Retina mediators in fresh-water mollusc Lymnaeae stagnalis

Retrograde staining of the Lymnaeae stagnalis retina with neurobiotin has shown that most photoreceptor cells send axons to optic nerve without intermediate contacts. A part of these photoreceptors have immunireactivity to glutamate that possibly provides synaptic transmission of visual signal to central neurons. Other photoreceptors stained through optic nerve seem to have different transmitter systems. In some retina cell, but not in optic nerve fibers, immunoreactivity to pigment-dispersing hormone has been revealed. In tissues surrounding the eye cup numerous serotonin-containing fibers are present, a part of them penetrating the retina basal layer. Some of them belong to central neurons responsible for efferent innervation of the pond snail eye. It is suggested that the serotoninergic innervation as well as the cell containing the pigment-dispersing hormone are included in the mechanism of regulation of light sensitivity of the mollusc eye.     

Neuroanatomical and immunocytochemical studies of the head retractor muscle innervation in the pond snail, Lymnaea stagnalis L

Axonal tracing and immunocytochemical techniques were used to study the innervation of the head retractor muscle (HRM) in the pond snail Lymnaea stagnalis L. Fibers of both the superior and inferior cervical nerves which innervate the HRM form endings that comply with the structure of chemical synapses. The somata of neurons with axons in these nerves are located in all except the buccal ganglia of the central nervous system, and this seems to be a special feature of the HRM motor system. By staining the filamentous actin with Oregon-green conjugated phalloidin, we demonstrated that the HRM has a multiterminal innervation and one muscle fiber can contain several synaptic endings which appear to be both morphologically and physiologically different. The morphological diversity of synaptic vesicles suggests a multiplicity of neurotransmitters acting on these nerve-muscle junctions. Immunocytochemical evidence was found for a strong serotonergic and FMRFamidergic innervation of muscle fibers through axons of the inferior cervical nerve. The thin fibers of the inferior cervical nerve possess immunoreactivity to glutamate, gamma-aminobutyric acid (GABA) and choline-acetyltransferase, and form sparser innervation patterns in the muscle. Our results indicate that several neurotransmitters are present in the nerves innervating the Lymnaea HRM and may therefore participate in the control of this muscle. The possible behavioral significance of such different neurotransmitter sets involved in the regulation of contractions is discussed.     

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.     

The expression pattern of CREB genes in the central nervous system of the pond snail Lymnaea stagnalis

To analyze the expression pattern of genes of cAMP responsive element binding protein (CREB), we performed in situ hybridization for the whole central nervous system (CNS) of the pond snail Lymnaea stagnalis. The CREB1 (activator) and CREB2 (repressor) homologues have already been cloned in L. stagnalis, and they are referred to as LymCREB1 and LymCREB2. Using the frozen sections and the whole mount preparations of the CNS, we mapped the distribution of LymCREB1 and LymCREB2 mRNA containing neurons. The present findings showed that the LymCREB1 mRNA containing neurons are a relatively few, whereas LymCREB2 mRNA is contained ubiquitously in the whole CNS of L. stagnalis.     

Swim pacemaker response to bath applied neurotransmitters in the cubozoan Tripedalia cystophora

The four rhopalia of cubomedusae are integrated parts of the central nervous system carrying their many eyes and thought to be the centres of visual information processing. Rhopalial pacemakers control locomotion through a complex neural signal transmitted to the ring nerve and the signal frequency is modulated by the visual input. Since electrical synapses have never been found in the cubozoan nervous system all signals are thought to be transmitted across chemical synapses, and so far information about the neurotransmitters involved are based on immunocytochemical or behavioural data. Here we present the first direct physiological evidence for the types of neurotransmitters involved in sensory information processing in the rhopalial nervous system. FMRFamide, serotonin and dopamine are shown to have inhibitory effect on the pacemaker frequency. There are some indications that the fast acting acetylcholine and glycine have an initial effect and then rapidly desensitise. Other tested neuroactive compounds (GABA, glutamate, and taurine) could not be shown to have a significant effect.     

