The Histochemical Journal 34: 281–289, 2002. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. Heterogeneity in olfactory neurons in mouse revealed by differential expression of glycoconjugates James A. St John 1 & Brian Key 1,2 1 Department of Anatomy and Developmental Biology, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Australia 2 Centre for Functional and Applied Genomics, University of Queensland, Brisbane 4072, Australia Received 4 October 2002 and in revised form 20 November 2002 Summary Cell surface glycoconjugates have been implicated in the growth and guidance of subpopulations of primary olfactory axons. While subpopulations of primary olfactory neurons have been identified by differential expression of carbohydrates in the rat there are few reports of similar subpopulations in the mouse. We have examined the spatiotemporal expression pattern of glycoconjugates recognized by the lectin from Wisteria floribunda (WFA) in the mouse olfactory system. In the developing olfactory neuroepithelium lining the nasal cavity, WFA stained a subpopulation of primary olfactory neurons and the fascicles of axons projecting to the target tissue, the olfactory bulb. Within the developing olfactory bulb, WFA stained the synaptic neuropil of the glomerular and external plexiform layers. In adults, strong expression of WFA ligands was observed in second- order olfactory neurons as well as in neurons in several higher order olfactory processing centres in the brain. Similar, although distinct, staining of neurons in the olfactory pathway was detected with Dolichos biflorus agglutinin. These results demonstrate that unique subpopulations of olfactory neurons are chemically coded by the expression of glycoconjugates. The conserved expression of these carbohydrates across species suggests they play an important role in the functional organization of this region of the nervous system. Introduction Primary olfactory neurons expressing the same odorant receptor project their axons to one to several glomeruli that are in topographically fixed positions in each olfactory bulb (Vassar et al. 1993, 1994, Ressler et al. 1994, Mombaerts et al. 1996, Royal & Key 1999). Odorant receptors are involved in the final targeting of axons to specific glomeruli; however other guidance cues are required to guide axons from the olfactory neuroepithelium to the region of nerve fibre layer proximal to the target glomerulus. The highly restricted expression of specific carbohydrates by subsets of primary olfactory axons suggests to us that a glycocode may contribute to the targeting of primary olfactory axons (Key & St John 2002, St John et al. 2002). We have demon- strated that at least one carbohydrate, novel carbohydrate-3 (NOC-3) is expressed by axons that consistently target the same topographically fixed glomeruli (St John & Key 2001), which is consistent with carbohydrates having a role in axon guidance. The development of monoclonal antibodies against car- bohydrate antigens led to the observations that sensory neu- rons expressed carbohydrates. The differential expression of carbohydrates on subsets of sensory neurons was first iden- tified by Dodd et al. (1984) in the somatosensory system. Within the olfactory system, Fujita et al. (1985) first demon- strated the expression of carbohydrates by subsets of primary olfactory axons. It was found that axons arising from the ventrolateral neuroepithelium projected to the ventrolateral bulb. The mosaic distribution of subsets of primary olfac- tory neurons was then described by Allen and Akeson (1985) who demonstrated that glycoproteins with sialic acid and D-galactosyl components were differentially and mosaically expressed by neurons, however, it was not shown that their axons terminated in specific glomeruli. Key and Giorgi (1986) showed for the first time that subsets of neurons mosaically distributed in the olfactory neuroepithelium projected to sub- populations of glomeruli. Together, these results established that carbohydrates were differentially expressed by subsets of olfactory neurons and therefore potentially could contribute to establishing the olfactory topographic map. How does the expression of cell surface carbohydrates contribute to axon guidance in the olfactory system? It is likely that these carbohydrates interact with endogenous lectins. Regan et al. (1986) first demonstrated that endoge- nous lectins were selectively expressed in the somatosensory system; however, a role for these lectins was not revealed until Mahanthappa et al. (1994) showed that the endogenous lectin, L-14 or galectin-1, can promote axonal adhesion and fasciculation in the olfactory system. This galectin (St John & Key 1999) is expressed by the specialized glial cells, the olfactory ensheathing cells, within the olfactory nerve path- way. The expression of specific carbohydrates by subsets of axons that are cross-linked by bivalent lectins would enable