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.     

Follow your nose: axon pathfinding in olfactory map formation

Two new studies report how discrete identities of olfactory sensory neurons are converted into a spatial map of axonal connections (Imai et al., 2006; Serizawa et al., 2006). They find that levels of cAMP signals derived from olfactory receptors (ORs) can direct targeting of axons along an axis, and that ORs and neural activity regulate expression of adhesion/guidance molecules in mosaic patterns that can sort axons into discrete locations.     

Interhemispheric olfactory circuit and the memory beyond

In mammals, olfactory sensory neurons project their axons exclusively to the ipsilateral olfactory bulb. It remains unclear how odor information interacts between the two hemispheres of the brain. In this issue of Neuron, Yan et al. describe the precise interbulbar connection through the anterior olfactory nucleus pars externa (AONpE), which links contralateral isotypic olfactory columns.     

Reciprocal interactions between olfactory receptor axons and olfactory nerve glia cultured from the developing moth Manduca sexta

In olfactory systems, neuron-glia interactions have been implicated in the growth and guidance of olfactory receptor axons. In the moth Manduca sexta, developing olfactory receptor axons encounter several types of glia as they grow into the brain. Antennal nerve glia are born in the periphery and enwrap bundles of olfactory receptor axons in the antennal nerve. Although their peripheral origin and relationship with axon bundles suggest that they share features with mammalian olfactory ensheathing cells, the developmental roles of antennal nerve glia remain elusive. When cocultured with antennal nerve glial cells, olfactory receptor growth cones readily advance along glial processes without displaying prolonged changes in morphology. In turn, olfactory receptor axons induce antennal nerve glial cells to form multicellular arrays through proliferation and process extension. In contrast to antennal nerve glia, centrally derived glial cells from the axon sorting zone and antennal lobe never form arrays in vitro, and growth-cone glial-cell encounters with these cells halt axon elongation and cause permanent elaborations in growth cone morphology. We propose that antennal nerve glia play roles similar to olfactory ensheathing cells in supporting axon elongation, yet differ in their capacity to influence axon guidance, sorting, and targeting, roles that could be played by central olfactory glia in Manduca.     

Perception without a thalamus how does olfaction do it?

The olfactory system has generated considerable interest in recent years, mainly focused on receptor genes and early olfactory processing. In this issue of Neuron, Mori et al. focus centrally, providing evidence for slow- and fast-wave states in olfactory cortex that appear to gate the inflow of information underlying conscious smell perception.     

Extraepithelial cells expressing distinct olfactory receptors are associated with axons of sensory cells with the same receptor type

During critical phases of mouse development, axons from olfactory sensory neurons grow out of the nasal neuroepithelium and navigate through the connective mesenchyme tissue towards their targets in the developing telencephalic vesicle. Between embryonic days E11 and E16, populations of cells are located in the mesenchyme which express distinct olfactory receptor genes along with the olfactory marker protein (OMP); thus they express markers characteristic for mature olfactory sensory neurons. These extraepithelial cells are positioned along the axon tracts, and each population expressing a given receptor gene is specifically associated with the axons of those olfactory sensory neurons with the same receptor type. The data suggest that they either might be guide posts for the outgrowing axons or migrate along the axons into the brain.     

Different isoforms of fasciclin II are expressed by a subset of developing olfactory receptor neurons and by olfactory-nerve glial cells during formation of glomeruli in the moth Manduca sexta

During development of the primary olfactory projection, olfactory receptor axons must sort by odor specificity and seek particular sites in the brain in which to create odor-specific glomeruli. In the moth Manduca sexta, we showed previously that fasciclin II, a cell adhesion molecule in the immunoglobulin superfamily, is expressed by the axons of a subset of olfactory receptor neurons during development and that, in a specialized glia-rich "sorting zone," these axons segregate from nonfasciclin II-expressing axons before entering the neuropil of the glomerular layer. The segregation into fasciclin II-positive fascicles is dependent on the presence of the glial cells in the sorting zone. Here, we explore the expression patterns for different isoforms of Manduca fasciclin II in the developing olfactory system. We find that olfactory receptor axons express transmembrane fasciclin II during the period of axonal ingrowth and glomerulus development. Fascicles of TM-fasciclin II+ axons target certain glomeruli and avoid others, such as the sexually dimorphic glomeruli. These results suggest that TM-fasciclin II may play a role in the sorting and guidance of the axons. GPI-linked forms of fasciclin II are expressed weakly by glial cells associated with the receptor axons before they reach the sorting zone, but not by sorting-zone glia. GPI-fasciclin II may, therefore, be involved in axon-glia interactions related to stabilization of axons in the nerve, but probably not related to sorting.     

