Glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria

We have investigated the pattern of glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria. Using the glia-specific marker Repo, we identified glia associated externally with such lineages, termed lineage-extrinsic glia, and glia located internally within the lineages, termed lineage-intrinsic glia. Populations of both glial types increase up to 60 % of embryogenesis, and thereafter decrease. Extrinsic glia change their locations over time, while intrinsic ones are consistently found in the more apical part of a lineage. Apoptosis is not observed for either glial type, suggesting migration is a likely mechanism accounting for changes in glial number. Proliferative glia are present both within and without individual lineages and two glial clusters associated with the lineages, one apically and the other basally, may represent sources of glia.     

Organization of a midline proliferative cluster in the embryonic brain of the grasshopper

We have studied a group of midline cells in the embryonic brain of the grasshopper by using immunocytochemical and intracellular dye injection techniques. This cluster of midline cells differentiates between the pars intercerebralis lobes of the protocerebrum during early embryogenesis, and is composed of putative midline progenitors as well as neuronal and glial cells. Annulin immunoreactive glial processes surround the borders of the midline cell cluster and also form a network of processes extending from there to the borders of proliferative clusters in the brain hemispheres. Among the cells that derive from the midline cluster are two bilaterally symmetrical pairs of identified primary commissure pioneer neurons. By navigating along the glial bound borders of the midline proliferative cluster, the axons of these pioneers establish an initial axonal bridge across the brain midline. This analysis identifies a glial-bound midline proliferative cluster in the brain and shows that neuronal and glial cells of this cluster are closely associated with neurons pioneering the primary brain commissure. Comparable features of midline cells in the ventral ganglia and similarities to other proliferative clusters in the brain hemispheres are discussed.     

Building the central complex of the grasshopper Schistocerca gregaria: temporal topology organizes the neuroarchitecture of the w,x, y,z tracts

The central complex is a brain specific structure involved in multimodal information processing and in coordinating motor behaviour. It possesses a highly organized neuroarchitecture, which is remarkably conserved across insect species. A prominent feature of this neuroarchitecture is the stereotypic projection of axons from clusters of neurons in the pars intercerebralis to the central body via the so-called w, x, y and z tracts. Despite extensive analyses of this neuroarchitecture in adults, little is known about its ontogeny in any insect. In this paper we use the expression pattern of the segment polarity gene engrailed to identify those neuroblasts belonging to the protocerebrum of the early embryonic brain of the grasshopper Schistocerca gregaria. We present a new map for this brain region in which the 95 protocerebral neuroblasts in each hemisphere are organized into seven rows, as they are in the neuromeres of the ventral nerve cord. We then identify a subset of four of these neuroblasts as being the progenitor cells for four clusters of neurons, some of whose axons we show project via discrete tracts (w, x, y, z) into the central complex. These tracts begin to form prior to 39% of embryogenesis. We show further, that the cells from one of these clusters (the Z cluster) are organized according to age, and direct axons topologically according to age into the appropriate z tract. This pattern is repeated in each of the other three clusters, thus establishing a clonally based modular system of fibre tracts consistent with the model proposed for this brain region in the adult.     

The effect of pretreatment or combined treatment of quercetin on menadione toxicity in rat primary mixed glial cells in vitro

Neurons and glia are highly susceptible to reactive oxygen species that play a key role in various neurodegenerative diseases. Menadione, a synthetic derivative of vitamin K, induces reactive oxygen generation. Quercetin one of the most ubiquitous bioflavonoids in food of plant origin, has strong antioxidant activities on different cell types, however recent studies demonstrated that it has also prooxidant and cytotoxic potentials. We examined the action of pre- and co-treatment of quercetin on menadione induced glial toxicity. The primary mixed glial cells obtained from 1 to 3 day old rat brain were pretreated with 10, 25, 100 or 250 μM quercetin for 1 h, washed out and 10, 25, 50, 75 or 100 μM menadione was added for 6 h. The other group of cells was treated with respective doses of quercetin combined simultaneously with the same doses of menadione for 6 h. The cells were washed and incubated for additional 24 h for recovery period and the viability was measured by using MTT assay. Menadione was dose-dependently toxic to glia cells and pretreatment with respective quercetin doses for 1 h could not eliminate this toxicity. Although 10 and 25 μM quercetin combined with 10 and 25 μM menadione could not change, 100 and 250 μM quercetin together with 10 or 25 μM menadione for 6 h increased further the menadione induced toxicity. We conclude that when combined with menadione, quercetin at high doses could be toxic to primary rat glia cells in culture.     

