Demonstration of insulin-related substances in the central nervous systems of pulmonates and Aplysia californica

Summary the occurrence of insulin-related substances in the central nervous system of pulmonates and Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Previous experiments have shown that, in Lymnaea stagnalis, the growth hormone-producing neurons in the cerebral ganglia (the so-called light green cells) express at least 5 genes that are related to the vertebrate insulin genes, i.e., they encode prohormones that are composed of a B- and A-chain and a connecting C peptide. These insulin related molecules also have the amino acids essential for their tertiary structure (viz. cysteines) at identical positions to those of the vertebrate insulins. In the investigated basommatophoran and stylommatophoran snails and slugs, neurons reacted with an antiserum raised against the C peptide of one of the molluscan insulin-related peptides. These neurons can be considered to be, based on morphological and endocrinological criteria, homologous to the light green cells of L. stagnalis. In A. californica, all central ganglia contain immunoreactive neurons. The highest number (about 50) was observed in the abdominal ganglion. The present results indicate that insulin-related substances are generally occurring neuropeptides in the central nervous system of molluscs.     

Immunocytochemical localization of histamine in flatworms

Summary Specific antibodies against histamine were used to demonstrate the occurrence and cellular distribution of histamine-like immunoreactivity in three species of flatworms (phylum Platyhelminthes). In the parasitic cestode Diphyllobothrium dendriticum, histamine-reactivity was found in neurons of the main nerve cords, and in cells lining the central and peripheral excretory ducts. In the free-living microturbellarian Microstomum lineare and in the planarian Polycelis nigra, histamine-immuno-reactivity was restricted to cells and fibres of the nervous system. The occurrence of histamine or a related substance in the nervous system of flatworms, which represent primary bilateria, indicates the importance of this neuroactive substance in the animal kingdom.     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

Immunocytochemical Distribution of 5-HT (Serotonin) in the Nervous System of the Gastrotrich Turbanella cornuta

The 5-HT (serotonin) distribution in the nervous system of the macrodasyoid gastrotrich Turbanella cornuta was studied using immunocytochemical methods. Positive immunoreaction was found in two pairs of neurons. The neurons of one pair had processes which extended peripherally to the surface of the body. Central processes of both pairs entered the brain commissure and proceeded into the longitudinal cords. Unlike other acoelomate worms studied so far, the Platyhelminthes and the Nematoda, in this gastrotrich no 5-HT positive perikarya or processes were present in the pharynx innervation, and no positive neurons sent processes directly to the nerve cords     

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

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).     

Small cardioactive peptide-like immunoreactivity and its colocalization with FMRFamide-like immunoreactivity in the central nervous system of the leech Hirudo medicinalis

Summary The distributions of small cardioactive peptide (SCP)- and FMRFamide-like immunoreactivities in the central nervous system of the medicinal leech Hirudo medicinalis were studied. A subset of neurons in the segmental ganglia and brains was immunoreactive to an antibody directed against SCP_B. Immunoreactive cell bodies were regionally distributed throughout the nerve cord, and occurred both as bilaterally paired and unpaired neurons. The majority of the unpaired cells displayed a tendency to alternate from side to side in adjacent ganglia. A small number of neurons were immunoreactive only in a minority of nerve cords investigated. Intracellular injections of Lucifer yellow dye and subsequent processing for immunocytochemistry revealed SCP-like immunoreactivity in heart modulatory neurons but not in heart motor neurons. FMRFamide-like immunoreactivity was also detected in cell bodies throughout the central nervous system. A subset of neurons contained both SCP- and FMRFamide-like immunoreactivities; others stained for only one or the other antigen. These data suggest that an antigen distinct from FMRFamide is responsible for at least part of the SCP-like immunoreactivity. This antigen likely bears some homology to the carboxyl terminal of SCP_A and SCP_B.     

Muscle fibers in the central nervous system of nemerteans: Spatial organization and functional role

