H. H. Boer

Axonal mapping of the giant peptidergic neurons VD1 and RPD2 located in the CNS of the pond snail Lymnaea stagnalis, with particular reference to the innervation of the auricle of the heart

VD1 and RPD2 are two giant neuropeptidergic neurons located respectively in the visceral and right parietal ganglion of the central nervous system (CNS) of the pond snail Lymnaea stagnalis. They are the most prominent representatives of a system of neurons expressing a gene that is similar to the gene expressed in R15 of Aplysia californica. Both neuronal systems are involved in the regulation of cardio-respiratory phenomena. In the present study the axonal branches of VD1 and RPD2 were mapped using immunocytochemical and tracer studies. To this end the alpha 1-antiserum (directed to one of the VD1/RPD2 neuropeptides) was used in combination with Lucifer yellow (LY) and Ni-lys tracers. In whole mount preparations of the CNS, immunostained axons of VD1 and RPD2 were observed to run to the pleural, cerebral and pedal ganglia and in several nerves. Upon LY injection of VD1 thin axon branches were observed in the internal right parietal nerve. These run to the skin in the mantle area near the pneumostome and osphradium. The skin of the lips appeared to receive a similar innervation via the lip nerves. Thick LY filled axons of VD1 and RPD2 were observed in the intestinal nerve. They could be traced to the heart region. The pericardial branch of the intestinal nerve innervates the pericardium and heart (Ni-lys tracing). Immunocytochemically, using the alpha 1-antiserum, it was demonstrated that this nerve branch carries the axons of VD1 and RPD2 to the venous side of the auricle, where they enter the pericardial cavity and ramify in the auricle (but not in the ventricle).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of four genes encoding molluscan insulin-related peptides in the central nervous system of the pond snail Lymnaea stagnalis

SummaryIn the pond snail Lymnaea stagnalis, the growth regulating system consists of (1) about 200 neuroendocrine light green cells, located in four clusters in the cerebral ganglia, and (2) the paired canopy cells, located in the lateral lobes. These cells express genes encoding the molluscan insulin-related peptides (MIPs). Six MIP genes have previously been identified. Four of these (I, II, III and V) are expressed in the light green cells and the canopy cells. The MIP-VI gene is a pseudogene. In the present in situ hybridization study, using oligonucleotide probes specific to the transcripts of MIP-I,-II,-III,-IV, and-V, no signal was obtained with the MIP-IV probe, indicating that gene IV is also a pseudogene. With the other four probes, two types of light green cells were distinguished. Type-A cells express all four MIP genes, whereas type-B cells do not (or only faintly) express the MIP-I gene. Gene III is relatively strongly expressed in type-B cells. Genes II and V are moderately expressed in both cell types. Type-A cells are mainly located in the periphery of the clusters, whereas type-B cells are present in the center. The canopy cell resembles type-A light green cells. The expression levels of the MIP-II and MIP-V genes are low in the canopy cell. The expression pattern of the MIP genes correlates with the staining pattern of the anti-MIP-C antibody, which has been raised to a synthetic C-fragment shared by MIP-I,-II and-V. Type-A cells stain more intensely with the antibody than type-B cells.

Ultrastructural neuropathologic effects of Taxol on neurons of the freshwater snailLymnaea stagnalis

SummaryCerebral ganglia of the freshwater snailLymnaea stagnalis were incubatedin vitro in 10 μM Taxol for 8 and 24 h. Cremophor EL (0.1%) was used as a diluant. The tissue was processed for electron microscopy. Various ultrastructural parameters were assessed quantitatively. Cremophor EL appeared to seriously affect the cell somata of the multipeptidergic caudodorsal cells. In the Cremophor-controls the mean area of Golgi zones, the percentage dense material (neuropeptides) in these zones, the number of large electron dense granules (these are involved in neuropeptide processing) and the mean nuclear heterochromatin clump size, were significantly smaller than in the Ringer-controls, whereas the number of lipid droplets was higher. All these parameters, except for the lipid droplets, were not different in the Cremophor-controls and the Taxol-treated specimens. After 24 h treatment, but not after 8 h, Cremophor EL furthermore induced an increase in the number of axonal microtubules. It is argued that the results might signify activation of the neurons by Cremophor EL. Taxol induced a significant increase in the number of microtubules in axons and cell somata. Furthermore an increase in the number of Golgi zones was observed, suggesting activated neuropeptide synthesis. In all groups immunostaining with antibodies to neuropeptides produced by the caudodorsal cells was normal. Release of neuropeptide (exocytosis) from axon endings was elevated after Taxol treatment, and exceptionally high in specimens cotreated with Taxol and Org 2766 (incubation time 22 h). The effect of Org 2766 and Taxol on the number of microtubules was cumulative. It is argued that transport of neuropeptide granules from the cell somata to the axon terminals was not affected by Taxol. It is concluded that Taxol neurotoxicity is probably not due to impeded microtubular axonal transport.

