Cell and Tissue Research

, Volume 319, Issue 3, pp 513–524 | Cite as

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

  • Wolf Huetteroth
  • Joachim Schachtner
Regular Article


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.


3D reconstruction AMIRA Brain Glomerular map Olfactory system Manduca sexta males, (Insecta) 



The authors thank Dr. Kaushiki Menon (Caltech, USA) for kindly providing the synaptotagmin antiserum and Dr. Robert Brandt (Zuse Institute, Berlin, Germany) for his excellent support with respect to the AMIRA software. This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Grant Scha 678/3-3) to JS.


  1. Anton S, Gadenne C (1999) Effect of juvenile hormone on the central nervous processing of sex pheromone in an insect. Proc Natl Acad Sci USA 96:5764–5767CrossRefGoogle Scholar
  2. Baumann PM, Oland LA, Tolbert LP (1996) Glial cells stabilize axonal protoglomeruli in the developing olfactory lobe of the moth Manduca sexta. J Comp Neurol 373:118–128Google Scholar
  3. Bell RA, Joachim FA (1978) Techniques for rearing laboratory colonies of the tobacco hornworm, Manduca sexta and pink ballworms. Ann Entomol Soc Am 69:365–373Google Scholar
  4. Berg BG, Galizia CG, Brandt R, Mustaparta H (2002) Digital atlases of the antennal lobe in two species of tobacco budworm moths, the Oriental Helicoverpa assulta (male) and the American Heliothis virescens (male and female). J Comp Neurol 446:123–134CrossRefGoogle Scholar
  5. Brown SM, Napper RM, Thompson CM, Mercer AR (2002) Stereological analysis reveals striking differences in the structural plasticity of two readily identifiable glomeruli in the antennal lobes of the adult worker honeybee. J Neurosci 22:8514–8522Google Scholar
  6. Brown SM, Napper RM, Mercer AR (2004) Foraging experience, glomerulus volume, and synapse number: a stereological study of the honey bee antennal lobe. J Neurobiol 60:40–50CrossRefGoogle Scholar
  7. Bucher D, Scholz M, Stetter M, Obermayer K, Pflüger HJ (2000) Correction methods for three-dimensional reconstructions from confocal images. I. Tissue shrinking and axial scaling. J Neurosci Methods 100:135–143CrossRefGoogle Scholar
  8. Chambille I, Rospars JP (1981) Le deutocérébron de la blatte Blaberus craniifer Burm. (Dictyoptera: Blaberidae). Etude qualitative et identification visuelle de glomérules. Int J Insect Morphol Embryol 10:141–165Google Scholar
  9. Chiang AS, Liu YC, Chiu SL, Hu SH, Huang CY, Hsieh CH (2001) Three-dimensional mapping of brain neuropils in the cockroach, Diploptera punctata. J Comp Neurol 440:1–11Google Scholar
  10. Daly KC, Christensen TA, Lei H, Smith BH, Hildebrand JG (2004) Learning modulates the ensemble representations for odors in primary olfactory networks. Proc Natl Acad Sci USA 101:10476–10481CrossRefGoogle Scholar
  11. Devaud JM, Masson C (1999) Dendritic pattern development of the honeybee antennal lobe neurons: a laser scanning confocal microscopic study. J Neurobiol 39:461–474CrossRefGoogle Scholar
  12. Devaud JM, Acebes A, Ferrus A (2001) Odor exposure causes central adaptation and morphological changes in selected olfactory glomeruli in Drosophila. J Neurosci 21:6274–6282Google Scholar
  13. Devaud JM, Acebes A, Ramaswami M, Ferrus A (2003) Structural and functional changes in the olfactory pathway of adult Drosophila take place at a critical age. J Neurobiol 56:13–23CrossRefGoogle Scholar
  14. Dubuque SH, Schachtner J, Nighorn AJ, Menon KP, Zinn K, Tolbert LP (2001) Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta. J Comp Neurol 441:277–287CrossRefGoogle Scholar
