Synapse-specific changes in serotonin signalling contribute to age-related changes in the feeding behaviour of the pond snail, Lymnaea

This study utilised the pond snail, Lymnaea to examine the contribution that alterations in serotonergic signalling make to age-related changes in feeding. Age-related decreases in 5-HIAA levels in feeding ganglia were positively correlated with a decrease in the number of sucrose-evoked bites and negatively correlated with an increase in inter-bite interval, implicating alterations in serotonergic signalling in the aged phenotype. Analysis of the serotonergic cerebral giant cell (CGC) input to the protraction motor neurone (B1) demonstrated that fluoxetine (10–100 nM) increased the amplitude/duration of the evoked EPSP in both young and middle aged but not in old neurones, suggesting an age-related attenuation of the serotonin transporter. 5-HT evoked a concentration-dependent increase in the amplitude/duration of B1 EPSP, which was greater in old neurones compared to both young and middle aged. Conversely, the 5-HT-evoked depolarisation and conditional bursting of the swallow motor neurone (B4) were attenuated in old neurones, functions critical for a full feeding rhythm. The CGCs' ability to excite B1 was blocked by cinanserin but not by methysergide. Conversely, the CGC to B4 connection was completely blocked by methysergide and only partially by cinanserin suggesting that age-related changes may be receptor-specific. In summary, synapse-specific attenuation of the CGC-B4 connection and enhancement of the CGC-B1 connection would slow the swallow phase and maintain protraction, consistent with behavioural observations.     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. I. Cholinergic receptors on feeding neurons.

All the identified feeding motoneurons of Lymnaea respond to bath or iontophoretically applied acetylcholine (ACh). Three kinds of receptors (one excitatory, one fast inhibitory and one slow inhibitory) were distinguished pharmacologically. The agonist TMA (tetramethylammonium) activates all three receptors, being weakest at the slow inhibitory receptor. PTMA (phenyltrimethylammonium) is less potent than TMA and is ineffective at the slow inhibitory receptor, which is the only receptor sensitive to arecoline. At 0.5 mM the antagonists HMT (hexamethonium) and ATR (atropine) selectively block the excitatory response, while PTMA reduces the response to ACh at all three receptors. d-TC (curare) antagonizes only the fast excitatory and the fast inhibitory responses, but MeXCh (methylxylocholine) blocks the fast excitatory and slow inhibitory responses solely. For each of the feeding motoneurons, the sign of the cholinergic response (excitation or inhibition) is the same as the synaptic input received in the N1 phase of the feeding rhythm.     

The octopamine-containing buccal neurons are a new group of feeding interneurons in the pond snail Lymnaea stagnalis

In the pond snail, Lymnaea stagnalis, the paired buccal ganglia contain 3 octopamine-immunoreactive neurons, which have previously been shown to be part of the feeding network. All 3 OC cells are electrically coupled together and interact with all the known buccal feeding motoneurons, as well as with all the modulatory and central pattern generating interneurons in the buccal ganglia. N1 (protraction) phase neurons: Motoneurons firing in this phase of the feeding cycle receive either single excitatory (depolarising) synaptic inputs (B1, B6 neurons) or a biphasic response (hyperpolarisation followed by depolarisation) (B5, B7 motoneurons). Protraction phase feeding interneurons (SO, N1L, NIM) also receive this biphasic synaptic input after OC stimulation. All of protraction phase interneurons inhibit the OC neurons. N2 (retraction) phase neurons: These motoneurons (B2, B3, B9, B10) and N2 interneurons are hyperpolarised by OC stimulation. N2 interneurons have a variable (probably polysynaptic) effect on the activity of the OC neurons. N3 (swallowing) phase: OC neurons are strongly electrically coupled to both N3 phase (B4, B4cluster, B8) motoneurons and to the N3p interneurons. In case of the interneuronal connection (OC<->N3) the electrical synapse is supplemented by reciprocal chemical inhibition. However, the synaptic connections formed by the OC neurons or N3p interneurons to the other members of the feeding network are not identical. CGC: The cerebral, serotonergic CGC neurons excite the OC cells, but the OC neurons have no effect on the CGC activity. In addition to direct synaptic effects, the OC neurons also evoke long-lasting changes in the activity of feeding neurons. In a silent preparation, OC stimulation may start the feeding pattern, but when fictive feeding is already occurring, OC stimulation decreases the rate of the fictive feeding. Our results suggest that the octopaminergic OC neurons form a sub-population of N3 phase feeding interneurons, different from the previously identified N3p and N3t interneurons. The long-lasting effects of OC neurons suggest that they straddle the boundary between central pattern generator and modulatory neurons.     

