Ontogeny of central serotonergic neurons in the directly developing frog, <Emphasis Type="Italic">Eleutherodactylus coqui</Emphasis>

Embryonic development of the central serotonergic neurons in the directly developing frog, Eleutherodactylus coqui, was determined by using immunocytochemistry. The majority of anuran amphibians (frogs) possess a larval stage (tadpole) that undergoes metamorphosis, a dramatic post-embryonic event, whereby the tadpole transforms into the adult phenotype. Directly developing frogs have evolved a derived life-history mode where the tadpole stage has been deleted and embryos develop directly into the adult bauplan. Embryonic development in E. coqui is classified into 15 stages (TS 1–15; 1 = oviposition / 15 = hatching). Serotonergic immunoreactivity was initially detected at TS 6 in the raphe nuclei in the developing rhombencephalon. At TS 7, immunopositive perikarya were observed in the paraventricular organ in the hypothalamus and reticular nuclei in the hindbrain. Development of the serotonergic system was steady and gradual during mid-embryogenesis. However, starting at TS 13 there was a substantial increase in the number of serotonergic neurons in the paraventricular, raphe, and reticular nuclei, a large increase in the number of varicose fibers, and a differentiation of the reticular nuclei in the hindbrain. Consequentially, E. coqui displayed a well-developed central serotonergic system prior to hatching (TS 15). In comparison, the serotonergic system in metamorphic frogs typically starts to develop earlier but the surge of development that transpires in this system occurs post-embryonically, during metamorphosis, and not in the latter stages of embryogenesis, as it does in E. coqui. Overall, the serotonergic development in E. coqui is similar to the other vertebrates.     

Ontogeny of GABA-immunoreactive neurons in the central nervous system in a teleost, Gasterosteus aculeatus L.

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is known to exert various neurotrophic actions in the developing nervous system, but little is known about its distribution in the central nervous system during early development. We have studied the development of GABA-immunoreactive (GABAir) neurons during embryogenesis of a teleost fish, the three-spined stickleback. As early as 51 h postfertilization (PF; hatching occurs 144–168 h PF, and the first monoaminergic neurons appear around 72 h PF) GABAir neurons appear in the ventral prosencephalon caudal to the optic recess, in the ventral meencephalon, and in the spinal cord. Then, there is a gradual addition of GABAir cell groups in the rostral prosencephalon and ventral rhombencephalon (66 h PF), dorsal and caudal hypothalamus and pretecturn (72 h PF), ventral hypothalamus (78 h PF), preoptic region, thalamus, and in the meencephalon and rhombencephalon (96 h PF). GABAir axons appear in the spinal cord already at 51 h PF, and then gradually appear in the various tracts of the early axonal scaffold of pathfinding fibers, so that by 96 h PF the entire axonal scaffold contains GABAir fibers. It appears likely that GABAergic axons contribute a major population to the formation of the axonal scaffold. Moreover, in the prosencephalon GABAir neurons are arranged in clusters that may reflect a neuromerec organization with six prosencephalic neuromeres.     

Mosaic evolution of neural development in anurans: acceleration of spinal cord development in the direct developing frog Eleutherodactylus coqui

Previous studies have shown that spinal cord development in direct developing frogs of the genus Eleutherodactylus, which have evolutionarily lost the tadpole stage, differs from that in biphasically developing anurans (with the larval and the adult stage separated by metamorphosis). The present study of spinal cord development in Eleutherodactylus coqui provides additional information about neurogenesis, neuronal differentiation and growth analyzed by immunostaining for proliferating cell nuclear antigen (PCNA), in situ hybridization for NeuroD, and morphometric measurements in various developmental stages. Furthermore, spinal cord development in the frogs Discoglossus pictus, Xenopus laevis, and Physalaemus pustulosus, which belong to different anuran families but all exhibit biphasic development, was similarly analyzed. This comparative analysis allows inference of the ancestral anuran pattern of spinal cord development and how it has been modified during the evolution of Eleutherodactylus. All biphasically developing frogs analyzed share a similar pattern of spinal cord development, suggesting that this is ancestral for anurans: after neural tube closure, levels of proliferation and neurogenesis in the spinal cord were low throughout embryogenesis until they were upregulated drastically at early larval stages followed by development of the lateral motor columns. In contrast, no such quiescent embryonic period exists in E. coqui, where rapid growth, high levels of proliferation and neurogenesis, and early formation of lateral motor columns occur shortly after neural tube closure, while other parts of the central nervous system develop more slowly. Thus, spinal cord development has been accelerated during the evolution of Eleutherodactylus relative to the development of other parts of the central nervous system, probably related to the precocious development of limbs in this lineage.     

