Light and electron microscopic histochemical observations on cholinesterase-containing sympathetic nerve fibres in the pineal body of the rat

Synopsis Pineal glands of adult albino rats were examined histochemically using, first, formaldehyde-induced fluorescence to study monoamines and, second, copper thiocholine or copper ferrocyanide methods to study acetylcholinesterase and non-specific cholinesterase by light and electron microscopy. Cholinesterase was determined quantitatively by a constant pH titration assay.Fluorescent and acetylcholinesterase-positive nerve nets formed identical patterns. Nonspecific cholinesterase was observed only in nerve trunks outside the pineal. Bilateral removal of superior cervical ganglia resulted in complete disappearance of fluorescence and acetylcholinesterase from nerve fibres. Electron microscopically, acetylcholinesterase was found on sympathetic axons containing small granular vesicles. Quantitative cholinesterase determinations suggested that the pineal activity was mainly due to acetylcholinesterase. Comparison of the incubation times required for equal histochemical acetylcholinesterase reactions suggested that the activity of the sympathetic nerve fibres in the pineal is of the same order of magnitude as that in the nerve fibres of the iris.     

FMRFamide-like immunoreactivity in the nervous system of hydra

Summary FMRFamide-like immunoreactivity has been localized in different parts of the hydra nervous system. Immunoreactivity occurs in nerve perikarya and processes in the ectoderm of the lower peduncle region near the basal disk, in the ectoderm of the hypostome and in the ectoderm of the tentacles. The immunoreactive nerve perikarya in the lower peduncle region form ganglion-like structures. Radioimmunoassays of extracts of hydra gave displacement curves parallel to standard FMRFamide and values of at least 8 pmol/gram wet weight of FMRFamide-like immunoreactivity. The immunoreactive material eluted from Sephadex G-50 in several components emerging shortly before or after position of authentic FMRFamide. The presence of FMRFamide-like material in coelenterates shows that this family of peptides is of great antiquity.     

Neurotensin-like immunoreactivity in the nervous system of hydra

Summary Neurotensin-like immunoreactivity is found in nerve fibers present in all body regions of hydra. The nerve fibers are especially numerous in the ectoderm at the bases of the tentacles and in the ectoderm at a site just above the foot. Radioimmunoassays of acetic-acid extracts of hydra, using various region-specific antisera towards mammalian neurotensin, show the presence of multiple neurotensin-related peptides. The amounts of these peptides vary between 1 and 350 pmol per gram wet weight. Gel filtration on Sephadex G-25 reveals a fraction of neurotensin-like peptides that crossreacts equally well with an antiserum directed against sequence 1–8 and an antiserum directed against sequence 6–13 of neurotensin. This fraction elutes also at the position of neurotensin and might closely resemble the mammalian peptide. A fraction eluting with the void volume crossreacts preferentially with antisera directed against sequences 1–8 and 10–13 of neurotensin. Several components of apparent lower molecular weight than neurotensin crossreact preferentially with an antiserum against sequence 10–13. These last peptides represent the major portion of the neurotensin-like peptides in hydra.     

Bombesin-like immunoreactivity in the nervous system of hydra

Summary With immunocytochemical methods, nerve cells have been detected in Hydra attenuata containing bombesin-like immunoreactivity. These nerve cells are located in the ectoderm of all body regions of the animal and are especially abundant in basal disk and tentacles. Radioimmunoassay of extracts of hydra demonstrated at least 0.2 pmol/g wet weight of bombesinlike immunoreactivity. The immunoreactive material elutes from Sephadex G-50 in a similar position to synthetic bombesin. The data show that bombesin-like peptides are among the phylogenetically oldest neuropeptides found so far.     

Substance P-like immunoreactivity in the nervous system of hydra

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Substance P-like immunoreactivity in the nervous system of hydra

Summary Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Chemical anatomy of hydra nervous system using antibodies against hydra neuropeptides: a review

Polyclonal antibodies against hydra neuropeptides allow us to visualize the hydra nerve net. Chemical anatomy of the hydra nerve net was archived by means of immunocytochemistry with various antibodies to hydra neuropeptides. Our goal is to describe the hydra nerve net both qualitatively and quantitatively. The present chemical anatomy results indicate (1) differences in peptide expression between ganglion cells and sensory cells, (2) large differences in neuropeptide expression between ectodermal nerve cells and endodermal nerve cells, and (3) the usefulness of quantitative chemical anatomy to analyze the entire nervous system of hydra.     

Neuronal evolution: analysis of regulatory genes in a first-evolved nervous system, the hydra nervous system

Cnidarians represent the first animal phylum with an organized nervous system and a complex active behavior. The hydra nervous system is formed of sensory-motoneurons, ganglia neurons and mechanoreceptor cells named nematocytes, which all differentiate from a common stem cell. The neurons are organized as a nerve net and a subset of neurons participate in a more complex structure, the nerve ring that was identified in most cnidarian species at the base of the tentacles. In order to better understand the genetic control of this neuronal network, we analysed the expression of evolutionarily conserved regulatory genes in the hydra nervous system. The Prd-class homeogene prdl-b and the nuclear orphan receptor hyCOUP-TF are expressed at strong levels in proliferating nematoblasts, a lineage where they were found repressed during patterning and morphogenesis, and at low levels in distinct subsets of neurons. Interestingly, Prd-class homeobox and COUP-TF genes are also expressed during neurogenesis in bilaterians, suggesting that mechanoreceptor and neuronal cells derive from a common ancestral cell. Moreover, the Prd-class homeobox gene prdl-a, the Antp-class homeobox gene msh, and the thrombospondin-related gene TSP1, which are expressed in distinct subset of neurons in the adult polyp, are also expressed during early budding and/or head regeneration. These data strengthen the fact that two distinct regulations, one for neurogenesis and another for patterning, already apply to these regulatory genes, a feature also identified in bilaterian related genes.     

