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Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver (Ag, 1-20 nm particles) on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed.

In vertebrates, the receptor neurons of the olfactory/vomeronasal systems express different receptor gene families and related G-protein types (in particular the G protein alpha subunit). There are no data in the literature about the molecular features of the olfactory/vomeronasal systems of Cladistia thus, in this work, the presence and distribution of different types of G protein alpha subunits were investigated in the olfactory organs of the bichir Polypterus senegalus, using immunohistochemistry. Gαo-like immunoreactivity was detected in the microvillous receptor neurons, with the cell body in the basal zone of the sensory epithelium, and in the crypt neurons. Gαo-like ir glomeruli were mainly localized in the anterior part of the olfactory bulb. Gαolf-like immunoreactivity in the sensory epithelium was detected in the ciliated receptor neurons, while the immunoreactive glomeruli in the olfactory bulb were mainly localized in the ventral-posterior part. No Gαq nor Gαi3 immunoreactivity was detected. These data are partially in agreement with studies that show the distribution of G protein alpha subunits in teleosts, allowing to hypothesize a common organization of the olfactory/vomeronasal systems in the group of Actinopterigians., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Here, we report the presence of the γ-aminobutyric acid (GABA)-ergic system in the calcisponge Leucandra aspera and examine the cellular localization of the components of this system, including GABA-like receptors using immunofluorescence and confocal microscopy. Furthermore, we demonstrate for the first time that GABA plays a functional role as a messenger in regulating sponge-feeding behavior. We found that both GABA(B) R1 and R2 subunits are present in the choanocytes of sponges as well as in the eso- and endopinacocytes. The functional role of GABA in the feeding behavior of this sponge was tested. The involvement of GABA receptors in the endocytic processes in L. aspera was demonstrated with dextran conjugated to Texas Red as a marker for material ingestion and by treating isolated sponge cells with a GABA(B) receptor agonist and an antagonist. The amount of dextran that was ingested increased in dissociated sponge cells when the GABA(B) receptor agonist baclofen was used, and this stimulatory effect was prevented by treatment with the GABA(B) receptor antagonist phaclofen. The baclofen effect on uptake was blocked by treatment with pertussis toxin, thus indicating a role for G proteins in modulating feeding behavior in L. aspera. Moreover, we found evidence that GABA receptors are involved in the consumption of dissolved organic matter by sponge cells. These findings suggest that GABA receptors and their functional role are highly conservative traits in the animal kingdom prenervous system evolution., (Copyright © 2010 Wiley-Liss, Inc., A Wiley Company.)

To date, no study has been published on cell renewal in the olfactory epithelium of Chondrichthyes. Our work aimed at detecting proliferating cells (by Proliferating Cell Nuclear Antigen - PCNA immunohistochemistry) and apoptotic cells (by terminal uridine deoxynucleotidyl transferase nick end labeling method) in the olfactory epithelium of the shark Scyliorhinus canicula. PCNA immunoreactivity and mitotic figures were localized almost exclusively at the basal and apical thirds of the epithelial thickness. Double immunofluorescence for PCNA and OMP (a marker of mature olfactory neurons) showed that PCNA immunoreactivity is lacking in mature olfactory neurons, with the exception of crypt neurons. Crypt neurons, a cell type peculiar to fish, often showed PCNA immunoreactivity in the nucleus and may be involved in repair processes. The role of PCNA in mature crypt neurons requires further investigation to be clarified. Apoptosis was observed in sensory neurons and in basal cells. Our data highlight the presence of cell proliferation at different levels within the epithelium and the occurrence of apoptosis in both mature and proliferating cells., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The ontogenetic cycle of the barnacle Balanus amphitrite (=Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

The mammalian hormone, leptin, is mainly synthesized in adipose tissue along with other tissues. Leptin plays a role in numerous processes such as in the control of food intake, homeostasis, immune function and reproduction. In this study, we detected and localized leptin immunoreactivity to the muscle of early juvenile sea bass (Dicentrarchus labrax) by Western blot analysis and immunohistochemistry. A leptin immunopositive band with a molecular weight of approximately 16 kDa, corresponding to mammalian leptin, was identified in trunk skeletal muscle homogenate. Furthermore, leptin immunopositive cells were detected in the endomysium of skeletal muscular fibers. These cells showed immunostained cytoplasmic granules and roundish and oval nuclei. The most intense immunostaining was observed in the endomysial space among the superficial red muscular fibers of the trunk. These findings suggest that in early juvenile sea bass, leptin is mostly produced by skeletal muscles. Therefore, during the developmental stage lacking adipose tissue, skeletal muscles can be considered an important source of leptin. As already suggested in mammals, we can hypothesize the potential roles of leptin not only in energy expenditure for muscle contraction but also during muscle differentiation and growth., ((c) 2010 Wiley-Liss, Inc.)

Receptor neurons in the olfactory and vomeronasal epithelia of vertebrates have dendritic specialization that is correlated to the receptor gene family they express and the G protein coupled with that receptor (in particular the G protein alpha subunit). There are not very many data in the literature about the morphological and molecular features of the olfactory epithelium of Chondrichthyes. In this work, the presence and distribution of different types of G protein alpha subunits (Galpha(o), Galpha(q) and Galpha(olf)) were investigated in the olfactory epithelium of the holocephalan Chimaera monstrosa using immunohistochemistry. Only Galpha(o)-like immunoreactivity was detected in the microvillous receptor neurons and in numerous axon bundles of the fila olfactoria. These preliminary data are in agreement with studies showing G protein alpha subunits in elasmobranchs and support the data present in the literature about putative odorant receptor families in the sequenced genome of the holocephalan Callorhinchus milii., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

In the present study, biochemical and immunochemical methods were used to investigate the presence and distribution of GABA, glutamate decarboxylase (GAD), GABA(B)R1 and GABA(A) gamma2 subunit receptors and the vesicular GABA transporter (vGAT) in the cyprid of Balanus amphitrite (=Amphibalanus amphitrite). GAD(65/67) immunoreactive neuron cell bodies and nerve fibers were detected in the central nervous system. Paired GAD(65/67) immunoreactive nerves running from the posterior ganglion to the body and limb muscles were detected. Thin GABA-immunoreactive nerve terminals were present on striated muscular fibers and in the antennules. Furthermore, GABA, GAD(65/67), GABA(B)R1 and GABA(A)gamma2 subunit receptors and vGAT were observed in the lateral compound eyes, and GABA(A)gamma2 subunit receptor immunoreactivity was seen in the naupliar eye. These results suggest a neurotransmitter/neuromodulatory role for GABA in thoracic muscle contraction and regulatory functions in compound eyes and antennules of B. amphitrite cyprids.

