Biomedical subjects

L P Tolbert

Publications and source records attributed to L P Tolbert.

At least 37 records · Page 2Linked to original sources

PubMed1996

Activity blockade does not prevent the construction of olfactory glomeruli in the moth Manduca sexta.

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.

Action Potentials↗

PubMed1995

20-Hydroxyecdysone stimulates proliferation of glial cells in the developing brain of the moth Manduca sexta.

The steroid hormone 20-hydroxyecdysone (20-HE) controls diverse aspects of neuronal differentiation during metamorphosis in the hawkmoth Manduca sexta. In the present study we have examined the effect of 20-HE on glial cells of the brain during the metamorphic period. The antennal (olfactory) lobe of Manduca provides an ideal system in which to study effects of hormones on glial cells, since three known classes of glial cells participate in its development, and at least one type is critically important for establishment of normal neuronal morphology. These glial cells, associated with the neuropil, form boundaries for developing olfactory glomeruli as a result of proliferation and migration. We determined whether glial cells proliferate in response to 20-HE by injecting a pulse of 20-HE into the hemolymph at different stages of development and monitoring proliferation of all three types of glial cells. Hormone injections at the beginning and end of metamorphic development, when hormone titers are normally low, did not stimulate proliferation of neuropil-associated glial cells. Injections during the period when hormone titers are normally rising produced significant increases in their proliferation. Injections when hormone titers are normally high were ineffective at enhancing their proliferation. One other class of glial cells, the perineurial cells, also proliferate in response to 20-HE. Thus, glial proliferation in the brain is under the control of steroid hormones during metamorphic development.

Animals↗

PubMed1995

Development of an identified serotonergic neuron in the antennal lobe of the moth and effects of reduction in serotonin during construction of olfactory glomeruli.

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.

Animals↗

PubMed1995

Normal glomerular organization of the antennal lobes is not necessary for odor-modulated flight in female moths.

A prominent hypothesis for the function of the glomerular structures in the primary olfactory neuropil of many groups of vertebrate and invertebrate animals is that they enable the processing and coding of information about the chemical compounds that compose complex odors. Previous studies have indicated that various degrees of glomerulus formation in the antennal lobes of the brain of the moth Manduca sexta can be effected by reducing the number of olfactory sensory axons that grow from the antenna into the antennal lobe during metamorphosis. To test the hypothesis that the presence of glomerular structure is necessary to process and identify odors, we substantially reduced, by surgery, the number of antennal segments in developing moths and upon metamorphosis we observed and quantified behavioral responses known to be elicited by odors. Intact and lesioned adult female moths were challenged to fly upwind to the source of an attractive host-plant odor in a wind tunnel. Some of the moths that had developed with reduced olfactory input flew upwind to the odor source. The flight behavior of these individuals was similar to the odor-mediated flight typically observed in moths that had developed normally. Histological analysis of the moths' antennal lobes revealed that the lobes of more than half of the respondents that had been lesioned during development lacked normal glomerular organization. The neuropil of these abnormally developed antennal lobes was mostly aglomerular, but with a few isolated, clearly abnormal glomerulus-like structures. This suggests either that even a few abnormal glomeruli are sufficient to mediate this specific behavior or that "canonical" glomerular organization per se is not necessary for this odor-mediated behavior.

Animals↗

PubMed1995

Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three-dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.

Animals↗

PubMed1994

Uniglomerular projection neurons participate in early development of olfactory glomeruli in the moth Manduca sexta.

Glomerular organization of the antennal (olfactory) lobe is initiated by the arrival of sensory axons from the antenna. Bundles of axon terminals coalesce into spheroidal knots of neuropil called protoglomeruli. Previous studies have suggested that the protoglomeruli form a template for the mature glomerular array, but an early role for projection neurons in establishing the template has not been excluded. We examined with the confocal laser scanning microscope the morphological development of the uniglomerular projection neurons during the stages in which glomeruli are constructed. Groups of projection neurons were stained with the lipophilic dye DiI to assess the development of the population as a whole; individual neurons were filled intracellularly with Lucifer Yellow to examine in detail the development of shape. In some preparations, sensory axons and glial cells also were labeled by using different fluorescent dyes to reveal possible interactions between projection neuron dendrites and sensory axons or glial cells. Protoglomeruli form in a wave beginning at the entry point of the antennal nerve and proceeding across the lobe to the opposite pole. A second wave follows in which projection neurons become tufted and innervate the newly formed glomeruli, sometimes extending into the glial border surrounding the protoglomeruli. In animals deprived of sensory axons, some projection neurons still form tufted dendritic trees and, in one region of the neuropil, a glomerulus-like structure. The early presence of projection neuron processes in the protoglomeruli and the formation of at least one glomerulus-like structure in unafferented lobes suggest that uniglomerular projection neurons play an active role in the construction of olfactory glomeruli.

