Expression of transforming growth factor beta-like molecules in normal and regenerating arms of the crinoid Antedon mediterranea: immunocytochemical and biochemical evidence

The phylum Echinodermata is well known for its extensive regenerative capabilities. Although there are substantial data now available that describe the histological and cellular bases of this phenomenon, little is known about the regulatory molecules involved. Here, we use an immunochemical approach to explore the potential role played by putative members of the transforming growth factor-beta (TGF-beta) family of secreted proteins in the arm regeneration process of the crinoid Antedon mediterranea. We show that a TGF-beta-like molecule is present in normal and regenerating arms both in a propeptide form and in a mature form. During regeneration, the expression of the mature form is increased and appears to be accompanied by the appearance of an additional isoform. Immunocytochemistry indicates that TGF-beta-like molecules are normally present in the nervous tissue and are specifically localized in both neural elements and non-neural migratory cells, mainly at the level of the brachial nerve. This pattern increases during regeneration, when the blastemal cells show a particularly striking expression of this molecule. Our data indicate that a TGF-beta-like molecule (or molecules) is normally present in the adult nervous tissues of A. mediterranea and is upregulated significantly during regeneration. We suggest that it can play an important part in the regenerative process.     

In vitro capsaicin-induced cytological changes and alteration in calcium distribution in giant serotonergic neurons of the snail Helix pomatia: a light- and electron-microscopic study

Morphological changes induced by capsaicin were studied in the serotonergic metacerebral giant neurons of the cerebral ganglia of Helix pomatia under in vitro conditions. Capsaicin at a concentration of 10^-4 M caused characteristic structural alterations in the giant serotonergic neurons but did not significantly influence serotonin immunoreactivity in the neurons. At the lightmicroscopic level, the most conspiciuous structural alterations were swelling of the cell bodies, which contained a swollen pale nucleus. Under the electron microscope, the nuclei,mitochondria and the cisternae of the endoplasmic reticulum were swollen in the capsaicin-affected metacerebral giant neurons. Electron-microscopic cytochemical techniques for calcium demonstration revealed electron-dense deposits in the swollen mitochondria and in the cisternae of the endoplasmic reticulum, suggesting an increased Ca²⁺ influx. The serotonergic metacerebral giant neurons could be labelled by cobalt (1 mM) in the presence of capsaicin (10^-4 M) suggesting that capsaicin opens the cation chanels of the capsaicin-sensitive neuronal membrane. The morphological and cytochemical alterations induced by capsaicin in the serotonergic metacerebral giant neurons of Helix pomatia closely resemble those induced in sensory neurons of mammalian dorsal root ganglion.This work was supported by OTKA grants No.: 2477, T016861, T017127 and ETT 587/93     

Changes in the concentration of enolase isozymes and S-100 protein in degenerating and regenerating rat sciatic nerve

Levels of enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) and S-100 protein in rat sciatic nerves were determined during their degeneration and regeneration processes. The sciatic nerves were unilaterally crushed or severed. The rats were killed 1, 2, 6, and 8-9 weeks later, and both the proximal and distal portions of the damaged nerves were dissected. Control samples were obtained from the untreated contralateral hindlimbs. Enolase isozymes and S-100 protein in the nerve segments were determined with the enzyme immunoassay method. The control nerves contained about 40, 90, and 30 pmol/mg protein of alpha alpha, alpha gamma, and gamma gamma enolases, respectively, and 0.85 microgram/mg protein of S-100 protein. These levels were not affected by repetitive electrical stimulation of the nerve fibers in vivo. The levels of the nervous system-specific forms of enolase (alpha gamma and gamma gamma) and S-100 protein decreased markedly within a week in the distal portion of the crushed nerve (alpha gamma, 27 pmol/mg; gamma gamma, 5.5 pmol/mg; S-100 protein, 0.36 microgram/mg) with apparently no change in the concentration of alpha alpha enolase. These levels in the proximal portion of the crushed nerve remained unaltered. The sensory and motor functions impaired by the sciatic nerve crush showed a recovery more or less after 4-9 weeks. This recovery was accompanied by a gradual regaining of the specific proteins in the distal portion of injured nerves (alpha gamma, 64 pmol/mg; gamma gamma, 13 pmol/mg; S-100 protein, 0.63 microgram/mg at the 8-9th week).     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Immunocytochemical demonstration of neurotransmitters in the nerve plexuses of the foot and the anterior byssus retractor muscle of the mussel,Mytilus galloprovincialis

