Cellular Mechanisms of axon guidance and targeting in the olfactory system

Olfactory sensenory neurons (OSNs) of the olfactory epithelium are located in the nasal cavity and project axons that synapse onto dendrites of second-order neurons within the olfactory bulb in neuropil structures termed glomeruli. Each OSN expresses one of the over 1000 odorant receptors (ORs) and are randomly dispersed across one of the four partially overlapping regions of the olfactory epithelium. OSNs expressing a single OR project their axons to typically two symmetrically bilateral glomeruli, one each on the medial and lateral surfaces of the olfactory bulb. The target choice of the OSN axons appears to depend on a combination of molecular determinants that first promote segregation of axons into broad regions of the olfactory bulb and then favor sorting and convergence into specific glomeruli. The role of ORs has clearly been demonstrated to play a significant role in axon sorting and targeting to the glomeruli, however other guidance molecules have clearly been shown to be required in the precise targeting of the OSN axons. During development of the mouse olfactory system, olfactory sensory axons first contact their terminal zone in the glomerular layer of the olfactory bulb during late embryonic period and target to their appropriate glomeruli over the next few days. During this period, many axons can branch inappropriately into several glomeruli and overshoot the target layer and enter deeper layers of the olfactory bulb. The aberrant axonal projections are normally detected within the deeper layers of the bulb up to postnatal day 12. By the end of the second postnatal week, axon trajectories are refined and axons over-projections are seldom observed in adult animals. A detailed understanding of the process of axon over-projections enabled the identification of directional cues that navigate the outgrowing axons to their defined destination. There are two aims of the present thesis, first, to investigate the role of cell surface carbohydrates in the molecular and cellular mechanisms underlying axon navigation in the developing olfactory system. To achieve this aim, transgenic mice expressing two glycosyltransferases (Mgat3 and 5) under the control of OMP promoter to drive expression in all mature OSNs have been generated. The results obtained from these transgenic mice showed that the transgene is expressed based on the ability to detect GFP reporter expression, however no apparent targeting defect in the olfactory pathway was observed from these transgenic mice. The purpose of the second study was twofold: 1) To examine the trajectory of olfactory sensory axons that penetrate past the glomerular layer in OMP-Zsgreen transgenic mice and 2) using in vitro cultures of the olfactory epithelium from OMP-Zsgreen mice which allowed visualization of mature OSN and their axons, together with extract prepared from inner layers of olfactory bulb to elucidate the impact of distinct molecular and cellular cues on defined OSN populations. Our results revealed that targeting of OSN axons was initially imprecise where extensive axons over-projection was observed in early postnatal animals. This targeting error was then refined later in development after the first two postnatal weeks and rarely observed in adult animals. The in vitro study using cultured OE explants to examine the effect of the extract prepared from the inner layers of the olfactory bulb on the axon outgrowth suggests the presence of a repellent activity in the inner layers of the olfactory bulb and that this inibition is stronger in P17.5 animals than P8.5 and P2.5 animals.