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

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

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: Possible involvement in reproduction

Peptides of the gonadotropin-releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH-like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH-like peptide plays a role in regulating reproduction in freshwater snails. J. Comp. Neurol. 404:427–437, 1999. © 1999 Wiley-Liss, Inc.     

Peptidergic neurons in the snail Helix pomatia: Distribution of neurons in the central and peripheral nervous systems that react with an Antibody raised to the insect neuropeptide,leucokinin I

In this study, antiserum raised against an insect myotropic peptide, leucokinin I (DPAFNSWGamide), was: used for mapping leucokinin-like immunoreactive (LK-LI) neurons in the gastropod mollusc, Helix pomatia. Immunocytochemistry performed on both whole-mounts and cryostat sections demonstrated LK-LI neurons in all ganglia of the central nervous system (CNS), except the visceral ganglion. Altogether about 700 immunolabelled neurons have been found, with nearly one-half (46%) in the cerebral ganglia. A large proportion of the LK-LI neurons have small cell bodies and are likely to be interneurons. The most prominent LK-LI cell group is represented by the entire neuron population of the mesocerebri, which is the major source of a thick fiber bundle system, encircling and innervating the whole CNS. One single LK-LI giant neuron was found, which is located in the left pedal ganglion and is termed GLPdLKC (giant left pedal leucokinin immunoreactive cell). This cell has not been identified previously. The ganglion neuropils are heavily innervated by varicose LK-LI fiber arborizations. Some integrative centers, such as the medullary neuropil of the procerebri, reveal an extreme density of LK-LI innervation. All major peripheral nerves contain a large number of LK-LI axons, and LK-LI innervation is found in the musculature of different peripheral organs (buccal mass, lip, tentacles, oviduct, intestine). Among the peripheral organs investigated, the intestine contains a rich varicose LK-LI network, composed of both intrinsic and extrinsic elements. Radioimmunoassay (RIA) demonstrates a very high content of LK-LI material in Helix ganglion extracts (about 50 pmol/CNS). This is the first report on the occurrence of a substance resembling the myotropic neuropeptide leucokinin I in a phylum outside arthropods. Based on our immunocytochemical observations, a role for leucokinin-like peptides in both central and peripheral regulatory processes in Helix is suggested. According to double-labelling experiments, only a small number of the LK-LI neurons are labelled with an antibody to the vertebrate tachykinin substance P.     

Distribution of a Y1 receptor mRNA in the brain of two lamprey species,the sea lamprey (Petromyzon marinus) and the river lamprey (Lampetra fluviatilis)

The neuropeptide Y system consists of several neuropeptides acting through a broad number of receptor subtypes, the NPY family of receptors. NPY receptors are divided into three subfamilies (Y1, Y2, and Y5) that display a complex evolutionary history due to local and large‐scale gene duplication events and gene losses. Lampreys emerged from a basal branch of the tree of vertebrates and they are in a key position to shed light on the evolutionary history of the NPY system. One member of the Y1 subfamily has been reported in agnathans, but the phylogenetic tree of the Y1 subfamily is not yet clear. We cloned the sequences of the Y1‐subtype receptor of Petromyzon marinus and Lampetra fluviatilis to study the expression pattern of this receptor in lampreys by in situ hybridization and to analyze the phylogeny of the Y1‐subfamily receptors in vertebrates. The phylogenetic study showed that the Y1 receptor of lampreys is basal to the Y1/6 branch of the Y1‐subfamily receptors. In situ hybridization showed that the Y1 receptor is widely expressed throughout the brain of lampreys, with some regions showing numerous positive neurons, as well as the presence of numerous cerebrospinal fluid–contacting cells in the spinal cord. This broad distribution of the lamprey Y1 receptor is more similar to that found in other vertebrates for the Y1 receptor than that of the other members of the Y1 subfamily: Y4, Y8, and Y6 receptors. Both phylogenetic relationship and expression pattern suggest that this receptor is a Y1 receptor. J. Comp. Neurol. 521:426–447, 2013. © 2012 Wiley Periodicals, Inc.     

