CalpA, a Drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells.

Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.

Peptidergic neurohormonal control systems in invertebrates.

The concerted activity of many neuropeptides has been implicated in the neurohormonal control of specific behaviors and various physiological functions in some invertebrate model systems. What are the functional consequences of this neuropeptide multiplicity? The distinct actions of closely related neuropeptides have been detected in molluscs and insects; however, recent work provides examples of systems in which some of the multiple isoforms may be functionally redundant. Groups of functionally distinct neuropeptides encoded by the same gene can be expressed in different neurons by alternative gene splicing or cell-specific post-translational processing; therefore, as shown recently, they can be targeted for release as 'cocktails' to act on specific sets of muscles or neurons. One prominent role of neuropeptides is to modulate the activity of rhythm-generating circuits, as exemplified by recent research on mollusc neural networks, the crab stomatogastric ganglion, and fly circadian pacemakers.

Neuropeptides, amines and amino acids in an elementary insect ganglion: functional and chemical anatomy of the unfused abdominal ganglion.

The insect ventral nerve cord consists of metamerically repeated ganglia subserving the thoracic and abdominal segments. The abdominal ganglia control basic functions such as respiration, circulation, heartbeat, diuresis, hindgut motility, functions of the genitalia and ovipositor and abdominal posture. Some of this control is by efferent innervation of target tissues but hormonal control also is exerted by abdominal neurosecretory cells via release from neurohemal organs or other release sites. The present review summarizes what is known about the distribution of neurotransmitters, monoamines and neuropeptides in the abdominal ganglia of different insect species. Special emphasis is on the unfused abdominal ganglion, since this is the least complex of all central ganglia and therefore may reveal the minimum number of neuroactive compounds utilized in neurotransmission, neuromodulation and neurohormonal control. Both GABA and glutamate are present in both interneurons and motoneurons, whereas biogenic amines such as serotonin, dopamine and histamine are found primarily in interneurons (although some cases of sensory cells and efferent neurons are known). Octopamine can be seen both in interneurons, efferent neurons and neurosecretory cells. A large number (about 20 different main types) of neuropeptides has been indicated in abdominal ganglia. Each peptide has a very specific distribution pattern. Depending on the peptide type, the localization is known to be in interneurons, neurosecretory cells or motoneurons, or combinations of these. The structure and known functions of the different neuropeptides in different insect species are summarized in some detail. Both GABA and glutamate appear to have roles as fast neurotransmitters, whereas amines and neuropeptides seem to have modulatory roles both within the CNS and at peripheral targets. After a comprehensive overview of different substances in studied insect species, the unfused abdominal ganglia from the moth Manduca sexta, locusts and cockroaches are dealt with in some detail and a comparison is made with insects possessing fused abdominal ganglia such as blowflies and Drosophila. Some emphasis is made of the presence of neuroactive compounds in neurosecretory cells and other identifiable neurons for which physiological analysis is feasible.

Tachykinin-related peptide and GABA-mediated presynaptic inhibition of crayfish photoreceptors.

Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

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.

Axonal projections from transplanted ectopic legs in an insect.

Ectopic legs were produced in the fleshfly Sarcophaga bullata by transplantation of leg imaginal discs. Cobalt or horseradish peroxidase (HRP) fills from these supernumerary legs showed that their sensory axons invariably innervate the metathoracic leg neuropil via an abdominal nerve. This was so irrespective of whether they differentiated from pro-, meso-, or metathoracic leg disc. Implantation of left leg discs on the right side and vice versa revealed that the site of implantation determined which side (left or right) of the ganglion was innervated. In general, the same results were obtained when the implantation was made after removal of a leg disc from the host, except in a few instances in which the implanted leg directly innervated the deafferented leg neuropil. The results indicate that axonal pathfinding from the imaginal discs in this insect is via contact guidance along preexisting nerves or imaginal disc stalks.

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.

Neuronal organization in fly optic lobes altered by laser ablations early in development or by mutations of the eye.

