Brain-derived neurotrophic factor promotes neurite growth and survival of antennal lobe neurons in brain from the silk moth, Bombyx mori in vitro

This study was conducted to investigate effects of brain-derived neurotrophic factor on the neurite growth and the survival rate of antennal lobe neurons in vitro, and secretion of brain-derived neurotrophic factor-like neuropeptide from brain into hemolymph in the silk moth, Bombyx mori. In primary culture of antennal lobe neurons with brain-derived neurotrophic factor, it promoted both a neurite extension of putative antennal lobe projection neurons and an outgrowth of branches from principal neurites of putative antennal interneurons with significance (p<0.05). Brain-derived neurotrophic factor also increased significantly a survival rate of antennal lobe neurons (p<0.05). Results from immunolabeling of brain and retrocerebral complex, and ELISA assay of hemolymph showed that brain-derived neurotrophic factor-like neuropeptide was synthesized by both median and lateral neurosecretory cells of brain, then transported to corpora allata for storage, and finally secreted into hemolymph for action. These results will provide valuable information for differentiation of invertebrate brain neurons with brain-derived neurotrophic factor.     

Regulation of cyclic GMP elevation in the developing antennal lobe of the Sphinx moth, Manduca sexta.

In the moth, Manduca sexta, 3',5'-guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20-hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity-dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal-lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal-lobe neurons.     

Effects of Brain-Derived Neurotrophic Factor on Neurite Growth of Deutocerebral Neurons in Brain of the Silk Moth Bombyx mori

This study was conducted to investigate effects of brain‐derived neurotrophic factor (BDNF) on the neurite growth of deutocerebral neurons in vitro, and production of BDNF‐like neuropeptide from brain of the silk moth, Bombyx mori. In primary culture of antennal lobe (AL) neurons with BDNF, it promoted a significant neurite extension of putative AL projection neurons and an outgrowth of branches from principal neurites of putative AL interneurons. Results from immunolabeling of brain and retrocerebral complex showed that BDNF ‐like neuropeptide labeled in brain was synthesized by median and lateral neurosecretory cells, then transported to corpora allata for storage.     

Immunocytochemical identification of neuroactive substances in the antennal lobe of the male silkworm moth Bombyx mori

As a first step towards understanding the functional role of neuroactive substances in the first olfactory center of the male silkworm moth Bombyx mori, we carried out an immunocytochemical identification of antennal lobe neurons. Antibodies against gamma-aminobutyric acid (GABA), FMRFamide, serotonin, tyramine and histamine were applied to detect their existence in the antennal lobe. In the present immunocytochemical study, we clarified four antenno-cerebral tracts from their origin and projection pathways to the protocerebrum, and revealed the following immunoreactive cellular organization in the antennal lobe. 1) Local interneurons with cell bodies in the lateral cell cluster showed GABA, FMRFamide and tyramine immunoreactivity. 2) Projection neurons passing through the middle antenno-cerebral tract with cell bodies in the lateral cell cluster showed GABA and FMRFamide immunoreactivity. Projection neurons passing through the outer antenno-cerebral tract with cell bodies in the lateral cell cluster showed FMRFamide immunoreactivity. 3) Centrifugal neurons passing through the inner antenno-cerebral tract b with cell bodies located outside the antennal lobe showed serotonin and tyramine immunoreactivity. Our results revealed basic distribution patterns of neuroactive substances in the antennal lobe and indicated that each projection pathway from the antennal lobe to the protocerebrum contains specific combination of neuroactive substances.     

