Serotonin-like Immunoreactive Cells Research Articles

Does the frontal sensory organ in adults of the hoplonemertean Quasitetrastemma stimpsoni originate from the larval apical organ?

BackgroundThe apical organ is the most prominent neural structure in spiralian larvae. Although it has been thoroughly investigated in larvae of the class Pilidiophora in phylum Nemertea, studies on its structure in other nemertean larvae are limited. Most adult hoplonemertean worms have a frontal organ located in a position corresponding to that of the larval apical organ. The development and sensory function of the frontal organ has not been thoroughly characterized to date.ResultsThe apical organ in the early rudiment stage of Quasitetrastemma stimpsoni larvae consists of an apical plate enclosed by ducts of frontal gland cells and eight apical neurons. The apical plate is abundantly innervated by neurites of apical neurons. During the late rudiment stage, the larval apical organ has external innervation from below by two subapical-plate neurons, along with 11 apical neurons, and its plate contains serotonin-like immunoreactive (5-HT-lir) cells. In the vermicular stage (free-swimming juvenile), the number of apical neurons is reduced, and their processes are resorbed. Serotonin is detected in the apical plate with no visible connection to apical neurons. In adult worms, the frontal organ has a small apical pit with openings for the frontal gland ducts. The organ consists of 8 to 10 densely packed 5-HT-lir cells that form the roundish pit.ConclusionsAlthough the ultrastructure of the Q. stimpsoni larval apical organ closely resembles that of the apical organ of Polycladida larvae, the former differs in the presence of flask-shaped neurons typical of Spiralia. Significant differences in the structure of the apical organs of hoplonemertean and pilidia larvae point to two different paths in the evolutionary transformation of the ancestral apical organ. Ultrastructural and immunoreactive analyses of the apical organ of a hoplonemertean larva in the late rudiment and vermicular stages and the frontal organ of the adult worms identified common morphological and functional features. Thus, we hypothesize that the larval apical organ is modified during morphogenesis to form the adult frontal organ, which fulfills a sensory function in the hoplonemertean worm. This unique developmental trait distinguishes the Hoplonemertea from other nemertean groups.

Distribution and evolution of serotonin-like immunoreactive cells in Thaliacea (Tunicata)

Thaliacea are marine planktonic animals within the taxon Tunicata. The species-poor taxon is characterized by diverse life cycles, with sexually (blastozooid) and asexually (oozooid) reproducing generations, that usually evolved polymorph phenotypes. While recent molecular phylogenetic studies indicate that tunicates might be closest living relatives to free-living craniates, additional insights into the evolution of the nervous systems of planktonic tunicates are overdue. To test the hypothesis that polymorphism in the different stages correlates with different distribution patterns of neurotransmitters, we comparatively conducted immunostaining experiments utilizing antibodies against serotonin and tyrosinated-α-tubulin, and confocal laser scanning microscopy in planktonic Thaliacea. We considerably increase the available data on serotonin-like immunoreactive (serotonin-lir) in Thaliacea and found that the pattern of distribution of serotonin-lir cells clearly differs between oozooid and blastozooid stages. Thereby, we reveal that the distribution of serotonin-lir cells in different tissues of thaliaceans can be considered as independent characters. Further, we test the potential phylogenetic signal present in serotonin-lir in thaliaceans, propose a phylogenetic mapping of conceptualized characters, and analyze the evolution of serotonin-lir cells. Comparing our results with data from previous studies indicated that it was necessary to reevaluate already described distribution patterns of serotonin-lir. Due to the complex life cycles of Salpida and Doliolida, erroneous descriptions of the localization of serotonin-lir occur in thaliacean literature. To facilitate the evaluation of data on serotonin-lir in thaliaceans for future readers, we suggest depicting whole-mount stainings of complete animals.

