Synaptic organization of a multifunctional interneuron in the central nervous system of Helix pomatia L.

Summary The morphology, axonal arborization and ultrastructure of synaptic connections of the V21 giant neuron in the visceral ganglion of the snail Helix pomatia has been investigated following intracellular labelling with horseradish peroxidase. The V21 neuron establishes several afferent and efferent axo-axonic connections, mainly along the first half of the primary axon. Collaterals of 200–300 m length originate from the primary axon, which shows further arborization, and both afferent and efferent synaptic contacts are formed on these fine axon profiles. Afferent and efferent contacts of the cell occur within very short distances of a few micrometers. On the basis of ultrastructure and vesicle and granule content, several afferent terminals can be distinguished on V21 labelled axon profiles. The majority of these afferent terminals resembles peptidergic-(neurosecretory)-like terminals. This finding supports the possible transmitter role of neuropeptides in the central nervous system of gastropods. Our results are consistent with and provide morphological evidence for recent electrophysiological observations suggesting that, in addition to integrating input, the V21 neuron functions as an interneuron in Helix central nervous system.     

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.     

Distribution of FMRFamide-like immunoreactive neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of FMRFamide-like immunoreactive (FLI) neurons and their morphological characteristics have been investigated in the central nervous system of the snail, Helix pomatia L. Approximately phageal ganglion complex. More than 50% of the FLI neurons were located in the cerebral ganglia. The FLI neurons could be divided into four groups according to size: (i) giant neurons (over 100 m); (ii) large neurons (80–100 m); (iii) medium-sized neurons (40–70 m); (iv) small neurons (12–30 m). They were distributed i) in groups or clusters, typical of small neurons and ii) in solitary form or in groups comprising 2–3 cells, typical of large and giant neurons. Giant and large neurons revealed only limited arborizations in the neuropil, but rich branching towards and in the peripheral nerves. Some of the small neurons had extensive arborizations of varicose fibers in the neuropil. They may therefore play some role in integratory processes. Varicose FLI fibers were visualized in the cell body layer of the different ganglia, and in the neural sheath of both the ganglia and the peripheral nerves. We propose a multifunctional involvement of FLI neurons and FMRFamide-like neuropeptides in the Helix nervous system: (i) a synaptic or modulatory role in axo-axonic interactions in the neuropil; (ii) a direct influence on neuronal cell bodies in the cortical layer, (iii) innervation of different peripheral organs; and (iv) remote neurohormonal control of peripheral events through the neural sheath.     

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.     

Granular cells in the connective tissue of Helix pomatia L. (Gastropoda,Pulmonata)

Summary Granular cells (cells crowded with colourless granules staining with paraldehyde fuchsin according to Gomori-Gabe and not containing calcium) are independent cells in the connective tissue of Helix pomatia. Histochemical data suggest that the granules are rich in sulfhydryl-containing proteins, but lack biogenic monoamines. Electron microscopic investigations confirm the supposed secretory activity of the granular cells. Secretory proteins are presumed to be synthetized in the endoplasmic reticulum and condensed in the Golgi apparatus giving rise to the granules. Extrusion occurs by exocytosis.Electrophoresis of homogenates, prepared from tissues containing numerous granular cells, results in the separation and identification of a secretory protein from the granular cells. An electrophoretically homologous protein is recognized in the hemolymph, but in very small quantities.Our findings and the work of others suggest the involvement of granular cells in neuroendocrine events.The author is indebted to Prof. Dr. D. Kuhlmann for suggesting the problem and for his valuable criticism during the investigation. I would like to thank Mr. J.N. Howell, who helped with the English.Part of this work has been supported by the Stiftung Volkswagenwerk and by the Deutsche Forschungsgemeinschaft     

Distribution and anatomy of GABA-like immunoreactive neurons in the central and peripheral nervous system of the snail Helix pomatia

Gamma-aminobutyric acid (GABA)-like immunoreactive neurons were studied in the central and peripheral nervous system of Helix pomatia by applying immunocytochemistry on whole-mount preparations and serial paraffin sections. GABA-immunoreactive cell bodies were found in the buccal, cerebral and pedal ganglia, but only GABA-immunoreactive fibers were found in the viscero-parietal-pleural ganglion complex. The majority of GABA-immunoreactive cell bodies were located in the pedal ganglia but a few could be found in the buccal ganglia. Varicose GABA-ir fibers could be seen in the neuropil areas and in distinct areas of the cell body layer of the ganglia. The majority of GABA-ir axonal processes run into the connectives and commissures of the ganglia, indicating an important central integrative role of GABA-immunoreactive neurons. GABA may also have a peripheral role, since GABA-immunoreactive fibers could be demonstrated in peripheral nerves and the lips. Glutamate injection did not change the number or distribution of GABA-immunoreactive neurons, but induced GABA immunoreactivity in elements of the connective tissue ensheathing the muscle cells and fibers of the buccal musculature. This shows that GABA may be present in different non-neural tissues as a product of general metabolic pathways.     