New observations on green hydra symbiosis

New observations on green hydra symbiosis are described. Herbicide norflurazon was chosen as a "trigger" for analysis of these observations. Green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. In its gastrodermal myoeptihelial cells it contains individuals of Chlorella vulgaris Beij. (KESSLER & HUSS 1992). Ultrastructural changes were observed by means of TEM. The newly described morphological features of green hydra symbiosis included a widening of the perialgal space, missing symbiosomes and joining of the existing perialgal spaces. Also, on the basis of the newly described mechanisms, the recovery of green hydra after a period of intoxication was explained. The final result of the disturbed symbiosis between hydra and algae was the reassembly of the endosymbiosis in surviving individuals.     

An electron microscopic and histochemical study of the secretory cells inHydra viridis

Summary The fresh water coelenterateHydra viridis possesses a unique distribution of mucous and serous secretory cells in the gastrodermis. The mucous cells are found only in the hypostome, a region devoid of the serous zymogen cells. On the other hand, the zymogen cells are found extending from the tentacles to the peduncle. Histochemical stains indicated that the two hypostomal mucous cells, spumous and granular, secreted an acid mucopolysaccharide, and incorporated radiosulfate. The radiosulfate label was not sensitive to hyaluronidase digestion, but was removed by acid methanolysis. In contrast, the secretory product of the zymogen cell was rich in proteins and a PAS-positive moiety (unsulfated).The ultrastructure of these cells was correlated with their histochemical staining properties. It was demonstrated that glutaraldehyde preserved the ultrastructure of the secretory granules better than osmium, and also preserved more components within the granules. The mucous cell granules contained an electrolucent and an electron dense component. The cells were both PAS-positive and alcianophilic. After osmium fixation the dense component was lost and the cells were primarily alcianophilic. Osmium also failed to preserve the electron dense component in the zymogen cells.Observations of corresponding thick and thin sections showed a cell containing granules similar to the granules seen in mouse Paneth cells. The dense core was osmiophilic and the lighter halo was alcianophilic.These results lead us to conclude that the electrolucent filamentous component is an alcianophilic acid mucopolysaccharide and the dense granular component is probably a PAS-positive material.

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Zusammenfassung Der FrischwassercölenteratHydra viridis weist eine einzigartige Verteilung von mukösen und serösen sekretorischen Zellen in der Gasterodermis auf. Die mukösen Zellen finden sich nur im Hypostom, in welchem seröse Zymogenzellen fehlen. Die Zymogenzellen andereseits finden sich von den Tentaklen bis zum Pedunkulus. Histochemische Methoden zeigten, daß die zwei Typen hypostomaler muköser Zellen, d.h. spumöse und granuläre, ein saures Mukopolysaccharid ausscheiden und radioaktives Sulfat einbauen. Der Radiosulfat-Markierer war nicht sensitiv gegenüber Hyaluronidase, konnte aber entfernt werden mit saurem Methanol. Im Gegensatz dazu war das Produkt der Zymogenzellen reich an Proteinen und enthielt PAS-positives Material.Die Feinstruktur dieser Zellen war korreliert mit diesen histochemischen Befunden. Glutaraldehyd erhielt die Feinstruktur der Sekretgranula besser und fixierte mehr Komponenten als Osmium. Die Granula der mukösen Zellen enthielten elektronendichte und -durchsichtige Komponenten; diese Zellen färbten sich mit PAS und Alcyan. Nach Osmium-Fixierung war die elektronendichte Komponente abwesend und die Zellen waren hauptsächlich alcyanophil. Auch in den Zymogenzellen vermochte Osmium nicht, die elektronendichte Komponente zu erhalten. Beobachtungen an alternierenden dicken und dünnen Schnitten zeigten eine Zelle mit Körnern ähnlich den Granulen von Maus Paneth-Zellen. Das dichte Zentrum dieser Granula war osmiophil, der hellere Halo alcyanophil.Diese Resultate lassen uns schließen, daß die elektronen-durchsichtige filamentöse Komponente ein alcyanophiles Mukopolysaccharid ist; das dichte, zentrale Material ist wahrscheinlich PAS-positiv.

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This paper was prepared from a thesis submitted in partial fulfillment for the degree of Master of Arts.

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This work was supported by the National Institutes of Health grant no. GM-11218.

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I wish to thank Dr.Marcus Singer for permission to use the E. M. facilities in the Dept. of Anatomy, Case Western Reserve University, and Dr.Joseph A.Grasso for instruction in the techniques of electron microscopy and the use of his facilities.