Charles A. Greer - US grants

Description (provided by applicant): Throughout the CNS diversity in the location and laminar organization of subpopulations of neurons and their synaptic circuits underlies functional specificity, as is evident in the visual pathways mediating low versus high resolution images. The degree of circuit specification in the olfactory bulb remains controversial. Functional analyses suggest a topography of odor-induced activity, but this has focused on the glomeruli where sensory neuron axons expressing the same odor receptor converge and terminate;conflicting data has been reported on the odor-specificity of mitral cells and the deeper radial and horizontal circuits in the bulb. Our proposal has two primary goals. First, there is little information on the innervation of individual glomeruli by subsets of mitral cells. Estimates suggest that each glomerulus is innervated by a subset of 25 mitral cells, but given the heterogeneity in the volume of individual glomeruli, this is likely a misleading generalization. Whether the mitral cells innervating single glomeruli share a common birth date, migratory timeframe or molecular phenotype are also not known. Second, while several studies have examined deafferentation in the bulb, we know comparatively little about trophic mechanisms influencing the fine structural organization of dendrites and their synaptic organization. In brief, our overarching goal is a better understanding of the embryonic and perinatal events underlying the primary cellular organization in the olfactory bulb. To achieve this goal our specific aims are summarized as: 1) Determine the principles underlying targeting of glomeruli by subsets of mitral cells;2) Establish the relationship between birth date, apoptotic cell death, and topography of olfactory bulb mitral cells;and 3) Test the hypothesis that trophic mechanisms, in particular BDNF, contribute to the differentiation and maturation of mitral cells. This work provides insight into determinants of early development relevant to diseases such as Kallman's syndrome and autism;the principles derived are also likely relevant to broad categories of anomalous cortical development and syndromes such as Fragile-X. PUBLIC HEALTH RELEVANCE: the mechanisms that regulate the development of organization and functional properties of the nervous system remain largely unknown. Developmental disorders such as Kallmans syndrome, autism and cortical dysplasia all represent likely aberrations in the development and differentiation of the central nervous system. The studies proposed here address questions related to the genesis of new neurons and the mechanisms that may influence their distribution, organization and connections in the brain. As such, these new studies are likely to be important in understanding and developing interventional strategies for syndromes such as those noted above, among others.

IBN: 9723978 PI: Greer The chemical senses include not only smell and taste, but some other chemical senses (the 'burning' or 'coolness' of spices such as pepper or mint, and the 'tingle' of carbonation). The International Symposium on Smell and Taste (ISOT) meets only once every four years, for nearly a week, bringing together researchers from all over the world to discuss current research results and plan future projects and collaborations. Partial support for this conference will help defray travel expenses for some of the invited speakers, and for an outreach program to involve the local San Diego area schools in workshops that will lead to more understanding chemosensory science. The symposium proceedings will become a published volume. Given past history, some aspects of this conference will be important to fields beyond chemosensory work alone, and these symposia will have an impact on the field for years to come.

Injury to axons, such as in the spinal cord or the central nervous system in generate initiates a cascade of complex events, the net effect of which is a functionally limiting neurological deficit. The magnitude of the deficit is determined not only by the extent of the initial injury but also, by variation within the inflammatory, degenerative and attempted regenerative events that follow the initial injury. In order to devise strategies that can improve the likelihood of a more favorable neurological outcome,.we must first understand the fundamental cellular mechanisms influence degenerative and regenerative events within the CNS. One potential mechanism that may improve the potential for axon regeneration is the presence of a favorable substrate. Recent reports suggest the hypothesis that specific subpopulations of glial cells, in particular the ensheathing cells of the olfactory nerve, may provide an optimal substrate. The ultimate goal of the projects described in this program is to test the hypothesis that transplants of specific glial suspensions can promote recovery in the injured spinal cord. This proposal seeks to develop a body of knowledge that will help us to identify basic mechanisms that influence regenerations in axons following injury. Building on that knowledge, we hope to identify specific mechanisms that may be susceptible to intervention strategies that would improve outcome following spinal cord injury. The program is divided into 3 major areas: 1) A Core facility providing support for electron microscopy and administration; 2) Studies designed to test specific hypotheses about the mechanisms underlying axonal responses to injury and growth properties and the degree to which these can be modified by different glial substrates; and 3) Studies on the degree to which injury-induced demyelination and axon transection can be overcome in the spinal cord with cell transplants. Pursuing these studies will fill critical gaps in our current understanding of degenerative/regenerative processes occurring within the central nervous system. This knowledge is essential for the development of treatment strategies directed not only at the spinal cord, but the central nervous system in general.