Parvalbumin-immunoreactive neurons in the brain of Helix pomatia

Parvalbumin-immunoreactive material was detected in the central nervous system of the snail, Helix pomatia. Each ganglion investigated contained parvalbumin-immunoreactive neurons. The molecular weight of Helix parvalbumin-immunoreactive material as determined by Western blots is about 40 kilodaltons. ⁴⁵Ca²⁺ overlays showed that this protein binds Ca²⁺. In contrast to vertebrates, in Helix neurons parvalbuminlike material was not colocalized with the neurotransmitter gamma-aminobutyric acid (GABA).     

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.     

Alterations in the fine structure of cytoplasmic organelles in the hepatopancreatic cells of shell-regenerating snail,Helix pomatia L.

Summary The investigation of hepatopancreatic cells following damage to the shell revealed hypertrophic alterations of several Cytoplasmic organelles. The network of endoplasmic reticulum was extensively developed within both digestive and calcium cells. The arrangement of tubules exhibited a peculiar hexagonal pattern. It is suggested that the reticulum is of agranular type and may be engaged in the transport of lipids and calcium ions. Remarkable alterations were observed in mitochondria, apparently as the result of their hyperfunction. Mitochondria with reduced cristae, with two-layered transverse septum, and with bleb-like protrusions, occurred frequently. In the calcium cells, helically entwined fibres appeared among the fragments of disintegrated calcium spherites. The results of the investigation and the possible presence of collagen in the fibres originating from disintegrated spherites are discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. The author wishes to thank Mrs. I. Rehnberg for technical assistance.     

The tubular endoplasmic reticulum in the amoebocytes of the shell-regenerating snail,Helix pomatia L.

Summary The tubular endoplasmic reticulum has been studied in the amoebocytes which are present in the connective tissue of the hepatopancreas of the snail, Helix pomatia. The reticulum is similar to that previously described within the glandular cells of the hepatopancreas. Two distinct components are recognizable in the reticulum—central main tubules approximately 100 m in diameter and connecting tubules about 20 m in width. The profile of this tubular network in cross-sections appears as a very regular, apparently crystalline array. The tubules are intimately associated with dense granular material, dense bodies and with mitochondria. The possible function of the tubular endoplasmic reticulum is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. I am indebted to Miss G. Drugge for her technical assistance.     

Nerve cells and synaptic connections in the intestinal nerve of the snail,Helix pomatia L.

Summary The ultrastructure of nerve cells and the finestructural organization of synaptic contacts have been investigated in the intestinal nerve in the snail Helix pomatia. Three types of nerve cells, occurring singly or in groups, can be distinguished on the basis of the ultrastructure of their perikaryon and content of granules. The peripheral output of these nerve cells has been verified by retrograde CoCl₂ and NiCl₂ staining. Both axosomatic and axo-axonic specialized synaptic contacts occur in the intestinal nerve. Presynaptic elements of these synaptic contacts contain 100–120 nm granular vesicles or 120–200 nm neurosecretory-like granules. Following intracellular horseradish peroxidase (HRP) labelling of identified central neurons responsible for peripheral regulatory processes, several labelled axons running toward the periphery can be followed throughout the branches of the intestinal nerve. These labelled axon processes (either primary axon or small collaterals) form specialized synaptic contacts, inside the intestinal nerve, and are always in a postsynaptic position. The occurrence of peripheral axo-somatic and axo-axonic synapses provides a morphological basis for integrative processes taking place in the intestinal nerve (peripheral nervous system) of Helix pomatia.     

Ultrastructural study of the shell-repair membrane in the snail,Helix pomatia L.

Summary The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted.This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensjö and Mrs. E. Hellmén for their technical assistance     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

Uptake of serotonin, 5-hydroxytryptophan and tryptophan by giant serotonin-containing neurones and other neurones in the central nervous system of the snail (Helix pomatia)

Summary Following exposure to tritiated 5-HTP, silver grains were observed over the perikarya of the GSCs (Giant serotonin cells) of Helix pomatia and other known serotonin-containing neurones in light and electron microscope autoradiograms. There was no indication that the 5-HTP was taken up by nerve endings or by non-nervous structures. The distribution of radioactivity was completely different in autoradiograms of tissue exposed to tritiated serotonin. Silver grains, often in very high concentrations, were observed only over certain fine axon branches and processes thought to be nerve endings. Electron microscope autoradiography showed that these processes contained small dense-cored vesicles, morphologically identical to those thought to sequester serotonin in the perikarya of the GSCs. The accumulation of tritiated tryptophan was less specific; all the neurone perikarya showed an accumulation of radioactivity after exposure to this substance.We are grateful to Professor J. F. Lamb for the use of the Scintillation Spectrometer.     

