Topographic patterns of odorant receptor expression in mammals: a comparative study

In a comparative study, molecular probes for various odorant receptor subtypes were employed in in situ hybridization experiments on tissue sections through the nose from different mammalian species. OR37 reactive neurons were found exclusively in the rodent species, where they were clustered in very similar position within the nasal cavities; an OR37-related receptor subtype was not detectable in the rabbit. All other subtypes tested, hybridized across species borders to neurons that were distributed within distinct zones of the olfactory epithelium. Most receptor types were found in the same zone in all species; however, a few subtypes which are expressed in the medial zone in rat were found in the dorsal zone in guinea pig.     

Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit

Axons of olfactory sensory neurons expressing a given odorant receptor converge to a few glomeruli in the olfactory bulb. We have generated mice with unresponsive olfactory sensory neurons by targeted mutagenesis of a cyclic nucleotide-gated channel subunit gene, OCNC1. When these anosmic mice were crossed with mice in which neurons expressing a given odorant receptor can be visualized by coexpression of an axonal marker, the pattern of convergence was affected for one but not another receptor. In a novel paradigm, termed monoallelic deprivation, axons from channel positive or negative neurons that express the same odorant receptor segregate into distinct glomeruli within the same bulb. Thus, the peripheral olfactory projections are in part influenced by mechanisms that depend on neuronal activity.     

Decline and recovery of olfactory receptor expression following unilateral bulbectomy

The effects of unilateral olfactory bulb ablation upon the odorant receptor expression were studied during the degeneration/regeneration process in the olfactory epithelium of adult rats. Using the in situ hybridization approach, we compared the time course of decay and recovery of expression for three different receptor subtypes (OR14, OR5, OR124). The number of neurons expressing receptor subtypes dramatically decreased in the olfactory epithelium on the lesioned side and reached a minimum at day 5 postsurgery. A progressive recovery was then observed from day 5 to day 15 postlesion, when a plateau was reached. Noticeable differences in the recovery level of receptor expression were observed according to the zonal patterning: the recovery level for neurons located in the lateral zone reached 70% of the control side value while the recovery levels in the dorsal and medial zones represented 35% and 53% of this value, respectively. Axotomy experiments suggest that zone-specific differences in receptor reexpression reported after bulbectomy might be related to the trophic influence of the olfactory bulb.     

Expression of olfactory receptors in the cribriform mesenchyme during prenatal development

Olfactory receptors (ORs) are expressed in sensory neurons of the nasal epithelium, where they are supposed to be involved in the recognition of suitable odorous compounds and in the guidance of outgrowing axons towards the appropriate glomeruli in the olfactory bulb. During development, some olfactory receptor subtypes have also been found in non-sensory tissues, including the cribriform mesenchyme between the prospective olfactory epithelium and the developing telencephalon, but it is elusive if this is a typical phenomenon for ORs. Monitoring the onset and time course of expression for several receptor subtypes revealed that 'extraepithelial' expression of ORs occurs very early and transiently, in particular between embryonic stages E10.25 and E14.0. In later stages, a progressive loss of receptor expressing cells was observed. Molecular phenotyping demonstrated that the receptor expressing cells in the cribriform mesenchyme co-express key elements, including Galpha(olf), ACIII and OMP, characteristic for olfactory neurons in the nasal epithelium. Studies on transgenic OMP/GFP-mice showed that 'extraepithelial' OMP/GFP-positive cells are located in close vicinity to axon bundles projecting from the nasal epithelium to the presumptive olfactory bulb. Moreover, these cells are primarily located where axons fasciculate and change direction towards the anterior part of the forebrain.     

Anatomical contributions to odorant sampling and representation in rodents: zoning in on sniffing behavior

Odorant sampling behaviors such as sniffing bring odorant molecules into contact with olfactory receptor neurons (ORNs) to initiate the sensory mechanisms of olfaction. In rodents, inspiratory airflow through the nose is structured and laminar; consequently, the spatial distribution of adsorbed odorant molecules during inspiration is predictable. Physicochemical properties such as water solubility and volatility, collectively called sorptiveness, interact with behaviorally regulable variables such as inspiratory flow rate to determine the pattern of odorant deposition along the inspiratory path. Populations of ORNs expressing the same odorant receptor are distributed in strictly delimited regions along this inspiratory path, enabling different deposition patterns of the same odorant to evoke different patterns of neuronal activation across the olfactory epithelium and in the olfactory bulb. We propose that both odorant sorptive properties and the regulation of sniffing behavior may contribute to rodents' olfactory capacities by this mechanism. In particular, we suggest that the motor regulation of sniffing behavior is substantially utilized for purposes of "zonation" or the direction of odorant molecules to defined intranasal regions and hence toward distinct populations of receptor neurons, pursuant to animals' sensory goals.     

