Distribution and action of some putative neurotransmitters in the stomatogastric nervous system of the earthworm, Eisenia fetida (Oligochaeta, Annelida).

The chemical neuroanatomy of the stomatogastric nervous system of the earthworm, Eisenia fetida, has been investigated, using antibodies raised against serotonin, tyrosine hydroxylase, octopamine, GABA, FMRFamide, proctolin, Eisenia tetradecapeptide and neuropeptide Y. Neurons immunoreactive to these antibodies can be observed in the stomatogastric ganglia. The labelled cells comprise altogether 95.4% of the total number of neurons in the ganglion. Immunoreactive projections were followed between stomatogastric individual ganglia as well as towards the enteric plexus. Intrinsic neurons containing the different signal molecules examined are present along the entire length of the enteric plexus, but serotonin immunoreactive perikarya were only found in the hindgut. The density of the different immunoreactive neurons, except the serotonin ones, is highest in the pharyngeal plexus, and the number of labelled neurons decreases along the alimentary canal towards the hindgut. A number of epithelial cells also reveal tyrosine hydroxylase, octopamine, GABA and Eisenia tetradecapeptide immunoreactivity. The action of some putative neurotransmitters, such as dopamine, octopamine, serotonin and proctolin was tested on foregut preparations. Dopamine and octopamine (10(-6)-10(-4) M) have an excitatory effect on the musculature, whereas the effect of serotonin depends on the actual muscle tension. Following precontraction evoked by acetylcholine, serotonin in low concentrations (10(-7)-10(-6) M) causes relaxation, whereas in higher (10(-4) M) concentration it evokes slight contractions. In preparations at basal tone, serotonin (10(-7)-10(-6) M) evokes contractions of the foregut. Atropine strongly inhibits the action of acetylcholine but is ineffective against serotonin, dopamine and octopamine. Similarly, the Na+ channel blocker tetrodotoxin fails to influence the contractile effect of dopamine, octopamine and serotonin. These results suggest that dopamine, octopamine and serotonin act directly on the muscle cells of the alimentary tract. Proctolin do not evoke any significant effect on the foregut.

Distribution of Eisenia tetradecapeptide immunoreactive neurons in the nervous system of earthworms.

A detailed mapping of Eisenia-tetradecapeptide-immunoreactive neurons in the central and peripheral nervous system combined with quantitative morphological measurements was performed in Eisenia fetida and Lumbricus terrestris. In Eisenia, most labelled neurons were observed in the ganglia of the ventral cord (20.38% of the total cell number of the ganglion) and 15.67% immunoreactive cells occurred in the brain, while 6% of the neurons could be shown in the subesophageal ganglion. In the case of Lumbricus, most immunoreactive cells were found in the subesophageal ganglion (16.17%) and in the ventral ganglia (12.54%). The brain contained 122 ETP-immunoreactive cells (5.6%). The size of the immunoreactive cells varied between 35-75 microm. A small number of Eisenia-tetradecapeptide immunoreactive fibres were seen to leave the ventral ganglia via segmental nerves, and labelled processes could also be observed in the stomatogastric system and the body wall. Labelled axon branches originating from the segmental nerves formed an immunoreactive plexus both between the circular and longitudinal muscle layer and on the inner surface of the longitudinal muscle layer. This inner plexus was especially rich in the setal sac. Among the superficial epithelial cells the body wall contained a significant number of immunoreactive cells. Only a few Eisenia-tetradecapeptide immunoreactive neurons and fibres occurred in the stomatogastric ganglia. In the enteric plexus the number of immunoreactive neurons and fibres decreased along the cranio-caudal axis of the alimentary tract. Eisenia-tetradecapeptide immunoreactive cells were also present among the epithelial cells in the alimentary canal. Some of these cells resembled sensory neurons in the foregut, while others showed typical secretory cell morphology in the midgut and hindgut.

Reorganization of peptidergic systems during brain regeneration in Eisenia fetida (Oligochaeta, Annelida).

After the extirpation of the brain reorganization of the peptidergic (FMRFamide, neuropeptide Y, proctolin) systems was studied in the newly forming cerebral ganglion of the annelid Eisenia fetida. During regeneration, all immunoreactive fibres appear on the 1st-2nd postoperative day. At the beginning of regeneration, immunoreactive neurons and fibres form a mixed structure in the wound tissue. On the 3rd postoperative day, FMRFamide positive and neuropeptide Y-immunoreactive, while on the 7th postoperative day proctolin-immunoreactive neurons appear in the loose wound tissue. From the 25th postoperative day a capsule gradually develops around it. The neurons of the preganglion move to the surface of the newly appearing preganglion. The number of these cells gradually increase, and by the 72th-80th postoperative days the localization and number of peptide-immunoreactive neurons is similar to that in the intact one. The neurons of all examined peptidergic systems may originate from the neuroblasts, situated on the inner and outer surface of the intact ganglia (e.g. suboesophageal and ventral cord ganglia). In addition FMRFamide and proctolin immunoreactive neurons may take their derive by mitotic proliferation from the pharyngeal neurons, too.

