In both instances, studies will determine the time course of c-fos induction and/or dye uptake and will examine the odor intensity and specificity required to produce this selective labelling. Compared to previously used ensemble-labelling techniques, either of these two methods has the capacity to provide superior Spatial and temporal resolution in labelling physiologically active neurons and would provide a powerful new way of investigating pattern discrimination in the olfactory system.

Data now clearly establish that odors activate at least two second messenger-mediated transduction pathways in olfactory receptor cells, and that they do so in phylogenetically diverse animals. As yet, the functional basis for having more than one way to activate olfactory receptor cells is unknown, even though this knowledge is fundamental to understanding the sense of smell. For example, having multiple transduction pathways co-expressed in the same cells would provide direct evidence that olfactory integration begins at the level of receptor cell, while having them expressed in different cells would indicate functional subclasses of olfactory receptor cells. Either of these heretofore unknown elements of peripheral olfactory organization have direct implications as to how odors are coded. It is proposed to combine whole cell and single channel electrophysiological recording and biochemical analyses of second messenger production to characterize cAMP and IP3-regulated olfactory transduction. The study uses an animal model for which preliminary data establish that the two transduction pathways co-exist in the same receptor cells and regulate distinct, opposing conductances that act in concert to determine the electrophysiological response of the receptor cell to complex odors. The study provides the first opportunity to investigate these two transduction pathways in a functionally defined context. Understanding olfactory transduction is fundamental to understanding the role of the receptor cell in the sense of smell and, in turn, olfactory dysfunctions caused by environmental and pathogenic insult to the receptor organ.

The anatomical organization of the sense of smell involves projections from the olfactory primary receptor cells to connect at synapses on particular cells within the central nervous system. The synapses form particular ball-like structures called glomeruli, composed of neuronal terminals, or neuropil, from incoming and central nerve cells. This fundamental plan of glomerular organization has been conserved in animals as diverse as snails, insects, fish, rodents and humans, suggesting it may be important in the recognition and discrimination of various odors detected by the multiple olfactory receptors. This work uses a multidisciplinary approach of electrophysiological, pharmacological and immunocytochemical techniques in an invertebrate model system to determine how regionalized processing within and among glomeruli shapes the odor sensitivity of the glomerulus. The basic organizational plan of this olfactory region suggests that results will be important for our general understanding of the poorly known mechanisms for chemosensory coding, and of the relation of central nervous system structure to function in sensory processing.

The sense of smell plays an increasingly appreciated role in our general quality of life and well being, a role that is compromised by the effect of disease, drugs, aging and environmental onslaught on our chemosensory competence. Receptor neurons in the nose serve the critical function of detecting and transducing odorants. Disruption of any of the intracellular events leading to receptor cell activation can result in olfactory dysfunction. Emerging evidence implicates phosphoinositide signaling and the transient receptor potential (TRP) ion channels commonly targeted by this signaling pathway in chemosensory transduction in taste receptor cells, vomeronasal receptor neurons, invertebrate olfactory receptor neurons, and possibly in vertebrate olfactory receptor neurons, where recent evidence suggests it can modulate the well established cyclic nucleotide signaling pathway. Phosphoinositide signaling is inherently complex, and this complexity has impeded a clear understanding of its role in many cellular systems. In this continuing project, we use an integrated, molecular, biochemical, and electrophysiologicalapproach to further define the cellular mechanisms of phosphoinositide signaling in an olfactory system. We use an animal model in which the functional role of phosphoinositide signaling in olfactory transduction is particularly well established, and has led to the evidence suggesting a potential role for phosphoinositide signaling in vertebrate olfaction. This project has the potential to identify new cellular and molecular events potentially involved in olfactory receptor cell activation, as well as contribute to a more general understanding of phospholipid signaling and TRP channel function in chemical sensing.