Rewiring the olfactory bulb: changes in odor maps following recovery from nerve transection

Recent studies have shown that axons from olfactory receptor subtypes converge onto glomeruli in fixed positions within the olfactory bulb. Different receptor subtypes project to different glomeruli, forming a spatial distribution of odor information or 'odor maps'. Olfactory receptor neurons are continuously replaced throughout the life span of an animal, yet they preserve this highly localized mapping of receptor subtypes. In this study we used a transgenic mouse (P2-IRES-tau-lacZ) to map axons from a single receptor subtype (P2 receptors) in order to determine if regenerating axons were able to re-establish the P2 receptor map following nerve transection. Results confirm that P2 receptor axons retain their capacity to grow back to the olfactory bulb and converge onto glomeruli following nerve transection. However, the location and number of convergence sites was significantly altered compared to the control map. This change in the spatial distribution of axons alters the topography of odor mapping and has important implications for the processing of olfactory information. Findings from this study may explain why animals recovering from nerve injury require odor training before odor discrimination is restored. Future studies of olfactory receptor mapping could prove helpful in planning strategies to rewire connections in the brain and to restore function following injury or neurological disease.     

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.     

LUSH shapes up for a starring role in olfaction

In the fruit fly Drosophila, odorant-binding proteins are secreted into the fluid that bathes olfactory neurons. Laughlin et al. (2008) now challenge the assumption that the odorant-binding protein LUSH passively transports its pheromone to a specific olfactory receptor. Instead, LUSH undergoes a conformational change upon pheromone binding that is sufficient for neuronal activation.     

Insect odorant receptors: channeling scent

Odorant detection in insects involves heterodimers between an odorant receptor (OR) and a conserved seven-transmembrane protein called Or83b, but the exact mechanism of OR signal transduction is unclear. Two recent studies in Nature (Sato et al., 2008; Wicher et al., 2008) now reveal that these OR-Or83b heterodimers form odorant-gated ion channels, revealing a surprising new mode of olfactory transduction.     

The bound leading the bound: target-derived receptors act as guidance cues

Developing axons are guided to their targets by chemoattractive and chemorepulsive ligands. Ledda et al., in this issue of Neuron, demonstrate that the target-derived receptor glial cell line-derived neurotrophic factor receptor alpha1 (GFRalpha1) can also act in trans as an axon guidance molecule for neurons.     

A diffusible signal attracts olfactory sensory axons toward their target in the developing brain of the moth

The signals that olfactory receptor axons use to navigate to their target in the CNS are still not well understood. In the moth Manduca sexta, the primary olfactory pathway develops postembryonically, and the receptor axons navigate from an experimentally accessible sensory epithelium to the brain along a pathway long enough for detailed study of regions in which axon behavior changes. The current experiments ask whether diffusible factors contribute to receptor axon guidance. Explants were made from the antennal receptor epithelium and co-cultured in a collagen gel matrix with slices of various regions of the brain. Receptor axons were attracted toward the central regions of the brain, including the protocerebrum and antennal lobe. Receptor axons growing into a slice of the most proximal region of the antennal nerve, where axon sorting normally occurs, showed no directional preference. When the antennal lobe was included in the slice, the receptor axons entering the sorting region grew directly toward the antennal lobe. Taken together with the previous in vivo experiments, the current results suggest that an attractive diffusible factor can serve as one cue to direct misrouted olfactory receptor axons toward the medial regions of the brain, where local cues guide them to the antennal lobe. They also suggest that under normal circumstances, in which the receptor axons follow a pre-existing pupal nerve to the antennal lobe, the diffusible factor emanating from the lobe acts in parallel and at short range to maintain the fidelity of the path into the antennal lobe.     

Glomerulus development in the absence of a set of mitral-like neurons in the insect olfactory lobe

Mitral cells are the first neurons in the mammalian olfactory bulb to synapse with olfactory receptor axons during glomerulus development, and in an invertebrate, the moth Manduca sexta, mitral-like neurons overlap very early with olfactory receptor axons as they begin to form protoglomeruli. The possibility for early interaction between receptor neurons and mitral-like neurons led us to ask whether such an interaction plays an essential role in glomerulus development. In the current study in the moth, we surgically removed a major class of these mitral-like neurons before glomeruli began to form and asked: (a) Is the formation of the array of olfactory glomeruli triggered by an interaction of the first-arriving receptor axons with the dendrites of mitral-like neurons? (b) At the level of individual glomeruli, must the mitral-like dendrites be in place either to maintain receptor axons in a glomerular arrangement, or to guide later-growing dendrites of other types into the developing glomeruli? Our results indicate that even without the participation of this group of mitral-like neurons, the array of sexually isomorphic ordinary glomeruli forms and the basic substructure of individual glomeruli develops apparently normally. We conclude that the mitral-like neurons in Manduca are not essential for the formation of ordinary olfactory glomeruli during development. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 41–52, 1998     