Multiple lines of evidence support the notion that the axons from olfactory receptor neurons expressing the same odor receptor (OR) largely converge onto only 2 or a few glomeruli in the olfactory bulb. To accomplish this there is an extraordinary reorganization of axons that occurs between the olfactory epithelium and the olfactory bulb. The pathfinding exhibited by axons during reorganization as well as their targeting of specific glomeruli appears to be influenced, at least in part, by the unique OR expressed by any single olfactory receptor neuron. Thus, ORS constitute a unique set of molecular tags/determinants that provide an exciting opportunity to study further the organization of the glomerulus and its role in odor processing. . Moreover, because of recent advances in protein tracking technology, the opportunity is presented to investigate the spatio-temporal proteins of ORS in olfactory receptor neurons in order to understand if their intracellular distribution is consistent with a determinant role in axon guidance. Three specific aims are proposed: 1) Does one specific gene, coding an OR sequence, uniquely define all of the afferent axons projecting to one glomerulus? Using Lac-Z constructs, the synaptic organization of axons expressing specific ORS will be studied. 2) What is the distribution of mitral cells whose apical dendrites innervate a specific glomerulus? While some evidence suggests that the somata of these mitral cells should be contiguous, the hypothesis has not been addressed. Green Fluorescent Protein constructs can be employed to identify the afferent axons converging on specific glomeruli. Identified glomeruli will be infected with fluorescent or light-dense tracers to map the distribution of mitral cells whose dendrites are innervating the identified glomerulus. 3) What is the intracellular distribution of OR protein? While the evidence favoring multiple roles for ORS is compelling, it is not evident in which intracellular compartments OR protein is found. Using Green Fluorescent Protein coding sequence fusions with OR coding sequences, the spatio- temporal characteristics of OR protein products will be assessed to address this significant gap in our knowledge. Collectively, these studies will shed new light on the organization of primary olfactory projections, the role of the glomerulus in odor processing, the topography of higher order projections emerging from glomeruli and finally, the mechanisms that may govern the intracellular distribution of OR proteins. These results may be significant for people suffering from anosmia and hyposmia, Kalman's Syndrome and related diseases of axon guidance.

DESCRIPTION (provided by applicant): The mammalian olfactory system is increasingly recognized as an attractive model system for studying the formation of neural circuits during development. An olfactory sensory neuron (OSN) expresses one the of approximately 1000 intact odorant receptor (OR) genes in the mouse genome. Cell bodies of OSNs expressing a given OR gene are dispersed in one of four zones within the olfactory epithelium, and their axons converge onto a few of the approximately 1800 glomeruli in the olfactory bulb of the mouse. A mapping problem is thus posed: 1000 populations of OSNs, each expressing a distinct OR, must be sorted onto approximately 1800 glomeruli. What defines the site in the bulb to which axons of OSNs expressing a given OR or responding to given odorants converge? Progress in our understanding of the parameters defining glomerular position has been slow. No systematic studies of the glomerular array have been undertaken, and the literature is fragmented across species, odorants, ORs and glomeruli. A productive approach to study glomerular convergence has been targeted mutagenesis of OR genes to express axonal markers in OSNs expressing a given OR gene. Such experiments have revealed that the OR is a critical determinant of axonal convergence to glomeruli. Here we propose a coordinated, systematic approach focusing on the mouse and based on meaningful subsets of ORs and glomeruli. The approach is an integrated combination of molecular biology and genetics, physiology, and anatomy. Our hypothesis is that parameters defining a neighborhood in the glomerular array of the mouse olfactory bulb are: OR sequence features, odorant responsiveness and onset and zone of OR expression. Specific Aim 1: How are neighboring glomeruli defined in terms of odorant receptor sequence and odorant responsiveness? Specific Aim 2: What is the glomerular organization of two families of related OR genes at a known genomic locus? Specific Aim 3: How are time and zone of OR expression related to glomerular position?

