Development of pigment-dispersing hormone-immunoreactive neurons in the nervous system of Drosophila melanogaster

An antiserum against the crustacean pigment-dispersing hormone (PDH) was used to identify PDH-immunoreactive neurons in the developing nervous systems of wild type Drosophila melanogaster and the brain mutant disconnected. Particular attention was paid to a group of PDH-immunoreactive neurons at the anterior margin of the medulla—the pigment-dispersing factor-containing neurons close to the medulla (PDFMe neurons)—that seem to be involved in the control of adult circadian rhythmicity. In adults, this group consists of four to six neurons with large somata (large PDFMe neurons) and of four neurons with small somata (small PDFMe neurons). Both subgroups were usually absent in adults of behaviorally arrhythmic mutants of disconnected. In the wild type, PDH immunoreactivity was seen first in the small PDFMe neurons of 4 hour old first-instar larvae. The small PDFMe neurons were found to persist unchanged into adulthood, whereas the large ones seemed to develop halfway through metamorphosis. Beside the PDFMe neurons, three other clusters of PDH-immunoreactive neurons were stained in the developing nervous systems of Drosophila and are described in detail. Two of them were located in the brain, and the third was located in the abdominal neuromeres of the thoracic nervous system. In the mutant disconnected, the larval and the adult set of PDFMe neurons were absent. The other clusters of PDH-immunoreactive neurons seemed to develop normally. The present results are consistent with the hypothesis that the PDFMe neurons are circadian pacemaker neurons that may control rhythmic processes in larvae, pupae, and adults. J. Comp. Neurol. 380:335–354, 1997. © 1997 Wiley-Liss, Inc.     

Myoinhibitory peptides in the brain of the cockroach Leucophaea maderae and colocalization with pigment-dispersing factor in circadian pacemaker cells

Myoinhibitory peptides (MIPs) are a family of insect W(X(6))Wamides with inhibitory effects on visceral muscles and juvenile hormone synthesis. Although MIPs are widely distributed within the nervous system, a detailed analysis of their distribution and function in insect brains is still missing. We analyzed the distribution of MIPs in the brain of the cockroach Leucophaea maderae. We focused on the accessory medulla (AMe), a small neuropil near the medulla that acts as the master circadian clock. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) and Nano-LC electrospray ionization (ESI) mass spectrometry revealed five Lem-MIPs in preparations of the AMe and corpora cardiaca. The complete sequences of two of these peptides were identified. Immunocytochemistry revealed wide distribution of MIP-related peptides in the cockroach brain. The superior median protocerebrum, parts of the central complex, and the tritocerebrum showed particularly dense immunostaining. In contrast, only a few local interneurons were stained in the antennal lobe and a few extrinsic neurons in the mushroom body, including a giant neuron innervating the calyces. The noduli of the AMe showed dense immunostaining, and neurons in all AMe cell groups except the anterior neurons were labeled. Pigment-dispersing factor- (PDF) and MIP immunostaining was colocalized in two neurons of the AMe. No colocalization of MIP- and PDF immunostaining was detected in the anterior optic commissure, but two small PDF-immunoreactive commissural fibers near the posterior optic commissure showed colocalized MIP immunostaining. The results suggest that several MIPs participate in different functional circuits of the circadian system and are involved in multiple brain circuits of the Madeira cockroach.     

Implementation of pigment-dispersing factor-immunoreactive neurons in a standardized atlas of the brain of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae is an established model in circadian rhythm research. Its circadian clock is located in the accessory medulla of the brain. Pigment-dispersing factor-immunoreactive (PDF-ir) neurons of the accessory medulla act as circadian pacemakers controlling locomotor activity rhythms. To characterize the neuronal network of the circadian system in L. maderae, the PDF-ir neurons were implemented into a standardized three-dimensional atlas of the cockroach brain. Serial confocal images from 20 wholemount brains were used for the construction of the atlas comprising 21 neuropils. Two different standardization protocols were employed: the iterative shape averaging (ISA) procedure using an affine transformation followed by iterative non-rigid registrations, and the virtual insect brain (VIB) protocol employing local non-rigid transformations after global and local rigid transformations. Quantitative analysis of the 20 brains revealed that volumes of the accessory medulla are directly correlated with the volumes of the medulla, the protocerebral bridge, and the upper division of the central body, suggesting functional connections among these neuropils. For a standardized reconstruction of the circadian pacemaker network, the ISA protocol was used to register PDF-ir neurons in the standard cockroach brain. The registration revealed that two PDF-ir arborization areas in the brain are highly interconnected with other PDF-ir projection sites and appear to be contacted both by fibers in the posterior and the anterior optic commissures. The distances between PDF-ir branching areas show specific numerical relationships that might be physiologically relevant for temporal encoding.     

