[Early Peripheral Sensory Neurons in the Development of Trochozoan Animals].

Neuronal development of the majority of trochozoan animals with biphasic pelago-bentic life cycle starts from transient peripheral neurons, which do not belong to the central nervous system and are mainly located in the apical sensory organ and in the hyposphere. Some of these neurons are pioneer and send neurites that form a scaffold upon which the adult central nervous system later develops. In representative species of molluscs and polychaetes, immunolabelling with the antibodies against neurotransmitters serotonin and FMRFamide, and acetylated α-tubulin revealed that the structure of almost all early peripheral neurons is typical for sensory, most probably chemosensory cells: flask shape, and cilia at the end of the apical dendrite or inside the distal ampoule. Morphology, transmitter specificity, location and projections of the early sensory cells differ in trochophores of different species thus suggesting different origin of these cells. In polychaete larvae, pharmacological inhibition of serotonin synthesis in early peripheral neurons did not affect the development, whereas its increase resulted in developmental arrest and neural malformations, suggesting that early peripheral sensory neurons are involved in developmental regulation.

Delayed action of serotonin in molluscan development.

Serotonin (5-HT) is known to induce a wide range of short-term and long-term (or delayed) effects. In the present paper we demonstrated that short time-window application of the 5-HT precursor 5-hydroxytryptophan during early cleavage stages results in both irreversible morphological malformation (exogastrulation) and distinct changes in behavior of young animals of the freshwater snail, Lymnaea stagnalis (Mollusca: Gastropoda). Pharmacological and immunocytochemical analysis confirmed that both the increase of intracellular 5-HT level within the cleaved blastomers and activation of membrane 5-HT2-like type receptors are required for the appearence of these phenomena.

[The golden age of comparative morphology: laser scanning microscopy and neurogenesis of trochophore animals].

Immunochemical labeling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology and allowed us to monitor the neurogenesis of various trochophore animals at the level of individual identified neurons and their projections. It has been demonstrated that many generally accepted concepts of the larval nervous system and the phylogenetic theories constructed on this basis are incorrect. Comparative analysis has demonstrated that the orthogonal brain is absent at all developmental stages in the representative Lophotrochozoa members. Fundamental differences in the structure and development of the nervous system have been found in the trochophores belonging to different taxonomic groups within Lophotrochozoa; these differences demonstrate that the trochophore larva in these groups are not homologous, while their similarity is most likely a result of convergence. Our results challenge the concept of trochophore as the ancestral form common for all trochophore animals. It is necessary to exclude from phylogenetic discussions the orthogon as a basic plan for the structure of the nervous system and the trochophore as an ancestral form for all Lophtrochozoa.

Adult-to-embryo chemical signaling in the regulation of larval development in trochophore animals: cellular and molecular mechanisms.

The regulation of larval development by starved adults occurs in both freshwater snails, Helisoma trivolvis and marine polychaetes, Platynereis dumerilii. Serotonin (5-HT) links this environmental signal which is detected by larval apical sensory neurons to changes in larval development. A profile of the stage-dependent expression of 5-HT receptors and coupled G proteins is essential in this regulatory mechanism. The final effect on development depends on the modulation of the activity of the larval digestive system.

[Role of chemical signalling in release of motor programs during embryogenesis of freshwater snails Lymnaea stagnalis and Helisoma trivolvis].

We have earlier found that freshwater pond snails Helisoma trivolvis and Lymnaea stagnalis, when reared under conditions of starvation, release chemical signals that reversibly suppress larval development of conspecific embryos. Here, we report that (i) these signals are not strictly conspecific and affect also embryos of a closely related species, which occupies a similar environmental niche; (ii) besides the development of embryos, the signals also affect the release of main motor programs, such as locomotion, feeding, and cardiac activity; (iii) action of the signals is bidirectional: they retard the development and release of motor programs at the early larval stages (trochophore to veliger) and accelerate them at later stages (late veliger to hatching). A possible adaptive significance of the described phenomena is discussed.

[Structure and electrophysiological properties of bursting neurosecretory cells in a peripheral sensory ganglion of the pond snail Lymnaea stagnalis]. / Morfologiia i élektrofiziologicheskie svoistva pachechnykh neirosekretornykh kletok v perifericheskom sensornom organe bol'shogo prudovika 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.

Novel, posterior sensory organ in the trochophore larva of Phyllodoce maculata (Polychaeta).