Summary Immunocytochemical methods were applied to study the distribution of putative neurotransmitters (5-HT, substance P, GABA, glutamate and aspartate) in the nerve plexuses of the foot and the anterior byssus retractor muscle (ABRM) of Mytilus galloprovincialis (Mollusca, Bivalvia). The foot presents extensive nerve plexuses containing 5-HT and substance P-like immunoreactive material with a similar distribution beneath the surface epithelium, around the vessels and in the glandular regions. Coexistence of the two putative neurotransmitters was observed in a few nerve fibers, Conversely, muscle fibers, both in the foot and in the ABRM, are innervated only by 5-HT-positive fibers, while substance P-like material is present only in the networks of the ABRM epimysial sheath. Immunoreactivity for glutamate and aspartate was not demonstrated, while rare GABA-positive nerve cells and fibers were found only in the foot. The results of this investigation provide a morphological background to previous physiological studies on 5-HT in the nervous system of bivalve molluscs. Moreover, they confirm that the nervous system of Mytilus contains a remarkable amount of a substance related to the vertebrate tachykinin family.     

Nitric oxide (NO) synthase immunoreactivity in the starfish Marthasterias glacialis

The neuroendocrine system of the starfish Marthasterias glacialis was investigated immunocytochemically using antisera specific for rat neuronal, bovine aortic endothelial, and mouse macrophage, nitric oxide (NO) synthases. Immunoreactivity was detected only with the antibodies specific for the neural enzyme, in the ectoneural and hyponeural tissues of the radial nerve cords and in the basiepithelial plexus and endocrine cells of the digestive tract. The pyloric stomach showed more immunoreactive structures than the other digestive organs, with the rectal caeca showing the least activity. Immunoreactive endocrine cells were located in the cardiac and pyloric stomachs and in the pyloric caeca. Co-localization of the enzyme immunoreactivity, and the staining for NADPH-diaphorase, demonstrate the presence of NO synthase in echinoderms. These results provide further evidence that NO is a neuronal messenger of early phylogenetic origin which has been conserved throughout evolution.     

Transmitter contents of cells and fibers in the cephalic sensory organs of the gastropod mollusc<Emphasis Type="Italic"> Phestilla sibogae</Emphasis>

While the central ganglia of gastropod molluscs have been studied extensively, relatively little is known about the organization and functions of the peripheral nervous system in these animals. In the present study, we used immunohistochemical procedures to examine the innervation of the rhinophores, oral tentacles and region around the mouth of the aeolid nudibranch, Phestilla sibogae. Serotonin-like immunoreactivity was found in an extensive network of efferent projections apparently originating from central neurons, but was not detected within any peripheral cell bodies. In contrast, large numbers of peripheral, and presumably sensory, somata exhibited reactivity to an antibody raised against tyrosine hydroxylase (the enzyme catalyzing the initial step in the conversion of tyrosine into the catecholamines). Additional tyrosine hydroxylase-like immunoreactivity was detected in afferent fibers of the peripheral cells and in several cells within the rhinophoral ganglia. The presence of serotonin, dopamine and norepinephrine in the rhinophores, tentacles and central ganglia was confirmed using high-performance liquid chromatography. Finally, FMRFamide-like immunoreactivity was detected in cells and tangles of fibers found within the rhinophore, possibly revealing glomerulus-like structures along olfactory pathways. FMRFamide-like immunoreactivity was also found in somata of the rhinophoral ganglia, in a small number of cells located in the body wall lateral to the tentacles and in what appeared to be varicose terminals of efferent projections to the periphery. Together, these results indicate several new features of the gastropod peripheral nervous system and suggest future experiments that will elucidate the function of the novel cells and innervation patterns described here.This research was supported by Natural Sciences and Research Council of Canada Grant #OPG38863 to R.P.C. and Office of Naval Research Grant #N00014-94-1-0524 to M.G.H.     

