Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles. Within the central ganglia many neurons, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata. Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Localization of a substance P-like material in the central and peripheral nervous system of the snail Helix aspersa

Summary A monoclonal antibody against substance P was used for immunocytochemical staining of the central ganglia of the snail Helix aspersa and several peripheral tissues including the gut, reproductive system, cardiovascular system, tentacle and other muscles.Within the central ganglia many neurones, and many fibres in the neuropile and the nerves entering the ganglia, were stained for the SP-like material. The largest numbers of reactive cell bodies were in the pleural ganglia and on the dorsal surfaces of the pedal ganglia. A group of cells was also found, surrounding the right pedal-cerebral connective, that did not fluoresce, but were enveloped by reactive processes terminating directly onto the neurone somata.Specific staining was observed in all peripheral tissues examined and always appeared to be concentrated in nerve terminals. Most particularly these occurred in the heart and aorta, the pharyngeal retractor muscle and the tentacle. Although mostly present in muscular tissues, some fluorescence was also observed in the nervous layer surrounding the retina. The tentacular ganglion also contained immunoreactive cell bodies.     

Actions of the molluscan neuropeptide FMRF-amide on neurones in the suboesophageal ganglia of the snail Helix aspersa

The effects of the molluscan neuropeptide FMRF-amide were tested on several neurones in the suboesophageal ganglia of the snail Helix aspersa. Almost all neurones tested responded to the peptide, some being hyperpolarized (H response) and others depolarized (D response). The H response is due primarily to an inward potassium current and may be blocked in 20 microM 4-aminopyridine. The hyperpolarizing actions of FMRF-amide and dopamine may be separated by ergometrine which blocks the response to dopamine but not to FMRF-amide. The D response is due mainly to an inward sodium current but this is not blocked by d-tubocurarine, morphine or TTX. It appears to be mediated by a distinct receptor/ionophore as excitation by ACh and 5-HT are both antagonized by d-tubocurarine. The Leu2-substituted analogue FLRF-amide was found to produce similar H responses to FMRF-amide, but was much less potent at producing D responses. It did, however, produce cross-desensitization of the D response to FMRF-amide, suggesting that it does bind to the FMRF-amide receptor.     

An analysis of the 5-hydroxytryptamine (serotonin) receptor subtypes of central neurones of Helix aspersa

1. Intracellular recordings were made from identified neurones in the central nervous system of Helix aspersa. Two types of cell were used, those excited by 5-hydroxytryptamine (5-HT) and acetylcholine and those inhibited by 5-HT and dopamine. The actions of a range of 5-HT agonists and antagonists were tested for their ability to interact with 5-HT receptors.2. 5-Carboxyamidotryptamine, α-methyl-5-HT and N-methyl-5-HT were active on cells excited by 5-HT, with similar potencies to 5-HT. Only 5-carboxyamidotryptamine and 5-methoxytryptamine were equiactive with 5-HT on cells inhibited by 5-HT. Most of the non-indole analogues were inactive or very weak agonists on both receptors.3. MDL 72222 was the most active antagonist tested against 5-HT excitation, showing some selectivity for 5-HT over acetylcholine. Cinanserin and ketanserin also showed selectivity for 5-HT over acetylcholine.4. Tryptamine was inhibitory on both cell types and was a potent antagonist of 5-HT excitation, showing selectivity for 5-HT over acetylcholine.5. It is concluded that the 5-HT excitatory receptor recognizes the indole nucleus with substitution on position 5, save for 5-fluorotryptamine which was inhibitory. It does not appear that these 5-HT receptors can be classified in terms of the vertebrate subtypes of 5-HT receptor. However, it should be noted that only two receptor subtypes located on a small number of neurones were studied in these experiments and other 5-HT receptor subtypes may be located on other groups of neurones and peripheral tissues. These receptors may recognize other 5-HT receptor ligands including non-indoles.     

An analysis of the 5-hydroxytryptamine (serotonin) receptor subtypes of central neurones of Helix aspersa.

