Modulation in firing pattern and oscillation in nerve cells of Lymnaea during network reconstruction

The modulation and reconstruction of the cardio-respiratory neural circuit of Lymnaea stagnalis L. was compared to that of Helix ponatia L. where the input variation and signal molecules were found to have primary importance in network reorganization. From the cardio-respiratory circuit only neurons connected by afferent or efferent pathways to the peripheral chemosensory organ, the osphradium, were used. It was shown that, the general principles of the network reorganization is similar in the two species. The firing pattern of the neurons altered in Lymnaea depending on the input activation or presence of signal molecules in the vicinity of the neurons. The responses of the neurons to the same sensory information, originating from osphradium varied depending on their firing patterns. On central neurones the generation of phasic pattern and/or oscillation was an indicator of network disintegration leading to insensibility to the osphradial sensory inputs. Co-application of signal molecules (5HT, DA, GABA with opioid peptides) to the neurons caused a phasic firing pattern and/or oscillation leading to disintegration of one network and activation of another one. The effect of mu-opioid peptides on GABA-induced and voltage activated ion currents were shown to be the cellular target in reconstruction of neural networks in Lymnaea. The neural network reconstruction in vertebrate brain evoked by signal molecules can be compared to that observed in the identified network of Lymnaea stagnalis making this latter a useful model in further studies, too.     

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.     

Central representation of internal and external sensory information in the CNS of Helix pomatia L. and Lymnaea stagnalis L

The central representation of intero- and exteroreceptors located in visceral organs and the osphradium were compared in the CNS of Helix pomatia L. (Gastropoda, Stylommatophora) and Lymnaea stagnalis L. (Gastropoda, Basommatophora), two pulmonate snail species inhabiting a terrestrial and anaquatic environment, respectively. Semi-intact preparations were used comprising the CNS connected by the corresponding nerves either to the cardio-renal, respiratory and genital systems or to the osphradium. Spike discharges of central neurons and the nerves were recorded simultaneously. The central representation of intero- and exteroreceptors was found to be distributed throughout the CNS and involved about 300 neurons. The majority of the neurons received sensory information from all the studied visceral organs and the osphradium. Among the neurons responding to intero- and exteroreceptors a multimodal reaction to tactile, chemical and osmotic stimuli prevailed while in the osphradium specific reactions also were demonstrated. Central neurons receiving sensory information from visceral organs and the osphradium form overlapping and reorganizing neural circuits using the same neurons in the regulation of heart activity, respiration or reproduction producing the appropriate behaviour. In the selection of sensory information the firing pattern appears to be the main determining factor as bursting neurons do not receive sensory information. The central representation of intero- and exteroreceptors and its variability can be a model system for cellular studies of motivational state and self-perception.     

Structure and electrophysiological properties of bursting neurosecretory cells in a peripheral sensory ganglion of the pond snail Lymnaea stagnalis

A group of peripheral neurosecretory oscillating neurons belonging to the type of parabolic bursters, were identified in the osphradium (peripheral putative chemosensory organ) of the pond snail Lymnaea stagnalis. The cells are unipolar, their process ramifies and terminates in the nerve. Applications of 5-HT caused long-lasting bursts with significantly increasing duration and frequency of spikes. GABA and FMRFamide inhibited the activity of these cells.     

The osphradium is involved in the control of egg-laying in the pond snail Lymnaea stagnalis

Summary

Ablation of the osphradium (a peripheral putative chemo-sensory organ) in Lymnaea stagnalis resulted in significant increase of egg-laying activity as compared with control animals (observed for 50 days). Cutting the osphradial nerve increased egg-laying to a lesser degree and only for the first 15–20 days. Water changes in the aquaria had a similar stimulating effect on egg-laying activity in both osphradium-deprived and control snails. Permanent aeration of the aquaria abolished this effect. Possible mechanisms of tonic inhibitory action of the osphradium on egg-laying are discussed.     