Goα expression in the vomeronasal organ and olfactory bulb of the tammar wallaby

The vomeronasal organ (VNO) detects pheromones via 2 large families of receptors: vomeronasal receptor 1, associated with the protein Giα2, and vomeronasal receptor 2, associated with Goα. We investigated the distribution of Goα in the developing and adult VNO and adult olfactory bulb of a marsupial, the tammar wallaby. Some cells expressed Goα as early as day 5 postpartum, but by day 30, Goα expressing cells were distributed throughout the receptor epithelium of the VNO. In the adult tammar, Goα appeared to be expressed in sensory neurons whose nuclei were mostly basally located in the vomeronasal receptor epithelium. Goα expressing vomeronasal receptor cells led to all areas of the accessory olfactory bulb (AOB). The lack of regionally restricted projection of the vomeronasal receptor cell type 2 in the tammar was similar to the uniform type, with the crucial difference that the uniform type only shows expression of Giα2 and no expression of Goα. The observed Goα staining pattern suggests that the tammar may have a third accessory olfactory type that could be intermediate to the segregated and uniform types already described.     

Small subfamily of olfactory receptor genes: structural features, expression pattern and genomic organization.

Olfactory receptors of the OR37 subfamily are characterized by distinct sequence features and are expressed in neurons segregated in a restricted area of the olfactory epithelium. In the present study, we have characterized the complement of OR37-like genes in the mouse. Five OR37-like genes were identified. They reside within only 60kb of DNA on chromosome 4. About 70kb distant from this cluster, two additional olfactory receptor genes are located, which are members of distinct receptor subfamilies. Phylogenetic analysis demonstrated that the two physically linked receptors are closely related to the OR37 subfamily. Studies of gene expression showed that both genes are also expressed in clustered neuron populations located in the typical OR37 region of the epithelium. These data suggest the involvement of locus-dependent mechanisms for the spatial control of OR gene expression.     

A long-term culture system for olfactory explants with intrinsically fluorescent cell populations

As a prerequisite for exploring the mechanisms which lead to the formation and maintenance of the precise wiring patterns in the olfactory system, organotypic cultures of olfactory tissue from transgenic mice expressing green fluorescent protein (GFP) under control of the olfactory marker protein promotor have been established. Tissue specimen from embryonic stage 14 were explanted and kept in culture for >1 week. Within the explants, numerous GFP-fluorescent olfactory sensory neurons assembled in an epithelial-like manner during this period. Under optimized culture conditions, strongly GFP-positive axons extended from these explants, fasciculated and formed bundles. When co-cultured with explants from the olfactory bulb, distinct axon populations were attracted by the target tissue; the fluorescent axon bundles invaded the bulbular explants and formed conglomerates at distinct spots. Explants from transgenic mice expressing GFP under control of a given olfactory receptor gene (mOR37A) also comprised labeled neurons that extended intensely fluorescent axonal processes, which all seemed to grow in a common fascicle. The results demonstrate that GFP-labeled olfactory sensory neurons differentiate in the established organotypic cultures, which thus appear to be a useful tool to monitor and to manipulate the processes underlying the axonal wiring between the olfactory epithelium and bulb.     

