The synaptic neuropil of the olfactory (antennal) lobe of the moth Manduca sexta is subdivided into histologically conspicuous structures called glomeruli that are typical of olfactory systems in vertebrates and invertebrates. Each glomerulus consists of the highly branched neuritic arbors of both primary olfactory axons and antennal-lobe neurons, bounded by a nearly complete envelope of glial cells. We have studied events occurring during the first half of metamorphic adult development. The first signs of organization of the neuropil into glomeruli are changes in glial cells. Prior to the ingrowth of olfactory axons from the antenna, glial cells form a continuous border around the neuropil. When olfactory axons begin to reach the lobe, glial cells embark on a stereotyped series of changes: the border becomes disrupted, glial cells begin to proliferate and extend processes into the outer regions of the neuropil, and some glial cells migrate toward the center of the neuropil. Shortly thereafter, glomeruli emerge from the neuropil, delineated by glial cells. If, however, afferent axons are prevented from ever reaching the antennal lobe, glomeruli never develop and the glial cells remain almost entirely restricted to a thick layer bordering the neuropil. Thus sensory axons have a direct influence not only on neuronal but also on glial differentiation. Our results lead us to suggest that the glial cells may be in a position to act as intermediaries in developmental interactions between sensory axons and antennal-lobe neurons.

Many insects possess a highly developed sense of smell. This paper summarizes the cellular and synaptic organization of the antennal (olfactory) lobe of the insect brain and then reviews morphological and fine-structural aspects of the development of the lobe. Visualization of synapses between classes of neurons identified by physiological, morphological, or transmitter-cytochemical properties has provided insights into arrangements of contacts and their possible roles in information processing. Studies of development have revealed the requirement for afferent axons from the antenna for the formation of olfactory glomeruli, where virtually all of the synapses in the lobe occur, and have suggested the possibility that glial cells play a role in the instructive influence of the axons on their target neurons in the lobe. The findings reviewed in this paper are primarily from one representative hemimetabolous insect, the American cockroach, and one representative holometabolous insect, a hawkmoth, and comparisons are made with vertebrate systems when appropriate.

During the metamorphosis of holometabolous insects, the larval nervous system is restructured to provide the circuitry needed by the developing adult. Prominent new centers in the brain, the antennal lobes, arise to receive olfactory afferent axons from the developing adult antennae and provide an excellent system in which to study the development of synapses in a central nervous system. We have examined the anatomy and physiology of developing synapses in the antennal lobes of the moth Manduca sexta during the 18 days of metamorphic adult development. On day 5, the neuropil of the newly emerging antennal lobe condenses into distinct glomeruli, in which intercellular junctional complexes have already begun to form. Although some junctions have associated synaptic vesicles, most complexes are desmosome-like until day 9, when the number of synaptic complexes begins to increase. Early synapses are characterized by membrane-associated densities in at least two abutting cellular processes and a small number of synaptic vesicles clustered near the membrane of one process. As adult development proceeds, the membrane-associated densities become denser and more extensive, and the number of synaptic vesicles in the clusters increases. At day 14 synapses appear ultrastructurally mature, and almost all junctions in the neuropil can be identified as synaptic. Not until day 9 do antennal lobe neurons begin to respond postsynaptically when the antennal nerve is stimulated electrically, suggesting that the earliest synapses observed in the electron microscope may not be made by antennal nerve axons. At first the postsynaptic responses are graded and fatigue rapidly. By day 11, the antennal lobe neurons respond with action potentials, but the fatigability does not decline to adult levels until day 13. Filling of antennal lobe neurons with cobalt reveals that the arborizations of both local interneurons and output neurons continue to mature morphologically until about day 13. Previous work (Schweitzer,

Olfactory receptor cells (ORCs) of a particular odor tuning are dispersed in the olfactory epithelium, but their axons converge on distinct glomeruli in primary olfactory centers. As a consequence, axon associations must change to bring axons of ORCs with the same odor specificity together. Studies in Manduca sexta have indicated that just before they enter the antennal lobe (AL), ORC axons undergo extreme reorganization, finally entering the AL in fascicles destined for subsets of glomeruli. This axon-sorting zone is heavily populated by glial cells, and ORC axon growth cones often are in close physical contact with the glia. In moths rendered glia deficient, ORC axons fail to fasciculate in this region. Using propidium iodide to label nuclei and 5-bromo-2'-deoxyuridine to monitor proliferation, we found that the glia in the sorting zone arise from the AL, appearing shortly after the first ORC axons arrive. Experimental removal of some or all of the sensory innervation revealed that proliferation of sorting-zone glia is triggered by ORC axons. A second set of glia arises in the antenna and migrates along the antennal nerve toward the brain, populating the nerve after the establishment of the sorting zone. Development of this type of glial cell is independent of contact of the ORC axons with their central targets. We conclude that the sorting zone arises from CNS glia in response to ingrowth of ORC axons, and a critical number of glia must be present in the sorting zone for axons to correctly establish new neighbor-neighbor associations.

Olfactory glomeruli in insects share many features of organization with their vertebrate counterparts, and yet offer distinct advantages for study of neuronal development. Previous studies have revealed that the olfactory lobes of the brain of the moth Manduca sexta arise postembryonically and that glomeruli in the lobe are induced by olfactory afferent axons (Hildebrand et al., 1979; Oland and Tolbert, 1987). In the present study, we have used the Golgi method, intracellular labeling of neurons with Lucifer yellow, and electron microscopy to follow neuronal development in the antennal lobe through the period when glomeruli develop. Our results, taken together with other results from our laboratory, suggest that olfactory sensory axons have the intrinsic ability to form protoglomeruli, and that an interaction between these axons and glial cells (but not the majority of the neurons of the antennal lobe) causes the glial cells to surround the protoglomeruli. Ingrowth of the neurites of most antennal-lobe neurons into the protoglomeruli occurs after a small delay and appears to be constrained to glomerular units by the presence of the glial boundaries. Synapses, initially not detected in the protoglomeruli, begin to appear as soon as the neurites of antennal-lobe neurons appear in the glomeruli. Thus, antennal axons, instead of immediately seeking out postsynaptic targets, first form the template for organization of future glomeruli. The neurites of most of the neurons of the antennal lobe grow outward to meet the olfactory sensory axons, and in doing so, join with these axons to form glomeruli. Preliminary results concerning the development of a second class of neuron in the lobe, the projection neurons, indicate that at least some of these neurons may arborize in the region of the protoglomeruli very early and therefore participate with the afferent axons in laying the foundation for glomeruli.