Different profiles of main and accessory olfactory bulb mitral/tufted cell projections revealed in mice using an anterograde tracer and a whole-mount, flattened cortex preparation

A whole-mount, flattened cortex preparation was developed to compare profiles of axonal projections from main olfactory bulb (MOB) and accessory olfactory bulb (AOB) mitral and tufted (M/T) cells. After injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin, mapping of labeled axons using a Neurolucida system showed that M/T cells in the AOB sent axons primarily to the medial and posterior lateral cortical amygdala, with minimal branching into the piriform cortex. By contrast, M/T cells in the MOB displayed a network of collaterals that branched off the primary axon at several levels of the lateral olfactory tract (LOT). Collaterals emerging from the LOT into the anterior piriform cortex were often observed crossing into the posterior piriform cortex. M/T cells in the dorsal MOB extended fewer collaterals from the primary axon in the rostral LOT than did M/T cells from the anterior or ventral MOB. MOB M/T cells that projected to the medial amygdala did not do so exclusively, also sending collaterals to the anterior cortical amygdala as well as to olfactory cortical regions. This arrangement may be related to the ability of social experience to modify the response of mice to volatile pheromones detected by the main olfactory system.     

Possible roles of robo1+ ensheathing cells in guiding dorsal‐zone olfactory sensory neurons in mouse

In the mouse olfactory system, the anatomical locations of olfactory sensory neurons (OSNs) correlate with their axonal projection sites along the dorsoventral axis of the olfactory bulb (OB). We have previously reported that Neuropilin‐2 expressed by ventral‐zone OSNs contributes to the segregation of dorsal and ventral OSN axons, and that Slit is acting as a negative land mark to restrict the projection of Robo2⁺, early‐arriving OSN axons to the embryonic OB. Here, we report that another guidance receptor, Robo1, also plays an important role in guiding OSN axons. Knockout mice for Robo1 demonstrated defects in targeting of OSN axons to the OB. Although Robo1 is colocalized with dorsal‐zone OSN axons, it is not produced by OSNs, but instead by olfactory ensheathing cells. These findings indicate a novel strategy of axon guidance in the mouse olfactory system during development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:828–840, 2013     

Slits and Robo-2 regulate the coalescence of subsets of olfactory sensory neuron axons within the ventral region of the olfactory bulb

Olfactory sensory neurons (OSNs) project their axons to second-order neurons in the olfactory bulb (OB) to form a precise glomerular map and these stereotypic connections are crucial for accurate odorant information processing by animals. To form these connections, olfactory sensory neuron (OSN) axons respond to axon guidance molecules that direct their growth and coalescence. We have previously implicated the axon guidance receptor Robo-2 in the accurate coalescence of OSN axons within the dorsal region of the OB (Cho et al., 2011). Herein, we have examined whether Robo-2 and its ligands, the Slits, contribute to the formation of an accurate glomerular map within more ventral regions of the OB. We have ablated expression of Robo-2 in OSNs and assessed the targeting accuracy of axons expressing either the P2 or MOR28 olfactory receptors, which innervate two different regions of the ventral OB. We show that P2-positive axons, which express Robo-2, coalesce into glomeruli more ventrally and form additional glomeruli in the OB of robo-2^lox/lox;OMP-Cre mice. We also demonstrate that Robo-2-mediated targeting of P2 axons along the dorsoventral axis of the OB is controlled by Slit-1 and Slit-3 expression. Interestingly, although MOR28-positive OSNs only express low levels of Robo-2, a reduced number of MOR28-positive glomeruli is observed in the OB of robo-2^lox/lox;OMP-Cre mice. Taken together, our results demonstrate that Slits and Robo-2 are required for the formation of an accurate glomerular map in the ventral region of the OB.     