Differential development of odorant receptor expression patterns in the olfactory epithelium: a quantitative analysis in the mouse septal organ

The rodent olfactory epithelium expresses more than 1000 odorant receptors (ORs) with distinct patterns, yet it is unclear how such patterns are established during development. In the current study, we investigated development of the expression patterns of different ORs in the septal organ, a small patch of olfactory epithelium predominantly expressing nine identified ORs. The presumptive septal organ first appears at about embryonic day 16 (E16) and it completely separates from the main olfactory epithelium (MOE) at about postnatal day 7 (P7). Using in situ hybridization, we quantified the densities of the septal organ neurons labeled by specific RNA probes of the nine abundant OR genes from E16 to postnatal 3 months. The results indicate that olfactory sensory neurons (OSNs) expressing different ORs have asynchronous temporal onsets. For instance, MOR256-17 and MOR236-1 cells are present in the septal organ at E16; however, MOR0-2 cells do not appear until P0. In addition, OSNs expressing different ORs show distinct developmental courses and reach their maximum densities at different stages ranging from E16 (e.g. MOR256-17) to 1 month (e.g. MOR256-3 and MOR235-1). Furthermore, early onset does not correlate with high abundance in adult. This study reveals a dynamic composition of the OSNs expressing different ORs in the developing olfactory epithelium.     

Shh‐proteoglycan interactions regulate maturation of olfactory glomerular circuitry

The olfactory system relies on precise circuitry connecting olfactory sensory neurons (OSNs) and appropriate relay and processing neurons of the olfactory bulb (OB). In mammals, the exact correspondence between specific olfactory receptor types and individual glomeruli enables a spatially precise map of glomerular activation that corresponds to distinct odors. However, the mechanisms that govern the establishment and maintenance of the glomerular circuitry are largely unknown. Here we show that high levels of Sonic Hedgehog (Shh) signaling at multiple sites enable refinement and maintenance of olfactory glomerular circuitry. Mice expressing a mutant version of Shh (Shh^Ala/Ala), with impaired binding to proteoglycan co‐receptors, exhibit disproportionately small olfactory bulbs containing fewer glomeruli. Notably, in mutant animals the correspondence between individual glomeruli and specific olfactory receptors is lost, as olfactory sensory neurons expressing different olfactory receptors converge on the same glomeruli. These deficits arise at late stages in post‐natal development and continue into adulthood, indicating impaired pruning of erroneous connections within the olfactory bulb. In addition, mature Shh^Ala/Ala mice exhibit decreased proliferation in the subventricular zone (SVZ), with particular reduction in neurogenesis of calbindin‐expressing periglomerular cells. Thus, Shh interactions with proteoglycan co‐receptors function at multiple locations to regulate neurogenesis and precise olfactory connectivity, thereby promoting functional neuronal circuitry. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1255–1267, 2014     

Rostro-caudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity

The rostro-caudal extent of odorant receptor expression zones in the rat olfactory epithelium was analysed by means of in situ hybridization. Three broad non-overlapping zones were identified that extended along almost the entire anterior-posterior axis; each zone was composed of several separate bands running anterior to posterior throughout the olfactory epithelium. Superimposed onto these broad zones was the expression area of a particular receptor subtype (OR37); it was restricted to a small region of the epithelial sheet with a high density of reactive neurones in the centre and declining numbers towards the periphery of the region. A quantitative evaluation of the reactive cells revealed that, despite their diferent distribution patterns, all receptor subtypes were expressed in an equal number of neurones.     

Conditional ablation of neurones in transgenic mice

Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Heterogeneous Sensory Innervation and Extensive Intrabulbar Connections of Olfactory Necklace Glomeruli

The mammalian nose employs several olfactory subsystems to recognize and transduce diverse chemosensory stimuli. These subsystems differ in their anatomical position within the nasal cavity, their targets in the olfactory forebrain, and the transduction mechanisms they employ. Here we report that they can also differ in the strategies they use for stimulus coding. Necklace glomeruli are the sole main olfactory bulb (MOB) targets of an olfactory sensory neuron (OSN) subpopulation distinguished by its expression of the receptor guanylyl cyclase GC-D and the phosphodiesterase PDE2, and by its chemosensitivity to the natriuretic peptides uroguanylin and guanylin and the gas CO₂. In stark contrast to the homogeneous sensory innervation of canonical MOB glomeruli from OSNs expressing the same odorant receptor (OR), we find that each necklace glomerulus of the mouse receives heterogeneous innervation from at least two distinct sensory neuron populations: one expressing GC-D and PDE2, the other expressing olfactory marker protein. In the main olfactory system it is thought that odor identity is encoded by a combinatorial strategy and represented in the MOB by a pattern of glomerular activation. This combinatorial coding scheme requires functionally homogeneous sensory inputs to individual glomeruli by OSNs expressing the same OR and displaying uniform stimulus selectivity; thus, activity in each glomerulus reflects the stimulation of a single OSN type. The heterogeneous sensory innervation of individual necklace glomeruli by multiple, functionally distinct, OSN subtypes precludes a similar combinatorial coding strategy in this olfactory subsystem.     

