Standard three-dimensional glomeruli of the Manduca sexta antennal lobe: a tool to study both developmental and adult neuronal plasticity

Abstract

The metamorphosing antennal lobe (AL) of the sphinx moth Manduca sexta serves as an established model system for studying neuronal development. To improve our understanding of mechanisms involved in neuronal plasticity, we have analyzed the size, shape, and localization of ten identified glomeruli at three different time points during development and in the adult, viz., (1) 13 days after pupal eclosion (P13), which reflects a time when the basic glomerular map has formed, (2) immediately after adult eclosion (A0), which represents a time when the newly formed glomeruli are uninfluenced by external odors, and (3) 4 days after adult eclosion (A4), which reflects a time when the animals have been exposed to surrounding odors. Our data from normally developing ALs of male M. sexta from P13 to A0 revealed an increase in size of all examined glomeruli of between 40% and 130%, with the strongest increases occurring in two of the three sex-specific glomeruli (cumulus, toroid). From A0 to A4, the cumulus and toroid increased significantly when correlated to AL volume, whereas the other glomeruli reached the sizes gained after A0. This study was based on antibody staining against the ubiquitous synaptic vesicle protein synaptotagmin, confocal laser scan microscopy, and the three-dimensional (3D) analysis tool AMIRA. Tissue permeability and therefore reliability of the staining quality was enhanced by using formalin/methanol fixation. The standard 3D glomeruli introduced in this study can now be used as basic tools for further examination of neuronal plasticity at the level of the identified neuropil structures, viz., the glomeruli of the AL of M. sexta.

Different growth patterns of two adjacent glomeruli responsible for sex-pheromone processing during postembryonic development of the cockroach Periplaneta americana

“…moths), in which glomerular development mostly occurs during the pupal and early adult stages [7,9], cockroach glomeruli continually develop in each molting after hatching concurrent with the growth of the antenna [15,18,19]. Histological studies have suggested that the MGC of the cockroach is formed during the last third of postembryonic development by fusion of several small glomeruli [15].…”

“…In the hawkmoth, glomerulus development depends on the arrival of a subset of sensory axons, and this occurs at different times for different glomeruli during the pupal stage [9]. However, two adjacent male-specific macroglomeruli (called "cumulus" and "toroid") have similar glomerular volumes and grow at similar rates, at least in their late developmental phases [7].…”

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

“…After fixation, brains were either embedded in gelatin/albumin, postfixed overnight in 4% buffered formaldehyde and cut at 40 lm with a vibrating blade microtome (Leica VT 1000S) in the frontal, sagittal or horizontal plane, or processed as wholemount. Immunostaining procedure was performed exactly as described in Schachtner et al (2004b) or, for the wholemounts, as described in Huetteroth and Schachtner (2005). For triple-labeling, the anti-Mas-AT, anti-GABA, and antisynapsin antibodies were applied simultaneously and, likewise, the corresponding secondary antisera.…”

“…Digitization and 3D reconstructions of wholemount ALs labeled with anti Mas-AT and anti-synapsin antisera, visualized by corresponding secondary antisera coupled to Cy2 and Cy5, respectively, were performed with the Leica TCS SP2 and the 3D reconstruction software AMIRA 3.1-4.1.1 (Mercury Computer Systems, Chelmsford, MA) according to the protocol described in Huetteroth and Schachtner (2005). The AL outlines and cell group borders were labeled based on background staining of either data channel.…”

“…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.…”