Octopamine-containing neurons in the alimentary tract of the earthworm (Eisenia fetida).

Octopamine-containing nerve cells have been demonstrated in the enteric plexus of the earthworm (Eisenia fetida), applying immunocytochemistry and HPLC assay. A few octopamine-immunoreactive neurons occurred in the fore- and hindgut, whereas their number in the midgut was considerably greater. Octopamine levels detected by HPLC correlated with the distribution of octopamine-containing nerve cells. A regulatory role for these intrinsic octopaminergic neurons is suggested in the enteric plexus in the earthworm alimentary tract. This is the first report on the occurrence of octopamine-containing nerve cells in the peripheral nervous system of an invertebrate.

The number of ganglion cells in the intact and regenerated nervous system in the earthworm (Lumbricus terrestris).

1. The nerve cell number is higher in the cerebral ganglion than in the subesophageal or segmental ganglia of Lumbricus terrestris. 2. The nerve cell number of the subesophageal ganglion is similar to that of the segmental ganglia in spite that the latter developed from a fusion of several ganglia. 3. Segmental ganglia in front and behind the clitellum can be considered as "typical" ganglia. 4. The most caudal segmental ganglia are less differentiated and resembles to the embryological state. 5. Nerve cells of the regenerating ganglia, at least in part, originate from undifferentiated nerve cells of the intact ganglia.

Proctolin immunoreactive elements in the nervous system of earthworm (Lumbricus terrestris).

Proctolin (H-Arg-Try-Leu-Pro-Thr-OH) immunoreactive elements in the nervous system of earthworm (Lumbricus terrestris, L.) were detected in paraffin sections using peroxidase-antiperoxidase method. Each part of the central nervous system contains proctolin immunoreactive cell bodies. The nerve cells, according to their locations are motoneurons, sensory neurons as well as interneurons. A part of the sensory epithelial cells of the body surface are proctolin immunoreactive, too. The body wall muscles receive a rich proctolin immunoreactive network. It is supposed that proctolin acts as a neuromodulator in the nervous system of Lumbricus terrestris.

Immunohistochemical and ultrastructural study of the enteric nervous system of earthworm, Lumbricus terrestris L.

Light and electronmicroscopic data reveal the presence of a well developed nerve plexus in the gut of the earthworm. The plexus contains subepithelial solitary nerve cells and fibers and an extensive neuropil among the muscle cells. There are two types of nerve cells in the enteric plexus. The first type contains mainly dense-core vesicles, and exhibits glyoxylic-acid induced fluorescence. Since none of these cells showed serotonin immunoreactivity, they are probably noradrenergic or dopaminergic. The second type contains large dense granules, suggesting that these cells are peptidergic (neurosecretory). A part of these cells are substance P immunoreactive, however no NPY, CGRP, or proctolin immunopositive cells were found. Ultrastructurally seven types of nerve fibers can be distinguished in the neuropil. Their distribution shows great variability within parts of the enteric canal. The observation that only two types of nerve cells are located within the gut makes it probable that some of the axons are extrinsic. According to immunohistological studies they may come from the stomatogastric system or from the segmental nerves. This is further supported by the fact that there is a well-developed subepithelial serotoninergic plexus in the fore-gut. Two types of neuromuscular junctions can be visualized in the muscular layer. The first type, representing a phylogenetically earlier form, exhibits wide junctional gap and pre- or postjunctional membrane thickening. The second type is the close contact. There are significantly more junctions observed in the fore-gut than in other parts of the gut.

Immunohistochemical study of the nervous system in earthworm (Lumbricus terrestris L.).

