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"Crickets in Space" was a Neurolab experiment by which the balance between genetic programs and the gravitational environment for the development of a gravity sensitive neuronal system was studied. The model character of crickets was justified by their external gravity receptors, identified position-sensitive interneurons (PSI) and gravity-related compensatory head response, and by the specific relation of this behavior to neuronal arousal systems activated by locomotion. These advantages allowed to study the impact of modified gravity on cellular processes in a complex organism. Eggs, 1st, 4th and 6th stage larvae of Acheta domesticus were used. Post-flight experiments revealed a low susceptibility of the behavior to micro- and hypergravity while the physiology of the PSI was significantly affected. Immunocytological investigations revealed a stage-dependent sensitivity of thoracic GABAergic motoneurons to 3 g-conditions concerning their soma sizes but not their topographical arrangement. The morphology of neuromuscular junctions was not affected by 3 g-hypergravity. Peptidergic neurons from cerebral sensorimotor centers revealed no significant modifications by microgravity (micro g). The contrary physiological and behavioral results indicate a facilitation of 1 g-readaptation originating from accessory gravity, proprioceptive and visual sense organs. Absence of anatomical modifications point to an effective time window of micro g or 3 g-expo-sure related to the period of neuronal proliferation. The analysis of basic mechanisms of how animals and man adapt to altered gravitational conditions will profit from a continuation of the project "Crickets in Space".

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"Crickets in Space" (CRISP) was a Neurolab experiment by which the balance between genetic programs and the gravitational environment for the development of a gravity sensitive neuronal system was studied. The model character of crickets was justified by their external gravity receptors, identified position-sensitive interneurons (PSI) and gravity-related compensatory head response, and by the specific relation of this behavior to neuronal activation systems. These advantages allowed us to study the impact of modified gravity on cellular processes in a complex organism. Eggs, 1st, 4th and 6th stage larvae of Acheta domesticus were used. Post-flight experiments revealed a low susceptibility of the behavior to microgravity and hypergravity (hg) while the physiology of the PSI was significantly affected. Immunocytological investigations revealed a stage-dependent sensitivity of thoracic GABAergic motoneurons to 3g-conditions concerning their soma sizes but not their topographical arrangement. Peptidergic neurons from cerebral sensorimotor centers revealed no significant modifications by microgravity. The contrary physiological and behavioral results indicate a facilitation of 1g-readaptation by accessory gravity. proprioceptive and visual sense organs. Absence of anatomical modifications point to an effective time window of microgravity or hg-exposure related to the period of neuronal proliferation. Grant numbers: 50WB9553-7.

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Crustacean cardioactive peptide (CCAP) stimulates the contractions of locust oviducts. CCAP increased the basal tonus and increased the frequency and amplitude of phasic contractions, as well as the amplitude of neurally-evoked oviduct contractions in a dose-dependent manner. Oviducts from Vth instar larvae and adult locusts aged 10 days or less, were more sensitive to CCAP than oviducts from adult locusts aged 12 days or more. This may be indicative of a differential expression of number or subtypes of CCAP receptors on the oviducts at different ages, and may be related to reproductive functions or to functions of CCAP on the oviducts during ecdysis. The oviducts appear more sensitive to CCAP when compared with previously published reports of CCAP actions on the hindgut. CCAP actions on the amplitude of neurally-evoked contractions of the oviducts are similar to those of proctolin, however, the oviducts are more sensitive to CCAP. No CCAP-like immunoreactive structures were discovered in the nerves innervating the oviducts, or on the oviducts themselves, confirming the previously published suggestion (Dircksen et al., 1991) that CCAP acts as a neurohormone at the oviducts. Cells showing CCAP-like immunoreactivity were discovered in the fat body associated with the oviducts and represent a potential source of CCAP, along with CCAP released from the transverse nerve and perivisceral organs.

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CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.