Peptide-immunocytochemistry of neurosecretory cells in the brain and retrocerebral complex of the sphinx moth Manduca sexta.

Antisera against a variety of vertebrate and invertebrate neuropeptides were used to map cerebral neurosecretory cells in the sphinx moth Manduca sexta. Intense immunoreactive staining of distinct populations of neurosecretory cells was obtained with antisera against locust adipokinetic hormone, bovine pancreatic polypeptide, FMRFamide, molluscan small cardioactive peptide (SCPB), leucine-enkephalin, gastrin/cholecystokinin, and crustacean beta-pigment dispersing hormone (beta PDH). Other antisera revealed moderate to weak staining. Each type of neurosecretory cell is immunoreactive with at least one of the antisera tested, and most of these neurons can be identified anatomically. The staining patterns provide additional information on the organization of cerebral neurosecretory cells in M. sexta. Based upon anatomical and immunocytochemical characteristics, 11 types of neurosecretory cells have been recognized in the brain, one type in the suboesophageal ganglion, and one in the corpus cardiacum. Extensive colocalization experiments show that many neurosecretory cells are immunoreactive with several different antisera. This raises the possibility that these cells may release mixtures of neuropeptides into the hemolymph, as has been demonstrated in certain other systems. The immunocytochemical data should be helpful in efforts to identify additional peptide neurohormones released from the brain of this and other insects.     

Substance P-, FMRFamide-, and gastrin/cholecystokinin-like immunoreactive neurons in the thoraco-abdominal ganglia of the flies Drosophila and Calliphora.

Immunocytochemical analysis of the thoraco-abdominal ganglia of the flies Drosophila melanogaster and Calliphora vomitoria revealed neurons displaying substance P- (SPLI), FMRFamide-(FLI), and cholecystokinin-like (CCKLI) immunoreactivity. It could be demonstrated that a number of neurons contain peptides reacting with antisera against all the three types of substances, others were either FLI or CCKLI alone. No neurons displayed only SPLI. Instead, the total number (about 30) of SPLI neurons constitute a subpopulation of the FLI/CCKLI neurons. Many of the identifiable immunoreactive neurons seem to be homologous in the two fly species. One set of six large neurons, termed ventral thoracic neurosecretory neurons (VTNCs), are among those that are SPLI, FLI, and CCKLI in both Drosophila and Calliphora. With the present immunocytochemical technique, the detailed morphology of the VTNCs could be resolved. These neurosecretory neurons supply the entire dorsal neural sheath of the thoraco-abdominal ganglia with terminals, thus forming an extensive neurohaemal area. The VTNCs also have processes connecting the thoracic neuromeres to the cephalic suboesophageal ganglion, as well as extensive arborizations in the thoracic ganglia, suggesting an important role in integrating and/or regulating large portions of the central nervous system, in addition to their neurosecretory function. Most of the other SPLI, FLI, and CCKLI neurons in the thoraco-abdominal ganglia seem to be interneurons. However, there are four FLI neurons that appear to be efferents innervating the hindgut and a few abdominal FLI and CCKLI neurons may be additional neurosecretory cells. From the present study it appears as if neuropeptides related to substance P, FMRFamide and CCK have roles as neurotransmitters/neuromodulators and circulating neurohormones in Drosophila and Calliphora.     

Proctolin-like immunoreactive neurons in the blowfly central nervous system

The pentapeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is a well-studied bioactive substance in insects. With an antiserum against proctolin we have mapped proctolinlike-immunoreactive (PLI) neurons in the nervous system of the blowfly Calliphora erythrocephala. In the brain, including the suboesophageal ganglia, 80-90 neurons were found to be PLI. A further 200-250 PLI neurons innervate the lobula of the optic lobe. The thoracic ganglia contain 100-130, and the abdominal ca. 60 PLI neurons. In the brain and ventral ganglia the immunoreactive neurons are of different types: interneurons, efferents (possibly some motorneurons), and neurosecretory cells. Some of these neurons are individually identifiable; others can be identified collectively as clusters. Identifiable neurons innervate protocerebral neuropil associated with the pars intercerebralis and the beta-lobes of the mushroom bodies as well as tritocerebral neuropil. Some of the prominent clusters innervate the central body of the protocerebrum, tritocerebrum, and possibly leg motor neurons. One abdominal cluster is of special interest because it consist of efferent neurons with processes in the lateral abdominal nerves. Some of these processes are located in the neural sheath in neurohaemal regions, and electron microscopy demonstrates that their terminals are outside the blood-brain barrier. The PLI processes in the protocerebrum contain large granular vesicles and form chemical synapses with different kinds of nonimmunoreactive neural elements. Thus, in Calliphora the proctolinlike substance may be used as a central transmitter/modulator, a neuromuscular transmitter, and a neurohormone released into the circulation.     

