Glial cells in the central nervous system of earthworm, Eisenia fetida.

Glial elements in the central nervous system of Eisenia fetida were studied at light- and electron microscopic level. Cells were characterized with the aid of toluidine blue, Glial Fibrillary Acidic Protein (GFAP), S100 staining. We identified neurilemmal-, subneurilemmal-, supporting-nutrifying- and myelinsheath forming glial cells. Both neuronal and non-neuronal elements are S100-immunoreactive in the CNS. Among glial cells neurilemmal and subneurilemmal cells are S100-immunopositive. With the antibody against the S100 protein one band is visible at 15 kDa. GFA P-immunopositive supporting-nutrifying glial cells are localized around neurons and they often appear as cells with many vacuoles. GFA P-positive cell bodies of elongated neurilemmal glial cells are also visible. Western blot analysis shows a single 57 kDa GFA P immunoreactive band in the Eisenia sample. At ultrastructural level contacts between neuronal and glial cells are recognizable. Glial cell bodies and their filopodia contain a granular and vesicular system. Close contacts between neuronal cell membranes and glial filopodia create a special environment for material transport. Vesicles budding off glial cell granules move towards the cell membranes, probably emptying their content with kiss and run exocytosis. The secreted compounds in return may help neuronal survival, provide nutrition, and filopodia may also support neuronal terminals.

Correlative morphometric and electrochemical measurements of serotonin content in earthworm muscles.

Distribution of serotonin (5-HT) content of nervous fibers in both the somatic and the visceral muscle of Eisenia fetida have been investigated using immunocytochemical staining and voltammetric measurements. The somatic muscles in the body wall are richer innervated with serotoninergic fibers than the visceral ones in the pharynx and gizzard. The relative density of immunopositive fibers in the circular muscle layer of the body wall was found to be 2.73% while in the prostomium it was 1.02%. In the case of the muscle in pharynx 1.12% and in gizzard 1.28% density values were found. Differential Pulse Voltammetric (DPV) measurements with carbon fiber electrodes in the above mentioned muscle layers gave 272.5 nA, 135.0 nA, 122.5 nA, 137.5 nA peak heights, respectively. In the statistical analysis T-test was used at a confidence level of 95% (p<0.05). DPV current peak (i(p)) values reflect clearly the 5-HT concentration differences. Significant correlation was found between the innervation density and the i(p) values recorded in different areas. The i(p) values recorded at different times in different locations are determined by instantaneous serotonin concentration of the living tissue. As far as we know this is the first report using in vivo voltammetry investigating serotonin content in earthworm, E. fetida.

Embryogenesis of the serotonergic system in the earthworm Eisenia fetida (Annelida, Oligochaeta): immunohistochemical and biochemical studies.

Organization of the serotonergic system and changes of the serotonin (5-HT) content were studied during the embryogenesis of the earthworm Eisenia fetida, using immunocytochemistry and HPLC. A gradual emergence of 5-HT immunoreactive (IR) cells and their axon projections in the several ganglia of the central (CNS) and peripheral nervous system are described in the context of a staged time-scale of development. The first 5-HT-IR neurons appear in the subesophageal ganglion at an early embryonic stage (E2), followed by neurons in some rostrally located ventral ganglia. In the cerebral ganglion, 5-HT-IR cells can be detected only from stage E5. The number of labeled cells in each ganglion of the embryo increases until hatching, when it is still considerably lower than that observed in adults. This shows that the development of the 5-HTergic system is far from complete by the end of embryogenesis. Organization of 5-HT-IR innervation of the body wall starts by stages E3 to E4. In the stomatogastric nervous system the first 5-HT-IR fibers can be detected by stage E5. By stage E9 5-HT immunopositive neurons can be observed in both the stomatogastric ganglia and the enteric plexus. Both 5-HT levels and the numbers of the labeled cells show a significant increase before hatching, which indicate a functional maturation of the 5-HTergic system. Based on the early appearance of 5-HT, we suppose that it may play a regulatory role in both the gangliogenesis and the maturation of peripheral functions necessary during postembryonic life.

Embryogenesis of GABAergic elements in the nervous system of Eisenia fetida (Annelida, Oligochaeta).

The appearance and development of the GABA-immunoreactive nervous elements in the central nervous system of Eisenia fetida were studied by immunocytochemistry. The nervous system originates from the neuroectoderm situated on the ventral side of the embryo. The organization of the circumpharyngeal ring starts earlier than that of the ventral cord. In the elementary ring the first GABA-immunopositive neurons can be observed (E1 stage) around the mouth. Later the cell number gradually increases and parallel to this process the elementary ring is separeted into a superficial and a deeper portion. The brain and the subesophageal ganglion will be organized from the superficial ring, while the nervous elements of the deeper ring will give rise for the first GABA-immunoreactive elements of the stomatogastric nervous system. In the early stages of the embryogenesis the immunoreactive cells of the developing brain appear solitary, while from the stage E4 they gradually are observed in groups. According to their position, these cell groups are similar to those observed in the brain of the adult earthworms. During embryogenesis the level of the ventral cord ganglia depends on their position in the ectodermal germ bands. It means, that the more organized ganglia are near the circumpharyngeal ring, mean while less developed ganglia are situated caudally from them. By the end of the embryogenesis all ganglia of the ventral cord will be equally well organized. The nerve tracts of the ganglia are built up from contra- and ipsilateral by projected fibres. From E3 stage the medial tracts, mean while from the E4 stage the lateral tracts begin to be formed. During the next stages, more and more fibres connect to the both tracts. At hatching, the development of the central nervous system of Eisenia fetida is not completed, the process is continued during the postembryonic development.