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A small, medial heterolateral neuropil in the brain of crustaceans has long been regarded as the central body of the crustacean brain. Its simplicity and the absence of clear layers within its neuropil have led to the question of its homology with the more complex central body that occupies an approximately equivalent position in the brain of insects. We have labelled neurons in the central body of the Australian freshwater crayfish Cherax destructor by the extracellular application of dextrans and by treating the brain with antibodies to anti-CCAP, anti-locustatachykinin, anti-perisulfakinin, anti-proctolin, anti-dip-allatostatin AI, anti-PEA-head-peptide, anti-serotonin, and anti-rabbit anti-substance P, all of which label neurons in the insect brain. The dextran and immunocytochemical labelling have revealed a neural complex associated with the crayfish central body that is very similar in overall anatomical architecture to the subset of neuropils that are incorporated in the central complex of the insects, and in particular to that of the locust. Similarities between the crayfish and locust central complexes extend to the number and position of the neuropils, the location of the cell body clusters of the neurons that belong to the central complex, the numbers of tracts that link some of the constituent neuropils together, and the form and immunoreactivity of many of the individual neuron classes. These similarities are taken as evidence to support a possible homology between the crustacean central complex and that of the insects.

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In the nervous system of embryos and adult Locusta migratoria, somata, neurites within the ganglia, and axons leaving the thoracic ganglia show allatostatin immunoreactivity. The immunoreactive efferent axons divide to follow different nerve branches and form varicose terminals on skeletal muscles. In the adult locust, one pair of motor neurons is particularly prominent among the allatostatin-immunoreactive neurons. The somata are located symmetrically in a lateral position in the first abdominal neuromere of the fused metathoracic ganglion. Each neuron gives rise to five axon branches projecting into ipsilateral nerves. Three axons project posteriorly and exit through the dorsal nerves of the abdominal neuromeres A1, A2, and A3. One axon extends into the metathoracic neuromere and exits through metathoracic nerve 1 (N1). The fifth axon extends anteriorly through the connective into the mesothoracic ganglion, where it leaves through the mesothoracic N1. The targets of this neuron, among them the mesothoracic and metathoracic muscles M87, M88, M116 and the dorsal longitudinal muscles M81 and M112, are located in five different segments. In addition to supplying skeletal muscles, the neuron forms neurohaemal-like structures in the sheath of nerve branches. The authors call this neuron the common lateral neuron (CLN). The innervation of several muscles by Diploptera allatostatin 7-immunoreactive axon branches with a common cellular origin and the anatomy of one of the corresponding motor neurons in adults, the CLN, suggest that allatostatin acts as a modulator of neuromuscular parameters in insects by multisegmental direct innervation of skeletal muscles.

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In the central and peripheral nervous system of the crayfish, Orconectes limosus, neuropeptides immunoreactive to an antiserum against allatostatin I (= Dipstatin 7) of the cockroach Diploptera punctata have been detected by immunocytochemistry and a sensitive enzyme immunoassay. Abundant immunoreactivity occurs throughout the central nervous system in distinct interneurons and neurosecretory cells. The latter have terminals in well-known neurohemal organs, such as the sinus gland, the pericardial organs, and the perineural sheath of the ventral nerve cord. Nervous tissue extracts were separated by reverse-phase high-performance liquid chromatography and fractions were monitored in the enzyme immunoassay. Three of several immunopositive fractions have been purified and identified by mass spectroscopy and microsequencing as AGPYAFGL-NH2, SAGPYAFGL-NH2, and PRVYGFGL-NH2. The first peptide is identical to carcinustatin 8 previously identified in the crab Carcinus maenas. The others are novel and are designated orcostatin I and orcostatin II, respectively. All three peptides exert dramatic inhibitory effects on contractions of the crayfish hindgut. Carcinustatin 8 also inhibits induced contractions of the cockroach hindgut. Furthermore, this peptide reduces the cycle frequency of the pyloric rhythms generated by the stomatogastric nervous system of two decapod species in vitro. These crayfish allatostatin-like peptides are the first native crustacean peptides with demonstrated inhibitory actions on hindgut muscles and the pyloric rhythm of the stomatogastric ganglion.

