Morphology and physiology of pleural-to-buccal neurons coordinating defensive retraction with feeding arrest in the pond snail Lymnaea stagnalis

1. We have studied morphology, physiology and chemistry of a bilateral pair of pleural-to-buccal projecting neurons (PlB cells) of the pond snail Lymnaea stagnalis. Intracellular dye fills revealed axon arborization within neuropiles of ipsilateral pedal and cerebral ganglia, as well as in both buccal ganglia. Terminal axons of the left and right PlBs showed close proximity within the buccal commissure. 2. The left and right PlB neurons have been found electrotonically coupled and, sometimes, generating synchronous spikes. 3. The results show that two PlB cells operate as a single unit, and that paired buccal networks responsible for feeding rhythm are treated by the PlBs as a single target.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

Sprouting and functional regeneration of an identified serotonergic neuron following axotomy

An identified serotonergic neuron (C1) in the cerebral ganglion of Helisoma trivolvis sprouts following axotomy and rapidly (seven to eight days) regenerates to recover its regulation of feeding motor output from neurons of the buccal ganglia. The morphologies of normal and regenerated neurons C1 were compared. Intracellular injection of the fluorescent dye, Lucifer Yellow, into neuron C1 was compared with serotonin immunofluorescent staining of the cerebral and buccal ganglia. The two techniques revealed different and complimentary representations of the morphology of neuron C1. Lucifer Yellow provided optimal staining of the soma, major axon branches, and dendritic arborization. Immunocytochemical staining revealed terminal axon branches on distant targets and showed an extensive plexus of fine fibers in the sheaths of ganglia and nerve trunks. In addition to C1, serotonin-like immunoreactivity was localized in approximately 30 other neurons in each of the paired cerebral ganglia. Only cerebral neurons C1 had axons projecting to the buccal ganglia. No neuronal somata in the buccal ganglia displayed serotonin-like immunoreactivity. Observations of regenerating neurons C1 demonstrated: Actively growing neurites, both in situ and in cell culture, displayed serotonin-like immunoreactivity; severed distal axons of C1 retained serotonin-like immunoreactivity for up to 28 days; axotomized neurons C1 regenerated to restore functional control over the feeding motor program.     

Segmental peptidergic innervation of abdominal targets in larval and adult dipteran insects revealed with an antiserum against leucokinin I

Summary An antiserum against the cockroach neuropeptide leucokinin I (LKI) was used to study peptidergic neurons and their innervation patterns in larvae and adults of three species of higher dipteran insects, the flies Drosophila melanogaster, Calliphora vomitoria, and Phormia terraenovae, as well as larvae of a primitive dipteran insect, the crane fly Phalacrocera replicata. In the larvae of the higher dipteran flies, the antiserum revealed three pairs of cells in the brain, three pairs of ventro-medial cells in the subesophageal ganglion, and seven pairs of ventro-lateral cells in the abdominal ganglia. Each of these 14 abdominal leucokinin-immunoreactive (LKIR) neurons innervates a single muscle of the abdominal body wall (muscle 8), which is known to degenerate shortly after adult emergence. Conventional electron microscopy demonstrates that this muscle is innervated by at least one axon containing clear vesicles and two axons containing dense-cored vesicles. Electronmicroscopical immunocytochemistry shows that the LKIR axon is one of these two axons with dense-cored vesicles and that it forms terminals on the sarcolemma of its target muscle. The abdominal LKIR neurons appear to survive metamorphosis. In the adult fly, the efferent abdominal LKIR neurons innervate the spiracles, the heart, and neurohemal regions of the abdominal wall. In the crane fly larva, dorso-medial and ventrolateral LKIR cell bodies are located in both thoracic and abdominal ganglia of the ventral nerve cord. As in the larvae of the other flies, the abdominal ventrolateral LKIR neurons form efferent axons. However, in the crane fly larva there are two pairs of efferent LKIR neurons in each of the abdominal ganglia and their peripheral targets include neurohemal regions of the dorsal transverse nerves. An additional difference is that in the crane fly, a caudal pair of LKIR axons originating from the penultimate pair of dorso-median LKIR cells in the terminal ganglion innervate the hindgut.     

