Chemical-neuroanatomical organization of peripheral sensory-efferent systems in the pond snail (Lymnaea stagnalis).

Perception and processing of chemical cues are crucial for aquatic gastropods, for proper elaboration of adaptive behavior. The pond snail, Lymnaea stagnalis, is a model species of invertebrate neurobiology, in which peripheral sensory neurons with different morphology and transmitter content have partly been described, but we have little knowledge regarding their functional morphological organization, including their possible peripheral intercellular connections and networks. Therefore the aim of our study was to characterize the sensory system of the tentacles and the lip, as primary sensory regions, and the anterior foot of Lymnaea with special attention to the transmitter content of the sensory neurons, and their relationship to extrinsic elements of the central nervous system. Numerous bipolar sensory cells were demonstrated in the epithelial layer of the peripheral organs, displaying immunoreactivity to antibodies raised against tyrosine hydroxylase, histamine, glutamate and two molluscan type oligopeptides, FMRFamide and Mytilus inhibitory peptide. A subepithelial plexus was formed by extrinsic serotonin and FMRFamide immunoreactive fibers, whereas in deeper regions axon processess of different origin with various immunoreactivities formed networks, too. HPLC-MS assay confirmed the presence of the low molecular weight signal molecules in the three examined areas. Following double-labeling immunohistochemistry, close arrangements were observed, formed by sensory neurons and extrinsic serotonergic (and FMRFamidergic) fibers at axo-dendritic, axo-somatic and axo-axonic levels. Our results suggest the involvement of a much wider repertoire of signal molecules in peripheral sensory processes of Lymnaea, which can locally be modified by central input, hence influencing directly the responses to environmental cues.

Serotonergic regulation of the buccal (feeding) rhythm of the pond snail, Lymnaea stagnalis. An immunocytochemical, biochemical and pharmacological approach.

Hatching is an important phase of the development of pulmonate gastropods followed by the adult-like extracapsular foraging life. Right before hatching the juveniles start to display a rhythmic radula movement, executed by the buccal complex, consisting of the buccal musculature (mass) and a pair of the buccal ganglia. In order to have a detailed insight into this process, we investigated the serotonergic regulation of the buccal (feeding) rhythm in 100% stage embryos of the pond snail, Lymnaea stagnalis, applying quantitative immunohistochemistry combined with the pharmacological manipulation of the serotonin (5-HT) synthesis, by either stimulating (by the 5-HT precursor 5-hydroxytryptophan, 5-HTP) or inhibiting (by the 5-HT synthesis blocker para-chlorophenylalanine, pCPA) it. Corresponding to the direction of the drug effect, significant changes of the fluorescence intensity could be detected both in the cerebral ganglia and the buccal complex. HPLC-MS assay demonstrated that 5-HTP increased meanwhile pCPA decreased the 5-HT content both of the central ganglia and the buccal complex. As to the feeding activity, 5-HTP induced only a slight (20%) increase, whereas the pCPA resulted in a 20% decrease of the radula protrusion frequency. Inhibition of 5-HT re-uptake by clomipramine reduced the frequency by 75%. The results prove the role of both central and peripheral 5-HTergic processes in the regulation of feeding activity. Application of specific receptor agonists and antagonists revealed that activation of a 5-HT1-like receptor depressed the feeding activity, meanwhile activation of a 5-HT6,7-like receptor enhanced it. Saturation binding plot of [3H]-5-HT to receptor and binding experiments performed on membrane pellets prepared from the buccal mass indicated the presence of a 5-HT6-like receptor positively coupled to cAMP. The results suggest that 5-HT influences the buccal (feeding) rhythmic activity in two ways: an inhibitory action is probably exerted via 5-HT1-like receptors, while an excitatory action is realized through 5-HT6,7-like receptors.

Neuronal Development in the Larvae of the Invasive Biofouler Dreissena polymorpha (Mollusca: Bivalvia), with Special Attention to Sensory Elements and Swimming Behavior.