The role of afferent and efferent connections in the differentiation of optic lobe interneurons was investigated by using laser ablations of neuronal precursors in the brain of Musca domestica and analysis of two eye mutants of the same species. The first mutant, split eye, had no connections between the retina and the optic lobes. In this case the optic lobes were drastically reduced in volume and the neural organization within the neuropil regions was altered. The other mutant, spindle, had reduced retinae that innervated reduced optic lobes with a normal-appearing orderly arrangement of neurons. In addition disordered neuropil, composed of identified visual interneurons, was found that had no afferent innervation. Three main types of alterations resulting from laser ablations were analyzed. These ablations removed entire neuropil regions or parts of these: (1) removal of the first optic neuropil region (the lamina) resulting in receptor axons projecting directly to the second neuropil (the medulla) and sprouting of medulla neurons toward the receptor layer; (2) removal of one part of the third optic neuropil (the lobula plate) and severe alteration of the other part (the lobula) resulting in sprouting of lobula neurons into the medulla neuropil; and (3) removal of the entire optic lobe resulting in reduction of the volume of the lateral midbrain and photoreceptor axons forming a tangle beneath the retina. Our findings confirm that afferent retinal input is essential for normal differentiation and maintenance of many optic lobe interneurons. Furthermore, it was seen that a normal columnar organization of the neuropils and the dendritic patterns of visual interneurons are dependent on afferent inputs. A common response to removal of inputs was a reorganization of axonal and dendritic projections.

Galanin immunoreactivity in the blowfly nervous system: localization and chromatographic analysis.

In this study chromatographic, immunochemical, and immunocytochemical methods provide evidence of a galanin-like peptide(s) in an invertebrate, the blowfly Phormia terraenovae. The major portion of the galanin-like immunoreactivity (GAL-LI) in fly heads was extractable in acetic acid but not in boiling water, which suggests that the peptide(s) may be highly basic in nature. GAL-LI was present both in the head and body portion of the blowfly in roughly the same amounts. Initial gel filtration data, using a G-50 Sephadex column and a weak phosphate-buffer (pH 6.5) as eluent, suggested that a fly GAL-LI peptide(s) from fly heads, eluting as an apparent single peak, was smaller than porcine GAL(1-29) and GAL(1-15). However, concomitant analysis using a G-25 Sephadex column and acetic acid (0.2 M) as eluent, spread the immunoreactive material over a great portion of the chromatogram, although the main portion of the material eluted in the same size range as porcine GAL(1-29). Taken together, the gel filtration data thus suggest that fly GAL-LI peptide(s) may be highly basic but presumably similar in size to vertebrate GAL(1-29). However, the hydrophobic properties of the fly GAL-LI peptide(s) differ from that of porcine GAL as demonstrated by the presence of several immunoreactive components eluting both early as well as late in the chromatogram when using reverse-phase high performance liquid chromatography (HPLC); early peaks may represent highly basic and/or possibly smaller GAL-immunoreactive peptide(s), whereas later peaks may represent less basic and possibly elongated forms. Immunocytochemistry indicated that GAL-LI was present in the nervous system of the blowfly. About 160 GAL-immunoreactive neurons were found in the brain and subesophageal ganglion, 26 in the fused thoracic ganglion and 30 in the fused abdominal ganglion. In the brain, GAL-immunoreactive fibers supply specific subdivisions of the central body, optic lobe, superior protocerebrum, and tritocerebrum as well as neuropil in the subesophageal ganglia. In the thoracico-abdominal ganglia, GAL-immunoreactive neuron processes are found inside synaptic neuropil as well as in the neural sheath of the ganglia and several of the dorsal nerve roots. Many of the GAL-immunoreactive neurons react also with an antiserum against porcine galanin message associated peptide, a peptide present in the preprogalanin protein. Immunocytochemical double-labeling indicated that some GAL-immunoreactive neurons also reacted with antisera against the molluscan peptides FMRFamide and SCPB, whereas no evidence could be found for colabeling with antisera against tyrosine hydroxylase, substance P and physalaemin.(ABSTRACT TRUNCATED AT 400 WORDS)

Histaminelike immunoreactive neurons innervating putative neurohaemal areas and central neuropil in the thoraco-abdominal ganglia of the flies Drosophila and Calliphora.