A new posterior sensory organ (PSO), located at the dorsal midline of the hyposphere, is described by immunocytochemical detection of acetylated alpha tubulin and serotonin (5-HT) in a laser-scanning microscope, as well as three-dimensional reconstructions after optical serial sectioning in the trochophore larva of the polychaete Phyllodoce maculata (Phyllodocidae). The unpaired PSO consists of five bipolar sensory cells, two of them being 5-HT immunopositive, which send axons to the cerebral ganglion and prototroch nerve. The dendrites of these cells project to the surface and bear one cilium each. A single neuronal fibre from the apical sensory organ innervates the PSO.

Slice culture of the olfactory bulb of Xenopus laevis tadpoles.

We report on the development of a slice culture of amphibian brain tissue. In particular, we cultured slices from Xenopus laevis tadpoles that contain the olfactory mucosae, the olfactory nerves, the olfactory bulb and the telencephalon. During 6 days in roller tubes the slices flattened, starting from 250 microm and decreasing to approximately 40 microm, corresponding to about three cell layers. Dendritic processes could be followed over distances as long as 200 microm. Neurons in the cultured slice could be recorded using the patch clamp technique and simultaneously imaged using an inverted laser scanning microscope. We characterized the main neuron types of the olfactory bulb, i.e. mitral cells and granule cells, by correlating their typical morphological features in the acute slice with the electrophysiological properties in both the acute slice and slice culture. This correlation allowed unambiguous identification of mitral cells and granule cells in the slice culture.

Noradrenergic modulation of calcium currents and synaptic transmission in the olfactory bulb of Xenopus laevis tadpoles.

Norepinephrine (NE) has various modulatory roles in both the peripheral and the central nervous systems. Here we investigate the function of the locus coeruleus efferent fibres in the olfactory bulb of Xenopus laevis tadpoles. In order to distinguish unambiguously between mitral cells and granule cells of the main olfactory bulb and the accessory olfactory bulb, we used a slice preparation. The two neuron types were distinguished on the basis of their location in the slice, their typical branching pattern and by electrophysiological criteria. At NE concentrations lower than 5 microM there was only one effect of NE upon voltage-gated conductances; NE blocked a high-voltage-activated Ca(2+)-current in mitral cells of both the main and the accessory olfactory bulbs. No such effect was observed in granule cells. The effect of NE upon mitral cell Ca(2+)-currents was mimicked by the alpha(2)-receptor agonists clonidine and alpha-methyl-NE. As a second effect, NE or clonidine blocked spontaneous synaptic activity in granule cells of both the main and the accessory olfactory bulbs. NE or clonidine also blocked the spontaneous synaptic activity in mitral cells of either olfactory bulb. The amplitude of glutamate-induced currents in granule cells was modulated neither by clonidine nor by alpha-methyl-NE. Taken together, the main effect of the noradrenergic, presynaptic, alpha(2)-receptor-mediated block of Ca(2)+-currents in mitral cells appeared to be a wide-spread disinhibition of mitral cells in the accessory olfactory bulb as well as in the main olfactory bulb.

Structure of the olfactory bulb in tadpoles of Xenopus laevis.

The structure of the olfactory bulb in tadpoles of Xenopus laevis (stages 54-56) was studied using axon tracing (with biocytin or low-weight dextran) and immunocytochemical techniques. Filling the olfactory nerve with biocytin made the nerve layer and the glomeruli visible. Dye injections into the glomerular layer labeled the lateral olfactory tract. Vice versa, dye injections into the lateral olfactory tract made mitral cells and their glomerular branching patterns visible. Anti-GABA antiserum stained periglomerular and granule cells, while the olfactory nerve and mitral cells were labeled by antiglutamate antiserum. We describe the layering, the numbers of cells and glomeruli, and their localization in both the main and the accessory olfactory bulb.

GABA-immunoreactive neurones and interactions of GABA with serotonin and FMRFamide in a peripheral sensory ganglion of the pond snail Lymnaea stagnalis.

The osphradium is a putative chemosensory organ of aquatic molluscs. Previously, we identified cells with serotonin (5-HT) and FMRFamide (FMRFa)-like immunoreactivity in the osphradial ganglion of Lymnaea stagnalis. The present investigation has established the presence of cells immunoreactive to gamma-aminobutyric acid (GABA). Some of these cells send processes to the sensory epithelium and are thus considered to be primary sensory neurones. Colocalisation of GABA and FMRFamide-like immunoreactivities was found in some of these and other neurones. The responses of the osphradial output electrical activity to the single and combined application of the above neuroactive substances were examined. 5-HT slightly increased and FMRFa decreased the activity. GABA alone was generally ineffective; however, it had a consistent stimulating effect after pretreatment with 5-HT. In its turn, pretreatment with GABA significantly increased the inhibitory action of FMRFa. Primary sensory neurones giving this kind of responses in the nerve were identified electrophysiologically and morphologically in the osphradial ganglion. Our results indicate that GABA takes part in relay of sensory signals into the central nervous system, and transmitter interactions involving GABA, 5-HT, and FMRFa are considerable for the final output pattern of the osphradial sensory network.