  15. Eisthen HL (2002) Why are olfactory systems of different animals so similar? Brain Behav Evol 59:273–293CrossRefGoogle Scholar
  16. Farris SM, Robinson GE, Fahrbach SE (2001) Experience- and age-related outgrowth of intrinsic neurons in the mushroom bodies of the adult worker honeybee. J Neurosci 21:6395–6404Google Scholar
  17. Flanagan D, Mercer AR (1989) An atlas and 3-D reconstruction of the antennal lobes in the worker honey bee, Apis mellifera L. (Hymenoptera: Apidae). Int J Insect Morphol Embryol 18:145–159CrossRefGoogle Scholar
  18. Gadenne C, Anton S (2000) Central processing of sex pheromone stimuli is differentially regulated by juvenile hormone in a male moth. J Insect Physiol 46:1195–1206CrossRefGoogle Scholar
  19. Gahm T, Witte S (1986) Measurement of the optical thickness of transparent tissue layers. J Microsc 141:101–110Google Scholar
  20. Galizia CG, McIlwrath SL, Menzel R (1999) A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquired by confocal microscopy. Cell Tissue Res 295:383–394CrossRefGoogle Scholar
  21. Gibson NJ, Hildebrand JG, Tolbert LP (2004) Glycosylation patterns are sexually dimorphic throughout development of the olfactory system in Manduca sexta. J Comp Neurol 476:1–18Google Scholar
  22. Greiner B, Gadenne C, Anton S (2002) Central processing of plant volatiles in Agrotis ipsilon males is age-independent in contrast to sex pheromone processing. Chem Senses 27:45–48CrossRefGoogle Scholar
  23. Greiner B, Gadenne C, Anton S (2004) Three-dimensional antennal lobe atlas of the male moth, Agrotis ipsilon: a tool to study structure-function correlation. J Comp Neurol 475:202–210CrossRefGoogle Scholar
  24. Guerenstein PG, Christensen TA, Hildebrand JG (2004) Sensory processing of ambient CO2 information in the brain of the moth Manduca sexta. J Comp Physiol [A] 190:707–725Google Scholar
  25. Haddad D, Schaupp F, Brandt R, Manz G, Menzel R, Haase A (2004) NMR imaging of the honeybee brain. J Insect Sci 4:1–7Google Scholar
  26. Hildebrand JG, Shepherd GM (1997) Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Annu Rev Neurosci 20:595–631CrossRefPubMedGoogle Scholar
  27. Hildebrand JG, Rössler W, Tolbert LP (1997) Postembryonic development of the olfactory system in the moth Manduca sexta: primary-afferent control of glomerular development. Cell Dev Biol 8:163–170CrossRefGoogle Scholar
  28. Homberg U, Hoskins SG, Hildebrand JG (1995) Distribution of acetylcholinesterase activity in the deutocerebrum of the sphinx moth Manduca sexta. Cell Tissue Res 279:249–259CrossRefPubMedGoogle Scholar
  29. Huetteroth W, Schachtner J (2003) 3D reconstructions of pupal and adult glomeruli in the antennal lobe of the sphinx moth Manduca sexta. Proc Fifth German Neurosci Soc Conf 689:735Google Scholar
  30. Jindra M, Huang JY, Malone F, Asahina M, Riddiford LM (1997) Identification and mRNA developmental profiles of two ultraspiracle isoforms in the epidermis and wings of Manduca sexta. Insect Mol Biol 6:41–53CrossRefGoogle Scholar
  31. Julian GE, Gronenberg W (2002) Reduction of brain volume correlates with behavioral changes in queen ants. Brain Behav Evol 60:152–164CrossRefGoogle Scholar
  32. Kent KS, Harrow ID, Quartararo P, Hildebrand JG (1986) An accessory olfactory pathway in Lepidoptera: the labial pit organ and its central projections in Manduca sexta and certain other sphinx moths and silk moths. Cell Tissue Res 245:237–245PubMedGoogle Scholar
  33. Kent KS, Oland LA, Hildebrand JG (1999) Development of the labial pit organ glomerulus in the antennal lobe of the moth Manduca sexta: the role of afferent projections in the formation of identifiable olfactory glomeruli. J Neurobiol 40:28–44CrossRefPubMedGoogle Scholar