The fused thoraco-abdominal ganglia of the flies Calliphora vomitoria and Drosophila melanogaster were investigated immunocytochemically with antisera against histamine. In both insect species, 18 histaminelike immunoreactive (HA-IR) neurons were resolved in these ganglia. Six of these neurons have cell bodies in the thoracic neuromeres and 12 in the fused abdominal neuromeres. All cell bodies are situated ventrally. In Calliphora all cell bodies are arranged in a segmental pattern. In Drosophila only the thoracic cell bodies have a segmental arrangement, whereas the abdominal ones are clustered anteriorly close to the last thoracic neuromere. In both species the six thoracic neurons supply processes to the synaptic neuropil in the thoracic neuromeres and to the dorsal neural sheath. The processes in the neural sheath run anteriorly in the lateral portions of the ganglion into the cervical connective. In a few regions laterally arborizing terminals are found in putative neurohaemal areas. These areas were investigated by electron microscopic immunocytochemistry in Calliphora. The HA-IR terminals (containing small granular vesicles) were found outside the "blood-brain barrier" below the acellular basal lamina of the neural sheath. Release of histamine into the circulation is therefore theoretically possible. The central processes of the six thoracic HA-IR neurons may interact synaptically with large numbers of other neurons in the neuropil, and the peripheral varicose fibers from the same HA-IR neurons possibly are neurohaemal release sites. The abdominal HA-IR neurons, in contrast, form extensive arborizations within the synaptic neuropil only. Both thoracic and abdominal neurons have ipsilateral and contralateral branches as well as processes that invade more than one neuromere. A single HA-IR neuron thus invades large volumes of synaptic neuropil. Histamine may be used by neurons of the ventral ganglia both as neurotransmitter (or neuromodulator) and as a circulating neurohormone released from the neural sheath.

Metamorphosis of identified neurons innervating thoracic neurohemal organs in the blowfly: transformation of cholecystokininlike immunoreactive neurons.

With antisera to gastrin/cholecystokinin, we studied the postembryonic development of neurons in the thoracic ganglia of the blowfly Calliphora erythrocephala. There are some changes in the population of thoracico-abdominal neurons displaying gastrin/CCK-like immunoreactivity (CCKLI): some CCKLI neurons cannot be found after pupariation; other neurons become immunoreactive during metamorphosis. Six large thoracic CCKLI neurons could, however, be followed through metamorphosis. These CCKLI neurons innervate neuropil in thoracic ganglia and segmental neurohemal organs in the larva. In the adult insect the same neurons innervate many regions of thoracic neuropil and extensive neurohemal areas dorsally in the fused thoracico-abdominal ganglia. The immunoreactive terminals are located in the neural sheath, and electron microscopy shows that only an extracellular basal lamina separates them from the circulating hemolymph. On the basis of the location of their terminals, it can be suggested that the six CCKLI neurons have functions as neurosecretory cells both in the larva and in the adult. In both developmental stages the neurons can interact with large portions of the thoracic nervous system and release bioactive substance into the circulation. A CCK-like substance may be used both as a transmitter/neuromodulator and as a neurohormone by the same neuron. The larval neurohemal organs are described here for the first time. They show characteristics of thoracic perisympathetic organs known to exist in more primitive insects. The adult neurohemal regions on the other hand are typical of higher insects. Since the neurohemal areas are continuously (during development) innervated by the six large CCKLI neurons, we conclude that the larval neurohemal organs metamorphose into the adult neurohemal area in the neural sheath.

Mapping and ultrastructure of serotonin-immunoreactive neurons in the optic lobes of three insect species.