Development of the serotonergic system in the central nervous system of the sea lamprey

Lampreys belong to the most primitive extant group of vertebrates, the Agnathans, which is considered the sister group of jawed vertebrates. Accordingly, characterization of neuronal groups and their development appears useful for understanding early evolution of the nervous system in vertebrates. Here, the development of the serotonergic system in the central nervous system of the sea lamprey, Petromyzon marinus, was investigated by immunohistochemical analysis of specimens ranging from embryos to adults. The different serotonin-immunoreactive (5-HT-ir) neuronal populations that are found in adults were observed between the embryonic and metamorphic stages. The earliest serotonergic neurons were observed in the basal plate of the isthmus region of late embryos. In prolarvae, progressive appearance of new serotonergic cell groups was observed: firstly in the spinal cord, then in the pineal organ, tuberal region, zona limitans intrathalamica, rostral isthmus, and the caudal part of the rhombencephalon. In early larvae a new group of serotonergic cells was observed in the mammillary region, whereas in the pretectal region and the parapineal organ the first serotonergic cells were seen in the middle and late larval stages, respectively. The first serotonergic fibres appeared in early prolarvae, with fibres that ascend and descend from the isthmic cell group, and the number of immunoreactive fibres increased progressively until the adult stage. The results reveal strong resemblances between lampreys and other vertebrates in the spatio-temporal pattern of development of brainstem populations. This study also reveals a shared pattern of early ascending and descending serotonergic pathways in lampreys and jawed vertebrates.     

Ontogeny of immunoreactive somatolactin, prolactin and growth hormone secretory cells in the developing pituitary gland of Cichlasoma dimerus (Teleostei, Perciformes)

Prolactin, growth hormone and somatolactin constitute a hormone family because they are structurally related and are secreted by acidophilic cells of different regions of the adenohypohyisis. In this work, we report the ontogeny of ir-prolactin, ir-growth hormone and ir-somatolactin cells in the developing pituitary gland of the cichlid fish Cichlasoma dimerus (Teleostei; Perciformes). Antisera raised against fish pituitary hormones were used. In this species hatching occurs 54 hs after fertilization and the three different cell types were recognized two days later. The neurohypophysis was recognized on day 14 after hatching and in later stages it began to show the characteristic deep interdigitations of the adults. On day 42 (juvenile stage) the distribution of ir-PRL, ir-GH and ir-SL showed the pattern described for adults of this species. The ir-SL cells were not PAS-positive in larvae as they are in adults. This would suggest the presence of a nonglycosilated form of somatolactin in early stages of development which may coexist in adults with a glycosilated form. The appearence of these hormones so early in development suggest their importance in the survival of fish larvae but further studies focused on the ontogeny of hypothalamic factors that regulate their synthesis and secretion must be performed. Accepted: 30 January 2001     

Morphological and immunocytochemical identification of macrophages in the developing corpus callosum

Summary The presence of macrophages in the developing corpus callosum of fetal and neonatal rats is confirmed using ultrastructural, immunocytochemical and histochemical techniques. The macrophages are first seen in the corpus callosum at about the time the callosal axons cross the midline and penetrate the cerebral hemispheres and they persist until about 10 days postnatal. The cells are found in the corpus callosum dorsal to the lateral ventricle, in the sub-ventricular zone and in the forming cavum septi pellucidi. The macrophages were identified at the light and electron microscopic level, including labelling of the cells following an intracerebral injection of horseradish peroxidase. The cells were also positive for an esterase stain specific for blood monocytes and macrophages and were labelled with a monoclonal antibody directed against macrophage cell surface polypeptides (Springer et al. 1979). These observations confirm the non-neuroepithelial origin of certain forms of phagocytic cells during cerebral development.     