Embryonic development of the pars intercerebralis/central complex of the grasshopper

 We have studied the embryonic development of the pars intercerebralis/central complex in the brain of the grasshopper using immunocytochemical and histochemical techniques. Expression of the cell-surface antigen lachesin reveals that the neuroblasts of the pars intercerebralis first differentiate from the neuroectoderm at around 26% of embryogenesis. Differentiation of medial and lateral neuroblasts occurs first. By the 28% stage a more or less uniform sheet of 20 neuroblasts has formed. As a result of both cell proliferation and cell translocation, the pars intercerebralis proliferative cluster in each hemisphere expands so that at 30% the most medial neuroblasts lie apposed at the midline. We followed the further development of the pars intercerebralis of each brain hemisphere using bromo-deoxy-uridine incorporation and osmium-ethyl-gallate staining. Within the pars intercerebralis itself, the neuroblasts redistribute into discrete subsets. The neuroblasts of each subset generate clusters of progeny which extend in a stereotypic, subset-specific direction in the brain. We have used this feature to identify one subset of four neuroblasts as being the likely progenitor cells for four clusters of embryonic neurons (W, X, Y, Z) which develop at around 55% of embryogenesis. We show that these progeny project axons via four discrete fascicles (w, x, y, z) into the embryonic central complex. At the single cell level, Golgi impregnation reveals that the axons from these neighbouring cell clusters remain discrete, and those from the same cluster tightly fasciculated, as they project into the central complex, consistent with a modular organization for this brain region. Received: 16 June 1997 / Accepted: 25 June 1997     

Glial cell lineage in vivo in the mouse cerebellum

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus-mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus-labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus-labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus-labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.     

Timelines in the insect brain: fates of identified neural stem cells generating the central complex in the grasshopper Schistocerca gregaria

This study employs labels for cell proliferation and cell death, as well as classical histology to examine the fates of all eight neural stem cells (neuroblasts) whose progeny generate the central complex of the grasshopper brain during embryogenesis. These neuroblasts delaminate from the neuroectoderm between 25 and 30 % of embryogenesis and form a linear array running from ventral (neuroblasts Z, Y, X, and W) to dorsal (neuroblasts 1-2, 1-3, 1-4, and 1-5) along the medial border of each protocerebral hemisphere. Their stereotypic location within the array, characteristic size, and nuclear morphologies, identify these neuroblasts up to about 70 % of embryogenesis after which cell shrinkage and shape changes render progressively more cells histologically unrecognizable. Molecular labels show all neuroblasts in the array are proliferative up to 70 % of embryogenesis, but subsequently first the more ventral cells (72–75 %), and then the dorsal ones (77–80 %), cease proliferation. By contrast, neuroblasts elsewhere in the brain and optic lobe remain proliferative. Apoptosis markers label the more ventral neuroblasts first (70–72 %), then the dorsal cells (77 %), and the absence of any labeling thereafter confirms that central complex neuroblasts have exited the cell cycle via programmed cell death. Our data reveal appearance, proliferation, and cell death proceeding as successive waves from ventral to dorsal along the array of neuroblasts. The resulting timelines offer a temporal blueprint for building the neuroarchitecture of the various modules of the central complex.     

Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. In this study, we use the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. Our results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. Our study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development.     

Morphogenesis and proliferation of the larval brain glia in Drosophila

Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.     

Neonatal separation stress reduces glial fibrillary acidic protein‐ and S100β‐immunoreactive astrocytes in the rat medial precentral cortex

The interactions between the mother/parents and their offspring provides socioemotional input, which is essential for the establishment and maintenance of synaptic networks in prefrontal and limbic brain regions. Since glial cells are known to play an important role in developmental and experience‐driven synaptic plasticity, the effect of an early adverse emotional experience induced by maternal separation for 1 or 6 h on the expression of the glia specific proteins S100β and glial fibrillary acidic protein (GFAP) was quantitatively analyzed in anterior cingulate cortex, hippocampus, and precentral medial cortex. Three animal groups were analyzed at postnatal day 14: (i) separated for 1 h; (ii) separated for 6 h; (iii) undisturbed (control). Twenty‐four hours after stress exposure, the stressed brains showed significantly reduced numbers of S100β‐immunoreactive (ir) cells in the anterior cingulate cortex (6‐h stress) and in the precentral medial cortex (1‐ and 6‐h stress). Significantly reduced numbers of GFAP‐ir cells were observed only in the medial precentral cortex (1‐ and 6‐h stress); no significant changes were observed in the anterior cingulate cortex. No significant changes of the two glial markers were observed in the hippocampus. Double‐labeling experiments with GFAP and pCREB revealed pCREB labeling only in the hippocampus, where the stressed brains (1 and 6 h) displayed significantly reduced numbers of GFAP/pCREB‐ir glial cells. The observed downregulation of glia‐specific marker proteins is in line with our hypothesis that emotional experience can alter glia cell activation in the juvenile limbic system. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Distribution and morphology of descending brain neurons in the cricket Gryllus bimaculatus