The ACTH/MSH(4–9) analogue ORG 2766 stimulates microtubule formation in axons of central neurons of the snail Lymnaea stagnalis

Central nervous systems of the pond snail Lymnaea stagnalis were incubated in vitro in different concentrations of ORG 2766 (10(-9)-2.5 x 10(-4) M) for 10 and 20 h. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons had increased after 10 h of incubation by approximately 50% (Experiment 1) and 30% (Experiment 2), respectively. No further increase was observed after 20 h of incubation. (The higher concentrations were studied.) Maximal stimulation was already found at a concentration of 10(-8) M. At a concentration of 10(-9) M control levels were observed. It is concluded that ORG 2766 stimulates microtubule formation already at very low concentrations. It is not clear whether the compound stimulates synthesis of tubulin, induces assembly of microtubules, or causes an increase in stability of microtubules. Nevertheless, ultrastructural data on the morphology of the glial cells indicate that these cells are activated by ORG 2766 treatment, which suggest that ORG 2766 has general trophic effects.

The sodium influx stimulating peptide of the pulmonate freshwater snail Lymnaea stagnalis

In Lymnaea stagnalis integumental Na+ uptake is stimulated by the sodium influx stimulating (SIS)-peptide. Its primary structure was determined as: SRTQSRFAS- YELMGTEGTECVTTKTISQICYQCATRHEDSFVQVYQECCKKEMGLREYCEEIYTELPIRSGLWQPN++ +. Antisera raised against parts of SIS-peptide stained neurons in the visceral, parietal, and pleural ganglia, and in the proximal parts of the intestinal, anal, and right internal pallial nerves. Locations and axon projection patterns of these neurons suggest that they represent the previously described neurosecretory yellow cells.

Functional morphology of the neuroendocrine sodium influx-stimulating peptide system of the pond snail, Lymnaea stagnalis, studied by in situ hybridization and immunocytochemistry

SummaryThe functional morphology of the neuroendocrine system producing sodium influx-stimulating (SIS) peptide in the pond snail, Lymnaea stagnalis, was studied by in situ hybridization and immunocytochemistry. The SIS-peptide, which is 76 amino acids long, stimulates sodium uptake from the ambient medium. Two synthetic DNA probes were used for in situ hybridization. The nucleotide sequences were chosen from the cDNA structure; they encode amino acids 8–17 and 64–73, respectively. SIS-peptide sequences 10–20 and 67–76 were synthesized and antibodies were raised to them and affinity-purified. In addition to these antibodies, a monoclonal antibody raised to a bioactive, high-pressure liquid chromatography (HPLC)-purified brain extract was used for immunocytochemistry. Paraffin sections of central nervous systems and of whole snails were studied. The SIS-peptide system could be identified as the previously described yellow cell (YC) system by comparing alternate sections treated with the DNA probes, stained with the antibodies, or stained with alcian blue-alcian yellow. SIS-peptide neurons (∼45) occur in the ganglia of the visceral ring and in the proximal parts of visceral nerves. Axons run in the nerves of these and in several nerves of other ganglia. Numerous axon branches penetrate the perineurium forming a vast central neurohemal area. The SIS-peptide system innervates the pericardium, the nephridial gland, the reno-pericardial canal, the ureter, the spermoviduct and gonadal acini, the anterior aorta, the ventral buccal artery, and the penis protractor muscle. The morphology of the system is discussed in relation to the process of sodium ion uptake from the ambient medium and from pro-urine, and to that of regulating blood pressure. In the central nervous system and other organs, neurons and axons not labeled with the DNA probes, but immunoreactive to one or two of the antibodies, were observed. It seems unlikely that these elements are functionally related to the SIS-peptide system.