In the olfactory and vomeronasal systems of vertebrates, the morphology of the receptor neurons, the receptor gene family they express, the G-protein coupled with the receptor (in particular the G-protein alpha subunit), and their projection to the olfactory bulb are correlated. Much information about this complicated system have been collected in different groups, but nothing is known about Chondrichthyes. In this work, the presence and distribution of immunoreactivity for different types of G-protein alpha subunit (Galpha(o), Galpha(q) and Galpha(s/olf)) were investigated in the olfactory mucosa and olfactory bulb of the shark Scyliorhinus canicula. Only Galpha(o)-like immunoreactivity was detected in the olfactory mucosa and bulb, both in tissues and homogenates. Its distribution was partially similar to that found in other vertebrates: it was localized in the microvillous receptor neurons, in numerous axon bundles of the fila olfactoria, in the stratum nervosum and in the most of glomeruli in the stratum glomerulosum. No immunoreactivity was instead observed in the crypt neurons, the second type of olfactory neurons present in cartilaginous fish. The projections of crypt neurons to olfactory bulb probably correspond to the few ventrally-located glomeruli which were negative to the antiserum against Galpha(o). These data suggest, in S. canicula, different olfactory neuron types send projections to the olfactory bulb with a segregated distribution, as observed in other vertebrates.

The mechanosensory lateral line (LL) is involved in many fish and amphibian behaviors, however little is known about the molecules involved in the signal transmission. Neuropeptide Y (NPY) has a number of functions in vertebrate physiology and also plays important roles in different sensory systems. The Antarctic nototheniods are a monophyletic radiation of fishes that have evolved under the extreme environmental conditions of low light and cold, where non-visual sensory structures, such as LL, are of importance. In this study we describe the presence of NPY-like immunoreactivity (IR) in LL of the Antarctic nototheniod fish, Trematomus bernacchii Boulenger. Differences in size and cellular composition between the two neuromasts were in compliance with previous descriptions of these sensory organs. Despite structural and functional differences between canal and superficial neuromasts, the distribution of NPY-like IR was similar within both the receptors classes. In particular, NPY IR was observed in all three cell types which constitute these sensory organs, allowing us to hypothesize the involvement of this molecule in the processing of the sensory information.

The aim of this work was to investigate the distribution of regulative molecules in the stomach of juvenile Dicentrarchus labrax during compensatory growth, using immunohistochemical methods. Antisera against galanin, neuropeptide Y, ghrelin, leptin, and serotonin were used on fasted and refed D. labrax. The results show a characteristic distributive pattern for the sought molecules in fish refed after 35 days of fasting, with a high increased presence of both ghrelin and leptin.

The presence and distribution of FMRFamide-like peptides (FLPs) in the cyprid larvae of the barnacle Balanus amphitrite were investigated using immunohistochemical methods. Barnacles are considered to be one of the most important constituents of animal fouling communities, and the cyprid stage is specialized for settlement and metamorphosis in to the sessile adult condition. FLPs immunoreactive (IR) neuronal cell bodies were detected in both the central and the peripheral nervous system. One bilateral group of neurons somata was immunodetected in the brain, and IR nerve fibers were observed in the neuropil area and optic lobes. Intense immunostaining was also observed in the frontal filament complex: frontal filament tracts leaving the optic lobes and projecting towards the compound eyes, swollen nerve endings in the frontal filament vesicles, and thin nerve endings in the external frontal filament. Thin IR nerve fibers were also present in the cement glands. Two pairs of neuronal cell bodies were immunodetected in the posterior ganglion; some of their axons appear to project to the cirri. FLPs IR neuronal cell bodies were also localized in the wall of the dilated midgut and in the narrow hindgut; their processes surround the gut wall and allow gut neurons to synapse with one another. Our data demonstrated the presence of FLPs IR substances in the barnacle cyprid. We hypothesize that these peptides act as integrators in the central nervous system, perform neuromuscular functions for thoracic limbs, trigger intestinal movements and, at the level of the frontal filament, play a neurosecretory role., ((c) 2008 Wiley-Liss, Inc.)

Gamma-amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. The presence of GABA in the marine demosponge Chondrilla nucula was verified using immunofluorescence detection and high-pressure liquid chromatography. A strong GABA-like immunoreactivity (IR) was found associated with choanocytes, exopinacocytes, endopinacocytes lining inhalant, and exhalant canals, as well as in archaeocytes scattered in the mesohyl. The capacity to synthesize GABA from glutamate and to transport it into the vesicles was confirmed by the presence in C. nucula of glutamate decarboxylase (GAD) and vesicular GABA transporters (vGATs), respectively. GAD-like and vGAT-like IR show the same distribution as GABA-like IR. Supporting the similarity between sponge and mammalian proteins, bands with an apparent molecular weight of about 65-67 kDa and 57 kDa were detected using antibodies raised against mammalian GAD and vGAT, respectively. A functional metabotropic GABA(B)-like receptor is also present in C. nucula. Indeed, both GABA(B) R1 and R2 isoforms were detected by immunoblot and immunofluorescence. Also in this case, IR was found in choanocytes, exopinacocytes, and endopinacocytes. The content of GABA in C. nucula amounts to 1225.75 +/- 79 pmol/mg proteins and GABA is released into the medium when sponge cells are depolarized. In conclusion, this study is the first indication of the existence of the GABA biosynthetic enzyme GAD and of the GABA transporter vGAT in sponges, as well as the first demonstration that the neurotransmitter GABA is released extracellularly.

