Leslie P. Tolbert

Organization and Synaptic Ultrastructure of Glomeruli in the Antennal Lobes of the Moth Manduca sexta: A Study Using Thin Sections and Freeze-Fracture

The antennal lobe of the brain of Manduca sexta comprises a central area of coarse neuropil surrounded by dense, spheroidal glomeruli, where all synaptic interactions between antennal-nerve axons and the second-order neurons of the lobe occur. Neuronal interactions in the glomeruli are complex, involving several types of neuritic profiles and mediated by synapses with a one-to-many ratio of pre- to postsynaptic elements. Presynaptic profiles in the glomeruli have been categorized into three types, containing round clear vesicles, large numbers of large dense-cored vesicles, and pleiomorphic clear vesicles, respectively. Preliminary studies of horseradish peroxidase-filled axons and neurons indicate that antennal-nerve axons form synapses without large numbers of dense-cored vesicles and that antennal-lobe neurons not only receive synapses but also may synapse onto other elements in the antennal lobe. A typical synaptic contact involves multiple postsynaptic elements apposed in pairs to an individual presynaptic element. The presynaptic element contains a bar-shaped membrane-associated density, which follows a shallow groove in the membrane and is flanked by synaptic vesicles. Postsynaptic elements are lined by membrane-associated densities in the region opposite to the synaptic bar, and may be observed to participate in serial synapses. Freeze-fracture replicas of the glomerular neuropil contain many membrane specializations that are thought to be presynaptic, some of which resemble those of vertebrate excitatory synapses. At these apparently presynaptic regions, large particles cluster in the P face of the membrane and are often surrounded by plasmalemmal deformations presumably representing sites of exo- or endocytosis. The shape of the predominant type of presynaptic membrane specialization (a plaque) does not match the shape of the presynaptic membrane-associated density (a bar); this raises the possibility that vesicle release occurs at isolated ‘active zones’ along the presynaptic bar. Postsynaptic sites are represented by clusters of large particles in the E face of the postsynaptic membrane.

The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Horseradish peroxidase labelling of identified cell types

Golgi impregnations of the posterior part of the cats anteroventral cochlear nucleus have revealed two types of neurons, bushy cells with short bush-like dendrites and stellate cells with long, tapered processes; Nissl stains have revealed globular and multipolar cell bodies with dispersed and clumped ribosomal patterns, respectively. In the present study, we injected horseradish peroxidase into the trapezoid body. Ipsilaterally, retrograde, diffuse labelling of neurons, presumably through damaged fibers, yielded Golgi-like profiles of numerous bushy cells with typical dendrites and with thick axons projecting toward the trapezoid body. Stellate cells were almost never labelled in this way. Anterograde diffuse labelling of thick axons demonstrated calyx endings in the contralateral medial nucleus of the trapezoid body. In the electron-microscope, the perikarya of diffusely-filled bushy neurons were found to have the dispersed ribosomal pattern and the kinds of synaptic endings typical of globular cells, including large profiles of end-bulbs from cochlear nerve axons. After injections restricted to the medial trapezoid nucleus, granularly-labelled cells in the cochlear nucleus were almost completely confined to the contralateral side; Nissl counterstaining showed them to be globular cells in the posterior part of the anteroventral cochlear nucleus. After larger injections, involving surrounding regions of the superior olivary complex, granular labelling occurred throughout the ventral cochlear nucleus on both sides. There is also evidence that stellate cells in Golgi impregnations correspond to multipolar cell bodies in Nissl stains. We conclude that bushy cells typically correspond to globular cells, which receive end-bulbs from the cochlea and send thick axons to the contralateral medial trapezoid nucleus, where they form calyces on principal cells. Principal cells, in turn, are known to project to the lateral superior olive and to one of the nuclei of origin of the crossed olivo-cochlear bundle, which feeds back to the cochlea. In this circuit, correlations between synaptic patterns and particular physiological signal transfer characteristics can be suggested. These could be related to binaural intensity interactions in the lateral superior olive and to a regulatory loop involving the olivo-cochlear bundles.