Morphology and evolution of the nervous system in Gnathostomulida (Gnathifera, Spiralia)

Within Spiralia, Gnathifera may represent the deepest branching lineage comprising the jaw worms Gnathostomulida and their sister group Micrognathozoa + Syndermata. Yet, very few nervous system studies have been conducted on this lineage of microscopic, jaw-bearing worms, limiting our understanding of the evolution of this organ system in Spiralia. The nervous system of representatives from all major groups of Gnathostomulida was here mapped using confocal laser scanning microscopy and immunohistochemistry. Their intra-epidermal, unsegmented nervous systems comprise an anterior brain and three to five ventral and two to four dorsal longitudinal nerves, connected by few transverse commissures. Neurites of the stomatogastric nervous system were found lining the pharynx and connecting to a prominent buccal ganglion. Supposedly, sensory ciliated cells in the pharynx and the gut were documented for the first time. Based on these morphological results, primary homologies of neural structures in Gnathostomulida and other Gnathifera were hypothesized and thereafter tested using parsimony. This first neurophylogeny of Gnathostomulida resulted in a topology congruent with molecular data, supporting the monophyly of Bursovaginoidea, Conophoralia, and Scleroperalia. From this topology, the evolution of the gnathostomulid nervous system was reconstructed. It suggests a specialization and diversification of cords and serotonin-like immunoreactive cell patterns from a plesiomorphic neuroarchitecture of three unsegmented nerve cords and a compact anterior brain and buccal ganglion. These plesiomorphic states resemble the nervous system of Micrognathozoa, and possibly the ancestral states of Spiralia.

Development of the Nervous System of Carinina ochracea (Palaeonemer-tea, Nemertea).

The various clades of Lophotrochozoa possess highly disparate adult morphologies. Most of them, including Nemertea (ribbon worms), are postulated to develop via a pelagic larva of the trochophora type, which is regarded as plesiomorphic in Lophotrochozoa. With respect to the nervous system, the trochophora larva displays a set of stereotypic features, including an apical organ and trochal neurites, both of which are lost at the onset of metamorphosis. In the investigated larvae of Nemertea, the nervous system is somewhat divergent from the postulated hypothetical trochophore-like pattern. Moreover, no detailed data is available for the “hidden” trochophore larva, the hypothetical ancestral larval type of palaeonemertean species. Therefore, the development of the nervous system in the larva of Carinina ochracea, a basally branching palaeonemertean species, was studied by means of immunofluorescence and confocal laserscanning microscopy. Like in the other investigated nemertean larvae, the prospective adult central nervous system in C. ochracea develops in an anterior to posterior direction, as an anterior brain with paired longitudinal nerve cords. Thus, development of the adult nervous system in Nemertea is largely congruent with currently accepted hypotheses of nervous system development in Spiralia. In early development, transitory apical, serotonin-like immunoreactive flask-shaped cells are initially present, but the trochal neurites that have been considered as pivotal to lophotrochozoan development, are absent. In the light of the above stated hypothesis, trochal neurites have to be interpreted as reduced in Nemertea. On the other hand, due to the unsettled systematic status of Palaeonemertea, more comparative data are desirable to answer the remaining questions regarding the evolution of nervous system development in Nemertea.

Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny.

BackgroundThaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea.ResultsAdults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida).ConclusionsComparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to Pyrosomella verticillata might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-016-0177-6) contains supplementary material, which is available to authorized users.

Comparative study of serotonin-like immunoreactivity in the branchial basket, digestive tract, and nervous system in tunicates

As one of the three major chordate taxa, the highly diverse taxon, Tunicata has always played a key role in considerations of evolutionary origins of vertebrates, especially since several larger-scaled molecular phylogenetic analyses support the hypothesis that tunicates are the sister group to vertebrates. Molecular phylogenetic studies of the relationships within Tunicata are highly contradictory, and cladistic analyses of morphological characters for Tunicata remained limited. In order to come to a better understanding of chordate evolution, we comparatively investigated nine different tunicate species belonging to five different higher tunicate taxa: Phlebobranchiata, Aplousobranchiata, Stolidobranchiata, Thaliacea, and Appendicularia using immunofluorescence labeling with antibodies against serotonin in conjunction with confocal laser scanning microscopy. We found that adult ascidians are comparable in regard to their respective patterns of serotonin-like immunoreactive (serotonin-lir) cells, whereas the planktonic tunicates differ in several aspects. The distribution patterns of serotonin-lir cells in tunicates suggest that serotonin-like immunoreactivity can behave as an independent character during evolution. While the distribution pattern of serotonin-lir cells agrees well with classical taxonomic groupings, phylogenetic interpretation remains inconclusive, as ad hoc hypotheses are always necessary to explain contradictory character distribution. Based on light-microscopically observed morphology, we could distinguish three different types of serotonin-lir cells, most probably functionally distinctive. These were approximately spherical serotonin-lir cells, possibly involved in the control of ciliary beating and mucus secretion, elongated serotonin-lir cells potentially involved in hormonal regulation of feeding, and serotonin-lir neurons that might be implicated in the initiation of locomotory behavior.