Dock and Pak regulate olfactory axon pathfinding in Drosophila

The convergence of olfactory axons expressing particular odorant receptor (Or) genes on spatially invariant glomeruli in the brain is one of the most dramatic examples of precise axon targeting in developmental neurobiology. The cellular and molecular mechanisms by which olfactory axons pathfind to their targets are poorly understood. We report here that the SH2/SH3 adapter Dock and the serine/threonine kinase Pak are necessary for the precise guidance of olfactory axons. Using antibody localization, mosaic analyses and cell-type specific rescue, we observed that Dock and Pak are expressed in olfactory axons and function autonomously in olfactory neurons to regulate the precise wiring of the olfactory map. Detailed analyses of the mutant phenotypes in whole mutants and in small multicellular clones indicate that Dock and Pak do not control olfactory neuron (ON) differentiation, but specifically regulate multiple aspects of axon trajectories to guide them to their cognate glomeruli. Structure/function studies show that Dock and Pak form a signaling pathway that mediates the response of olfactory axons to guidance cues in the developing antennal lobe (AL). Our findings therefore identify a central signaling module that is used by ONs to project to their cognate glomeruli.     

Multiple axon guidance cues establish the olfactory topographic map: how do these cues interact?

Each primary olfactory neuron stochastically expresses one of approximately 1000 odorant receptors. The total population of these neurons therefore consists of approximately 1,000 distinct subpopulations, each of which are mosaically dispersed throughout one of four semi-annular zones in the nasal cavity. The axons of these different subpopulations are initially intermingled within the olfactory nerve. However, upon reaching the olfactory bulb, they sort out and converge so that axons expressing the same odorant receptor typically target one or two glomeruli. The spatial location of each of these approximately 1800 glomeruli are topographically-fixed in the olfactory bulb and are invariant from animal to animal. Thus, while odorant receptors are expressed mosaically by neurons throughout the olfactory neuroepithelium their axons sort out, converge and target the same glomerulus within the olfactory bulb. How is such precise and reproducible topographic targeting generated? While some of the mechanisms governing the growth cone guidance of olfactory sensory neurons are understood, the cues responsible for homing axons to their target site remain elusive.     

Guidepost neurons for the lateral olfactory tract: Expression of metabotropic glutamate receptor 1 and innervation by glutamatergic olfactory bulb axons

The guidepost neurons for the lateral olfactory tract, which are called lot cells, are the earliest‐generated neurons in the neocortex. They migrate tangentially and ventrally further down this tract, and provide scaffolding for the olfactory bulb axons projecting into this pathway. The molecular profiles of the lot cells are largely uncharacterized. We found that lot cells specifically express metabotropic glutamate receptor subtype‐1 at a very early stage of development. This receptor is functionally competent and responds to a metabotropic glutamate receptor agonist with a transient increase in the intracellular calcium ion concentration. When the glutamatergic olfactory bulb axons were electrically stimulated, lot cells responded to the stimulation with a calcium increase mainly via ionotropic glutamate receptors, suggesting potential neurotransmission between the axons and lot cells during early development. Together with the finding that lot cells themselves are glutamatergic excitatory neurons, our results provide another notable example of precocious interactions between the projecting axons and their intermediate targets. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

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     

Development and topography of the lateral olfactory tract in the mouse: imaging by genetically encoded and injected fluorescent markers

In mammals, conventional odorants are detected by OSNs located in the main olfactory epithelium of the nose. These neurons project their axons to glomeruli, which are specialized structures of neuropil in the olfactory bulb. Within glomeruli, axons synapse onto dendrites of projection neurons, the mitral and tufted (M/T) cells. Genetic approaches to visualize axons of OSNs expressing a given odorant receptor have proven very useful in elucidating the organization of these projections to the olfactory bulb. Much less is known about the development and connectivity of the lateral olfactory tract (LOT), which is formed by axons of M/T cells connecting the olfactory bulb to central neural regions. Here, we have extended our genetic approach to mark M/T cells of the main olfactory bulb and their axons in the mouse, by targeted insertion of IRES-tauGFP in the neurotensin locus. In NT-GFP mice, we find that M/T cells of the main olfactory bulb mature and project axons as early as embryonic day 11.5. Final innervation of central areas is accomplished before the end of the second postnatal week. M/T cell axons that originate from small defined areas within the main olfactory bulb, as visualized by localized injections of fluorescent tracers in wild-type mice at postnatal days 1 to 3, follow a dual trajectory: a branch of tightly packed axons along the dorsal aspect of the LOT, and a more diffuse branch along the ventral aspect. The dorsal, but not the ventral, subdivision of the LOT exhibits a topographical segregation of axons coming from the dorsal versus ventral main olfactory bulb. The NT-GFP mouse strain should prove useful in further studies of development and topography of the LOT, from E11.5 until 2 weeks after birth.