Temporally staggered glomerulus development in the moth Manduca sexta.

Glomeruli, neuropilar structures composed of olfactory receptor neuron (ORN) axon terminals and central neuron dendrites, are a common feature of olfactory systems. Typically, ORN axons segregate into glomeruli based on odor specificity, making glomeruli the basic unit for initial processing of odorant information. Developmentally, glomeruli arise from protoglomeruli, loose clusters of ORN axons that gradually synapse onto dendrites. Previous work in the moth Manduca sexta demonstrated that protoglomeruli develop in a wave across the antennal lobe (AL) during stage 5 of the 18 stages of metamorphic adult development. However, ORN axons from the distal segments of the antenna arrive at the AL for several more days. We report that protoglomeruli present at stage 5 account for only approximately two or three of adult glomeruli with the number of structures increasing over subsequent stages. How do these later arriving axons incorporate into glomeruli? Examining the dendritic projections of a unique serotonin-containing neuron into glomeruli at later stages revealed glomeruli with immature dendritic arbors intermingled among more mature glomeruli. Labeling ORN axons that originate in proximal segments of the antenna suggested that early-arriving axons target a limited number of glomeruli. We conclude that AL glomeruli form over an extended time period, possibly as a result of ORNs expressing new odorant receptors arriving from distal antennal segments.

Bidirectional influences between neurons and glial cells in the developing olfactory system.

Olfactory systems serve as excellent model systems for the study of numerous widespread aspects of neural development and also for the elucidation of features peculiar to the formation of neural circuits specialized to process odor inputs. Accumulated research reveals a fine balance between developmental autonomy of olfactory structures and intercellular interactions essential for their normal development. Recent findings have uncovered evidence for more autonomy than previously realized, but simultaneously have begun to reveal the complex cellular and molecular underpinnings of key interactions among neurons and glial cells at several important steps in olfactory development. Striking similarities in the functional organization of olfactory systems across vertebrate and invertebrate species allow the advantages of different species to be used to address common issues. Our own work in the moth Manduca sexta has demonstrated reciprocal neuron-glia interactions that have key importance in two aspects of development, the sorting of olfactory receptor axons into fascicles targeted for specific glomeruli and the creation of glomeruli. Studies in vertebrate species suggest that similar neuron-glia interactions may underlie olfactory development, although here the roles have not been tested so directly. Similar cellular interactions also are likely to play roles in development of some other systems in which axons of intermixed neurons must sort according to target specificity and systems in which reiterated modules of synaptic neuropil develop.

Cell surface carbohydrates and glomerular targeting of olfactory sensory neuron axons in the mouse.

Cell surface carbohydrates have been implicated in axon guidance and targeting throughout the nervous system. We have begun to test the hypothesis that, in the olfactory system, a differential distribution of cell surface carbohydrates may influence olfactory sensory neuron (OSN) axon targeting. Specifically, we have examined the spatial distribution of two different plant lectins, Ulex europaeus agglutinin (UEA) and Dolichos biflorus agglutinin (DBA), to determine whether they exhibit differential and reproducible projections onto the main olfactory bulb. Each lectin exhibited a unique spatial domain of glomerular labeling that was consistent across animals. UEA labeling was strongest in the ventral aspect of the olfactory bulb; DBA labeling was strongest in the dorsal aspect of the olfactory bulb. Some evidence for colocalization was present where these two borders intersected. Large areas of the glomerular layer were not labeled by either lectin. To determine whether patterns of lectin labeling were reproducible at the level of individual glomeruli, UEA labeling was assessed relative to M72-IRES-taulacZ- and P2-IRES-taulacZ-labeled axons. Although glomeruli neighboring these two identified glomeruli were consistently labeled with UEA, none of the lacZ positive axons was lectin labeled. Labeling of vomeronasal sensory neuron axons in the accessory olfactory bulb was more uniform for the two lectins. These data are the first to show a differential distribution of UEA vs. DBA labeling in the main olfactory bulb and are consistent with the hypothesis that a differential distribution of cell surface carbohydrates, a glycocode, may contribute to the targeting of OSN axons.

Cell surface carbohydrates reveal heterogeneity in olfactory receptor cell axons in the mouse.

Cell surface carbohydrates, both in the olfactory system and elsewhere, have been proposed to play critical roles in axon guidance and targeting. Recent studies have used plant lectins to study the heterogeneous distribution of carbohydrates in the olfactory system. One lectin, Dolichos biflorus agglutinin (DBA), heterogeneously labels subsets of glomeruli. In the olfactory epithelium DBA labeled a subset of olfactory sensory neurons (OSNs) including their cilia, dendrites, and somata. OSN axons were also labeled and readily observed in the olfactory nerve and bulb. The patterns of glomerular innervation by DBA labeled (DBA(+)) axons were diverse; some glomeruli contained many labeled axons, while others contained few or no labeled axons. To characterize the heterogeneous innervation of glomeruli, we double labeled olfactory bulbs with DBA and an antibody to olfactory marker protein (OMP). OMP colocalized in most, but not all, DBA(+) axons. To determine if those axons that did not express OMP were immature, we double labeled olfactory bulbs with DBA and anti-GAP-43. GAP-43 rarely colocalized with DBA, suggesting that DBA(+) axons are not, as a population, immature. Triple labeling with all three markers revealed a small subset of DBA(+) axons which did not express either OMP or GAP-43. Electron microscopy established that DBA labels axons in the olfactory nerve and DBA-labeled axons form typical glomerular axodendritic synapses.

Novel microglomerular structures in the olfactory bulb of mice.

The murine olfactory system consists of two primary divisions: (1) a main olfactory system, in which olfactory sensory neurons (OSNs) located in the main olfactory epithelium (MOE) send their axons to glomeruli in the main olfactory bulb (MOB); and (2) an accessory olfactory system, in which OSNs located in the vomeronasal organ send their axons to glomeruli in the accessory olfactory bulb (AOB). In labeling studies using the lectin Ulex europaeus agglutinin (UEA), we discovered a novel subset of small neuropilar structures in the MOB that are distinct from other glomeruli both in the MOB and AOB. These "microglomeruli" are morphologically similar to MOB glomeruli in many respects: they receive innervation from processes present in the olfactory nerve layer and are isolated from other glomeruli by juxtaglomerular cells; in addition, the compartmental pattern of UEA labeling suggests the presence of UEA (-) processes within their neuropil. Microglomeruli contained processes that express the olfactory marker protein, a marker common to mature OSN axons. However, unlike other glomerular structures, the microglomeruli did not contain neural cell adhesion molecule-labeled processes. Within microglomeruli, UEA(+) processes interdigitated with MAP2(+) dendrites, some of which likely originate from interneurons, as indicated by glutamic acid decarboxylase labeling. Synaptophysin labeling in microglomeruli strongly suggested that synapses occur between UEA(+) processes and dendrites. Anterograde labeling of OSNs, by injection of rhodamine-dextran into one naris, demonstrated that UEA(+) processes in microglomeruli originated in the MOE. The unique morphology, protein expression, and location of microglomeruli have led us to hypothesize that they represent a novel class of glomerular structures in the murine olfactory system.