Octopaminergic modulation of the membrane currents in the central feeding system of the pond snail Lymnaea stagnalis

Octopamine is released by the intrinsic OC interneurons in the paired buccal ganglia and serves both as a neurotransmitter and a neuromodulator in the central feeding network of the pond snail Lymnaea stagnalis. The identified B1 buccal motoneuron receives excitatory inputs from the OC interneurons and is more excitable in the presence of 10 microM octopamine in the bath. This modulatory effect of octopamine on the B1 motoneuron was studied using the two electrode voltage clamp method. In normal physiological saline depolarising voltage steps from the holding potential of -80 mV evoke a transient inward current, presumably carried by Na(+) ions. The peak values of this inward current are increased in the presence of 10 microM octopamine in the bath. In contrast, both the transient (IA) and delayed (IK) outward currents are unaffected by octopamine application. Replacing the normal saline with a Na(+)-free bathing solution containing K(+) channel blockers (50 mM TEACl, 4 mM 4AP) revealed the presence of an additional inward current of the B1 neurons, carried by Ca(2+). Octopamine (10 microM) in the bath decreased the amplitudes of this current. These results suggest that the membrane mechanisms which underlie the modulatory effect of octopamine on the B1 motoneuron include selective changes of the Na(+)- and Ca(2+)-channels.     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. III. Pharmacological dissection of the feeding rhythm.

The feeding activity of the pond snail Lymnaea stagnalis was stimulated by depolarization of a modulatory interneuron (SO) or of a N1 pattern-generating interneuron. The cholinergic antagonists phenyltrimethylammonium (PTMA), methylxylocholine (MeXCh), hexamethonium (HMT) and atropine (ATR) were applied at 0.5 mM in the bath and their effects on the rhythmic feeding pattern were monitored. Each of the antagonists slowed or blocked the feeding rhythm. The block was due to interference in the pattern generating network, not to disturbance of modulatory inputs. The experimental results favour a model in which the alternation of protraction (N1) and retraction (N2) phases occurs by recurrent inhibition. The results would be more difficult to explain on the reciprocal inhibition model. When all the N1 output was blocked, the N1 neurons fired rhythmic bursts endogenously.     

Leaf mechanical properties modulate feeding movements and ingestive success of the pond snail, Lymnaea stagnalis

We examined the mechanical properties of Butterhead and Iceberg lettuce leaves, and the rate at which they were eaten by the pond snail Lymnaea stagnalis. The outer part of Butterhead leaves were less robust than either the inner Butterhead or outer Iceberg leaves (Young’s modulus 2.8, 5.2, 7.7 MPa respectively; ultimate tensile stress 0.18, 0.34 0.51 MPa) which were also thicker. Snails ingested inner Butterhead and Iceberg strips more slowly (36 and 32%) than outer Butterhead. This was not due to differences in latency to first bite or biting rate. Rather, the drop was due to a decrease in the proportion of successful bites (inner Butterhead 84%; Iceberg 86%), to a shorter length ingested per bite (inner Butterhead 55%; Iceberg 45%) and to increased handling time (inner Butterhead 30%). We conclude that sensory input from the mechanically more robust lettuce slows the buccal central pattern generator.     

Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

Respiratory behavior in the pond snail Lynmaea stagnalis

Summary Previously (Syed et al. 1991) we described the ventilatory behavior of the pond snail Lymnaea stagnalis and identified motor neurons that innervate various muscles involved in this behavior. In the present study we describe an interneuronal network that controls ventilatory behavior in Lymnaea. An identified interneuron, termed the input 3 interneuron (Ip.3.I), was found to be involved in the opening movement of the pneumostome (expiration), whereas another identified interneuron known as visceral dorsal 4 (V.D.4) caused its closure (inspiration). These cells have reciprocal inhibitory connections with each other, which accounts for their opposing effects on common follower motor neurons. In isolated brain preparations a third identified interneuron, right pedal dorsal 1 (R.Pe.D.1) initiated the respiratory cycle by the excitation of Ip.3.I. Whereas Ip.3.I in turn excited R.Pe.D.1, the connections between R.Pe.D.1 and V.D.4 were mutually inhibitory. Both Ip.3.I and V.D.4 were active during spontaneously occurring respiratory behavior as recorded from semi-intact preparations, and selective hyperpolarization of V.D.4 during such spontaneous activity disrupted the respiratory behavior. Regarding peripheral feedback, the mechanical stimulation of the pneumostome during its opening movements not only caused closure but also inhibited Ip.3.I in the middle of its discharge. Ip.3.I and V.D.4 were also found to be multifunctional, inhibiting both locomotor and whole body withdrawal neural networks. We conclude from these results that the rhythmic patterned activity underlying respiratory behavior in Lymnaea is generated centrally, and that the network described here therefore comprises a central pattern generator.     

Predation and the distribution and abundance of a pulmonate pond snail

Summary The abundances of a freshwater pulmonate snail, Lymnaea elodes were studied in a temporary pond and a permanent, more productive pond in northeastern Indiana, USA. When snails from both populations were reared in each of the ponds in containers excluding predators, snails grew to be 1.3 to 2 times as large in the more productive pond, and laid 9 times as many eggs. However, field sampling data showed adults to be more abundant in the temporary pond. The only obvious difference between the two ponds was the presence of the molluscivorous central mudminnow (Umbra limi) in the permanent pond. These fish fed upon L. elodes when eggs and juvenile snails were abundant. In an experiment in the temporary pond, addition of mudminnows lowered egg and juvenile snail survival in pens where snail abundances had been increased. We suggest that vertebrate predators like the mudminnow can be significant sources of mortality for thin shelled species like L. elodes, possibly excluding them from habitats like lakes and rivers.Address for offprint requests     

Cholinergic interneurons in the feeding system of the pond snail Lymnaea stagnalis. II. N1 interneurons make cholinergic synapses with feeding motoneurons.

The N1 neurons are a population of interneurons active during the protraction phase of the feeding rhythm. All the N1 neurons are coupled by electrical synapses which persist in a high Mg/low Ca saline which blocks chemical synapses. Individual N1 spikes produce discrete electrotonic postsynaptic potentials (PSPS) in other N1 cells, but the coupling is not strong enough to ensure 1:1 firing. Bursts of N1 spikes generate compound PSPS in the feeding motoneurons. The sign (excitation or inhibition) of the N1 input corresponds with the synaptic barrage recorded during the protraction phase. Discrete PSPS are only resolved in a Hi-Di saline. Their variation in latency and number can be explained by variation in electrotonic propagation within the electrically coupled network of N1 cells. The excitatory postsynaptic potentials (ESPS) in the 1 cell are reduced by 0.5 mM antagonists hexamethonium (HMT), atropine (ATR), curare (d-TC) and by methylxylocholine (MeXCh), all of which block the excitatory cholinergic receptor (Elliott et al. (Phil. Trans. R. Soc. Lond. 336, 157-166 (Preceding paper.) (1992)). The 1 cell EPSPS were transiently blocked by phenyltrimethylammonium (PTMA), which is both an agonist and antagonist at the 1 cell excitatory acetylcholine (ACh) receptor (Elliott et al. 1992). The inhibitory postsynaptic potential (IPSP) in the 3 cell is blocked by bath applications of MeXCh and PTMA, which both abolish the response of the 3 cell to ACh (Elliott et. al. 1992). The effects of the cholinergic antagonists on the response of 4 cluster and 5 cells to N1 stimulation matches their response to ACh (Elliott et al. 1992). It is concluded that the population of N1 cells are multiaction, premotor cholinergic interneurons.     