Immunocytochemical demonstration of peptidergic neurons in the central and peripheral nervous systems of the flatworm Microstomum lineare with antiserum to FMRF-amide

Summary The central nervous system (CNS) and the peripheral nervous system (PNS) of the flatworm Microstomum lineare were studied by means of the peroxidase-antiperoxidase (PAP) immunocytochemical method, with the use of antisera to the molluscan cardioactive peptide FMRF-amide. FMRF-amide immunoreactive perikarya and nerve fibres are observed in the CNS and the PNS. In the CNS, immunoreactive perikarya and nerve fibres occur in the brain, in the epithelial lining and the mesenchymal surroundings of the ciliated pits, and positive fibres in the longitudinal nerve cords. In the PNS, immunoreactive fibre bundles with variocosities occur in the pharyngeal nerve ring, in symmetrical groups of perikarya on each side of the pharynx, and in the mouth area. Positive perikarya and meandering nerve fibres appear in the intestinal wall. A few immunoreactive cells and short nerve processes are observed at the male copulatory organ and on both sides of the vagina. Some immunoreactive peptidergic cells do not correspond to cells previously identified by histological techniques for neurosecretory cells. The distribution of immunoreactivity suggests that the FMRF-amide-like substance in CNS and PNS in this worm has roles similar to those of the brain-gut peptides in vertebrates. The status of FMRF-amide-like peptides as representatives of an evolutionarily old family of peptides is confirmed by the positive immunoreaction to anti-FMRF-amide in this primitive microturbellarian.     

Monoclonal antibodies recognize localized antigens in the eye and central nervous system of the marine snail Bulla gouldiana

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.     

Postembryonic changes in the optic primordia and optic bud in the flesh fly Sarcophaga ruficornis fabr. (Diptera: Sarcophagidae)

Differentiation of the optic lobe anlagen begin in the brain of second instar. Each is an elongated disc of cortical cells placed on the dorsolateral border of each protocerebrum. In the late second instar the disc elongates and its two ends bend inwards which gradually separate from the central region, thus giving three imaginal discs. The protocerebral neuropile extends into these discs and medulla interna and externa are formed. The rudiments of compound eyes (cephalic complex) appear in the early laid larva. These are attached with the brain and pharyngeal wall separately. The posterior portion of cephalic complex (optic bud), after establishing a nervous association with the central optic lobe anlage (lamina ganglionaris), forms the compound eye. Ech optic bud is attached to the brain by a non-nervous stalk. The epiblast cells of the optic bud do not migrate into the brain and the lamina is formed by the proliferation of the central imaginal disc. The reorientation of the optic lobe anlagen starts in the late third instar and the medulla interna divides into two unequal lobes. In 2 day pupa the nerve fibres from the lamina travel into the optic stalk and the optic nerve is formed. The epiblast cells of the optic bud differentiate to form a peripheral epithelial layer which becomes pigmented and gets apposed to the lateral boundary of the brain. The central epiblast cells of the optic bud form several ommatidia. The optic nerve degenerates gradually and various components of the compound eye are formed by the epiblast cells. Chiasm internum is present but chiasm externum is absent.     

The actions of halothane on spontaneous activity, action potential shape and synaptic connections of the giant serotonin-containing neurone of Lymnaea stagnalis (L.)

1. Cerebral giant cells (CGCs) in the isolated central nervous system (CNS) of the pond snail Lymnaea stagnalis (L.) exhibit bursting activity when superfused with anaesthetic concentrations of halothane. 2. Calcium-dependent components of the CGC action potential appear more sensitive to halothane than do other ionic mechanisms. 3. Higher concentrations of halothane block the chemical synaptic connection between CGC and buccal motoneurone B1, but have no effect on the strong electrotonic coupling between the CGCs. 4. The mechanisms underlying CGC bursting and synaptic block in the presence of halothane are discussed.     