With antibodies to serotonin (5-HT) we have mapped immunoreactive neurons in the optic lobes of three species, the blowfly Calliphora, the desert ant Cataglyphis, and the worker bee Apis. The main emphasis in this investigation is on a system of 5-HT-positive neurons connecting the most peripheral neuropil of the optic lobes, the lamina, to more central neuropil regions. To aid in electron microscopical identification of these neurons we used immunocytochemistry at the EM-level and Golgi-EM for Calliphora and horseradish peroxidase (HRP) labelling for the other two insects. The immunoreactive terminals in Calliphora and the HRP-labelled ones in the other insects contain large (c. 100 nm) granular vesicles and smaller (c.60 nm) clear vesicles. In Cataglyphis and Apis the profiles with granular vesicles are presynaptic to second order neurons of the lamina, whereas in Calliphora no synaptic contacts were found. In this animal the 5-HT-positive terminals are situated distal to the synaptic layer of the lamina, in a region of retinal photoreceptor axons and perikarya of the lamina monopolar neurons. In Catagylphis and Apis the interactions of the 5-HT-neurons with the laminar neurons might occur through chemical synapses, whereas in Calliphora neuroactive substance could be released non-synaptically from varicosities distal to the synaptic layer. The possible involvement of 5-HT in control of neuronal activity in the optic lobes is discussed.

Postembryonic development of corazonin-containing neurons and neurosecretory cells in the blowfly, Phormia terraenovae.

An antiserum against the cockroach cardioactive peptide corazonin was used to investigate the distribution of immunoreactive neurons and neurosecretory cells in the nervous system of the blowfly, Phormia terraenovae, during postembryonic development. A small number of corazonin-immunoreactive neurons was found at larval, pupal, and adult stages. At all postembryonic stages two cell groups were found in the protocerebrum of the brain: 1) two lateral cell clusters and 2) two median cells. In the larva eight bilateral cell pairs were found in thoracic and abdominal neuromeres of the fused ventral ganglion. The lateral brain neurons are located in the lateral neurosecretory cell group and extend axons with branches in several components of the retrocerebral neuroendocrine complex, in the stomatogastric nervous system of larvae and adults, and additionally in muscles of the alimentary canal in the adult. The most prominent element of these peripheral processes is a large plexus of varicose fibers located in the wall of the aorta, the main site for the release of neurohormones produced in the brain of blowflies. The presence of corazonin-immunoreactive material in the aortic plexus suggests that this peptide functions as a neurohormone. During metamorphosis, the immunoreactive neurons found in the thoracic-abdominal ganglion of the larva disappear, and in the brain new immunoreactive neurons are added to those that persist from larval stages. The bulk of the corazonin-immunoreactive material extracted from adult brains and corpora cardiaca-aorta complexes was found to co-elute with synthetic corazonin in reversed-phase high-performance liquid chromatography as monitored with enzyme-linked immunosorbent assay.

Postembryonic differentiation of serotonin-immunoreactive neurons in fleshfly optic lobes developing in situ or cultured in vivo without eye discs.

The differentiation of serotonin-immunoreactive (5-HTi) neurons in the optic lobes of fleshflies was studied during in situ development and in in vivo cultures. All 5-HTi neurons with cell bodies in the imaginal optic lobes differentiate during postembryonic (pupal) development. These are local anaxonal neurons. In addition there are two large 5-HTi bilateral neurons that connect all optic lobe neuropil regions on both sides of the brain and have their cell bodies in the midbrain proper. Deafferentation of optic lobes cultured in vivo leads to drastic reduction in optic lobe volume and increased cell death. All the 5-HTi neurons differentiate after deafferentation but their morphology changes. The neuropil receiving the photoreceptor inputs, the lamina, degenerates but a disorganized "pseudolamina" is formed by the processes of the two large 5-HTi neurons. The layering of the optic lobe neuropils cannot be distinguished and 5-HTi processes form novel projectional patterns. Hence, the 5-HTi neurons do not require afferent inputs from the retina for their differentiation and survival, but the effect on other optic lobe interneurons is reflected in the morphological plasticity of the 5-HTi neurons.

