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.     

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.     

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 beta-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.     

Optical imaging of odorant representations in the mammalian olfactory bulb.

We adapted the technique of intrinsic signal imaging to visualize how odorant concentration and structure are represented spatially in the rat olfactory bulb. Most odorants activated one or more glomeruli in the imaged region of the bulb; these optically imaged responses reflected the excitation of underlying neurons. Odorant-evoked patterns were similar across animals and symmetrical in the two bulbs of the same animal. The variable sensitivity of individual glomeruli produced distinct maps for different odorant concentrations. Using a series of homologous aldehydes, we found that glomeruli were tuned to detect particular molecular features and that maps of similar molecules were highly correlated. These characteristics suggest that odorants and their concentrations can be encoded by distinct spatial patterns of glomerular activation.     

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.     

Differential but competitive binding of Nogo protein and class i major histocompatibility complex (MHCI) to the PIR-B ectodomain provides an inhibition of cells

Binding of class I MHC molecules (MHCI) to an inhibitory receptor, PIR-B, expressed on B cells and myeloid cells provides constitutive cellular inhibition, thus ensuring peripheral tolerance. Recent unexpected findings pointed to a novel inhibitory role of PIR-B in neurite regeneration through binding to three axonal outgrowth inhibitors of myelin, including Nogo. Thus, it becomes interesting to determine whether the actions of the inhibitory myelin proteins and MHCI could coexist independently or be mutually exclusive as to the PIR-B-mediated immune and neural cell inhibition. Here, we present data supporting the competition of Nogo- and MHCI-mediated inhibition where they coexist. Kinetic analyses of Nogo and MHCI binding to the whole or a part of the recombinant PIR-B ectodomain revealed that PIR-B binds with higher affinity to Nogo than MHCI and that the MHCI binding only occurred with the N-terminal domains of PIR-B, whereas Nogo binding occurred with either the N- or C-terminal ectodomains. Importantly, kinetic tests indicated that the binding to PIR-B of Nogo and MHCI was competitive. Both endogenous and exogenous Nogo intensified the PIR-B-mediated suppression of interleukin-6 release from lipopolysaccharide-stimulated wild-type, but not PIR-B-deficient, cultured mast cells, indicating that PIR-B mediates Nogo-induced inhibition. Thus, we propose a novel mechanism by which PIR-B-mediated regulation is achieved differentially but competitively via MHCI and Nogo in cells of the immune system.     

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.     

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.     

Functional Deficiency of MHC Class I Enhances LTP and Abolishes LTD in the Nucleus Accumbens of Mice

Major histocompatibility complex class I (MHCI) molecules were recently identified as novel regulators of synaptic plasticity. These molecules are expressed in various brain areas, especially in regions undergoing activity-dependent synaptic plasticity, but their role in the nucleus accumbens (NAc) is unknown. In this study, we investigated the effects of genetic disruption of MHCI function, through deletion of β2-microblobulin, which causes lack of cell surface expression of MHCI. First, we confirmed that MHCI molecules are expressed in the NAc core in wild-type mice. Second, we performed electrophysiological recordings with NAc core slices from wild-type and β2-microglobulin knock-out mice lacking cell surface expression of MHCI. We found that low frequency stimulation induced long-term depression in wild-type but not knock-out mice, whereas high frequency stimulation induced long-term potentiation in both genotypes, with a larger magnitude in knock-out mice. Furthermore, we demonstrated that knock-out mice showed more persistent behavioral sensitization to cocaine, which is a NAc-related behavior. Using this model, we analyzed the density of total AMPA receptors and their subunits GluR1 and GluR2 in the NAc core, by SDS-digested freeze-fracture replica labeling. After repeated cocaine exposure, the density of GluR1 was increased, but there was no change in total AMPA receptors and GluR2 levels in wild-type mice. In contrast, following repeated cocaine exposure, increased densities of total AMPA receptors, GluR1 and GluR2 were observed in knock-out mice. These results indicate that functional deficiency of MHCI enhances synaptic potentiation, induced by electrical and pharmacological stimulation.     

Role of IGF signaling in olfactory sensory map formation and axon guidance

Olfactory neurons project their axons to spatially invariant glomeruli in the olfactory bulb, forming an ordered pattern of innervation comprising the olfactory sensory map. A mirror symmetry exists within this map, such that neurons expressing a given receptor typically project to one glomerulus on the medial face and one glomerulus on the lateral face of the bulb. The mechanisms underlying an olfactory neuron's choice to project medially versus laterally remain largely unknown, however. Here we demonstrate that insulin-like growth factor (IGF) signaling is required for sensory innervation of the lateral olfactory bulb. Mutations that eliminate IGF signaling cause axons destined for targets in the lateral bulb to shift to ectopic sites on the ventral-medial surface. Using primary cultures of olfactory and cerebellar neurons, we further show that IGF is a chemoattractant for axon growth cones. Together these observations reveal a role of IGF signaling in sensory map formation and axon guidance.     