Dishevelled Proteins Are Associated with Olfactory Sensory Neuron Presynaptic Terminals

Olfactory sensory neurons (OSNs) project their axons from the olfactory epithelium toward the olfactory bulb (OB) in a heterogeneous and unsorted arrangement. However, as the axons approach the glomerular layer of the OB, axons from OSNs expressing the same odorant receptor (OR) sort and converge to form molecularly homogeneous glomeruli. Axon guidance cues, cell adhesion molecules, and OR induced activity have been implicated in the final targeting of OSN axons to specific glomeruli. Less understood, and often controversial, are the mechanisms used by OSN axons to initially navigate from the OE toward the OB. We previously demonstrated a role for Wnt and Frizzled (Fz) molecules in OSN axon extension and organization within the olfactory nerve. Building on that we now turned our attention to the downstream signaling cascades from Wnt-Fz interactions. Dishevelled (Dvl) is a key molecule downstream of Fz receptors. Three isoforms of Dvl with specific as well as overlapping functions are found in mammals. Here, we show that Dvl-1 expression is restricted to OSNs in the dorsal recess of the nasal cavity, and labels a unique subpopulation of glomeruli. Dvl-2 and Dvl-3 have a widespread distribution in both the OE and OB. Both Dvl-1 and Dvl-2 are associated with intra-glomerular pre-synaptic OSN terminals, suggesting a role in synapse formation/stabilization. Moreover, because Dvl proteins were observed in all OSN axons, we hypothesize that they are important determinants of OSN cell differentiation and axon extension.     

A contextual model for axonal sorting into glomeruli in the mouse olfactory system

No models fully account for how odorant receptors (ORs) function in the guidance of axons of olfactory sensory neurons (OSNs) to glomeruli in the olfactory bulb. Here, we use gene targeting in mice to demonstrate that the OR amino acid sequence imparts OSN axons with an identity that allows them to coalesce into glomeruli. Replacements between the coding regions of the M71 and M72 OR genes reroute axons to their respective glomeruli. A series of M71-M72 hybrid ORs uncover a spectrum of glomerular phenotypes, leading to the concept that the identity of OSN axons is revealed depending on what other axons are present. Naturally occurring amino acid polymorphisms in other ORs also produce distinct axonal identities. These critical amino acid residues are distributed throughout the protein and reside predominantly within transmembrane domains. We propose a contextual model for axon guidance in which ORs mediate homotypic interactions between like axons.     

BDNF promoter-mediated beta-galactosidase expression in the olfactory epithelium and bulb

The neurotrophin brain-derived neurotrophic factor (BDNF) has been implicated in the generation and differentiation of new olfactory sensory neurons (OSNs) and in the regulation of branching of OSN axons in their target glomeruli. However, previous reports of BDNF mRNA and protein expression in olfactory epithelium and olfactory bulb (OB) have been inconsistent, raising questions on the proposed roles for BDNF. Here, we report on beta-galactosidase (beta-gal) expression in adult gene-targeted mice where the BDNF promoter drives expression of the Escherichia coli lacZ gene (BDNF(lacZneo) mice). We find that beta-gal is expressed in a small subset of OSNs with axons that reach the olfactory nerve layers throughout the OB. In the OB, we find expression of beta-gal in gamma-aminobutyric acidergic but not dopaminergic periglomerular cells and external tufted cells and in interneurons located in the mitral cell layer. Our results are inconsistent with the regulation of generation and differentiation of new OSNs elicited by the release of BDNF from horizontal basal cells. The results are consistent with a role for BDNF in competitive branching of OSN axons within the glomeruli of the OB.     

Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice

The septal organ represents one of the three chemosensory subsystems found in most vertebrate species. Analyzing the projection pattern of septal organ neurons using the OMP-GFP transgenic mouse line revealed that axons navigate in highly variable fiber tracks across the main olfactory epithelium toward the main olfactory bulb. All septal organ axons cross through the cribriform plate at a spatially defined site and terminate exclusively in the posterior, ventromedial aspect of the bulb. Here, one portion of axons forms a dense network on the medial side where they apparently enter glomeruli which are mainly innervated by axons of olfactory sensory neurons from the main olfactory epithelium. Another significant portion of the axons targets a few glomeruli which appear to receive input exclusively from the septal organ neurons.     