Crypt neurons are olfactory receptor cells located in the olfactory epithelium of fishes. They exhibit a peculiar and well-recognizable morphology, although their odorant specificity is still unknown. Data on their appearance during development are few and far between. This study set out to identify the time at which crypt neurons appeared in the skate, Raja clavata, using histological and immunohistochemical methods. For this purpose, embryos and juveniles at different stages of development, from 13 weeks after laying (11 weeks before hatching) to 24 weeks after hatching, were examined. The crypt neurons were identified on a morphological basis. An anti-alpha-tubulin antibody and two lectins (wheat germ agglutinin and peanut agglutinin) were used to highlight morphological details. The olfactory marker protein was detected by immunohistochemistry, because this protein is a marker of neuronal maturity in vertebrates. The crypt neurons could be detected by their morphology at 15 weeks after laying and became strongly olfactory marker protein immunoreactive 22 weeks after laying. Although involvement of crypt neurons in reproductive behavior has been inferred in various studies on bony fishes, their early presence in skate embryos and juveniles may suggest that they are not exclusively involved in sexual behavior., ((c) 2007 Wiley-Liss, Inc.)

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Olfactory marker protein (OMP) is a protein expressed in the mature olfactory and vomeronasal neurons of many vertebrates, such as mammals, amphibians and bony fishes. Aim of this work was to investigate the OMP expression in the olfactory epithelium of the shark Scyliorhinus canicula (Linnaeus, 1758), by immunohistochemistry (IHC). Immunoreactivity was detected in the olfactory receptor neurons, in the crypt neurons and in the nerve fibers below the epithelium. Although very little is known about the OMP's function, its involvement in synaptogenesis, transduction cascade, neurogenesis and development of olfactory system has been suggested. The present work shows for the first time OMP's presence in a cartilaginous fish.

To date only peptidergic innervation has been described in the alimentary tract of barnacles. In the present work the presence and distribution of choline acetyltransferase (ChAT), the acetylcholine (ACh) synthesizing enzyme, was investigated by immunohistochemistry in the alimentary tract of the adult barnacle Balanus amphitrite. Numerous ChAT-immunoreactive (IR) cells and a net of ChAT-IR cytoplasmic processes were localized inside the epithelium of the posterior midgut, close to the basement membrane; no IR nerve endings were detected in the midgut longitudinal and circular muscle bundles. Epithelial neurons or endocrine cells in the gut epithelium have been described in some invertebrate species belonging to different taxa and their peptidergic features are reported in the literature. Our results point out the presence of neuroepithelial cells also in the gut epithelium of barnacles; moreover, for the first time, a cholinergic feature is suggested for this cell type. These data seem to indicate the involvement of ACh in the gut functions of barnacle and suggest that the barnacle alimentary tract is more complex than previously thought and requires further study.

A new receptor neuron (RN) type was recently described in bony fish olfactory epithelium (OE): the crypt receptor neuron. This name is due to its main feature: the presence, at the apical part, of a deep invagination into which cilia protrude. The presence of this receptor neuron type is well documented in different species of bony fishes but it has never been described in cartilaginous fishes. In this study we demonstrate that crypt neuron-like cells are present in the olfactory epithelium of the elasmobranch Scyliorhinus canicula (Linnaeus, 1758). Histological observations allowed us to detect the presence of a few egg-shaped cells, characterized by a crypt like zone; alpha-tubulin immunoreactivity suggested the presence of cilia in the same area; fluorocrome conjugated lectin bindings suggested a distinctive mucus composition inside the presumptive crypt. The possible presence of crypt neuron-like cells in chondrichthyes would represent an interesting common feature between bony and cartilaginous fishes.

In this study, the presence and distribution of FMRFamide-like immunoreactivity in the alimentary tract of barnacle Balanus amphitrite were investigated. A net of nerve fibers strongly immunoreactive to FMRFamide-like molecules was localized in the posterior midgut and hindgut. Positive varicose nerve terminals were also localized close to the circular muscle cells and, in the hindgut, close to the radial muscular fibers. Besides this nerve fibers network, one pair of contralateral ganglia was localized in the hindgut, each of them constituted by two strongly FMRFamide-labeled neurons and one nonlabeled neuron. Their immunoreactive axons directed toward the hindgut and posterior midgut suggest an involvement of FMRFamide-like substances in adult B. amphitrite gut motility. The hindgut associated ganglia of barnacles seem to correspond to the terminal abdominal ganglia of the other crustaceans. Since they are the only residual gut ganglia in the barnacle's reduced nervous system, we can hypothesize that gut motility needs a nervous system regulation partially independent of the central nervous system.

The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of intracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. We have previously shown that GABAB receptors display a punctuate vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Here we have studied constitutive GABAB receptor trafficking after internalization in Paramecium primaurelia by confocal laser scanning microscopy and multiple immunofluorescence analysis. After internalization, receptors are targeted to the early endosomes characterized by the molecular markers EEA1 and rab5. Some of these receptors, destined for recycling back to the plasma membrane, traffic from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4-immunoreactivity (IR). Receptors that are destined for degradation exit the endosomal pathway at the early endosomes and traffic to the late endosome-lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP-1. GABAB vesicle structures also colocalize with TGN38-IR and rab11-IR. TGN38 and rab11 are proteins found in association with post-Golgi and recycling endosomes, respectively., ((c) 2005 Wiley-Liss, Inc.)

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.

In this work, the presence and distribution of serotonin in the cyprid of the barnacle Balanus amphitrite were investigated by immunohistochemical methods. Serotonin-like immuno-reactive neuronal cell bodies were detected in the central nervous system only. Various clusters of immunoreactive neuronal cell bodies are distributed in the brain (protocerebrum, deutocerebrum, optical lobes), and at least, four pairs of neuronal cell bodies were detected in the centrally positioned neuropil of the posterior ganglion. Rich plexuses of immunoreactive nerve fibers in the neuropil area were also observed. Furthermore, bundles of strongly immunoreactive nerve fibers surrounding the gut wall were localized, and immunoreactive nerve terminals in the antennules and compound eyes were observed. These data demonstrate the presence of a serotonin-like immunoreactive substance in the barnacle cyprids; furthermore, its immunolocalization in the cephalic nerve terminals allows us to postulate the involvement of this bioactive molecule in substrate recognition during the settlement process.