Neural markers reveal a one-segmented head in tardigrades (water bears).

BackgroundWhile recent neuroanatomical and gene expression studies have clarified the alignment of cephalic segments in arthropods and onychophorans, the identity of head segments in tardigrades remains controversial. In particular, it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure. To clarify this, we applied a variety of histochemical and immunocytochemical markers to specimens of the tardigrade Macrobiotus cf. harmsworthi and the onychophoran Euperipatoides rowelli.Methodology/Principal FindingsOur immunolabelling against serotonin, FMRFamide and α-tubulin reveals that the tardigrade brain is a dorsal, bilaterally symmetric structure that resembles the brain of onychophorans and arthropods rather than a circumoesophageal ring typical of cycloneuralians (nematodes and allies). A suboesophageal ganglion is clearly lacking. Our data further reveal a hitherto unknown, unpaired stomatogastric ganglion in Macrobiotus cf. harmsworthi, which innervates the ectodermal oesophagus and the endodermal midgut and is associated with the second leg-bearing segment. In contrast, the oesophagus of the onychophoran E. rowelli possesses no immunoreactive neurons, whereas scattered bipolar, serotonin-like immunoreactive cell bodies are found in the midgut wall. Furthermore, our results show that the onychophoran pharynx is innervated by a medullary loop nerve accompanied by monopolar, serotonin-like immunoreactive cell bodies.Conclusions/SignificanceA comparison of the nervous system innervating the foregut and midgut structures in tardigrades and onychophorans to that of arthropods indicates that the stomatogastric ganglion is a potential synapomorphy of Tardigrada and Arthropoda. Its association with the second leg-bearing segment in tardigrades suggests that the second trunk ganglion is a homologue of the arthropod tritocerebrum, whereas the first ganglion corresponds to the deutocerebrum. We therefore conclude that the tardigrade brain consists of a single segmental region corresponding to the arthropod protocerebrum and, accordingly, that the tardigrade head is a non-composite, one-segmented structure.

Development of the nervous system in Phoronopsis harmeri (Lophotrochozoa, Phoronida) reveals both deuterostome- and trochozoan-like features

BackgroundInferences concerning the evolution of invertebrate nervous systems are often hampered by the lack of a solid data base for little known but phylogenetically crucial taxa. In order to contribute to the discussion concerning the ancestral neural pattern of the Lophotrochozoa (a major clade that includes a number of phyla that exhibit a ciliated larva in their life cycle), we investigated neurogenesis in Phoronopsis harmeri, a member of the poorly studied Phoronida, by using antibody staining against serotonin and FMRFamide in combination with confocal microscopy and 3D reconstruction software.ResultsThe larva of Phoronopsis harmeri exhibits a highly complex nervous system, including an apical organ that consists of four different neural cell types, such as numerous serotonin-like immunoreactive flask-shaped cells. In addition, serotonin- and FMRFamide-like immunoreactive bi- or multipolar perikarya that give rise to a tentacular neurite bundle which innervates the postoral ciliated band are found. The preoral ciliated band is innervated by marginal serotonin-like as well as FMRFamide-like immunoreactive neurite bundles. The telotroch is innervated by two neurite bundles. The oral field is the most densely innervated area and contains ventral and ventro-lateral neurite bundles as well as several groups of perikarya. The digestive system is innervated by both serotonin- and FMRFamide-like immunoreactive neurites and perikarya. Importantly, older larvae of P. harmeri show a paired ventral neurite bundle with serial commissures and perikarya.ConclusionsSerotonin-like flask-shaped cells such as the ones described herein for Phoronopsis harmeri are found in the majority of lophotrochozoan larvae and therefore most likely belong to the ground pattern of the last common lophotrochozoan ancestor. The finding of a transitory paired ventral neurite bundle with serially repeated commissures that disappears during metamorphosis suggests that such a structure was part of the “ur-phoronid” nervous system, but was lost in the adult stage, probably due to its acquired sessile benthic lifestyle.

