A tissue-specific marker of Ecdysozoa.

Over the past few years, molecular studies of phylogeny have challenged the traditional view of evolutionary relationships among protostomian animal phyla. Based on analysis of 18S ribosomal RNA gene sequences, it has been suggested that some traditional groups, like the articulata and the pseudocoelomata, should be completely abandoned and that instead the protostomians should be split into two major clades: the Ecdysozoa and the Lophotrochozoa. However, this new molecular phylogeny still awaits confirmation by independent methods. In this study, we present a cytological feature that supports the new classification. The carbohydrate epitope that is recognised by antisera against the plant glycoprotein horseradish peroxidase (HRP) is known to be selectively expressed by membrane proteins on the surface of neural tissue in insects. We found that the major ecdysozoan phyla show neural expression of HRP immunoreactivity, which is completely absent in the nervous tissue of lophotrochozoans, deuterostomians, and cnidarians. This suggests that the presence of anti-HRP-reactive glycoproteins in neural tissue is an ecdysozoan autapomorphy.

Kinetic characterization of the changes in protein tyrosine phosphorylation of membranes, cytosolic Ca2+ concentration and viability in boar sperm populations selected by binding to oviductal epithelial cells.

On reaching the oviduct, spermatozoa are retained in the isthmic region of the oviduct until ovulation occurs. The essential steps of capacitation are co-ordinated in this region. In this study, a primary cell culture system of oviductal epithelial cells was established to investigate sperm binding to oviductal epithelium and modulation of sperm function during incubation under capacitating conditions in co-culture with oviductal epithelial cells. Epithelial cells were stripped from the oviducts of sows and cultivated for 5-7 days on Lab-Tek Chamber slides on Matrigel. The preparations on chamber slides and suspensions of control spermatozoa were incubated for 3 h in Tyrode's albumin lactate pyruvate (TALP) medium. At 3, 30, 60, 90 and 180 min the free-swimming spermatozoa were collected by washing, and membrane integrity, tyrosine phosphorylation patterns and [Ca(2+)](i) of bound, unbound and control spermatozoa were assessed with fluorescent probes (propidium iodide, Cy-3 and fluo-3-AM). The cells bound to oviductal epithelial cells showed reduced cytosolic Ca(2+) concentration, reduced and almost absent tyrosine phosphorylation of membrane proteins and higher viability at the time of the first sampling. Increases in Ca(2+) concentration and cell death occurred much more slowly during incubation in cells bound to oviductal epithelial cells compared with free-swimming spermatozoa, and no changes in tyrosine phosphorylation were observed. The preferential binding of viable, low-Ca(2+) cells with suppressed tyrosine phosphorylation and slower functional modulation of boar spermatozoa attached to oviductal epithelial cells might represent a mechanism for selecting functionally competent spermatozoa and prolonging their lifespan by delaying capacitation in the oviductal reservoir.

Nitric oxide: an unconventional messenger in the nervous system of an orthopteroid insect.

Nitric oxide (NO) is a membrane-permeant messenger molecule generated from the amino acid L-arginine. NO can activate soluble guanylyl cyclase leading to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This paper examines biochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of the locust, a commonly used neurobiological preparation. Biochemical investigations suggest that an identical enzyme is responsible for both NO synthase (NOS) and NADPH-diaphorase activity after tissue fixation. Immunocytochemical staining of an olfactory center in the locust brain shows that NOS-immunoreactivity colocalizes with NADPH-diaphorase at the cellular level. The cytochemical staining of NO donor and target cells in adult animals suggests functions in olfaction, vision, and sensorimotor integration. During development, NO is implicated in axonal outgrowth and synaptogenesis. The cellular distribution of NO-responsive cells in neural circuits reflects potential functions of NO as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger independent of conventional synaptic connectivity.

Sources and targets of nitric oxide signalling in insect nervous systems.