Synaptic and nonsynaptic release of neuromediators in the central nervous system

Different types of release site were studied ultrastructurally with tannic acid and immunohistochemical techniques in the central nervous system (CNS) of the invertebrate pond snail Lymnaea stagnalis and in two neuromediator rich core regions in the CNS of the rat, viz., the median eminence (ME) and the mesencephalic central grey substance (MCG). In the CNS of the snail, release of the contents of the secretory granules could be clearly demonstrated in (1) neurohaemal axonterminals, (2) synapses and (3) in nonsynaptic release sites: neuronal processes without morphological synaptic specializations. In the ME, release of secretory products by exocytosis was found in neurohaemal axonterminals in the external part of the palisade layer and in nonsynaptic release sites in all other layers of the median eminence. It was found that oxytocine and vasopressin were released by exocytosis into the extracellular space from such (preterminal) nonsynaptic release sites. Serial section analysis revealed three types of fibre in the MCG, viz. (1) varicose fibres that made synaptic contacts with MCG dendrites on every varicosity, (2) fibres with two types of varicosity, viz. synapse-bearing varicosities and varicosities without synaptic specializations, and (3) varicose fibres without any synaptic specializations. It has been discussed that the nonsynaptic release sites in the CNS of the snail Lymnaea stagnalis, and the nonsynaptic varicosities in the rat brain are the morphological correlates of nonsynaptic communication in the CNS. The results further indicate that particular peptidergic neuromediators are released from such nonsynaptic varicosities, and may reach via the extracellular space receptors located at some distance.     

Transmitter-dependent involvement of the respiratory interneurons in the locomotor rhythm in the pulmonate mollusc Lymnaea

Pedal cell RPeD1 of the pond snail L. stagnalis becomes involved in a central rhythm identified as an activity of the central pattern generator (CPG) for locomotion. The RPeD1 rhythm developed as driven by a synaptic input in isolated CNS preparations treated with 0.05 mM serotonin (5HT) or 0.1 mM 5-hydroxytryptophan (5HTP). The 5HT-induced co-ordinated rhythmic activity was retained by each of two pedal ganglia after complete isolation thus suggesting that the respective CPG lies entirely within the pedal portion of the CNS and is paired. The findings suggest that the RPeD1 switching from one network to another represents a neurotransmitter-dependent phenomenon.     

Distribution of serotonin-containing neurons in the central nervous system of the snail Helix pomatia

Summary The distribution of serotonin (5HT)-containing neurons in the central nervous system of the snail Helix pomatia has been determined in whole-mount preparations by use of immunocytochemical and in vivo 5,6-dihydroxy-tryptamine labelling. 5HT-immunoreactive neuronal somata occur in all but the buccal and pleural ganglia. Immunoreactive fibres are present throughout the central nervous system. The 5HT-immunoreactive neuronal somata characteristically appear in groups, located mainly in the cerebral, pedal, visceral and right parietal ganglia. The majority of 5HT-immunoreactive neurons is located in the pedal ganglia. Additionally a dense network of 5HT-immunoreactive varicose fibres is found in the neural sheath of the central nervous system including all the nerves and ganglia. The number and distribution of 5HT-immunoreactive neurons correlates with that demonstrated by 5,6-dihydroxytryptamine labelling method.     

The distribution of NADPH diaphorase activity and immunoreactivity to nitric oxide synthase in the nervous system of the pulmonate mollusc Helix aspersa

Enzyme histochemistry and immunocytochemistry were used to determine the distribution of neurons in the snail Helix aspersa which exhibited nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase activity and/or immunoreactivity to nitric oxide synthase (NOS). NADPH diaphorase-positive cells and fibres were distributed extensively throughout the central and peripheral nervous system. NADPH diaphorase-positive fibres were present in all neuropil regions of the central and peripheral ganglia, in the major interganglionic connectives and in peripheral nerve roots. NADPH diaphorase-positive cell bodies were found consistently in the eyes, the lips, the tentacular ganglia and the procerebral lobes of the cerebral ganglia; staining of cell bodies elsewhere in the nervous system was capricious. The distribution of NOS-like immunoreactivity differed markedly from that of NADPH diaphorase activity. Small clusters of cells which exhibited NOS-like immunoreactivity were present in the cerebral and pedal ganglia; fibres which exhibited NOS-like immunoreactivity were present in restricted regions of the neuropil of the central ganglia. The disjunct distributions of NADPH diaphorase activity and NOS-like immunoreactivity in the neurvous system of Helix suggest that the properties of neuronal NOS in molluscs may differ sigificantly from those described previously for vertebrate animals.     