Receptor guanylyl cyclases in mammalian olfactory function

The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system.     

Odorant-dependent, spatially restricted induction of c-fos in the olfactory epithelium of the mouse

Volatile odorous chemicals are detected by around a thousand different G protein-coupled odorant receptors in the mouse. We demonstrated that exposure of the behaving mouse to odorant for a few minutes led to induction of the immediate early gene c-fos for several hours in a fraction of the olfactory sensory neurones in the nasal cavity. Associated with this odorant-specific induction event was activation of extracellular-regulated kinase (ERK)1/2 that preceded increased c-fos expression. The distribution of odorant-activated neurones mimicked the scattered and spatially limited distribution of neurones expressing a single odorant receptor gene. A small change in odorant chemical structure caused a zonal shift in the spatial distribution of activated neurones, suggesting that the gene expression change resulted from specific receptor interaction. Repeated exposure to odorant or use of different concentrations did not change the pattern of c-fos induction. These results indicate that odorant-induced c-fos expression can be used to visualize odorant representations in the olfactory epithelium that reflect late cellular events regulated by adequate odorant receptor stimulation.     

Roles of odorant receptors in projecting axons in the mouse olfactory system

In the mouse olfactory epithelium, there are about ten million olfactory sensory neurons, each expressing a single type of odorant receptor out of approximately 1000. Olfactory sensory neurons expressing the same odorant receptor converge their axons to a specific set of glomeruli on the olfactory bulb. How odorant receptors play an instructive role in the projection of axons to the olfactory bulb has been one of the major issues of developmental neurobiology. Recent studies revealed previously overlooked roles of odorant receptor-derived cAMP signals in the axonal projection of olfactory sensory neurons; the levels of cAMP and neuronal activity appear to determine the expression levels of axon guidance/sorting molecules and thereby direct the axonal projection of olfactory sensory neurons. These findings provide new insights as to how peripheral inputs instruct neuronal circuit formation in the mammalian brain.     

Subfamily of Olfactory Receptors Characterized by Unique Structural Features and Expression Patterns

Abstract: The complex chemospecificity of the olfactory system is probably due to the large family of short-looped, heptahelical receptor proteins expressed in neurons widely distributed throughout one of the several zones within the nasal neuroepithelium. In this study, a subfamily of olfactory receptors has been identified that is characterized by distinct structural features as well as a unique expression pattern. Members of this receptor family are found in mammals, such as rodents and opossum, but not in lower vertebrates. All identified subtypes comprise an extended third extracellular loop that exhibits amphiphilic properties and contains numerous charged amino acids in conserved positions. Olfactory sensory neurons expressing these receptor types are segregated in small clusters on the tip of central turbinates, thus representing a novel pattern of expression for olfactory receptors. In mouse, genes encoding the new subfamily of receptors were found to be harbored within a small contiguous segment of genomic DNA. Based on species specificity as well as the unique structural properties and expression pattern, it is conceivable that the novel receptor subfamily may serve a special function in the olfactory system of mammals.     

Axon Guidance Events in the Wiring of the Mammalian Olfactory System

The detection of odorant signals from the environment and the generation of appropriate behavioral outputs in response to these signals rely on the olfactory system. Olfactory sensory neurons (OSNs) of the olfactory epithelium are located in the nasal cavity and project axons that synapse onto dendrites of second-order neurons in the olfactory bulb (OB) that in turn relay the information gathered to higher order regions of the brain. The connections formed are remarkably accurate such that axons of OSNs expressing the same olfactory receptor innervate specific glomeruli within the complex three-dimensional structure that represents the OB. The molecular determinants that control this complex process are beginning to be identified. In this review, we discuss the role of various families of axon guidance cues and of recently characterized families of adhesion molecules in the formation of stereotypic connections in the olfactory system of mice. Cho and Prince contributed equally.