Multiple transmitter neurons in Tritonia. I. Biochemical studies

The buccal ganglia of the marine mollusc Tritonia control a variety of movements associated with feeding, including gut motility. The buccal ganglia and gut contain a class of peptides termed small cardioactive peptides (SCPs). Cobalt backfilling of the nerve which innervates the gut stains several buccal neurons including two pairs of reidentifiable cells, B11 and B12. Both appear white under epiillumination, a characteristic of peptidergic neurons in gastropods. Enzymatic and biochemical analyses of extracts from microdissected B11 cell bodies demonstrate that this neuron contains two species of SCPs. Labeling in organ culture followed by dissection and extraction of cell bodies indicates that these peptides were synthesized in B11. One of these peptides appears to be identical to SCPB, one of two SCPs that have been sequenced. The other SCP present in these neurons is novel. Less extensive analyses of extracts of B12 somata suggest that it also contains the same SCPs. In addition to the peptides, B11 also contains large quantities of acetylcholine (ACh) as determined by a radioenzymatic assay of cell body extracts. B12 does not contain measureable ACh. The concentration of the two peptides and ACh in the B11 cytoplasm is approximately 1 mM. Neuron B11 appears to be an appropriate model system for studying the biochemical and physiological properties of multiple transmitter neurons.     

Properties of the serotonergic cerebral ganglion neurons of the gastropod mollusc, Philine aperta

The characteristics of a pair of identified neurons found in the cerebral ganglia of the gastropod mollusc Philine aperta have been examined. Because they appear to contain serotonin, and since they probably also use serotonin as a neurotransmitter, these neurons were named the serotonergic cerebral neurons (SCNs). Each SCN sent an axon out of the ipsilateral cerebro-buccal connective to the buccal ganglia. The SCNs also had extensive projections to all the ipsilateral, and most of the contralateral, buccal nerve trunks. Stimulating the SCNs produced the hyperpolarization of a pair of identified buccal ganglion mechano-sensory neurons (the S-cells), and had an excitatory action on the electrical activity of acinar cells of the salivary glands. A comparison of the properties of the Philine SCNs with those of similar serotonin-containing cerebral ganglion neurons in other gastropod molluscs provides evidence of homology with these neurons.     

Distribution of tyrosine-hydroxylase-immunoreactive and dopamine-immunoreactive neurons in the central nervous system of the snail Helix pomatia

The distribution and characterization of dopamine-containing neurons are described in the different ganglia of the central nervous system of Helix on the basis of the distribution of tyrosine hydroxylase immunoreactive (TH-ir) and dopamine immunoreactive (DA-ir) neurons. Both TH-ir and DA-ir cell bodies of small diameter (10–25 m) can be observed in the buccal, cerebral and pedal ganglia, dominantly on their ventral surface, and concentrated in small groups close to the origin of the peripheral nerves. The viscero-parietal-pleural ganglion complex is free of immunoreactive cell bodies but contains a dense fiber system. The largest number of TH-ir and DA-ir neurons can be detected in the pedal, and cerebral ganglia. The average number of TH-ir and DA-ir neurons significantly differs but all the identifiable groups of TH-ir neurons also show DA-immunoreactivity. Therefore, we consider the TH-ir neurons in those groups as being DA-containing neurons. The amounts of DA in the different ganglia assayed by high performance liquid chromatography correspond to the distribution and number of TH-ir and DA-ir neurons in the different ganglia. The axon processes of the labeled small-diameter neurons send thin proximal branches toward the cell body layer but only rarely surround cell bodics, whereas distally they give off numerous branches in the neuropil and then leave the ganglion through the peripheral nerves. In the cerebral ganglia, the analysis of the TH-ir pathways indicates that the largest groups of labeled neurons send their processes through the peripheral nerves in a topographic order. These results furnish morphological evidence that DA-containing neurons of Helix pomatia have both central and peripheral roles in neuronal regulation.     