Odors play an increasingly appreciated role in our general quality of life and well being, a role that is compromised by the effect of disease, drugs, aging and environmental onslaught on the olfactory epithelium. Primary receptor neurons in the olfactory epithelium serve the critical function of detecting and transducing odor stimuli. Disruption of any of the cellular processes leading to receptor cell activation would impair olfactory function. New findings on which this project is built underscore earlier, inconclusive evidence that the process by which odorants activate mammalian olfactory receptor cells may be more complex than originally appreciated. These findings show that blocking the phosphoinositide-3 kinase (PI3K)-mediated arm of the phosphoinositide signaling pathway enhances odorant-evoked increases in intracellular calcium in rat olfactory receptor cells in an odorant-specific manner, and suggest that phosphoinosotide signaling modulates odorant-evoked excitation of mammalian olfactory receptor cells in the context of odorant coding. The presence of a second intracellular signaling pathway activated in an odorant-specific manner would allow the receptor cell to actually integrate the signal that establishes the combinatorial code on which odor recognition is based. A series of focused, primarily electrophysiological and imaging-based experiments address this novel idea by further characterizing the effect of blocking PI3K on the response of rat and mouse olfactory receptor neurons, by beginning to characterize the cellular mechanisms by which PI3K modulates the output of the cells, and by further characterizing the functional significance of this modulation. The results of the project can be expected to clarify the long-suspected, but elusive role of phosphoinositide signaling in mammalian olfaction, and provide a more complete understanding of the role of the olfactory periphery in how odorants are encoded by the nervous system.

DESCRIPTION (provided by applicant): Support is requested to integrate the basic research expertise in the chemical senses at the University of Florida (UF) and Florida State University (FSU) by creating the UF/FSU Chemosensory Research Core Center;the first such center in the Southeast. The proposal creates an administrative Core and two research Cores, one in Engineering and one in Genetics, to provide 'value added'to the mostly NIDCD-funded chemical senses research at the two institutions. The research base consists of six qualifying and eight supporting chemosensory investigators, together with three other auditory investigators who will also benefit from the research Cores. Two related research themes unite the research base, chemosensory processing controlling ingestive behavior and the detection and processing of olfactory input. Two scientific leaders in the field who have a proven history of working effectively together anchor the initiative at the respective institutions. Interaction is facilitated by establishing office space at each institution for use by scientists from the other institution, by establishing dedicated videoconferencing facilities at each institution, and by using UF's NIDCD-supported secure data sharing network. The conceptual framework of the Core Center closely aligns it with that of the UF Center for Smell and Taste's NIDCD-supported Chemosensory Clinical Research Program, which focuses on studying the functional role of chemosensory systems in ingestive behavior and its impact on human health.

DESCRIPTION (provided by applicant): The existing University of Florida Center for Smell and Taste (UFCST) will provide the basis for a Biomedical Research Core Center for Smell and Taste at the University of Florida (Core Center). The existing research infrastructure and environment of the UFCST, including its human resources, will be incorporated into the Core Center. In addition to its inherent research infrastructure, the Core Center has access to the vast research infrastructure of the University of Florida. The proposal is targeted at hiring a new faculty member, Dr. Cedrick Dotson, whose qualifications will strengthen the scientific capacity of the Core Center in both clinical and animal research. The Core Center will contribute to Dr. Dotson's academic development by fostering strong links across the University of Florida. Dr. Dotson will receive a tenure track faculty position in the Department of Neuroscience. The Chairperson of the Department will be responsible for mentoring and monitoring the academic progress of Dr. Dotson with the help of the Director of the Core Center. Award of the P30 will be instrumental in leveraging this position from the university, which like many public universities is undergoing significant, permanent cuts in State funding that have curtailed new hiring. The ability of the P30 to 'bridge'the position for two years until the current financial crisis is likely to abate will be instrumental in the ability to hire Dr. Dotson. The Director of the Core Center will be responsible for evaluating the overall success of the P30 and reporting progress to the NIDCD. PUBLIC HEALTH RELEVANCE: The senses of smell and taste play an increasingly appreciated role in our general quality of life as well as in diseases of growing importance such as dementia, diabetes, and obesity. This proposal reflects this awareness by proposing to establish a Biomedical Research Core Center for Smell and Taste around the existing University of Florida Center for Smell and Taste to study chemosensory deficits and their genetic basis in humans and animals.