淡水蜗牛视觉系统的输入与输出通路(英文)

通过神经生物素在视神经上的逆行性传输对淡水蜗牛(Planorbarius corneus)视网膜及中央神经节的输入、输出神经元进行标记。由于没有发现突触联系,所以至少一部分光感受细胞的轴突可被视为直接参与形成视神经。这些神经元的轴突进入大脑神经节形成密集的细传入神经纤维束-视神经堆。传出神经元则存在于除颊部以外的所有神经节。一些上行轴突在大脑神经节处分叉,通过脑-脑联合,到达对侧眼并在眼杯处形成分枝。部分传出神经元的轴突也投射于不同的外周神经,如:n.n.intestinalis,pallialis dexter,pallialis sinister internus et externus。五羟色胺能纤维和FMRF-酰胺能纤维均存在于视神经上,且这些纤维隶属于只投射在同侧眼的中央神经元。它们形成了位于眼杯处的丰富曲张结构及视网膜核心层,并且可能有助于调节视网膜对光的敏感性。     

<Emphasis Type="Italic">Engrailed</Emphasis> is expressed in larval development and in the radial nervous system of <Emphasis Type="Italic">Patiriella</Emphasis> sea stars

We documented expression of the pan-metazoan neurogenic gene engrailed in larval and juvenile Patiriella sea stars to determine if this gene patterns bilateral and radial echinoderm nervous systems. Engrailed homologues, containing conserved En protein domains, were cloned from the radial nerve cord. During development, engrailed was expressed in ectodermal (nervous system) and mesodermal (coeloms) derivatives. In larvae, engrailed was expressed in cells lining the larval and future adult coeloms. Engrailed was not expressed in the larval nervous system. As adult-specific developmental programs were switched on during metamorphosis, engrailed was expressed in the central nervous system and peripheral nervous system (PNS), paralleling the pattern of neuropeptide immunolocalisation. Engrailed was first seen in the developing nerve ring and appeared to be up-regulated as the nervous system developed. Expression of engrailed in the nerve plexus of the tube feet, the lobes of the hydrocoel along the adult arm axis, is similar to the reiterated pattern of expression seen in other animals. Engrailed expression in developing nervous tissue reflects its conserved role in neurogenesis, but its broad expression in the adult nervous system of Patiriella differs from the localised expression seen in other bilaterians. The role of engrailed in patterning repeated PNS structures indicates that it may be important in patterning the fivefold organisation of the ambulacrae, a defining feature of the Echinodermata.     

Alterations in Extracellular and Tissue Levels of Biogenic Amines in Rat Brain Induced by the Serotonin2 Receptor Antagonist, Ritanserin

Systemic administration of ritanserin elicited rapid changes in dopamine (DA) and serotonin (5-HT) levels in both dialysate and neuronal tissue extracts. These effects occurred in both a site-selective and a dose-related manner. Increases in extracellular levels of DA and 5-HT in the nucleus accumbens were maximal at 120-140 min after treatment. A dose of 0.63 mg/kg of ritanserin elicited larger and more prolonged increases in extracellular DA and 5-HT levels than did the 0.3 mg/kg dose. By contrast, 0.63 mg/kg of ritanserin elicited no changes in either DA or 5-HT levels with dialysate collected from the striatum. Ritanserin also induced dose-related decreases in tissue levels of DA and 5-HT from the nucleus accumbens. The site specificity of action was again noted in that there were no dose-dependent decreases in tissue levels of DA or 5-HT measured from the striatum. Ritanserin exerted little effect on metabolite levels from either dialysate or tissue extracts. Taken together, these findings show that selective 5-HT2 receptor antagonism modulates DA and 5-HT neurotransmission in a specific manner. These actions appear to involve increased release of DA and 5-HT rather than significant changes in metabolism. These findings add further weight to the importance of 5-HT2 receptor interactions as an important component of antipsychotic activity.     

