Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta

Abstract

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.

Mas‐allatotropin in the developing antennal lobe of the sphinx moth Manduca sexta: Distribution, time course, developmental regulation, and colocalization with other neuropeptides

“…The specificity of the anti-TM-MFas II antiserum for Manduca nervous tissue was shown by Western blot analysis (Wright and Copenhaver, 2000). Anti-synaptotagmin (1:4000, kindly provided by Dr. K. Menon, Caltech, USA) and anti-synapsin antibodies (1:100, kindly provided by Dr. E. Buchner, University of Würzburg, Germany) were used to selectively label neuropil structures including olfactory glomeruli (Dubuque et al, 2001;Berg et al, 2002). M. sexta synaptotagmin was previously cloned and the specificity of the monoclonal anti-synaptotagmin antibody in Manduca brain tissue was shown by Western blot analysis and by cross comparison with a polyclonal anti-synaptotagmin antiserum recognizing a different region of the protein (Dubuque et al, 2001).…”

“…Anti-synaptotagmin (1:4000, kindly provided by Dr. K. Menon, Caltech, USA) and anti-synapsin antibodies (1:100, kindly provided by Dr. E. Buchner, University of Würzburg, Germany) were used to selectively label neuropil structures including olfactory glomeruli (Dubuque et al, 2001;Berg et al, 2002). M. sexta synaptotagmin was previously cloned and the specificity of the monoclonal anti-synaptotagmin antibody in Manduca brain tissue was shown by Western blot analysis and by cross comparison with a polyclonal anti-synaptotagmin antiserum recognizing a different region of the protein (Dubuque et al, 2001). Western blot analysis with the monoclonal anti-Synapsin I antiserum revealed one double band at a molecular weight of about 70 kDa in pupal (P8) and adult (A0) M. sexta brain tissue which is similar to the situation in Drosophila larval brain tissue (supplementary material Fig.…”

“…During this time, the whole brain undergoes reorganization and small larval antennal centers (LACs) develop into the adult ALs. AL development in M. sexta can be roughly divided into three phases Dubuque et al, 2001). The preparation phase (phase I) lasts about 7-8 days beginning at pupal formation.…”

“…The glomeruli formation phase (phase II) lasts about 5 days and is characterized by massive synaptogenesis between the involved neurons. It is assumed, that during phase II a basic network of synaptic contacts within and between the glomeruli is established, which ensures the principal correlation of in and output components of the AL (Dubuque et al, 2001). In contrast to phase II, in phase III, which lasts about 8 days up to adult eclosion, only little synaptogenesis occurs.…”

“…In contrast to phase II, in phase III, which lasts about 8 days up to adult eclosion, only little synaptogenesis occurs. During this last phase, the glomeruli grow in size between 40 and 130% (Huetteroth and Schachtner, 2005), probably because of the increasing neurite diameters, and the synaptic wiring in the glomeruli is thought to undergo further refinement and maturation (Tolbert et al, 1983;Tolbert, 1989;Dubuque et al, 2001). None of the neurons of the ALs undergo programmed cell death during phases I to III (Schachtner et al, 2004a), and, as all neurons of the ALs are born early in AL development , the neuronal composition of the ALs does not change throughout formation of the ALs.…”