Enhancement by serotonin of the growth in vitro of antennal lobe neurons of the sphinx moth Manduca sexta

Cell culture experiments have been used to examine the effects of serotonin [5-hydroxytryptamine (5-HT)] on the morphological development of antennal lobe (AL) neurons in the brain of the sphinx moth, Manduca sexta. The majority of cells used in this study were from animals at stage 5 of the 18 stages of metamorphic adult development. 5-HT did not affect the survival of M. sexta AL neurons in culture, but did increase the numbers of cells displaying features characteristic of certain cell types. Three morphologically distinct cell types were examined in detail. The principal effect of 5-HT on these neurons was enhancement of cell growth. The magnitude of responses to this amine was cell-type specific. Site-specific responses to 5-HT were apparent also in one cell type. Our results suggest that the effects of 5-HT can change during the course of metamorphic development. These changes coincide temporally with the development of fast, sodium-based action potentials. © 1996 John Wiley & Sons, Inc.     

Morphology and physiology of the serotonin-immunoreactive putative antennal lobe feedback neuron in the male silkmoth Bombyx mori

In the male silkmoth Bombyx mori, olfactory information is relayed from olfactory receptor neurons in the antennae to the antennal lobe, and then to a variety of protocerebral neuropils. Currently, very little is known about neuromodulators that may affect the dynamics of this olfactory neural network. Immunocytochemical studies have revealed the presence of a serotonin-immunoreactive (SI) neuron that, in several insect species, is thought to provide feedback to the antennal lobe. To date, no studies have revealed details of this neuron's physiology. Using intracellular recording and staining, the silkmoth SI neuron (in two individuals) was first characterized physiologically and then stained with Lucifer Yellow to reveal morphological details. Immunocytochemical methods were also used to confirm the presence of serotonin. The silkmoth SI neuron branched in many important brain neuropils such as the mushroom body, central body, lateral accessory lobe and antennal lobe. The SI neuron in both individuals fired spontaneous, long duration action potentials, and responded to mechanosensory stimuli to the antennae.     

Effects of the steroid hormone 20-hydroxyecdysone and prior sensory input on the survival and growth of moth central olfactory neurons in vitro

Neurons in the developing (antennal) olfactory lobe of the moth Manduca sexta undergo a period of extensive process outgrowth and branching that coincides temporally with both a rising titer of the steroid hormone 20-hydroxyecdysone and the ingrowth of sensory axons from receptors in the antenna. To evaluate the contribution of these two influences to the morphological development of antennal-lobe neurons, we placed the neurons in cell culture. Antennal-lobe neurons were dissociated from normal and chronically unafferented lobes at different stages of development and were exposed to different doses of hormone. Six neuronal cell types with distinctive and stable morphologies appeared in cultures from all stages of pupal development. Morphological changes in these neuronal types were examined quantitatively by comparison of the total length and number of branches. We found that 20-hydroxyecdysone had little direct effect on the morphological development of antennal-lobe neurons, but brief exposure to sensory axons in vivo prior to dissociation significantly enhanced subsequent outgrowth in culture. © 1993 John Wiley & Sons, Inc.     

A novel serotonin-immunoreactive neuron in the antennal lobe of the sphinx moth Manduca sexta persists throughout postembryonic life

A single serotonin-immunoreactive neuron in the antennal lobe (AL) of the brain of the sphinx moth Manduca sexta is present in larval, pupal, and adult stages. This neuron has a neurite that extends to the contralateral AL, where it forms sparse arborizations in each glomerulus. Other neurites from this neuron project into the ipsilateral and contralateral protocerebrum. This cell is morphologically very different from other neurons previously characterized in the adult AL. The neuron maintains the same basic profile in the adult as in the larva, although fine processes such as the arborizations within the AL neuropil appear to be restructured to conform to the larger, more anatomically differentiated regions of the adult brain.     