The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Electron microscopy

We have studied the posterior division of the anteroventral cochlear nucleus, where the cochlear nerve root enters the brain, in the cat. In Nissl preparations, this region contains two types of neuronal cell bodies: globular and multipolar. The two types can be identified in the electron-microscope by comparing Nissl substance and rough endoplasmic reticulum. Globular cell bodies receive many synaptic terminals, which cover 85% of the surface. In contrast, multipolar cell bodies are almost entirely wrapped by thin glial sheets--synaptic terminals contact less than 15% of the surface and tend to cluster at the bases of dendrites. Synaptic terminals are of three kinds, types 1, 2, and 3, which contain large round, small round-to-oval, and small flattened synaptic vesicles, respectively. Terminals of all three kinds synapse on both types of cell bodies. However, only globular cell bodies receive the largest type 1 terminals, which correspond to end-bulbs, seen in Golgi impregnations to arise from cochlear nerve axons. Cochlear ablation leads to degeneration of type 1, but not type 2 or 3 terminals. We conclude that neurons with globular cell bodies receive heavy somatic input from the cochlear nerve, as well as from other sources. Neurons with multipolar cell bodies receive very little input to their perikarya--giving their dendrites a more important role in determining their response properties. We suggest a morphological basis for correlating individual kinds of neurons with certain electrophysiological response types.

Multiple factors shape development of olfactory glomeruli: Insights from an insect model system

The antennal system of the moth Manduca sexta is a useful model for studies of the development of olfactory glomeruli, the complex synaptic structures that typically underlie the initial processing of olfactory input in vertebrates and invertebrates. In this review, we summarize cellular events in the construction of glomeruli in Manduca and highlight experiments that reveal factors that influence glomerulus development. By methodically manipulating each of various cell types, both neuronal and glial, that contribute to glomerular architecture, we have found that: olfactory receptor axons lay a template for developing glomeruli, stabilization of the template by glial cells is necessary to permit subsequent steps in development of the glomeruli, and the hormone that regulates adult development causes production of adequate numbers of glial cells. Neither electrical activity nor the presence of a serotonin-containing neuron that persists throughout development is required for a glomerular pattern to develop; these factors might, however, influence the synaptic organization of individual glomeruli.

The neuronal architecture of the anteroventral cochlear nucleus of the cat in the region of the cochlear nerve root: Golgi and Nissl methods

This report characterizes the cells and fibers in one part of the cochlear nucleus, the posterior division of the anteroventral cochlear nucleus. This includes the region where the cochlear nerve root enters the brain and begins to form endings. Nissl stains reveal the somata of globular cells with dispersed Nissl substance and those of multipolar cells with coarse, clumped Nissl bodies. Both parts of the posterior division contain cells with each Nissl pattern, but in different relative numbers and locations. Golgi impregnations demonstrate two types of neurons: bushy cells, with short bush-like dendrites, and stellate and elongate cells, with long tapered dendrites. Several varieties of bushy cells, differing in the morphology of the cell body and in the size and extent of the dendritic field, can be distinguished. Comparison of the distributions of these cell types, as well as cellular morphology, suggest that the globular cells recognized in Nissl stains correspond to bushy neurons, while the multipolar cells correspond to stellate and elongate neurons. Golgi impregnations reveal large end-bulbs and smaller boutons from cochlear nerve fibers, as well as boutons from other, unidentified sources, ending in this region. The particular arrangements of the dendritic fields of the different cell types and the axonal endings associated with them indicate that these neurons must have different physiological properties, since they define different domains with respect to the cochlear and non-cochlear inputs.

Synaptic organization of the uniglomerular projection neurons of the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study.

The detailed branching pattern and synaptic organization of the uniglomerular projection neurons of the antennal lobe, the first processing center of the olfactory pathway, of the moth Manduca sexta were studied with laser scanning confocal microscopy and a technique combining laser scanning confocal microscopy and electron microscopy. Uniglomerular projection neurons, identified electrophysiologically or morphologically, were stained intracellularly with neurobiotin or biocytin. Brains containing the injected neurons were treated with streptavidin‐immunogold to label the injected material for electron microscopy and with Cy3‐streptavidin to lable the neurons with fluorescence for laser scanning confocal microscopy, and then embedded in Epon. Labeled neurons were imaged and reconstructed with laser scanning confocal microscopy (based on the retained fluorescence of the labeled neuron in the Epon block), and thin sections were cut at selected optical levels for correlation of light microscopic data and electron microscopic detail. Each neuron had a cell body in one of the three cell‐body clusters of the antennal lobe, a primary neurite that extended across the coarse neuropil at the center of the antennal lobe and then formed a dense tuft of processes within a single glomerulus, and an axon that emanated from the primary neurite and projected from the antennal lobe via the antenno‐cerebral tract to the lateral horn of the ipsilateral protocerebrum and, collaterally, to the calyces of the mushroom body. In the electron microscope, the fine dendritic branches in the apical zones of the glomeruli, where sensory axons terminate, were found to receive many input synapses. In deeper layers across the glomeruli, the processes participated in both input and output synapses, and at the bases of the glomeruli, the most proximal, thickest branches formed output synapses. In both of the protocerebral areas in which axonal branches terminated, those branches formed exclusively output synapses. Our findings indicate that, in addition to conveying olfactory information to the protocerebrum, uniglomerular projection neurons in the antennal lobes of M. sexta participate in local intraglomerular synaptic circuitry. J. Comp. Neurol. 379:2‐20, 1997.