The presence of FMRFamide-related peptides (FaRPs) was investigated, by immunohistochemical methods with a polyclonal FMRFamide antiserum, in the sea-fan Eunicella cavolini (Van Koch 1887), a representative of the cnidarians (octocorallians). The identification of FaRP-immunoreactive elements as neuronal cells and a nerve net was performed by double immunohistochemical methods with the monoclonal anti-beta-tubulin antibody. A strong and widely distributed FaRPs immunoreactivity was detected: FaRPs-immunoreactive nerve cells were observed among and underlying gastrodermal epithelial cells, epidermal cells lining tentacles, muscular septs and gonophores. A diffuse FaRPs-immunoreactive nerve net was also found between epithelia and mesoglea and in the stalk of the gonophore. These results improve our knowledge of the gorgonian nervous system and demonstrate that most of the immunoreactive cells belong to neural elements.

The anatomical distribution of PACAP-like immunoreactivity was investigated in sensory and peripheral organs of the zebrafish, Danio rerio, during the pharyngula, hatching and larval periods, by using indirect immunofluorescence methods. First PACAP-like immunoreactive (ir) elements appeared during the pharyngula period, at 24 hours post fertilization (hpf), within the most superficial layer of the retina and the dorsal aorta. At 48 hpf, additional ir cells were found in the olfactory placode and esophagus. At 72 hpf (hatching period), PACAP-like immunoreactivity was first detected in the ganglion cell layer of the retina, the otic sensory epithelium, pharyngeal arches, swim bladder and pancreatic progenitor cells. During day 5 of larval development, new groups of ir cells appeared in the liver, whereas no ir elements were observed in the olfactory placode. Subsequently, at day 13 of larval development, additional ir elements were found for the first time in some gut epithelial cells while those previously observed in the retina and otic sensory epithelium were absent. The transient expression of PACAP-like ir material in sensory organs suggests that the peptide could be implicated in neurotrophic activities and neurosensorial connections in the migration and/or differentiation processes. The appearance of PACAP-like ir elements in peripheral organs at different developmental stages, indicates that this peptide could be involved in the control of more specific functions as soon as these peripheral structures begin to operate.

The aim of the present study was to investigate the presence and distribution of cholinergic molecules in Balanus amphitrite cyprids and their possible involvement in settlement and adhesion. Acetylcholinesterase (AChE, the lythic enzyme of acetylcholine) activity was detected, for the first time, by biochemical and histoenzymological methods, in the thoracic muscles, gut wall and cement gland. The immunodetection of choline acetyltransferase-like (ChAT) molecules in the same area and in the neuropil of the central nervous system suggests the presence of a cholinergic innervation, and the involvement of acetylcholine in muscular contraction and cement gland exocytosis. The binding of FITC-conjugate alpha-bungarotoxin in the cement gland cells confirms the latter hypothesis. Acetylcholine involvement in the settlement process was also investigated by laboratory tests employing cholinergic antagonists and agonists. An increase of available acetylcholine due to the partial inhibition of AChE activity produced an increase in cyprid settlement. The data presented support the hypothesis that acetylcholine has a neurotransmitter/neuromodulator role in settlement and adhesion of barnacle cyprids.

The anatomical distribution of neuropeptide tyrosine (NPY)-like immunoreactivity was investigated in the brain, olfactory organ and retina of the zebrafish, Danio rerio, during development and in juvenile specimens, by using the indirect immunofluorescence and the peroxidase-antiperoxidase methods. In 60 h post fertilization (hpf) embryos, NPY-like immunoreactive cell bodies appeared in the hypothalamus, within the posterior periventricular nucleus. Few positive nerve fibers were found in the hypothalamus and in the tegmentum of the mesencephalon. In 72 hpf embryos, a new group of NPY-like immunoreactive cells was found in the olfactory pit. At day 4 of development, NPY-like immunoreactive cell bodies were detected between the olfactory pit and the olfactory organ. In the hypothalamus the location of positive cell bodies was similar to that reported in the previous developmental stages. A few positive nerve fibers appeared in the tegmentum of the rhombencephalon. At days 7 and 15 of development, the distribution of NPY-like immunoreactivity was very similar to that reported at day 4. However, at day 15, NPY-like immunoreactivity appeared for the first time in amacrine cells of the retina and in nerve fibers of the tectum of the mesencephalon. In 1-month/3-month-old animals, additional groups of NPY-like immunoreactive cell bodies appeared in the glomerular layer of the olfactory bulbs, the terminal nerve, the lateral nucleus of the ventral telencephalic area, the entopeduncular nucleus and in the medial region of the reticular formation of the rhombencephalon. These results show that NPY-like immunoreactive structures appear early during ontogeny of zebrafish. The distribution of the immunoreactive system increases during the ontogeny, the juvenile stages, and reaches the complete development in mature animals. The location of NPY-like immunoreactivity indicates that, during development, NPY could be involved in several neuromodulatory functions, including the processing of visual and olfactory information. In 1-month/3-month-old animals, NPY-like immunoreactive nerve fibers are present in the pituitary, suggesting that, from these stages onward, NPY may influence the secretion of pituitary hormones.

Cultured astrocytes were submitted to simulated microgravity using a Fokker clinostat under continuous rotation (60 rpm) for 15', 30', 1h, 20h and 32h. Samples processing included (i) nuclear stainings using Propidium Iodide and 4,6-diamidino-2-phenilindole, dihydro chloride, (ii) immunohistochemical identification of Caspase-7, (iii) identification of DNA fragmentation using the terminal dUTP nick end labelling and (iv) Scanning Electron Microscope analysis. After 30' at simulated microgravity the glial cells showed morphological evidence of apoptosis: cell shrinkage, chromatin condensation, nuclear blebs and fragmentation. The enzyme caspase-7 was present and DNA fragmentation was evident. After 32h the density of the cell population was much lower than that observed in controls.