Nitric oxide (NO) is a membrane permeant signalling molecule which activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP) in target cells. In the nervous system, NO/cGMP signalling is thought to play essential roles in synaptic plasticity during development and also in the mature animal. This review summarizes neurochemical, cell biological, and physiological investigations of NO/cGMP signalling in the nervous system of insects. The anatomical localization of donor and target cells suggests functions in olfaction, vision, and mechanosensation. Behavioural assays have uncovered contributions of NO signalling in oxygen sensing, habituation to chemosensory stimuli, and associative memory formation. During development, NO regulates cell proliferation, axonal outgrowth, and synaptic maturation. The cellular distribution of NO-responsive cells suggests that NO can serve as a retrograde synaptic messenger, as an intracellular messenger, and as a lateral diffusible messenger irrespective of conventional synaptic connectivity.

NADPH diaphorase is developmentally regulated in rat olfactory epithelium.

In vertebrate olfactory receptor neurons, NO synthase (NOS) has been detected in embryonic and early postnatal stages. However, expression of the enzyme in the mature epithelium is still controversial. We analyzed the developmental expression pattern of the histochemical NOS-marker NADPH diaphorase (NADPHd) in the olfactory epithelium of young rats. NADPHd was expressed in a small subset of olfactory receptor neurons as early as P0. Between P0 and P24 the number of labeled neurons increased 10-fold, stabilizing thereafter. Whereas NADPHd was generally found in the somata, a transitory dendritic expression was observed between P2 and P5. This dynamic postnatal regulation of the cellular distribution of NADPHd appears to reflect developmental processes within the olfactory epithelium.

Nitric oxide and cGMP influence axonogenesis of antennal pioneer neurons.

The grasshopper embryo has been used as a convenient system with which to investigate mechanisms of axonal navigation and pathway formation at the level of individual nerve cells. Here, we focus on the developing antenna of the grasshopper embryo (Schistocerca gregaria) where two siblings of pioneer neurons establish the first two axonal pathways to the CNS. Using immunocytochemistry we detected nitric oxide (NO)-induced synthesis of cGMP in the pioneer neurons of the embryonic antenna. A potential source of NO are NADPH-diaphorase-stained epithelial cells close to the basal lamina. To investigate the role of the NO/cGMP signaling system during pathfinding, we examined the pattern of outgrowing pioneer neurons in embryo culture. Pharmacological inhibition of soluble guanylyl cyclase (sGC) and of NO synthase (NOS) resulted in an abnormal pattern of pathway formation in the antenna. Axonogenesis of both pairs of pioneers was inhibited when specific NOS or sGC inhibitors were added to the culture medium; the observed effects include the loss axon emergence as well as retardation of outgrowth, such that growth cones do not reach the CNS. The addition of membrane-permeant cGMP or a direct activator of the sGC enzyme to the culture medium completely rescued the phenotype resulting from the block of NO/cGMP signaling. These results indicate that NO/cGMP signaling is involved in axonal elongation of pioneer neurons in the antenna of the grasshopper.

Enantiomeric separation of enzymatic hydrolysis products of dihydropyrimidinone methyl ester with cationic cyclodextrin by capillary electrophoresis.

The achiral separation of dihydropyrimidinone (DHP) methyl ester and its corresponding carboxylic acid and the chiral separation of their respective enantiomers were achieved in a single analysis using capillary electrophoresis (CE) with quaternary ammonium-beta-cyclodextrin (QA-beta-CD) as a chiral buffer additive. Separation of the DHP methyl ester from the corresponding carboxylic acid was achieved because the acid was negatively charged at pH 8.3 of the running buffer and the ester is neutral. Upon the addition of QA-beta-CD, the enantiomers of the acid and ester were well resolved before and after the electroosmotic flow, respectively. In addition, the minor DHP methyl ester enantiomer (R isomer) was well separated from several impurities. This CE system was used to monitor the progress of a bioresolution reaction that utilizes an enzyme to convert the R isomer of the ester to its corresponding acid. The quantities of all four enantiomers can be determined using a single set of CE conditions. In addition, it is demonstrated that samples can be directly injected into the capillary without sample pretreatment due to the fact that the coating of the cationic CD on the capillary surface prevents adsorption of the positively charged enzyme. The effects of other experimental parameters such as type of CDs, concentration of CDs, pH, temperature, and the preconditioning of capillary were also studied.

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein.

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.

Nitric oxide and cyclic GMP induce vesicle release at Drosophila neuromuscular junction.