A role for glia in the development of organized neuropilar structures

Intercellular interactions are critical in the development of the nervous system. In the olfactory system of a moth, sensory axons induce the formation of large synaptic glomeruli, each surrounded by a glial envelope, in the antennal lobe of the brain. During development, the sensory axons cause changes in glial shape and disposition one day before glomeruli are recognized. Early removal of glial cells prevents the development of glomeruli despite the presence of afferent axons. Thus, the glial cells appear to play a role as intermediaries in the induction of glomeruli by afferent axons. Recent findings in the mammalian somatosensory cortex suggest a similar role for glia there.

Axons of olfactory receptor cells of transsexually grafted antennae induce development of sexually dimorphic glomeruli in Manduca sexta

The influence of olfactory receptor cell (ORC) axons from transsexually grafted antennae on the development of glomeruli in the antennal lobes (ALs), the primary olfactory centers, was studied in the moth Manduca sexta. Normally during metamorphic adult development, the pheromone-specific macroglomerular complex (MGC) forms only in the ALs of males, whereas two lateral female-specific glomeruli (LFGs) develop exclusively in females. A female AL innervated by ORC axons from a grafted male antenna developed an MGC with three glomeruli, like the MGC of a normal male AL. Conversely, a male AL innervated by ORC axons from a grafted female antenna lacked the MGC but exhibited LFGs. ORC axons from grafted male antenna terminated in the MGC-specific target area, even in cases when the antennal nerve (AN) entered the AL via an abnormal route. Within ectopic neuromas formed by ANs that had become misrouted and failed to enter the brain, male-specific axons were not organized and formed terminal branches in many areas. The results suggest the presence of guidance cues within the AL for male-specific ORC axons. Depending on the sex of the antennal innervation, glial borders formed in a pattern characteristic of the MGC or LFGs. The sex-specific number of projection neurons (PNs) in the medial group of AL neurons remained unaffected by the antennal graft, but significant changes occurred in the organization of PN arborizations. In gynandromorphic females, LFG-specific PNs extended processes into the induced MGC, whereas in gynandromorphic males, PNs became restricted to the LFGs. The results indicate that male-and female-specific ORC axons play important roles in determining the position, anatomical features, and innervation of sexually dimorphic glomeruli.

Glial cells stabilize axonal protoglomeruli in the developing olfactory lobe of the moth Manduca sexta

Odor information is processed in spherical structures called glomeruli, which in all animals with differentiated olfactory systems are sites of densely spaced synaptic contacts between olfactory sensory axons and target central nervous system (CNS) neurons. Glomerulus development in the antennal (olfactory) lobe of the moth brain, which is initiated by the arrival of antennal receptor axons, requires interaction among three elements: glial cells, receptor axons, and their targets, the antennal‐lobe neurons. Receptor axons form an array of protoglomeruli that become surrounded by glia and serve as a template for mature glomeruli. Previous experiments showed that when the number of glial cells is sharply reduced during development either by irradiation or by an anti‐mitotic agent, receptor axons form protoglomeruli, but in the mature lobes, glomeruli are absent and central neurons lack the characteristic glomerular tufting of their arbors. The current investigation was conducted to determine which cellular events in the process of glomerulus formation are disrupted by severe reduction in glial‐cell number. The branching patterns of receptor axons and antennal‐lobe neurons were examined in animals that had been irradiated to produce glia‐deficient antennal lobes at stages during which glomeruli normally would develop. We found that the receptor axons did form protoglomeruli, but that the protoglomeruli quickly disintegrated in glia‐deficient antennal lobes; the receptor axons branched diffusely, except where several neighboring glia survived irradiation and together formed a wall of processes that appeared to block the passage of neuronal processes. Multi‐glomerular antennal‐lobe neurons never developed tufted arbors even at early stages. These results suggest that maintenance of protoglomeruli depends on the border of glia that forms around each protoglomerulus and that the subsequent tufting of antennal‐lobe neurons depends on maintenance of the protoglomerular template during the period of dendritic growth.

Early formation of sexually dimorphic glomeruli in the developing olfactory lobe of the brain of the moth Manduca sexta.

The antennal lobes (ALs), the primary olfactory centers, of the moth Manduca sexta are sexually dimorphic. Only ALs of males possess the macroglomerular complex (MGC), the site of primary processing of information about the females sex pheromone. To understand the development of identified, odor‐specific olfactory glomeruli, we investigated the cellular events involved in the morphogenesis of the MGC by means of various fluorescence staining techniques and laser‐scanning confocal microscopy.