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During development, olfactory bulb axons navigate a complex microenvironment composed of myriad molecules to construct a bundle called the lateral olfactory tract. The axons themselves also express thousands of different molecules. In the present study, we produced and characterized six monoclonal antibodies that label the lateral olfactory tract and its surroundings in a unique pattern. The labeling profiles suggested that the antigen molecules recognized by each antibody are heterogeneously distributed around the developing lateral olfactory tract. We developed an efficient screening method to identify the antigen molecules by combining expression of a cDNA library in COS-7 cells and the subsequent immunohistochemical staining of the cells. The systematic screening successfully identified specific cDNA clones for all of the monoclonal antibodies, which highly probably coded for the antigen molecules, and therefore unveiled the molecular nature of local components that embrace the developing lateral olfactory tract in mice.

The arrangement of axons in a tract can have a specific effect on the organization of functional neuronal circuits. Here we describe olfactory bulb axons chronologically arranged in the lateral olfactory tract. Newly differentiated projection neurons over the whole olfactory bulb are similarly marked with transient expression of c-kit protein. Their axons are assembled together and project into the ventral superficial part of the tract, displacing the older axons. This special assembly of the axons explains the nontopographic relationships between the olfactory bulb and the lateral olfactory tract axons that have been described in previous studies and could possibly influence the subsequent selection of the olfactory target areas by these axons. J. Comp. Neurol. 475:247–260, 2004. © 2004 Wiley-Liss, Inc.

After the initial primary projection, axons undergo various structural and functional changes to establish mature neural circuits. The changes in protein expression associated with this maturation were investigated in lateral olfactory tract axons using two-dimensional gel electrophoresis. The most prominent group upregulated during the period consisted of calcium-dependent membrane-binding proteins including VILIP1, neurocalcin delta, copine 6, and annexin A6 from three structurally different families. During maturation of primary cultured neurons, annexin A6 gradually became concentrated on the axon initial segment, and its overexpression significantly enhanced axon branching. On the other hand, overexpression of VILIP1 and neurocalcin delta reduced axon outgrowth and branching. The second group upregulated during axon maturation comprised tubulin- and microtubule-binding proteins including CRMP2, guanine deaminase, MAP1B, and fibronectin type3 SPRY domain-containing protein. Because the maturation of lateral olfactory axons involves massive extension of secondary collateral branches, the augmentation of these proteins during these stages may underlie the drastic restructuring of the axon cytoskeleton.

cis -Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45°C. The K m value for cis -aconitic acid was determined as 2.45 mM (pH 6.2, 37°C). The enzyme was completely inactivated by Hg + , Cu 2+ , Zn 2+ , p -chloromercuribenzoate, and 5,5′-dithio- bis (2-nitrobenzoate).

In the mammalian olfactory system, each olfactory neuron expresses only one of about 1,000 different odorant receptor genes. In the olfactory epithelium, the olfactory neurons expressing the same receptor are scattered, but their axons converge into a few topographically fixed glomeruli, the specialized synaptic structures in the olfactory bulb (OB). Consequently, a stereotyped spatial map is constructed on the surface of the OB, in which about 1,800 glomeruli are orderly arranged in mice. The physiological and anatomical studies show that the glomeruli receiving similar odorant molecules are grouped together and form a special domain on the OB. The information converged in the glomeruli is transmitted to the second-order neurons, mitral and tufted cells in the OB. These neurons project axons caudally and construct the lateral olfactory tract (LOF) on the surface of the ventrolateral telencephalon. The LOT axons eventually sprout collateral branches invading the olfactory cortex and form connections with the third-order neurons. The olfactory cortex consists of morphologically distinct areas such as the anterior olfactory nucleus, the piriform cortex (PC), the olfactory tubercle (OT), the entorhinal cortex and the amygdala. Each second-order neuron projects to the multiple target areas with massive sprouting of collateral branches. In contrast to the clear rule of the peripheral olfactory projection, the principal of central olfactory projection is obscure. Although a few studies suggest specific targeting of the projections from second-order neurons in the olfactory cortex, there is not a point-to-point topographic relationship between the spatial map on the OB to any of the olfactory cortical areas, as other sensory systems. For example, the retrograde axonal labeling shows that a single cortical region receives inputs from the mitral/tufted cells scattering over the OB. The anterograde axonal tracing also shows that the spatial arrangement in the OB is not reflected in the olfactory cortical areas. Furthermore, there is the evidence that the spatial representation of the OB is already lost in the LOT, in which mitral/tufted cell axons are intermingled randomly regardless of the position of their cell bodies. In the present study, I demonstrated that c-kit receptor tyrosine kinase is expressed on a fasciculated subset of axons in the LOT at each developmental stage. Their c-kit-expressing cell bodies were distributed in the intermediate zone flanked between the ventricular zone and the mitral cell layer in the OB. BrdU-labeling experiment showed that the newly-differentiated mitral/tufted cells radially migrating in the intermediate zone transiently expressed c-kit so that, the expression of c-kit is always fixed in the intermediate zone. These results indicate that mitral/tufted cell axons projecting at the same developmental stage are grouped together and constitute a special assembly within the LOT bundle, regardless of the position of their cell bodies in the OB. The newly elongating c-kit-positive axons usually occupied the ventral surface area in the LOT. Therefore, there seems to be a developmental gradient in the organization of LOT axons from the dorsal depth to ventral surface. These results together with the previous axonal tracing studies suggest, that arrangement of LOT axons is not, based on the topographical position of the cell bodies but the developmental status of the axons. The OT is one of the olfactory cortical areas that receive the late innervation by OB axons. The OT receives a heavier projection from tufted cells the late-born projection neurons in the OB. In later developmental stages, many c-kit-positive axons were observed to project into the OT selectively and directly. This observation further supports that the OT receives the selective projection from the late-born projection neurons in the OB. In the LOT, c-kit-positive axons were always fasciculated and segregated from c-kit-negative axons. When OB explants were cultured in the dish, c-kit-positive neturites did not fasciculate but randomly mixed with c-kit-negative neurites. Even in the organotypic co-culture, c-kit-positive axons did not choose the superficial part but randomly elongated within the LOT bundle, intermingling with c-kit-negative axons. Thus, the selective fasciculation of c-kit-positive axons in the LOT was not, reproduced in culture and might require developmentally ordered projection of LOT axons. The c-kit is encoded by the Wlocus in mice and the ligand for c-kit is stem cell factor(SCF), which encoded by the Sllocus in mice. The interaction between c-kit and SCF is considered to be essential for the development of melanocytes, erythrocytes, mast cells and germ cells, because mutations in either the W or Sl locus result in serious defects in differentiation of these cells. I examined whether c-kit or SCF was involved in the projection of LOT axons. However, I did not detect any abnormality in the LOT projection of W/W or Sl/Sl d mutant mice, although it is still possible that more detailed analyses reveal some function of this signaling.