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.     

Distribution of GluR1 is altered in the olfactory bulb following neonatal naris occlusion

The olfactory system is well suited for studies of glutamate receptor plasticity. The sensory neurons are glutamatergic, and they turn over throughout life, and the olfactory bulb neurons that process their inputs express many of the known glutamate receptor subunits. Neonatal naris occlusion alters olfactory bulb development and the expression of certain neuroactive substances and receptors, at least in part due to loss of the sensory inputs. We therefore postulated that neonatal naris occlusion might alter glutamate receptor expression during postnatal development. Single nares of newborn mice were occluded on postnatal days 1-2, and the distribution of glutamate receptor subunits was evaluated using immunoperoxidase methods. Light microscopic examination on postnatal day 6 failed to reveal adult-like staining of neuronal cell bodies in the olfactory bulbs. By day 12, cell bodies that were immunoreactive (-IR) for the GluR1 subunit were visible in the external plexiform layer (EPL) of both sides. By day 18, many of the GluR1-IR cell bodies could be identified as cell types that had previously been reported to express homomeric GluR1 receptors. Analysis of single, mid-dorsal sections from 18-25-day-old mice showed that the medial EPL of the occluded side had a significantly lower density of these cell bodies. The GluR1 staining of the adjacent mitral cell layer (MCL) was also heavier on the occluded side, but no gross differences in staining for other glutamate receptor subunits were observed. Neonatal naris occlusion therefore appears to provide a new model for studying expression of GluR1 receptors during the development of a discrete population of olfactory bulb neurons.     

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.     

Olfactory axon pathfinding: who is the pied piper?

Mammalian olfactory sensory neurons that express a particular odorant receptor (OR) project axons to the same few glomeruli in the olfactory bulb. In this issue of Neuron, Vassalli et al. use OR minigenes that coexpress histochemical markers and show that the determinants in the sensory neurons required to generate the stereotyped olfactory bulb map are the same as those needed for appropriate expression of the OR.     

Adenovirus-mediated gene transfer in olfactory neurons in vivo

We used recombinant adenoviruses as a means of expressing exogenous genes in olfactory neurons in vivo. A replication incompetent adenovirus (type 5, Ad5) carrying the reporter gene lacZ, which codes for the enzyme β-galactosidase (β-Gal), was applied in solution to the olfactory epithelia of rats. The expression of lacZ was controlled by the cytomegalovirus immediate-early promoter/enhancer. β-Gal expression was observed 1 day postinfection and was maximal at 3–10 days, although it could be detected for at least 21 days postinfection. Expression patterns were heterogeneous, ranging from a few percent to over 25% of the cells in different regions of both turbinate and septal epithelium. Staining was stronger in the olfactory versus respiratory epithelia. In olfactory epithelium staining was almost entirely restricted to olfactory neurons. β-Gal staining was also observed in the olfactory axons so that nerve bundles could be traced to their targets in the glomerular layer of the olfactory bulb. Intense staining of some glomeruli was evident as long as 21 days postinfection. There was no evidence of cell loss or tissue damage due to viral infection. These results demonstrate that it is possible to use recombinant Ad5 for expressing foreign genes in olfactory neurons. This technique could be used in olfactory neurons to increase expression levels of olfactory specific genes, including the odor receptor, putative guidance and growth molecules, or elements of the transduction cascade, in order to elucidate their biological functions in vivo. © 1996 John Wiley & Sons, Inc.     

Olfactory marker protein during ontogeny: Immunohistochemical localization

Specific immunohistochemical staining for the olfactory marker protein (OMP) is first demonstrated in rat olfactory receptor neurons at embryonic day 18, at which age no OMP can be seen in the olfactory bulb or vomeronasal epithelium. At 21 days OMP staining in the olfactory epithelium is more extensive and is evident in the fibrous and glomerular layers of the bulb as well. Staining intensity increases progressively until the full adult pattern is seen by 1 month postnatally. In the vomeronasal organ, staining is not observed until the fourth postnatal day and, even then, only with higher antiserum concentrations. In mice, very similar results are obtained, except for a much earlier appearance of OMP, on embryonic day 14. Olfactory epithelium from 12- and 13-day rat embryos maintained in organ culture for up to 2 weeks did not exhibit OMP staining, nor did several neural or nonneural tissues from adult animals. The temporal and causal interrelationships between OMP and other indicators of olfactory receptor cell maturation are considered.     

BIG-2 mediates olfactory axon convergence to target glomeruli

Olfactory sensory neurons expressing a given odorant receptor converge axons onto a few topographically fixed glomeruli in the olfactory bulb, leading to establishment of the odor map. Here, we report that BIG-2/contactin-4, an axonal glycoprotein belonging to the immunoglobulin superfamily, is expressed in a subpopulation of mouse olfactory sensory neurons. A mosaic pattern of glomerular arrangement is observed with strongly BIG-2-positive, weakly positive, and negative axon terminals in the olfactory bulb, which is overlapping but not identical with those of Kirrel2 and ephrin-A5. There is a close correlation between the BIG-2 expression level and the odorant receptor choice in individual sensory neurons. In BIG-2-deficient mice, olfactory sensory neurons expressing a given odorant receptor frequently innervate multiple glomeruli at ectopic locations. These results suggest that BIG-2 is one of the axon guidance molecules crucial for the formation and maintenance of functional odor map in the olfactory bulb.     

