Leonid Nezlin

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.

Neuronal development in larval mussel Mytilus trossulus (Mollusca: Bivalvia)

Although our understanding of neuronal development in Trochozoa has progressed substantially in recent years, relatively little attention has been paid to the bivalve molluscs in this regard. In the present study, the development of FMRFamide-, serotonin- and catecholamine-containing cells in the mussel, Mytilus trossulus, was examined using immunocytochemical and histofluorescent techniques. Neurogenesis starts during the trochophore stage at the apical extreme with the appearance of one FMRFamide-like immunoreactive (lir) and one serotonin-lir sensory cell. Later, five FMRFamide-lir and five serotonin-lir apical sensory cells appear, and their basal fibres form an apical neuropil. Fibres of two lateral FMRFamide-lir apical cells grow posteriorly and at the time that they reach the developing foot, the first FMRFamide-lir neurons of the pedal ganglia also appear. Subsequently, FMRFamide-lir fibres grow further posteriorly and reach the caudal region where neurons of the developing visceral ganglia then begin to appear. In contrast, the five apical serotonin-lir neurons do not appear to project outside the apical neuropil until the late veliger stage. Catecholamine-containing cells are first detected in the veliger stage where they appear above the oesophagus, and subsequently in the velum, foot, and posterior regions. Though neural development in M. trossulus partly resembles that of polyplacophorans in the appearance of the early FMRFamidergic elements, and of scaphopods in the appearance of the early serotonergic elements, the scenario of neural development in M. trossulus differs considerably from that of other Trochozoa (bivalves, gastropods, polyplacophorans, scaphopods and polychaetes) studied to date.

Apical sensory neurones mediate developmental retardation induced by conspecific environmental stimuli in freshwater pulmonate snails

Freshwater pond snails Helisoma trivolvis and Lymnaea stagnalis undergo larval development and metamorphosis inside egg capsules. We report that their development is permanently under slight tonic inhibitory influence of the anterior sensory monoaminergic neurones, which are the remnants of the apical sensory organ. Conspecific juvenile snails, when reared under conditions of starvation and crowding, release chemical signals that are detected by these neurones in encapsulated larvae and reversibly suppress larval development, thus providing a link between environmental signals and developmental regulation. Induced retardation starts from the trochophore stage and results in up to twofold prolongation of the larval lifespan. Upon stimulation with the signal, the neurones increase synthesis and release of monoamines [serotonin (5-HT) in Helisoma and dopamine in Lymnaea] that inhibit larval development acting via ergometrine-sensitive internal receptors. Thus, the novel regulatory mechanism in larval development of molluscs is suggested and compared with the phenomenon of dauer larvae formation in the nematode Caenorhabditis elegans.

Structure of the olfactory bulb in tadpoles of Xenopus laevis.

Abstract. 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.

Serotonin- and FMRFamide-immunoreactive nerve elements in the chiton, Lepidopleurus asellus (Mollusca, Polyplacophora)

The distribution of serotonin-like and FMRFamide-like immunoreactive (5HT-ir and FMRFa-ir, respectively) neurons in the nervous system of the chiton Lepidopleurus asellus (Mollusca, Polyplacophora) was studied using an immunocytochemical technique. The neurons were distributed in characteristic patterns in the central nervous system, the 5HT-ir neurons predominating in the ventral (pedal) cords and FMRFa-ir neurons in the lateral cords. In the body wall including the foot, a tight network of 5HT-ir and FMRFa-ir nerve fibers is found, the former being mostly attributed to the musculature whereas the latter seems to be associated with the blood sinuses. Intraepithelial neurons of both types are abundant in the fore-and hindgut. The presence and general distribution in the central and peripheral nervous system of the 5HT-ir and FMRFa-ir elements seems thus to be similar in simple and advanced molluscs. The relationship between these neurons and their targets in the body also appears to be well conserved in molluscs.

Patterns of enkephalin immunolabeling in the pulmonate snailCepaea 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.