1. Intracellular recordings were made from identified neurones in the central nervous system of Helix aspersa. Two types of cell were used, those excited by 5-hydroxytryptamine (5-HT) and acetylcholine and those inhibited by 5-HT and dopamine. The actions of a range of 5-HT agonists and antagonists were tested for their ability to interact with 5-HT receptors. 2. 5-Carboxyamidotryptamine, alpha-methyl-5-HT and N-methyl-5-HT were active on cells excited by 5-HT, with similar potencies to 5-HT. Only 5-carboxyamidotryptamine and 5-methoxytryptamine were equiactive with 5-HT on cells inhibited by 5-HT. Most of the non-indole analogues were inactive or very weak agonists on both receptors. 3. MDL 72222 was the most active antagonist tested against 5-HT excitation, showing some selectivity for 5-HT over acetylcholine. Cinanserin and ketanserin also showed selectivity for 5-HT over acetylcholine. 4. Tryptamine was inhibitory on both cell types and was a potent antagonist of 5-HT excitation, showing selectivity for 5-HT over acetylcholine. 5. It is concluded that the 5-HT excitatory receptor recognizes the indole nucleus with substitution on position 5, save for 5-fluorotryptamine which was inhibitory. It does not appear that these 5-HT receptors can be classified in terms of the vertebrate subtypes of 5-HT receptor. However, it should be noted that only two receptor subtypes located on a small number of neurones were studied in these experiments and other 5-HT receptor suptypes may be located on other groups of neurones and peripheral tissues. These receptors may recognize other 5-HT receptor ligands including non-indoles.     

The possible site of action of 5-hydroxytryptamine, 6-hydroxytryptamine, tryptamine and dopamine on identified neurons in the central nervous system of the snail, Helix aspersa

Intracellular recordings were made from identified neurons in the right parietal ganglion of the snail, Helix aspersa. Cells F 4, 5 and 6 were excited by 5-hydroxytryptamine (5-HT) and inhibited by dopamine while cells in the F 30 area were inhibited by both compounds. Low doses of both tryptamine and 6-HT produced weak excitation of cells F 4, 5 and 6 while higher doses of both compounds inhibit the activity of these cells. In terms of the inhibitory responses, tryptamine and 6-HT are approximately equipotent but between 10 and 100 times less potent than dopamine. d-Tubocurarine reversibly antagonized the excitatory action of 5-HT on cells F 4, 5 and 6 and converted tryptamine and 6-HT excitation to inhibition. In the presence of the antagonist, ergometrine, the dopamine inhibitory response was almost completely abolished while the inhibitory responses to tryptamine and 6-HT were converted to weak excitation. All four agonists inhibited cells in the F 30 area with the following potency ratios: dopamine much greater than tryptamine/6-HT greater than 5-HT. Tubocurarine had no antagonist effects on these responses while ergometrine reduced or blocked all four, often irreversibly. In potassium-free Ringer the inhibitory responses to all four agonists were enhanced. It is concluded that on cells F 4, 5 and 6, low concentrations of tryptamine and 6-HT act on 5-HT receptors while higher concentrations of both agonists act on dopamine receptors. On cells in the F 30 area, 5-HT, 6-HT and tryptamine all act on a dopamine receptor.     

The actions of FxRFamide related neuropeptides on identified neurones from the snail, Helix aspersa

Intracellular recordings were made from identified neurones in the suboesophageal ganglia of the snail, Helix aspersa. The actions of the eight FxRFamide analogues were investigated on these neurones. These peptides included ones isolated from arthropods and nematodes. All the peptides excited certain neurones while inhibiting others, though their relative potencies varied. Overall on neurones inhibited by these peptides the potency order was: DNFLRFamide > FMRFamide > PDVDHVFLRFamide = KNEFIRFamide > FLRFamide > SDRNFLRFamide = SDPNFLRFamide > KHEYLRFamide. However, if the responses are compared on individual cell types, then the picture becomes more complex. For example, on cell F-2, KNEFIRFamide proved to be potent with an EC-50 value of 0.54 microM. On neurones F-13/16 and E-16, PDVDHVFLRFamide was inhibitory while FMRFamide, FLRFamide, SDRNFLRFamide and SDPNFLRFamide were excitatory. In terms of overall excitatory actions, the data are less complete but an approximate order of potency is: FMRFamide > DNFLRFamide > SDPNFLRFamide > PDVDHVFLRFamide > KNEFIRFamide = KHEYLRFamide = SDRNFLRFamide. However this again varies between specific neurones. These results demonstrate that peptides from insects, crustacea and nematodes are active on Helix neurones and may activate specific receptor subtypes, indicating the possible presence of endogenous analogues of these non-molluscan peptides in the Helix nervous system.     