Although understanding of the neuronal development of Trochozoa has progressed recently, little attention has been paid to freshwater bivalves, including species with a strong ecological impact, such as the zebra mussel (Dreissena polymorpha). Therefore, an important question might concern how the developing nervous system is involved in the formation of the rapid and successful invasive behavior of this species. Our aim was to reveal the neuronal development of trochophore and veliger larvae of Dreissena, with special attention to the organization of sensory structures and their possible involvement in detecting environmental cues. After applying serotonin and FMRFamide immunocytochemistry, the first serotonin immunoreactive sensory elements appeared 16-18 hours after fertilization, whereas the first FMRFamide immunoreactive sensory cell was seen only at 32 hours of development (trochophore stage). Later, sensory elements were found in three parts of the larval body, including the apical organ, the posterior region, and the stomach. Although differences in the timing of appearance and the morphology of cells were observed, the two signaling systems showed basic similarity in their organization pattern until the end of the veliger stage. Pharmacological, physiological, and quantitative immunocytochemical investigations were also performed, suggesting the involvement of both the serotoninergic system and the FMRFamidergic system in sensomotor processes. Manipulation of the serotonin synthesis by para-chloroplenylalanine and 5-hydroxytryptophane, as well as application of increased salinity, influenced larval swimming activity, both accompanied by changes in immunofluorescence intensity. We concluded that these two early sensory systems may play an important role in the development of settlement competency of this biofouling invasive bivalve, Dreissena.

Nitric oxide-coupled signaling in odor elicited molecular events in the olfactory center of the terrestrial snail, Helix pomatia.

Olfaction, a chemosensory modality, plays a pivotal role in the orientation and behavior of invertebrates. The central olfactory processing unit in terrestrial stylomatophoran snails is the procerebrum, which contains NO synthesizing interneurons, whose oscillatory currents are believed to be the base of odor evoked memory formation. Nevertheless, in this model the up- and downstream events of molecular cascades that trigger and follow NO release, respectively, have not been studied. Immunocytochemistry and flow cytometry studies performed on procerebral neural perikarya isolated from the snail Helix pomatia revealed cell populations with discrete DAF-2 fluorescence, indicating the release of different amounts of NO. Glutamate increased the intensity of DAF-2 fluorescence, and the number of DAF-2 positive non-bursting interneurons, through a mechanism likely to involve an NMDA-like receptor. Similarly to glutamate, NO activation induced an increase in intracellular cGMP levels through activation of soluble guanylyl cyclase. Immunohistochemical localization of proteins possessing the phosphorylated target sequence of AGC family kinases (RXXS/T-P), among them protein kinase A (RRXS/T-P), showed striking similarities to the distribution of NOS/cGMP. Activators of cyclic nucleotide synthesis increased the AGC-kinase-dependent phosphorylation of discrete proteins with 28, 45, and 55kDamw. Importantly, exposure of snails to an attractive odorant induced hyperphosphorylation of the 28kDa protein, and increased levels of cGMP synthesis. Protein S-nitrosylation and intercellular activation of protein kinase G were also suggested as alternative components of NO signaling in the snail procerebrum. The present results from Helix pomatia indicate an important role for procerebrum NO/cGMP/PKA signaling pathways in the regulation of olfactory (food-finding) behavior.

Organization of the procerebrum in terrestrial pulmonates (Helix, Limax) reconsidered: cell mass layer synaptology and its serotonergic input system.

The synaptology of the cell body layer of the olfactory center, procerebrum, was investigated in two prominent terrestrial pulmonate gastropod species, Helix pomatia and Limax valentianus. In addition, the analysis of the 5-HT-immunoreactive innervation, including ultrastructural level, was performed at high resolution in H. pomatia. A highly complex system of synaptic and non-synaptic connections was found in the procerebrum of both species connected to local neuropil areas of different size. The procerebral (globuli) cell perikarya were richly innervated by varicosities meanwhile the axon profiles also established contacts with each other. Synaptic configurations including convergence, divergence and presynaptic modulation were also revealed. The frequent occurrence of unspecialized but close axo-somatic and axo-axonic membrane contacts referring to the modulatory forms of transmitter release were also accompanied by membrane configurations indicative of active exocytosis. In H. pomatia, the cell mass layer was shown to receive a rich 5-HT-immunoreactive innervation, forming a dense network around the cell bodies. At ultrastructural level, 5-HT-immunoreactive varicosities contacted both cell bodies and different unlabeled axon profiles. Our results suggest that the local neuropil regions in the cell body layer are site of local circuits, which may play a decisive role in olfactory integrative processes bound to the procerebrum. The pattern and form of the 5-HT-immunoreactive innervation of extrinsic origin suggest an overall modulatory role in the cell body layer. The results may serve a basis for considering the role of local intercellular events, connected to microcircuits, within the procerebrum cell body layer involved in oscillation activities.