The regulation of neurite outgrowth, growth cone motility, and electrical synaptogenesis by serotonin

Identified neurons of the buccal ganglion of the snail Helisoma when isolated from their ganglionic environment and plated in cell culture grow new neurites that are tipped with motile growth cones. Addition of the neurotransmitter serotonin to the culture medium surrounding actively growing neurons causes an immediate, premature cessation of neurite elongation in specific identified neurons. Serotonin selectively inhibits neurite extension of neurons B19 and P5 while having no effect on the extension of neuron B5. Coincident with the serotonin evoked inhibition of neurite elongation is an inhibition of growth cone motile activities and a retraction of growth cone filopodia and lamellipodia. One site of serotonin's growth inhibitory actions is directly at the growth cone rather than at the neurites or cell body. A second area of this study concerns connectivity. In Helisoma neurons the formation of electrical synaptic connections critically relies on both potential partner neurons having a mutual interaction of actively growing neurites. Neurons in a nongrowing state do not form electrical synapses (Hadley et al., 1983). As a result of inhibiting neurite extension, serotonin is able to affect synaptogenesis by preventing certain neurons (neurons B19) from forming electrical synaptic connections with other neurons (neurons B5) that are themselves competent to interconnect. Thus, by inhibiting neurite extension, serotonin is capable of regulating both the development of arborizations and the formation of connectivity.     

Modulatory actions of dopamine and serotonin on insect antennal lobe neurons: insights from studies in vitro

Biogenic amines play diverse roles in the development and modulation of invertebrate neurons and ultimately also, in the regulation of animal behaviour. Here we examine the contribution that analyses of antennal lobe neurons in vitro have made towards our understanding of the mechanisms through which dopamine and serotonin operate in primary olfactory centres of the brain of the moth, Manduca sexta and the honey bee, Apis mellifera. This chapter reviews evidence suggesting that these biogenic amines function as regulators of neuronal development and as mediators of cellular and behavioural plasticity, in part at least, through the modulation of K(+) conductances in the cells. Insect neurons in vitro provide an excellent model for exploring basic principles of amine function and their impact on neuronal excitability.     

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. © 1995 John Wiley & Sons, Inc.     

The effects of 20-hydroxyecdysone on the differentiationin vitro of cells from the eye imaginal disc fromDrosophila melanogaster

We have examined the effects of the insect ecdysteroid, 20-hydroxyecdysone, on the differentiation of neuronal and non-neuronal elements in the developing adult visual system, usingin vitro methods inDrosophila. We examined the differentiation of early neuronal markers in the presence and absence of 1 μg/ml 20-hydroxyecdysone. Immunoreactivity to 22C10, a marker of an early neuronal antigen, as well as to the photoreceptor-specific antibody 24B10, suggests that differentiation of neuronal and photoreceptor antigens does not require 20-hydroxyecdysone. In eye-discs cultured from animals 5 hours after the white prepupa (P+5), ommochrome pigmentation first appeared after 2 days in 1 μg/ml 20-hydroxyecdysone, but cultures lacked pigmentation without 20-hydroxyecdysone. Our culture conditions failed to support the formation of the second screening pigment, drosopterins, even with 20-hydroxyecdysone. Eye discs from P+5 also formed lenses and interommatidial bristles in culture when 20-hydroxyecdysone was added but not in cultures devoid of the hormone. The differentiation of synaptotagmin and the elongation of extending photoreceptor neurites from eye disc fragments both occur in the absence of 20-hydroxyecdysone in cultures, but adding the hormone increased average neurite length. The threshold for enhanced neurite length was less than 125 ng/ml 20-hydroxyecdysone. Eye-disc cultures also developed immunoreactivity to histamine, the photoreceptor transmitter, from synthesis not re-uptake, in both the presence and in the absence of 20-hydroxyecdysone. These findings suggest that photoreceptor axons may be able to release transmitterin vivo both when they grow into the optic lobe and during the subsequent events in synapse formation.     

Conserved synteny of genes between chromosome 15 of Bombyx mori and a chromosome of Manduca sexta shown by five-color BAC-FISH.