DESCRIPTION (provided by applicant): Odor information is generally assumed to be encoded by olfactory receptor neurons (ORNs) in which odorants evoke a tonic discharge. Exciting new evidence extends hints in the earlier literature that some ORNs are not tonically active, but rather are inherently rhythmically active or 'bursting'ORNs (bORNs) in which the rhythmic bursting is entrained by odorants. bORNs open up an entirely new possibility for encoding odor information, in particular information relative to the spatiotemporal characteristics of the odor signal. Experiments are proposed using an animal model in which both tonically active and bORNs are well characterized physiologically to compare the potential of both types of ORNs to encode odor information. This will be done by first using computational modeling to test the hypothesis that synchronization of large ensembles of bORNs selectively favors (1) the detection and amplification of weak odor signals and (2) the detection and coding of the temporal characteristics of the odor signal itself compared to tonically active ORNs. Combined optical and electrophysiological recording from small ensembles of actual ORNs will then be used to experimentally test the predictions of the computational model. This project has the capacity to identify a heretofore unappreciated way in which olfactory information is encoded. PUBLIC HEALTH RELEVANCE: This project has the potential to derive a heretofore unappreciated principle of olfactory information encoding and processing that can be used not only to better understand olfactory dysfunction in humans but also to better design artificial systems capable of analyzing and localizing complex chemosensory signals.

Most animals survive in turbulent air or water environments and are living proof that it is possible to quantify odor signals in complex turbulent flow conditions to track and find sources of odors (such as food, mates, etc.). However, our engineering knowledge is still incapable of formulating simple and effective measurements that will enable man-made systems to predict, navigate and utilize properties of this turbulent flow to locate sources of chemical release. This project builds on recent exciting computational modeling of the neurobiology of organisms by the proposers, which predict that lobsters are capable of estimating not only the concentration of odors but also the time since the last odor was encountered. Lobsters accomplish this by using ensemble competition across a population of olfactory receptor neurons (ORNs), called "bursting ORNs". Bursting ORNs function to compute the time since last encounter of an odor that, along with concentration, can provide a measure of the distance to the odor source. This research will seek to increase understanding of how ORNs perceive odor concentration and intermittency measured within an odor plume, and how this information is integrated within the lobster?s brain. An additional goal is to develop new neurobiology-based theories in the search for odor sources that can be implemented within human-engineered autonomous underwater vehicles that have the ability to navigate in turbulent chemical plumes.

The broader implications of this work stem from the large potential market for defense and civilian applications of a new generation of electronic noses for tracking chemicals in natural or man-initiated disasters. Through this project, there are also excellent resources and outreach opportunities for integrated education and training of students at the intersection of fluid dynamics, neuroscience, computer engineering and information processing. Outreach will be coordinated through the Center of Innovative Brain Machine Interfaces at the University of Florida and will provide opportunities for undergraduate and graduate research, promote neurotechnology innovations, and foster entrepreneurship activities in order to create potential future start-up companies.

The research will include laboratory experiments of chemical plume mixing and ORN responses to odor encounters by lobsters, theoretical analysis of search optimization, as well as numerical simulations and novel system architecture for electronic noses. This research brings together a multidisciplinary and complementary team of experts, including a fluid dynamicist, a neurobiologist, and an electrical engineer with the very clear goal of understanding and exploiting olfactory scene analysis in turbulent flow. In this new light, neurobiologists will understand new sensing strategies for olfaction, and engineers can improve the quantification of turbulent mixing and replicate these sensory strategies to propose novel bio-inspired sensors capable of quantifying the dispersion of chemical plumes and improve the search for the source.