On the structure of the pulmonate osphradium

Summary The osphradium of Planorbarius consists of a blindly-ending ciliated canal, formed by an infolding of the mantle epithelium, and a basal ganglion of nerve cells which is comparable in complexity with ganglia of the central nervous system. The distribution of cell types in the osphradial epithelium is specialised so that three regions can be recognised; the ciliated, the secretory and the sensory regions. The basal sensory region of the canal epithelium consists of ciliated cells and is innervated by sensory neurones of the osphradial ganglion. The middle secretory region contains mainly of mucus-secreting cells and the epithelium adjacent to the osphradial aperture of ciliated cells and secretory cells of a second type. The sensory neurones of the osphradial ganglion are bipolar or of a modified monopolar type. Other monopolar neurones, similar to those common in the central nervous system are of non-sensory function. The osphradium of Paludina, although of typical prosobranch form, possesses ciliated pits similar to the single canal of Planorbarius, which may indicate a shared modality of receptor function. A definite function cannot be ascribed to the pulmonate osphradium based on morphological evidence alone.     

The presence and specificity of crustacean cardioactive peptide (CCAP)-immunoreactivity in gastropod neurons

CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.     

Morphologic characteristics and responses of the neurons of the right parietal ganglion in Lymnaea stagnalis to stimulation of the sensory structures

Intracellular recordings have been made of the responses of 22 neurons of the central part of the dorsal surface of the right parietal ganglion of the snail Lymnaea stagnalis to adequate stimulation of chemo-, photo- and mechanoreceptor cells of the mantle and head skin including tentacles and lips. It was shown that the main bulk of the neurons investigated has broad receptive fields in the body wall and mantle, being able to respond to all types of the applied stimuli. Alongside, single neurons were revealed which receive single-mode input, either a mechanosensory or chemosensory one. Morphological studies indicate that the neurons are unipolar and have usually one, sometimes several projections. They differ in the pattern of branching as well as in the projections to peripheral nerves. However, almost all of them have vast dendritic regions in the central nervous system including central sensory nucleus of the right parietal ganglion.     

Ultrastructure of the osphradium of Siphonaria grisea (Mollusca, Pulmonata)

By means of scanning and transmissive electron microscopic methods osphradium of Siphonaria grisea has been studied. The osphradium of the animal is presented as a small torulus formed by supporting ciliated cells. Among them bodies of receptory cells are situated; they are of smaller size and decorated with a bundle of microvilli. Central processes of the receptory cells, penetrating through a thick layer of the connective tissue, reach the subepithelial neural trunk. Concentration of the neural cells in the periphery of the trunk is small, and in the central area no chemical synapses are revealed. The data presented demonstrate a primitive structure of the osphradial chemoreceptory organ in Siphonaria.     

Octopamine-containing (OC) interneurons enhance central pattern generator activity in sucrose-induced feeding in the snail <Emphasis Type="Italic">Lymnaea</Emphasis>

In the pond snail Lymnaea stagnalis octopamine-containing (OC) interneurons trigger and reconfigure the feeding pattern in isolated CNS by excitation of the central pattern generator. In semi-intact (lip–mouth—CNS) preparations, this central pattern generator is activated by chemosensory inputs. We now test if sucrose application to the lips activates the OC neurons independently of the rest of the feeding central pattern generator, or if the OC interneuron is activated by inputs from the feeding network. In 66% of experiments, sucrose stimulated feeding rhythms and OC interneurons received regular synaptic inputs. Only rarely (14%) did the OC interneuron fire action potentials, proving that firing of OC interneurons is not necessary for the sucrose-induced feeding. Prestimulation of OC neurons increased the intensity and duration of the feeding rhythm evoked by subsequent sucrose presentations. One micromolar octopamine in the CNS bath mimicked the effect of OC interneuron stimulation, enhancing the feeding response when sucrose is applied to the lips. We conclude that the modulatory OC neurons are not independently excited by chemosensory inputs to the lips, but rather from the buccal central pattern generator network. However, when OC neurons fire, they release modulatory octopamine, which provides a positive feedback to the network to enhance the sucrose-activated central pattern generator rhythm.     

