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 s mell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of ol factory glomeruli has been studied particularly heavily in the antennal lob e of the moth Manduca sexta. In the current study, we address the developme nt of synapses within the antennal lobe of M. sexta by reporting on the loc alization of synaptotagmin, a ubiquitous synaptic vesicle protein, througho ut development. A cDNA clone coding for M. sexta synaptotagmin was characte rized and found to encode a protein that shares 67% amino acid identity wit h Drosophila synaptotagmin and 56% amino acid identity with human synaptota gmin I. Conservation was especially high in the C2 domains near the C-termi nus and very low near the N-terminus. A polyclonal antiserum (MSYT) was rai sed against the unique N-terminus of M. sexta synaptotagmin, and a monoclon al antibody (DSYT) was raised against the highly conserved C-terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labe led a 60 kD protein, which very likely corresponds to synaptotagmin. On sec tions, both antibodies labeled known synaptic neuropils in M. sexta and yie lded similar labeling patterns in the developing antennal lobe. In addition , DSYT detected synaptotagmin-like protein in three other insect species ex amined. Analysis of synaptotagmin labeling at the light microscopic level d uring development of the antennal lobe of M. sexta confirmed and extended p revious electron microscopic studies. Additional synapses in the coarse neu ropil and a refinement of synaptic densities in the glomeruli during the la st one-third of metamorphic development were revealed.(C) 2001 Wiley-Liss, Inc.