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PubMed1992

Interaction of sulfhydryl reagents with A-type channels of Lymnaea neurons.

The effect of sulfhydryl reagents on macroscopic inactivation of A-current in internally perfused Lymnaea neurons under voltage-clamp conditions was investigated. It was found that the binding of Hg2+ rather than PHMB with channel proteins resulted in a strong decrease of the peak current and the inactivation rate. Hg2+ markedly influenced the steady-state inactivation but did not change the rate of recovery from inactivation. It was found that both reagents reacted with the same groups of the channel protein and that those are most likely sulfhydryl groups. These groups seemed not to be involved in the gating charge movement. Hg2+ ions can immobilize some part of the gating charge thereby resulting in strong changes of the steady-state inactivation.

Animals↗
PubMed1993

Kinetic study of A-type current inactivation in Lymnaea neurons.

Macroscopic inactivation of A-current was studied in internally perfused Lymnaea neurons under voltage clamp conditions. Inactivation kinetics were satisfactorily described by the sum of two exponentials, suggesting the presence of two type inactivation. The kinetics of recovery from inactivation were exponential. The rate constants of the fast phase of inactivation gamma f(V) rose steeply with depolarization exposing the pronounced plateau in the range from -30 to 0 mV. The time course of inactivation in this potential range was more closely approximated with the sum of three exponentially decaying components. Calcium and hydrogen ions strongly affected the fast phase of inactivation. Calcium gave a positive shift of a part of the gamma f(V) curve on the left of the plateau. Raising the pH caused a negative shift of the right-hand branch of the gamma f(V) curve. It was shown that these effects are associated with Ca2+ and H+ binding to some specific sites of the channel protein. Two models give good fits with the experimental data. They include two pathways for fast inactivation. Calcium and hydrogen ions are assumed to selectively affect the voltage-dependent transitions related to these pathways of inactivation.

Animals↗
PubMed1982

Suppression of egg-laying during starvation in the snail Lymnaea stagnalis by inhibition of the ovulation hormone producing caudo-dorsal cells.

Starvation inhibits egg-laying in the snail, Lymnaea stagnalis. In starved animals the neurosecretory Caudo-Dorsal Cells (CDC), which produce the egg-laying hormone, are hyperpolarized as compared to the CDC of controls. However, they are more responsive to repetitive intracellular stimulation, which induces the hormone releasing discharge. Hyperpolarization is not found in the non-neurosecretory Cerebral Giant Cells, which indicates that the effect is specific for the CDC. It is also a characteristic effect of starvation as compared to another treatment (dirty water) inhibiting egg-laying.

Animals↗
PubMed1985

Heterogeneity of peptides released by electrically active neuroendocrine caudodorsal cells of Lymnaea stagnalis.

The peptidergic caudodorsal cells (CDC) are located in the cerebral ganglia (CG) of the snail Lymnaea stagnalis. They have the periphery of the intercerebral commissure (COM) as the neurohaemal area. Release of the ovulation hormone (CDCH) and other presumed peptides by electrically active CDC was studied in vitro using CG/COM preparations which were preincubated with radioactive amino acids. Electrical activity of the CDC was induced by electrical stimulation of these cells, and by adenosine 3',5'-cyclic phosphate (cAMP) or potassium depolarization. Four classes of radioactively-labelled peptides are released by active CDC. They have molecular weights of greater than or equal to 6000, approximately 5500, approximately 4500 and approximately 2500 daltons, respectively. Released CDCH bioactivity co-eluted with the approximately 4500 dalton radioactive peptides. Selective removal of CDC demonstrated that the CDC release these peptides. cAMP and potassium depolarization also induce release of these peptides. The release after potassium depolarization is dependent upon the presence of calcium ions. CG/COMs from juvenile snails are not capable of releasing the peptides. With Bio-Gel P-6 chromatography, sodium dodecyl sulphate urea polyacrylamide electrophoresis, isoelectric focusing and proteolytic enzymes it was found that released CDCH is a peptide with the same molecular weight and pI as CDCH stored in the COM (molecular weight approximately 4700; pI approximately 9.3).

Animals↗
PubMed1986

Age-related decrease in electrical coupling of two identified neurons in the mollusc Lymnaea stagnalis.

Electrophysiological characteristics of two identified giant electrotonically coupled neurosecretory cells in the central nervous system of the mollusc Lymnaea stagnalis were studied in mature animals of different age. The coupling coefficient of the neurons decreased considerably with age. The possibility that the decrease is due to an increase in the junctional resistance between the cells is discussed.