The successful assignment of the existing genetic linkage groups (LGs) to individual chromosomes and the second-generation linkage map obtained by mapping a large number of bacterial artificial chromosome (BAC) contigs in the silkworm, Bombyx mori, together with public nucleotide sequence databases, offer a powerful tool for the study of synteny between karyotypes of B. mori and other lepidopteran species. Conserved synteny of genes between particular chromosomes can be identified by comparatively mapping orthologous genes of the corresponding linkage groups with the help of BAC-FISH (fluorescent in situ hybridization). This technique was established in B. mori for 2 differently labeled BAC probes simultaneously hybridized to pachytene bivalents. To achieve higher-throughput comparative mapping using BAC-FISH in Lepidoptera, we developed a protocol for five-color BAC-FISH, which allowed us to map simultaneously 6 different BAC probes to chromosome 15 in B. mori. We identified orthologs of 6 B. mori LG15 genes (RpP0, RpS8, eIF3, RpL7A, RpS23, and Hsc70) for the tobacco hornworm, Manduca sexta, and selected the ortholog-containing BAC clones from an M. sexta BAC library. All 6 M. sexta BAC clones hybridized to a single M. sexta bivalent in pachytene spermatocytes. Thus, we have confirmed the conserved synteny between the B. mori chromosome 15 and the corresponding M. sexta chromosome (hence provisionally termed chromosome 15).     

Mas-allatotropin in the developing antennal lobe of the sphinx moth Manduca sexta: distribution, time course, developmental regulation, and colocalization with other neuropeptides

The paired antennal lobes (ALs) of the sphinx moth Manduca sexta serve as a well-established model for studying development of the primary integration centers for odor information in the brain. To further reveal the role of neuropeptides during AL development, we have analyzed cellular distribution, developmental time course, and regulation of the neuropeptide M. sexta allatotropin (Mas-AT). On the basis of morphology and appearance during AL formation, seven major types of Mas-AT-immunoreactive (ir) cells could be distinguished. Mas-AT-ir cells are identified as local, projection, and centrifugal neurons, which are either persisting larval or newly added adult-specific neurons. Complementary immunostaining with antisera against two other neuropeptide families (A-type allatostatins, RFamides) revealed colocalization within three of the Mas-AT-ir cell types. On the basis of this neurochemistry, the most prominent type of Mas-AT-ir neurons, the local AT neurons (LATn), could be divided in three subpopulations. The appearance of the Mas-AT-ir cell types occurring during metamorphosis parallels the rising titer of the developmental hormone 20-hydroxyecdysone (20E). Artificially shifting the 20E titer to an earlier developmental time point resulted in the precocious occurrence of Mas-AT immunostaining. This result supports the hypothesis that the pupal rise of 20E is causative for Mas-AT expression during AL development. Comparing localization and developmental time course of Mas-AT and other neuropeptides with the time course of AL formation suggests various functions for these neuropeptides during development, including an involvement in the formation of the olfactory glomeruli.     

Primary culture of antennal mechanoreceptor neurons of Manduca sexta

We have developed a primary cell culture system of antennal mechanoreceptor neurons from early-stage pupal sphinx moth Manduca sexta. Dissociated neurons from the moth antennae differentiated, grew and survived for several weeks in a conditioned culture medium. Bipolar neurons with soma diameters of 10-25 microns from the basal portion of the antennae could be positively identified as mechanoreceptor neurons, presumably derived from Johnston's organ, using a monoclonal antibody that recognizes neurofilaments in these neurons. The immunoreactivity was clear and specific from the first day after dissociation and became stronger during several days in culture. These neurons appeared healthy and showed normal whole-cell properties only a few days after plating. We found numerous mechanosensitive ion channels responding to both negative and positive pressures on the somata and neurites of differentiated neurons. This new culture system provides access to mechanoreceptor neurons that has never been possible before, allowing the use of both mechanical and electrical stimuli on neurons that are free from the accessory structures surrounding them in intact preparations.     

