Elena E. Voronezhskaya

Neuronal development in larval chiton Ischnochiton hakodadensis (Mollusca: Polyplacophora).

Chitons are the most primitive molluscs and, thus, a matter of considerable interest for understanding both basic principles of molluscan neurogenesis and phylogeny. The development of the nervous system in trochophores of the chiton Ischnochiton hakodadensis from hatching to metamorphosis is described in detail by using confocal laser scanning microscopy and antibodies raised against serotonin, FMRFamide, and acetylated α‐tubulin. The earliest nervous elements detected were peripheral neurons located in the frontal hemisphere of posthatching trochophores and projecting into the apical organ. Among them, two pairs of unique large lateral cells appear to pioneer the pathways of developing adult nervous system. Chitons possess an apical organ that contains the largest number of neurons among all molluscan larvae investigated so far. Besides, many pretrochal neurons are situated outside the apical organ. The prototroch is not innervated by larval neurons. The first neurons of the developing adult central nervous system (CNS) appear later in the cerebral ganglion and pedal cords. None of the neurons of the larval nervous system are retained in the adult CNS. They cease to express their transmitter content and disintegrate after settlement. Although the adult CNS of chitons resembles that of polychaetes, their general scenario of neuronal development resembles that of advanced molluscs and differs from annelids. Thus, our data demonstrate the conservative pattern of molluscan neurogenesis and suggest independent origin of molluscan and annelid trochophores. J. Comp. Neurol. 444:25–38, 2002.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: I. Embryonic development of dopamine-containing neurons and dopamine-dependent behaviors

The embryonic development of the catecholaminergic system of the pond snail, Lymnaea stagnalis, was investigated by using chromatographic and histochemical methods. High performance liquid chromatography suggested that dopamine was the only catecholamine present in significant concentrations throughout the embryonic development of Lymnaea. Dopamine first became detectable at about embryonic stage (E) 15 (15% of embryonic development) and then increased in amount during early development to reach about 120–140 fmol per animal by around E40. Dopamine content remained stable during mid‐embryogenesis (E40–65), increased slowing for the next couple of days, and then increased rapidly to culminate at about 400 fmol per animal by hatching. The detection of aldehyde‐ and glyoxylate‐induced fluorescence and of tyrosine hydroxylaselike immunoreactivity indicated that the first catecholaminergic cells appeared in the late trochophore or early veliger stage of embryonic development (E32–35). The paired perikarya of these transient apical catecholaminergic (TAC) neurons were located beneath the apical plate, remained outside of the central ganglia during embryogenesis, and no longer contained detectable catecholamines close to hatching. TAC neurons bore cilia on the ends of short processes that penetrated the overlying epithelium; their long processes branched repeatedly under the ciliated apical plate. Several smaller catecholaminergic cells first appeared in the anterior margin of the foot at a stage when the embryos began to metamorphose from the veliger form (E55). Similar bipolar cells later appeared in the tentacle and lips. The axons of all of these small peripheral cells projected centrally and terminated within the neuropil of different central ganglia. Central catecholaminergic neurons, including RPeD1, differentiated only after metamorphosis was complete (E75). Development of locomotor, respiratory, and feeding behaviors correlated with maturation of catecholaminergic neurons, as indicated by histology and chromatography. J. Comp. Neurol. 404:285–296, 1999.

Neuronal development in larval polychaete Phyllodoce maculata (Phyllodocidae)

