Specific insulin binding sites in snail (Helix aspersa) ganglia

1. Insulin binding sites were characterized and quantified in snail (Helix aspersa) ganglia by incubation of tissue sections with 125I-porcine insulin, autoradiography with [3H]Ultrofilm, image analysis coupled to computer-assisted microdensitometry, and comparison with 125I-standards. Cellular localization was performed in the same sections by emulsion autoradiography. 2. Specific insulin binding sites were demonstrated in discretely localized groups of neurons of the cerebral, pleural, parietal, visceral, and pedal ganglia and in nerves. Scatchard analysis performed with consecutive sections from single animals revealed a single class of high-affinity insulin binding sites (Kd, 0.13 +/- 0.01 nM; Bmax, 157 +/- 10 fmol/mg protein). 3. Our results suggest that insulin may play a role as a neurotransmitter or neuromodulator in snail ganglia.     

Nervous system of the snail Helix aspersa

Summary The fine structure of vascular channels and amebocytes associated with the sheath of the infraesophageal ganglion of Helix aspersa, is described. The extracellular stroma of the sheath, together with the hemocoel and blood vessels, forms an interconnected system of pathways which appears to be involved in the transport of metabolites, amebocytes, hemocyanin and experimentally introduced opaque tracers. The hemocoel, blood capillaries and precapillaries are lined by a discontinuous layer of single muscle cells whose luminal aspect is covered by a lamina of extracellular material named the vascular coat. This coat consists of a ground substance that forms a basement membrane and filamentous elements some of which are collagenous. Gaps in the blood vessel wall seem to provide the main routes for the movement of cells and large molecules to the hemocoel. Tracer experiments have given support to the idea that a diffusion barrier may be absent at the sheath-ganglion junction. Amebocytes have phagocytic properties; they appear associated in groups or scattered singly within the extracellular space of the sheath and the lumen of blood vessels. Single amebocytes have features of mobile cells and may function in the transport of hemocyanin as well as other proteins.This work has been supported by the Rockefeller Foundation and grants NB 06662 (from the U.S. Public Health Service) and N-105 (from Conicyt, Santiago, Chile). The continuous advice and encouragement of Drs. R. W. Guillery and D. B. Slautterback are gratefully acknowledged.     

DARPP-32 like protein in specific snail (Helix aspersa) neurones

Summary Monoclonal antibodies to DARPP-32 recognise an antigen which is present in specific neurones in the snail (Helix aspersa). Consecutive sections 10-m-thick processed for the localisation of DARPP-32 and tyrosine-hydroxylase immunoreactivity did not show a coexistence in any neuronal structures. DARPP-32 positive cells were, however, often morphologically closely associated with tyrosine-hydroxylase positive cells, implying a functional relationship consistent with the proposed role of DARPP-32. Immunochemical analysis of the DARPP-32 immunoreative material in the snail nervous system shows that the substance has a molecular weight of 28 kDa and therefore different from the DARPP-32 protein found in the rat brain.     

DARPP-32 like protein in specific snail (Helix aspersa) neurones.

Monoclonal antibodies to DARPP-32 recognise an antigen which is present in specific neurones in the snail (Helix aspersa). Consecutive sections 10-microns-thick processed for the localisation of DARPP-32 and tyrosine-hydroxylase immunoreactivity did not show a coexistence in any neuronal structures. DARPP-32 positive cells were, however, often morphologically closely associated with tyrosine-hydroxylase positive cells, implying a functional relationship consistent with the proposed role of DARPP-32. Immunochemical analysis of the DARPP-32 immunoreactive material in the snail nervous system shows that the substance has a molecular weight of 28 kDa and therefore different from the DARPP-32 protein found in the rat brain.     

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.     

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.     

The fine structure of the collar cells in the optic tentacles of Helix aspersa

Summary At the base of the optic tentacular ganglion there is a group of large monopolar cells containing numerous secretory inclusions. These are the collar cells. Secretory material can be seen accumulating in swollen portions of the granular endoplasmic reticulum. It is postulated that this material is transported to the Golgi bodies and thus the limiting membrane of the inclusions is derived from the Golgi membranes. The Golgi bodies appear to be polarized and small vesicles resembling secretory inclusions are associated with one face of these organelles. The secretory inclusions fuse together to form large membrane-bound secretory pools in the perikaryon. The collar-cell processes are packed with secretory inclusions. These processes traverse the digital extensions of the tentacular ganglion and pass into the epithelium covering the tip of the tentacle. The secretory inclusions do not resemble neurosecretory inclusions in other situations. The collar cell processes receive a nerve supply from single axons containing granular and agranular vesicles. The evidence that these cells may be modified neurons is only minimal.This work was supported by the Australian Research Grants Committee.     