Effect of enkephalins on the plasticity of pond snail

The effects of introduction of leu- and met-enkephalins (LE and ME) into the perfusion medium on the characteristics of electrical activity, spontaneous firing and habituation of single neurones to repeated intracellular electrical stimulation were studied on isolated CNS of molluscs. The character, speed, and degree of development of endoneuronal habituation changed significantly after application of LE in 67 per cent of the neurones studied and in 25 per cent of neurones after ME application. As a rule the changes of habituation dynamics occurred at constant levels of membrane potential, excitability, and reactivity of the neurone. LE and ME exerted different modulating effects on the initial electrical activity in 60 per cent of neurones. The obtained data on the independence of the effects of enkephalins on different parameters of activity of one and the same neurone give evidence of a mosaic character of excitable neuronal membrane. A suggestion is made about the possible role of the revealed opiate dependence of the endoneuronal functional plasticity in realization of opiate influences at the behavioural level.     

Role of aminergic (serotonin and dopamine) systems in the embryogenesis and different embryonic behaviors of the pond snail, Lymnaea stagnalis

A detailed biochemical and pharmacological analysis of the dopaminergic (DAergic) and serotonergic (5-HTergic) systems was performed during the embryogenesis of Lymnaea stagnalis, to monitor their role in development and different behaviors. The dopamine (DA) level and the synthesizing decarboxylase enzyme activity showed a continuous increase, whereas the serotonin (5-HT) concentration remained low until late postmetamorphic development, when they all showed a rapid and significant increase. Application of monoamine precursors increased, whereas enzyme inhibitors and neurotoxins reduced monoamine levels; all treatments resulting in a prolongation of embryogenesis. Following, p-chlorphenylalanine (pCPA) and 3-hydroxybenzylhydrazine (Nsd-1015) treatments, no 5-HT immunoreactivity could be detected in the embryonic nervous system. These findings suggest that changes of monoamine levels in either (negative or positive) direction cause slowing of embryogenesis. Embryonic rotation and radula protrusion rate was enhanced following both serotonin and dopamine application, whereas frequency of gliding was increased by serotonin treatment. These results clearly indicate the involvement of 5-HT and DA in the regulation of a broad range of embryonic behaviors. Pharmacological characterization of a 5-HT receptor associated with the L. stagnalis embryonic behaviors studied revealed that a mammalian 5-HT(1)-like receptor type is involved in the 5-HTergic regulation of locomotion activity.     

Multiple populations of serotonin receptors may modulate the behavioral effects of serotonergic agents

In order to establish a functional role for the various populations of serotonin (5-HT) receptors, behavioral studies have been conducted over the past decade with serotonergic agonists and antagonists. And, although there is reason to believe that certain behavioral effects may be mediated via particular populations of 5-HT receptors, evidence now suggests that some serotonin-mediated behaviors may be modulated by the interaction of serotonergic agents at multiple subtypes of 5-HT receptors. The generality of these effects, and the exact mechanism(s) by which they occur, have yet to be elucidated. Nevertheless, over the past year, results from several different laboratories provide a growing recognition of this novel phenomenon.     

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.     

Shell attachment in the pond snail Lymnaea stagnalis (L.)

The attachment of the body of the snail Lymnaea stagnalis to the shell was studied by histochemistry and light and electron microscopy. Muscles of the body wall insert into the connective tissue by way of long thin projections of sarcolemma. The muscle cells end under the basement membrane of a specialised area of the epidermis, the adhesive epithelium. The cells of this epithelium are filled with microfilaments and possess characteristic knob-like microvilli. The epithelium is attached to the shell by way of an adhesive substance containing proteins and mucopolysaccharides.This research was made possible by a grant from the Netherlands Organization for Pure Research (Z.W.O.)     

Bioaccumulation and toxicity of aluminium in the pond snail at neutral pH

The low solubility of aluminium (Al) at neutral pH means that it largely exists as colloidal particulates in aquatic systems. However, the pond snail Lymnaea stagnalis accumulates significant amounts of Al following exposure to water containing added Al (up to 500 microg l(-1)) at pH 7. This is accompanied by depression of behavioural activity (locomotion, feeding) which subsequently recovers, suggesting tolerance to the metal. The presence of silica ameliorates behavioural toxicity of Al, but does not prevent uptake of the metal. In vitro studies using the isolated central nervous system demonstrate toxicity at the cellular level. Extracellular application of Al (100 microM) led to membrane depolarisation, bursts of action potentials and action potential broadening. The chemical form in which Al is applied influences the extent of bioaccumulation and toxicity. Detailed knowledge of its solution chemistry is therefore essential.     