  34. Laissue PP, Reiter C, Hiesinger PR, Halter S, Fischbach KF, Stocker RF (1999) Three-dimensional reconstruction of the antennal lobe in Drosophila melanogaster. J Comp Neurol 405:543–552Google Scholar
  35. Lee JK, Strausfeld NJ (1990) Structure, distribution and number of surface sensilla and their receptor cells on the olfactory appendage of the male moth Manduca sexta. J Neurocytol 19:519–538Google Scholar
  36. Malun D, Oland LA, Tolbert LP (1994) Uniglomerular projection neurons participate in early development of olfactory glomeruli in the moth Manduca sexta. J Comp Neurol 350:1–22Google Scholar
  37. Mechaber WL, Capaldo CT, Hildebrand JG (2002) Behavioral responses of adult female tobacco hornworms, Manduca sexta, to hostplant volatiles change with age and mating status. J Insect Sci 2:1–8Google Scholar
  38. Oland LA, Tolbert LP (1988) Effects of hydroxyurea parallel the effects of radiation in developing olfactory glomeruli in insects. J Comp Neurol 278:377–387Google Scholar
  39. Oland LA, Tolbert LP (1996) Multiple factors shape development of olfactory glomeruli: insights from an insect model system. J Neurobiol 30:92–109Google Scholar
  40. Oland LA, Tolbert LP, Mossman KL (1988) Radiation-induced reduction of the glial population during development disrupts the formation of olfactory glomeruli in an insect. J Neurosci 8:353–367Google Scholar
  41. Oland LA, Orr G, Tolbert LP (1990) Construction of a protoglomerular template by olfactory axons initiates the formation of olfactory glomeruli in the insect brain. J Neurosci 10:2096–2112Google Scholar
  42. Oland LA, Marrero HG, Burger I (1999) Glial cells in the developing and adult olfactory lobe of the moth Manduca sexta. Cell Tissue Res 297:527–545CrossRefGoogle Scholar
  43. Ott SR, Elphick MR (2003) New techniques for whole-mount NADPH-diaphorase histochemistry demonstrated in insect ganglia. J Histochem Cytochem 51:523–532Google Scholar
  44. Rein K, Zöckler M, Heisenberg M (1999) A quantitative three-dimensional model of the Drosophila optic lobes. Curr Biol 9:93–96CrossRefGoogle Scholar
  45. Rein K, Zöckler M, Mader MT, Grubel C, Heisenberg M (2002) The Drosophila standard brain. Curr Biol 12:227–231CrossRefGoogle Scholar
  46. Rohlfing T, Brandt R, Menzel R, Maurer CR Jr (2004) Evaluation of atlas selection strategies for atlas-based image segmentation with application to confocal microscopy images of bee brains. Neuroimage 21:1428–1442CrossRefGoogle Scholar
  47. Rospars JP (1983) Invariance and sex-specific variations of the glomerular organization in the antennal lobes of a moth, Mamestra brassicae, and a butterfly, Pieris brassicae. J Comp Neurol 220:80–96Google Scholar
  48. Rospars JP, Hildebrand JG (1992) Anatomical identification of glomeruli in the antennal lobes of the male sphinx moth Manduca sexta. Cell Tissue Res 270:205–227CrossRefGoogle Scholar
  49. Rospars JP, Hildebrand JG (2000) Sexually dimorphic and isomorphic glomeruli in the antennal lobes of the sphinx moth Manduca sexta. Chem Senses 25:119–129CrossRefPubMedGoogle Scholar
  50. Ruffins SW, Jacobs RE, Fraser SE (2002) Towards a Tralfamadorian view of the embryo: multidimensional imaging of development. Curr Opin Neurobiol 12:580–586CrossRefGoogle Scholar
  51. Sadek MM, Hansson BS, Rospars JP, Anton S (2002) Glomerular representation of plant volatiles and sex pheromone components in the antennal lobe of the female Spodoptera littoralis. J Exp Biol 205:1363–1376Google Scholar