Leucokinin and callitachykinin immunoreactive neurons during postembryonic development of calliphora vomitoria (L.) (DIPTERA : CALLIPHORIDAE)

Neurons containing 2 types of myotropic neuropeptides were investigated by immunocytochemistry during postembryonic development of the brain and ventral nerve cord of the blowfly Calliphora vomitoria (Diptera : Calliphoridae). Antisera raised against the insect neuropeptides Callitachykinin II (CavTK II), Locustatachykinin I (LomTK I), and Leucokinin I (LK I) were used. Callitachykinin immunoreactive (CavTK–IR) neurons were detected from the 1st-instar larva throughout development to adult. The number of CavTK–IR cell bodies in the brain was 4–16 in larval stages, 10–84 in pupal stages, and over 140 neurons in the newly emerged fly. With the CavTK antiserum, the fibers of only 4 descending neurons were detected in thoracico–abdominal ganglia throughout development. The antiserum to LomTK displayed the same neurons as that to CavTK II as well as a small number of additional neurons. Notably, there were seen about 14–20 locustatachykinin-like immunoreactive (LomTK-LI) cell bodies in the thoracico–abdominal ganglia throughout development. Leucokinin-like immunoreactive (LK-LI) neurons were labeled throughout postembryonic development. In the brain, 2–4 LK-LI cell bodies were labeled from 1st-instar larva to 8-day-old pupa, and 6 LK-LI cell bodies were labeled in the adult brain. In the abdominal ganglia, 7 pairs of LK-LI cell bodies were labeled from 1st-instar larva to 96-h-old pupa, 8 pairs in 8-day-old pupa, and 9 pairs in newly emerged fly, respectively. The CavTK containing neurons in the brain displayed a drastic increase in numbers from larval stages to adult, which indicates an addition of functional roles for this type of peptide. During earlier pupal stages, the number of CavTK–IR neurons decreased. The LK-LI neurons, however, were strongly immunoreactive throughout postembryonic development. Only one additional pair of cells appeared in the brain and 2 additional pair of cells appeared in the abdominal ganglia of the adult as compared with larvae. The continuous high expression of LK-LI material may suggest a functional role for this type of peptide during development.     

Peripheral Actions of the SCPs in Aplysia and Other Gastropod Molluscs

The SCPs are a family of neuropeptides found in many gastropodspecies. Two SCPs with similar sequences have been characterizedin Aplysia. These peptides are potent modulators of centraland peripheral synapses. They also enhance ongoing contractileactivity in spontaneously active tissues such as heart and gut.Their distribution in central ganglia suggests that their predominantrole is in the regulation of feeding behavior. There is goodevidence that the identified SCP-containing neurons, B1 andB2, provide the major central regulation of gut motility duringfeeding through the release of the SCPs from their terminalsin gut. The SCPs have also been localized to motor neurons thatinnervate buccal muscles which generate biting and swallowingmovements. In many of these neurons, the SCPs have been shownto coexist with conventional transmitters such as ACh, or otherpeptides such as FMRFamide. The SCPs appear to be released alongwith conventional transmitters from these neurons to modulatethe effectiveness of the conventional transmitter. In all cases,the SCPs cause an enhancement of the amplitude of contractionsproduced by motor neuron stimulation. The precise mechanismsunderlying this effect vary from muscle to muscle. All of theeffects of the SCPs are mediated by increased cAMP levels intarget tissue. At many sites of action, serotonin produces actionsthat are qualitatively similar to those of the SCPs. This islikely to involve a convergence at the level of the adenylylcyclase. In addition to these peripheral effects, the SCPs alsohave multiple central effects on feeding and other behaviorsin gastropods.     

