Laminar Organization of the Developing Lateral Olfactory Tract Revealed by Differential Expression of Cell Recognition Molecules KOICHIRO INAKI, 1 SACHIKO NISHIMURA, 2 TOSHIAKI NAKASHIBA, 2 SHIGEYOSHI ITOHARA, 2 AND YOSHIHIRO YOSHIHARA 1 * 1 Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Saitama 351-0198, Japan 2 Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Saitama 351-0198, Japan ABSTRACT The projection neurons in the olfactory bulb (mitral and tufted cells) send axons through the lateral olfactory tract (LOT) onto several structures of the olfactory cortex. However, little is known of the molecular and cellular mechanisms underlying establishment of functional connectivity from the bulb to the cortex. Here, we investigated the developmental process of LOT formation by observing expression patterns of cell recognition molecules in embryonic mice. We immunohistochemically identified a dozen molecules expressed in the developing LOT and some of them were localized to subsets of mitral cell axons. Combinatorial immu- nostaining for these molecules revealed that the developing LOT consists of three laminas: superficial, middle, and deep. Detailed immunohistochemical, in situ hybridization, and 5-bromodeoxyuridine labeling analyses suggested that the laminar organization reflects: 1) the segregated pathways from the accessory and main olfactory bulbs, and 2) the different maturity of mitral cell axons. Mitral cell axons of the accessory olfactory bulb were localized to the deep lamina, segregated from those of the main olfactory bulb. In the main olfactory pathway, axons of mature mitral cells, whose somata is located in the apical sublayer of the mitral cell layer, were localized to the middle lamina within LOT, while those of immature mitral cells that located in the basal sublayer were complementarily localized to the super- ficial lamina. These results suggest that newly generated immature axons are added to the most superficial lamina of LOT successively, leading to the formation of piled laminas with different maturational stages of the mitral cell axons. J. Comp. Neurol. 479:243–256, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: axon guidance molecule; cell adhesion molecule; lateral olfactory tract; mitral cell; olfactory bulb; olfactory cortex The olfactory system possesses sophisticated molecular and cellular equipment to distinguish enormous numbers of odorant molecules and to build up odor images of ob- jects. In mice, this feat is accomplished partly by dedicat- ing more than 3% of the genome for generation of a thou- sand odorant receptors that are expressed by the olfactory sensory neurons (OSNs) in the olfactory epithelium (Buck and Axel, 1991) and by establishing the refined neuronal circuitry from the nose to the brain for odor information transfer, processing, and coding (Mori et al., 1999; Buck, 2000; Mombaerts, 2001). OSNs in the olfactory epithelium project their axons onto glomeruli in the main olfactory bulb (MOB), where Grant sponsor: Japan Science and Technology Corporation (JST)/Special Coordination Funds for Promoting Science and Technology; Grant sponsor: JST/Grant-in-Aid for Scientific Research on Priority Areas (C); Grant spon- sor: Ministry of Education, Culture, Sports, Science and Technology of Japan/Advanced Brain Science Project. *Correspondence to: Yoshihiro Yoshihara, Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. E-mail: yoshihara@brain.riken.go.jp Received 12 April 2004; Revised 17 June 2004; Accepted 18 June 2004 DOI 10.1002/cne.20270 Published online in Wiley InterScience (www.interscience.wiley.com). THE JOURNAL OF COMPARATIVE NEUROLOGY 479:243–256 (2004) © 2004 WILEY-LISS, INC.