21+ years old; 5-BrdU; 5-Bromo-2'-deoxyuridine; 5-Bromodeoxyuridine; 5-Bromouracil deoxyriboside; 5-Bromouracil-2-deoxyriboside; 5-Budr; Address; Adult; Affect; Afferent Neurons; Age; Aged 65 and Over; Aging; Ammon Horn; Auditory system; Axon; BUdR; Behavior; Brain; BrdU; Bromodeoxyuridine; Bromodeoxyuridine (BUDR); Bromouracil Deoxyriboside; Broxuridine; Cell Death; Cells; Characteristics; Chromosome Pairing; Cognitive Discrimination; Computer information processing; Connector Neuron; Consensus; Cornu Ammonis; Cortex, Olfactory; Diet Habits; Discrimination; Discrimination (Psychology); Dysfunction; Elderly; Elderly, over 65; Encephalon; Encephalons; Epithelium, Olfactory; Feedback; Functional disorder; GFP; Generations; Green Fluorescent Proteins; Habits, Dietary; Hippocampus; Hippocampus (Brain); Human, Adult; In Situ Nick-End Labeling; Intercalary Neuron; Intercalated Neurons; Interneurons; Internuncial Cell; Internuncial Neuron; Knockout Mice; Label; Lateral; Lead; Light; MT-bound tau; Mammals, Mice; Measures; Mediating; Mice; Mice, Knock-out; Mice, Knockout; Mice, Mutant Strains; Modeling; Molecular; Murine; Mus; Mutant Strains Mice; NRVS-SYS; Nature; Nerve Cells; Nerve Unit; Nervous System; Nervous System, Brain; Nervous system structure; Neural Cell; Neural Growth; Neural Transmission; Neurocyte; Neurologic Body System; Neurologic Organ System; Neuronal Growth; Neurons; Neurons, Afferent; Neurons, Sensory; Null Mouse; Numbers; Nutritional; Odor Receptor Protein; Odorant Receptors; Olfaction; Olfactions; Olfactory Bulb; Olfactory Cortex; Olfactory Epithelium; Olfactory Pathways; Olfactory Receptor Proteins; Olfactory tract; PSA-NCAM; Pathway interactions; Pb element; Phenotype; Photoradiation; Physiopathology; Process; Processing, Information; Proteins; Racial Segregation; Receptor Proteins, Odorant; Receptors, Odorant; Reporting; Senescence; Sensory; Sensory Cell Afferent Neuron; Series; Sight; Smell; Smell Perception; Specificity; Staining method; Stainings; Stains; Stream; Structure of olfactory bulb; Study models; Synapses; Synapsis; Synapsis, Chromosomal; Synaptic; Synaptic Transmission; System; System, LOINC Axis 4; TUNEL; Testing; Tubulin; Uridine, 5-bromo-2'-deoxy-; Vision; Work; adult human (21+); advanced age; age dependent; age effect; age related; aging effect; density; eating habit; elders; gene product; geriatric; heavy metal Pb; heavy metal lead; hippocampal; late life; later life; microtubule associated protein tau; microtubule bound tau; microtubule-associated protein tau; microtubule-bound tau; mouse mutant; necrocytosis; neocortical; nervous system development; neural circuit; neural circuitry; neuroblast; neurogenesis; neuronal; older adult; older person; olfactory bulb; pathophysiology; pathway; polysialyl NCAM; polysialyl neural cell adhesion molecule; postnatal; segregation; senescent; senior citizen; tau; tau Proteins; tau factor; terminal nick end labeling

DESCRIPTION (provided by applicant): The perception of odorous molecules begins in the olfactory epithelium when odorant ligands bind to molecular receptors expressed on the cilia of the olfactory sensory neurons (OSNs). Buck and Axel (1991) were the first to describe the large family of genes coding for the odorant receptors, now known to number ~1,200 in mice. An OSN expresses only 1 odorant receptor. OSNs expressing the same receptor do not cluster together but rather are broadly distributed across the epithelium. Thus the olfactory epitheium is a complex mosaic of neurons each of which expresses only 1 of 1,200 possible odorant receptors. As their axons exit the epithelium they initially fasciculate with nearest neighbors, no necessarily with axons from other neurons expressing the same odorant receptor. However, as they progress over the surface of the olfactory bulb and a point of glomerular convergence, the axons undergo a profound topographical reorganization such that all of the axons coming from neurons expressing the same odorant receptor converge into only 2/3 glomeruli/olfactory bulb. This process of reorganization of axons and convergence into specific glomeruli poses a significant wiring problem, perhaps the most complex wiring problem found among sensory systems. It is widely accepted that the odorant receptors themselves contribute to the convergence of homotypic axons but the process of fasciculation and axon behavior remains poorly understood. Despite a concerted effort to identify the molecular substrates of sensory axon growth, coalescence and targeting, we remain woefully ignorant of the most fundamental aspects of OSN axon organization: When does the initial fasciculation of axons begin, the processes of defasciculation and reorganization? When do homotypic axons expressing the same odorant receptor show evidence of irreversible adhesion? What is the relationship of individual axons to the olfactory ensheathing cells along the course of the olfactory nerve and olfactory nerve layer? How are growth cones distributed and organized in the fascicles both during early development when the pathway is established and in the adult during ongoing axogenesis? Where and when is the odorant receptor mRNA expressed in axons? Does local axonal translation of mRNA occur, and if so under what conditions and where? To begin addressing these significant gaps in our knowledge we are proposing 3 specific aims: Aim 1 - Test the hypothesis that adhesion of homotypic axons does not occur until they are proximal to the site of glomerular convergence; Aim 2 - Test the hypothesis that the growth cones of olfactory sensory neuron axons are not homogeneously distributed within fascicles; and Aim 3 - Test the hypotheses that odorant receptor mRNAs and the translational components are locally compartmentalized in the olfactory nerve/sensory axons.