The existing view on neuronal development in polychaetes, as undergoing neurogenesis beginning in the rudiments of central ganglia and then extending peripherally, has been contrasted with the latest findings in molluscs, their sister trochozoan group, which show a peripheral to central mode of neurogenesis. The current study addresses this issue by examining early neuronal development in the polychaete Phyllodoce maculata using immunolabeling against acetylated α‐tubulin, serotonin, and the FMRFamide. The first nervous cell was detected 20 hours before hatching, at the early trochophore stage. A solitary serotonergic neuron was located at the posterior‐dorsal extreme of the larva and issued anterior projecting fibers, which outline the future ventral nerve cords and prototroch nerve. Two more serotonergic dorsal peripheral cells and three peripheral FMRFamidergic cells appeared soon thereafter. The fibers of these early cells formed a scaffolding, which prefigured the future adult nervous system (cerebral ganglion, ventral cords, prototroch and esophageal nerve rings) in prehatched trochophores. Shortly before hatching, the larval nervous system developed, including the apical organ, meridianal nerves in the episphere, and posttrochal nerves that innervate the feeding apparatus. After hatching, the rudiments of the adult nervous system started to develop along the paths already established by the earliest peripheral neurons. Thus, the general strategy of neurogenesis in a representative polychaete trochophore appears to resemble that of molluscs. The first neuronal cells to appear are peripheral in origin, located near the posterior margins of the embryo. Their similar anatomical appearance suggests that they share a similar functional role in trochophore development and behavior. J. Comp. Neurol. 455:299–309, 2003.

Development of catecholaminergic neurons in the pond snail, Lymnaea stagnalis: II. Postembryonic development of central and peripheral cells

Catecholamines have long been thought to play important roles in different mollusc neural functions. The present study used glyoxylate‐ and aldehyde‐induced histofluorescence to identify central and peripheral catecholaminergic neurons in the snail Lymnaea stagnalis. The majority of these cells were also found to react to antibodies raised against tyrosine hydroxylase. A minority of the catecholaminergic neurons, however, exhibited no such immunoreactivity. The number of central catecholaminergic neurons nearly doubled (from about 45 to about 80 cells) during the first 2–3 days of postembryonic development. Thereafter, catecholaminergic neurons again doubled in number and generally grew by about 100–200% in soma diameter as the snails grew by 1,000% in overall linear measurements. In contrast to the relatively meager addition of central catecholaminergic neurons, several thousand catecholaminergic somata were added to different peripheral tissues during postembryonic development. These small, centrally projecting neurons were particularly concentrated in the lips, esophagus, anterior margin of the foot, and different regions of the male and female reproductive tracts. Chromatographic analyses indicated that dopamine was the major catecholamine present in the central ganglia, foot, and esophagus, although detectable levels of norepinephrine (approximately 20% of dopamine levels) were also found in the ganglia. The total content but not the concentration of dopamine increased within the tissue samples during postembryonic development. The companion study (Voronezhskaya et al. [1999] J. Comp. Neurol. 404:285–296) and the present study furnish a complete description of central and peripheral catecholaminergic neurons from their first appearance in early embryonic development to adulthood. J. Comp. Neurol. 404:297–309, 1999.

Development of Embryonic Cells Containing Serotonin, Catecholamines, and FMRFamide-Related Peptides in Aplysia californica

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.

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.

Localization of serotonin and its possible role in early embryos of Tritonia diomedea (Mollusca: Nudibranchia)

A classical neurotransmitter serotonin (5-HT) was detected immunochemically using laser scanning microscopy at the early stages of Tritonia diomedea development. At the one- to eight-cell stages, immunolabeling suggested the presence of 5-HT in the cytoplasm close to the animal pole. At the morula and blastula stages, a group of micromeres at the animal pole showed immunoreactivity. At the gastrula stage no immunoreactive cells were detected, but they arose again at the early veliger stage. Antagonists of 5-HT2 receptors, ritanserin and cyproheptadine, as well as lipophilic derivatives of dopamine blocked cleavage divisions or distorted their normal pattern. These effects were prevented by 5-HT and its highly lipophilic derivates, serotoninamides of polyenoic fatty acids, but not by the hydrophilic (quaternary) analog of 5-HT, 5-HTQ. The results confirm our earlier suggestion that endogenous 5-HT in pre-nervous embryos acts as a regulator of cleavage divisions in nudibranch 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.

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.