D-Mannitol oxidation in the land snail, Helix aspersa

Respiration by mitochondrial preparations from hepatopancreas tissue of the land snail Helix aspersa is stimulated by D-mannitol. The rate of mannitol-stimulated respiration is approximately one-half that given by succinate, the most effective substrate thus far tested with these preparations. Mannitol-stimulated respiration is cyanide-insensitive but is not inhibited by salicylhydroxamate. The product of the membrane-bound mannitol-oxidizing activity was shown to be D-mannose by thin layer chromatography, high voltage electrophoresis of the germanate and borate complexes, gas chromatography of the trimethylsilyl derivative, low resolution mass spectrometry of the trimethylsilyl derivative, and by an enzymatic method dependent upon phosphomannose isomerase. The reaction mannitol + O2 leads to mannose is stoichiometric; however, it is not known whether O2 is the immediate electron acceptor. The activity in Helix mitochondria is thus unique among most alditol-oxidizing enzymes in not being pyridine nucleotide linked and in acting on carbon 1 rather than carbon 2.     

Transport of pinocytic vesicles in the eye of a snail,Helix aspersa

The heads of small adult snails, Helix aspersa, were injected with horseradish peroxidase (HRP) for one to five hours before extirpating the eyes and preparing them cytochemically for electron microscopy. There was internalization of tracer by pinocytic vesicles (pinosomes) at the bases of types-I and -II sensory cells, ganglion cells and, in lesser amounts, by pigmented supportive cells. Vesicles and vacuoles filled with HRP were transported in two directions: lensward as far distad as the ends of the cells (retrograde) and brainward down the optic nerve (anterograde). We believe that the numerous reacted vacuoles in the cell somata are formed by fusion of vesicles, tubules and C-shaped organelles filled with tracer; we present evidence that they become secondary lysosomes. Sensory cell type II possesses more HRP-reacted vacuoles distally than the other retinal cells. Other vesicles are also described. There was no uptake of tracer by the distal ends of the retinal cells following injection HRP into the hemolymph. The swelling of the optic nerve, immediately behind the eye, contains more HRP-filled pinosomes and vacuoles than does the nerve below the dilatation. The significance of endocytosis and transport of pinosomes within the eye and down the optic nerve is discussed.     

Surfactant in the gas mantle of the snail Helix aspersa

Surfactant occurs in cyclically inflating and deflating, gas-holding structures of vertebrates to reduce the surface tension of the inner fluid lining, thereby preventing collapse and decreasing the work of inflation. Here we determined the presence of surfactant in material lavaged from the airspace in the gas mantle of the pulmonate snail Helix aspersa. Surfactant is characterized by the presence of disaturated phospholipid (DSP), especially disaturated phosphatidylcholine (PC), lavaged from the airspace, by the presence of lamellated osmiophilic bodies (LBs) in the airspaces and epithelial tissue, and by the ability of the lavage to reduce surface tension of fluid in a surface balance. Lavage had a DSP/phospholipid (PL) ratio of 0.085, compared to 0.011 in membranes, with the major PL being PC (45.3%). Cholesterol, the primary fluidizer for pulmonary surfactant, was similar in lavage and in lipids extracted from cell homogenates (cholesterol/PL: 0.04 and 0. 03, respectively). LBs were found in the tissues and airspaces. The surface activity of the lavage material is defined as the ability to reduce surface tension under compression to values much lower than that of water. In addition, surface-active lipids will vary surface tension, increasing it upon inspiration as the surface area expands. By these criteria, the surface activity of lavaged material was poor and most similar to that shown by pulmonary lavage of fish and toads. Snail surfactant displays structures, a biochemical PL profile, and biophysical properties similar to surfactant obtained from primitive fish, teleost swim bladders, the lung of the Dipnoan Neoceratodus forsteri, and the amphibian Bufo marinus. However, the cholesterol/PL and cholesterol/DSP ratios are more similar to the amphibian B. marinus than to the fish, and this similarity may indicate a crucial physicochemical relationship for these lipids.