Reorganization of the GABAergic system following brain extirpation in the earthworm (Eisenia fetida, Annelida, Oligochaeta).

The reorganization of the GABAergic system was studied by means of immunohistochemistry after the symmetrical and asymmetrical (unilateral) extirpation of the brain of the annelid Eisenia fetida. GABA-immunoreactive neurons were first observed in the wound tissue on the 3rd postoperative day. Thereafter the number of labelled cells gradually increased, and by postoperative days 76-80 all GABA-immunoreactive cells (approx. 140 neurons) could be found in their final positions in the symmetrically regenerated brain. After asymmetrical brain extirpation, nearly all cells (70-75) could be detected in the regenerating hemisphere by postoperative days 50-56. In the early stages of the asymmetrical regeneration of the brain, more GABAergic cells were concentrated dorsally and laterally in the preganglion than during the symmetrical type of regeneration. In both types of regeneration, the immunoreactive neurons in the regenerated brain originated in part from undifferentiated neuroblasts situated in different parts of the body, and in part from dividing neurons localized mainly in the pharyngeal nerve plexus. Both exogenous GABA and picrotoxin, applied during the early stages (days 10- 12) of brain regeneration, inhibited the development of the wound tissue and the migration of the neuroblasts and the enteric neurons. At the same time, exogenous GABA application accelerated the proliferation of the pharyngeal neurons. No effect on the process of regeneration could be demonstrated when exogenous GABA and picrotoxin were given together.

Evidence for the presence of thyroid stimulating hormone, thyroglobulin and their receptors in Eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues.

The present study describes the localization and distribution of thyroid-stimulating hormone (TSH), thyroglobulin (TGB) and their receptors in Eisenia fetida (Annelida, Oligochaeta) as revealed by immunohistological methods. Immunopositive neuronal and non-neuronal cells are present in both the central nervous system and some peripheral organs (e.g. foregut and coelomocytes). TSH- and TGB-immunopositive neurons in the various ganglia of the central nervous system are differentially distributed. Most of the immunoreactive cells are found in the suboesophageal ganglion. The stained cells also differ in their shapes (round, oval, pear-shaped) and sizes (small, 12-25 microm; medium, 20-35 microm; large, 30-50 microm). In all ganglia of the central nervous system, TSH-positive neurons additionally show gamma aminobutyric acid (GABA) immunopositivity. Non-neuronal cells also take part in hormone secretion and transport. Elongated TSH-positive cells have been detected in the capsule of the central ganglia and bear granules or vacuoles in areas lacking neurons. Many of capillaries show immunoreactivity for all four tested antibodies in the entire central nervous system and foregut. Among the coelomocytes, granulocytes and eleocytes stain for TSH and its receptor and for TGB but not for thyroid hormone receptor. Most of the granulocytes are large (25-50 microm) but a population of small cells (10-25 microm) are also immunoreactive. None of the coelomocytes stain for GABA. We therefore suggest that the members of this hormone system can modify both metabolism and immune functions in Eisenia. Coelomocytes might be able to secrete, transport and eliminate hormones in this system.

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies.

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological-physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus sub-muscularis is composed of nerve fibres only, whereas the plexus sub-epithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT(3) and GABA(B) receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.

Reorganization of monoaminergic systems in the earthworm, Eisenia fetida, following brain extirpation.

The present study describes the major aspects of how monoaminergic (serotonin, dopamine) systems change in the course of regeneration of the brain in the earthworm (Eisenia fetida), investigated by immunocytochemistry, HPLC assay, and ligand binding. Following brain extirpation, the total regeneration time is about 80 days at 10 degrees C. On the 3rd postoperative day serotonin, and on the 11th postoperative day tyrosine hydroxylase-immunoreactive neurons can be observed in the wound tissue. Thereafter the number of the immunoreactive cells increases gradually, and by the 76th-80th postoperative days all serotonin- and tyrosine hydroxylase-immunopositive neurons can be found in their final positions, similarly to those observed in the intact brain. Labeled neurons located in the dorsal part of the regenerated brain appear earlier than the cells in lateral and ventral positions. Both serotonin- and tyrosine hydroxylase-immunoreactive neurons of the newly formed brain seem to originate from undifferentiated neuroblasts situated within and around the ventral ganglia and the pleura. Dopaminergic (tyrosine hydroxylase-immunoreactive) elements may additionally derive from the proliferation of neurons localized in the subesophageal ganglion and the pharyngeal nerve plexus. Following brain extirpation, both serotonin and dopamine levels, assayed by HPLC, first increase in the subesophageal ganglion; by the 25th day of regeneration, the monoamine content decreases in it and increases in the brain. Hence it is suggested that monoamines are at least partly transported from this ganglion to the regenerating brain. At the same time, (3)H-LSD binding can be detected in the regenerating brain from the 3rd postoperative day, showing a continuous increase until the 80th postoperative day, suggesting a guiding role of postsynaptic elements in the monoaminergic reinnervation of the newly formed brain.