Distinct Responses of Osphradial Neurons to Chemical Stimuli and Neurotransmitters in Lymnaea stagnalis L.

1. In Lymnaea stagnalis L. (Pulmonata, Basommatophora) the neurons in the osphradium were visualized by staining through the inner right parietal nerve by 5,6-carboxyfluorescein (5,6-CF). Three types of neurons were identified: three large ganglionic cells (GC1-3; 80–100 m), the small putative sensory neurons (SC; 20 m) and very small sensory cells (3–5 m).2. The ganglionic and putative sensory neurons were investigated by whole cell patch-clamp method in current-clamp condition. The three giant ganglionic neurons (GC1-3) located closely to the root of osphradial nerve, had a membrane potential (MP) between –30 and –70 mV and showed tonic or bursting activities. The small putative sensory cells (SCs) scattered throughout the osphradial ganglion, possessed a MP between –25 and –55 mV and showed an irregular firing pattern with membrane oscillations. At resting MP the GC1-3 cells were depolarized and increased the frequency of their firing, while the SCs were hyperpolarized and inhibited by NaCl (10^–2 M) and L-aspartate (10^–5 M) applied to the osphradium.3. 5-Hydroxytryptamine (5HT, 10^–6 M), -aminobutyric acid (GABA; 10^–6 M) and the GABA_B agonist baclofen (10^–6 M) depolarized the neurons GC1-3 and increased their firing frequency. In contrast, on the GC1-3 neurons, acetylcholine (Ach; 10^–6 M) and FMRFamide (10^–6 M) caused hyperpolarization and cessation of the firing activity. The 5HT effect was blocked by mianserin (10^–6 M) but picrotoxin (10^–5 M) failed to block the GABA-induced effect on the GC1-3 cells.4. The small putative sensory neurons (SCs) were excited by Ach (10^–6 M) and 5HT (10^–6 M) but were inhibited by GABA (10^–6 M). FMRFamide (10^–6 M) had a biphasic response. The Ach effect was blocked by hexamethonium (10^–6 M) and tetraethylammonium (10^–6 M), indicating the involvement of nicotinic cholinergic receptors.5. The distinct responses of the two populations of osphradial neurons to chemical stimuli and neurotransmitters suggest that they can differently perceive signals from environment and hemolymph.     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Electrical activity in the chemoreceptors of the blowfly. II. Responses to electrical stimulation

An analysis of the various parts of the electrical responses to the chemical and electrical stimulation of a single labellar chemosensory hair of the blowfly, Phormia regina, indicates that the recording conditions for the spike potentials approximate the intracellular recordings made in other types of sense cells. The large positive resting potential probably arises from the basement membrane of the hypodermal cells and neurilemma rather than from the neurons at the base of the chemosensory hair. The responses to polarizing currents passed through single chemosensory hairs support this analysis. The behavioral responses to similar polarizing currents are shown to result from the action of the current on the neurons at the bases of the adjacent chemosensory hairs. The reported neural interaction of the two chemosensory neurons associated with the chemosensory hair is probably due to the physical-chemical attributes of the stimulating solution rather than to any real neural interaction. Observations on the latency of the initial nerve impulse in response to chemical stimulation indicate that the chemosensory neurons are normally free from spontaneous spike activity.     