Organization of projection neurons and local neurons of the primary auditory center in the fruit fly Drosophila melanogaster

Acoustic communication between insects serves as an excellent model system for analyzing the neuronal mechanisms underlying auditory information processing. The detailed organization of auditory neural circuits in the brain has not yet been described. To understand the central auditory pathways, we used the brain of the fruit fly Drosophila melanogaster as a model and performed a large‐scale analysis of the interneurons associated with the primary auditory center. By screening expression driver strains and performing single‐cell labeling of these strains, we identified 44 types of interneurons innervating the primary auditory center. Five types were local interneurons whereas the other 39 types were projection interneurons connecting the primary auditory center with other brain regions. The projection neurons comprised three frequency‐selective pathways and two frequency‐embracive pathways. Mapping of their connection targets revealed that five neuropils in the brain—the wedge (WED), anterior ventrolateral protocerebrum, posterior ventrolateral protocerebrum (PVLP), saddle (SAD), and gnathal ganglia (GNG)—were intensively connected with the primary auditory center. In addition, several other neuropils, including visual and olfactory centers in the brain, were directly connected to the primary auditory center. The distribution patterns of the spines and boutons of the identified neurons suggest that auditory information is sent mainly from the primary auditory center to the PVLP, WED, SAD, GNG, and thoracico‐abdominal ganglia. Based on these findings, we established the first comprehensive map of secondary auditory interneurons, which indicates the downstream information flow to parallel ascending pathways, multimodal pathways, and descending pathways. J. Comp. Neurol. 524:1099–1164, 2016. © 2016 Wiley Periodicals, Inc.     

Peptidergic clock neurons in Drosophila: Ion transport peptide and short neuropeptide F in subsets of dorsal and ventral lateral neurons

About 150 clock neurons are clustered in different groups in the brain of Drosophila. Among these clock neurons, some pigment‐dispersing factor (PDF)‐positive and PDF‐negative lateral neurons (LNs) are principal oscillators responsible for bouts of activity in the morning and evening, respectively. The full complement of neurotransmitters in these morning and evening oscillators is not known. By using a screen for candidate neuromediators in clock neurons, we discovered ion transport peptide (ITP) and short neuropeptide F (sNPF) as novel neuropeptides in subpopulations of dorsal (LN_ds) and ventral (s‐LN_vs) LNs. Among the six LN_ds, ITP was found in one that coexpresses long neuropeptide F (NPF) and cryptochrome. We detected sNPF in two LN_ds that also express cryptochrome; these cells are distinct from three LN_ds expressing NPF. Thus, we have identified neuropeptides in five of the six LN_ds. The three LN_ds expressing cryptochrome, with either ITP or sNPF, are the only ones with additional projections to the accessory medulla. Among the five s‐LN_vs in the adult brain, ITP was detected in the fifth neuron that is devoid of PDF and sNPF in the four neurons that also express PDF. By using a choline acetyltransferase (Cha) Gal4, we detected Cha expression in the two sNPF producing LN_ds and in the fifth s‐LN_v. In the larval brain, two of the four PDF‐producing s‐LN_vs coexpress sNPF. Our findings emphasize that the LN_ds are heterogeneous both anatomically and with respect to content of neuropeptides, cryptochrome, and other markers and suggest diverse functions of these neurons. J. Comp. Neurol. 516:59–73, 2009. © 2009 Wiley‐Liss, Inc.     