Enhancement to the RANKPEP resource for the prediction of peptide binding to MHC molecules using profiles

We introduced previously an on-line resource, RANKPEP that uses position specific scoring matrices (PSSMs) or profiles for the prediction of peptide-MHC class I (MHCI) binding as a basis for CD8 T-cell epitope identification. Here, using PSSMs that are structurally consistent with the binding mode of MHC class II (MHCII) ligands, we have extended RANKPEP to prediction of peptide-MHCII binding and anticipation of CD4 T-cell epitopes. Currently, 88 and 50 different MHCI and MHCII molecules, respectively, can be targeted for peptide binding predictions in RANKPEP. Because appropriate processing of antigenic peptides must occur prior to major histocompatibility complex (MHC) binding, cleavage site prediction methods are important adjuncts for T-cell epitope discovery. Given that the C-terminus of most MHCI-restricted epitopes results from proteasomal cleavage, we have modeled the cleavage site from known MHCI-restricted epitopes using statistical language models. The RANKPEP server now determines whether the C-terminus of any predicted MHCI ligand may result from such proteasomal cleavage. Also implemented is a variability masking function. This feature focuses prediction on conserved rather than highly variable protein segments encoded by infectious genomes, thereby offering identification of invariant T-cell epitopes to thwart mutation as an immune evasion mechanism.     

Spatially and temporally regulated expression of specific heparan sulfate epitopes in the developing mouse olfactory system

Heparan sulfate (HS) comprises a structurally diverse group of glycosaminoglycans present ubiquitously on cell surfaces and in the extracellular matrix. The spatially and temporally regulated expression of specific HS structures is essential for various developmental processes in the nervous system but their distributions in the mouse olfactory system have not been explored. Here, we examined the spatiotemporal distribution of particular HS species in the developing mouse olfactory system using three structure‐specific monoclonal antibodies (HepSS‐1, JM403 and NAH46). The major findings were as follows. (i) During olfactory bulb morphogenesis, the HepSS‐1 epitope was strongly expressed in anterior telencephalic cells and coexpressed with fibroblast growth factor receptor 1. (ii) In early postnatal glomeruli, the JM403 epitope was expressed at different levels among individual glomeruli. The expression pattern and levels of the JM403 epitope were both associated with those of ephrin‐A3. (iii) In the vomeronasal system, the JM403 epitope was expressed in all vomeronasal axons but became increasingly restricted to vomeronasal axons terminating in the anterior region of the accessory olfactory bulb by 3 weeks of age. Our results demonstrate that each HS epitope exhibits a unique expression pattern during the development of the mouse olfactory system. Thus, each HS epitope is closely associated with particular developmental processes of the olfactory system and might have a particular role in developmental events.     

Sequence variability analysis of human class I and class II MHC molecules: functional and structural correlates of amino acid polymorphisms

Major histocompatibility complex class I (MHCI) and class II (MHCII) molecules display peptides on antigen-presenting cell surfaces for subsequent T-cell recognition. Within the human population, allelic variation among the classical MHCI and II gene products is the basis for differential peptide binding, thymic repertoire bias and allograft rejection. While available 3D structural analysis suggests that polymorphisms are found primarily within the peptide-binding site, a broader informatic approach pinpointing functional polymorphisms relevant for immune recognition is currently lacking. To this end, we have now analyzed known human class I (774) and class II (485) alleles at each amino acid position using a variability metric (V). Polymorphisms (V>1) have been identified in residues that contact the peptide and/or T-cell receptor (TCR). Using sequence logos to investigate TCR contact sites on HLA molecules, we have identified conserved MHCI residues distinct from those of conserved MHCII residues. In addition, specific class II (HLA-DP, -DQ, -DR) and class I (HLA-A, -B, -C) contacts for TCR binding are revealed. We discuss these findings in the context of TCR restriction and alloreactivity.     

Developmental regulation of olfactory circuit formation in mice

In mammals, odorants induce various behavioral responses that are critical to the survival of the individual and species. Binding signals of odorants to odorant receptors (ORs) expressed in the olfactory epithelia are converted to an odor map, a pattern of activated glomeruli, in the olfactory bulb (OB). This topographic map is used to identify odorants for memory-based learned decisions. In the embryo, a coarse olfactory map is generated in the OB by a combination of dorsal-ventral and anterior-posterior targeting of olfactory sensory neurons (OSNs), using specific sets of axon-guidance molecules. During the process of OSN projection, odor signals are sorted into distinct odor qualities in separate functional domains in the OB. Odor information is then conveyed by the projection neurons, mitral/tufted cells, to various regions in the olfactory cortex, particularly to the amygdala for innate olfactory decisions. Although the basic architecture of hard-wired circuits is generated by a genetic program, innate olfactory responses are modified by neonatal odor experience in an activity-dependent manner. Stimulus-driven OR activity promotes post-synaptic events and dendrite selection in the responding glomeruli making them larger. As a result, enhanced odor inputs in neonates establish imprinted olfactory memory that induces attractive responses in adults, even when the odor quality is innately aversive. In this paper, I will provide an overview of the recent progress made in the olfactory circuit formation in mice.     