Neurons in the cockroach nervous system reacting with antisera to the neuropeptide leucokinin I.

Antisera were raised against the myotropic neuropeptide leucokinin I, originally isolated from head extracts of the cockroach Leucophaea maderae. Processes of leucokinin I immunoreactive (LKIR) neurons were distributed throughout the nervous system, but immunoreactive cell bodies were not found in all neuromeres. In the brain, about 160 LKIR cell bodies were distributed in the protocerebrum and optic lobes (no LKIR cell bodies were found in the deuto- and tritocerebrum). In the ventral ganglia, LKIR cell bodies were seen distributed as follows: eight (weakly immunoreactive) in the subesophageal ganglion; about six larger and bilateral clusters of 5 smaller in each of the three thoracic ganglia, and in each of the abdominal ganglia, two pairs of strongly immunoreactive cell bodies were resolved. Many of the LKIR neurons could be described in detail. In the optic lobes, immunoreactive neurons innervate the medulla and accessory medulla. In the brain, three pairs of bilateral LKIR neurons supply branches to distinct sets of nonglomerular neuropil, and two pairs of descending neurons connect the posterior protocerebrum to the antennal lobes and all the ventral ganglia. Other brain neurons innervate the central body, tritocerebrum, and nonglomerular neuropil in protocerebrum. LKIR neurons of the median and lateral neurosecretory cell groups send axons to the corpora cardiaca, frontal ganglion, and tritocerebrum. In the muscle layer of the foregut (crop), bi- and multipolar LKIR neurons with axons running to the retrocerebral complex were resolved. The LKIR neurons in the abdominal ganglia form efferent axons supplying the lateral cardiac nerves, spiracles, and the segmental perivisceral organs. The distribution of immunoreactivity indicates roles for leucokinins as neuromodulators or neurotransmitters in central interneurons arborizing in different portions of the brain, visual system, and ventral ganglia. Also, a function in circuits regulating feeding can be presumed. Furthermore, a role in regulation of heart and possibly respiration can be suggested, and probably leucokinins are released from corpora cardiaca as neurohormones. Leucokinins were isolated by their myotropic action on the Leucophaea hindgut, but no innervation of this portion of the gut could be demonstrated. The distribution of leucokinin immunoreactivity was compared to immunolabeling with antisera against vertebrate tachykinins and lysine vasopressin.

Allatotropin-like neuropeptide in the cockroach abdominal nervous system: myotropic actions, sexually dimorphic distribution and colocalization with serotonin.

Allatotropin (AT) was isolated from the moth Manduca sexta as a peptide stimulating biosynthesis of juvenile hormone in the corpora allata, but has also been shown to be cardioactive in the same species. Here, we have investigated the presence and biological activity of AT-like peptide in the cockroaches Leucophaea maderae and Periplaneta americana with focus on abdominal ganglia and their target tissues. An antiserum to M. sexta AT was used for immunocytochemical mapping of neurons in the abdominal ganglia. A small number of interneurons and efferent neurons were found AT-like immunoreactive (AT-LI) in each of the abdominal ganglia. A prominent sexual dimorphism was detected in the terminal abdominal ganglion: in L. maderae the male ganglion there are approximately 18 AT-LI neurons with cell bodies posteriorly and efferent axons in the genital nerves; in the female ganglion 4-5 AT-LI cell bodies (with efferent axons) were found in the same region. Correlated with the extra efferents in males, the male accessory glands are richly supplied by AT-LI fibers and in females a less prominent innervation was seen in oviduct muscle. A similar dimorphism was seen in abdominal ganglia of P. americana. A sexual dimorphism was also detected in the abdominal ganglia A4-A6 of L. maderae. In each of these ganglia, approximately 8-10 large AT-LI neuronal cell bodies were found along the midline; in females these neurons have significantly larger cell bodies than in males. In both sexes, and both cockroach species, two large dorsal midline neurons were detected in A-5 and 6, which seem to send axons to the hindgut: the rectal pads of the hindgut are supplied by arborizing AT-LI axons. In males and females of both species, efferent AT-LI axons from midline neurons in A3-A6 supply the lateral heart nerves and other neurohemal release sites with arborizations. The efferent midline neurons of females contain colocalized serotonin-immunoreactivity. We tested the in vitro actions of M. sexta AT on muscle contractions in the L. maderae hindgut and the abdominal heart of both species. The frequency of contractions in the hindgut increased dose dependently when applying AT at 5 x 10(-8) to 5 x 10(-6) M (maximal response at 5 x 10(-7) M). Also the frequency of contractions of the heart increased by application of AT (threshold response at 5 x 10(-9) M). This effect was more prominent in males of both species (maximal response was a 35-40% increase in males and 10-20% in females). In conclusion, an AT-like peptide is present in neurons and neurosecretory cells of cockroach abdominal ganglia and seems to play a role in control of contractions in the hindgut and heart and also to have some function in male accessory glands and oviduct.