The 5-HT immunoreactive innervation of the Helix procerebrum.

In the procerebrum of terrestrial snails, 5-HT is a key modulatory substance of the generation of synchronous oscillatory activity and odor learning capability. In this study, we have analyzed the characteristics of the 5-HT-immunoreactive (5-HT-IR) innervation of the distinct anatomical regions of the procerebrum of Helix pomatia, applying correlative light- and electron microscopic immunocytochemistry. A dense network of 5-HT-IR innervation was demonstrated in the cell body layer, meanwhile a varicose fiber system of different density occurred in the different neuropil regions. At the ultrastructural level, labeled varicosities were found to contact both procerebral cell bodies, and different unlabeled axon profiles in the neuropils. The labeled structures established mostly close non-specialized membrane contacts with the postsynaptic profiles. The overall dense distribution of 5-HT-IR innervation supports a general modulatory role of 5-HT in processing different olfactory events.

Potassium channels in the Helix central nervous system: preliminary immunohistochemical studies. Short communication.

Distribution of the potassium channel of Kv4.3 type was investigated in the central nervous system (CNS) of Helix pomatia by immunohistochemistry. Immunopositive neurons were found widely distributed in the CNS, present mostly in smaller groups in the different central ganglia but not in the visceral ganglion. Labeled fibers were characteristic for not only the neuropils of all ganglia but also the connective tissue sheath around the CNS and the aorta wall were richly innervated. Western blot analysis revealed a clear identity with the mammalian Kv4.3 subunit, suggesting an evolutionary conserved structure of this channel type. Our preliminary results provide a steady basis for further experiments aiming partly at the identification of other potassium channel types and partly the ultrastructural localization of Kv4.3.

Subcellular distribution of nitric oxide synthase isoforms in the rat duodenum.

AIM: To study the cell-type specific subcellular distribution of the three isoforms of nitric oxide synthase (NOS) in the rat duodenum. METHODS: Postembedding immunoelectronmicroscopy was performed, in which primary antibodies for neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), were visualized with protein A-gold-conjugated secondary antibodies. Stained ultrathin sections were examined and photographed with a Philips CM10 electron microscope equipped with a MEGAVIEW II camera. The specificity of the immunoreaction in all cases was assessed by omitting the primary antibodies in the labeling protocol and incubating the sections only in the protein A-gold conjugated secondary antibodies. RESULTS: Postembedding immunoelectronmicroscopy revealed the presence of nNOS, eNOS, and iNOS immunoreactivity in the myenteric neurons, the enteric smooth muscle cells, and the endothelium of capillaries running in the vicinity of the myenteric plexus of the rat duodenum. The cell type-specific distributions of the immunogold particles labeling the three different NOS isozymes were revealed. In the control experiments, in which the primary antiserum was omitted, virtually no postembedding gold particles were observed. CONCLUSION: This postembedding immunoelectronmicroscopic study provided the first evidence of cell-type-specific differences in the subcellular distributions of NOS isoforms.

Spatial pattern analysis of nitrergic neurons in the myenteric plexus of the duodenum of different mammalian species.

Nitrergic myenteric neurons are especially susceptible to the development of neuropathy in functional gastrointestinal disorders. Investigations of the similarities and dissimilarities in the organization of nitrergic neurons in the various mammalian species are therefore important in an effort to determine the extent to which the results obtained in different animal models can be generalized. In the present work, the density and the spatial organization of the nitrergic neurons in the myenteric plexus of the duodenum were investigated in 7 mammalian species. After nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, the Plexus Pattern Analysis software (PPAs) was applied to count the nuclei of nitrergic neurons, calculate the proportions of the areas covered by the plexus and perform randomization analysis. All 7 species exhibited a large population of nitrergic myenteric neurons, with densities in the range 12-56 cells/mm 2 . The distribution patterns of these neurons differed markedly in the different species, however, the rat was the only species in which the nitrergic neurons appeared to be randomly distributed. The PPAs in conjunction with NADPH-d histochemistry proved to be a simple and fast tool with which to reveal similarities and dissimilarities in the spatial arrangement of the nitrergic neurons in the different species.