The number and distribution of descending brain neurons have been investigated in the cricket. The results are based on retrograde labeling of these cells with either Lucifer yellow or Neurobiotin via whole or small split portions of the cervical connectives. Various groups of cells and single neurons have been identified, and the morphology of more than 40 cells is described. Nearly 200 descending brain neurons can be stained via one cervical connective. Their perikarya are concentrated in clusters that occur ipsi- and contralateral to the filled connective and that lie dorsal and ventral in the brain. Descending cells only arborize in the nonglomerular neuropils of the brain and never branch in the optic lobe. Cells descending ipsilaterally never arborize in the contralateral hemisphere, whereas contralateral descending neurons often branch in both hemispheres. Irrespective of soma position, cells can arborize in the ventral and/or dorsal neuropils of the brain. Neurons with somata in the protocerebrum often have branches in the deutocerebrum and vice versa. The main arborizations of the cells from the prominent ventral i5 group are found in the same part of the protocerebrum. In contrast, various cells arborize in the ventral posterior deutocerebrum, but their somata are not located in different clusters. Thus, neurons from the same cluster may, but need not necessarily, arborize in the same brain area.     

A developmental study of serotonin-immunoreactive neurons in the embryonic brain of the Marbled Crayfish and the Migratory Locust: Evidence for a homologous protocerebral group of neurons

It is well established that the brains of adult malacostracan crustaceans and winged insects display distinct homologies down to the level of single neuropils such as the central complex and the optic neuropils. We wanted to know if developing insect and crustacean brains also share similarities and therefore have explored how neurotransmitter systems arise during arthropod embryogenesis. Previously, Sintoni et al. (2007) had already reported a homology of an individually identified cluster of neurons in the embryonic crayfish and insect brain, the secondary head spot cells that express the Engrailed protein. In the present study, we have documented the ontogeny of the serotonergic system in embryonic brains of the Marbled Crayfish in comparison to Migratory Locust embryos using immunohistochemical methods combined with confocal laser-scan microscopy. In both species, we found a cluster of early emerging serotonin-immunoreactive neurons in the protocerebrum with neurites that cross to the contralateral brain hemisphere in a characteristic commissure suggesting a homology of this cell cluster. Our study is a first step towards a phylogenetic analysis of neurotransmitter system development and shows that, as for the ventral nerve cord, traits related to neurogenesis in the brain can provide valuable hints for resolving the much debated question of arthropod phylogeny.     

Astrocyte-like glia associated with the embryonic development of the central complex in the grasshopper <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

In this study we employed the expression of the astrocyte-specific enzyme glutamine synthetase, in addition to the glia-specific marker Repo, to characterize glia cell types associated with the embryonic development of the central complex in the grasshopper Schistocerca gregaria. Double labeling experiments reveal that all glutamine synthetase-positive cells associated with the central complex are also Repo-positive and horseradish peroxidase-negative, confirming they are glia. Early in embryogenesis, prior to development of the central complex, glia form a continuous population extending from the pars intercerebralis into the region of the commissural fascicles. Subsequently, these glia redisperse to envelop each of the modules of the central complex. No glial somata are found within the central complex neuropils themselves. Since glutamine synthetase is expressed cortically in glia, it allows their processes as well as their soma locations to be visualized. Single cell reconstructions reveal one population of glia as directing extensive ensheathing processes around central complex neuropils such as the central body, while another population projects columnar-like arborizations within the central body. Such arborizations are only seen in central complex modules after their neuroarchitecture has been established suggesting that the glial arborizations project onto a prior scaffold of neurons or tracheae.     