Vasopressin- and proctolin-like immunoreactive efferent neurons in blowfly abdominal ganglia: development and ultrastructure

In the neural sheath of the fused thoracicoabdominal ganglia of the blowfly Calliphora erythrocephala, extensive neurohaemal areas can be seen in the electron microscope. A separate set of neurohaemal areas located in the sheath of the lateral abdominal nerve roots contain neural terminals of at least three morphological types. To determine which bioactive substances are stored and possibly released from the neurons supplying these neurohaemal areas, we applied a large number of antisera raised against different neuropeptides of invertebrate and mammalian type. Antisera to two types of neuropeptides react with neurons innervating the sheath of the abdominal nerve roots: antisera to lysine-vasopressin and proctolin. There are only 14-24 vasopressin-like immunoreactive (VPLI) neurons in the entire nervous system of Calliphora. These are all restricted to a bilateral cluster in the fused abdominal ganglia. From this cluster, the neurohaemal areas in abdominal nerve roots are supplied. Proctolin-like immunoreactivity (PLI) can be seen in a large number of neurons in the nervous system of blowflies. The supply of PLI terminals to the abdominal nerve roots is from 12 to 14 neurons in a bilateral cluster of abdominal PLI neurons. It is clear from light- and electron-microscopic immunocytochemistry that the two antisera label two separate populations of neurons that form overlapping terminals in the neural sheath. The immunoreactive terminals are located just below the permeable acellular basal lamina of the neural sheath. Hence, it is likely that at least two different bioactive peptides can be released neurohormonally into the circulation. An additional set of four efferent PLI neurons send axons into the medial abdominal nerve. These do not form neurohaemal terminals in the nerve root, but may innervate the hindgut. Also in the larval nervous system, VPLI and PLI neurons can be recognized. In the larva, the peptide-containing neurons are segmentally arranged. The 14 larval VPLI neurons supply segmental abdominal nerves with axons that run inside the nerves to their targets. During metamorphosis, the segmental nerves fuse and the VPLI axons invade the neural sheath where they arborize and form varicose terminals. About the same number of PLI neurons could be detected in the abdominal ganglia of larval and adult flies. Only for a set of four caudal PLI neurons could efferent axons be traced in the larva. These axons run inside the medial abdominal nerves. The same four PLI neurons, with the same axonal projections, can be recognized in the adults.     

Vasopressin-immunoreactive neurons and neurohemal systems in cockroaches and mantids.

Vasopressin-like neuropeptides of insects are of special interest because of their possible function as hormones and neuromodulators. Therefore, this study was undertaken by using whole-mount immunofluorescent staining by two antisera that recognize different types of vasopressin-like immunoreactive groups of neurons in the cockroaches Periplaneta americana, Leucophaea maderae, Nauphoeta cinerea, Diploptera punctata, and Blaberus discoidalis and in the mantids Litaneuria minor and Tenodera aridifolia sinensis. Using an antiserum to Arg/vasopressin, only two cells, the paired ventral paramedian (PVP) neurons, were immunostained in the central nervous system (CNS) of the cockroaches. These cells are located in the subesophageal ganglion, project throughout the CNS, and appear to be neurosecretory. Their varicose collaterals extend into the dorsal (motor) neuropil of the segmental ganglia, and this neuropil may be the principal site of the release of their neurosecretion. The PVP neurons were also stained by an antiserum to Lys/vasopressin; in addition, this antiserum stained several other groups of neurons, most of which appeared to be neurosecretory. Two pairs of Lys/vasopressin-immunoreactive cells are similar to the PVP neurons in that they are located in the subesophageal ganglion, extend through the ventral nerve cord, have collaterals in the dorsal neuropil of the segmental ganglia, and appear to be neurosecretory within the CNS. In addition, midventral and anteroventral clusters of Lys/vasopressin-immunoreactive neurosecretory neurons in the subesophageal ganglion project neurohemal release sites on the corpora allata. Other types of Lys/vasopressin-immunoreactive neurons include median and lateral neurosecretory cells of the protocerebrum and neurosecretory cells in the tritocerebrum, all of which project to the corpora cardiaca. In the abdominal ganglia there are posterolateral clusters of Lys/vasopressin neurosecretory neurons, and these cells extend to neurohemal release sites on the transverse and lateral cardiac nerves. In mantids the anti-Arg/vasopressin and anti-Lys/vasopressin antisera stained most of the same groups of neurons that these antisera recognized in cockroaches. The results of this study suggest that there are two or more vasopressin-like peptides in cockroaches and mantids and that these peptides may be released either as hormones in the blood or as neurosecretions within the CNS. Their function(s) in these insects remains to be determined.     

Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system

Drosophila melanogaster is a long‐standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor‐labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s‐LN_v) and one ITP positive dorsolateral neuron (LN_d). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre‐ and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF‐neurons (l‐LN_vs). We could show that the four hardly distinguishable l‐LN_vs consist of two subgroups with different innervation patterns. While three of the neurons reflect the well‐known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla‐surface. We named this neuron “extra” l‐LN_v (l‐LN_vx). We suggest the anatomical findings reflect different functional properties of the two l‐LN_v subgroups.     

Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin-related peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol. 118:161-172), including the original [Asn(13)]-orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da). The fifth peptide is a hitherto unknown orcokinin variant: [Ala(8)-Ala(13)]-orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well-studied system.     

Histamine-immunoreactive neurons in the midbrain and suboesophageal ganglion of sphinx moth Manduca sexta

This paper describes the distribution of histamine-like immunoreactivity in the midbrain and suboesophageal ganglion of the sphinx moth Manduca sexta. Intense immunocytochemical staining was detected in ten bilateral pairs of neurons in the median protocerebrum and in one pair of neurons in the suboesophageal ganglion. Whereas most areas of the brain and suboesophageal ganglion are innervated by one or more of these neurons, typically no immunoreactive fibers were found in the mushroom bodies, the protocerebral bridge, and the lateral horn of the protocerebrum. The 11 histamine-immunoreactive neurons were reconstructed from serial sections. Ten neurons have bilateral arborizations, often with axonal projections in symmetric areas of both hemispheres. One neuron, whose soma resides in the lateral protocerebrum, has only unilateral projections. Of the 11 neurons, 6 occur in pairs with similar morphological features. In addition to these neurons, weak histamine-like immunoreactivity was detected in 7-13 interneurons that were not reconstructed individually. The central projections of the ocellar nerves from the intracranial ocelli also exhibit histamine-like immunoreactivity. The single-cell reconstructions reveal similarities between the organization of histamine- and serotonin-immunoreactive neurons in the brain and suboesophageal ganglion of this insect.     

Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster

The clock-gene-expressing lateral neurons are essential for the locomotor activity rhythm of Drosophila melanogaster. Traditionally, these neurons are divided into three groups: the dorsal lateral neurons (LN(d)), the large ventral lateral neurons (l-LN(v)), and the small ventral lateral neurons (s-LN(v)), whereby the latter group consists of four neurons that express the neuropeptide pigment-dispersing factor (PDF) and a fifth PDF-negative neuron. So far, only the l-LN(v) and the PDF-positive s-LN(v) have been shown to project into the accessory medulla, a small neuropil that contains the circadian pacemaker center in several insects. We show here that the other lateral neurons also arborize in the accessory medulla, predominantly forming postsynaptic sites. Both the l-LN(v) and LN(d) are anatomically well suited to connect the accessory medullae. Whereas the l-LN(v) may receive ipsilateral photic input from the Hofbauer-Buchner eyelet, the LN(d) invade mainly the contralateral accessory medulla and thus may receive photic input from the contralateral side. Both the LN(d) and the l-LN(v) differentiate during midmetamorphosis. They do so in close proximity to one another and the fifth PDF-negative s-LN(v), suggesting that these cell groups may derive from common precursors.     

Serotonin-containing neurons in Drosophila melanogaster: development and distribution

Antibodies made against serotonin (5HT) were used to identify the serotonin neuronal system in the developing and adult nervous system of Drosophila melanogaster. The 5HT neuronal pattern is composed of a small number of neurons, 84 in larvae and 106 in adults, distributed in clusters composed of one to five neurons in the CNS; 5HT immunoreactive (5HT-IR) neurons appear to be predominantly intrasegmental interneurons; however, intersegmental 5HT-IR fibers are observed and at least some neurons send peripheral fibers. Acquisition of 5HT immunoreactivity in the CNS occurs late in embryogenesis, by 16-18 hours, and most if not all the 5HT neurons appear to persist into adulthood. During early metamorphosis, the intensity of 5HT-IR neuropil transiently decreases. Other changes in the CNS during this period are reflected in the appearance of two new 5HT clusters and 5HT-IR neuropil in the developing optic lobes. Comparison of the 5HT-IR pattern with other transmitter systems in Drosophila as well as comparison of the 5HT-IR pattern within different insect species is presented.     