Distribution of serotonergic neurons and processes in the lamprey spinal cord

The distribution of serotonin in the spinal cord in two species of lamprey, Ichthyomyzon unicuspis and Petromyzon marinus, was studied by indirect immunofluorescence techniques. Multipolar cell bodies containing serotonin-like immunoreactivity were found along the length of the spinal cord, along the midline and slightly ventral to the central canal. These cell bodies send a diffuse projection of processes throughout the spinal cord, including: (1) a dense projection to the ventral surface; (2) a strong projection to the ventromedial longitudinal fiber tracts; (3) a less intense projection to the dorsal longitudinal fiber tracts; and (4) a weak projection to the lateral fiber tracts. Lesion experiments showed that processes descending from the brain or rostral spinal cord provide a major projection to the lateral fiber tracts and smaller contributions to the dorsal and ventromedial fiber tracts. Fluorescent processes were also observed in the dorsal roots and serotonergic peripheral cell bodies were seen adjacent to the dorsal roots.Our results suggest that the serotonergic innervation of the lamprey spinal cord arises from three sources: spinal interneurons, descending tracts and peripheral (possibly sensory) input. This provides an anatomical substrate for our recent finding^12,13 that serotonin modulates the central pattern generator for locomotion in the lamprey spinal cord.     

Ultrastructural localisation of serotonin and substance P in vascular endothelial cells of rat femoral and mesenteric arteries

Summary An electron microscopic-immunocytochemical study has been made on the localisation of serotonin and substance P in endothelial cells of femoral and mesenteric arteries of the rat. In the femoral and mesenteric arteries, less than 10% of the endothelial cells showed positive immunolabelling for these substances. The distribution of serotonin and substance P in endothelial cells was similar in both arteries. The immunoprecipitate was confined to the cytoplasm, including subcellular organelles. The results are discussed in terms of endothelial mechanisms of local control of blood flow.     

Distribution of OMP-, PGP 9.5- and CaBP-like immunoreactive chemoreceptor neurons in the developing human olfactory epithelium

We have examined the distribution of olfactory marker protein (OMP), protein gene product 9.5 (PGP 9.5) and calcium-binding protein D-28k (CaBP) in the olfactory epithelium of mid- to late fetal and newborn humans using immunocytochemistry. Olfactory chemoreceptor neurons (ORNs) in a 24-week-old female fetus, a 31-week-old male fetus and a newborn male were examined. OMP-like immunoreactivity (-LI) and PGP 9.5-LI were distributed throughout ORNs at all ages. CaBP-like immunoreactivity, however, was found only in clustered or isolated fetal ORNs; in the newborn, CaBP-LI was seen only in isolated ORNs sparsely distributed throughout the OE. These findings demonstrate that human ORNs express OMP-LI nearly 4 weeks earlier in development than previously reported. PGP 9.5-LI is coincidentally abundant within these cells, suggesting it may have an important role in mature ORNs. Because the number of ORNs expressing CaBP-LI decreases during perinatal development, CaBP may be important in intracellular calcium regulation during ORN growth and maturation in the developing OE.     

Ontogeny of sensory nerves in the developing skeleton

Background: Previous studies have shown that the neuropeptide calcitonin gene-related peptide (CGRP) have an influence on osteoclastic bone resorption and that CGRP and substance P (SP), both wellknown markers for sensory neurons, behave as growth factors. Materials and Methods: The ontogeny of the sensory nerves in the hindlimb skeleton of the rat was studied from gestational day (GD) 15 to neonatal day (ND) 24 by immunohistochemistry. Neurofilaments and nerve terminals were labelled with protein gene-product 9.5 (PGP 9.5) and synaptophycin (SYN) respectively. Results: PGP 9.5 appeared at GD 15 and SYN at GD 19, both in the perichondrial tissue of the long bones. One week later, at ND 4 nerve fibre, immunoreactive to PGP 9.5 and SYN were observed within the bone organ. Sensory nerves, indicated by CGRP and SP, were first discerned at GD 18–19 in the periosteal tissue of the diaphyseal and metaphseal regions and in the bone organ at ND 4. Approximately at ND 6, vascular as well as non-vascular nerves extended into the metaphyses and at ND 8 into the epiphyses, concomitant with the first signs of mineralization. Conclusions: The study shows that a functional sensory nerve supply of the developing bone organ occurs immediatly prior to partus, apparently parallel with an increasing mineralization. The combined findings may indicate a sensory influence on developmental processes in the skeleton. © 1995 Wiley-Liss, Inc.     

The immunocytochemical localization of neuronal nitric oxide synthase in the developing rat retina

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina.     