Tyrosine hydroxylase-negative, dopaminergic neurons are targets for transmitter-depleting action of haloperidol in the snail brain.

1. The effects of long term administration of micromolar concentrations of the D2 antagonist haloperidol upon monoaminergic neurons in the snail Lymnaea stagnalis was investigated. 2. Treatment by bath application with 0.5-2.0 micromolar haloperidol, caused a significant, continuous depletion of dopamine levels in the nervous system as revealed by high performance liquid chromatography. 3. A transient depletion of serotonin was also observed, but DOPA and norepinephrine levels were unaffected. Similar depletion of dopamine was observed after the land snail, Achatina fulica, was injected with haloperidol on each of 4 consecutive days. 4. The depletion of dopamine as revealed with glyoxylate-induced fluorescence in Lymnaea appears to be restricted to a subpopulation of catecholaminergic neurons which are immuno-negative for tyrosine hydroxylase, the synthetic enzyme responsible for the conversion of tyrosine to DOPA. 5. The results thus demonstrate a depleting action of low micromolar doses of chronic haloperidol on specific subsets of dopaminergic neurons and a novel preparation for studying catecholaminergic mechanisms operating across the animal kingdom.

Primary sensory neurons and their central projections in the pond snail Lymnaea stagnalis.

We describe the location of primary sensory neurons in the tentacles, lips, pneumostome area and osphradium of the pond snail Lymnaea stagnalis after Nickel-lysine and biocytin dye-filling into the corresponding nerves. In addition, central projections of the osphradial sensory system were determined. Most of the neurons in the osphradial ganglion are primary sensory cells that send processes directly to the CNS. Fibres originating from these neurons are located in the neuropil of all central ganglia except buccal and pedal. Back-filling the CNS from the osphradial nerve showed that only two pairs of neurons in the cerebral ganglia, two neurons in the right parietal ganglion, and one cell in the left pleural ganglion send processes into the osphradium. Back-filling with biocytin showed that only five to seven neurons in the right parietal and visceral ganglia are dye-coupled with osphradial neurons. Such a small number of central neurons projecting to the osphradial sensory system represents a large difference from the tentacular and lip systems, where hundreds of central neurons innervate the pheriphery. We propose that two different systems with either central or peripheral processing of sensory information may be involved in chemoreception in Lymnaea. Our results provide a basis for physiological studies in this field.

Chronic haloperidol: neural correlates of motor disorders in an invertebrate model.

To analyse the mechanisms underlying chronic effects of antipsychotic drugs, a simpler invertebrate preparation is proposed. Bathing of leeches for three days in a 1.0 microM aqueous haloperidol (HAL) solution resulted in (i) discoordination of locomotion, including the inability of the posterior sucker to successfully attach to the substratum, and (ii) transmitter depletion in all putative dopamine (DA) neurones of the sucker and in some, but not all, DA neurones of middle body segments. Both behavioural restoration and cellular DA recovery were observed in disabled animals after bathing in 1.25 mM L-DOPA solution. We conclude that chronic HAL-produced motor disorders are causally related to DA deficiency.

Transmitter-Specific Subsets of Sensory Elements in the Prosobranch Osphradium.

The osphradium is a putative chemosensory organ of aquatic molluscs. Previously, we identified two distinct types of primary sensory neurons in the osphradial ganglion of freshwater pulmonates, one immunoreactive to leucine-enkephalin (LEnk-ir) and another to FMRFamide (FMRFa-ir). In addition, NADPH diaphorase (NADPHd)-positive elements apparently producing nitric oxide (NO) were demonstrated in the organ. In the present study, prosobranch molluscs, which have retained the osphradial sensory neurons within the epithelium, were studied. Both types of peptidergic neurons, as well as NADPHd-positive cells, were found within the epithelium or in a basiepithelial position in the relatively simple osphradium of the mesogastropod Littorina littorea and in the complex, bipectinate osphradium of the neogastropod Buccinum undatum. Similar evidence was also obtained for another mesogastropod, Ampullarius sp. Transmitter-specific sensory cell types like those discovered in the osphradium are also present as single neuroepithelial cells in other organs of the mantle complex in prosobranchs and in the pelecypod Anodonta cygnea. We suggest that evolutionarily conservative, transmitter-specific types of epithelial and neuroepithelial sensory cells predated the osphradium, which developed as the site of their concentration, while retaining characteristic subsets of sensory neurons.