Presence and distribution of serotonin immunoreactivity in the cyprids of the barnacle Balanus amphitrite

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.

Histological and immunocytochemical localization of serotonin‐like immunoreactivity in the brain and optic ganglia of the Indian white shrimp, Fenneropenaeus indicus

Serotonin is one of the important neurotransmitter and neuromodulator so far studied in crustacean models. With its secretory sites well-studied in higher crustaceans, its function in controlling the release of metabolic hormones from their storage and release sites has been well proved. The present study attempts to localize serotonin-like immunoreactivity in Fenneropenaeus indicus, a commercially important shrimp species and a natural inhabitant of the Indian oceans. Histological studies were employed to visualize the different types of neurosecretory cells and their regions of occurrence in brain and optic ganglia on the basis of their size, shape, and tinctorial properties. Immunocytochemical studies were performed in the brain and optic ganglia with specific antisera against serotonin in combination with peroxidase anti-peroxidase to map the serotonin-like immunoreactive cells. Variations in the immunoreactivity were observed on comparing the cells of brain and optic ganglia. Medulla terminalis region had intense serotonin immunoreactivity suggesting it to be the primary source of the neurotransmitter.

Distribution of serotonin-like immunoreactive neurons in the slug Limax valentianus

Immunohistochemical techniques were used to study the distribution of serotonin-containing neurons in the nervous system of the slug Limax valentianus. Approximately 350 serotonin-like immunoreactive cell bodies were found in the central nervous system. These were located in the cerebral, pedal, visceral and right parietal ganglia. Most serotonin-like immunoreactive neurons had small cell bodies, which were aggregated into discrete clusters. A pair of previously identified metacerebral giant cells were found on the anterior side of the cerebral ganglion, and two additional pairs of uniquely identifiable, serotonin-like immunoreactive cells were found on the posterior side of the cerebral ganglion. The whole-mount maps of these stained neurons will be useful in further physiological and biochemical studies of olfactory learning at the cellular level in Limax valentianus.

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.

Synaptic contacts between serotonergic and cholinergic neurons in the rat dorsal raphe nucleus and laterodorsal tegmental nucleus

We examined synaptic connectivity between cholinergic and serotonergic neurons in the dorsal raphe nucleus and the laterodorsal tegmental nucleus of the rat. To this purpose we employed two variations (the combination of pre-embedding immunogold–silver intensification with avidin–biotin-peroxidase complex technique and the combination of avidin–biotin–peroxidase/3,3′-diaminobenzidine/silver–gold intensification with avidin–biotin–peroxidase/3,3′-diaminobenzidine reaction) of a double pre-embedding immunoelectron procedure, using primary antibodies against vesicular acetylcholine transporter and serotonin. At the light-microscopic level, serotonin-like immunoreactive neurons in the dorsal raphe nucleus appeared as reddish black and vesicular acetylcholine transporter-like immunoreactive axon terminals were brown colored using a combination of pre-embedding immunogold-silver technique and avidin–biotin–peroxidase complex technique. Serotonin-like immunoreactive fibers projected to the laterodorsal tegmental nucleus. At the electron microscopy level, with both methods we observed in the dorsal raphe nucleus vesicular acetylcholine transporter-immunopositive axon terminals in synaptic contact with serotonin-like immunoreactive dendrites and, to a lesser degree, with serotonin-like immunoreactive cell bodies. These synapses usually were of the symmetrical type. Occasionally we noted, next to vesicular acetylcholine transporter-immunopositive axon terminals, also immunonegative terminals synapsing with the serotonin-like immunoreactive dendrites. In the laterodorsal tegmental nucleus we found serotonin-like immunoreactive axon terminals and immunonegative terminals forming synapses with vesicular acetylcholine transporter-immunoreactive dendrites. Most synapses formed by the serotonin-like immunopositive terminals were of the asymmetrical type. Our results suggest that serotonergic neurons in the dorsal raphe nucleus and cholinergic neurons in the laterodorsal tegmental nucleus may reciprocally influence each other by means of synaptic connectivity. Such connectivity may serve to regulate pain sensation, or be involved in the regulation of the sleeping–waking cycle.