An olfactory subsystem that mediates high-sensitivity detection of volatile amines

Olfactory stimuli are detected by over 1,000 odorant receptors in mice, with each receptor being mapped to specific glomeruli in the olfactory bulb. The trace amine-associated receptors (TAARs) are a small family of evolutionarily conserved olfactory receptors whose contribution to olfaction remains enigmatic. Here, we show that a majority of the TAARs are mapped to a discrete subset of glomeruli in the dorsal olfactory bulb of the mouse. This TAAR projection is distinct from the previously described class I and class II domains, and is formed by a sensory neuron population that is restricted to express TAAR genes prior to choice. We also show that the dorsal TAAR glomeruli are selectively activated by amines at low concentrations. Our data uncover a hard-wired, parallel input stream in the main olfactory pathway that is specialized for the detection of volatile amines.     

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.     

A sexually dimorphic group of atypical glomeruli in the mouse olfactory bulb

Atypical glomeruli (AtG) are clearly distinguishable from typical ones because of their strong cholinergic innervation. AtG are located in defined positions in the caudal half of the main olfactory bulb of rodents. The AtG partially overlap with other specialized olfactory subsystems, such as the modified glomerular complex, which is close to the accessory olfactory bulb. So far, possible sex differences in these specialised olfactory systems have not been investigated. In this work we have identified AtG in the mouse by means of acetylcholinesterase histochemistry and compared the number and size of these glomeruli between the sexes and also between the two strains that demonstrate intraglomerular synaptic differences, i.e. BALB/c and CD-1 mice. First, we divided the AtG into three types according to their position (I, rostral-most; II, around the accessory olfactory bulb; III, caudal-most) or their reactivity to acetylcholinesterase histochemistry (AtG type II being the least reactive glomeruli). ANOVA analyses revealed differences in the maximum diameter of glomeruli among the three types, but not in their sectional areas, indicating that all three types have different shapes. Moreover, both morphoplanimetric parameters were seen to be different between the two strains studied and also between the sexes: male mice and BALB/c animals had the largest glomeruli. The number of AtG was also significantly different between the sexes and strains, although these factors presented a strong interaction. Thus, the males had higher numbers of AtG in the CD-1 strain whereas in the BALB/c mice males demonstrated fewer AtG than females. These differences in number were largely due to AtG type II. The present work is evidence that AtG type II is a sexually dimorphic group of specialized glomeruli located in the main olfactory bulb.     

Precise circuitry links bilaterally symmetric olfactory maps

Olfactory sensory neurons expressing a common receptor gene converge onto one or a few glomeruli with stereotyped positions within the mouse main olfactory bulb (MOB), producing a map of approximately 1800 olfactory columns representing approximately 1000 odorant receptors. Here, we report that this precise olfactory map is maintained over several synapses that ultimately cross MOB hemispheres to link bilateral isofunctional olfactory columns. Focal injection of tracer into genetically identified glomeruli revealed an exquisite topography that involves a bilateral connection via the anterior olfactory nucleus pars externa (AONpE) that links isofunctional olfactory columns in the contralateral MOB. Physiological and behavioral assays revealed an important role for the AONpE in bilateral exchange of odorant-specific information. These results indicate that the interbulbar link through the AONpE integrates bilateral olfactory sensory maps and exchanges olfactory information, positioning it as a unique model system for studying interhemispheric connections.     

Vertebrate slit, a secreted ligand for the transmembrane protein roundabout, is a repellent for olfactory bulb axons

The olfactory bulb plays a central role in olfactory information processing through its connections with both peripheral and cortical structures. Axons projecting from the olfactory bulb to the telencephalon are guided by a repulsive activity in the septum. The molecular nature of the repellent is not known. We report here the isolation of vertebrate homologs of the Drosophila slit gene and show that Slit protein binds to the transmembrane protein Roundabout (Robo). Slit is expressed in the septum whereas Robo is expressed in the olfactory bulb. Functionally, Slit acts as a chemorepellent for olfactory bulb axons. These results establish a ligand-receptor relationship between two molecules important for neural development, suggest a role for Slit in olfactory bulb axon guidance, and reveal the existence of a new family of axon guidance molecules.