During and after spaceflight astronauts experience neurophysiological alterations. To investigate if the impairment observed might be traced back to cytomorphology, we undertook a ground based research using a random positioning machine (clinostat) as a simulation method for microgravity. The outcome of the study was represented by cytoskeletal changes occurring in cultured glial cells (C(6) line) after 15 min, 30 min, 1 h, 20 h and 32 h under simulated microgravity. Glia is fundamental for brain function and it is essential for the normal health of the entire nervous system. Our data showed that after 30 min under simulated microgravity the cytoskeleton was damaged: microfilaments (F-actin) and intermediate filaments (Vimentin, Glial Fibrillary Acidic Proteins GFAP) were highly disorganised, microtubules (alpha-tubulin) lost their radial array, the overall cellular shape was deteriorated, and the nuclei showed altered chromatin condensations and DNA fragmentation. This feature got less dramatic after 20 h of simulated microgravity when glial cells appeared to reorganise their cytoskeleton and mitotic figures were present. The research was carried out by immunohistochemistry using antibodies to alpha-tubulin, vimentin and GFAP, and cytochemical labelling of F-actin (Phalloidin-TRIC). The nuclei were stained with propidium iodide or 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). The cells were observed at the conventional and/or the confocal laser scanning microscope. Samples were also observed at the scanning electron microscope (SEM). Our data showed that in weightlessness alterations occur already visible at the scale of the single cell; if this may lead to the neurophysiological problems observed in flight is yet to be established.

In this study we investigated the relationship between nuclear and cell surface modifications (i.e. blebbing, phosphatidylserine [PS] and sugar residues exposure) in a monocytic cell line, U937, during apoptosis induced by oxidative stress (1 mM H2O2) or inhibition of protein synthesis (10 microg/ml puromycin). Dying cells were simultaneously observed for nuclear modifications, presence of superficial blebs and plasma membrane alterations. Morphological analysis performed by conventional fluorescence microscopy, or by transmission and scanning electron microscopy showed that the courses of nuclear and membrane alterations occured concomitantly, but the phenotype was dependent on the stage of the apoptotic process and the type of apoptogenic inducer used. The progression of apoptosis in U937 cells beyond early stages resulted in the extensive formation of blebs which concomitantly lost some typical markers of apoptosis, such as PS and sugar residues. Therefore, the modality by which the nucleus condenses, or the amount and the pattern of distribution of PS on the cell surface were, for each cell line, strictly related to the apoptogenic inducer. The morphological data reported in the present paper should lead to a more precise quantification of apoptosis by improving the detection of apoptotic cells in vivo (i.e. in tissue, organs), which is a crucial point in the evaluation of efficiency of antiproliferative drugs, such as antiblastic or immunosuppressive compounds.

Apoptosis is a form of naturally occurring cell death that plays fundamental roles during embryonic developement. In adults, it neatly disposes of cells damaged by injuries provoked by external causes such as UV radiation, ionisation and heat shock. Alteration of the gravity vector may be one of the external apoptosis inducers. Neurophysiological impairment signs were seen during space flights in astronauts, but very few studies were carried out on the nervous system and none at the cellular level. In this study, we submitted cultured C6 glioma cells to microgravity (0xg) of varying duration, obtained by clinorotation in a Fokker three-dimensional clinostat for 15 min, 30 min, 1h, 20h or 32h. After 30 min at 0xg, numerous nuclei underwent the classical morphological alterations (chromatin condensation, nuclear fragmentation, apoptotic bodies) that lead to the programmed cell death. After 30 min at 0xg, immunostaining for the enzyme caspase-7 was present in the cytoplasm of many cells concurrently with DNA fragmentation identified by the TUNEL method. At 32h, the number of apoptotic nuclei was much reduced indicating the ability of glial cells to adapt to altered gravity.

To investigate whether the signs of neurophysiological impairment observed in flight may be traced back to cytomorphology, we undertook a ground-based study focusing upon the architecture of cultured glial cells under simulated microgravity obtained by three-dimensional clinorotation.

The localization of vasoactive intestinal peptide (VIP)-like immunoreactivity was investigated in the brain, olfactory system and retina of the zebrafish, Danio rerio, during development and in juvenile specimens, by using the indirect immunofluorescence and the peroxidase-antiperoxidase methods. In 24 h post fertilization (hpf) embryos, VIP-like immunoreactive cells were present in the olfactory pit, the retina, and several regions of the brain, including the dorsal telencephalon, the diencephalon, the tegmentum of the mesencephalon, the caudal rhombencephalon and the anterior pituitary. In 48 hpf embryos, additional VIP-like immunoreactive cell bodies were found in the ventral telencephalon, whereas in the diencephalon VIP-like immunopositive cells were more concentrated within the ventro-caudal hypothalamus. During the 7 day larval period, a dense plexus of VIP-like immunoreactive fibers first appeared in the olfactory bulbs. In 15-day-old larvae, two new groups of positive cells were observed in the periventricular preoptic nucleus and in the dorsal rhombencephalon. In 1 month/2 months old animals, VIP-like immunoreactive elements were confined to the olfactory organ, the olfactory bulbs, the periventricular preoptic nucleus and the pituitary, pars distalis. At 3 months stage, a large number of cells was observed in the periventricular preoptic nucleus. Western immunoblot analysis confirmed that VIP-like peptides, with molecular weight similar to that of synthetic VIP, are present early during the development of zebrafish. These results show that VIP-like immunoreactive structures appear early during ontogeny both in the olfactory pit, retina and brain. Transient expression of positive cells was found in the retina, telencephalon, diencephalon and brainstem. The location of VIP-like immunoreactivity indicates that, during development, VIP could be involved in several neuromodulatory functions, including the processing of visual and olfactory informations, as well as growth or survival promotion activities. The presence of VIP-like immunopositive cells in the pituitary, pars distalis, suggest that, during development, VIP may influence the secretion of pituitary hormones.