Distribution of myomodulin-like immunoreactivity in the adult and developing ventral nervous system of the locust Schistocerca gregaria

The distribution of myomodulin-like immunoreactivity in the ventral nervous system of an insect, the locust Schistocerca gregaria, both in the adult and during development, is described. The result suggest the presence of a novel modulatory system in insects which uses myomodulin-like neuropeptides. The study also indicates that the myomodulins, which were first identified in mollusks, may represent another interphyletic family of neuropeptides. In the suboesophageal ganglion, immunoreactive cells occur in five groups. The processes from the two anterior ventral midline groups of cells projects to the corpora allata via nervi corpora allata II. Thus myomodulin-like neuropeptides may be involved in the control of the release of juvenile hormone from the corpora allata. The thoracic ganglia contain three groups of immunoreactive cells, including a bilaterally symmetrical group of 12–15 posterior lateral cells, which project to the median nerve and its neurohaemal organs, suggesting a possible neurohaemal role for myomodulin-like peptides. Each thoracic neuromere also contains a single, intensely stained, dorsal unpaired median (DUM) cell that may correspond to the so-called H cell. In the abdominal ganglia, the staining shows sexual dimorphism, both in terms of the number of dorsal and ventral midline cells stained and in terms of the distribution of their immunoreactive processes. Myomodulin-like immunoreactivity is one of the earliest neurotransmitter/neurohormone phenotypes detectable during the development of the locust nervous system. It first appears in the single DUM cells in each of the thoracic neuromeres at 50% development, and the complete adult pattern of staining is present at 85%-90% of development. © 1994 Wiley-Liss, Inc.     

Development of locustatachykinin immunopositive neurons in the central complex of the beetle Tenebrio molitor

Locustatachykinin-immunoreactive (LomTK-IR) interneurons were found to be associated with the central complex, a prominent neuropil region of the insect brain. The structures and development of this set of brain interneurons was studied from the embryo onward in the beetle Tenebrio molitor, showing individual neurons that persist from the late embryo to the adult stage. Their essential structural characteristics were already present in the late embryo, but distinct parts of their arborization patterns became newly formed throughout development. Using a combination of immunohistochemistry and single-cell injection, we demonstrated minute structural changes, allowing a characterization of structural plasticity of identifiable, persistent, neuropeptidergic neurons throughout ontogenesis. Furthermore, this study has provided new information about basic principles of central brain neuroanatomy and the development of a distinct midbrain region of the insect brain, the central complex. The development of its basic connections, the connections between the fan-shaped body and the protocerebral bridge, and the compartmentation of these neuropil regions were shown, using LomTK-IR neurons as marker structures. These basic features of the central complex-associated LomTK-immunopositive neurons were formed in the embryonic brain, whereas in metamorphosis, reorganization of these persistent interneurons was restricted to the formation of a precisely defined projection of their side branches. © 1996 Wiley-Liss, Inc.     

Locustatachykinin immunoreactivity in the blowfly central nervous system and intestine

An antiserum raised against locustatachykinin I, one of four myotropic peptides that have been isolated from the locust brain and corpora cardiaca, was characterized by enzyme-linked immunosorbent assay (ELISA) and used for immunocytochemical detection of neurons and endocrine cells in the nervous system and intestine of the blowfly Calliphora vomitoria. The ELISA characterization indicated that the antiserum recognizes the common C-terminus sequence of the locustatachykinins I-III. Hence, the cross reaction with locustatachykinin IV is less, and in competitive ELISAs no cross reaction was detected with a series of vertebrate tachykinins tested. It was also shown that the antiserum recognized material in extracts of blowfly heads, as measured in ELISA. In high-performance liquid chromatography the extracted locustatachykinin-like immunoreactive (Lom TK-LI) material eluted in two different ranges. A fairly large number of LomTK-LI neurons was detected in the blowfly brain and thoracicoabdominal ganglion. A total of about 160 LomTK-LI neurons was seen in the proto-, deuto-, and tritocerebrum and subesophageal ganglion. Immunoreactive processes from these neurons could be traced in many neuropil regions of the brain: superior and dorsomedian protocerebrum, optic tubercle, fan-shaped body and ventral bodies of the central complex, all the glomeruli of the antennal lobes, and tritocerebral and subesophageal neuropil. No immunoreactivity was seen in the mushroom bodies or the optic lobes. In the fused thoracicoabdominal ganglion, 46 LomTK-LI neurons could be resolved. The less evolved larval nervous system was also investigated to obtain additional information on the morphology and projections of immunoreactive neurons. In neither the larval nor the adult nervous systems could we identify any efferent or afferent immunoreactive axons or neurosecretory cells. The widespread distribution of LomTK-LI material in interneurons suggests an important role of the native peptide(s) as a neurotransmitter or neuromodulator within the central nervous system. Additionally a regulatory function in the intestine is indicated by the presence of immunoreactivity in endocrine cells of the midgut.