Structure of visual pathways in the nervous system of fresh water pulmonary molluscs

The central nervous system of freshwater pulmonary molluscs Lymnaea stagnalis and Planorbarins corneus was stained by the method of neurobiotin retrograde transport along optic nerve fibers. In the animals of both species, bodies and fibers of stained neurons are found in all ganglia except for the buccal ones. Afferent fibers of the optic nerve form a dense sensor neuropil located in a small volume of cerebral ganglia. Characteristic groups of neurons sending their processes into optic nerves both of ipsi- and of contralateral half of the body are described. Revealed among them are neurons of visceral and parietal ganglia, which simultaneously innervate both eyes as well as give projections into peripheral nerves. It is suggested that these neurons can perform function of integration of sensor signals and, on its base, regulate photosensitivity of retina as well as activity of peripheral organs. There is established the presence of bilateral connections of the mollusc eye with cells of pedal ganglia and statocysts, which seems to be the structural basis of manifestation of the known behavior forms associated with stimulation of visual inputs of the studied gastropod molluscs.     

On the structure of the pulmonate osphradium

Summary The cellular organisation of the osphradium of Planorbarius as revealed by the previously described light microscope studies is confirmed in the present study. A third epithelial cell type, the basal cell, is described. Perception is carried out by dendritic processes from osphradial sensory neurones, forming free nerve-endings in the sensory region of the osphradial epithelium. Ciliated cells and secretory cells of the osphradial epithelium are concerned with the transport of material in the osphradial canal. Many of the ultrastructural features of molluscan central ganglia are present in the osphradial ganglion, including inter-axonal chemical synapses. Neuromuscular junctions are present in the sheath surrounding the organ and these may be involved in a behavioural response of the organ. The need for further electrophysiological studies is emphasised.     

Single neurone responses to tactile stimulation of the heart in the snail,Helix pomatia L.

Summary The electrical activity of the heart nerve and of single neurons in the suboesophageal ganglia were recorded during tactile stimulation of the heart. 15 neurons were identified which responded to heart stimulation by inhibiting or accelerating activity. Cells influenced by heart afferents are scattered in the visceral and in the right and left parietal ganglia.In most of the cases both decrease and increase of cell activity are caused by synaptic potentials, in some cases, however, the neuron is assumed to have a sensory character.The activity of three neurons influenced by heart stimulation was conducted into the heart nerve. These cells are central neurons of a heart-CNS-heart reflex.Some of the neurons located in the right parietal and visceral ganglia have no connection with the mechanoreceptors of the heart. Since their spikes propagate into the heart nerve, they probably take part in the extracardial regulation of heart activity.One of the neurons located in the visceral ganglion (cell V12) sends its axon into the heart nerve. The response of this neuron to heart stimulation was an increase in activity and an inhibition of the heart rate. This is an inhibitory neuron of the extracardial heart regulatory system.     

Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.     

Localization of glutamate-like immunoreactive neurons in the central and peripheral nervous system of the adult and developing pond snail, Lymnaea stagnalis

We investigated the distribution and projection patterns of central and peripheral glutamate-like immunoreactive (GLU-LIR) neurons in the adult and developing nervous system of Lymnaea. Altogether, 50-60 GLU-LIR neurons are present in the adult central nervous system. GLU-LIR labeling is shown in the interganglionic bundle system and at the varicosities in neuropil of the central ganglia. In the periphery, the foot, lip, and tentacle contain numerous GLU-LIR bipolar sensory neurons. In the juvenile Lymnaea, GLU-LIR elements at the periphery display a pattern of distribution similar to that seen in adults, whereas labeled neurons increase in number in the different ganglia of the central nervous system from juvenile stage P1 up to adulthood. During embryogenesis, GLU-LIR innervation can be detected first at the 50% stage of embryonic development (the E50% stage) in the neuropil of the cerebral and pedal ganglia, followed by the emergence of labeled pedal nerve roots at the E75% stage. Before hatching, at the E90% stage, a few GLU-LIR sensory cells can be found in the caudal foot region. Our findings indicate a wide range of occurrence and a broad role for glutamate in the gastropod nervous system; hence they provide a basis for future studies on glutamatergic events in networks underlying different behaviors.