Different Ca2+ signalling cascades manifested by mastoparan in the prothoracic glands of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori

Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and the silkworm, Bombyx mori, resulted in increases in intracellular Ca(2+) ([Ca(2+)](i)). In M. sexta, Gi proteins are involved in the mastoparan-stimulated increase in [Ca(2+)](i). However, there is no involvement of Gi proteins in the mastoparan-stimulated increase in [Ca(2+)](i) in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic glands mastoparan increases [Ca(2+)](i) even in the absence of extracellular Ca(2+). Pharmacological manipulation of the Ca(2+) signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan's first site of action is influx of Ca(2+) through plasma membrane Ca(2+) channels while in B. mori prothoracic glands, mastoparan's first site of action is mobilization of Ca(2+) from intracellular stores. In M. sexta, the combined results indicate the presence of mastoparan-sensitive plasma membrane Ca(2+) channels, distinct from those activated by prothoracicotropic hormone or the IP(3) signalling cascade, that coordinate spatial increases in [Ca(2+)](i) in prothoracic gland cells. We propose that in B. mori, mastoparan stimulates Ca(2+) mobilization from ryanodine-sensitive intracellular Ca(2+) stores in prothoracic gland cells.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Local interneurons and information processing in the olfactory glomeruli of the moth Manduca sexta

Intracellular recordings were made from the major neurites of local interneurons in the moth antennal lobe. Antennal nerve stimulation evoked 3 patterns of postsynaptic activity: (i) a short-latency compound excitatory postsynaptic potential that, based on electrical stimulation of the antennal nerve and stimulation of the antenna with odors, represents a monosynaptic input from olfactory afferent axons (71 out of 86 neurons), (ii) a delayed activation of firing in response to both electrical- and odor-driven input (11 neurons), and (iii) a delayed membrane hyperpolarization in response to antennal nerve input (4 neurons).Simultaneous intracellular recordings from a local interneuron with short-latency responses and a projection (output) neuron revealed unidirectional synaptic interactions between these two cell types. In 20% of the 30 pairs studied, spontaneous and current-induced spiking activity in a local interneuron correlated with hyperpolarization and suppression of firing in a projection neuron. No evidence for recurrent or feedback inhibition of projection neurons was found. Furthermore, suppression of firing in an inhibitory local interneuron led to an increase in firing in the normally quiescent projection neuron, suggesting that a disinhibitory pathway may mediate excitation in projection neurons. This is the first direct evidence of an inhibitory role for local interneurons in olfactory information processing in insects. Through different types of multisynaptic interactions with projection neurons, local interneurons help to generate and shape the output from olfactory glomeruli in the antennal lobe.Abbreviations AL antennal lobe - EPSP excitatory postsynaptic potential - GABA -aminobutyric acid - IPSP inhibitory postsynaptic potential - LN local interneuron - MGC macroglomerular complex - OB olfactory bulb - PN projection neuron - TES N-tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid     

Regulation of cyclic GMP elevation in the developing antennal lobe of the sphinx moth,Manduca sexta

In the moth, Manduca sexta, 3′,5′‐guanosine monophosphate (cGMP) is transiently elevated during adult development in about 100 neurons of the antennal lobe. We demonstrate that nearly all of these neurons are local interneurons of the lateral cluster I, that their capacity to show a strong cGMP response during development is regulated by the steroid hormone 20‐hydroxyecdysone, and that in a subpopulation of these neurons cGMP elevation seems to be controlled directly by the gaseous messenger molecule nitric oxide (NO). Treatment with the acetylcholine esterase inhibitor eserine, antennal nerve transection, and electrical stimulation of the antennae suggest that NO/cGMP signaling during development is an activity‐dependent process. Besides input from the antennae, input from the central brain and the ventral ganglia is involved in upregulating cGMP in the antennal‐lobe neurons. Possible sources are centrifugal aminergic neurons, since application of serotonin and histamine enhances the GMP signal in local interneurons. Comparing the time course of cGMP elevation with events occurring during development leads us to the hypothesis that the NO/cGMP signaling pathway might be involved in synapse formation of a subset of antennal‐lobe neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 359–375, 1999