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Published 2001 | public
Journal Article

Immunolocalization of Synaptotagmin for the Study of Synapses in the Developing Antennal Lobe of Manduca sexta


In the mature olfactory systems of most organisms that possess a sense of smell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of olfactory glomeruli has been studied particularly heavily in the antennal lobe of the moth Manduca sexta. In the current study, we address the development of synapses within the antennal lobe of M. sexta by reporting on the localization of synaptotagmin, a ubiquitous synaptic vesicle protein, throughout development. A cDNA clone coding for M. sexta synaptotagmin was characterized and found to encode a protein that shares 67% amino acid identity with Drosophila synaptotagmin and 56% amino acid identity with human synaptotagmin I. Conservation was especially high in the C2 domains near the C-terminus and very low near the N-terminus. A polyclonal antiserum (MSYT) was raised against the unique N-terminus of M. sexta synaptotagmin, and a monoclonal antibody (DSYT) was raised against the highly conserved C-terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labeled a 60 kD protein, which very likely corresponds to synaptotagmin. On sections, both antibodies labeled known synaptic neuropils in M. sexta and yielded similar labeling patterns in the developing antennal lobe. In addition, DSYT detected synaptotagmin-like protein in three other insect species examined. Analysis of synaptotagmin labeling at the light microscopic level during development of the antennal lobe of M. sexta confirmed and extended previous electron microscopic studies. Additional synapses in the coarse neuropil and a refinement of synaptic densities in the glomeruli during the last one-third of metamorphic development were revealed.

Additional Information

© 2001 Wiley-Liss, Inc. Received 17 January 2001; Revised 6 July 2001; Accepted 19 September 2001. Article first published online: 28 Nov 2001. The first two authors contributed equally to this work. The authors thank Dr. Achim Paululat for providing D. melanogaster embryos and prepupa and for his support in immunostaining D. melanogaster embryos. The authors also thank Dr. Nicholas Gibson for his insight and technical assistance and Dr. A.A. Osman for assistance in animal rearing. The study was supported by grants from the National Institutes of Health to L.P.T. (NS20040) and A.J.N. (DC04292) and a grant from the Deutsche Forschungsgemeinschaft to J.S. (Scha 678/3-1).

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