Ultrastructural localization of monoamines in the epidermis of Lumbricus terrestris (L.)

Summary Receptor cells in the epithelium and the basiepithelial nerve net of the prostomium of Lumbricus terrestris were investigated with electron microscope with special regard to the presence of monoamines. The receptor cells are found in groups of about 40 intermingled with supportive cells. After pretreatment with -methyl-noradrenaline and fixation with potassium permanganate a few receptor cells in each group and some nerve fibres in the basiepithelial nerve net contain small granular vesicles (about400 Å) characteristic for monoaminergic neurons. The distribution and relative number of these receptor cells and nerve fibres coincide well with previous reports on fluorescent receptor cells and varicose fibres. That the monoamine-storing small granular vesicles not are visualized until pretreatment with -methyl-noradrenaline is in accordance with recent microspectrofluorometric analysis, which shows that dopamine is the only primary monoamine present in the epithelium.In the epithelium there are occasional receptor cells and nerve fibres containing large vesicles (1000–1800 Å) which resemble the neurosecretory vesicles in the central nervous system. Photoreceptor cells having an intracellular cavity with microvilli and cilia have infrequently been observed at the base of the epithelium.No synapses on the mucous cells have been noticed. Nor have any synaptic specializations been observed in the basiepithelial nerve net. The morphological conditions necessary for the existence of possible axo-axonal synapses are briefly discussed.This work was supported by grants from the Helge Ax: son Johnson Foundation and the Magn. Bergvall Foundation.     

Fine structure of photoreceptor cells in the earthworm,Lumbricus terrestris

Summary Photoreceptor cells in the epidermis and nerve branches of the prostomium and in the cerebral ganglion of Lumbricus terrestris were investigated with the electron microscope. The photoreceptor cell is similar to the visual cell of Hirudo by having a central intracellular cavity (phaosome) filled with microvilli. Besides microvilli, several sensory cilia can also be found in the phaosome but they are structurally independent of the microvilli. A gradual branching of the phaosome cavity into smaller cavities makes its sectional profile extremely labyrinthic. Flattened smooth-surfaced cisternae in stacks of 2 to 5 are frequently observed around the phaosome. Characteristic constituents of the cytoplasm are vesicles and vacuoles filled with a substance of varying density. The photoreceptor cell is covered by glial cells or by their processes which at many places deeply invaginate the cell surface (trophospongium).

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Zusammenfassung Die Feinstruktur der Photorezeptorzellen in der Epidermis, in kleineren Nervenästen und im Zerebralganglion von Lumbricus terrestris wurde untersucht. Das Vorhandensein eines zentralen, intrazellulären Lumens (Phaosom), das mit Mikrozotten gefüllt ist, erinnert in der Struktur der Photorezeptorzelle des Regenwurms an Lichtsinneszellen von Hirudo. Außer Mikrozotten findet man im Phaosom einige Zilien vom Typ 9×2+0; solche Zilien sind von den Mikrozotten strukturell unabhängig. Durch eine Aufzweigung des Phaosoms in kleinere Buchten, die tief in das umgebende Zytoplasma eindringen, erhält es ein labyrinthartiges Aussehen.Glatte Zisternen in Gruppen von 2 bis 5 wurden oft um das Phaosom im Zytoplasma beobachtet. Charakteristische Bestandteile der Zelle sind noch Vesikel und Vakuolen, die mit einer Substanz von wechselnder Elektronendichte gefüllt sind. Die Photorezeptorzellen werden von Gliazellen und Gliafortsätzen umgeben, die an vielen Stellen die Zelloberfläche tief einstülpen (Trophospongium).