We have implemented a simple program to solve three of the problems related to 3D reconstruction (3D-Rec) of soft tissues: alignment of sections, distortions, and estimation of the spatial position of elements of interest inside the tissues. As a model, we chose the distribution of FMRFamide-like immunopositive neurons in the ventral ganglion of the barnacle Balanus amphitrite collected during different seasonal periods. Images of immunostained sections were acquired by means of a CCD-camera-equipped microscope and a PC and the reference points were taken inside the sections. The FMRFamide-like immunopositive neurons detected in the barnacle ventral ganglion were grouped into four different classes according to size, shape and staining intensity. More numerous FMRFamide-like immunopositive neurons were detected in the autumn-collected barnacle than in the summer counterpart. The two 3D reconstructions obtained from transverse and longitudinal ventral ganglion sections were efficaciously compared after 90 degrees rotation of one of them. Comparison of these two 3D-Rec suggests the presence of at least two groups of FMRFamide-like immunopositive neurons that are seasonally-related and probably involved in reproduction.

The chromatin structure dynamics of the Colpoda inflata macronucleus have been investigated in relation to its functional condition, concerning chromatin body extrusion regulating activity. Samples of 2- and 25-day-old resting cysts derived from a standard culture, and of 1-year-old resting cysts derived from a senescent culture, were examined by means of histogram analysis performed on acquired optical microscopy images. Three groups of histograms were detected in each sample. Histogram classification, clustering and matching were assessed in order to obtain the mean histogram of each group. Comparative analysis of the mean histogram showed a similarity in the grey level range of 25-day- and 1-year-old cysts, unlike the wider grey level range found in 2-day-old cysts. Moreover, the respective mean histograms of the three cyst samples appeared rather similar in shape. All this implies that macronuclear chromatin structural features of 1-year-old cysts are common to both cyst standard cultures. The evaluation of the acquired images and their respective histograms evidenced a dynamic state of the macronuclear chromatin, appearing differently condensed in relation to the chromatin body extrusion regulating activity of the macronucleus. The coexistence of a chromatin-decondensed macronucleus with a pycnotic extrusion body suggests that chromatin unable to decondense, thus inactive, is extruded. This finding, along with the presence of chromatin structural features common to standard and senescent cyst populations, supports the occurrence of 'rejuvenated' cell lines from 1-year-old encysted senescent cells, a phenomenon which could be a result of accomplished macronuclear renewal.

The endocrine pancreas of three red frogs was studied immunohistochemically. It consisted of islets and diffuse endocrine cells. The islets showed a mammalian-like arrangement with a central core of B cells and a peripheral mantle of A/PP cells. A few D and VIP cells were also present. Several regulatory peptides were co-localized in the same endocrine cells by consecutive sections and double-labeling studies. The A/PP cells were formed by subpopulations of cells showing various types of immunoreactivity and varying degrees of immunolabeling. Generally, glucagon/pancreatic polypeptide, glucagon/pancreatic polypeptide/peptide tyrosine tyrosine and glucagon/pancreatic polypeptide/neuropeptide tyrosine immunoreactivities were present in the islets and in the endocrine cells scattered throughout the exocrine parenchyma (the diffuse component). Some specimens, mainly belonging to Rana dalmatina, showed evident periinsular halos around the islets. The diffuse component was abundant, and mainly contained A/PP cells. It formed a net across the exocrine parenchyma; its interrelationship with the latter might occur by a paracrine mechanism.

The presence, localization and distribution of some regulatory peptides and serotonin were investigated by single and double immunohistochemical methods in the digestive system of two reptiles, Chalcides chalcides and Zonosaurus madascariensis. Both immunoreactive (IR) cells and nerve elements were demonstrated, showing different distributions according to the antisera tested. Similar results were observed in the two saurian species. Chromogranin-SPI-, serotonin-, somatostatin14-, pancreatic polypeptide (PP)-, and gastrin-IR cells were present along the gut epithelium. Cholecystokinin (CCK)-, and insulin-IR cells seemed to be more concentrated in the intestinal portion, while very few glucagon containing cells were observed. Bombesin-IR cells were found in the stomach and they constituted the only endocrine cells of the closed type in the gut. Vasoactive intestinal polypeptide (VIP)-, insulin-, and bombesin-IR nerve terminals were also seen. In the pancreatic duodenal portion of Z madascariensis, the insulin-, glucagon-, PP-, and somatostatin 14-IR cells were present as single elements or grouped in endocrine islets showing a typical topographical distribution. By double immunohistochemical techniques, chromogranin-SPI was found co-localized with the serotonin- and somatostatin-immunoreactivity, but CCK-IR cells were always negative to chromogranin-SP1 antiserum. The present work demonstrates that the chromogranin antiserum is not useful for identifying all the gut endocrine cell types; furthermore, the presence of insulin-immunoreactivity in the endocrine cells is confirmed, and, for the first time, insulin-immunoreactivity is shown in reptilian gut nerve fibres.

The presence, distribution, and interrelationships of the four typical pancreatic islet hormones were investigated in the digestive system of Protopterus annectens by single and double immunohistochemical methods. Insulin-, glucagon-, and somatostatin-immunoreactive (IR) elements were detected in both the pancreas and the gut. Pancreatic polypeptide (PP)-IR endocrine cells were always present in the gut, but were only present in the pancreas of a few specimens. Some of the latter cells also seemed to react with glucagon antiserum. In the pancreas the immunopositive cells were organized into islets of different sizes, and their organizations were studied by the double immunohistochemical techniques. In the few large islets insulin-IR cells were present in the central zone, glucagon- and PP-IR cells at the periphery, and somatostatin-IR cells intermingled with both the peripheral and the central endocrine cells. In the smaller islets, the number and the staining intensity of glucagon- and PP-IR endocrine cells varied markedly. In the gut, insulin-, somatostatin-, and PP-IR cells were of the open type; glucagon-containing cells were very few and had no luminal contact. They were differently distributed along the intestinal epithelium. Somatostatin-IR nerve fibers and somatostatin-IR neuron cell bodies were also observed in the intestinal wall. The organization of pancreatic endocrine cells in P. annectens is similar to that observed in the majority of teleosts even if a different topographical association can be found. Furthermore, islets of different sizes seem to display a different metabolic turnover, and the detection of pancreatic PP-immunoreactivity varied according to the specimens utilized. In the intestinal portion insulin-IR cells, in addition to PP-, glucagon- and somatostatin-IR cells are present: this suggests that intestinal insulin-like immunoreactivity may be more widespread than previously supposed.