Aging↗
PubMed1986

Role of cAMP in electrical and secretory activity of the neuroendocrine caudo-dorsal cells of Lymnaea stagnalis.

The peptidergic neuroendocrine caudo-dorsal cells (CDC) in the cerebral ganglia of the freshwater snail Lymnaea stagnalis L. produce an ovulation-stimulating neurohormone (CDCH). Release occurs by exocytosis in a calcium-dependent way from axon terminals in the periphery of the intercerebral commissure, particularly during a period of electrical activity (the 'discharge'). An important factor in electrical and, hence, secretory activity of the CDC appears to be cyclic adenosine 3', 5'-monophosphate (cAMP). Incubation of cerebral ganglia in snail Ringer with the cAMP-analogue 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) or with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) leads to activation of the CDC: electrophysiological, quantitative electron microscopic and bioassay studies show that incubation results in the onset of intense electrical activity, a marked reduction in the number of secretory granules in the axon terminals, an enormous increase in the number of exocytosis phenomena and a strong stimulation of CDCH-release. It is assumed that treatment with IBMX or with cpt-cAMP mimics a rise in the cytoplasmic level of cAMP when the CDC become activated by a physiological stimulus. This rise most likely effectuates a permeability change of the axolemma for ions involved in the discharge. As a consequence of the depolarization of the axolemma during the discharge, calcium ions would enter the axon terminal and induce exocytotic release of CDCH.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Quantitative immunoelectron microscopy and tannic acid study of dynamics of neurohaemal and non-synaptic peptide release by the caudodorsal cells of Lymnaea stagnalis.

The caudodorsal cells (CDCs) of the freshwater snail Lymnaea stagnalis control egg laying and egg-laying associated behaviours by releasing various peptides including the ovulation hormone CDCH. Previously it has been shown that release occurs (1) into the haemolymph from neurohaemal axon terminals in the outer compartment of the cerebral commissure, and (2) into the intercellular space of the central nervous system from non-synaptic release sites of axon collaterals in the inner compartment of the commissure. Outer and inner compartments are separated by a sheath of glial cells. In the present study the secretory dynamics of neurohaemal and collateral release have been studied. Immunoelectron microscopy with an antibody against a synthesized fragment of the egg-laying hormone [CDCH] indicates that CDCH is released by exocytosis from both sites: positive immunoreaction was found for the contents of secretory granules and contents that underwent exocytosis, and furthermore in the intercellular spaces of the inner and outer compartments. Quantitative (immunogold) electron microscopy combined with either the tannic acid-glutaraldehyde-osmium tetroxide (TAGO) method or the tannic acid-Ringer incubation (TARI) method for the visualization and quantification of exocytosis of CDCH, shows different dynamics of neurohaemal and collateral CDCH release. Neurohaemal release is strongly increased during electrical activity of the CDCs (active state). This increase does not only appear from an increased number of (immunopositive) exocytoses (3X) but also from increases in (1) the percentages of all stationary and all exocytosing granule contents that are immunopositive (both increase from 70% to 85%), (2) the degree of immunopositivity per exteriorized granule content (2X) and (3) the degree of immunopositivity in the intercellular space of the neurohaemal area (5X). Collaterals show a different picture: CDCH release particularly occurs during electrical silence (resting and inhibited states). No effect was noted of the electrical state of the CDCs on the percentages of CDCH-immunoreactive stationary or exteriorized granule contents, nor on the degree of immunopositivity of the exteriorized contents. Furthermore, the degree of immunopositivity in the intercellular space of the inner compartment is drastically decreased (8X). Finally, both in the resting and the active state, the percentage of CDCH-positive exocytosing contents in the collaterals is smaller than that of CDCH-positive stationary contents whereas in the neurohaemal area these percentages do not differ.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis.

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an after discharge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Distribution of catecholamines and of immunoreactivity to substances like vertebrate enzymes for the synthesis of catecholamines within the central nervous system of the snail, Lymnaea stagnalis.

Catecholamines (CAs) were detected histochemically within over 185 cell bodies in the central nervous system (CNS) of juvenile and young adult Lymnaea. This distribution of CA-containing cells in all central ganglia except the pleural ganglia is more widespread than previously described but is consistent with other reports suggesting numerous roles for CAs within the nervous system. This study also describes the distribution of substances which are antigenically similar to four bovine enzymes for catecholamine synthesis, but the distribution patterns showed little or no overlap with each other or with CA. These results suggest the need for caution in the interpretation of such immunohistochemical studies.