Immunocytochemistry of peptidergic systems in the pond snail Lymnaea stagnalis.

Evidence suggests that there exists in the animal kingdom a family of biologically active peptides whose members are related to the molluscan cardio-active tetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2). Immunocytochemical and ultrastructural studies indicate that several family-members occur in the pond snail Lymnaea stagnalis. Monoclonal antibodies were raised to whole brain homogenates of the pond snail. Selection of antibody producing hybridomas was carried out by staining sections of the central nervous system of the snail with the supernatants of the hybridomas. Certain antibodies stain selectively known (neuro)endocrine centres of the snail, others are directed against particular groups of neurons. It is argued that these antibodies were raised against biologically active peptides and/or their precursors. The antibodies may be used for the isolation of these peptides.

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I. Our observations support the hypothesis that light yellow cells are involved in maintaining the shape of the animal via the regulation of ion- and waterbalance processes and blood pressure.

Functional morphology of the neuropeptidergic light-yellow-cell system in pulmonate snails.

The light yellow neuropeptidergic cell system of the basommatophoran snail Lymnaea stagnalis is homologous to the R3-R14 system of the opisthobranch Aplysia californica, and produces three different neuropeptides. Systems homologous to the light yellow cells of Lymnaea stagnalis have been investigated morphologically in two Basommatophora (Lymnaea ovata, Bulinus truncatus) and three Stylommatophora (Helix aspersa, Cepaea nemoralis, Deroceras reticulatum). To this end, an antibody to synthetic light-yellow-cell peptide-II and oligonucleotides to mRNAs encoding parts of peptide-I and peptide-III, were used. The in situ hybridization probes gave negative results. On the other hand, neuronal cell clusters were observed in the central nervous system of all specias studied by immunocytochemistry. These clusters were located in the ganglia of the visceral complex. The neurons project axons into all nerves of these ganglia, especially into the pallial nerves, into the connective tissue of the central nervous system, and into the neuropile of various ganglia. The morphology of the systems is similar to that of the light-yellow-cell system of Lymnaea stagnalis. In all species, the wall of the aorta was innervated by immunoreactive axons. Peripheral innervation by the light-yellow-cell system was investigated in Helix aspersa and Deroceras reticulatum. Serial and alternate sections of whole snails were studied. Reconstructions were made of the heart-kidney-lung complex of these animals. In both species, the muscular vessels of the pulmonary system at the right side of the body were strongly innervated by immunoreactive axons. Furthermore, immunopositive innervation was observed to muscles in the secondary ureter-pneumostome area. The light-yellow-cell system of pulmonates is thus probably involved in the regulation of blood pressure and urine release.

In vivo modulation of vincristine-induced neurotoxicity in Lymnaea stagnalis, by the ACTH(4–9)analogue Org 2766

SummaryThe use of the cytostatic agent vincristine (VCR) is limited by the occurrence of peripheral neuropathy. This side-effect is probably caused by interference with axonal microtubules. VCR depolymerizes microtubules and reacts with tubulin to form paracrystals. The potential of a neurotrophic ACTH(4–9) analogue, Org 2766, to counteract peripheral neuropathy caused by cytostatic agents is being investigated. In the present ultrastructural study, modulatory effects of Org 2766 on VCR-induced neurotoxicity were studied in vivo in neurons of the pond snail Lymnaea stagnalis, which has been shown previously to be a suitable test system to investigate neurotoxic side-effects of cytostatic agents. 24 h after treatment with VCR (25 μM), 68.4 ± 34.7 paracrystals were counted per cross-section of the cerebral commissure and the number of microtubules in the axons had been lowered to 46% of the control level. After a survival period of two weeks all paracrystals had disappeared. By that time, no recovery of the axonal microtubular system could be observed. However, post-treatment with Org 2766 (10−6 M) on day 6 after VCR treatment had induced a significant increase in the number of microtubules (+ 55%) on day 7. This beneficial effect lasted for the rest of the experimental period (14 days). These results suggest that post-treatment with Org 2766, i.e. after VCR clearance, can induce long-lasting beneficial effects on VCR-induced neurotoxicity in vivo.