Glycosylation Patterns Are Sexually Dimorphic throughout Development of the Olfactory System in Manduca sexta NICHOLAS J. GIBSON, * JOHN G. HILDEBRAND, AND LESLIE P. TOLBERT Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, Arizona 85721 ABSTRACT In the moth Manduca sexta, development of the adult olfactory system depends on complex interactions between olfactory receptor neurons in the antenna, antennal-lobe neu- rons in the brain, and several classes of glial cells. As one approach to characterizing molecules that may play roles in these interactions, we used lectins to screen antennae and antennal lobes at different stages of adult development. We find that each of the major neural cell types has a distinct pattern of labeling by lectins. Effects of enzymatic and other treatments on lectin labeling lead us to conclude that the predominant lectin ligands are: glycosphingolipids and an O-linked, fucose-containing glycoprotein on axons of olfactory receptor neurons, O-linked glycoproteins on antennal-lobe neurons, and N-linked glycopro- teins on all classes of glial cells in the primary olfactory pathway. Wheat germ agglutinin labels all olfactory axons uniformly during much of development, but labeling becomes restricted to the pheromone-responsive olfactory receptor neurons in the adult male. Succi- nylated WGA reveals differences in these axon classes earlier, as glomerului develop from protoglomeruli. The adult female displays a less pronounced difference in labeling of axons targeting ordinary and sexually dimorphic glomeruli. Differences in labeling of receptor axons targeted to ordinary and sexually dimorphic glomeruli may be correlated with differ- ences in function or connectivity in different regions of the antennal lobe. J. Comp. Neurol. 476:1–18, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: glycosphingolipids; glycoproteins; lectins; antennal system One of the most important questions in neurobiology concerns the mechanisms by which discrete populations of neurons and glial cells establish neural circuitry via a combination of proliferation and migration, outgrowth of axons to specific targets, and formation of synaptic con- nections. Development of the olfactory system during metamorphosis from larva to adult in the moth Manduca sexta has served as a valuable model for these develop- mental processes. Numerous studies of the formation of the antennal (olfactory) lobe (AL) of the brain have re- vealed that complex interactions between olfactory recep- tor neurons (ORNs) and several types of glial cells are required (see Oland and Tolbert, 1996, 2003; Hildebrand et al., 1997, for reviews). Previous work indicates that critical steps in the formation of synaptic glomeruli in the AL are: 1) ingrowth of ORN axons from the olfactory epithelium to the AL; 2) outward migration of a subset of glial cells along the ingrowing axons to form an axon sorting zone; 3) interaction of ORN axons with glial cells in the sorting zone, which causes the axons to respecify their association and targeting based on glomerular spec- ificity; 4) formation of a protoglomerular template by the ORN terminals; 5) establishment and maintenance of glo- merular boundaries by neuropil-associated glial cells in response to signals from the ORN axons; and 6) develop- ment of synapses between ORN axon terminals and cen- trally derived interneurons and output neurons (Fig. 1). The developmental roles played by AL interneurons (whose cell bodies are found in the lateral group of AL cell Grant sponsor: National Institutes of Health; Grant number: P01- NS28495 *Correspondence to: Nicholas J. Gibson, Arizona Research Laboratories Division of Neurobiology, Room 611 Gould-Simpson Bldg., 1040 E. 4th St., University of Arizona, Tucson, Arizona 85721-0077. E-mail: njgibson@neurobio.arizona.edu Received 20 November 2003; Revised 10 February 2004; Accepted 24 March 2004 DOI 10.1002/cne.20178 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 476:1–18 (2004) © 2004 WILEY-LISS, INC.