     

Histochemical localization of production and secretion of trypsin in the earthworm Lumbricus terrestris

Summary The origin of trypsin in the digestive tract of Lumbricus terrestris was localized by using the chromogenic tryptic substrate carbobenzyloxy-L-arginine--napthylamide HCl (CANA) coupled with the azo dye Fast Garnet GBC. The specificity of earthworm trypsin toward the naphthylamide substrate was confirmed by disc gel electrophoresis of homogenates of the digestive tract and of intestinal fluid. Eluted fractions were assayed for tryptic activity using the synthetic substrates benzoyl-DL-arginine-p-nitroanilide (BAPA) and p-toluenesulfonyl-L-arginine methyl ester (TAME). The peak of activity toward these substrates corresponded in electrophoretic mobility to the band of CANA activity and all activities were abolished in the presence of the tryptic inhibitor N-tosyl-L-lysyl chloromethane HCl (TLCK).In frozen sections of the pharynx, esophagus, crop, gizzard and intestine, tryptic (CANA) activity was localized consistently only in the ventral and lateral fold epithelium of the pharynx. The chromogenic reaction was completely inhibited by pre-incubation of frozen sections with TLCK.Tissues from three regions of the pharynx were fixed and studied in an attempt to correlate electron microscopic observations with the histochemical results. Whereas mucousproducing cells are generally distributed in pharyngeal epithelium, heterogeneous spherical granules were found only in the ventral and lateral fold epithelium. Observations concerning the spherical secretory granules closely paralleled those of the histochemical reaction product suggesting that the spherical secretory granules may contain the tryptic enzyme.The authors wish to thank Ruth D. Merz and Sandra K. Elson for their technical assistance. The investigation was supported in part by funds to the Instrumentation Laboratories and from the Faculty Research Committee of Miami University.     

Patterns of serotonin-immunoreactive neurons in the central nervous system of the earthworm Lumbricus terrestris L.

Summary The distribution patterns of serotonin-immunoreactive somata in the cerebral and subpharyngeal ganglion, and in the head and tail ganglia of the nerve cord of Lumbricus terrestris are described from whole-mount preparations. A small number of serotonin-immunoreactive neurons occurs in the cerebral ganglion, in contrast to the large population of serotonin-immunoreactive neurons that exists in all parts of the ventral nerve cord. From the arrangement of serotonin-immunoreactive somata in the subpharyngeal ganglion, we suggest that this ganglion arises from the fusion of two primordial ganglia. In head and tail ganglia, the distribution of serotonin-immunoreactive somata resembles that in midbody segments. Segmental variations in the pattern and number of serotonin-immunoreactive somata in the different body regions are discussed on the background of known developmental mechanisms that result in metameric neuronal populations in annelids and arthropods.Abbreviations CG1, CG2 cerebral soma group 1, 2 - CNS central nervous system - GINs giant interneurons - 5-HT 5-hydroxytryptamine, serotonin - 5-HTi 5-HT-immunoreactive - N side nerve - SG19 subpharyngeal soma group 1–9 - SN segmental nerve     

Fine structure of isolated fibrils from the cuticle of Lumbricus sp.

A method has been developed to isolate and purify cuticular fibrils of Lumbricus. Polarizing microscopy confirms the collagenous nature of the isolated fibrils. Study in the electron microscope of isolated fibrils, negatively or positively stained, shows that they are cylindrical, unbranching and without periodic structure. Enzymatic treatment of cuticles with alpha-amylase and trypsin results in a more or less complete dissociation of the fibrils which appear clearly to be made up of helically wound bundles of filaments (30-40 A). The biophysical data and compared to the ultrastructural organization of other periodically cross-banded fibrils.     

Localization of monoamine-containing neurones in the nervous system ofStrophocheilus oblongus (Gastropoda)