Serotonergic sensory-motor neurons mediate a behavioral response to hypoxia in pond snail embryos

Oxygen (O(2)) is one of the most important environmental factors that affects both physiological processes and development of aerobic animals, yet little is known about the neural mechanism of O(2) sensing and adaptive responses to low O(2) (hypoxia) during development. In the pond snail, Helisoma trivolvis, the first embryonic neurons (ENC1s) to develop are a pair of serotonergic sensory-motor cells that regulate a cilia-driven rotational behavior. Here, we report that the ENC1-ciliary cell circuit mediates an adaptive behavioral response to hypoxia. Exposure of egg masses to hypoxia elicited a dose-dependent and reversible acceleration of embryonic rotation that mixed capsular fluid, thereby facilitating O(2) diffusion to the embryo. The O(2) partial pressures (Po(2)) for threshold, half-maximal, and maximal rotational response were 60, 28, and 13 mm Hg, respectively. During hypoxia, embryos relocated to the periphery of the egg masses where higher Po(2) levels occurred. Furthermore, intermittent hypoxia treatments induced a sensitization of the rotational response. In isolated ciliary cells, ciliary beating was unaffected by hypoxia, suggesting that in the embryo, O(2) sensing occurs upstream of the motile cilia. The rotational response of embryos to hypoxia was attenuated by application of the serotonin receptor antagonist, mianserin, correlated to the development of ENC1-ciliary cell circuit, and abolished by laser-ablation of ENC1s. Together, these data suggest that ENC1s are unique oxygen sensors that may provide a good single cell model for the examination of mechanistic, developmental, and evolutionary aspects of O(2) sensing.     

Ultrastructure of neuromuscular contacts in the embryonic pond snail Lymnaea stagnalis L

Ultrastructural characteristics of muscle fibers and neuromuscular contacts were investigated during two stages of embryogenesis of the pulmonate snail Lymnaea stagnalis. The first muscle cells appear as early as during metamorphosis (50-55% of embryonic development), whereas previously, in the trochophore/veliger stages (25-45%), muscular elements cannot be detected at all. The first muscle fibers contain large amounts of free numbers, a well-developed rER system and only a few irregularly arranged contractile elements. The nucleus is densely packed with heterochromatine material. At 75% adult-like postmetamorphic stage, the frequency of muscle fibers increases significantly, but, bundles of muscle fibers cannot yet be observed. Furthermore the muscle cells are characterized by large numbers of free ribosomes and numerous rER elements. Fine axon bundles and single axon processes, both accompanied by glial elements, can already be found at this time. Axon varicosities with different vesicle and/or granule contents form membrane contacts with muscle fibers, but without revealing membrane specialization on the pre- or postsynaptic side. The late development of the muscle system and neuromuscular contacts during Lymnaea embryogenesis correlates well with the maturation of different forms of behavior of adult, free-living life, and also with the peripheral appearance of chemically identified components of the embryonic nervous system of central origin.     

Ultrastructural immunocytochemical evidence for peptidergic neurotransmission in the pond snail Lymnaea stagnalis

Summary Three neuronal systems of the pond snail Lymnaea stagnalis were immunocytochemically investigated at the ultrastructural level with the unlabeled peroxidase-antiperoxidase technique. Preliminary electrophysiological and cell-filling investigations have shown that a cluster of neurons which reacts positively with an antiserum against the molluscan cardio-active peptide FMRFamide, sends axons to the penis retractor muscle. In this muscle anti-FMRF-amide (aFM) positive axons form neuro-muscular synapses with (smooth) muscle fibers. The morphological observations suggest the aFM immunoreactive system to be involved in peptidergic neurotransmission. In the right parietal ganglion a large neuron (LYAC) is penetrated by aFM positive axons which form synapse-like structures (SLS) with the LYAC. The assumption that the SLS represent the morphological basis for peptidergic transmission is sustained by the observation that iontophoretical application of synthetic FMRFamide depolarizes the LYAC. The axons of a group of pedal anti-vasopressin (aVP) positive cells run in close vicinity to the cerebral ovulation (neuro-)-hormone producing cell system (CDC system) Synapses or SLS between the two systems were not observed. The fact that (bath) application of arg-vasopressin induces bursting in the CDC, may indicate that the vasopressin-like substance of the aVP cells is released non-synaptically.