The system of muscle fibers associated with the brain and lateral nerve cords is present in all major groups of enoplan nemerteans. Unfortunately, very little is known about the functional role and spatial arrangement of these muscles of the central nervous system. This article examines the architecture of the musculature of the central nervous system in two species of monostiliferous nemerteans (Emplectonema gracile and Tetrastemma cf. candidum) using phalloidin staining and confocal microscopy. The article also briefly discusses the body‐wall musculature and the muscles of the cephalic region. In both species, the lateral nerve cords possess two pairs of cardinal muscles that run the length of the nerve cords and pass through the ventral cerebral ganglia. A system of peripheral muscles forms a meshwork around the lateral nerve cords in E. gracile. The actin‐rich processes that ramify within the nerve cords in E. gracile (transverse fibers) might represent a separate population of glia‐like cells or sarcoplasmic projections of the peripheral muscles of the central nervous system. The lateral nerve cords in T. cf. candidum lack peripheral muscles but have muscles similar in their position and orientation to the transverse fibers. The musculature of the central nervous system is hypothesized to function as a support system for the lateral nerve cords and brain, preventing rupturing and herniation of the nervous tissue during locomotion. The occurrence of muscles of the central nervous system in nemerteans and other groups and their possible relevance in taxonomy are discussed. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Distribution of tyrosine-hydroxylase-immunoreactive and dopamine-immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution and characterization of dopamine-containing neurons are described in the different ganglia of the central nervous system of Helix on the basis of the distribution of tyrosine hydroxylase immunoreactive (TH-ir) and dopamine immunoreactive (DA-ir) neurons. Both TH-ir and DA-ir cell bodies of small diameter (10–25 m) can be observed in the buccal, cerebral and pedal ganglia, dominantly on their ventral surface, and concentrated in small groups close to the origin of the peripheral nerves. The viscero-parietal-pleural ganglion complex is free of immunoreactive cell bodies but contains a dense fiber system. The largest number of TH-ir and DA-ir neurons can be detected in the pedal, and cerebral ganglia. The average number of TH-ir and DA-ir neurons significantly differs but all the identifiable groups of TH-ir neurons also show DA-immunoreactivity. Therefore, we consider the TH-ir neurons in those groups as being DA-containing neurons. The amounts of DA in the different ganglia assayed by high performance liquid chromatography correspond to the distribution and number of TH-ir and DA-ir neurons in the different ganglia. The axon processes of the labeled small-diameter neurons send thin proximal branches toward the cell body layer but only rarely surround cell bodics, whereas distally they give off numerous branches in the neuropil and then leave the ganglion through the peripheral nerves. In the cerebral ganglia, the analysis of the TH-ir pathways indicates that the largest groups of labeled neurons send their processes through the peripheral nerves in a topographic order. These results furnish morphological evidence that DA-containing neurons of Helix pomatia have both central and peripheral roles in neuronal regulation.     

Distribution of catch-relaxing peptide (CARP)-like immunoreactive neurons in the central and peripheral nervous system of Helix pomatia

Immunocytochemistry was performed on the nervous system of Helix by the use of an antibody raised against a myotropic neuropeptide, the catch-relaxing peptide (CARP), isolated from Mytilus edulis. In each ganglion of the central nervous system of Helix pomatia, numerous CARP-immunoreactive cell bodies and a dense immunoreactive fiber system could be observed with a dominancy in the cerebral and pedal ganglia. The majority of the immunoreactive neurons are unipolar, although multipolar neurons also occur. In the neuropil areas, CARP-immunoreactive fibers show extensive arborization, which may indicate a central role of CARP. CARP-immunoreactive elements could be observed in each investigated peripheral nerve and peripheral areas, namely in the intestine, heart, aorta, buccal mass, lips, and foot. However, CARP-immunoreactive cell bodies could only be demonstrated in the intestine and the foot musculature. Thin varicose CARP-immunoreactive fibers were observed over both muscle and gland cells in the different peripheral organs, suggesting a peripheral role of CARP. In vivo CARP injection into the body cavity (10^-3, 10^-4, 10^-5 M) altered the general behavioral state of the animals and induced the relaxation of the musculature of the whole body wall indicating that CARP has a significant role in the regulation of muscle contraction.     

Histamine is a major mechanosensory neurotransmitter candidate in Drosophila melanogaster

Histamine is known to be the neurotransmitter of insect photoreceptors. Histamine-like immunoreactivity is also found in a number of interneurons in the central nervous system of various insects. Here, we demonstrate by immunohistochemical techniques that, in Drosophila melanogaster (Acalypterae), most or all mechanosensory neurons of imaginal hair sensilla selectively bind antibodies directed against histamine. The histamine-like staining includes the cell bodies of these neurons as well as their axons, which form prominent fibre bundles in peripheral nerves, and their terminal projections in the central neuropil of head and thoracic ganglia. The specificity of the immunostaining is demonstrated by investigating a Drosophila mutant unable to synthesize histamine. Other mechanosensory organs, such as campaniform sensilla or scolopidial organs, do not stain. In the calypteran flies, Musca and Calliphora, we find no comparable immunoreactivity associated with either hair sensilla or the nerves entering the central nervous system, observations in agreement with earlier studies on Calliphora. Thus, histamine seems to be a major mechanosensory transmitter candidate of the adult nervous system of Drosophila, but apparently not of Musca or Calliphora.     

