Prenatal Stress and Adaptive Behavior of Offspring: The Role of Placental Serotonin.

The effect of mild prenatal stress in mice, leading to an increase in the placental serotonin level, on the formation of adaptive behavior in male offspring at the age of 35 days was studied. It was shown that, in BalbC mice, daily immobilization for 1 h during the period from 11 to 14 days of pregnancy led to an increase in placental and fetal serotonin levels on the 15th day of prenatal development. According to "resident-intruder" behavioral test, the prenatally stressed mice showed more reactive behavior in adulthood and low tendency to defend their territory. Thus, placental serotonin, formed under the stress condition, may act as a mediator between the environment and the fetuses and determine the adaptive behavior of offspring.

[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.

Expression of components of the serotonergic system in the developing rat thymus.

The developing thymus of rat fetuses contains all components of the serotonergic system: receptors, enzymes of synthesis, and membrane transporters. The expression of receptors suggests the possibility of a direct influence of serotonin on thymic development. The presence of tryptophan hydroxylase (the key rate-limiting enzyme of serotonin synthesis) and aromatic l-amino acid decarboxylase indicates the ability of fetal thymic cells to synthesize serotonin. It was shown that the cells of a developing thymus can actively uptake extracellular monoamines. The results of this study suggest different functions of the intrathymic and circulating serotonin pools in the regulation of thymic development.

Fine morphology of frontal filaments in nauplii of cirriped crustaceans.

Fine morphology of the frontal filaments (FFs) at all nauplius stages of two barnacle species (Verruca stroemia and Hesperibalanus hesperius) has been investigated by scanning electron microscopy. FFs have been detected at the second nauplius stage and persist during all stages. FFs contain a wide proximal and a fine distal parts, but they are not actually separated as segments of the limbs, and the area between them looks like a single cuticular crease. Apical and subapical pores have been found at the top of each FF in the larvae of both species, which may indicate the chemoreceptor function of these organs.

[ENGAGEMENT OF SEROTONIN-MODULATING ANTICONSOLIDATION PROTEIN IN REGULATION OF EMBRYOGENESIS OF LYMNEAE STAGNALIS AND LEWIS SARCOMA IN HYBRID MICE Fl C57B2/6 X DBA].

The article concerns study of the effects of a novel serotonin-modulating anticonsolidation protein (SMAP) being in a linear relationship with serotonin level, on embryogenesis of Lymneae stagnalis and Lewis sarcoma in hybrid mice Fl C57B2/6 X DBA. Inhibition of embryogenesis of Lymneae stagnalis on the stage of four blastomers and late blastula, lack of changes on the stage of trochofora and acceleration of metamorphosis under the effects of SMAP in a dose-dependent manner was observed. Short-term retardation (during the first 10 days) of development of Lewis sarcoma in mice and survival of 25% of transferring animals under high doses of SMAP was revealed. Cytostatic activity for high doses of SMAP and their effects on the duration of single phases of the cell cycle is proposed.

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.

Expression of FMRFamide gene encoded peptides by identified neurons in embryos and juveniles of the pulmonate snail Lymnaea stagnalis.

Immunoreactivity to antibodies (ABs) against FMRFamide, CARP, and three FMRFamide gene encoded peptides, i.e., EFLRIamide, the non-FMRFamide peptide "SEEPLY," and the 35-amino-acid "acidic peptide," were investigated in developing embryos and juveniles of Lymnaea stagnalis. Five early transient embryonic neurons revealed immunoreactivity to EFLRIamide. One of the early neurons, the central posterior, also expressed SEEPLY and CARP immunoreactivity. Two neurons in the anlage of the left and right parietal ganglia coexpressed immunoreactivity to EFLRIamide (type 1 transcript) and acidic peptide (type 2 transcript). Within the developing ganglia altogether 30 neurons expressed the type 1 transcript, and three expressed the type 2 transcript. No peripheral cells immunoreactive to SEEPLY or acidic peptide ABs were found, whereas bipolar EFLRIamide- and CARP-immunoreactive cells were abundant in the lip, mantle and foot. After hatching, the number of immunoreactive neurons in ganglia increased up to 223 and the neurons expressing tetrapeptides were dominant (91%). No neurons coexpressing type 1 transcript and type 2 transcript could be detected in juveniles and adults. At this time, an extensive innervation is developed in the periphery, including foot, mantle, buccal mass, salivary glands and alimentary tract, established mainly by EFLRIamide-immunoreactive cells and varicose fibers of extrinsic and intrinsic origin. It is suggested that both sensory and regulatory function can be attributed to the FMRFamide gene encoded tetrapeptides throughout embryonic and juvenile development in Lymnaea, whereas heptapeptides are presumed to play a modulatory role.

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.

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.

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.

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.

Serotonin depletion after prolonged chlorpromazine treatment in a simpler model system.

1. Prolonged exposure of the pond snail Lymnaea stagnalis to micromolar concentrations of chlorpromazine (CPZ) results in marked changes in the serotonin (5-HT) content of the central nervous system. 2. High-performance liquid chromatography with electrochemical detection indicates that levels of 5-HT, but not those of dihydroxyphenyl-alanine (DOPA), dopamine or norepinephrine, were significantly decreased (e.g., to less than 40% of normal after 30 days of exposure to 1 microM CPZ in the bathing water). 3. Glyoxylate-induced fluorescence was depressed to undetectable levels in central, serotonergic neurons. 4. Performance of 5-HT-dependent motor behaviors was impaired. 5. The present results, in accord with earlier studies on the effects of chronic exposure to haloperidol, suggest that previously overlooked mechanisms of monoamine downregulation may contribute to long-term effects of antipsychotic drugs.