Analysis of training-induced changes in ethyl acetate odor maps using a new computational tool to map the glomerular layer of the olfactory bulb

Odor quality is thought to be encoded by the activation of partially overlapping subsets of glomeruli in the olfactory bulb (odor maps). Mouse genetic studies have demonstrated that olfactory sensory neurons (OSNs) expressing a particular olfactory receptor target their axons to a few individual glomeruli in the bulb. While the specific targeting of OSN axons provides a molecular underpinning for the odor maps, much remains to be understood about the relationship between the functional and molecular maps. In this article, we ask the question whether intensive training of mice in a go/no-go operant conditioning odor discrimination task affects odor maps measured by determining c-fos up-regulation in periglomerular cells. Data analysis is performed using a newly developed suite of computational tools designed to systematically map functional and molecular features of glomeruli in the adult mouse olfactory bulb. This suite provides the necessary tools to process high-resolution digital images, map labeled glomeruli, visualize odor maps, and facilitate statistical analysis of patterns of identified glomeruli in the olfactory bulb. The software generates odor maps (density plots) based on glomerular activity, density, or area. We find that training up-regulates the number of glomeruli that become c-fos positive after stimulation with ethyl acetate.     

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.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with β-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

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.     

Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb

An olfactory sensory neuron (OSN) expresses selectively one member from a repertoire of approximately 1000 odorant receptor (OR) genes and projects its axon to a specific glomerulus in the olfactory bulb. Both processes are here recapitulated by MOR23 and M71 OR minigenes, introduced into mice. Minigenes of 9 kb and as short as 2.2 kb are selectively expressed by neurons that do not coexpress the endogenous gene but coproject their axons to the same glomeruli. Deletion of a 395 bp upstream region in the MOR23 minigene abolishes expression. In this region we recognize sequence motifs conserved in many OR genes. Transgenic lines expressing the OR in ectopic epithelial zones form ectopic glomeruli, which also receive input from OSNs expressing the cognate endogenous receptor. This suggests a recruitment through homotypic interactions between OSNs expressing the same OR.     

Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons

Olfactory sensory neurons (OSNs) are individually specified to express one odorant receptor (OR) gene among approximately 1000 different and project with precision to topographically defined convergence sites, the glomeruli, in the olfactory bulb. Although ORs partially determine the location of convergence sites, the mechanism ensuring that axons with different OR identities do not co-converge is unknown. RNCAM (OCAM, NCAM2) is assumed to regulate a broad zonal segregation of projections by virtue of being a homophilic cell adhesion molecule that is selectively expressed on axons terminating in a defined olfactory bulb region. We have identified NADPH diaphorase activity as being an independent marker for RNCAM-negative axons. Analyses of transgenic mice that ectopically express RNCAM in NADPH diaphorase-positive OSNs show that the postulated function of RNCAM in mediating zone-specific segregation of axons is unlikely. Instead, analyses of one OR-specific OSN subpopulation (P2) reveal that elevated RNCAM levels result in an increased number of P2 axons that incorrectly co-converge with axons of other OR identities. Both Gpi-anchored and transmembrane-bound RNCAM isoforms are localized on axons in the nerve layer, while the transmembrane-bound RNCAM is the predominant isoform on axon terminals within glomeruli. Overexpressing transmembrane-bound RNCAM results in co-convergence events close to the correct target glomeruli. By contrast, overexpression of Gpi-anchored RNCAM results in axons that can bypass the correct target before co-converging on glomeruli located at a distance. The phenotype specific for Gpi-anchored RNCAM is suppressed in mice overexpressing both isoforms, which suggests that two distinct RNCAM isoform-dependent activities influence segregation of OR-defined axon subclasses.     

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.     

