Peptidergic neurons in the snail Helix pomatia: Distribution of neurons in the central and peripheral nervous systems that react with an Antibody raised to the insect neuropeptide,leucokinin I

In this study, antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was: used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.     

Control of locomotion in marine mollusc — Clione limacina. V. Photoinactivation of efferent neurons

Summary Efferent neurons in isolated pedal ganglia of the pteropodial mollusc Clione limacina were filled with Lucifer Yellow through the wing nerves. Then the ganglia were illuminated with intense blue light which resulted in the complete inactivation of these neurons. After inactivation of efferent neurons, interneurons of the pedal ganglia continued to generate the locomotor rhythm.     

Control of locomotion in the marine mollusc Clione limacina

The locomotor activity in the marine mollusc Clione limacina has been found to be strongly excited by serotonergic mechanisms. In the present study putative serotonergic cerebropedal neurons were recorded simultaneously with pedal locomotor motoneurons and interneurons. Stimulation of serotonergic neurons produced acceleration of the locomotor rhythm and strengthening of motoneuron discharges. These effects were accompanied by depolarization of motoneurons, while depolarization of the generator interneurons was considerably lower (if it occurred at all). Effects of serotonin application on isolated locomotor and non-locomotor pedal neurons were studied. Serotonin (5×10^-7 to 1×10^-6 M) affected most pedal neurons. All locomotor neurons were excited by serotonin. This suggests that serotonergic command neurons exert direct influence on locomotor neurons. Effects of serotonin on nonlocomotor neurons were diverse, most neurons being inhibited by serotonin. Some effects of serotonin on locomotor neurons could not be reproduced by neuron depolarization. This suggests that, along with depolarization, serotonin modulates voltage-sensitive membrane properties of the neurons. As a result, serotonin promotes the endogenous rhythmical activity in neurons of the C. limacina locomotor central pattern generator.     

Spiral and target patterns in bivalve nacre manifest a natural excitable medium from layer growth of a biological liquid crystal

Nacre is an exquisitely structured biocomposite of the calcium carbonate mineral aragonite with small amounts of proteins and the polysaccharide chitin. For many years, it has been the subject of research, not just because of its beauty, but also to discover how nature can produce such a superior product with excellent mechanical properties from such relatively weak raw materials. Four decades ago, Wada [Wada K (1966) Spiral growth of nacre. Nature 211:1427] proposed that the spiral patterns in nacre could be explained by using the theory Frank [Frank F (1949) The influence of dislocations on crystal growth. Discuss Faraday Soc 5:48–54] had put forward of the growth of crystals by means of screw dislocations. Frank''s mechanism of crystal growth has been amply confirmed by experimental observations of screw dislocations in crystals, but it is a growth mechanism for a single crystal, with growth fronts of molecules. However, the growth fronts composed of many tablets of crystalline aragonite visible in micrographs of nacre are not a molecular-scale but a mesoscale phenomenon, so it has not been evident how the Frank mechanism might be of relevance. Here, we demonstrate that nacre growth is organized around a liquid-crystal core of chitin crystallites, a skeleton that the other components of nacre subsequently flesh out in a process of hierarchical self-assembly. We establish that spiral and target patterns can arise in a liquid crystal formed layer by layer through the Burton–Cabrera–Frank [Burton W, Cabrera N, Frank F (1951) The growth of crystals and the equilibrium structure of their surfaces. Philos Trans R Soc London Ser A 243:299–358] dynamics, and furthermore that this layer growth mechanism is an instance of an important class of physical systems termed excitable media. Artificial liquid crystals grown in this way may have many technological applications.     

Pronase Acutely Modifies High Voltage-activated Calcium Currents and Cell Properties of Lymnaea Neurons

Pronase E ('pronase') is one of the proteolytic enzymes that are used in preparative procedures such as cell isolation and to soften the sheath of invertebrate ganglia. Although several effects of proteolytic enzymes on the physiology of non-neuronal tissues have been described, the effects of these enzymes on central neurons have received little attention. We examined the effects of bath-applied pronase on neurons in the Lymnaea central nervous system and in vitro . Pronase caused action potential broadening in neurons that exhibit a shoulder on the repolarization phase of their action potentials. This effect of pronase was accompanied by, although unrelated to, a depolarization and decrease in action potential interval. Some, but not all, effects of pronase in the central nervous system were reversible. For example, the decreases in membrane potential and action potential interval were both reversed after ∼1 h of washing with saline. However, the effect of pronase on the action potential duration was not reversed after a period of 90 min. The modulation of action potential width prompted us to examine Ca²⁺ currents. Exposure to pronase resulted in an increase in both peak and late high voltage-activated Ca²⁺ currents in isolated neurons. Pronase neither changed the inactivation rate nor caused a shift in the current-voltage relationship of the current. The changes in action potential duration could be prevented by application of 0.1 mM Cd²⁺, indicating that the action potential broadening caused by pronase depends on Ca²⁺ influx. This is the first systematic study of the acute and direct actions of pronase on Ca²⁺ currents and cell properties both in the CNS and in vitro .     

An insight on the neuropharmacological activity of Telescopium telescopium– a mollusc from the Sunderban mangrove

The present study was carried out to evaluate the biological properties of the tissue extract of a marine snail Telescopium telescopium, collected from the coastal regions of West Bengal India. On extensive pharmacological screening, it was found that the biological extract of T. telescopium (TTE) produced significant central nervous system (CNS)‐depressant activity as observed from the reduced spontaneous motility, potentiation of pentobarbitone induced sleeping time, hypothermia and respiratory depression with transient apnoea. The extract significantly decreased both residual curiosity and also muscle coordination. The fraction, obtained following saturation with 60–80% ammonium sulphate (80S), was also found to demonstrate predominant CNS‐depressant activity. It was observed that both TTE and the 80S fraction significantly altered the brain noradrenaline and homovanillic acid levels without affecting the brain gamma amino butyric acid (GABA) concentration. Based on the present observations, it can be suggested that the CNS‐depressant effects produced by TTE and 80S could be attributable to modified catecholamine metabolism in the brain.     