Neurohormones as putative circadian clock output signals in the central nervous system of two cricket species

Antisera to the neuropeptides corazonin (Crz) and crustacean cardioactive peptide (CCAP) and to the diapause hormone (DH) react with small sets of neurones in the cephalic ganglia of the crickets Dianemobius nigrofasciatus and Allonemobius allardi. The distribution of their immunoreactivities is similar in the two species and overlaps with the locations of presumed circadian clock components in the optic lobes, protocerebrum, tritocerebrum, suboesophageal ganglion (SOG) and frontal ganglion. D. nigrofasciatus contains two Crz-immunoreactive (Crz-ir) cells in each optic lobe, six cell groups in the protocerebrum, four in the tritocerebrum, and one in SOG, whereas A. allardi harbours only five Crz-ir groups in the protocerebrum and four in the tritocerebrum. CCAP immunoreactivity occurs in both species in four protocerebrum cell clusters, four tritocerebrum cell clusters, four SOG cell clusters, one frontal ganglion cell cluster, and two optic lobe cell clusters; D. nigrofasciatus possesses two additional cells with unique links to the lamina in the optic lobe. DH-related antigens are present in four cell clusters in the optic lobe, six (D. nigrofasciatus) or eight (A. allardi) in the protocerebrum, four in the tritocerebrum, and three (A. allardi) or five (D. nigrofasciatus) in the SOG. Some of the detected cells also react with antibody to the clock protein Period (PER) or lie close to PER-ir cells. Crickets reared at two different photoperiods do not differ in the distribution and intensity of immunoreactivities. No changes have been detected during the course of diurnal light/dark cycles, possibly because the antisera react with persistent prohormones, whereas circadian fluctuations may occur at the level of their processing or of hormone release. The projection of immunoreactive fibres to several brain regions, the stomatogastric nervous system and the neurohaemal organs indicates multiple functions of the respective hormones. The work was supported by the “Research for Future” program of the Japan Society for the Promotion of Science (JSPS, 99L01205) and by the JSPS Postdoctoral Fellowship for Foreign Researchers (no. P 04197).     

Proliferative cell types in embryonic lineages of the central complex of the grasshopper Schistocerca gregaria

The central complex of the grasshopper Schistocerca gregaria develops to completion during embryogenesis. A major cellular contribution to the central complex is from the w, x, y, z lineages of the pars intercerebralis, each of which comprises over 100 cells, making them by far the largest in the embryonic protocerebrum. Our focus has been to find a cellular mechanism that allows such a large number of cell progeny to be generated within a restricted period of time. Immunohistochemical visualization of the chromosomes of mitotically active cells has revealed an almost identical linear array of proliferative cells present simultaneously in each w, x, y, z lineage at 50% of embryogenesis. This array is maintained relatively unchanged until almost 70% of embryogenesis, after which mitotic activity declines and then ceases. The array is absent from smaller lineages of the protocerebrum not associated with the central complex. The proliferative cells are located apically to the zone of ganglion mother cells and amongst the progeny of the neuroblast. Comparisons of cell morphology, immunoreactivity (horseradish peroxidase, repo, Prospero), location in lineages and spindle orientation have allowed us to distinguish the proliferative cells in an array from neuroblasts, ganglion mother cells, neuronal progeny and glia. Our data are consistent with the proliferative cells being secondary (amplifying) progenitors and originating from a specific subtype of ganglion mother cell. We propose a model of the way that neuroblasts, ganglion mother cells and secondary progenitors together produce the large cell numbers found in central complex lineages.     

Postembryonic development of astrocyte-like glia of the central complex in the grasshopper Schistocerca gregaria

Central complex modules in the postembryonic brain of the grasshopper Schistocerca gregaria are enveloped by Repo-positive/glutamine-synthetase-positive astrocyte-like glia. Such cells constitute Rind-Neuropil Interface glia. We have investigated the postembryonic development of these glia and their anatomical relationship to axons originating from the w, x, y, z tract system of the pars intercerebralis. Based on glutamine synthetase immunolabeling, we have identified four morphological types of cells: bipolar type 1 glia delimit the central body but only innervate its neuropil superficially; monopolar type 2 glia have a more columnar morphology and direct numerous gliopodia into the neuropil where they arborize extensively; monopolar type 3 glia are found predominantly in the region between the noduli and the central body and have a dendritic morphology and their gliopodia project deeply into the central body neuropil where they arborize extensively; multipolar type 4 glia link the central body neuropil with neighboring neuropils of the protocerebrum. These glia occupy type-specific distributions around the central body. Their gliopodia develop late in embryogenesis, elongate and generally become denser during subsequent postembryonic development. Gliopodia from putatively type 3 glia within the central body have been shown to lie closely apposed to individual axons of identified columnar fiber bundles from the w, x, y, z tract system of the central complex. This anatomical association might offer a substrate for neuron/glia interactions mediating postembryonic maturation of the central complex.     