Summary The histochemical fluorescence method of Falck and Hillarp was used to locate monoamine-containing neurones in the nervous system ofStrophocheilus, a Brazilian land snail. Green-fluorescing (catecholamines) cell bodies were found only in cerebral ganglia. Neuropiles of all ganglia contained many green-fluorescing fibers most of which appeared to enter from the periphery. Giant (up to 500 ) non-fluorescing cells were seen in most ganglia of the suboesophageal mass. Green-fluorescing fibers surrounded, and apparently ended on, their axon hillocks and proximal segments of axons. Green-fluorescing sensory cells were seen in the eyeless tentacles. Cells fluorescing yellow (5-hydroxytryptamine) or yellow fading to green on exposure to ultraviolet light were found in cerebral, visceral and right palliai ganglia. Ligature of the cardiac nerve for 24 hrs resulted in an accumulation of yellow or yellow green-fluorescing masses in axons proximal to the constriction. The distribution of monoamine-containing neurones inStrophocheilus is rather similar to that found in other pulmonate molluscs such asHelix.The authors are eager to acknowledge the constant interest of Dr. Sonia Rude while the experiments were taking place. This work was jointly supported by OAS, Capes (Brazil), U. F. R. G. S. (Brazil), and Grant No. NB 00623 (USPHS).     

Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.)

Summary The localization and intraneuronal distribution of the monoaminergic transmitters in the nervous system of the earthworm, Lumbricus terrestris, have been investigated in detail with the aid of the histochemical fluorescence method of Falck and Hillarp.In the ventral nerve cord, many yellow fluorescent, 5-hydroxytryptamine containing neurons are found, but only few green fluorescent noradrenaline containing cell bodies, which, however, are numerous in the peripheral nervous system. There is an abundance of both fibre types in the neuropile.The 5-hydroxytryptaminergic neurons probably have a motor (possibly inhibitor) function; the adrenergic neurons in the body segments are supposed to have a receptor (exteroceptive and possibly proprioceptive) function.In the cerebral ganglion, both 5-hydroxytryptamine and noradrenaline containing neurons are found in large numbers, and there are closely packed numerous fibres of both types in the neuropile. Their function is more obscure, though an associative function can be presumed for some adrenergic neurons; smaller 5-hydroxytryptaminergic neurons might have a motor (perhaps inhibitor) function.Adrenergic sensory cells are found in the body integument, most frequently in the clitellum segments, in the prostomium, and in the roof of the buccal cavity. These cells give off varicose fibres that form a basi-epithelial network which is in communication with the green fluorescent sensory fascicles in the ventral nerve cord via the epidermal nerves, the ring nerves, and the segmental nerves. No direct adrenergic sensory-effector innervation of either circular and/or longitudinal musculature or gland cells seems to exist. No adrenergic free nerve endings in the body integument have been observed. Instead, there must be a synaptic contact with the motoneurons, either directly in the neuropile or via an interjacent neuron.No synaptic contacts have been observed in the ventral nerve cord between adrenergic or 5-hydroxytryptaminergic fibres and either the giant fibres or fluorescent or nonfluorescent perikarya.An adrenergic innervation of the pharynx musculature has been found, and sensory cells of a different type are present in and below the epithelium; here, a direct senso-motoric innervation of the pharyngeal musculature cannot be excluded. It is established that the adrenergic neurons in the stomatogastric nervous system have an exciting function on the pharynx, whereas a direct monoaminergic influence of the muscular movements of the intestine probably does not exist.Abbreviations Used A adrenaline - CA catecholamine - DA dopamine - 5-HT 5-hydroxytryptamine - MA monoamine - NA noradrenaline The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the Swedish Natural Science Research Council (99-34, 6627), and by the Swedish Medical Research Council (B67-12X-712-02A).     

Ultrastructural studies on neurophysine-containing vesicles of the neurosecretory system of vertebrates

Summary A new method described in the present paper allows identification of neurophysine-containing (NP) vesicles of the magnocellular neurosecretory system of vertebrates. After oxidation of ultrathin sections, the content of NP vesicles can be dissolved specifically in an alkaline medium. This procedure marks NP vesicles selectively in all portions of the classical neurosecretory system.Dedicated to Professor Dr. Dres h.c. W. Bargmann on the occasion of his 70^th birthday.Granted by the Ministerium für Wissenschaft und Technik der DDR.     