Animals↗

PubMed1994

Spatiotemporal pattern of expression of tenascin-like molecules in a developing insect olfactory system.

During the development of the olfactory (antennal) lobe of the moth Manduca sexta, olfactory sensory axons induce glomerular branching patterns in their target neurons. Glial cells, by surrounding the developing glomerular template, are thought to mediate the developmental influence of olfactory axons on these branching patterns. Previous studies have demonstrated that, in the absence of glia, neurons in the antennal lobe branch in an aglomerular fashion, even in the presence of competent antennal axons (Oland and Tolbert, 1988, J. Comp. Neurol. 278:377-387; Oland et al., 1988, J. Neurosci. 8:353-367). We have begun to explore the molecular basis by which glial cells could influence patterns of neurite branching. For this work, we have utilized immunocytochemical techniques and a partial biochemical analysis to demonstrate that molecules antigenically similar and comparable in size to mammalian tenascin are localized on the neuropil-associated glial cells that form borders around glomeruli in the developing antennal lobe. These tenascin-like molecules associated with neuropilar glia are present at critical stages of glomerulus development; tenascin-like immunoreactivity declines after glomeruli form and become stabilized. Neither the arrival nor the absence of antennal axons in the lobe induces changes in either the molecular forms or the amounts of tenascin-like molecules. The spatiotemporal pattern of expression of tenascin-like molecules suggests that they are in a position to participate in the formation of a glomerular neuropil and could form a molecular barrier that constrains neurite outgrowth strictly to glomeruli.

Animals↗

PubMed1994

In vitro analyses of neurite outgrowth indicate a potential role for tenascin-like molecules in the development of insect olfactory glomeruli.

Tenascin-like material is associated with glial cells that form borders around developing glomerular units in the olfactory (antennal) lobe of the moth Manduca sexta and is present at critical stages of glomerulus formation (Krull et al., 1994, J. Neurobiol. 25:515-534). Tenascin-like immunoreactivity declines in the mature lobe, coincident with a wave of synapse formation within the glomeruli and glomerulus stabilization. Tenascin-like molecules associated with neuropilar glia are in the correct position to influence the branching patterns of growing neurites by constraining them to glomeruli. In this study, we examine the growth of cultured moth antennal-lobe neurons in response to mouse CNS tenascin. Uniform tenascin provides a poor substrate for cell-body attachment and neurite outgrowth. Neuronal cell bodies provided with a striped substratum consisting of tenascin and concanavalin-A (con-A)/laminin attach preferentially to con-A/laminin lanes. Most neurons restrict their branching to con-A/laminin lanes both at early and later times in culture but others send processes across multiple tenascin and con-/laminin lanes in an apparently indiscriminate manner. Tenascin can inhibit the neuritic outgrowth of most antennal-lobe neurons, and this raises the possibility that the tenascin-like molecules associated with neuropilar glia in vivo act to constrain growing neurites to glomeruli. Thus, glial cells, acting in concert with olfactory axons, might act to promote glomerular patterns of branching by antennal-lobe neurons.

Animals↗

PubMed1994

Ultrastructural examination of the insemination reaction in Drosophila.

The insemination reaction is a swelling of the female vagina caused by the male ejaculate. This postmating phenomenon is common among species in the genus Drosophila. It could act as a plug securing male paternity. It is not clear, however, what benefits it provides to the female. The structure formed in the female vagina is expelled in some species and disappears gradually in others suggesting different phenomena. Based on ultrastructural examination of the vaginal contents of five Drosophila species (D. mettleri, D. nigrospiracula, D. melanogaster, D. mojavensis, and D. hexastigma), we propose three terms to describe these vaginal structures: the sperm sac, the mating plug, and the true insemination reaction. Each term describes a distinct structure associated with a specific female postmating behavior. This study questions the concept of the insemination reaction as a single phenomenon and discusses its possible functions from an evolutionary perspective.