Based on the general cross-reactivity of the cytokeratins in vertebrates, we describe the immunoreactivity for some mammalian cytokeratins of both the epidermis and gill of H. fossilis. The following monoclonal antibodies, commercially supplied, were tested: K8.13, KL1, AE1 and AE3, which have a wide spectrum of specificity, and LDS-68, M 20, K8.60, KS-B17.2, K4.62, which are more narrowly specific. The reaction of the epithelial cells of the skin to K8.13 was negative in the basal layer, weakly positive in the layers above but strongly positive in some cells of the superficial layer. KL1 was negative in the basal layer, positive in the outer layers. AE1 was strongly positive in the basal layer, negative in the superficial cells. AE3 gave a general but weak reaction in the epithelial cells. K8.60 was negative for the epithelial cells, but reacted positively in the club cells. Club cells also reacted to K8.13 and AE1, and were strongly positive to KL1 and AE3. Goblet mucous cells were negative to all antibodies tested. In the gills, K8.13 labelled cells of both filament and lamellar epithelia. The reaction to AE3 was similar but less intense. KL1 was positive in the basal layer of the filament epithelium but not elsewhere, and K8.60 was negative throughout. AE1 and KS- B17.2 specifically labelled small cells scattered in the filament and lamellar epithelia, which are tentatively identified as neuroendocrine cells.

The endocrine pancreas of Rana arvalis studied by immunochemistry consisted of islets and diffuse endocrine cells. The islets showed a mammalian-like arrangement with a central core of B cells and a peripheral mantle of A/PP cells. A few D and vasoactive intestinal polypeptide cells were also present. Consecutive sections or double-labeling studies allowed us to detect several regulatory peptides colocalized in the same endocrine cells. The so-called A/PP cells contained subpopulations of cells showing various types of immunoreactivity and varying degrees of immunolabeling. Generally, glucagon/pancreatic polypeptide, glucagon/pancreatic polypeptide/phe-met-arg-phe-amide, glucagon/pancreatic polypeptide/peptide tyrosine tyrosine, glucagon/pancreatic polypeptide/peptide tyrosine tyrosine/phe-met-arg-phe-amide immunoreactivities were present in the islets, while peptide tyrosine tyrosine/neuropeptide tyrosine colocalization was also found in the parenchyma.

Immunohistochemical methods identified serotonin, and the peptides bombesin and caerulein, in the skin of the teleosts Lepadogaster candollei and Mastacembelus erytrotaenia. In both species, the secretory content of epidermal sacciform cells reacted positively for all three substances. These results are compared with studies on the skin glands of amphibians, which also contain multiple active compounds, and on various neuroendocrine cells of fish. The precise functions of the secretions are not known.

The presence and distribution of eleven different types of immunoreactive endocrine cells and nine types of immunoreactive nerve elements were immunohistochemically identified in the gut and pancreas of the italian cave salamander, Hydromantes ambrosii. The majority of gastrointestinal endocrine cells were of open-type, often presenting basal cytoplasmic processes. Gastrin- and substance P-immunoreactive cells in the fundus and bombesin-immunoreactive cells in the intestinal portion were instead of closed type. Immunoreactive nerve fibres were particularly numerous in the muscular layers and blood vessel wall; bombesin- and substance P-immunoreactive nerve fibres were also abundant beneath gastro-intestinal epithelium. Besides substance P-, caerulein- and cholecystokinin-immunoreactive nerve fibres, all the other immunopositive nerve fibres seemed to be of intrinsic types. By the use of four different gastrin/cholecystokinin antisera three variously distributed subpopulations of endocrine cells and nerve elements were detected. Most of the pancreatic endocrine cells were organised in chord-like islets, polarized in the direction of blood vessels. A sparse network of bombesin-immunoreactive fibres was also found in the pancreas. The distribution of bombesin- and of the gastrin/cholecystokinin-immunoreactive material in the stomach and the presence of closed type endocrine cells indicate a more evoluted organization of the gastroenteropancreatic neuroendocrine system thus confirming the position of Hydromantes ambrosii among the higher urodeles.

Light and electron microscopy of the lungs of Ambystoma tigrinum (Urodela) revealed a relatively complex pattern of the neuroendocrine (NE) cells. In the apical parts of smaller septa single NE cells not associated with nerve fibres were covered and surrounded by pneumocytes. The larger septa possessed small areas of ciliated epithelium, in which the NE cells were grouped in a form of neuroepithelial bodies (NEB) consisting of 3-5 cells and covered by goblet cells. NE cells possessed a large nucleus with patches of condensed chromatin, clear cytoplasm, and membrane-bound vesicles of variable morphology and size, containing an electron dense interior surrounded by a lucent space. The size of these dense core vesicles (DCV) ranged from 70-140 nm, while rarely the larger ones exhibited a diameter of 300-600 nm. In some NEB a second type of NE cells was observed for the first time in an amphibian species: these cells communicated with the air space and exhibited on their surface microvilli and a single modified cilium with a 8 + 1 microtubule arrangement. Their cytoplasm contained two types of DCV: dense core granules with a diameter of 140-260 nm and vesicles 320-700 nm in diameter with a moderately electron dense interior. The NEB were associated with intracorpuscular, sensory nerve terminals morphologically afferent and efferent. By immunocytochemistry, the NE cells revealed the presence of serotonin, met-enkephalin, and leu-enkephalin. A paracrine and chemoreceptor role is proposed for NEB of Ambystoma tigrinum.