Development of parvalbumin immunoreactive neurons in normal and intracranially transplanted retinas in the rat

Summary Retinas from embryonic day 14 Sprague-Dawley rats were transplanted to the midbrain or cerebral cortex of newborn (P0) rats of which the right eye was enucleated at the time of transplantation. Parvalbumin immunoreactive (PV-I) neurons were studied in the developing retinal transplants, and in the remaining retina of the host, as well as in normal retinas. PV-I neurons were identifiable in retinas of normal and host rats from postnatal day 5 (P5) onward, with the PV-I somata primarily in the inner half of the inner nuclear layer and in the ganglion cell layer. An adult-like distribution of PV-I neurons was attained at P35, as judged by cell packing density, intensity of immunostaining, laminar distribution and soma size of subpopulations of PV-I cells. A similar time course of development and distribution of PV-I somata was observed in the retinal transplants, except for some minor differences such as a slight delay in PV-I cells achieving their final distribution. These findings provide evidence that PV-I neurons can survive, differentiate and mature according to pre-determined programmes intrinsic to the retinal tissue following transplantation to a new and foreign environment.Visiting research fellow on leave from the Neurosciences Research Institute, Guangzhou Medical College, People's Republic of China     

Embryonic exposure to thimerosal, an organomercury compound, causes abnormal early development of serotonergic neurons

Even though neuronal toxicity due to organomercury compounds is well known, thimerosal, an organomercury compound, is widely used in pediatric vaccine preservation. In the present study, we examined whether embryonic exposure to thimerosal affects early development of serotonergic neurons. Thimerosal (1 mg Hg/kg) was intramuscularly administered to pregnant rats on gestational day 9 (susceptible time window for development of fetal serotonergic system), and fetal serotonergic neurons were assessed at embryonic day 15 using anti-serotonin antibodies. A dramatic increase in the number of serotonergic neurons localized to the lateral portion of the caudal raphe was observed in thimerosal group (1.9-fold increase, p < 0.01 compared to control). These results indicate that embryonic exposure to thimerosal affects early development of serotonergic neurons.     

Basic fibroblast growth factor (bFGF, FGF-2) immunoreactivity exists in the noradrenaline, adrenaline and 5-HT nerve cells of the rat brain

By means of two colour immunofluorescence procedures it has been possible to demonstrate in the rat brain the coexistence of TH and bFGF immunoreactivities (IRs) in the perikarya of large numbers of noradrenaline (NA) nerve cells of the locus coeruleus and of the NA cell groups A1, A5 and A7 and in many perikarya of the adrenaline (A) cell groups C1, C2 and C3. The coexistence of 5-hydroxytryptamine (5-HT) and bFGF IRs was demonstrated in the perikarya of large numbers of 5-HT nerve cells of practically all raphe nuclei. These results open up the possibility that bFGF may have a trophic role in the NA, A and 5-HT cell groups of the rat brain.     

Ontogeny of ( -Ala)-deltorphin I-like immunoreactive neurones in foetal rat brain

Foetal rat brain from embryonic day (ED) 12–22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of ( -Ala²)-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15–17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.     

Ontogeny of substance P-containing neurons in relation to serotonin-containing neurons in the central nervous system of the mouse

In order to begin to investigate the developmental factors influencing co-localized neurotransmitters in the central nervous system, we have studied the ontogenesis of coexisting transmitters, substance P and serotonin, in cells of the medullary raphe of the fetal mouse using double-label immunocytochemistry. The results indicate that initial detection of these neurotransmitters occurs at embryonic day 12 in non-overlapping cell populations. Substance P-like immunoreactivity is first co-localized with serotonin in cells of the caudal medullary raphe (raphe pallidus and raphe obscurus) at embryonic day 13. The percentage of serotonin cells containing substance P was estimated at embryonic day 13 and subsequent gestational ages both by double-label immunofluorescence using fluorescein- and rhodamine-conjugated secondary antibodies and also by the elution technique. Double-label immunofluorescence routinely and reliably yielded the highest proportion of co-localized cells. At embryonic day 13, 89% of serotonin cells contained substance P-like immunoreactivity. A consistently high degree of co-localization occurred throughout embryonic development (87% at postnatal day 3) and into adulthood (87%). Colchicine treatment was required at older ages to elicit these data. Unexpectedly, we found that neurons containing substance P-like immunoreactivity in the piriform cortex and in the hypothalamus transiently expressed serotonin immunoreactivity during normal ontogenesis.     