Nitric oxide (NO) diffuses as short-lived messenger through the plasma membrane and serves, among many other functions, as an activator of the cGMP synthesizing enzyme soluble guanylyl cyclase (sGC). In view of recent genetic investigations that postulated a retrograde signal from the larval muscle fibers to the presynaptic terminals, we looked for the presence of an NO/cGMP signaling system at the neuromuscular junction (NMJ) of Drosophila melanogaster larvae. Application of NO donors induced cGMP immunoreactivity in the presynaptic terminals but not the postsynaptic muscle fibers at an identified NMJ. The NO-induced cGMP immunoreactivity was sensitive to a specific inhibitor (ODQ) of the sGC. Since presynaptic terminals which were surgically isolated from the central nervous system are capable of synthesizing cGMP, we suggest that an NO-sensitive guanylyl cyclase is present in the terminal arborizations. Using a fluorescent dye that is known to stain recycling synaptic vesicles, we demonstrate that NO donors and membrane permeant cGMP analogues cause vesicle release at the NMJ. Moreover, the NO-induced release could be blocked by the specific inhibitor of the sGC. A destaining of synaptic terminals after NO exposure in Ca2+-free solution in the presence of cobalt chloride as a channel blocker suggested that NO stimulates Ca2+-independent vesicle release at the NMJ. The combined immunocytochemical and exocytosis imaging experiments imply the involvement of cGMP and NO in the regulation of vesicle release at the NMJ of Drosophila larvae.

Histochemistry of classical neurotransmitters in antennal lobes and mushroom bodies of the honeybee.

This paper summarizes histochemical and immunocytochemical investigations of cholinergic, GABAergic, and glutamatergic pathways in the central brain and suboesophageal ganglion of the honeybee. Acetylcholinesterase histochemistry, immunocytochemical staining for nicotinic acetylcholine receptors, and mapping for alpha-bungarotoxin binding sites indicate cholinergic synaptic interactions in the antennal lobe and a cholinergic pathway via a subset of olfactory projection neurons into the mushroom bodies. Calcium imaging experiments in cell cultures prepared from mushroom bodies demonstrate the expression of nicotinic cholinergic receptors on Kenyon cells. Neurons synthesizing GABA and glutamate are stained with well-defined polyclonal antisera against the amino acids. GABA-immunoreactivity is mainly localized in local interneurons of the antennal lobe and in extrinsic neurons innervating the mushroom bodies. High levels of glutamate-immunoreactivity are found in motoneurons of the suboesophageal ganglion, the dorsal lobe, and also in interneurons. A subgroup of the Kenyon cells shows distinct but weaker levels of glutamate-immunoreactivity. The detailed knowledge about the chemical neuroanatomy of the bee provides a framework for behavioral pharmacological approaches, which implicate the involvement of cholinergic mechanisms in olfactory learning and GABAergic mechanisms in odor discrimination.

Biogenic amines in the brain of the honeybee: cellular distribution, development, and behavioral functions.

This review provides a summary of the cellular distribution of amine-containing neurons and the organization of aminergic pathways in the brain and suboesophageal ganglion of the honeybee. Neurons synthesizing the biogenic amines serotonin, dopamine, octopamine, and histamine are stained with well-defined polyclonal antisera. Since some of these aminergic neurons are uniquely identifiable, it is possible to follow their morphogenesis during brain development. Pharmacological studies show that aminergic mechanisms are involved in various behavioral modifications including associative learning. The immunocytochemical approach resolves at a single cell level the neural pathways that mediate adaptive behavioral changes.

Developmental expression of nitric oxide/cyclic GMP synthesizing cells in the nervous system of Drosophila melanogaster.

