Thomas Bozza, PhD - US grants

Studies of information coding in the vertebrate olfactory system have been hindered by the vast number of possible stimuli and the large number of sensory neuron types. In mice there are perhaps as many as 1,000 different classes of olfactory sensory neurons, that differ by the olfactory receptor gene they express. An outstanding issue is the contribution of these olfactory receptor gene products in determining odorant responsiveness of sensory neurons. They hypothesis is that the olfactory receptor solely determines the functional properties of olfactory sensory neurons. The proposed experiments integrate genetic, molecular and physiological approaches to determine 1) if neurons that express the same olfactory receptor gene respond similarly to odorants and 2) if expression of an olfactory receptor gene is necessary and sufficient to confer and operant response phenotype on individual sensory neurons. A significant advance is that odorant sensitivity will be studied in sensory neurons that are homogenous with respect to the olfactory receptor gene they express.

DESCRIPTION (provided by applicant): The olfactory system is emerging as an attractive model for studying neuronal wiring and information processing in the mammalian brain. This system represents information about molecular stimuli, in part, as spatial patterns of activation in the olfactory bulb of the brain. The molecular basis for these odor representations is being elucidated by studies on the genetic basis of olfactory function. In mammals, olfaction is mediated by a large family of odorant receptors expressed in chemosensory neurons. The current hypothesis is that odorant receptors have two distinct roles in olfactory function: mediating odorant responsiveness and influencing the wiring of axonal projections to the olfactory bulb. This idea raises important questions as to what information is mapped onto the surface of the brain. The objective of these studies is to begin to elucidate the relationship between these two roles. The first aim is to determine whether the distinct functions of ORs (ligand binding and axon guidance) can be separated. This will be done by creating mouse strains with targeted genetic mutations in a defined endogenous odorant receptor locus, assaying the odorant specificity of fluorescently tagged neurons expressing defined receptors, and correlating these data with observed axon pathfinding behavior in vivo. The second aim is to determine whether odorant specificity or receptor sequence correlate with the location of neuronal projections dictated by a set of odorant receptors expressed from a defined genetic locus. Odorant response profiles will be measured, and expressed receptors sequenced from afferents projecting to local regions of the olfactory bulb defined by the projections of genetically tagged neurons. Addressing these issues provides a first step towards understanding how the mammalian nervous system represents chemical information. The combination of gene targeting and optical imaging of neuronal activity provides powerful tools for investigating information coding in the mammalian olfactory system. The general approach of targeting expression of exogenous proteins (e.g., genetically encoded optical indicators) to defined neuronal populations could provide new methods to monitor neuronal activity in intact neuronal circuits.

[unreadable] DESCRIPTION (provided by applicant): The goal is to explore genetically-based methods for mapping activity in defined populations of neurons in the mouse olfactory system. The approach makes use of a genetically-encoded indicator for neuronal activity (synapto-pHluorin) in the intact mouse nervous system, and novel fluorescent proteins that can be spectrally separated from the indicator. The approach will be used to address fundamental questions of sensory processing by olfactory circuits. Specific Aim 1 is to test the physiological relevance of odorant receptor ligands by establishing an in vivo assay to image glomerular activity. Gene targeting will be used to label the glomeruli formed by olfactory sensory neurons that express defined odorant receptors. Responses from targeted glomeruli will be compared with responses from other glomeruli in the dorsal bulb to determine relative affinities for defined ligands. Previously identified odorants will be used and new chemical will be screened to identify high-affinity ligands in vivo. The experiments will provide a necessary context in which to place functional OR-ligand data from a variety of assays, and will pave the way for future experiments that aim to manipulate odorant receptor expression and olfactory function in mice. Specific Aim 2 is to target synaptopHluorin to mitral cells of the olfactory bulb to study lateral inhibition in the glomerular layer in vivo. This will permit, for the first time, selective imaging of synaptic release from mitral cell primary dendrites in vivo. The approach addresses a fundamentally important question regarding olfactory processing by olfactory bulb circuits that can not be addressed using currently available methods. These studies explore a new technique to set up experimental systems for studying neuronal function in vivo. Further development of these technologies promise to provide a novel window into brain function by allowing optical imaging of activity in genetically-defined neuronal populations that were previously difficult to isolate functionally in intact nervous tissue. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Studies of sensory systems have contributed greatly to our understanding of normal brain function. The mouse olfactory system is a genetically tractable experimental model in which to study how sensory information is encoded by neuronal circuits. In this system, 1000 populations of olfactory sensory neurons project axons to the brain where they sort out by identity and form an array of glomeruli on the surface of the olfactory bulb. The spatial arrangement of glomeruli is thought to play a role in odor coding. We have recently discovered that the olfactory epithelium contains phenotypically distinct types of sensory neurons, and that these cell types play a role in mapping Class I and Class II odorant receptors to domains in the olfactory bulb. Generally, our hypothesis is that these sensory neuron types play a key role in organizing the glomerular array. The first two Specific Aims seek to define the identity of these cell types. The third Specific Aim seeks to examine the functional properties of one Class of these sensory neurons. These experiments will allow us to test and expand our cell-type hypothesis and to address the mechanisms by which neurons make specific connections in the olfactory system. PUBLIC HEALTH RELEVANCE: A fundamental question in neuroscience is how the brain processes information under normal and pathological conditions. The olfactory system is an excellent model in which to study how chemical information is represented in genetically tractable neuronal circuits. The experiments described in this proposal examine a new model for understanding how neurons in the peripheral olfactory system organize circuits in the olfactory bulb. We hope that the insights from this work will lead to a better understanding of how neurons self-assemble into functional neuronal networks.