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Crustacean cardioactive peptide (CCAP) elicited expression of the motor pattern that drives coordinated swimmeret beating in crayfish and modulated this pattern in a dose-dependent manner. In each ganglion that innervates swimmerets, neurons with CCAP-like immunoreactivity sent processes to the lateral neuropils, which contain branches of swimmeret motor neurons and the local pattern-generating circuits. CCAP affected each of the four functional groups of motor neurons, power-stroke excitors (PSE), return-stroke excitors (RSE), power-stroke inhibitors (PSI), and return-stroke inhibitors (RSI), that innervate each swimmeret. When CCAP was superfused, the membrane potentials of these neurons began to oscillate periodically about their mean potentials. The mean potentials of PSE and RSI neurons depolarized, and some of these neurons began to fire during each depolarization. Both intensity and durations of PSE bursts increased significantly. The mean potentials of RSE and PSI neurons hyperpolarized, and these neurons were less likely to fire during each depolarization. When CCAP was superfused in a low Ca2+ saline that blocked chemical transmission, these changes in mean potential persisted, but the periodic oscillations disappeared. These results are evidence that CCAP acts at two levels: activation of local premotor circuits and direct modulation of swimmeret motor neurons. The action on motor neurons is differential; PSEs and RSIs are excited, but RSEs and PSIs are inhibited. The consequences of this selectivity are to increase intensity of bursts of impulses that excite power-stroke muscles.

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The median neuroendocrine cells of the subesophageal ganglion, important components of the neuroendocrine system of the tobacco hawkmoth, Manduca sexta, have not been well investigated. Therefore, we studied the anatomy of these cells by axonal backfills and characterized their peptide immunoreactivities. Both larvae and adults were examined, and developmental changes in these neuroendocrine cells were followed. Processes of the median neuroendocrine cells project to terminations in the corpora cardiaca via the third and the ventral nerves of this neurohemal organ, but the ventral nerve of the corpus cardiacum is the principal neurohemal surface for this system. Cobalt backfills of the third cardiacal nerves revealed lateral cells in the maxillary neuromere and a ventro-median pair in the labial neuromere. Backfills of the ventral cardiacal nerves revealed two ventro-median pairs of cells in the mandibular neuromere and two ventro-median triplets in the maxillary neuromere. The efferent projections of these cells are contralateral. The anatomy of the system is basically the same in larvae and adults. The three sets of median neuroendocrine cells are PBAN- and FMRFamide-immunoreactive, but only the mandibular and maxillary cells are proctolin-immunoreactive. During metamorphosis, the mandibular and maxillary cells also acquire CCK-like immunoreactivity and the labial cells become SCP- and sulfakinin-immunoreactive. Characteristics of FMRFamide-like immunostaining suggest that the median neuroendocrine cells may contain one or more of the FLRFamides that have been identified in M. sexta. The mandibular and maxillary neuroendocrine cells appear to produce the same set of hormones, and a somewhat different set of hormones is produced by the labial neuroendocrine cells. Two pairs of interneurons immunologically related to the neurosecretory cells are associated with the median maxillary neuroendocrine cells. These cells are PBAN-, FMRFamide-, SCP-, and sulfakinin-immunoreactive and project to arborizations in the brain and all ventral ganglia. These interneurons appear to have extensive modulatory functions in the CNS.

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The distribution and morphology of neurons containing allatostatin-related substances in the brain of the locust Schistocerca gregaria was investigated using an antiserum against Diploptera punctata allatostatin I (Dip-allatostatin I, APSGAQRLYGFGL-amide). In each brain hemisphere, about 550 neurons in the midbrain and 500 neurons in the optic lobe exhibit Dip-allatostatin I-like immunoreactivity, including about eight lateral neurosecretory cells with processes to the retrocerebral complex. All major brain areas except the antennal lobe, the mushroom body, and large parts of the lamina, are innervated by Dip-allatostatin I-immunoreactive processes. Immunostaining in the central complex was studied in detail. The central complex is innervated by more than 260 Dip-allatostatin I-immunoreactive neurons belonging to six different cell types, four sets of tangential neurons and two sets of columnar neurons. These neurons give rise to intense immunostaining in the protocerebral bridge, in several layers of the upper division of the central body, and in the dorsalmost layer of the lower division of the central body. Double-label experiments show colocalization of Dip-allatostatin I- and serotonin-like immunoreactivities in one type of columnar and one type of tangential neurons of the central complex. The similar patterns of Dip-allatostatin I- and galanin message-associated peptide-like immunoreactivities result from cross-reactivity of the anti-galanin message-associated peptide antiserum with Dip-allatostatin I. The results provide further insight into the anatomical and neurochemical organization of the locust central complex and suggest a prominent neuroactive role for Dip-allatostatin I-related peptides in this brain area.