In the present work it we describe serotonin, noradrenaline, proctolin, neuropeptide Y, substance P, and calcitonin gene related peptide immunoreactive structures in earthworm. A few large serotonin immunoreactive perikarya are located in brain and in the stomatogasric ganglia and many of them in the segmental ganglia. A serotonin immunoreactive fiber plexus can be seen beneath the epithelium of the body wall, both the sensory papillae and chaetae contain serotonin immunoreactive elements. Some of the sensory cells are serotonin immunoreactive, too. A serotonin immunoreactive network can ben found in the enteric network of the fore- and mid-gut. Only a few noradrenaline immunoreactive cells are observed in the caudal part of the brain, while their number in the segmental ganglia is high. Proctolin-, and substance P immunoreactive cells are small and numerous in the brain without any preference in their location. Such nerve cells are widely distributed in the ganglia of the stomatogastric system, in the subesophageal and in segmental ganglia. Many surface epithelial (probably sensory) cells are proctolin immunoreactive. Substance P immunoreactive nerve cells can also be located in the entire length of the enteric plexus together with substance P immunoreactive fibers. No neuropeptide Y- or calcitonin gene related peptide immunoreactive structures can be seen in the brain. A relative small number of neuropeptide Y- and calcitonin gene related peptide immunoreactive perikarya, and a rich network of neuropeptide Y- and calcitonin gene related peptide immunoreactive fibers can be detected in the subesophageal ganglion. According to preliminary studies, neuropeptide Y is probably co-localized with serotonin.

Some morphological and histochemical features of the midgut myenteric plexus of the common European frog, Rana esculenta.

The neuron morphology and distribution of four putative transmitters were investigated in the myenteric plexus of frog (Rana esculenta) midgut. The gross morphology was revealed by NADH-diaphorase histochemistry, and the shape of the neurons by silver impregnation. Nerve cells had heterogeneous distribution: they either formed ganglia or placed as solitary neurons in the duodenum, while in the rest of the midgut only solitary neurons were observed. Three morphologically distinct cell types were revealed by silver impregnation: mainly type I and type II neurons cells were seen in the duodenum, while the rest of the intestine contained type II and III cells. Catecholamine fluorescence was revealed in nerve fibres in the duodenum, while few small nerve cells were observed in the small intestinal region. Acetylcholinesterase histochemistry showed strongly reactive nerve cells that were associated with the main fibre bundles in the duodenum. Only longitudinally oriented fibres and occasionally stained neurons were seen in the small intestine. Substance P immunocytochemistry revealed an extensive plexus, which contained a moderate number of stained perikarya in the full length of the midgut. Gamma-aminobutyric acid showed non-uniform distribution in the two parts of the midgut: a stronger and more regular fibre staining was found in the duodenum then in the rest of the intestine. Ultrastructural observations demonstrated that intrinsic neurons received synaptic inputs from the profiles contained agranular vesicles, while "P"-type profiles established close contacts with neurons. Both profile types formed close contacts with the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical characterization of myenteric plexus in domestic fowl small intestine.

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.

Myenteric plexus of frog large intestine: light and electron microscopy of fiber system and neurons.

The myenteric plexus of the frog large intestine was studied by means of silver impregnation, NADH-diaphorase histochemistry and electron microscopy. The main fiber bundles of the myenteric plexus are situated between the longitudinal and circular smooth muscle layers. Most of the branches of this main plexus could be followed into the circular muscle layer. The diameter of the large nerve bundles was demonstrated by impregnation to be 7-8 microns. NADH-diaphorase histochemistry revealed a random distribution of the nerve cells; the nerve cells did not form ganglia. The maximal profile of these mostly multipolar cells was 75-100 microns 2. The packing density of nerve cells was 710 +/- 23 per cm2. At the electron microscopic level, the plexus consisted of a number of unmyelinated nerve profiles, a few myelinated axons, Schwann cells and neurons. The proportion of myelinated fibers did not exceed 1%. The nerve profiles contained vesicles, small agranular vesicles or mixed vesicle populations. Synapses were extremely rare in the neuropil. The possibility and importance of non-synaptic transmitter release and presumed transmitter substances are discussed.

Fine structure of the neuromuscular junctions in the alimentary tract of phylogenetically different animal species.

Authors studied the fine structural characteristics of the neuromuscular junctions in the alimentary tract of phylogenetically different animal species. Nearly in each studied species the so-called close contacts were observable, where the sarcolemma and axolemma establish a junction; the gap of this contact is 10-100 nm wide, and the neurotransmitters can affect the muscles through non-synaptic release (exocytosis). The junctional gap is widest in the gut wall of earthworm: 100-200 nm. Only close contacts are to be found in the alimentary canal of snail, and here, the junctional gap is as narrow as 10-15 nm. Beside close contacts synaptic neuromuscular junctions also occur in the locust gut. Their fine structural organization refer to that of chemical synapses. As in the tench intestine both striated and smooth muscular elements can be found, motor endplates as well as close contacts take part in their innervation. Only close contacts are in the smooth muscular layer of domestic fowl. Authors emphasize the role of close contacts in the regulation of gut peristalsis and only secondary importance is attached to other different junctions.