Catecholamine-Containing Cells in Larval and Postlarval Bivalve Molluscs.

Previous studies have suggested an involvement of catecholamines in the control of several larval behaviors, such as feeding, locomotion, and induction of settling and metamorphosis. In the present study we employed aldehyde-induced, blue-green fluorescence to indicate catecholamines in cells within representatives of two bivalve families, the Pectinidae (Placopecten magellanicus) and the Mytilidae (Mytilus edulis). Larvae were examined at different stages of development before and also shortly after settlement. The general distribution of fluorescent cells was similar in the two species. By midveliger stage, several fluorescent cells and fibers were located along the outer rim of each velar lobe, and a pair of flask-shaped cells was located lateral to the mouth. A single fiber from near the mouth projected to a region beneath the apical tuft. In the pediveliger, the cells by the mouth were joined by an additional two to four fluorescent cells. The developing foot also contained numerous such cells, some of which had processes that penetrated the epithelium on the "sole" and bore ciliated terminals. Fluorescent somata were also located around the edge of the mantle. Centrally projecting fibers appeared to terminate in the pedal and abdominal ganglia, which also contained a few fluorescent somata. After settlement, the velar lobes and resident fluorescent somata disappeared, but fluorescent cells in the foot persisted as this latter organ grew. Fluorescent cells within the developing gill were connected with the abdominal ganglia by means of fibers. Control preparations labeled with antibodies raised against serotonin indicated that the aldehyde-induced fluorescence was not due to the presence of indoleamines. The present study not only confirms previous chromatographic evidence suggesting the presence of catecholamines in the larvae of bivalve molluscs, but also identifies putative neuronal circuits that may control various larval behaviors.

Transient and sustained expression of FMRFamide-like immunoreactivity in the developing nervous system of Lymnaea stagnalis (Mollusca, Pulmonata).

1. In the present study we have investigated the ontogeny of FMRFamide expression in the snail, Lymnaea stagnalis, from its first appearance to its distribution in young adults. 2. The first FMRFamide-like immunoreactive (FaLI) cells within CNS appear by E45 embryonic stage (premetamorphic veliger). The number of FaLI neurons increases throughout both pre- and post-hatching development. 3. Both transient and sustained expression of FMRFamide-like immunoreactivity by specific sets of neurons occurs. Two cells which transiently express immunoreactivity appear outside the future CNS by the stage E45. Other population of transient FaLI neurons includes bilaterally symmetric groups of cells in the cerebral and pedal ganglia during posthatching stages P1 (hatchlings) to P5 (juveniles). All other immunostained cells which appear during development maintain their transmitter phenotype into adulthood. 4. The possible role of FMRFamide-related peptides in the processes of morpho- and neurogenesis is discussed.

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.

Early elements in gastropod neurogenesis.

The first elements of the nervous system of pond snails appear in the very early veliger stage of development at much earlier times than any previously described neurons. The first three cells are reactive to antibodies raised against both the neuropeptide FMRFamide and tubulin and their somata are located posteriorly within the embryo, not in anterior regions, as would be consistent with current concepts of gastropod neurogenesis. Furthermore, the extensive, anteriorly directed fibers from these cells appear to form a scaffold upon which the central ganglia and interconnecting pathways later develop. These findings challenge current thoughts on the origins of early embryonic neurons and on possible inductive cues and mechanisms of axonal navigation important in the development of the molluscan nervous system.

Octopamine in the developing nervous system of the pond snail, Lymnaea stagnalis L.

Development of the octopaminergic system in the pond snail, Lymnaea stagnalis, was investigated by means of immunocytochemistry and radioenzymatic assay. The earliest octopamine-immunoreactive neurons appear at a late embryonic stage (E85) of development following metamorphosis. At this moment of development, the adult-like pattern of distribution and projection (arborization) characteristics of octopamine-immunoreactive neurone can already be observed. During hatching and postembryonic (juvenile) development the number of labelled neurons increases only within the ventro-medial cell groups of the cerebral ganglia, whereas the extent of varicose axon arborization of the labelled neurons increases gradually. No peripheral projections of the embryonic and postembryonic octopamine-immunoreactive neurons were observed. The postembryonic increase in number of immunoreactive neurons and development of axonal arborization is accompanied by a rapid, exponential enhancement of octopamine content of Lymnaea CNS, as detected radioenzymatically. A possible role of the octopaminergic neurons in the regulation of certain physiological function(s), active only from a late stage of embryonic development, is suggested.

Mode of action of antipsychotic drugs: lessons from simpler models.

The monoaminergic system of the pond snail Lymnaea stagnalis changed markedly following prolonged exposure to chlorpromazine (CPZ). HPLC-ED indicated that levels of serotonin (5-HT), but not those of dopamine, were significantly decreased (e.g., to less than 40% after 30 days of treatment with 1 microM CPZ). Glyoxylate-induced fluorescence was depressed to undetectable levels in selected subpopulations of 5-HT neurons. Performance of 5-HT-dependent motor behaviors was impaired, and a considerably decreased firing activity was observed in affected 5-HT neurons. The present results, in accord with past ones with haloperidol, suggest that a previously overlooked mechanism of monoamine down-regulation may contribute to affects of antipsychotic drugs.