Distribution of tyrosine-hydroxylase-immunoreactive and dopamine-immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution and characterization of dopamine-containing neurons are described in the different ganglia of the central nervous system of Helix on the basis of the distribution of tyrosine hydroxylase immunoreactive (TH-ir) and dopamine immunoreactive (DA-ir) neurons. Both TH-ir and DA-ir cell bodies of small diameter (10–25 m) can be observed in the buccal, cerebral and pedal ganglia, dominantly on their ventral surface, and concentrated in small groups close to the origin of the peripheral nerves. The viscero-parietal-pleural ganglion complex is free of immunoreactive cell bodies but contains a dense fiber system. The largest number of TH-ir and DA-ir neurons can be detected in the pedal, and cerebral ganglia. The average number of TH-ir and DA-ir neurons significantly differs but all the identifiable groups of TH-ir neurons also show DA-immunoreactivity. Therefore, we consider the TH-ir neurons in those groups as being DA-containing neurons. The amounts of DA in the different ganglia assayed by high performance liquid chromatography correspond to the distribution and number of TH-ir and DA-ir neurons in the different ganglia. The axon processes of the labeled small-diameter neurons send thin proximal branches toward the cell body layer but only rarely surround cell bodics, whereas distally they give off numerous branches in the neuropil and then leave the ganglion through the peripheral nerves. In the cerebral ganglia, the analysis of the TH-ir pathways indicates that the largest groups of labeled neurons send their processes through the peripheral nerves in a topographic order. These results furnish morphological evidence that DA-containing neurons of Helix pomatia have both central and peripheral roles in neuronal regulation.     

Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain

Locustatachykinin I is one of four closely related myotropic neuropeptides isolated from brain and corpora-cardiaca complexes of the locust Locusta migratoria. Antiserum was raised against locustatachykinin I for use in immunocytochemistry. It was found that the antiserum recognizes also locustatachykinin II and hence probably also the other two locustatachykinins due to their similarities in primary structure. Locustatachykinin-like immunoreactive (LomTK-LI) neurons were mapped in the brain of the locust, L. migratoria. A total of approximately 800 Lom TK-LI neurons were found with cell bodies distributed in the proto-, deutoand tritocerebrum, in the optic lobes and in the frontal ganglion. Processes of these neurons innervate most of the synaptic neuropils of the brain and optic lobes, as well as the frontal ganglion and hypocerebral ganglion. The widespread distribution of LomTK-LI neurons in the locust brain indicates an important role of the locustatachykinins in signal transfer or regulation thereof. As a comparison neurons were mapped with an antiserum against the cockroach myotropic peptide leucokinin I. This antiserum, which probably recognizes the native peptide locustakinin, labels a population of about 140 neurons distinct from the LomTK-LI neurons (no colocalized immunoreactivity). These neurons have cell bodics that are distributed in the proto- and tritocerebrum and in the optic lobe. The processes of the leucokinin-like immunoreactive (LK-LI) neurons do not invade as large areas in neuropil as the Lom TK-LI neurons do and some neuropils, e.g. the mushroom bodies, totally lack innervation by LK-LI fibers. In some regions, however, the processes of the Lom TK-LI and LK-LI neurons are superimposed: most notably in the central body and optic lobes. A functinal relation between the two types of neuropeptide in the locust brain can, however, not be inferred from the present findings.     

The distribution of glutamate-like immunoreactivity in the thoracic and abdominal ganglia of the locust (Schistocerca gregaria)

The distribution of glutamate-like immunore-activity in the thoracic and abdominal ganglia of the locust was studied using two polyclonal antibodies against glutamate. Because glutamate is a precursor of the inhibitory transmitter -amino butyric acid (GABA) the distribution of immunostaining by antibodies against glutamate and GABA was closely compared in adjacent serial sections. When the antibodies were used at optimal dilutions there was no overlap in the distribution of immunostaining for glutamate and GABA. In the pro- and mesothoracic ganglia 360–400 somata are immunoreactive for glutamate, while in the metathoracic ganglion about 600 somata were stained. These range in diameter from 10–100 m in diameter and include the majority of the large somata in these ganglia. Bundles of primary neurites emerging from these large somata can be traced through the neuropile. Most of the bundles correspond to the known paths of motor neurone primary neurites. In addition the T-tract is also immunolabelled. The free abdominal ganglia each contain 80–100 somata ranging in size from 10–45 m while the terminal ganglion contains about 250 somata, 10–60 m in diameter.     