Grueneberg Glomeruli in the Olfactory Bulb are Activated by Odorants and Cool Temperature

Neurons of the Grueneberg ganglion respond to cool temperatures as well as to distinct odorants and extend axonal processes to the olfactory bulb of the brain. Analyses of transgenic mice, in which Grueneberg ganglion neurons and their axons are labeled, revealed that these axons innervated nine distinct glomeruli distributed in a characteristic topographical pattern in dorsal, lateral, ventral, and medial regions of rather posterior areas in the bulb. To assess activation of these glomeruli (hereinafter designated as Grueneberg glomeruli) upon stimulation of Grueneberg ganglion neurons, mice were exposed to the odorant 2,3-dimethylpyrazine (2,3-DMP) and the expression of the activity-dependent marker c-Fos in juxtaglomerular cells of the relevant glomeruli was monitored. It was found that all of these glomeruli were activated, irrespective of their localization in the bulb. To verify that the activation of juxtaglomerular cells in Grueneberg glomeruli was indeed based on stimulation of Grueneberg ganglion neurons, the 2,3-DMP-induced responses in these glomeruli were investigated in mice lacking the cyclic nucleotide-gated channel CNGA3 which is critical for chemo- and thermosensory signal transduction in Grueneberg ganglion neurons. This approach revealed that elimination of CNGA3 led to a reduction of the odorant-induced activity in Grueneberg glomeruli, indicating that the activation of these glomeruli is based on a preceding stimulation of the Grueneberg ganglion. Analyzing whether Grueneberg glomeruli in the bulb might also process thermosensory information, it was found that upon exposure to coolness, Grueneberg glomeruli were activated. Investigating mice lacking CNGA3, the activation of these glomeruli by cool temperatures was attenuated.     

Positional cues that are strictly localized in the telencephalon induce preferential growth of mitral cell axons

In mice, mitral cells are the major efferent neurons of the main olfactory bulb and elongate axons into a very narrow part of the telencephalon to form a fiber bundle referred to as the lateral olfactory tract (LOT). To clarify the mechanisms responsible for guidance of mitral cell axons along this particular pathway, we co-cultured mouse embryo main olfactory bulbs with the telencephalons, and analyzed the pathways taken by mitral cell axons. Ingrowth of mitral cell axons into the telencephalon was observed in those co-cultures in which the olfactory bulbs had been exactly combined to their normal pathway (the LOT position) of the telencephalon. The axons grew preferentially along the LOT position, and formed a LOT-like fiber bundle. When the olfactory bulbs were grafted at positions apart from their normal pathway, however, no mitral cell axons grew into the telencephalon. Neocortical fragments combined with the telencephalon projected fibers into the telencephalon in random directions. These results suggest that the LOT position of the telencephalon offers a guiding pathway for mitral cell axons and that guiding cues for mitral cell axons are extremely localized. © 1996 John Wiley & Sons, Inc.     

Lactosamine differentially affects olfactory sensory neuron projections to the olfactory bulb

During embryonic development, olfactory sensory neurons extend axons that form synapses with the dendrites of projection neurons in glomeruli of the olfactory bulb (OB). The glycosyltransferase beta3GnT1 regulates the expression of 1B2-reactive lactosamine glycans that are mosaically distributed among glomeruli. In newborn beta3GnT1-/- mice, lactosamine expression is lost, and many glomeruli fail to form. To determine the role of lactosamine in OB targeting, we analyzed the trajectories of specific OR axon populations and their reactivity with 1B2 in beta3GnT1-/- mice. mI7 axons and P2 axons, both of which are weakly 1B2+ in wild-type mice, fail to grow to their normal positions in the glomerular layer during early postnatal development and never recover in adult mutant mice. In contrast, many M72 axons, which are always lactosamine negative in wild-type mice, survive but are misguided to the extreme anterior OB in neonatal mutant mice and persist as heterotypic glomeruli, even in adult null mice. These results show that the loss of lactosamine differentially affects each OR population. Those that lose their normal expression of lactosamine fail to form stable connections with mitral and tufted cells in the OB, disappear during early postnatal development, and do not recover in adults. Neurons that are normally lactosamine negative, survive early postnatal degeneration in beta3GnT1-/- mice but extend axons that converge on inappropriate targets in the mutant OB.     

Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium

During development, primary olfactory axons typically grow to their topographically correct target zone without extensive remodelling. Similarly, in adults, new axons arising from the normal turnover of sensory neurons essentially project to their target without error. In the present study we have examined axon targeting in the olfactory pathway following extensive chemical ablation of the olfactory neuroepithelium in the P2-tau:LacZ line of mice. These mice express LacZ in the P2 subpopulation of primary olfactory neurons whose axons target topographically fixed glomeruli on the medial and lateral surfaces of the olfactory bulb. Intraperitoneal injections of dichlobenil selectively destroyed the sensory neuroepithelium of the nasal cavity without direct physical insult to the olfactory neuron pathway. Primary olfactory neurons regenerated and LacZ staining revealed the trajectory of the P2 axons. Rather than project solely to their topographically appropriate glomeruli, the regenerating P2 axons now terminated in numerous inappropriate glomeruli which were widely dispersed over the olfactory bulb. While these errors in targeting were refined over time, there was still considerable mis-targeting after four months of regeneration.     

Volumetric and horseradish peroxidase tracing analysis of rat olfactory bulb following reversible olfactory nerve lesions

Olfactory receptor neurons can regenerate from basal stem cells. Receptor neuron lesion causes degenerative changes in the olfactory bulb followed by regeneration as new olfactory receptor axons innervate the olfactory bulb. To our knowledge, parametric analyses of morphometric changes in the olfactory bulb during degeneration and regeneration do not exist except in abstract form. To better characterize olfactory bulb response, we performed morphometric analysis in rats following reversible olfactory nerve lesion with diethyldithiocarbamate. We also performed anterograde tracing of the olfactory nerve with wheatgerm agglutinin linked to horseradish peroxidase. Results of morphometry and tracing were complementary. The glomerular layer and external plexiform layer showed shrinkage of 45 and 26%, respectively, at 9 days. No significant shrinkage occurred in any other layer. Individual glomeruli shrank by 40-50% at 3 and 9 days following lesion. These data show that degenerative changes occur both in the glomeruli and transneuronally in the external plexiform layer. Olfactory nerve regeneration (identified by WGA-HRP transport) paralleled volumetric recovery. Recovery occurred first in ventral and lateral glomeruli between 9 and 16 days followed by recovery in medial and dorsal glomeruli. These data indicate substantial transynaptic degeneration in the olfactory bulb and a heretofore unrecognized gradient in olfactory nerve regeneration that can be used to systematically study recovery of a cortical structure.     

Effects of in utero odorant exposure on neuroanatomical development of the olfactory bulb and odour preferences

Human babies and other young mammals prefer food odours and flavours of their mother's diet during pregnancy as well as their mother's individually distinctive odour. Newborn mice also prefer the individual odours of more closely related--even unfamiliar--lactating females. If exposure to in utero odorants-which include metabolites from the mother's diet and the foetus's genetically determined individual odour-helps shape the neuroanatomical development of the olfactory bulb, this could influence the perception of such biologically important odours that are preferred after birth. We exposed gene-targeted mice during gestation and nursing to odorants that activate GFP-tagged olfactory receptors (ORs) and then measured the effects on the size of tagged glomeruli in the olfactory bulb where axons from olfactory sensory neurons (OSNs) coalesce by OR type. We found significantly larger tagged glomeruli in mice exposed to these activating odorants in amniotic fluid, and later in mother's milk, as well as significant preferences for the activating odour. Larger glomeruli comprising OSNs that respond to consistently encountered odorants should enhance detection and discrimination of these subsequently preferred odours, which in nature would facilitate selection of palatable foods and kin recognition, through similarities in individual odours of relatives.