Nitric oxide (NO) is a membrane-permeant signaling molecule which activates soluble guanylyl cyclase and leads to the formation of cyclic GMP (cGMP). The NO/cGMP signaling system is thought to play essential roles during the development of vertebrate and invertebrate animals. Here, we analyzed the cellular expression of this signaling pathway during the development of the Drosophila melanogaster nervous system. Using NADPH diaphorase histochemistry as a marker for NO synthase, we identified several neuronal and glial cell types as potential NO donor cells. To label NO-responsive target cells, we used the detection of cGMP by an immunocytochemical technique. Incubation of tissue in an NO donor induced cGMP immunoreactivity (cGMP-IR) in individual motoneurons, sensory neurons, and groups of interneurons of the brain and ventral nerve cord. A dynamic pattern of the cellular expression of NADPHd staining and cGMP-IR was observed during embryonic, larval, and prepupal phases. The expression of NADPH diaphorase and cGMP-IR in distinct neuronal populations of the larval central nervous system (CNS) indicates a role of NO in transcellular signaling within the CNS and as potential retrograde messenger across the neuromuscular junction. In addition, the presence of NADPH diaphorase-positive imaginal discs containing NO-responsive sensory neurons suggests that a transcellular NO/cGMP messenger system can operate between cells of epithelial and neuronal phenotype. The discrete cellular resolution of donor and NO-responsive target cells in identifiable cell types will facilitate the genetic, pharmacological, and physiological analysis of NO/cGMP signal transduction in the developing nervous system of Drosophila.

Nitric oxide and cyclic GMP modulate photoreceptor cell responses in the visual system of the locust

Nitric oxide (NO) is a membrane-permeant messenger molecule which activates the cyclic GMP (cGMP)-synthesizing enzyme soluble guanylyl cyclase. Using cytochemical techniques, we recently reported NO-induced cGMP immunoreactivity in the photoreceptor cells of the compound eye of the locust Schistocerca gregaria and also detected NADPH diaphorase staining, a marker of NO synthase, in a subset of the monopolar cells of the lamina. By recording the corneal electroretinogram (ERG), we found that the application of neurochemicals that raise NO/cGMP levels in the optic lobe increased the ERG amplitude, whereas the experimental reduction of NO levels caused a decrease in the response to light. An increase in the light response was also found in intracellular recordings after application of a NO donor, suggesting that the NO-induced changes in the ERG are not caused by changes in the resistive isolation of the retina. Our cytochemical and electrophysiological data are both consistent with the hypothesis that NO synthesized in monopolar cells is a retrograde messenger to the presynaptic photoreceptor neurones.

Determination of bis(tributyltin) oxide by GC--MS with on-line hydride derivatization: application to drug substance analysis.

We report the determination of residual bis(tributyltin) oxide in a drug substance by GC-MS after extraction and on-line conversion to tributyltin hydride. Gas chromatography was performed using a 15 m x 0.25 mm i.d. DB-5 HT column with a temperature program from 100 to 160 degrees C at 15 degrees C min(-1). A mass range of 165-185 amu was monitored with the MS detector. Hydride generation is performed by placing a small amount of solid sodium borohydride in the injection port of a gas chromatograph and injecting samples and standards through this material. Conversion to tributyltin hydride is shown to be quantitative and linear for levels of bis(tributyltin) oxide between 1 and 100 ppm in the drug substance. The use of GC-MS provides sensitive and selective detection of tin containing species and the tin isotope pattern allows for confirmation of the presence of tin in chromatographic peaks. Recovery at 6 ppm was 89% with an injection precision of 6%. The limit of detection for bis(tributyltin) oxide in drug substance is 1 ppm.

NO news from insect brains.

In nerve cells,the short-lived signalling molecule nitric oxide (NO) is generated by Ca2+-calmodulin-stimulated NO synthases. Nitric oxide activates soluble guanylate cyclase in target cells, leading to the formation of cGMP. Biochemical investigations have shown the presence of a Ca2+-calmodulin-regulated NO-cGMP signalling mechanism in the nervous system of insects. Using NADPH-diaphorase staining as a marker for the enzyme NO synthase and an antiserum against cGMP,the cellular organization of NO donor and target cells has so far been resolved in the locust and fruit fly. This paper provides an overview of the cellular organization of NO signalling in the insect nervous system as well as highlighting its functions in olfactory information processing, formation of olfactory memory, vision, and neuronal development. The resolution of discrete donor and NO-responsive target cells in the developing nervous system of Drosophila will facilitate the genetic and pharmacological analysis of NO-cGMP signal transduction.

Colocalization of NADPH-diaphorase and GABA-immunoreactivity in the olfactory and visual system of the locust.