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The midgut of the female mosquito Aedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.

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Both allatostatin immunoreactivity (AS-IR) and FMRFamide immunoreactivity (FMRFa-IR) have been demonstrated light-microscopically in the lateral heart nerve of Periplaneta americana. The identical labeling of some fibers suggests the coexistence of the two antigens. Electron-microscopically, six granule types in the peripheral part of the lateral heart nerve can be distinguished according to their size and density (types 1-6). These granule types can be subdivided immunocytochemically by means of a new mirror-section technique. Granules of types 4 and 5 always exclusively show FMRFa-IR. In the populations of fibers containing granules of types 1 and 6, axon profiles can be found that contain granules colocalizing FMRFa-IR and AS-IR. Other axon profiles of these populations only contain immunonegative granules of the same ultrastructure. Granules of type 2 can be differentiated immunocytochemically in three forms in the same section: In some fibers, they are nonreactive; in other fibers of the same section, they show FMRFa - IR, whereas in a third fiber type, granules show AS - IR. Finally, granules of type 3 can be observed with FMRFa-IR. In other fibers, they occur with the same ultrastructure but exhibit no immunoreactivity. Two soma types occur in the lateral heart nerve. Soma type I is characterized by the production of electron-dense granules that show FMRFa-IR. Type II is in close contact with various fibers, forming different types of axosomatic synapses, hitherto unknown in Insecta.

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Comparative aspects of arthropod peptidergic systems--in principle--can be studied on the level of precursor sequences (genes, preprohormones), peptide sequences (peptide families), and peptide expression patterns within the nervous system. The number of known arthropod neuropeptide precursor sequences is as yet far too small to provide a reasonably large basis for extended comparative studies. Comparative studies of peptide sequences have shown that many peptides belong to families with homologous members in both invertebrates and vertebrates. Comparative research on peptide expression has to find out whether phylogenetic necessities lead to "hard wired" neurochemical identities, i.e., a more or less fixed "Bauplan" that not only determines the lineage and morphology of a neuron but also its transmitter(s), or whether these necessities demand greater flexibility (plasticity), and hence cause great variability that would complicate comparative studies. As will be shown here, both possibilities appear to exist. On the one hand, peptidergic neurons may exist in comparable form in different groups of arthropods. On the other hand, the neurochemical identity of cells may vary in segmented organisms when comparing serially homologous sets of nerve cells in different segments. As a further complication, identical or similar peptides may serve different functions, even in closely related species. In view of these functional aspects in particular, it appears that peptidergic signalling pathways represent rapidly evolving systems. This conclusion, although very interesting in itself, reduces the use of such systems for general comparisons. However, arthropod nervous systems represent excellent model systems for the study of homology. At least for morphological and ontogenetic aspects arthropods provide numerous opportunities to study homology on the level of the individually identified peptidergic nerve cell.