DESCRIPTION (provided by applicant): Information about chemical structure is encoded by 1000 populations of olfactory sensory neurons, each of which projects to defined glomeruli in the olfactory bulb. Each glomerulus and associated olfactory bulb neurons are thought to be functional units in olfactory processing. Understanding the functional connections among neurons in the olfactory bulb is critical to understanding how the olfactory system works. While traditional anatomical techniques can reveal the structure of neuronal circuits, determining which nerve cells actually communicate with one another in a complex circuit is more challenging. The goal of this proposal is to develop an optogenetic approach to map functional circuits in the mouse olfactory bulb. Using gene targeting, we are expressing a light-activated channel in genetically-defined populations of neurons, allowing us to selectively activate glomeruli using light. Using this photostimulation-based approach, we are mapping the receptive fields of olfactory bulb neurons and determining whether neurons associated with a specific glomerulus are functionally similar. The research described here will allow us to address fundamental questions about sensory processing that are not possible to address using available techniques. The methods that we develop will also be useful map circuits in other parts of the mammalian brain. PUBLIC HEALTH RELEVANCE: We propose to apply novel optogenetic technologies to map neuronal circuits in the mammalian olfactory system using light. The research described here will allow us to address fundamental questions about sensory processing that were previously not possible to address using traditional techniques.

DESCRIPTION (provided by applicant): Human olfaction provides a unique window into understanding normal brain function. Odorous chemicals in the environment are detected by a large family of canonical odorant receptors, and a much smaller family of Trace Amine-Associated Receptors (TAARs). There are only 6 intact TAARs in humans and the TAARs are conserved in many vertebrate species, suggesting a common, important function. However, very little is known about the functional properties of human TAARs due to the limitations of current methods to express odorant receptors in cell culture systems. Here we propose to circumvent these issues by functionally expressing all of the human TAARs in mouse olfactory sensory neurons in vivo. Specific Aim 1 is to generate a set of gene-targeted mouse strains in which each of the human TAARs is expressed in a defined population of fluorescently labeled olfactory sensory neurons. Specific Aim 2 is to functionally characterize human TAARs by recording electrophysiological responses from TAAR-expressing olfactory sensory neurons in these mice. These studies should allow us to identify for the first time ligands for the human TAARs, and should shed light on how the TAAR genes contribute to human olfaction.

DESCRIPTION (provided by applicant): The olfactory system provides a unique window into how genetically defined neuronal circuits give rise to normal brain function. Volatile odorants are detected by a large family of olfactory receptor genes, each of which is represented by a specific functional input to the brain. Progress towards understanding mammalian olfaction has been hindered by difficulties in identifying main olfactory receptor genes that contribute significantly to odor perception. This proposal addresses how individual members of a small main olfactory receptor family, the Trace Amine-Associated Receptors (TAARs), contribute to odor perception in mammals. The TAARs are conserved in humans, mice and other vertebrates suggesting an important function. We propose a combination of genetics, physiology and behavior to test the hypothesis that the TAARs are the most sensitive receptors in a distinct pathway that mediates innate aversion to amines-a biologically relevant class of odorants that are produced by decay and microbial action. Specific Aim 1 is to determine whether the TAARs contribute significantly to setting behavioral detection thresholds to amines. Specific Aim 2 is to determine whether selective activation of TAAR inputs can drive aversive behaviors. Specific Aim 3 is to determine whether remapping the location of TAAR inputs to the olfactory bulb alters odor perception. Achieving these aims will advance our understanding of how this novel chemosensory gene family contributes to olfaction, and how the organization of mammalian olfactory circuits influences odor perception in mammals, including humans.