Type 1 vomeronasal receptors expressed in the olfactory organs of two African lungfish,Protopterus annectens and Protopterus amphibius

Lungfish are the fish related most closely to tetrapods. The olfactory organ of lungfish contains two distinct sensory epithelia: the lamellar olfactory epithelium (OE) and the recess epithelium (RecE). Based on their ultrastructural and histological characteristics, the lamellar OE and the RecE are considered to correspond respectively to the teleost OE and a primitive vomeronasal organ (VNO). In tetrapods, the OE and VNO have been shown to express different families of olfactory receptors; for example, in mammals, the OE expresses odorant receptors and trace amine-associated receptors, while the VNO expresses type 1 (V1Rs) and type 2 (V2Rs) vomeronasal receptors. In the present study, we examined the expression of V1Rs in the olfactory organs of two African lungfish, Protopterus annectens and Protopterus amphibius. RNA sequencing and phylogenetic analyses identified 29 V1R genes in P. annectens and 50 V1R genes in P. amphibius. Most V1Rs identified in these lungfish were classified as the tetrapod-type V1Rs initially found in tetrapods and distinct from fish-type V1Rs. In teleost, which all lack a VNO, all olfactory receptors are expressed in the OE, while in Xenopus V1Rs are expressed exclusively in the OE, and not in the VNO. In situ hybridization analysis indicated that lungfish V1Rs were expressed mainly in the lamellar OE and rarely in the RecE. These results imply that V1R expression in lungfish represents an intermediate step toward the complete segregation of V1R expression between the OE and VNO, reflecting the phylogenetic position of lungfish between teleosts and amphibians.     

Brain-derived neurotrophic factor is expressed in a gradient in the superior colliculus during development of the retinocollicular projection

Abstract Theoretical models of topographic map formation have postulated a gradient of attractant in addition to a gradient of repulsion in the target. In species where many axons grow past their correct positions initially, it has also been argued that a parallel gradient of attractant or branching signal is required to ensure collateral formation at the correct position (O'Leary et al., 1999). Brain-derived neurotrophic factor (BDNF) is a known attractant and promotes branching of retinal axons. We have examined its distribution in the superior colliculus and that of its receptor, trkB, in the retina, using immunohistochemistry and in situ hybridization, respectively, during the development of the topographic retinocollicular projection in the wallaby, a marsupial mammal. The number of glial endfeet expressing BDNF at the surface of the colliculus was found to be in a high caudal-to-low rostral gradient during the time when the retinocollicular projection was developing. When the projection was mature the rostrocaudal gradient had disappeared and the number of detectable endfeet expressing BDNF was very low. Messenger RNA for TrkB was expressed in the retinal ganglion cell layer throughout the time when the retinocollicular projection was developing, with no difference in expression across the nasotemporal axis of the retina. The low rostral to high caudal distribution of BDNF in glial endfeet supports the idea that it is providing a parallel gradient of attractant or branching signal in the colliculus.     

Molecular forms and solubility of acetylcholinesterase during the embryonic development of rat and human brain

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) form homologous sets of multiple molecular forms. The central nervous system of mammals contains mostly tetramers (G4) and monomers (G1). Their proportions have been shown to vary during maturation in rat brain. In order to examine whether a similar evolution occurs in the human, we performed parallel studies of the activity, solubility and molecular forms of acetylcholinesterase in rat and human brains at various stages. We find both similarities and differences: in rat brain, the enzyme increases mostly postnatally but in human brain acetylcholinesterase reaches a maximum at birth. There is an increase in the proportion of G4 and a decrease in the solubility of this from in the absence of detergent in human as well as in rat brain. These changes occur around birth in rat, but during early pregnancy, before 11 weeks in human brain. In both species, the solubility of the enzyme in detergent-free buffers decreases progressively from more than 50% before birth to about 10-20% in the adult. In addition we analyzed butyrylcholinesterase as well as the levels of the neuron-specific enolase and of the glial S-100 protein. In human, gamma gamma-enolase rises to its adult level after birth, but before the S-100 protein.     

Comparative analysis of dopamine and tyrosine hydroxylase immunoreactivities in the brain of two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii.