An immunohistochemical study of serotonin development in the opossum cerebellum

Summary In the present study we have used the indirect antibody peroxidase anti-peroxidase technique to analyze the development of serotonin in the cerebellum of pouch young opossums ranging in age from birth (postnatal day (PD) 1) — to PD 47. The pathways by which serotoninergic axons enter the cerebellum appear to change during development. Between PD 1 and PD 11 varicose serotoninergic fibers course though the continuity between the tectum and the dorsal medial aspect of the cerebellar plate and distribute primarily to the intermediate zone. By PD 11 serotoninergic fibers enter the cerebellum via a connecting band between the cerebellum and the dorsal lateral aspect of the rostral medulla. Fibers entering the cerebellum via this later route course around the lateral recess of the fourth ventricle and initially distribute to ventral and lateral areas of the cerebellum. At later developmental ages (PD 14–PD 47) serotoninergic fibers are present in: 1) the cellular zone of migration between the Purkinje cell layer and deep nuclei; 2) the Pukinje cell layer; 3) the internal granule cell layer; and 4) the deep cerebellar nuclei. The external granule cell and the molecular layers rarely contain serotoninergic fibers.The present study has shown that serotonin is present in the cerebellar anlage on PD 1 (within 13 days of conception). This is prior to the arrival of other major afferent systems (King et al. 1982; Bishop et al. 1983; Martin et al. 1983; Morgan et al. 1983). Further, serotoninergic axons reach the cerebellum via different routes at different stages of development. The presence of serotonin in the intermediate zone early in development supports the hypothesis that it may influence neuronal migration and differentiation (Lander and Krebs 1976). Axons containing serotonin and arriving later in development may function in synaptic transmission, a role proposed for this indoleamine in the adult (Strahlendorf et al. 1979).     

Ontogeny of the Vertebral Column of Eleutherodactylus johnstonei (Anura: Eleutherodactylidae) Reveals Heterochronies Relative to Metamorphic Frogs

Over the last century, the morphogenesis of the vertebral column has been considered as a highly conserved process among anurans. This statement is based on the study of few metamorphic taxa, ignoring the role of developmental mechanisms underlying the evolution of specialized life‐histories. Direct development in anurans has been regarded as evolutionarily derived and involves developmental recapitulation and repatterning at different levels in all amphibian taxa studied so far. Herein, we analyze the vertebral column morphogenesis of the direct‐developing frog Eleutherodactylus johnstonei, describing the sequence of chondrification and ossification, based on cleared and double‐stained specimens from early stage embryos to adults. In general, our results show that the morphogenesis of the vertebral column in E. johnstonei recapitulates the ancestral tadpole‐like pattern of development. However, the analysis of the sequence of events using heterochrony plots shows important heterocronies relative to metamorphic species, such as a delay in the chondrification of the vertebral centra and in osteogenesis. These ontogenetic peculiarities may represent derived traits in direct‐developing frogs and are possibly correlated with its unusual life history. In addition, several features of the vertebral column of E. johnstonei are highly variable from its typical morphology. We report some malformations and small deviations, which do not seem to affect the survival of individuals. These anomalies have also been found in other frogs, and include many vertebral defects, such as vertebral fusion, and vertebral preclusion and/or induction. Anat Rec, 296:00000–#2013, 2013. © 2013 Wiley Periodicals, Inc.     

Distribution,morphology, and central projections of mesencephalic trigeminal neurons in the frog Rana ridibunda

The distribution, morphology, and central projections of the mesencephalic trigeminal neurons in the frog Rana ridibunda were studied with tracing techniques. Retrograde tracing with horseradish peroxidase (HRP) or the fluorescent tracer Fluorogold, and anterograde tracing by means of Phaseolus vulgaris leucoagglutinin, the fluorescent dye Dil, and HRP were used. The mesencephalic trigeminal nucleus (MesV) of Rana ridibunda is formed by a population of 100 to 125 unipolar or multipolar cells that are scattered on both sides of the rostral mesencephalic tectum. Subpopulation of Mes V cells were labeled after tracer application to ophthalmic, maxillary, and mandibular trigeminal branches, separately. Differences in the morphology and distribution of cells in these experiments were not evident but the number of neurons labeled via the maxillary nerve was always the highest. Mes V cells have a single central branch that courses caudally in the brainstem. At different levels, it bifurcates into a peripheral branch, which leaves the brain via the trigeminal root, and a descending branch, which terminates in a region in, or close to, the trigeminal motor nucleus and in a supratrigeminal location. The lack of a distinct somatotopy in the distribution of Mes V cells and the lack of projections caudal to the trigeminal motor nucleus as revealed in this study with a wide variety of tracers are in striking contrast to previous data provided for other amphibians. © 1993 Wiley-Liss, Inc.