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Cockroach allatostatins are neuropeptides that have been isolated from the brain of Diploptera punctata and shown to inhibit juvenile hormone production by the corpora allata. Enzyme-linked immunoassay and immunocytochemistry with antisera to two allatostatins, ASB2 (AYSYVSEYKRLPVYNFGL-NH2) and ASAL (APSGAQRLYGFGL-NH2), revealed that allatostatins were located not only in the insect brain but also in several peripheral tissues including the cockroach midgut and hindgut. Allatostatin-like immunoreactivity was found in nerve fibers of the stomatogastric nervous system as well as in intrinsic endocrine cells of the midgut. Midgut extracts were shown to be biologically active in an allatostatin bioassay and to contain several allatostatin-like peptides, including the octadecapeptide ASB2, which was identified by mass spectrometry following HPLC purification. Reverse transcription of brain mRNA followed by PCR with degenerate oligonucleotides for ASB2 and ASAL yielded a 338-bp fragment of the allatostatin gene that encoded six allatostatins. In situ hybridization with this probe confirmed that an allatostatin gene is expressed in intrinsic endocrine cells of the midgut. Reverse transcription of midgut mRNA followed by PCR and sequencing of the product revealed that the same gene is expressed in the midgut and in the brain. Allatostatins are thus an example of insect "brain-gut peptides" and we suggest that their function may not be restricted to the regulation of juvenile hormone production.

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Innervation of the antennal heart, an independent accessory circulatory motor in the head of insects, was investigated in the cockroach Periplaneta americana by use of axonal cobalt filling and transmission electron microscopy. The muscles associated with this organ are innervated by neurones located in a part of the suboesophageal ganglion, generally considered to be formed by the mandibular neuromere. Dorsal unpaired median (DUM) and paired contralateral neurones were stained. The axons of all these neurones run along the circumoesophageal connectives and through the paired nervus corporis cardiaci III into the corpora cardiaca. They pass through these organs forming fine arborizations there and exit anteriorly as a small pair of nerves which terminate at the antennal heart-dilator muscles. Numerous branches of these nerves extend beyond the lateral borders of the large transverse dilator muscle and terminate in the ampullar walls of the antennal heart. These neurosecretory fibres form neurohaemal areas which obviously release their products into the haemolymph, which is pumped into the antennae. The possible functions of the neurones associated with the antennal heart are discussed with respect to both, their role as a modulatory input for the circulatory motor and as a neurohormonal release site.

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In the American cockroach, the distribution and connections of neuronal elements of the terminal ganglion-proctodeal nerve-hindgut system were investigated by means of immunohistochemical methods and axonal CoCl2 iontophoresis. Proctolinlike immunoreactivity was localized within neurons of the terminal ganglion projecting into the proctodeal nerve on the one hand, and in nerve cells without a direct connection to this system on the other. Immunohistochemically, in whole mount preparations fibres of the proctodeal nerve and terminal structures in the hindgut musculature exhibit strong proctolinlike immunoreactivity. At the light- and electron-microscopic levels the pathways of about 30 somata of the proctodeal neural system were characterized by cobalt chloride iontophoresis. The relationships of cobalt filled and immunoreactive neuronal structures are discussed.

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The course of the Nervus connectivus (N.c.), its branches, and synaptic connections within the frontal ganglion (FG) were investigated electron microscopically after cobalt iontophoresis of the N.c. The subsequent treatment of ultrathin sections with Timm's method was found to be very suitable for identifying the smallest branches. In the neuropil, fibers of the N.c. form Gray-I-type synapses, but also dyads are abundant, whereby the N.c. fibers occur exclusively in postsynaptic position with neurosecretory fibers. The possible role of these relationships is discussed.

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At first the allround etiology of premature birth is explained in the paper. There are to distinguish three groups: 1. Causes with known etiologic mechanism. 2. Causes with partly known etiologic mechanism. 3. Dispositions for premature birth. These are concluded from statistically investigations. In the last group are collected the patients from which are established some known scores for diagnosis of the risk of premature birth. All the scores but have a detriment. If they want to detect about 90% from premature births one must carry out examination and observation about 40% from all pregnant women. For this the scores are not suitable for selection of patients to observe in a special consultation. The organised care for pregnant women must be in such a way that all the criterias of imminent prematurity will be detected. This way has been successfull in our hospital.

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A balanced translocation was found in a normal female with a history of four abortions. On the basis of the Giemsa-banding pattern the abnormality was interpreted as to be a translocation of a part of the long arm of chromosome 13 to the short arm of chromosome some 7:t(7;13)(7qter leads to 7p22::13q14 leads to 13qter;13q14 leads to 13pter::7p22 leads to 7 pter). Problems in genetic counseling are discussed with respect to this case.

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