Neurotransmitters in the nervous system of Macoma balthica (Bivalvia)

The distribution of histamine-, octopamine-, gamma-aminobutyric acid- (GABA) and taurine-like immunoreactivity in the bivalve mollusc Macoma balthica was studied immunocytochemically with antisera produced in rabbits. Histamine levels in the ganglia and whole animals were also measured by high-performance liquid chromatography using a postcolumn derivatization method. Immunoreactivity for these substances, except for taurine, is found in the central nervous system of this species. The most extensive neuronal system is revealed with the antiserum against histamine. All the main ganglia contain histamine-immunoreactive cell bodies, and a dense network of nerve fibers is seen in the ganglia and nerve roots. Histamine-immunoreactive nerve fibers project to the mantle edge, lips and oesophagus. The basal part of the inhalant siphon is rich in histamine-immunoreactive fibers. Unlike histamine, octopamine- and GABA-like immunoreactivities are restricted to the central nervous system. Taurine-like immunoreactivity is not found in the nervous system of this species. In the nervous system, histamine-immunoreactive cell bodies and fibers are more numerous than those that are octopamine- and GABA-immunoreactive. The distribution of these substance in the ganglia is different. GABA-immunoreactive cells are typically smaller than most of the histamine- and octopamine-immunoreactive cells. Most GABA- and octopamine-immunoreactive cells and fibers are located in the pedal ganglion. Histamine is distributed more evenly in the ganglia and nerve roots. The biochemical measurements of histamine correlate well with the immunohistochemical findings and confirm the predominant location of the amine in the nervous tissue. These results suggest that histamine is more widespread than some other putative transmitters, and support the concept that histamine may have an important role in many physiological processes in molluscs. © 1993 Wiley-Liss, Inc.     

Input-output relationships of identified buccal neurones involved in feeding control in Aplysia

A group of about 28 neurones located in the lateral portion of the caudal face of Aplysia buccal ganglion and projecting into the cerebro-buccal connective were identified by retrograde cobalt staining, and designated as L neurones. It was found that the L neurones did not establish synaptic relations with the known buccal neurones, which are mainly involved in the production of the consummatory phase of feeding, nor with several cerebral neurones tested, including the well-known serotonin giant cell. Neither did they show responses to stimulation of the nerves directed to the buccal mass. On the other hand, the L neurones showed depolarizing responses, with the possible addition of a weak, slower hyperpolarizing phase, to stimulation of the ipsi- and contralateral oesophageal nerves, which innervate the portion of the gut posterior to the buccal mass. These findings, together with several properties of the oesophageal nerve input, suggest that one function of the L cells is to transmit information about gut regions posterior to the buccal mass towards the cerebral ganglia, and that they may mediate the inhibitory influence which in Aplysia is known to be exerted upon feeding by the presence of bulk in the anterior gut. The L neurones showed synaptic responses - consisting mainly or exclusively of depolarizations - to stimulation of the cerebro-buccal connectives. Besides this, large, tonic EPSPs, which often occurred in the 'spontaneous' activity of the L neurones, were found to be generated by spikes that travelled in the cerebro-buccal connective towards the buccal ganglion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The number and size of neurons in the CNS of gastropod molluscs and their suitability for optical recording of activity

Several factors that affect the suitability of opticaL methods for monitoring neuron activity were evaluated in several species of gastropod molluscs. The mean cell body diameter and the total number of cells in the central nervous system were determined In 6 preparations and qualitative evaluations were made for an additional 25 species. There was a factor of 10 difference in mean diameter between species with the smallest cells (prosobranchs) and those with the largest (certain opisthobranchs). Several opisthobranchs had about 5000 central neurons; we estimate that the prosobranchs and pulmonates had at least 5 times as many neurons. When the opacity to transmitted light was measured the percent transmissions of circumesophageal ganglia were between 4% and 40%. We attempted to measure optical signals associated with spike activity in 20 gastropod species; in most species signals were readily detected in single trials.     

Distribution of the peptide Ala-Pro-Gly-Trp-NH2 (APGWamide) in the nervous system and periphery of the snail Lymnaea stagnalis as revealed by immunocytochemistry and in situ hybridization.

Immunocytochemistry and in situ hybridization were used to identify 200-250 central neurons that synthesize and contain the peptide APGWamide in the central nervous system of Lymnaea. The majority of these cells reside within the right anterior lobe of the cerebral ganglion and most appear to have projections to the ventral lobe of the ganglion. The neurites then branch to innervate the lobe and to send further projections into the penial nerve and to the PeIb cluster of the right pedal ganglion. The right ventral lobe also contains a cluster of about 30-40 somata, which apparently synthesize and contain APGWamide. Other populations of cells found in both the right and left anterior lobes project ipsilaterally to the pleural, parietal, and visceral ganglia. Prominent somata are also located in clusters in the visceral and right parietal ganglia. These cells project ipsilaterally into caudal neuropilar regions of the cerebral ganglia. Peripheral projections of central neurons form a dense plexus of varicose, APGWamide-containing fibres in superficial layers of the penis and preputium. Other peripheral projections were noted in the prostate and vas deferens. No peripherally located cell bodies appeared to contain or synthesize the peptide. The results show that APGWamide is widely present in the central nervous system and male reproductive organs and suggest that it plays a major role in control of reproduction.