Each olfactory (antennal) lobe of the moth Manduca sexta contains a single serotonin (5-HT) immunoreactive neuron whose processes form tufted arbors in the olfactory glomeruli. To extend our present understanding of the intercellular interactions involved in glomerulus development to the level of an individual, identified antennal lobe neuron, we first studied the morphological development of the 5-HT neuron in the presence and absence of receptor axons. Development of the neuron's glomerular tufts depends, as it does in the case of other multiglomerular neurons, on the presence of receptor axons. Processes of the 5-HT neuron are excluded from the region in which the initial steps of glomerulus construction occur and thus cannot provide a physical scaffolding on which the array of glomeruli is organized. Because the neuron's processes are present in the antennal lobe neuropil throughout postembryonic development, 5-HT could provide signals that influence the pattern of development in the lobe. By surgically producing 5-HT-depleted antennal lobes, we also tested the importance of 5-HT in the construction of olfactory glomeruli. Even in the apparent absence of 5-HT, the glomerular array initiated by the receptor axons was histologically normal, glial cells migrated to form glomerular borders, and receptor axons formed terminal branches in their normal region within each glomerulus. In some cases, 5-HT-immunoreactive processes from abnormal sources entered the lobe and formed the tufted intraglomerular branches typical of most antennal lobe neurons, suggesting that local cues strongly influence the branching patterns of developing antennal lobe neurons.

Olfactory receptor axons in many species terminate centrally in an array of distinct glomeruli that are thought to encode the molecular features of odors. Particular molecular attributes are detected by receptor neurons widely distributed over the sensory epithelium, but these neurons then project to a small number of glomeruli in the olfactory bulb. This raises perplexing questions about olfactory axon guidance, especially how axons sort by odor specificity and how they find their appropriate targets in the brain. Taking advantage of the relative cellular simplicity of the moth antennal system, we have examined receptor axons in normally developing animals and also in preparations in which the nerve was experimentally misrouted. Just before they enter the antennal lobe, receptor axons undergo a dramatic reorganization in a discrete zone filled with glial cells. Here they shed neighbor relationships and become associated with axons that have common targets and presumably share common odor specificities. Electron microscopy revealed that the growth cones of early arriving axons travel preferentially next to glial processes. The growth cones of receptor axons were relatively simple except as they entered newly forming glomeruli. Misrouted nerves turned and ran along the surface of the brain until they reached the region of the antennal lobe. In only 6% of cases did misrouted axons enter the brain ectopically, never forming glomeruli. Our results suggest that olfactory receptor axons are attracted to the antennal lobe by soluble or surface-bound cues and sort by odor specificity by using a mechanism that may involve glial cells.

During metamorphic development, the arrival at the olfactory (antennal) lobe of olfactory receptor axons initiates the process of glomerulus formation. The glomeruli are discrete spheroidal regions of neuropil that are the sites of synaptic interactions among receptor neurons and their target antennal-lobe neurons. The process of glomerulus formation begins as groups of receptor axons form protoglomeruli. These dense clusters of terminal branches mostly are discrete entities from the time they can be recognized, although a few branches from neighboring protoglomeruli overlap laterally. A previous study by Schweitzer et al. [Schweitzer E. S., Sanes J. R. and Hildebrand J. G. (1976) Ontogeny of electroantennogram responses in the moth, Manduca sexta. J. Insect Physiol. 22, 955-960] has shown that odor-induced activity in the receptor neurons can be detected first in recordings from the axons in the antennal nerve only in the last few days of metamorphic development and thus could not influence the process of glomerulus formation. In this study, we have tested directly the possibility that an earlier presence of spontaneous activity in either the receptor axons or the antennal-lobe neurons could affect the process. Tetrodotoxin, a Na(+)-channel blocker, was injected into the hemolymph prior to the onset of glomerulus formation to block any spontaneous Na(+)-dependent activity. Subsequent intracellular recordings from antennal-lobe neurons revealed no spike activity. Comparison with vehicle-injected control animals at stages during and after glomerulus formation revealed no differences in the localization of receptor-axon terminal branches in the glomeruli, in the border of glial cells that forms around each glomerulus, or in the morphology of the tufted glomerular arbors of one of the antennal-lobe neurons. We conclude that: (1) the process of glomerulus formation is largely independent of activity; and (2) glomeruli as modular units of the CNS more closely resemble cortical barrels than cortical columns, both in their ontogeny and in the lack of an obvious effect of activity on the morphology of the neurons arborizing within them.

Olfactory receptor axons in many species terminate centrally in an array of distinct glomeruli that are thought to encode the molecular features of odors. Particular molecular attributes are detected by receptor neurons widely distributed over the sensory epithelium, but these neurons then project to a small number of glomeruli in the olfactory bulb. This raises perplexing questions about olfactory axon guidance, especially how axons sort by odor specificity and how they find their appropriate targets in the brain. Taking advantage of the relative cellular simplicity of the moth antennal system, we have examined receptor axons in normally developing animals and also in preparations in which the nerve was experimentally misrouted. Just before they enter the antennal lobe, receptor axons undergo a dramatic reorganization in a discrete zone filled with glial cells. Here they shed neighbor relationships and become associated with axons that have common targets and presumably share common odor specificities. Electron microscopy revealed that the growth cones of early arriving axons travel preferentially next to glial processes. The growth cones of receptor axons were relatively simple except as they entered newly forming glomeruli. Misrouted nerves turned and ran along the surface of the brain until they reached the region of the antennal lobe. In only 6% of cases did misrouted axons enter the brain ectopically, never forming glomeruli. Our results suggest that olfactory receptor axons are attracted to the antennal lobe by soluble or surface-bound cues and sort by odor specificity by using a mechanism that may involve glial cells.

The signals that olfactory receptor axons use to navigate to their target in the CNS are still not well understood. In the moth Manduca sexta, the primary olfactory pathway develops postembryonically, and the receptor axons navigate from an experimentally accessible sensory epithelium to the brain along a pathway long enough for detailed study of regions in which axon behavior changes. The current experiments ask whether diffusible factors contribute to receptor axon guidance. Explants were made from the antennal receptor epithelium and co-cultured in a collagen gel matrix with slices of various regions of the brain. Receptor axons were attracted toward the central regions of the brain, including the protocerebrum and antennal lobe. Receptor axons growing into a slice of the most proximal region of the antennal nerve, where axon sorting normally occurs, showed no directional preference. When the antennal lobe was included in the slice, the receptor axons entering the sorting region grew directly toward the antennal lobe. Taken together with the previous in vivo experiments, the current results suggest that an attractive diffusible factor can serve as one cue to direct misrouted olfactory receptor axons toward the medial regions of the brain, where local cues guide them to the antennal lobe. They also suggest that under normal circumstances, in which the receptor axons follow a pre-existing pupal nerve to the antennal lobe, the diffusible factor emanating from the lobe acts in parallel and at short range to maintain the fidelity of the path into the antennal lobe.