An ultrastructural in vitro study on the regulation of neurosecretory activity in the freshwater snail Lymnaea stagnalis (L.) with particular reference to Caudo-dorsal cells

The neurosecretory Caudo-Dorsal Cells (CDC) in the cerebral ganglia of the freshwater pulmonate snail Lymnaea stagnalis produce an ovulation stimulating hormone. Previously it has been shown that neuronal and non-neuronal inputs are involved in the regulation of their activity. The degree of autonomy of these cells has been investigated by studying with morphometric methods the ultrastructure of CDC maintained in vitro. CDC of isolated cerebral ganglia which were cultured for 7 days show a considerable rate of synthesis, transport and release of neurohormone. Apparently these processes can proceed in the absence of neuronal and hormonal inputs from outside the cerebral ganglia. Completely isolated CDC, however, do not show neurosecretory activity in vitro; active Golgi zones, indicating the formation of neurosecretory elementary granules, are absent from such cells. Isolation does not seem to affect general cell functions such as protein synthesis and respiration. It is suggested that a neuronal input, originating within the cerebral ganglia, is necessary for the stimulation of CDC neurosecretory activity. Techniques are described for the isolation and culture of neurosecretory cells of L. stagnalis.     

Neuronal and non-neuronal control of the neurosecretory Caudo-Dorsal cells of the freshwater snail Lymnaea stagnalis (L.)

The cerebral ganglia of the freshwater snail Lymnaea stagnalis contain two clusters of neurosecretory Caudo-Dorsal Cells (CDC). These cells produce a neurohormone which stimulates ovulation. Ganglion transplantation and quantitative electron microscopy show that neuronal isolation of the cerebral ganglia complex (CCC) results in an activation of the CDC. It was, therefore, concluded that the CDC are controlled by an inhibitory neuronal input originating outside the cerebral ganglia. Ultrastructural studies on synaptic degeneration in the CCC suggest that this input reaches the CDC via a special type of synapse-like structure, the type C-SLS.Furthermore, transplantation of CCC into acceptor snails leads to a reduced release and an increased intracellular breakdown of neurohormone in the CDC of the nervous system of the acceptors. It is supposed that these phenomena are caused by the release of an (unknown) factor from the transplanted CCC. Special attention was given to the formation and degradation of a peculiar type of neurohormone granule, the large electron dense granule.     

Morphometric in vitro analysis of the control of the activity of the neurosecretory dark green cells in the freshwater snail Lymnaea stagnalis (L.)

Summary The intracellular localization of calcium by means of cytochemical techniques was studied in smooth muscle cells of mouse intestine. When the lead acetate method according to Carasso and Favard (1966) was used calcium was found in mitochondria and sarcoplasmic reticulum and occasionally between the myofilaments. The active ATP-dependent accumulation of calcium into cell structures was investigated by the oxalate method (Heumann and Zebe, 1967). After appropriate treatment the only structures of smooth muscle cells which contained calcium oxalate (identified by microprobe analysis) were elements of the sarcoplasmic reticulum.The results are discussed in relation to the role of calcium in the control of muscle activity during the contraction-relaxation cycle.The electron probe microanalysis was carried out at SIEMENS (Berlin) in collaboration with Dr. von Muschwitz. I thank Miss M. Schlatter for her skillful assistance. The investigation was supported by the Deutsche Forschungsgemeinschaft.     

Immunoreactivity to molluskan neuropeptides in the central and stomatogastric nervous systems of the earthworm, Lumbricus terrestris L.

Polyclonal antisera against two related command neuropeptides (CNP2 and CNP4) described in neurons of the terrestrial snail Helix were used in a study of the nervous system of the earthworm Lumbricus. The CNP-like peptides belong to the same neuropeptide subfamily and bear a C-terminal signature sequence Tyr-Pro-Arg-X. The distribution patterns of immunoreactive (IR) neurons were studied in the central nervous system (CNS), skin, and stomatogastric nervous system of the earthworm. IR neurons were found in all CNS ganglia, the patterns being similar for both antibodies used. Several clusters of IR cells were observed in the cerebral and subesophageal ganglia. In the ventral cord ganglia, the number of IR cells decreased in the rostro-caudal direction, and the IR cells sent their fibers mostly into the median fiber bundle. Segmental nerves contained no IR fibers. After injury of the worm body, the number of IR neurons in the CNS significantly increased. In the skin, IR sensory neurons were present in sensory buds. The stomatogastric ganglia only contained IR fibers. Numerous scattered IR neurons were found in the inner subepithelial layer of the esophagus and formed the enteric plexus in which the cell bodies displayed a segmentally repeated pattern. Possible involvement of CNP-like-IR neurons in central integratory processes, sensory processes, and the regulation of feeding is discussed.This work was supported by INTAS (grant 01-2117), CRDF (grant RB1-2321-MO-02), and the Russian Foundation for Basic Research (grants 05-04-48724 and 03-04-48179).     