Differentiation of fly visual interneurons after laser ablation of their central targets early in development.

To test the differentiation of visual interneurons that had their targets removed before axogenesis, we ablated neuronal precursors in brains of first instar fly larvae by using a laser microsurgery unit. We describe ablations that resulted in the elimination of the third neuropil region (the lobula complex) of the optic lobes. Neural differentiation in the more peripheral second and first neuropil regions (the medulla and the lamina) was thus studied in the absence of the lobula complex. It was found that the medulla neuropil differentiated with normal columnar and layered organization. The neuropil, however, folded along its central surface. The only connection between the medulla and more central neuropil (the midbrain) was via a bundle of axons (the Cuccati bundle) present also in the normal optic lobes. Some types of neurons that normally connect the medulla and the lobula complex could be identified. These appeared to end in a disorganized neuropil mass in the center of the folded medulla. The differentiation of the lamina neuropil also appeared normal in flies with the lobula complex eliminated and the medulla folded. Also, in optic lobes where the medulla was severely disorganized and/or reduced due to laser ablations, the lamina neuropil appeared more or less normal. The results suggest that lamina and medulla nerve cells can differentiate and develop normal neuropil patterns in absence of their appropriate targets.

Pigment-dispersing hormone-like peptide in the nervous system of the flies Phormia and Drosophila: immunocytochemistry and partial characterization.

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab (Rao et al., '85) is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects were limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons was performed for the entire nervous systems of Phormia and the fruitfly Drosophila with a beta-PDH antiserum. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neurophils of the brain of both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies were found (only six were found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. We also found that some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hind-gut.

Primary commissure pioneer neurons in the brain of the grasshopper Schistocerca gregaria: development, ultrastructure, and neuropeptide expression.

The bilaterally paired primary commissure pioneer neurons in the median domain of the grasshopper brain are large, descending interneurons that uniquely express the TERM-1 antigen, even in the adult. After pioneering the primary interhemispheric brain commissure, these neurons extend TERM-1-immunoreactive collaterals into most parts of the brain except the mushroom bodies. In this report, the authors show that the TERM-1 antigen is located in the cell body cytoplasm of these neurons and not on the membranes. Screening with antisera to insect neuropeptides reveals that an antiserum recognizing peptides of the leucokinin family labels the cell body cytoplasm of the primary commissure neurons. Leucokinin-related peptides are known to modulate motility of visceral muscle, play a role in diuresis, and are likely to be neuromodulators in the insect nervous system. The primary commissure neurons differ ultrastructurally from median neurosecretory cells in that their cell body cytoplasm is more extensive, contains high numbers of mitochondria and extensive endoplasmic reticulum, but does not contain neurosecretory granules. In the adult, the cell somata are enveloped by multiple glia membranes and associated trophospongia. According to these ultrastructural characteristics, the primary commissure pioneers are not classical neurosecretory cells.

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.