Axonal branching pattern and coupling mechanisms of the cerebral giant neurones in the snail,lymnaea stagnalis

The axonal branching pattern of the two cerebral giant neurones (CGCs) of Lymnaea stagnalis was studied with intrasomatically applied horseradish peroxidase. The cells are symmetrical. Each CGC projects to the ipsilateral n. labialis medius and n. arteriae labialis, the subcerebral commissure, and to all ipsi- and contralateral buccal nerves. The contralateral buccal nerves are reached via the ipsilateral cerebro-buccal connective and the buccal commissure. The CGC fire action potentials 1:1 in a driver-follower relationship. Each cell is capable of both driving and following. The relationship depends on the membrane potentials of the somata. In driving CGC spikes are initiated in a cerebral spike trigger zone located near the soma. In following cells spikes are initiated in a distal zone located in the buccal ganglia. The buccal zone is only affected by the partner CGC. CGC are synchronized by three coupling mechanisms: mutual excitatory chemical synapses, electrotonic coupling, and common input. The chemical and electrotonic connections are located in the buccal ganglia. All spikes are relayed to the partner cell via the chemical synapses. The electrotonic coupling improves the efficiency of the chemical synapses. The dual connection selectively synchronizes the CGC-axonal spikes from each side of the buccal mass. Common excitatory input affects the cerebral spike trigger zones and can initiate simultaneous spikes in both cells. This results in bilateral synchrony of spikes in the CGC-axons in both the buccal and the lip nerves.     

Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria

The anatomy and cellular organization of serotonergic neurons in the echinoderm apical organ exhibits class-specific features in dipleurula-type (auricularia, bipinnaria) and pluteus-type (ophiopluteus, echinopluteus) larvae. The apical organ forms in association with anterior ciliary structures. Apical organs in dipleurula-type larvae are more similar to each other than to those in either of the pluteus forms. In asteroid bipinnaria and holothuroid auricularia the apical organ spans ciliary band sectors that traverse the anterior-most end of the larvae. The asteroid apical organ also has prominent bilateral ganglia that connect with an apical network of neurites. The simple apical organ of the auricularia is similar to that in the hemichordate tornaria larva. Apical organs in pluteus forms differ markedly. The echinopluteus apical organ is a single structure on the oral hood between the larval arms comprised of two groups of cells joined by a commissure and its cell bodies do not reside in the ciliary band. Ophioplutei have a pair of lateral ganglia associated with the ciliary band of larval arms that may be the ophiuroid apical organ. Comparative anatomy of the serotonergic nervous systems in the dipleurula-type larvae of the Ambulacraria (Echinodermata+Hemichordata) suggests that the apical organ of this deuterostome clade originated as a simple bilaterally symmetric nerve plexus spanning ciliary band sectors at the anterior end of the larva. From this structure, the apical organ has been independently modified in association with the evolution of class-specific larval forms.     

Activation by dopamine of patterned motor output from the buccal ganglia of Helisoma trivolvis

The buccal ganglia of the snail, Helisoma trivolvis, contain an intrinsic system of dopamine-containing neurons (Trimble, Barker, and Bullard, 1983). Dopamine, when bath applied to the isolated buccal ganglia, activates patterned motor output in a dose-dependent fashion. Haloperidol blocks the activating effect of dopamine, but the similar activation evoked by serotonin is not blocked by haloperidol. We suggest that there are two separate mechanisms for activating patterned motor output from the buccal ganglia. One is serotonergic, emanating from identified cerebral ganglion cells (Granzow and Kater, 1977), while the other is dopaminergic, involving neurons intrinsic to the buccal ganglia.     

Ontogenesis of the snail,Helix aspersa: Embryogenesis timetable and ontogenesis of GABA-like immunoreactive neurons in the central nervous system