Fine structural analysis of the basal epidermal receptor cells in the earthworm (Lumbricus terrestris L.)

Summary A fine structural investigation was performed on receptor cells lying at the base of the epidermis in the earthworm, Lumbricus terrestris. Two types of receptor cells with many similarities, but also with major differences, were discriminated.One receptor is of the microvillar receptor type, that appears to be identical with the photoreceptor cell described earlier by Röhlich et al. (1970). Proximal to the nucleus is a large vacuole (phaosome, Binnenkörper) with many daughter cavities containing a large number of microvilli and several cilia with the 9 × 2 + 0 microtubular pattern. The intracellular cavity has no connection with the surface membrane, in contrast to that in hirudineans (White and Walther, 1969) and pogonophores (Nørrevang, 1974).The other receptor is the ciliated receptor type, that is presently described for the first time. This receptor also has a comparatively large uniform cavity, few microvilli and about 20 cilia with the 9 × 2 + 2 microtubular pattern. The cilia leave the cell in the proximal part through a wide opening, make a turn of 180 °, and proceed toward the epidermal surface. Receptors of a similar type have been described by Golding and Whittle (1975) in the cerebral ganglion of four limicole oligochaete annelids; they presumed that these cells have an osmoreceptor function. The new epidermal receptor type described in the present investigation probably has a chemoreceptor function of hitherto unknown kind.This investigation was supported by the Royal Physiographic Society at Lund, Sweden. The author would like to express his thanks to Mrs. Lena Sandell for skilful technical assistance     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

Immunocytochemical studies on the central nervous system of the earthworm, Lumbricus terrestris

There are numerous aldehyde fuchsin (AF)-positive, neurosecretory cells of medium size (A cells) and a small number of large, AF-negative neurons (B cells) in the cortical layer of the cerebral ganglion. In the subesophageal ganglion, symmetrical groups of AF-positive cells lie ventrally. The peroxidase--antiperoxidase (PAP) method was used for the immunocytochemical study of substance P and ACTH in these ganglia. In addition, the presence of L-enkephalin and alpha endorphin could be confirmed. Using rabbit antibodies to substance P we found small immunoreactive neurons among negative A and B cells in the cerebral ganglion. The processes of these immunoreactive cells could be traced to the subcortical synaptic neuropil. With antibodies to ACTH, activity was visible in perikarya similar in size to A neurons. A part of the nerve terminals of the synaptic zone, some of the B neurons and further several nerve cells of the subesophageal ganglion reacted positively. Successive demonstration of substance P and ACTH on the same section showed that the two materials occurred in different cell types. Using antiopsin antibody in an indirect immunocytochemical test we observed strong reaction in numerous medium-sized perikarya and in nerve fibres of the synaptic zone of the cerebral ganglion, further in some neurons of the subesophageal and abdominal ganglia. In contrast to this result, the photoreceptor cells of the prostomium and cerebral ganglion were negative. Presumably, substance P is present in a perikaryon type hitherto unrecognized while ACTH and antiopsin reactions seem to be located first of all in A cells.     

Multisegmental cobalt filling of the dorsal giant fibers in the nervous system of the earthworm,Lumbricus terrestris

Summary The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.Abbreviations CL collateral of lateral giant fiber - CM collateral of medial giant fiber - GIN giant interneuron - LGF lateral giant fiber - MGF medial giant fiber - SN segmental nerve