Animals↗

PubMed1993

Ramification pattern and ultrastructural characteristics of the serotonin-immunoreactive neuron in the antennal lobe of the moth Manduca sexta: a laser scanning confocal and electron microscopic study.

The two antennal lobes, the primary olfactory centers of the brain, of the moth Manduca sexta each contain one neuron that displays serotonin immunoreactivity. The neuron projects out of the antennal lobe and sends branches into ipsi- and contralateral protocerebral areas. An axon-like process extends from the contralateral protocerebrum to, and terminates in, the contralateral antennal lobe. In order to begin to investigate the possible role of this unique neuron in olfactory information processing, we have used laser scanning confocal microscopic and electron microscopic immunocytochemical techniques to study the ramification pattern, ultrastructural characteristics, and synaptic connections of the neuron in the antennal lobes of female adult Manduca sexta. The neuron ramifies extensively in the antennal lobe contralateral to the cell body. The ramifications, mainly in the base and center of each glomerulus, do not overlap with those of the sensory axons from the antenna. This finding suggests that the serotonin-immunoreactive neuron may not receive direct input from sensory neurons, and that it may modulate the activity of the neurons of the antennal lobe rather than that of the sensory neurons. In the electron microscope, the neuron exhibits large dense-cored vesicles and small, clear round vesicles. In the antennal lobe ipsilateral to the cell body, the primary neurite of the serotonin-immunoreactive neuron is unbranched and lacks detectable synaptic connections. The ramifications in the contralateral antennal lobe, however, participate in synaptic connections. At very low frequency, contralateral branches form synapses onto unlabeled processes and also receive synapses from unidentified neurons in the glomeruli, indicating that the neuron may participate directly in synaptic processing of olfactory information. The high ratio of output to input synapses made by the serotonin-immunoreactive processes in the contralateral antennal lobe is consistent with the idea that this neuron may receive synaptic input via its bilateral branches in the protocerebrum and then send information to the contralateral antennal lobe where the neuron may exert feedback or modulatory influences on olfactory information processing in the glomeruli.

Animals↗

PubMed1993

Synaptic organization and development of the antennal lobe in insects.

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.

Animals↗

PubMed1990

Requirement for olfactory axons in the induction and stabilization of olfactory glomeruli in an insect.

The role of antennal sensory axons in the induction and stabilization of olfactory glomeruli has been explored in the moth Manduca sexta. First, we asked the question: how many axons are necessary to induce glomerulus formation within the first-order olfactory neuropil of the brain? Axons from as few as 10 of the normal 70-80 repeating antennal segments were sufficient to induce glomeruli. However, there was a dose dependence in the number of glomeruli that developed in partially innervated lobes. When only 11 segments of the antenna were allowed to provide innervation to the lobe, only 37 of the normal 59 +/- 2 glomeruli developed; over 20 segments were necessary to induce the normal number of glomeruli. In a second set of experiments, we asked: for how long must antennal axons be present to stabilize newly formed glomeruli? We found that antennal axons must be intact for at least 2 to 4 stages (roughly equivalent to 2 to 4 days) for glomeruli to be stable even if the axons are subsequently severed. This finding, taken in the light of other recent findings in our laboratory, suggests that the formation of synapses may be a crucial element in the stabilization of glomerular structure. All together, the results of the present study indicate that induction and stabilization of glomeruli are separable events with different underlying cellular bases.

Animals↗

PubMed1990

Glial cells form boundaries for developing insect olfactory glomeruli.

Like many other first-order olfactory centers, the olfactory lobe of the moth brain is organized into histologically conspicuous synaptic glomeruli delimited by glial cells. These glomeruli in the moth offer an advantageous system in which to study the parcellation of central neuropils during development. In this article, we review the evidence that glial cells play an essential role in the induction of glomeruli by olfactory sensory axons, and we present new evidence that leads us to propose that a major role for glia in the development of glomeruli is to define for ingrowing dendrites of second-order olfactory neurons edges that lead to the formation of these cells' characteristic morphology.

Animals↗

PubMed1990

Construction of a protoglomerular template by olfactory axons initiates the formation of olfactory glomeruli in the insect brain.

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.