Glia–Neuron Interactions in the Sensory-Motor Cortex of Warm-Blooded Animals (Guinea Pigs and Ground Squirrels) with Different Habitat Conditions and the M-Cholinergic Reaction of the Brain

The glia–neuron interactions were analyzed in the sensory-motor cortex of guinea pigs and ground squirrels (Spermophilus undulatus) during the active summer months. The glial cells were more concentrated in close proximity (15–25 μm) to neurons (38% in guinea pigs and 22.4% in ground squirrels). A more concentrated distribution of glial cells might be very necessary for spontaneous inactive nerve cells (37.2% in guinea pigs and 23% in ground squirrels), since these neurons are associated with the highest energy demand during their functioning and are most susceptible to disturbances of ion homeostasis. The network structure of glia and the close contact between glial cells and brain capillaries provide additional energy for neurons and stabilize the ion balance in the extracellular medium. Glial density in the sensory-motor cortex of ground squirrels is 3 times higher than that in the cortex of guinea pigs. The high content of glial cells in the ground-squirrel cortex is the most important protective factor for survival of animals during long-term hibernation, when the diffusion of K⁺ ions from nerve cells drastically increases due to the high temperature sensitivity of the M-cholinergic response.     

Morphometric characteristics of neuron-glial relations in brain edema caused by stimulation of hypothalamic nuclei

Morphologic characteristics and certain changes in cell composition of the cortex and the white substance of the brain have been studied at experimentally produced brain edema by stimulation of the lateral hypothalamic field, the indefinite zone, Forel's fields H1 and H2. It has been stated that diffuse edema of the white substance and perivascular edema dominate in histopathological changes. Morphometrical analysis of the structural changes had demonstrated a certain increase in the cortex thickness, decreased density in the arrangement of the neurons and increased volume of their nuclei at more moderate enlargement of their body volumes, as well as increased volume of the nuclei in the cortical glial cells and the white substance cells. In the cortex cells is observed, that is accompanied with increased glial index and average number of perineuronal gliocytes per one neuron. Simultaneously, in both hemispheres, the character in the arrangement of the perineuronal glia as regards the neuron changes. At the same time, in the white substance, the density of the glial arrangement sharply decreases. The changes have demonstrated that wider perivascular spaces predominate in small vessels. All the changes mentioned are more pronounced in the contralateral hemisphere.     

Diversification of indigenous gene- pool by using exotic germplasm in lentil (Lens culinaris Medikus subsp. culinaris)

Genetic diversity was studied among 21 accessions of lentil using SSR markers and morphological traits in order to assess the diversification of Indian gene-pool of lentil through introgression of exotic genes and introduction of germplasm. Among these , 16 genotypes either had ‘Precoz’ gene, an Argentine line in their pedigree or genes from introduced lines from ICARDA. Sixty five SSR markers and eight phenotypic traits were used to analyse the level of genetic diversity in these genotypes. Forty three SSR markers (66 %) were polymorphic and generated a total of 177 alleles with an average of 4.1 alleles per SSR marker. Alleles per marker ranged from 2 to 6. The polymorphic information content ranged 0.33 to 0.80 with an average of 0.57, suggesting that SSR markers are highly polymorphic among the studied genotypes. Genetic dissimilarity based a dendrogram grouped these accessions into two main clusters (cluster I and cluster II) and it ranged 33 % to 71 %, suggesting high level of genetic diversity among the genotypes. First three components of PCA based morphological traits explained higher variance (95.6 %) compared to PCA components based on SSR markers (42.7 %) of total genetic variance. Thus, more diversity was observed for morphological traits and genotypes in each cluster and sub-cluster showed a range of variability for seed size, earliness, pods/plant and plant height. Molecular and phenotypic diversity analysis thus suggested that use of germplasm of exotic lines have diversified the genetic base of lentil germplasm in India. This diversified gene-pool will be very useful in the development of improved varieties of lentil in order to address the effect of climate change, to adapt in new cropping systems niches such as mixed cropping, relay cropping, etc. and to meet consumers’ preference.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.