TYROSINE HYDROXYLASE-NEGATIVE, DOPAMINERGIC NEURONS ARE TARGETS FOR TRANSMITTER-DEPLETING ACTION OF HALOPERIDOL IN THE SNAIL BRAIN

Summary1.The effects of long term administration of micromolar concentrations of the D2 antagonist haloperidol upon monoaminergic neurons in the snailLymnaea 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 inLymnaea 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.

Immunolabeled neuroactive substances in the osphradium of the pond snail Lymnaea stagnalis

The osphradium of molluscs is assumed to be a sensory organ. The present investigation in Lymnaea stagnalis has established two ultrastructurally different types of dendrites in the sensory epithelium. Cells immunoreactive to leucine-enkephalin and FMRFamide send processes to the sensory epithelium. These neurons of the osphradial ganglion are thus considered to be part of the sensory system, as are methionine-enkephalin-immunoreactive cells in the mantle wall in the vicinity of the osphradium. The complexity of the osphradial ganglion is further demonstrated by serotonin-immunoreactive neurons innervating the muscular coat around the osphradial canal and methionine-enkephalin-immunoreactive cells sending projections to the central nervous system.

Mechanisms underlying dual effects of serotonin during development of Helisoma trivolvis (Mollusca)

BackgroundSerotonin (5-HT) is well known as widely distributed modulator of developmental processes in both vertebrates and invertebrates. It is also the earliest neurotransmitter to appear during neuronal development. In aquatic invertebrates, which have larvae in their life cycle, 5-HT is involved in regulation of stages transition including larval metamorphosis and settlement. However, molecular and cellular mechanisms underlying developmental transition in aquatic invertebrate species are yet poorly understood. Earlier we demonstrated that in larvae of freshwater molluscs and marine polychaetes, endogenous 5-HT released from the neurons of the apical sensory organ (ASO) in response to external stimuli retarded larval development at premetamorphic stages, and accelerated it at metamorphic stages. Here we used a freshwater snail Helisoma trivolvis to study molecular mechanisms underlying these dual developmental effects of 5-HT.ResultsLarval development of H. trivolvis includes transition from premetamorphic to metamorphic stages and shares the main features of metamorphosis with free-swimming aquatic larvae. Three types of 5-HT receptors (5-HT1-, 5-HT4- and 5-HT7-like) are functionally active at premetamorphic (trochophore, veliger) and metamorphic (veliconcha) stages, and expression patterns of these receptors and respective G proteins undergo coordinated changes during development. Stimulation of these receptors modulated cAMP-dependent regulation of cell divisions. Expression of 5-HT4- and 5-HT7-like receptors and their downstream Gs protein was down-regulated during the transition of pre- to metamorphic stage, while expression of 5-HT1 -like receptor and its downstream Gi protein was upregulated. In accordance with relative amount of these receptors, stimulation of 5-HTRs at premetamorphic stages induces developmental retardation, while their stimulation at metamorphic stages induces developmental acceleration.ConclusionsWe present a novel molecular mechanism that underlies stage-specific changes in developmental tempo of H. trivolvis larvae in response to endogenous 5-HT produced by the neurons of the ASO. We suggest that consecutive changes in expression patterns of different receptors and their downstream partners in the course of larval development represent the molecular base of larval transition from premetamorphic (non-competent) to metamorphic (competent) state.

The golden age of comparative morphology: Laser scanning microscopy and neurogenesis in trochophore animals

Immunochemical labelling of neuronal elements and laser confocal microscopy have considerably expanded the capacity of comparative morphology, and allowed us to study neurogenesis in various trochophore animals at the level of identified neurons. The data obtained have impeached some popular phylogenetic theories. Thus, the comparative study has shown that in the representative Trochozoan species, the orthogonal brain is absent at all developmental stages. Fundamental differences in neurogenesis and neuroarchitecture between trochophores from different taxa suggest that trochophore-like larvae are not homologous, and their likeness is most probably the result of convergence. Our data support the “intercalation hypothesis” of the origin of indirect development with a trochophore-like larva. It seems reasonable to exclude from phylogenetic discussions the orthogon and the trochophore as an ancestral form common for Lophotrochozoa.