Patterns of enkephalin immunolabeling in the pulmonate snail Cepaea nemoralis and related molluscs.

In the land pulmonate snail Cepaea nemoralis, immunocytochemical localization of methionine- and leucine-enkephalin-like substances was demonstrated in specific populations of brain neurons. Methionine-enkephalin reactivity is also present in the peripheral nervous system as (i) abundant axonal projections of central neurons to the sole of the foot, and (ii) sparse local neurons of the head and mantle wall. Similar peripheral methionine-enkephalinergic elements were found immunocytochemically in all pulmonates (Helix aspersa, Lymnaea stagnalis) and prosobranchs (Littorina littorea, Acmaea testudinalis) examined and seem therefore conserved among gastropods. Their distribution was different from those of nervous elements labeled by antisera against serotonin and FMRFamide. The chiton Lepidopleurus asellus considered to be a more primitive mollusc, had both types of enkephalin-immunoreactive elements in the central nervous system whereas peripheral methionine-enkephalin-immunoreactive elements were lacking. It is suggested on the morphological evidence that enkephalinergic (especially methionine-enkephalinergic) neurons are part of a sensory system mediating analgetic effects in molluscs.

Is nitric oxide (NO) produced by invertebrate neurones?

NADPH-diaphorase (NADPHd) is known to be identical to nitric oxide (NO) synthase in the mammalian nervous system, and is therefore used as a marker of NO-producing neurones. Using the histochemical reaction for NADPHd, we searched for such neurones in a selection of invertebrates. Special emphasis was given to molluscs. No selective neuronal staining was found in representatives of coelenterates, turbellarians, nematodes and urochordates. In all annelids, arthropods and molluscs examined, with the exception of a chiton, specific neurones were selectively stained. The reaction was particularly strong in pulmonate molluscs where scattered positive neurones were found in various ganglia and clustered symmetrically in the paired buccal ganglia. Biochemical assay of NO synthase in osphradia of the gastropod mollusc Lymnaea stagnalis revealed a formation of citrullin that was inhibited by the specific NO synthase N omega-nitro-L-arginine (NO2Arg). Both histochemical and biochemical methods indicate that NO can be used as a signal molecule by specific neurones in advanced invertebrates.

Nervous System of the Tornaria Larva (Hemichordata: Enteropneusta). A Histochemical and Ultrastructural Study.

Transmission electron microscopy (TEM) and histochemical approaches were used to investigate the topology and ultrastructure of the nervous system of the tornaria larva of an enteropneust, Balanoglossus proterogonius. Cholinesterase activity was detected in the epithelium of the pre- and postoral ciliary bands. Groups of catecholamine-containing cells (CA) were detected at the anterior tip of larva, in the ventral epidermis behind the mouth, and in the stomach wall near its junction with the intestine. Single CA neurons were detected in the telotroch epithelium. Axon tracts are described in ciliary band epithelia. At the base of the aboral plate, epithelial nerve cells form a ganglion-like cluster. Single neuron-like cells and single axons and axonal tracts were found in the epithelium of digestive tract. The data were compared with ones from the literature and with those obtained from other marine invertebrate larvae. The properties of the neural elements and their possible functions are discussed.

[The parasite-host relationships between the glochidia of the European pearl oyster Margaritifera margaritifera (Margaritiferidae: Bivalvia) and mass fish species in the European north of the USSR]. / Vzaimootnosheniia parazit--khoziain u glokhidiev Evropeiskoi zhemchuzhnitsy Margaritifera margaritifera (Margaritiferidae: Bivalvia) i massovykh vidov ryb evropeiskogo severa SSSR.

Narrow specificity of larvae (glochidia) of Margaritifera margaritifera to salmon in the rivers of the Kola Peninsula was proved experimentally. It was found that in the gills of minnow, the other mass fish in the northern rivers of the USSR, larvae of M. margaritifera cannot develop and perish. Reasons causing the narrow specificity of M. margaritifera to Salmonidae are discussed.

[A histochemical study of the nervous system in trematode parthenitae]. / Gistokhimicheskoe izuchenie nervnoi sistemy partenit trematod.

Localization of catecholamines in the nervous system of 12 species of Trematodes parthenitae from marine mollusks has been studied using the method of glyoxilic acid-induced fluorescence. Unlike primitive species with orthogon-like distribution of catecholamines, specialized ones have diffuse nervous plexus, which can be reduced in most specialized forms.