Late stages of embryogenesis in the terrestrial snail Helix aspersa L. were studied and a developmental timetable was produced. The distribution of gamma-aminobutyric acid-like immunoreactive (GABA-ir) elements in the CNS of the snail was studied from embryos to adulthood in wholemounts. In adults, approximately 226 GABA-ir neurons were located in the buccal, cerebral and pedal ganglia. The population of GABA-ir cells included four pairs of buccal neurons, three neuronal clusters in the pedal ganglia, two clusters and six single neurons in the cerebral ganglia. GABA-ir fibers were observed in all ganglia and in some nerves. The first detected pair of GABA-ir cells in the embryos appeared in the buccal ganglia at about 63–64% of embryonic development. Five pairs of GABA-ir cell bodies were observed in the cerebral ganglia at about 64–65% of development. During the following 30% of development three more pairs of GABA-ir neurons were detected in the buccal ganglia and over fifteen cells were detected in each cerebral ganglion. At the stage of 70% of development, the first pair of GABA-ir neurons was found in the pedal ganglia. In the suboesophageal ganglion complex, GABA-ir fibers were first detected at about 90% of embryonic development. In the posthatching period, the quantity of GABA-ir neurons reached the adult status in four days in the cerebral ganglia, and in three weeks in the pedal ganglia. In juveniles, transient expression of GABA was found in the pedal ganglia (fourth cluster).     

C. elegans dystroglycan coordinates responsiveness of follower axons to dorsal/ventral and anterior/posterior guidance cues

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle‐shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN‐1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn‐1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC‐6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN‐1 in unc‐6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN‐1 does not mediate follower/pioneer adhesion. Instead, DGN‐1 appears to block premature responsiveness of follower axons to a preanal ganglion‐directed guidance cue, which mediates ventral‐to‐anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N‐terminal DGN‐1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

The tritocerebral commissure ''dwarf'' (TCD): a major GABA-immunoreactive descending interneuron in the locust

Summary The minor branch of the tritocerebral commissure of the locust,Locusta migratoria, contains only two axons which are from interneurons in the brain descending to the ventral cord ganglia. The smaller of these two neurons, the tritocerebral commissure dwarf (TCD), is immunoreactive to GABA, suggesting that it may be an inhibitory interneuron. We have exploited the accessibility of its axon in the commissure, first, to fill it with cobalt to define its morphology, and second, to record its input characteristics. It has a cell body and arborization of fine branches in the deutocerebrum of the brain, its axon passes contralateral through the tritocerebral commissure and it forms bilateral arborizations in the suboesophageal and three thoracic ganglia. It receives mechanosensory input from many regions of the ipsilateral body and head, and it is sensitive to illumination levels, generally showing greater spontaneous activity in the dark.It is one of the largest GABA-immunoreactive descending interneurons in the locust, suggesting it plays a prominent role in behaviour. Since it is easily accessible for physiological recording, its roles in circuits for particular components of behaviour should be amenable to investigation.     

Mapping of serotonin-immunoreactive neurons in the larval nervous system of the flies Calliphora erythrocephala and Sarcophaga bullata

Summary Serotonin-immunoreactive (5-HTi) neurons were mapped in the larval central nervous system (CNS) of the dipterous flies Calliphora erythrocephala and Sarcophaga bullata. Immunocytochemistry was performed on cryostat sections, paraffin sections, and on the entire CNS (whole mounts).The CNS of larvae displays 96–98 5-HTi cell bodies. The location of the cell bodies within the segmental cerebral and ventral ganglia is consistent among individuals. The pattern of immunoreactive fibers in tracts and within neuropil regions of the CNS was resolved in detail. Some 5-HTi neurons in the CNS possess axons that run through peripheral nerves (antenno-labro-frontal nerves).The suboesophagealand thoracico-abdominal ganglia of the adult blowflies were studied for a comparison with the larval ventral ganglia. In the thoracico-abdominal ganglia of adults the same number of 5-HTi cell bodies was found as in the larvae except in the metathoracic ganglion, which in the adult contains two cell bodies less than in the larva. The immunoreactive processes within the neuropil of the adult thoracico-abdominal ganglia form more elaborate patterns than those of the larvae, but the basic organization of major fiber tracts was similar in larval and adult ganglia. Some aspects of postembryonic development are discussed in relation to the transformation of the distribution of 5-HTi neurons and their processes into the adult pattern.     

