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.

The distribution of neurons immunoreactive for γ-aminobutyric acid was studied in the nervous system of Lumbricus terrestris (Oligochaeta). In the cerebral ganglion, the 86 cells immunoreactive for γ-aminobutyric acid represented 4.0% of the nerve cells in the brain, had a diameter of 12-50 µm, and were arranged in seven groups. Small-sized (18-30 µm) immunoreactive neurons occurred in the circumpharyngeal connectives. The axons of most immunoreactive neurons of the cerebral ganglion richly arborized in the ventral part of the neuropil and some could also be traced in the circumpharyngeal connectives. The subesophageal ganglion contained 94 immunoreactive cells (6.7% of the cells of this ganglion), also divided into seven groups, and with a diameter of 8-55 µm. The axons of the labeled neurons ran to the central neuropil giving both contra-and ipsilateral processes. Altogether 108 neurons in each ganglion (8.0% of their cells) of the ventral cord were immunopositive. Four labeled cell groups were present in the rostral and caudal part of each ganglion. Axons of these immunoreactive cells arborized in the central neuropil and projected to the segmental nerves. The stomatogastric ganglia and the enteric plexus also contained immunoreactive neurons. Many small elongated immunoreactive cells occurred in the gut epithelium. Postembedding immunogold electron microscopy revealed that immunoreactive varicosities mainly contained small pleomorphic (24 nm) agranular synaptic vesicles and some small granular (50 nm) vesicles.& k w d :

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.

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.

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.