Aldehydes↗
PubMed1991

Differential responses of two identified neurons of the pond snail Lymnaea stagnalis to the convulsant drug pentylenetetrazol.

The convulsant drug pentylenetetrazol (PTZ) induces tonic depolarization in the identified B1 neuron of Lymnaea stagnalis. Another identified neuron, right parietal dorsal 1 (RPD1), gives the opposite response: tonic hyperpolarization and cessation of spontaneous firing. These effects are mimicked by intracellular injection of calcium ions and reversed following injection of ethylene glycol-bis(beta-aminoethyl ether) N,N,N,'N'-tetraacetic acid (EGTA). Responses to PTZ are retained in the presence of extracellular cobalt ions to block calcium influx. Under voltage clamp, PTZ or injection of calcium ions induces a slow inward current in B1, which is abolished in zero sodium saline. In RPD1 the current response to PTZ or intracellular calcium ions is outward, and is blocked by potassium channel blockers. Thus, differential responses of the two neurons to PTZ appear to be mediated via increased intracellular calcium ions, leading to activation of specific ion currents in each cell type.

Action Potentials↗
PubMed1991

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)

Animals↗
PubMed1994

Peptide processing and release by the neuroendocrine caudodorsal cells of Lymnaea stagnalis during an egg-laying cycle.

The freshwater mollusc Lymnaea stagnalis exhibits an egg-laying cycle that takes about 24 h. The cycle is under control of the cerebral neuroendocrine Caudodorsal Cells (CDC), which release various peptides, one of which is the ovulation-inducing hormone, CDCH. During the egg-laying cycle the CDC reveal three consecutive states of electrical activity, the resting, the active and the inhibited state. Processing and release of CDCH have been studied in relation to the states of electrical (and secretory) activity of the CDC, with quantitative immuno-electron microscopy and a newly raised CDCH antiserum. In somata of CDC in the resting state 1 (just before activation of the CDC), in the active state (30 min after activation) and in the inhibited state (3 h after activation) more secretory granules are immunolabelled, and to a higher degree, than in somata of CDC in the resting state 2 (10 h after activation). Axon terminals of CDC in resting states 1 and 2 are equally immunoreactive, whereas in terminals in the active state more granules are labelled, and to a higher degree, than in the resting states. Secretory granules in terminals in the inhibited state are intermediate in both respects. Immuno-electron microscopy combined with the tannic acid method for the demonstration of exocytosis showed that in terminals in the active state, the percentage of immunolabelled exocytosed granule contents is much higher than in the other states. The same holds for the degree of immunopositivity of these contents.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A neuronal network from the mollusc Lymnaea stagnalis.

The morphology, electrophysiology, and synaptic inputs of a ventrally located neuronal network from the CNS of the pond snail Lymnaea stagnalis was investigated. Three large, previously identified neurons [55] known as right parietal ventral one, two, and three (RPV1,2,&3) were found to be electrically coupled to one another. Coupling between either RPV1&2 or RPV1&3 was weak while coupling between RPV2&3 was strong. Consistent bursting activity was observed in neuron RPV1 while neurons RPV2&3 were either silent or fired tonically. When isolated in vitro, similar patterns of activity could be elicited in neurons RPV1-3. Lucifer yellow staining revealed that these cells send axons through nerves innervating musculature involved in locomotion, whole-body withdrawal, and cardio-respiratory function. Neurons RPV1-3 were found to be inhibited by an identified interneuron, visceral dorsal four, known to be directly involved in cardio-respiratory behavior [43]. Furthermore, neurons RPV1-3 were also inhibited by a wide-acting synaptic input, known as Input three [9], which is associated with respiratory pattern generation [43]. An interneuron, identified as right pedal dorsal eleven (RPeD11), which coordinates locomotory and withdrawal behavior [44], was found to excite neuron RPV1. When neurons RPeD11 and RPV1 were isolated in vitro and allowed to extend neurites, they formed a synaptic connection similar to that observed in the isolated brain. In vitro work on these neurons may make them an attractive model to study synapse formation and bursting activity.

Animals↗
PubMed1987

Further pharmacological characterization of a D-2-like dopamine receptor on growth hormone producing cells in Lymnaea stagnalis.