Project Summary ? Abstract The perception of odors begins in the olfactory epithelium when odorant ligands bind to molecular receptors expressed on the cilia of the olfactory sensory neurons, each of which expresses only 1 of ~1200 candidate receptors. As the sensory neuron axons exit the epithelium they progress over the surface of the olfactory bulb and all of the axons coming from neurons expressing the same odorant receptor converge into only 2/3 glomeruli/olfactory bulb. However, the convergence primary afferent axons within the glomerulus and the discrete local circuitry modulating afferent input remains uncertain Despite a long history of interest, we remain woefully ignorant of the most fundamental features of cellular and synaptic organization within glomeruli: What is the distribution of the dendritic processes of projection neurons versus interneurons within the glomerulus? What is the 3-dimensional topography of synapses along dendrites and in the core versus the periphery of the glomerulus? What is the nature and composition of non-synaptic interactions among dendrites? With the recent development of Serial Block-Face Scanning Electron Microscopy (sbSEM) the ability to serially reconstruct glomeruli at the ultrastructural level, to establish the glomerular connectome, these and other questions are within our grasp. To begin addressing the utility of sbSEM in understanding olfactory bulb circuitry we begin with with the following: 1) The hypothesis that the dendrodendritic synapses between glomerular interneurons and projection neurons are reciprocal. The dendritic Gray Type I and II synapses in glomeruli appear unipolar and isolated. They are presumed to be reciprocal based on physiology, but if there is reciprocity, it is not known if the ratio is 1:1 or unequal. Moreover, it is not clear if they mediate self- or lateral inhibition. 2) The hypothesis that axodendritic (excitatory) and dendrodendritic (inhibitory) synapses differentially localize to the distal versus the proximal dendritic segments of projection neurons. Gate-keeping inhibitory synapses are often found at dendritic branch points, but whether that holds true in glomeruli must be empirically determined using comprehensive 3D dendritic reconstructions. 3) The hypothesis that single OSN axon establish divergent synaptic connections with dendrites from different neurons. Does a single axon repeatedly contact the same dendrite, or is it divergent? While the question seems simple, the answer will provide new insight into the roles of feed- forward and feed-back processing. 4) The location(s) of presynaptic inhibition on OSN axons. Electrophysiological analyses support presynaptic inhibition by showing, for example, that GABAB agonists decrease Ca++ influx into the axon terminals. However, the site of synaptic apposition is not known. Using sbSEM to address these questions will resolve fundamental controversies regarding processing of odors within the glomerulus and provide the foundation for further studies of development and plasticity.  

Project Summary ? Abstract The perception of odors begins in the olfactory epithelium when odorant ligands bind on olfactory sensory neurons, each of which expresses only 1 of ~1200 candidate receptors. Axons coming from neurons expressing the same odorant receptor converge into only 2/3 glomeruli/olfactory bulb where they synapse onto projection neurons, each of which innervates a single glomerulus. Deep to the glomeruli, within the external plexiform layer (EPL), odor coding is tuned by local synaptic circuits. Deciphering connectivity with the EPL is the first step toward understanding the mechanisms of central odor processing. Several hypotheses of local processing within the EPL have been proposed and its synaptic organization is often presented as canonical. However, we lack a fundamental understanding of the how the diversity in interneuron, granule cell (GC), structural, molecular, and topographical organization affects EPL local circuits, which is an impediment to interpreting functional studies. Here we propose a series of testable hypotheses and experiments on factors that may influence the organization of GCs and their local synaptic circuits. These studies build on our prior developmental and ultrastructural work as well as a systematic review of the current literature and the recognition that fundamental features of EPL organization and connectivity have been presumed, but not empirically tested. First, we propose to address the hypotheses that the clonal history, timing and order of GC neurogenesis are determinants of their organization/distribution in the olfactory bulb. Using multiple strategies to track neurogenesis, cell lineage/fate we will assess the spiny dendritic arbors of GCs beginning in the embryo and continuing at regular intervals to those generated up 200 days of age. In addition, and not among prior studies, we propose to carefully assess the organization of the GC basal dendrites which are the primary recipients of incoming centrifugal modulation (Rothermei and Wachowiak, 2014; Kapoor et al., 2016; de Almeida et al., 2015). Second, we propose to test the hypothesis that the synaptology and molecular features of the dendrodendritic synapses in the EPL vary as a function of age. Presently, little is known of the structural features that regulate dendrodendritic synapses or how the molecular properties of the mitral to GC and the reciprocal GC to mitral cell synapses may differ. The analyses will address that fundamental problem and provide a sound foundation for the interpretation of functional analyses of odor processing. In addition, we will address the spatial distribution of synaptic appositions along 2o dendrites and the degree to which the features of the synaptic specialization change with age. Because the questions we propose to address are important throughout the nervous system, we anticipate that the results will have broad implications for understanding targeting, laminar specificity, and synaptic connectivity of neurons throughout the brain.