Non-uniform distribution of cellular phenotypes in the optic tectum of the leopard frog.

Regional specialization in the retina have been described in a number of species. We have investigated whether such specializations can be found in the optic tectum, an area of the brain responsible for the processing of visual information. Using the tectum of Rana pipiens, we have examined the distribution of three different cell types defined on the basis of their immunoreactivity to somatostatin, substance P, and serotonin antibodies. These three immunoreactive cell populations had differing, nonuniform distributions in the optic tectum. Somatostatin-line immunoreactive cells were largely restricted to the caudal one-third of the tectum, whereas both substance P-like immunoreactive (SP-ir) and serotonin-like immunoreactive (5-HT-ir) cells were found unequally represented throughout the tectum. The percentage of SP-ir cells decreased significantly in both the posterior and medial directions from its high in the anterior lateral tectum. Although serotonin-like immunoreactivity was also greatest in the lateral tectum and decreased significantly medially, it was largely constant in the anterior-to-posterior dimension. The populations of SP-ir and 5-HT-ir cells were nonoverlapping. Our results suggest that information may be processed differently in different regions of the optic tectum.

Origins of serotonin-like immunoreactivity in the optic tectum of Rana pipiens.

We have previously identified a population of serotonin-like immunoreactive (5-HT-ir) retinal ganglion cells in Rana pipiens. In this study, we examined serotonin-like immunoreactivity (5-HTLI) in a probable target of those cells, the optic tectum. We observed both 5-HT-ir fibers and cell bodies in this structure. 5-HT-ir cells were located in the cellular layers of the tectum, layers 2, 4, and 6, and scattered in its superficial layers. 5-HT-ir fibers in the tectum displayed a laminated organization and were located in tectal layers 3, 5, 6, 7, and 9. Retrograde labelling experiments showed that 5-HT-ir retinal ganglion cells projected to the optic tectum. However, these experiments also demonstrated that serotonergic neurons in the midbrain tegmentum, the nucleus isthmi, and the medulla did so as well. 5-HT-ir fibers seen in lamina A of layer 9 were very much reduced in density in animals in which the optic nerve had been lesioned for 3-6 months. Immunoreactive fibers in lamina B of layer 9 were not affected by the lesion. Our results suggest that 5-HT-ir fibers in lamina A of layer 9 are mainly of retinal origin, whereas those in lamina B originate from other brain areas. The 5-HT-ir tectal cells located in the cellular layers probably contribute the 5-HT-ir fibers seen in layers 3, 5, 6, and 7.

Immunohistochemical analyses of the human olfactory bulb

These studies explore the distribution of putative neuroactive peptides in the human olfactory bulb. Localization of synaptophysin-, serotonin-, cholecystokinin-, substance P-, and somatostatin-like staining was examined by immunocytochemical protocols. The results provide new insights into the composition and laminar segregation of subpopulations of neurons and neuronal processes in the human olfactory bulb. The prominent synaptophysin-like immunoreactivity observed in the glomeruli of the human olfactory bulb is consistent with the notion that the density of synapses, and hence the density of synaptic vesicles, is highest in the glomeruli. Serotonin-like immunoreactivity suggested a variable innervation of glomeruli ranging from a dense tangled ball of fibers within the glomerulus to a sparse innervation by a single immunoreactive fiber. There was no evidence of serotonin-like immunoreactive cell bodies in either the olfactory bulb proper, anterior olfactory nucleus, or proximal regions of the lateral olfactory tract. Cholecystokinin-like immunoreactivity was limited to fibers found largely in the juxtaglomerular region of the glomerular layer. In the deeper layers of the olfactory bulb, cholecystokinin-like immunoreactive fibers did not show any of branching or arborization that was evident in the juxtaglomerular region. Substance P-like immunoreactivity was seen in varicose fibers distributed in all of the human olfactory bulb laminae. In addition, stained multipolar neurons were found in the area of the anterior olfactory nucleus. Somatostatin-like immunoreactivity was similar to that of substance P in that a plexus of stained fibers was found in all laminae of the olfactory bulb. Also, somatostatin-like immunoreactive cell bodies were found in the area of the anterior olfactory nucleus. However, as compared to substance P, somatostatin had a less dense plexus of immunoreactive fibers in the olfactory bulb. These results increase our understanding of the fundamental organization of the human olfactory system. The current data, coupled with prior studies, provide a foundation from which to study the cellular pathology of diseases with known olfactory system sequelae such as Alzheimer's, Parkinson's, and schizophrenia.