A preliminary study has revealed that a mammalian D-2-like dopamine (DA) receptor mediates hyperpolarization of the neuroendocrine growth hormone-producing cells (GHCs) in the snail Lymnaea stagnalis. An extensive pharmacological characterization of this receptor was performed in the present study. Several mammalian D-2 receptor agonists (e.g. aminotetralins) and antagonists (e.g.(-)-sulpiride) showed agonistic and antagonistic effects, respectively. However, some selective D-2 receptor agonists (e.g. N 0437) and antagonists (e.g. domperidone) failed to show agonistic or antagonistic effects, respectively. It is concluded that the dopamine receptor mediating hyperpolarization of the GHCs displays, besides some similarities, several differences from the mammalian D-2 receptor.

Animals↗
PubMed1996

Functional characterisation of a 5-HT2 receptor cDNA cloned from Lymnaea stagnalis.

A G-protein-coupled receptor (5-HT2Lym) resembling members of the 5-HT2 receptor subfamily was cloned from the mollusc Lymnaea stagnalis. Serotonin induces a concentration-dependent increase in intracellular inositol phospates in HEK293 cells expressing this receptor (EC50 = 114 nM). 5-HT2Lym differs from mammalian 5-HT2 receptors by the presence of a large amino-terminal region. This large domain appears to preclude an adequate level of expression of 5-HT2Lym in HEK293. Therefore, we constructed a cDNA encoding an amino-terminally truncated receptor (delta N-5-HT2Lym) that appeared to be much better expressed in HEK293 cells. delta N-5-HT2Lym-expressing cells exhibit a serotonin-induced stimulation of phosphatidylinositol bisphosphate hydrolysis (EC50 = 11.4 nM) and a high-affinity binding of the 5-HT2-selective antagonist [3H]mesulergine (Kd = 4 nM). Inhibition of this binding by several 5-HT2 antagonists and agonists revealed a pharmacological profile most closely resembling those of 5HT2Dro, 5-HT2B and 5-HT2C.

Amino Acid Sequence↗
PubMed1988

Trichobilharzia ocellata: interference with endocrine control of female reproduction of Lymnaea stagnalis.

Calfluxin (CaFl), one of the gonadotropic hormones of Lymnaea stagnalis, stimulates the influx of Ca2+ into the mitochondria of the cells of the albumen gland, one of the accessory sex organs of the snail. This effect is suppressed in glands of noninfected snails by an agent (schistosomin) present in the hemolymph of snails infected by Trichobilharzia ocellata as shown in in vitro experiments. The agent is present from 6 weeks postinfection onward. Ca2+ deposits in the mitochondria were demonstrated with the ultracytochemical antimonate precipitation technique. The percentage of Ca2+-positive mitochondria was taken as a measure for the effects of CaFl. This percentage appeared to be greatly reduced when glands were incubated in serum of infected snails (Sinf). The data showed that Ringer incubations can serve as controls for experiments with serum: no differences were found between Ringer incubations and incubations in either fresh or frozen serum of noninfected snails. Schistosomin was not affected by freezing, which enables cold storage of Sinf. The dose-response relationship of schistosomin shows that at a 1:2 dilution of Sinf with Ringer the response to CaFl was reduced more than 50%. Schistosomin is heat-stable and Pronase-labile, which indicates that it has a peptide nature. Probably schistosomin(s) is responsible for the reduction/cessation of fecundity in trematode-infected snails.

Animals↗
PubMed1989

Trichobilharzia ocellata: influence of infection on the interaction between the dorsal body hormone, a female gonadotropic hormone, and the follicle cells in the gonad of the intermediate snail host Lymnaea stagnalis.

Activation of adenylate cyclase (AC)-cAMP system in follicle cells of Lymnaea stagnalis by the gonadotropic dorsal body hormone (DBH) is inhibited by schistosomin, an agent present in hemolymph of snails infected with Trichobilharzia ocellata. AC activation was determined enzyme cytochemically. This conclusion is based on the observation that the percentage of oocytes with AC-positive follicle cells in gonads incubated in the presence of schistosomin, i.e., in serum of infected snails (IS) with DBH, is significantly lower than that in gonads incubated in the absence of schistosomin, i.e., in serum of noninfected snails (NS) with DBH. Follicle cells in gonads preincubated in the absence of schistosomin, i.e., in NS, and subsequently incubated with freshly dissolved DBH showed a considerably lower response to DBH than those in not preincubated gonads. This indicates that the number of receptors for DBH on follicle cells had decreased during preincubation. The response to DBH also appeared to decrease when the hormone was preincubated in NS. This indicates that the activity of DBH decreases during preincubation. These data make it impossible to answer the question of whether or not schistosomin acts as an antagonist of DBH at the receptor level.

Adenylyl Cyclases↗