In vitro recalcification of the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The role of the amoebocytes in the calcification process of the shell-repair membrane of the snail, Helix pomatia, was investigated in vitro. The shell-repair membranes were demineralized with 0.5 M EDTA at pH 7.4. For the recalcification of the demineralized membranes two substrates were chosen: (i) Tris-buffered Helix pomatia-saline, pH 7.4, and (ii) Helix pomatia-saline supplemented with 5 mM CaCl₂ and 5 mM NaHCO₃. The membranes were incubated in 2 ml substrate at 37° C and examined after 2 h, 24 h, and 3, 5 and 7 days. Calcium deposition and crystal formation were observed within the membrane incubated in the salt-supplemented substrate. The control membranes were either heat-inactivated or deprived of lipids. No calcium precipitation was observed in control membranes. The experiments show that the recalcification of the shell-repair membrane is under strict cellular control and that the granules released from the amoebocytes serve as sites for calcium deposition. The role of phospholipids in the calcification process is discussed.     

Heart rate and hemocyte number as stress indicators in disturbed hibernating vineyard snails, Helix pomatia

In humans and other vertebrates, mental or physical stressors may trigger a variety of symptoms generally referred to as the fight/flight response (Cannon 1929). The processes also include variability of the heart frequency as well as leukocytosis. We monitored both body responses in disturbed hibernating vineyard snails, H. pomatia, to obtain information on the stress sensitivity of these “sleeping” invertebrates. The first mild stressor, a 100 meter transport of hibernating snails from the cold room to the laboratory, caused cardiac arrhythmia in the animals. This reaction could have been provoked by mechanical disturbances and/or by raising the body temperature to room temperature. But a change in the ambient temperature did not trigger an abnormal heart rhythm. Different from this observation, we recorded instant heart rate changes in response to knocking on the shell and a very irregular heartbeat occurred when a hole was punched in the shell. With a short time delay upon damaging the shell, a large increase in the number of circulating cells also occurred. This was not observed after knocking on the shell or when snails were adapted to different temperatures for each 48 h. Thus, hibernating snails sense environmental variations which cause an immediate change of the heart frequency and an elevated stimulus level initiates in addition leukocytosis which occurs at a post-stimulus latency. This disparity could indicate the activity of two different stress regulation pathways.Furthermore, the assays demonstrate an increasing linear relation between rising temperature and frequency of heart pulsations (y = 1.01x − 2.1); but the results do not indicate a correlation between heartbeat frequency and the number of cells in circulation. Consequently, neither temperature nor heart frequency seems to influence the number of circulating cells in hibernating H. pomatia.     

Zur Innervation des glatten Penisretraktormuskels von Helix pomatia: Allgemeine Histologie und Histochemie des monoaminergen Nervensystems

Summary The penis retractor muscle of Helix pomatia is passed by thick nerve trunks, which probably are nerve nets. In the contracted muscle, they are folded meanderlike, in the extended they are pulled smooth. Four types of vesicles different in size and contents can be distinguished in the nerves.Varicose fibres accompany the muscle cells. Frequently the terminals are running in a groove of the muscle fibre. In certain axon types occur presynaptic accumulations of vesicles and thickenings of the terminal membrane. Terminal and muscle cell are separated by a cleft of 300 AE width. The muscle fibre membrane has no subsynaptic infoldings. Beside these axons thin, naked neurites are running through the connective tissue. They are characterized by a high content of neurotubuli.One part of the axons presumably possesses a monoaminergic transmitter. After the glutaraldehyde-dichromate-reaction they contain dense grana, whose diameters are mainly below 1000 AE. The nerve trunks fluoresce after exposure to paraformaldehyde vapour, excited with UV-light, green to green-yellow. The maximum of the excitation was determined at 413 nm and the maximum of emission at 496 respectively at 510 nm. It is concluded, that both, a catecholamine and 5-HT are responsible for the fluorescence. Extraction and paperchromatographic separation lead to the opinion, that the catecholamine is dopamine.

Auszug aus der Dissertation Histologische und histochemische Untersuchungen zur Innervation des glatten Penisretraktormuskels der Weinbergschnecke Helix pomatia L. (Göttingen, 1970). Auf Anregung von Herrn Prof. Dr. Fr.-W. Schlote, mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Akademie der Wissenschaften.     

The effect of carbonic anhydrase,urea and urease on the calcium carbonate deposition in the shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of carbonic anhydrase (CA), urea and urease on the CaCO₃ deposition in the shell-repair membrane of the snail, Helix pomatia, was studied by injection of CA separately or in combination with urease. This treatment resulted in increased deposits of CaCO₃ and apparent crystal formation within the shell-repair membranes compared with those of the controls. The reactions to CA combined with urea were not uniform. Formation of organic crystalline structures and dendritic spherulites was observed in some of these membranes, whereas the deposition of CaCO₃ crystals was suppressed. Administration of urea alone inhibited the formation of large CaCO₃ crystals, whereas urease stimulated this process. The reaction of young snails was greater compared to adults. The membranes of young snails contained tighly packed, small CaCO₃ crystals and organic crystalline structures, which indicated increase of the calcifying centra and their successive mineralization. The results support the assumption that carbonic anhydrase and urease enhance the rate of calcium carbonate deposition and crystal formation in Helix pomatia.