Projections and actions of tachykininergic, cholinergic, and serotonergic neurones in the intestine of the Atlantic cod

The native tachykinins cod neurokinin A and cod substance P, serotonin and acetylcholine have excitatory effects on the circular smooth muscle of the cod intestine. Furthermore, immunoreactivities to the cod tachykinins, serotonin and two markers for cholinergic neurones, viz. choline acetyltransferase and vesicular acetylcholine transporter, have been demonstrated in myenteric neurones of the cod intestine. In order to elucidate whether the neurones containing these substances project orally and thus might be involved in the ascending excitatory reflex of peristalsis, myotomy operations have been performed on the cod intestine. The immunoreactive areas of the myenteric plexus immediately oral and anal to the myotomy operations have been measured by using confocal laser scanning microscopy. Large accumulations of immunoreactivity to the tachykinins are found on the anal side of the myotomies, indicating oral projections of tachykininergic neurones. The areas immunoreactive to serotonin and choline acetyltransferase are of equal size on the oral and anal sides. Since the tachykinin containing neurones of the intestine project orally, and since cod neurokinin A and cod substance P have excitatory effects on circular smooth muscle, we conclude that tachykininergic neurones are involved in the ascending excitatory reflex of peristalsis in the cod intestine. Received: 6 March 1997 / Accepted: 15 September 1997     

Unusual distribution of tubulin isoforms in the snail Lymnaea stagnalis

Immunocytochemistry and Western blotting techniques demonstrated that the nervous system and foot of the pond snail Lymnaea stagnalis are rich sources of tubulin, which can be extracted and assembled in vitro in the presence of taxol. Various broad-spectrum antibodies raised against -tubulin and -tubulin yielded qualitatively similar results. One monoclonal antibody to trypanosome -tubulin, however, labelled -tubulin more strongly on both probed sections and Western blots. Cytochemistry and immunoblotting revealed that tyrosinated tubulin constitutes a large proportion of total -tubulin in locomotor cilia of the foot and in axons of the nervous system. Detyrosinated tubulin also appeared to be abundant in the foot cilia but only a very faint band of detyrosinated tubulin was found on protein blots extracted from the central ganglia, and staining was barely detectable in central ganglia or peripheral nerves. Similarly, acetylated tubulin appeared to be abundant in foot cilia, but Western blotting indicated only low levels of acetylated tubulin in the nervous system. Immunocytochemistry indicated that, while most neurons possessed little or no acetylated tubulin, a small number of axons contained significant amounts of this isoform. Thus, while a large amount of tubulin was expected in the nervous system and locomotor cilia of L. stagnalis, the observed distribution of isoforms was unanticipated. Specifically, neurons of other organisms have generally been reported to contain substantial amounts of both detyrosinated -tubulin and acetylated -tubulin. Our results indicate that such findings cannot be generalized across all species. L. stagnalis, with its well studied nervous system and unusual distribution of tubulin isoforms, may prove to be particularly useful for studying the roles of tubulin isoforms in microtubule function and cell activity.     

A comparative study of plasma membrane Mg2+-ATPase activities in normal,regenerating and malignant cells

Plasma membranes have been prepared from rat normal liver cells, regenerating liver cells and Yoshida ascites hepatoma 66 cells after intact cells were first bound to polylysine-coated polyacrylamide beads, and the membrane-associated Mg²⁺-ATPase activity was assayed directly on beads with membrane attached. With plasma membranes from normal liver cells, K_m for ATP and V were found to be higher than those in regenerating liver cells and hepatoma cells. Vanadate caused a different sensitivity of the activity, without an effect in normal liver cells and with an inhibition in regenerating liver cells and hepatoma cells. The activity in normal and regenerating liver cells decreased with increasing temperature above 24–30°C, while the activity in hepatoma cells continued to increase linearly to 37°C. Unlike the enzyme in normal and regenerating liver cells, the hepatoma enzyme was shown to have a higher phase transition temperature and lower activation energies. In all three kinds of cells the activity was increased by the dephosphorylation of plasma membranes and unaffected by the phosphorylation. By means of histochemical Mg²⁺-ATPase staining applied on polyacrylamide gels, at least three major bands which show the enzymic activity were visible in normal and regenerating liver and a single band was detected in hepatoma cells.     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.