Large serotonin-like immunoreactive amacrine cells in the retina of developing Xenopus laevis

The earliest appearance of serotonin-like immunoreactivity (SLI) in different cell types and the development of large SLI amacrine cells were studied in the retina of Xenopus laevis from stage 33/34 to adult. Intense SLI was first found in the somas of large amacrine cells at stage 39. The somas of small amacrine cells showed weak SLI at stage 41, followed by bipolar cells at stages 43. The number of large SLI amacrine cells in the inner nuclear layer of the retina increased from 57 at stage 40 to 774 in adult. Over the same period, retinal area increased from 0.19 mm 2 to 24.57 mm 2 with an accompanying decrease of cell density from 301/mm 2 to 32/mm 2. In adult animals large SLI amacrine cells were non-uniformly distributed. Peak cell density of 50–60/mm 2 was located in the center of the ventrotemporal quadrant and a trough of 8–15/mm 2 in the dorsal periphery of the retina. Peak cell density region of the adult retina correspond to part of the retina formed at early developmental stages where the rate of cell generation of large SLI amacrine cells was higher. These observations indicate that (1) SLI is expressed first by large amacrine cells, followed by small amacrine and bipolar cells; (2) large SLI amacrine cells are generated continuously throughout life, (3) the non-uniform retinal distribution of large cells results from a spatio-temporally differential cell generation at the ciliary margin.

Development of serotoninlike immunoreactivity in the embryonic nervous system of the snail Lymnaea stagnalis.

In our initial effort to study the ontogeny of the gastropod nervous system, we used histological techniques to examine the post-embryonic development of cells which exhibit serotoninlike immunoreactivity in Lymnaea (Croll and Chiasson, J. Comp. Neurol. 230:122-142, '89). The present study complements that report by examining the embryonic development of these neurons. The first serotoninlike immunoreactive (SLIR) cells to be detected in the embryos are the paired C4 neurons of the cerebral ganglia. These cells are faintly visible at about 37-38% of embryonic development and have already produced axons which traverse the cerebral commissure. By about 2-3% later the axon tips reach the pedal ganglia and appose the next SLIR cells to appear, the EPe1 neurons. Over the next 30% of development four more pairs of cerebral neurons are added adjacent to the C4 neurons and over ten cells are added to each of the pedal ganglia. At about 70% of development SLIR fibers are first detected in the parietal and visceral ganglia forming the abdominal ring. Around this time the somata of the C1 neurons also first appear in the cerebral ganglia together with their prominent axons projecting to the buccal ganglia. The last 30% of development is marked by a massive addition of SLIR cells (up to 60) in each pedal ganglion. The early appearance of the first SLIR cells suggests that they may be among the first nerve cells to differentiate and that they may play central roles in the formation of the CNS. We hypothesize that most of the animal's neural circuitry is laid down during embryogenesis by a stereotypic ontogenetic program with post-embryonic neurogenesis subserving mostly compensatory and modulatory purposes.

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.

Enkephalinergic synaptic axon terminals on serotoninergic neurons in the dorsal raphe nucleus of the rat: an electron microscopic study by the double immunostaining method

Met-enkephalinergic synaptic axon terminals on serotoninergic neurons in the dorsal raphe nucleus of the rat were observed with double immunostaining at the electron microscopic level. Met-enkephalin-like immunoreactive axon terminals were found to make asymmetrical or symmetrical synaptic contacts with serotonin-like immunoreactive nerve cell bodies and dendrites. The findings suggest that opioid-containing neurons modulate serotoninergic neurons through synapses in the dorsal raphe nucleus.