DESCRIPTION (provided by applicant): The sense of smell in humans is pivotal for stimulating appetite, guiding food selection, avoiding spoiled foods and noxious chemicals, and enhancing overall quality of life. Disorders in the sense of smell are commonly observed in Alzheimer's disease and Parkinson's disease, with early accumulation of neuropathological lesions in the peripheral olfactory system including sensory neurons of the nasal mucosa and their projections to the olfactory bulb. Despite the critical contributions of these structures to olfactory perceptual processing, and their widespread involvement in neurodegenerative disorders, we have only a rudimentary understanding of the molecular and cellular components of the human peripheral olfactory system. Moreover, it remains unclear whether the wealth of information available about olfaction in model organisms applies to the anatomical, physiological, and functional properties of the human olfactory system. In research proposed here, we will leverage our complementary strengths in mouse olfactory genetics (Dr. Bozza) and human olfactory neurobiology (Dr. Gottfried) to comprehensively characterize the human peripheral olfactory system with a research breadth and specificity not previously attempted. This work will exploit advanced next-generation sequencing, novel trans vivo gene targeting, immunohistochemistry, electrophysiology, in vivo calcium imaging, and access to human biopsy and post-mortem tissue samples, to study the expression, function, and topographical mapping of human odorant receptor genes. Specific experiments will (1) measure the expression and determine the structure of chemosensory genes including olfactory receptors (ORs) and trace amine- associated receptors (TAARs) in the human olfactory epithelium, (2) characterize the odor response profiles of human odorant receptors when expressed in mouse olfactory sensory neurons, and (3) define the spatial distribution of receptor-specific projections of human olfactory sensory neurons from the epithelium to the olfactory bulb. Together these studies will advance our understanding of the functional organization of the human olfactory system, and will set the stage for clarifying how individual chemoreceptor genes influence odor perception. Finally, by defining the functional organization of olfactory pathways in neurologically intact individuals, this work will serve as a valuable starting point for investigating the impact of neurodegenerative disease on olfactory gene expression, circuit anatomy and sensory function.

Summary (Project 1: The peripheral representation of odor space) Progress towards an understanding of olfactory coding has been hampered by long-standing hurdles created by the nature of the stimulus and the complexity of the underlying sensory biology. At the same time, a description of olfactory coding in mammals would provide a unique window into how multidimensional stimuli are represented by the mammalian brain. Olfactory systems use large families of odorant receptors to detect a vast number of chemical stimuli. An important challenge that must be overcome to better understand olfaction is to establish a comprehensive description of what features of olfactory stimuli are represented by the system. Doing so requires that we overcome previously insurmountable technical challenges in identifying the stimulus specificity of a large number of receptors to a large number of odorants, and that we generate a theoretical framework for quantifying and exploring the multidimensional ?space? of odors and receptors. Here, we propose an interdisciplinary effort to comprehensively characterize the odorant response properties of a large number of odorant receptors in vivo, and to use this information to explicitly and rigorously test novel models of odor coding. This project exploits the one-to-one correspondence between odorant receptors and glomeruli in the olfactory bulb of mice. Aim 1 will characterize the sensitivities of a large number of receptors (glomeruli) in awake, intact animals using functional imaging. Aim 2 will map these glomerular responses to specific receptors using emerging spatial transcriptomics methods. Aim 3 will use a powerful genomics-based assay to identify the highest affinity receptors for a large set of individual odors. Aim 4 will test a novel theoretical framework for understanding how odor features are represented. This large-scale in vivo multidisciplinary approach will provide long-sought data and analytical tools to rigorously explore potential models of odor coding.

Project Summary Odorant receptor (OR) genes exhibit an unusual form of random monoallelic expression, commonly referred to as monogenic or singular expression. In the mouse, olfactory sensory neurons express one allele of one OR gene from a large repertoire of >1,000 genes scattered throughout the genome. Current models for OR choice involve epigenetic mechanisms orchestrated by a genome-wide network of distributed enhancers. The Trace Amine Associated Receptors (TAARs) constitute a small olfactory receptor gene family that is also subject to singular expression. However, the epigenetic properties of TAARs differ from ORs in a number of key ways, calling into question whether both share a common mechanism of singular expression. The goal of this proposal is to characterize the mechanisms of singular expression of TAAR genes. We have identified the local enhancers that govern TAAR gene expression and will exploit this knowledge to test hypotheses about how these regulator elements orchestrate choice. The Aims of the proposal are to 1) identify the sequence motifs that are required for enhancer function, 2) elucidate the specificity and spatial extent of enhancer function, and 3) characterize the three-dimensional interaction landscape of the TAAR gene cluster. These experiments exploit unique aspects of the TAAR cluster to test and extend current models of monoallelic expression?a phenomenon that plays a critical role in myriad biological processes including dosage compensation, cell-type specification, phenotypic variation and disease pathogenesis.