Neurochemical characterization of nervous elements innervating the body wall of earthworms (Lumbricus, Eisenia): immunohistochemical and pharmacological studies

The distribution and chemical neuroanatomy of nervous elements and certain pharmacological–physiological characteristics of the innervation of the body wall in earthworms are described. Solitary sensory bipolar cells can be found among the epithelial cells. These bipolar cells contain serotonin, tyrosine hydroxylase, histamine, gamma-amino-butyric acid (GABA), Eisenia tetradecapeptide, proctolin or rhodopsin in various combinations. In the body wall, the plexus submuscularis is composed of nerve fibres only, whereas the plexus subepithelialis and muscularis also contain solitary nerve cells. These cells display histamine, GABA or neuropeptide Y immunoreactivity. The fibres of the three plexuses are reactive to serotonin, histamine, Eisenia tetradecapeptide, proctolin, GABA and neuropeptide Y antibodies. FMRFamide-immunoreactive fibres of the plexus muscularis originate from the central nervous system, whereas axons containing the other studied molecules are derived from both peripheral and central structures. High pressure liquid chromatography assays have revealed serotonin, dopamine and histamine in the body wall. Contractions of the body wall musculature can be elicited with serotonin and FMRFamide. Serotonin-evoked contractions are suppressed by the application of GABA. Serotonin acts both directly on the muscle cell receptors and indirectly through initiating transmitter release from the nervous elements, whereas the FMRFamide-induced contractions seem to be mediated through the muscle cell receptors only. The pharmacological profiles of the serotonin and GABA receptors resemble those of the vertebrate 5-HT₃ and GABA_B receptor types. Our findings indicate that both the sensory and efferent system of the annelid body wall operate by means of a variety of neuroactive compounds, suggesting a complex role of signalling systems in the regulation of this organ.This work was supported by the Hungarian Scientific Research Fund (OTKA; grant nos. T 34106 and T 34160). Márta Wilhelm is in receipt of a János Bolyai Scholarship.     

Immunocytochemical demonstration of peptidergic cells in the pond snail Lymnaea stagnalis with an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide

Summary With an antiserum to the molluscan cardioactive tetrapeptide FMRF-amide immunoreactive perikarya and nerve fibers were identified in the central and peripheral nervous system of the pond snail Lymnaea stagnalis. Their localization is described. The same antiserum yielded reactive product in particular cells of the epithelium of the alimentary tract. The use of two different fixatives, glutaraldehyde, and a mixture of glutaraldehyde, picric acid, and acetic acid (GPA) showed that certain nerve cells can be identified only in material fixed with either the one or the other of these two fixatives, a result which indicates that in Lymnaea more than one FMRF-amide-like substance may occur.Positive axon endings were found in the periphery of various nerves, i.e., in places where neurohormones are released into the blood. Other fibers were found to end, probably synaptically, on other neurons, on epithelial cells in the stomach, and between muscle cells in various parts of the body, e.g., in the heart. In these cases the FMRF-amide-like substance may function as a neurotransmitter or a neuromodulator.     

The development of the serotonergic and FMRF-amidergic nervous system in<Emphasis Type="Italic"> Antalis entalis</Emphasis> (Mollusca,Scaphopoda)

The morphogenesis of serotonin- and FMRF-amide-bearing neuronal elements in the scaphopod Antalis entalis was investigated by means of antibody staining and confocal laser scanning microscopy. Nervous system development starts with the establishment of two initial, flask-like, serotonergic central cells of the larval apical organ. Slightly later, the apical organ contains four serotonergic central cells which are interconnected with two lateral serotonergic cells via lateral nerve projections. At the same time the anlage of the adult FMRF-amide-positive cerebral nervous system starts at the base of the apical organ. Subsequently, the entire neuronal complex migrates behind the prototroch and the six larval serotonergic cells lose transmitter expression prior to metamorphic competence. There are no strictly larval FMRF-amide-positive neuronal structures. The development of major adult FMRF-amide-containing components such as the cerebral system, the visceral loop, and the buccal nerve cords, however, starts before the onset of metamorphosis. The anlage of the putative cerebral system is the only site of adult serotonin expression in Antalis larvae. Establishment of the adult FMRF-amidergic and serotonergic neuronal bauplan proceeds rapidly after metamorphosis. Neurogenesis reflects the general observation that the larval phase and the expression of distinct larval morphological features are less pronounced in Scaphopoda than in Gastropoda or Bivalvia. The degeneration of the entire larval apical organ before metamorphic competence argues against an involvement of this sensory system in scaphopod metamorphosis. The lack of data on the neurogenesis in the aplacophoran taxa prevent a final conclusion regarding the plesiomorphic condition in the Mollusca. Nevertheless, the results presented herein shed doubts on general theories regarding possible functions of larval "apical organs" of Lophotrochozoa or even Metazoa.     