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.     

Unpredictable responses of garden snail (Helix aspersa) populations to climate change

We studied the impact of climate change on the population dynamics of the garden snail (Helix aspersa) in the Ecotron controlled environment facility. The experimental series ran for three plant generations, allowing the snails to reproduce. We investigated the isolated and combined effects of elevated CO₂ (current + 200 μmol mol^–1) and warming (current + 2ºC) in three consecutive runs (CO₂, Temperature and Combined). In the CO₂ Run, the number of juvenile snails recorded at the end of the experiment did not differ between ambient and elevated CO₂, whereas in the Temperature Run, fewer juveniles were found at elevated temperatures. An opposite response was observed in the Combined Run, where significantly more juveniles were found in elevated temperature and CO₂ compared to elevated CO₂ on its own. Within each run, juvenile emergence was not affected by treatments but juvenile presence was first observed about 70 days earlier in the Combined Run than in the Temperature Run. The differences in snail performance in the different runs were not correlated with differences in community structure or leaf quality measured as C:N ratios and neither with the abundance of the most preferred host plant species, Cardamine hirsuta. The abundance of this species, however, was significantly altered in all runs. The results illustrate clearly the degree of difficulty in making predictable generalisations about the consequences of climate change for certain species.     

Dart shooting influences paternal reproductive success in the snail Helix aspersa (Pulmonata, Stylommatophora)

Although animal courtship behaviors are generally understoodwithin the context of sexual selection, the relevance of manysexual behaviors to sexual selection, and vice versa, remainsunexplained. For example, the adaptive function of the "lovedart" used in the precopulatory behavior of hermaphroditicland snails is only now becoming apparent. Contrary to previous assumptions, dart shooting is unlikely to function as a stimulusfor copulation. In searching for a more ultimate explanationof the dart's function, we tested whether variation in dartshooting influences reproductive fitness in Helix aspersa.Individual mother snails were mated sequentially to two potentialfathers. Dart shooting was closely observed and quantifiedfor all pairings, and percentages of offspring sired by each potential father were determined using allozymes. The resultsindicate that snails that shoot darts effectively have significantlygreater paternal reproductive success than snails that shootpoorly. In contrast, there was no significant effect of matingorder on either dart shooting or paternal reproductive success.     

Brain cells that command sexual behavior in the snail Helix aspersa

Evidence is presented indicating that the mesocerebrum of the terrestrial snail, Helix aspersa, has a major role in the control of sexual behavior. Morphological and physiological results demonstrate a right-sided bias in the mesocerebrum that is consistent with the fact that sexual behavior is executed almost entirely on the animal's right side. Thus, the right lobe has 23% more neurons than the left lobe, and they are 24% larger. Excitatory synaptic inputs derive predominately from neurons on the right side. The axons of right-side mesocerebral neurons go to the right pedal ganglion almost without exception, and even the axons of left-side neurons travel mostly in right-side connective nerves. Direct evidence for a role of the mesocerebrum in commanding sexual behavior comes from experiments with electrical stimulation. Extracellular stimulation of the right mesocerebrum, but not the left mesocerebrum, resulted in movements of the "love dart" sac and the penis. Intracellular stimulation of neurons in the right mesocerebrum evoked measurable movements of either the dart sac or the penis, or both, in 17% of the cells tested. The latencies ranged between 5 and 50 s. In an intact animal, these movements would cause a release of the dart and an eversion of the penis. The motor effects were mediated through the right cerebropedal connective and the pedal nerve NCPD, with the motorneurons probably situated in the right pedal ganglion.     

Neuronal background of positioning of the posterior tentacles in the snail Helix pomatia

The location of cerebral neurons innervating the three recently described flexor muscles involved in the orientation of the posterior tentacles was investigated by applying parallel retrograde Co- and Ni-lysine tracing via the olfactory and the peritentacular nerves. Their innervation patterns in the flexor muscles were studied by applying anterograde neurobiotin tracings via these nerves. The labeled neurons are clustered in eight groups in the cerebral ganglion. They send both common and distinct innervation pathways to the flexor and the tegumental muscles and to the tentacular retractor muscle. The common pathway reaches the muscles via the olfactory nerve, whereas the distinct pathways innervate via the internal and external peritentacular nerves. The three anchoring points of the three flexor muscles at the base of the tentacle outline the directions of three force vectors generated by the contraction of the muscles and enable the protracted tentacle to bend around a basal pivot. In the light of earlier physiological and the present anatomical findings, we suggest that the common innervation pathway to the muscles is required for tentacle withdrawal and the retractor mechanism, whereas the distinct pathways primarily serve the bending of the protracted posterior tentacles during foraging.