Serotonergic immunoreactivity in the pedal ganglia of the pulmonate snail Megalobulimus abbreviatus after thermal stimulus: A semi-quantitative analysis

Using an immunohistochemical procedure and optical densitometry, the distribution of neurons containing serotonin (5-HT) was investigated in the pedal ganglia of Megalobulimus abbreviatus after thermal "non-functional stimulus" (22 degrees C) and stressful thermal conditions (50 degrees C).The animals were sacrificed at different times (3 h, 6 h and 24 h) following these stimuli. In control animals, the results showed the location of these serotonergic immunoreactive elements (5HT-ir) in this ganglion to be similar to those shown in other studies, where the anterior region of ventral sections showed the largest number of 5HT-ir neurons. In the anterior neurons, significant differences (p < 0.01) were observed between the groups of animals stimulated at 50 degrees C and 22 degrees C and sacrificed after 6 h. In the medial neurons, significant differences (p < 0.05) were observed between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 6 and 24 h. Neuropilar area 1 showed differences (p < 0.01) in 5HT-ir between the control group and the groups of animals stimulated at 50 degrees C and sacrificed after 3 and 24 h. Neuropilar area 2 showed a significant difference (p < 0.05) between the groups of animals stimulated at 22 degrees C and sacrificed after 3 and 24 h. These results suggest the involvement of 5-HT in the nociceptive circuit of M. abbreviatus, mainly that of the medial neurons and neuropilar area 1, which showed increases in 5HT-ir after thermal aversive stimuli. These results could be helpful in drawing cellular homologies with other gastropods.     

The nervous system of Bothriomolus balticus (Proseriata) —a contribution to the knowledge of the orthogon in the Plathelminthes

Summary The nervous system (NS) of Bothriomolus balticus (Proseriata) was studied by the immunocytochemical (ICC) method with antisera to RFamide, SALMFamide and serotonin and with the histochemical GAIF method. The use of the ICC technique provided a much more precise morphological account of the nervous system than had previously been possible. The obtained data are discussed in connection with the comparative morphology of the nerve cords of the Plathelminthes. A similar position does not grant direct correspondence between nerve cords in the taxon Seriata. Marginal cords had probably an independent origin in the Monocelididae and Otoplanidae. The ventral (main) cords of B. balticus seem to correspond to the lateral (main) cords of the Monocelididae. It can be hypothesized that both: (1) a shift of the main cords accompanied by formation of new cords from the plexus and fusion of other cords and (2) a redistribution of nerve processes and perikarya between the cords, take part in the evolution of cords in the Plathelminthes. The first hypothesis seems to explain the difference in the position of main cords in proseriates, though, the second hypothesis might dominate, for example, in the Neorhabdocoela and the Neodermata. The correctness of the evolutionary analysis of the nerve cords in plathelminths can only be provided by neurons or neuron groups marking these structures.     

Anatomy of the sensory nervous system in Craspedella pedum (Plathelminthes, Temnocephalida): DiO staining after fixation and in vivo

 The microanatomy of the nervous system of Craspedella pedum is described based on staining of the sensory nerves with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate) in whole worms after fixation. The high resolution and reproducibility of this method revealed that the microanatomy of the nervous system is uniform within this species to rather minute details. Although the lateral and ventral cords have a similar diameter, the ventral nerve cords are very poor in sensory fibres. Such a high level of functional differentiation of nerve cords has not been known in representatives of the Plathelminthes. In vivo staining reveals numerous DiO-accumulating sensory neurons in the anterior portion of the body and a few pairs in its posterior half. The morphology of some neurons is described. Several neurons were identified and our data suggest that most, if not all, sensory neurons are identificable cells. Accepted: 16 December 1997     

Distribution of 5-HT (serotonin) immunoreactivity in the central nervous system of the inshore hagfish,Eptatretus burgeri

Summary Immunohistochemical techniques were used to study the distribution of serotonin in the central nervous system of the hagfish,Eptatretus burgeri, in order to produce a detailed map of serotonin-containing structures. In the hypothalamus, many serotonin-containing neurons contacted the cerebrospinal fluid. Most of the serotonin-containing cell bodies were located in the raphe region, where they were compactly distributed at the level of the nucleus motorius tegmenti pars anterior but more diffusely distributed at the level of the nucleus motorius tegmenti pars posterior. Serotonin-containing cell bodies and varicose fibers were widely distributed throughout the brain and upper spinal cord segments, but the distribution density was not even. On the basis of its abundance, serotonin can be judged to have an important function in the control of the hagfish central nervous system. From a phylogenetic point of view, serotonin-containing neurons in the raphe region appear to be a common property of all classes of vertebrates studied except the lampreys, whereas serotonin-containing cerebrospinal fluid-contacting neurons may be considered to be a primitive condition in all nonmammalian vertebrates.     