Gastrin-cholecystokinin immunoreactivity in the central nervous system of Helix aspersa during rest and activity

The immunostaining pattern for the peptide gastrin/cholecystokinin 8 (gastrin/CCK8) in the molluscan central nervous system has been considered. The changes in the distribution of gastrin/CCK8 immunoreactivity were analyzed in the neurons of different areas of the cerebral ganglia (mesocerebrum and metacerebrum) and in the buccal ganglia of the terrestrial snail Helix aspersa, during rest and active phases. During the period of inactivity and after one day of activity, there were several immunoreactive neurons in the mesocerebrum and metacerebrum of the snails and in the buccal ganglia, whereas after 7 days of activity the number of labeled neurons decreased. Data suggested a storage of gastrin/CCK8 in the neurons when behavioral activities in which the peptide is involved (such as feeding-related behavior) are suppressed or reduced. The different percentage of gastrin/CCK8 immunoreactive neurons in the left and right mesocerebrum provides information about the activities controlled by these neurons, which could be related to the adaptive evolution and plasticity of the brain in terrestrial pulmonates.     

Multiple transmitter neurons in Tritonia. III. Modulation of central pattern generator controlling feeding

Feeding behavior in the gastropod mollusc Tritonia diomedea is controlled by a central pattern generator (CPG) in the buccal ganglia. The medially located, large dorsal white cells (B11) have been shown to contain two small cardioactive peptides (SCPs). A smaller nearby neuron (B12) also appears to contain the SCPs. B11's have also been shown to contain acetylcholine (ACh), whereas B12's do not. We have shown earlier that intracellular stimulation of B11's drives contractions of the foregut. Here we show that intracellular electrical stimulation of B11's also elicits excitation of neurons B5 and stimulates the patterned motor output of the CPG. We showed earlier that B12's also stimulate contractions in the foregut, but they are in the opposite direction from those elicited by B11. We show here that electrical stimulation of B12's inhibits the output of the CPG. We showed earlier that superfusion of the isolated gut with SCPB enhances peristalsis, and here we report that superfusion of the buccal ganglion with SCPB elicits enhanced coordinated motor output from the CPG. The peptide has two effects on the bursting output of motor neurons. It produces an increase in (1) the rate of bursting and (2) the spike frequency during each burst. On the other hand, we reported earlier that ACh applied directly to isolated foregut inhibits ongoing peristalsis. Here we demonstrate that ACh superfusion of the buccal ganglion also inhibits the CPG output. Our evidence supports the view that in addition to stimulating foregut contractility, B11's modulate the output of the swallowing CPG by releasing a peptide from central terminals. We suggest roles for B11, B12, the SCPs, and ACh in controlling both central and peripheral aspects of feeding behavior.     

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.     

Multilevel inhibition of feeding by a peptidergic pleural interneuron in the mollusc<Emphasis Type="Italic"> Lymnaea stagnalis</Emphasis>

The pleural interneuron PlB is a white neuron in the pleural ganglion of the snail Lymnaea. We test the hypothesis that it inhibits neurons at all levels of the feeding system, using a combination of anatomy, physiology and pharmacology. There is just one PlB in each pleural ganglion. Its axon traverses the pedal and cerebral ganglia, running into the buccal ganglia. It has neuropilar branches in the regions of the cerebral and buccal ganglia where neurons that are active during feeding also branch. Activation of the PlB blocks fictive feeding, whether the feeding rhythm occurs spontaneously or is driven by a modulatory interneuron. The PlB inhibits all the neurons in the feeding network, including protraction and retraction motoneurons, central pattern generator interneurons, buccal modulatory interneurons (SO, OC), and cerebral modulatory interneurons (CV1, CGC). Only the CV1 interneuron shows discrete 1:1 IPSPs; all other effects are slow, smooth hyperpolarizations. All connections persist in Ca²⁺/Mg²⁺-rich saline, which reduces polysynaptic effects. The inhibitory effects are mimicked by 0.5 to 100 mol l^–1 FMRFamide, which the PlB soma contains. We conclude that the PlB inhibits neurons in the feeding system at all levels, probably acting though the peptide transmitter FMRFamide.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00359-004-0503-x