  52. Schachtner J, Huetteroth W, Nighorn A, Honegger HW (2004a) Copper/zinc superoxide dismutase-like immunoreactivity in the metamorphosing brain of the sphinx moth Manduca sexta. J Comp Neurol 469:141–152CrossRefGoogle Scholar
  53. Schachtner J, Trosowski B, D’Hanis W, Stubner S, Homberg U (2004) Development and steroid regulation of RFamide immunoreactivity in antennal-lobe neurons of the sphinx moth Manduca sexta. J Exp Biol 207:2389–2400CrossRefGoogle Scholar
  54. Schwartz LM, Truman JW (1983) Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta. Dev Biol 99:103–114CrossRefGoogle Scholar
  55. Scott EK, Reuter JE, Luo L (2003a) Dendritic development of Drosophila high order visual system neurons is independent of sensory experience. BMC Neurosci 4:14CrossRefGoogle Scholar
  56. Scott EK, Reuter JE, Luo L (2003b) Small GTPase Cdc42 is required for multiple aspects of dendritic morphogenesis. J Neurosci 23:3118–3123Google Scholar
  57. Sigg D, Thompson CM, Mercer AR (1997) Activity-dependent changes to the brain and behavior of the honey bee, Apis mellifera (L.). J Neurosci 17:7148–7156Google Scholar
  58. Smid HM, Bleeker MA, Loon JJ van, Vet LE (2003) Three-dimensional organization of the glomeruli in the antennal lobe of the parasitoid wasps Cotesia glomerata and C. rubecula. Cell Tissue Res 312:237–248Google Scholar
  59. Strausfeld NJ, Hildebrand JG (1999) Olfactory systems: common design, uncommon origins? Curr Opin Neurobiol 9:634–639CrossRefPubMedGoogle Scholar
  60. Tolbert LP (1989) Afferent axons from the antenna influence the number and placement of intrinsic synapses in the antennal lobes of Manduca sexta. Synapse 3:83–95CrossRefGoogle Scholar
  61. Tolbert LP, Sirianni PA (1990) Requirement for olfactory axons in the induction and stabilization of olfactory glomeruli in an insect. J Comp Neurol 298:69–82CrossRefGoogle Scholar
  62. Tolbert LP, Matsumoto SG, Hildebrand JG (1983) Development of synapses in the antennal lobes of the moth Manduca sexta during metamorphosis. J Neurosci 3:1158–1175PubMedGoogle Scholar
  63. Tolbert LP, Oland LA, Tucker ES, Gibson NJ, Higgins MR, Lipscomb BW (2004) Bidirectional influences between neurons and glial cells in the developing olfactory system. Prog Neurobiol 73:73–105CrossRefGoogle Scholar
  64. Truman JW (1996) Metamorphosis of the insect nervous system. In: Gilbert LI (ed) Metamorphosis: postembryonic reprogramming of gene expression in amphibian and insect cells. Academic Press, Orlando, pp 283–320Google Scholar
  65. Van Essen DC (2002) Windows on the brain: the emerging role of atlases and databases in neuroscience. Curr Opin Neurobiol 12:574–579CrossRefGoogle Scholar
  66. Weevers RD (1966) A lepidopteran saline: effects of inorganic cation concentrations on sensory, reflex and motor responses in a herbivorous insect. J Exp Biol 44:163–175Google Scholar
  67. Willis MA, Butler MA, Tolbert LP (1995) Normal glomerular organization of the antennal lobes is not necessary for odor-modulated flight in female moths. J Comp Physiol [A] 176:205–216Google Scholar
  68. Winnington AP, Napper RM, Mercer AR (1996) Structural plasticity of identified glomeruli in the antennal lobes of the adult worker honey bee. J Comp Neurol 365:479–490CrossRefGoogle Scholar
  69. Yuste R, Bonhoeffer T (2004) Genesis of dendritic spines: insights from ultrastructural and imaging studies. Nat Rev Neurosci 5:24–34CrossRefGoogle Scholar
  70. Zöckler M, Rein K, Brandt R, Stalling D, Hege H (2001) Creating virtual insect brains with AMIRA. ZIB Report 01–32:1–11Google Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Fachbereich Biologie, TierphysiologiePhilipps-UniversitätMarburgGermany

Personalised recommendations