Distribution and development of the serotonin-and RFamide-like immunoreactive neurons in the arrowworm,Paraspadella gotoi (Chaetognatha)

Summary The distribution and development of serotonin-and RFamide-like immunoreactivities in the nervous system of Chaetognatha, Paraspadella gotoi, were examined in whole-mount preparations. In adults, a single serotonin-like immunoreactive (5HTLI) neuron and numerous RFamide-like immunoreactive (RFaLI) neurons were found in the central nervous system. Based on the structure of the fins, hooks, and eyes, seven postembryonic developmental stages were recognized. The most obvious features of the stages are: stage 1, newly hatched young; stage 2, elongation of a continuous lateral tail fin; stage 3, separation of the lateral and tail fins; stage 4, appearance of hooks; stage 5, pigmentation of eyes, stage 6, attachment by tail adhesive fins; stage 7, prey capture. Stage 1 did not show any immunoreactivity. The 5HTLI neuron first appeared at stage 4 and its axonal pathway became similar to the adult at stage 6. On the other hand, the RFaLI neurons appeared at stage 3 in the ventral ganglion. Some of their somata disappeared at stage 5 and the neuronal architecture resembled the adult at stage 7 although the RFaLI neurons in the cerebral ganglion were complete at the juvenile stage.We are sad to announce that Dr. M. Yoshida died on 29 October 1988     

Immunocytochemical demonstration of neurotransmitters in the nerve plexuses of the foot and the anterior byssus retractor muscle of the mussel,Mytilus galloprovincialis

Summary Immunocytochemical methods were applied to study the distribution of putative neurotransmitters (5-HT, substance P, GABA, glutamate and aspartate) in the nerve plexuses of the foot and the anterior byssus retractor muscle (ABRM) of Mytilus galloprovincialis (Mollusca, Bivalvia). The foot presents extensive nerve plexuses containing 5-HT and substance P-like immunoreactive material with a similar distribution beneath the surface epithelium, around the vessels and in the glandular regions. Coexistence of the two putative neurotransmitters was observed in a few nerve fibers, Conversely, muscle fibers, both in the foot and in the ABRM, are innervated only by 5-HT-positive fibers, while substance P-like material is present only in the networks of the ABRM epimysial sheath. Immunoreactivity for glutamate and aspartate was not demonstrated, while rare GABA-positive nerve cells and fibers were found only in the foot. The results of this investigation provide a morphological background to previous physiological studies on 5-HT in the nervous system of bivalve molluscs. Moreover, they confirm that the nervous system of Mytilus contains a remarkable amount of a substance related to the vertebrate tachykinin family.     

Immunoreactivity to a specific echinoderm neuropeptide in the nervous system of the flatworm Macrostomum hystricinum marinum (Turbellaria,Macrostomida)

Immunocytochemical characterization of the neuropeptides FMRF-amide and serotonin (5-HT) is a well-known method successfully applied to demonstrate nervous-system morphology in several platyhelminths (see Wikgren & Reuter, 1985, and Reuter, 1988, for review). We have immunolabeled whole-mount preparations of Macrostomum hystricinum marinum Rieger from cultures (see Rieger et al., 1988) with anti-SALMF-amide, an antibody specific for the C-terminal pentapeptide sequence of the neuropeptide GNFSALMF-amide recently isolated from echinoderms (source M. Thorndyke, England). Immunoreactivity to SALMF-amide gave a more detailed picture of the nervous system of M. hystricinum than FMRF-amide. Conventional light microscopy (Luther, 1905) shows this nervous system to consist of a bilobed brain, a pharyngeal nerve-ring system, a posterior commissure, and two main ventrolateral nerve cords. Immunostaining reveals, in addition, two thin paired longitudinal nerve strands and fine subepithelial and submuscular nerve nets. Anti-SALMF-amide labels a distinct class of neurons, causing the main lateral longitudinal cords and pharyngeal nerve-ring system to appear more filamentous than with other techniques. Recent fine-structural investigations on the nervous system of Macrostomum hystricinum marinum revealed several axon types with characteristic vesicles and neurotubules (D. R. pers. obs.). Partly supported by FWF grant P7816.     

Distribution of serotonin-like immunoreactive neurons in the slug<Emphasis Type="Italic"> Limax valentianus</Emphasis>

Immunohistochemical techniques were used to study the distribution of serotonin-containing neurons in the nervous system of the slug Limax valentianus. Approximately 350 serotonin-like immunoreactive cell bodies were found in the central nervous system. These were located in the cerebral, pedal, visceral and right parietal ganglia. Most serotonin-like immunoreactive neurons had small cell bodies, which were aggregated into discrete clusters. A pair of previously identified metacerebral giant cells were found on the anterior side of the cerebral ganglion, and two additional pairs of uniquely identifiable, serotonin-like immunoreactive cells were found on the posterior side of the cerebral ganglion. The whole-mount maps of these stained neurons will be useful in further physiological and biochemical studies of olfactory learning at the cellular level in Limax valentianus.This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Science, Sports and Technology, Japan (nos. 12307053 and 13771353)