Cellular analysis of appetitive learning in invertebrates

In recent years significant progress has been made in the analysis of the cellular mechanisms underlying appetitive learning in two invertebrate species, the pond snail Lymnaea stagnalis and the honeybee Apis mellifera. In Lymnaea, both chemical (taste) and tactile appetitive conditioning paradigms were used and cellular traces of behavioural classical conditioning were recorded at several specific sites in the nervous system. These sites included sensory pathways, central pattern generator and modulatory interneurones as well as motoneurones of the feeding network. In the honeybee, a chemical (odour) appetitive conditioning paradigm resulted in cellular changes at different sites in the nervous system. In both the pond snail and the honeybee the activation of identified modulatory interneurones could substitute for the use of the chemical unconditioned stimulus, making these paradigms even more amenable to more detailed cellular and molecular analysis.     

Immunocytochemical demonstration of neuropeptides and serotonin in the tapeworm Diphyllobothrium dendriticum

Summary The present immunocytochemical study concerns the distribution of four neuropeptides, FMRF-amide, vasotocin, leu-enkephalin and neurotensin, and of the bioamine serotonin in the plerocercoid larva of Diphyllobothrium dendriticum. Anti-FMRF-amide and vasotocin-reactivity occurs in perikarya and nerve fibres in the CNS and PNS of this worm. The peptide-containing fibres surround and seem to innervate the musculature and to terminate beneath the basal lamina of the tegument at the inner surface of the bothridia, suggesting a neurotransmitter function. Antileu-enkephalin reaction occurs in perikarya and fibres in the main nerve cords and in the PNS. Anti-neurotensin reactive fibres were observed in the neuropile of the nerve cords. Serotonin immunoreactivity was found in neurons in the ganglionic commissure of the brain and along the main nerve cords. This study is the first immunocytochemical identification of neuropeptides and serotonin in a parasitic flatworm and the information gained may be of importance for the development of new antihelminthics.     

Histochemical reaction to biogenic amines of the nervous system of the larval and mature cestode Microsomacanthus microskrjabini

Histochemical reaction with glyoxylic acid has revealed a specific fluorescence caused by the presence of indolamine (apparently serotonin) in the nervous system of larva and mature cestode. Fluorescence manifests itself in neurons and nerve fibres of the central ganglion and its commissure, in nerve cells of the proboscis, in longitudinal trunks and transverse commissures, and in the nerve elements connected with genital system.     

淡水蜗牛视觉系统的输入与输出通路(英文)

通过神经生物素在视神经上的逆行性传输对淡水蜗牛(Planorbarius corneus)视网膜及中央神经节的输入、输出神经元进行标记。由于没有发现突触联系,所以至少一部分光感受细胞的轴突可被视为直接参与形成视神经。这些神经元的轴突进入大脑神经节形成密集的细传入神经纤维束-视神经堆。传出神经元则存在于除颊部以外的所有神经节。一些上行轴突在大脑神经节处分叉,通过脑-脑联合,到达对侧眼并在眼杯处形成分枝。部分传出神经元的轴突也投射于不同的外周神经,如:n.n.intestinalis,pallialis dexter,pallialis sinister internus et externus。五羟色胺能纤维和FMRF-酰胺能纤维均存在于视神经上,且这些纤维隶属于只投射在同侧眼的中央神经元。它们形成了位于眼杯处的丰富曲张结构及视网膜核心层,并且可能有助于调节视网膜对光的敏感性。