The present immunocytochemical study concerns the distribution of serotonin in the epidermis of three species of teleost fish. Serotonin-like immunoreactivity was found in the club cells of Heteropneustes fossilis and Carapus acus but not in those from the sea eel Conger conger. This study is the first immunocytochemical identification of serotonin in the club cells of teleost epidermis. By comparing data from the literature (Zaccone et al. 1986, 1987, 1988) regarding the occurrence of serotonin and GRP/bombesin in the exocrine sacciform gland cells of piscine skin, it is worthy mentioning here that the serotonin contained in the club cells of the species studied may have the ability to affect the pheromonal or other possible functions of these cells. The presence of serotonin in these systems has been correlated with the capacity of the exocrine glands of fish skin to secrete, ectopically, amine messengers in contrast to those produced eutopically i.e. in the neuron-paraneuron system in some vertebrates (Fujita et al. 1988).

The presence of different kinds of cells in the gastric tubular glands of the Elasmobranchian Cartilaginous fish "Raja asterias" was investigated by histochemical and ultrastructural methods. Our study demonstrates that the gastric glands, unlike those of the other non-Mammalian Vertebrates, contain in the distal portion, near the neck zone, the oxyntic-like cells, and in the proximal portion the zymogenic-like cells. The former presents histochemical and ultrastructural patterns similar to the Mammalian oxyntic cells, the latter to the Mammalian peptic cells. Between these two types there are always cells with intermediate aspects. These features support the presence of two different stages in the secretory activity: at first the gastric tubular cell is implicated in the secretion of hydrochloric acid, later on, in the secretion of pepsinogen.

Neuroendocrine cells (NE) occurring in the pulmonary epithelium of the fishes Polypterus delhezi and P. ornatipinnis are studied by electron microscopy and by immunostaining for serotonin which is often present in such cells in the mammalian lung. With the electron microscopy NE are found to occur single, resting upon the basement membrane and forming a narrow cytoplasmic extension towards the air lumen. They contain dense-cored vesicles of 80-165 nm which form exocytotic profiles at the level of the basal membrane. An immunoreactivity for serotonin is demonstrated for the first time in the NE of these species. The role of this mediator may involve a paracrine or endocrine function as postulated for the respiratory neuroendocrine mammalian cells. NE of the species studied are considered similar to those found within the wall of lung airways in mammals and submammalian vertebrates. Although much immunocytochemical investigations remain to be executed, they may also be included in the APUD (or DNES) cell system.

The pulmonary mucosa of three species of ancient fish was studied immunohistochemically to show the distribution of serotonin, regarded as the main monoamine of mammalian bronchopulmonary paraneurons. Serotonin-like immunoreactive cells, dispersed through the airway epithelium as single cells, were found in all the fish species studied. They are presumably equivalent to the neuroendocrine cells reported in the lungs of mammalian and submammalian vertebrates. However, the precise role and the function of these cells remain unknown. Since the species studied belong to the most primitive extant groups of ancient fish, the present investigation suggests that serotonin is widely distributed in the lungs of the vertebrates. Several peptides, known to be specific cytochemical markers for the identification of the pulmonary neuroendocrine cells of mammals, are being investigated in the lungs of the fish species studied. They may help to trace the phylogeny of the pulmonary neuroendocrine cell system and to elucidate its function in lower vertebrates.

The ontogeny and distribution of glucagon-like cells were studied in the gastrointestinal tract of embryos, neonates, and adults of the cartilaginous fish Scyliorhinus stellaris (L.) by immunocytochemistry. The results indicate that they appear early during embryonic development, and, in some portion of the gastrointestinal tract, even before the mucosa morphological differentiation. Immunoreactive glucagon-like cells were observed both in gastric and intestinal epithelium, being present in the pyloric portion only at a particular period of its differentiation. Some differences were observed between the embryonic and adult distributive pattern. They were more numerous in proliferative zone and sometimes were situated near other endocrine epithelial cells. These findings together with available information on trophic effects of some gastrointestinal hormonal peptides suggest a possible regulatory role of this peptide on the growth and differentiation of the gastrointestinal tract.

The presence, distribution and development of vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the gastro-entero-pancreatic system of a cartilaginous fish Scyliorhinus stellaris (L.) was investigated by immunohistochemical methods utilizing mammalian VIP antisera. In the gut VIP-like immunoreactivity was observed in both nerves and endocrine cells. Endocrine cells with VIP-like material were only detected in the intestinal epithelium while nerve fibres containing VIP-like material were noted along the whole gastro-entero-pancreatic system, being more numerous in the pyloric sphincter and in the intestinal portion. Immunoreactive nerve cell bodies were encountered in the stomach and intestinal portions localized in the submucosa and in the myenteric plexus. Intestinal immunoreactive endocrine cells were already present in the first developmental stage considered (embryos aged 4 months). They grow in number and before birth reach a frequency higher than in adults. Nerves and cell bodies showing VIP-like immunoreactivity, appear later, before birth, as a few elements in the smooth muscular layer, but only after birth their distribution and frequency are similar to those found in adults. The faint immunofluorescence shown by the immunoreactive endocrine cells and their developmental pattern, which is always different from that observed in nervous elements, suggest the presence of at least two VIP-like substances in the gastro-entero-pancreatic system of S. stellaris.

The comparative distribution of somatostatin- and glucagon-like-containing cells in the histomorphologically different gastric mucosae of the cartilaginous fishes Heptranchias perlo, Raja asterias, Scyliorhinus canicula, Squatina aculeata, and Torpedo marmorata was immunocytochemically studied to demonstrate a possible interrelationship between these endocrine cells and/or other endocrine or nonendocrine cells. In the gastric mucosa, these open-type glucagon and somatostatin immunoreactive cells show a double localization with different morphology and interrelationships. At the bottom of gastric pits, which corresponds to a proliferative zone, spindle or pear-shaped immunopositive cells appear rather numerously and are often in close proximity to each other. In gastric glands, triangular or oval immunopositive cells never in contact with each other were detected; their numeric ratio seems to be rather constant even if their numeric frequency and distribution vary according to the histomorphological aspect of selachian gastric glands. Glucagon immunoreactive cells seem to be more related to pepsinogenic cells, while somatostatin immunoreactive cells seem to be more ubiquitous. Both cell types can present basal cytoplasmic processes. From our results we can suggest a possible regulative role exerted by these two peptides on gastric secretion and cell proliferation.