Information processing in mammalian olfactory system

In recent years, considerable progress has been made in understanding how the olfactory system uses neural space to encode sensory information. In this review, we focus on recent studies aimed at understanding the organizational strategies used by the mammalian olfactory system to encode information. The odorant receptor gene family is discussed in the context of its genomic organization as well as the specificity of olfactory sensory neurons. These data have important consequences for the mechanisms of odorant receptor gene choice by a given sensory neuron. Division of the olfactory epithelium into zones that express different sets of odorant receptors is the first level of input organization. The topographical relationship between periphery and olfactory bulb represents a further level of processing of information and results in the formation of a highly organized spatial map of information in the olfactory bulb. There, local circuitry refines the sensory input through various lateral interactions. Finally, the factors that may drive the development of such a spatial map are discussed. The onset of expression and the establishment of the zonal organization of odorant receptor genes in the epithelium are not dependent upon the presence of the olfactory bulb, suggesting that the functional identity of olfactory sensory neurons is determined independently of target selection. © 1996 John Wiley & Sons, Inc.     

Functional correlates of degeneration and renewal of cilia and knobs of olfactory receptor neurons in the frog

Changes in surface structures of the olfactory epithelium, olfactorynerve and olfactory nerve layer in the olfactory bulb followingolfactory nerve section were studied, by scanning electron microscopy,in the frog. Correlative neurophysiological responses were recordedfrom the olfactory epithelium in response to odor stimulation.Examination of the epithelial surface showed degeneration andloss of the dense ciliary matrix and olfactory knobs by day10, which exposed the microvillar surface of the sustentacularcells. The amplitude of slow voltage transients recorded fromthe epithelial surface systematically decreased through day10. By day 40, the olfactory epithelium became responsive toodor stimulation. At this time partial renewal of the ciliarymatrix on the epithelial surface and bundles of receptor cellaxons in the olfactory nerve layer of the olfactory bulb wereobserved. There was substantial replacement of the ciliary matrixby day 100; in contrast, considerably less recovery of the slowvoltage transient was evident. Recovery of odor-evoked responsivity lagged behind recovery of the ciliary matrix. Therefore,these data imply that the reappearance of olfactory knobs andcilia is causally related to the recovery of the slow voltagetransients.     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.     

Does intranasal application of zinc sulfate produce anosmia in the mouse? An olfactometric and anatomical study

Mice pre-trained in an olfactometer were tested daily on odor detection and discrimination tasks after irrigation of their olfactory epithelium in each naris with 50 microl of 5% zinc sulfate or saline. Anterograde transport of a wheatgerm agglutinin-horseradish peroxidase (WGA-HRP) conjugate from the epithelium to the olfactory bulb was used to assess anatomical connectivity in these and in mice that were used only for histological analyses. One day after treatment, saline controls performed at high levels of accuracy in detecting vapor from solutions of 5-0.01% ethyl acetate and in an odor discrimination task but most ZnSO4-treated mice performed at chance for 5-30 days before showing recovery. Although dense WGA-HRP reaction product was found in the accessory olfactory bulb, there was little or no evidence for axonal transport to glomeruli of the main olfactory bulb in the first 4-8 days after treatment. These results demonstrate that intranasal application of ZnSO4 to mice produces a brief but essentially total disruption of functional connections from the olfactory epithelium to the main olfactory bulb and a corresponding transient anosmia.     

Axonal ephrin-As and odorant receptors: coordinate determination of the olfactory sensory map

Olfactory sensory neurons expressing a given odorant receptor (OR) project with precision to specific glomeruli in the olfactory bulb, generating a topographic map. In this study, we demonstrate that neurons expressing different ORs express different levels of ephrin-A protein on their axons. Moreover, alterations in the level of ephrin-A alter the glomerular map. Deletion of the ephrin-A5 and ephrin-A3 genes posteriorizes the glomerular locations for neurons expressing either the P2 or SR1 receptor, whereas overexpression of ephrin-A5 in P2 neurons results in an anterior shift in their glomeruli. Thus the ephrin-As are differentially expressed in distinct subpopulations of neurons and are likely to participate, along with the ORs, as one of a complement of guidance receptors governing the targeting of like axons to precise locations in the olfactory bulb.     

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.     

Development of the vertebrate main olfactory system

Olfactory receptor neurons project from the sensory epithelium to stereotyped targets within the olfactory bulb. Recent studies suggest that the generation of this precise spatial map probably involves a hierarchy of guidance events, as receptor neurons integrate information present in the epithelium and bulb to reach their target.     

The alarm reaction in crucian carp is mediated by olfactory neurons with long dendrites

In the present study, we applied a lipophilic tracer, Dil (1,1-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), to the synaptic region of the medial olfactory bulb in formaldehyde-fixed preparations from the crucian carp. We observed staining both in the axons of secondary neurons leading to the brain and in the olfactory receptor neurons (ORNs) of the olfactory epithelium. In those preparations, where staining of the tract was restricted to axons of the medial part of the medial olfactory tract, the majority (86-98%) of the somata of the sensory neurons were found in the deep layers of olfactory epithelium. Since the medial bundle of the medial olfactory tract mediates alarm behaviour in the crucian carp, we conclude that the sensory neurons with long dendrites participate in the reception of alarm pheromones.     

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.     

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.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.