Nitric oxide synthesizing neurons of the locust CNS have been identified by NADPH-diaphorase staining. However, the conventional transmitters of these neurons are unknown. Here we use double labelling for NADPH-diaphorase and GABA-immunofluorescence on sections of the brain to investigate a potential coexpression of both markers. The antennal lobe is innervated by a cluster of about 45-50 NADPH-diaphorase positive local interneurons which express GABA-immunofluorescence. The mushroom bodies are a higher order olfactory center which receive an extrinsic innervation from GABA-immunoreactive and NADPH-diaphorase positive fiber systems. Each optic lobe contains about 4500 GABA-immunoreactive cell bodies. In the visual system, identifiable GABA-immunoreactive neurons arborize in the external plexiform layer of the lamina, in several strata of the medulla, and in the lobula complex. A survey of all NADPH-diaphorase positive cell groups detected a colocalization of GABA-immunoreactivity in a small subpopulation of somata along the anterior rim of the medulla. These cytochemical findings suggest that nitric oxide may be a characteristic cotransmitter of GABAergic circuits of the antennal lobe, while in mushroom bodies and the visual system the majority of nitric oxide and GABA releasing neurons are distinct populations.

Micellar enhanced cyanide ion determination in samples from synthetic organic processes.

A method was developed for the recovery and determination of cyanide ion in organic sample matrices. To facilitate the solubilization of cyanide ions, cetyltrimethylammonium bromide (CTAB) was added at concentrations above the critical micelle concentration. Sample cyanation reaction products consisted of solvent mixtures of a hydroxynitrile in DMF-toluene or DMF-isopropylacetate (IPAC). Spectrophotometric determination of cyanide ion at 578 nm by the pyridine-barbituric acid method was automated by flow injection analysis. Recovery of cyanide ion from spiked samples was 93.2% in DMF-IPAC solvent matrix and 93.9% in DMF-toluene. Low alkali concentration was observed to favor solubilization of cyanide ion in the micellar solution.

Cytochemical evidence for nitric oxide/cyclic GMP signal transmission in the visual system of the locust.

Nitric oxide is a membrane-permeant messenger molecule which activates soluble guanylyl cyclase. Using NADPH diaphorase staining as a marker for the enzyme nitric oxide synthase and an antiserum against cyclic GMP (cGMP) we investigated the possible sites of nitric oxide and cGMP synthesis in the retina and lamina of Schistocerca gregaria. The photoreceptor cells did not express NADPH diaphorase staining but monopolar cells of the lamina were strongly stained. After inhibition of phosphodiesterase activity and incubation of tissue in a nitric oxide donor, the photoreceptor cells showed cGMP immunoreactivity. In contrast to the photoreceptors, the monopolar cells of the lamina were not stained. Since the presynaptic photoreceptors were cGMP-immunoreactive and the postsynaptic targets of the monopolar cells did not express immunoreactivity, it is conceivable that nitric oxide released by monopolar cells may play a role as a retrograde messenger in visual information processing.

Embryonic brain tract formation in Drosophila melanogaster.

During embryogenesis in insects, the axonscaffold of the brain is built around the embryonic foregut which separates the anlagen of the brain hemispheres. Here, we investigate this process in Drosophila and show that the major longitudinal and horizontal tracts of the embryonic brain form superficially near the interface between the foregut and embryonic brain hemispheres. We identify three types of cellular structures which might be involved in tract formation. These are rows of glial cells at the medial brain margin, cellular bridges composed of neuronal somata and the epithelial surface of the foregut itself. The close proximity to the outgrowing axons suggests that the structures at the brain-foregut interface may play a role in the morphogenesis of embryonic brain tracts in Drosophila.

Glial patterning during postembryonic development of central neuropiles in the brain of the honeybee.

Glial cells are involved in several functions during the development of the nervous system. To understand potential glial contributions to neuropile formation, we examined the cellular pattern of glia during the development of the mushroom body, antennal lobe and central complex in the brain of the honeybee. Using an antibody against the glial-specific repo-protein of Drosophila, the location of the glial somata was detected in the larval and pupal brain of the bee. In the early larva, a continuous layer of glial cell bodies defines the boundaries of all growing neuropiles. Initially, the neuropiles develop in the absence of any intrinsic glial somata. In a secondary process, glial cells migrate into defined locations in the neuropiles. The corresponding increase in the number of neuropile-associated glial cells is most likely due to massive immigrations of glial cells from the cell body rind using neuronal fibres as guidance cues. The combined data from the three brain regions suggest that glial cells can prepattern the neuropilar boundaries.