Afferent Pathways↗

PubMed1989

Patterns of glial proliferation during formation of olfactory glomeruli in an insect.

Partitioning of the first-order olfactory neuropil into glomeruli in the developing brain of the moth Manduca sexta occurs only in the presence of olfactory sensory axons and appears to be mediated by changes in glial cells (Oland et al.: J. Neurosci., 8:353-367, 1988). The arrival of sensory axons in the brain triggers changes in glial shape and position that lead to the formation of a glial scaffolding for the developing glomeruli. The presence of mitotic figures in glial cells at stages before glomeruli emerge (Oland and Tolbert: J. Comp. Neurol., 255:196-207, 1987) suggested that glial proliferation might also contribute to the formation of the glomerular envelopes. To determine whether glial proliferation is induced by olfactory axons, we have used 3H-thymidine to label dividing cells before, during, and after the formation of glomeruli and have compared the patterns of proliferation in normal and chronically unafferented olfactory neuropils. We found significant differences in mitotic indices only after glomerular walls had been established, indicating that the sensory axons induce the formation of glomerular envelopes primarily via the changes in glial morphology and distribution, not by stimulating glial proliferation.

Animals↗

PubMed1989

Afferent axons from the antenna influence the number and placement of intrinsic synapses in the antennal lobes of Manduca sexta.

The present study compares the placement and frequency of occurrence of synapses in normally developing antennal lobes of the brain of the moth Manduca sexta and in lobes developing in the absence of afferent axons from the antenna. The antennal lobes develop during the metamorphosis from larva to moth, and require antennal afferent input to develop their characteristic knots of synaptic neuropil, the glomeruli, that are arrayed around a central core of coarse neuropil. Synapses begin to form throughout the neuropil of the antennal lobe before ingrowth of antennal axons into the lobe. During normal development, almost all synapses (those among intrinsic neurons as well as those made by antennal afferent axons) become segregated into glomeruli as the glomeruli are established. Thus, in normal mature lobes, the radial segregation between synaptic (glomerular) and nonsynaptic (central) regions is virtually complete. In lobes that have never been allowed to receive antennal axons, an outer ring of "protoglomerular" neuropil develops in place of the glomeruli. A similar segregation of synaptic and nonsynaptic regions occurs, with two major differences: the frequency of synapses that develops in the outer ring of fine-textured neuropil is almost 50% higher than in normal glomeruli, and the small number of synapses that occur in the coarse central neuropil now occur on aberrant fine processes in that neuropil. Apparently, in the absence of their normal massive input from the antenna, elements intrinsic to the antennal lobe are induced to form extra synapses with each other, and to send aberrant fine branches to synapse in a region of neuropil normally containing predominantly large-caliber neurite trunks and few synapses.

Afferent Pathways↗

PubMed1989

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.

Animals↗

PubMed1988

Effects of hydroxyurea parallel the effects of radiation in developing olfactory glomeruli in insects.

Previous observations (Oland and Tolbert: J. Comp. Neurol. 255:196-207, '87, Soc. Neurosci. Abstr. 13:1144, '87; Oland et al.: J. Neurosci. 8:353-367, '88) have provided evidence that the afferent-axon-induced development of synaptic glomeruli in the antennal lobe of the moth Manduca sexta depends upon an interaction between ingrowing sensory axons and the glial cells of the antennal lobe. In order to differentiate between the roles of glial cells and of afferent axons on the partitioning of the lobe into glomeruli, we have used the antimitotic agent hydroxyurea to produce lobes deficient in glial cells but retaining sensory input. The resulting lobes were analyzed in the light and electron microscopes, and the integrity of their antennal input was evaluated by examining the gross and microscopic structure of the antennae, the number of antennal afferent axons, and electroantennogram responses to odors. Our results with hydroxyurea show that in treated animals with adequate antennal input the degree to which the antennal-lobe neuropil becomes glomerular varies with the number of glial cells remaining in the lobe; when less than approximately one-quarter of the normal glial complement is present, glomeruli do not develop at all. These experiments complement and extend previous experiments in which the number of glial cells was reduced with radiation (Oland et al.: J. Neurosci. 8:353-367, '88). The fact that the present results mimic the previous results with radiation strongly suggest that glial cells do mediate the afferent-axon-induced formation of olfactory glomeruli in the moth.

Animals↗