To gain more insight into the dopaminergic system of amphibians and the evolution of catecholaminergic systems in vertebrates in general, the distribution of dopamine and tyrosine hydroxylase immunoreactivity was studied in the brains of the anuran Rana ridibunda and the urodele Pleurodeles waltlii. In both species, dopamine-immunoreactive (DAi) cell bodies were observed in the olfactory bulb, the preoptic area, the suprachiasmatic nucleus, the nucleus of the periventricular organ and its accompanying cells, the nucleus of the posterior tubercle, the pretectal area, the midbrain tegmentum, around the solitary tract, in the ependymal and subependymal layers along the midline of the caudal rhombencephalon, and ventral to the central canal of the spinal cord. Tyrosine hydroxylase (TH) immunohistochemistry revealed a similar pattern, although some differences were noted. For example, with the TH antibodies, additional cell bodies were stained in the internal granular layer of the olfactory bulb and in the isthmal region, whereas the same antibodies failed to stain the liquor contacting cells in the nucleus of the periventricular organ. Both antisera revealed an almost identical distribution of fibers in the two amphibian species. Remarkable differences were observed in the forebrain. Whereas the nucleus accumbens in Rana contains the densest DAi plexus, in Pleurodeles the dopaminergic innervation of the striatum prevails. Moreover, cortical structures of the newt contain numerous DAi fibers, whereas the corresponding structures in the frog are devoid of immunoreactivity. The dopaminergic system in amphibians appears to share many features not only with other anamniotes but also with amniotes.     

Molecular characterization and comparative localization of the mRNAs encoding two glutamic acid decarboxylases (GAD65 and GAD67) in the brain of the African lungfish, Protopterus annectens

The existence of two distinct genes encoding two isoforms of glutamic acid decarboxylase (GAD65 and GAD67) has been demonstrated in most vertebrate classes, yet little is known about their differential distributions and functions in the central nervous system in nonmammalian vertebrates. In the present study, we have partially sequenced the cDNAs encoding GAD65 and GAD67 in the lungfish Protopterus annectens and determined their relative distributions in the adult brain by in situ hybridization histochemistry. The expression patterns of the GAD65 and GAD67 mRNAs were globally similar; the highest expression levels being observed in the granular layer of the olfactory bulb, the pallium, the subpallium, the anterior preoptic area, the thalamus, the hindbrain central gray, and the rhombencephalic visceral areas. However, striking differential expression was noticed in several structures. Very high to high concentrations of GAD67 mRNA were seen in the dorsal and ventral aspects of the anterior olfactory nucleus, which is in marked contrast to the very low expression of GAD65 in this region. Similarly, high levels of GAD67 mRNA were observed in the intermediate and ventral parts of the medial pallium that were virtually devoid of GAD65 mRNA. In contrast, GAD65 mRNA was found in the periaqueductal gray that did not express GAD67 mRNA. The differential expression of GAD65 and GAD67 mRNAs in these regions of the lungfish CNS indicates that the two GAD isoforms can be differentially regulated and that they may have distinct physiological roles.     

A comparative analysis of the development of the primary somatosensory cortex: interspecies similarities during barrel and laminar development

The development of the barrels and layers II-V was examined in Nissl-stained preparations of the primary somatosensory cortex in six species--hamster, mouse, rat, gerbil, rabbit, and guinea pig--that have increasingly longer gestation periods. The barrels and layers II-V begin to differentiate postnatally during the first week postpartum in the hamster, mouse, rat and gerbil; perinatally in the rabbit; and approximately 4 weeks prenatally in the guinea pig. The structure of the barrels and layers II-V is similar at the onset of their differentiation in each species, even though there are interspecies differences in the mature structure of the barrels and layer V. The rate of the initial differentiation of the barrels and layers II-V is also similar in each species, even though there are considerable interspecies differences in the duration of the preceding period of development. In each species, layer V begins to differentiate first from the cortical plate and, within 1 or 2 days, contains sublayers that eventually disappear in the rabbit and guinea pig. About 3 days after the initial differentiation of layer V, layers II-IV begin to differentiate, seemingly simultaneously, causing the cortical plate to have a trilaminar appearance. Barrels are first evident just before the appearance of the trilaminar plate in hamsters; concomitant with the trilaminar plate in mice, rats, and guinea pigs; and just after the trilaminar plate in gerbils and rabbits. Septa appear 1 or 2 days after the barrels except in rabbits, which never have septa. Barrel maturation proceeds rapidly after the initial appearance in all species except the hamster, in which continued maturation seems to be delayed until the appearance of the trilaminar plate. The barrels in immature rats and rabbits become more prominent than they will eventually be in the adults. Our results indicate a close and rapid developmental affiliation between layers II-V, especially layers II-IV, that seems quite separate from the development of layers I and VI. However, barrel development and differentiation of layers II-IV seem to be closely, but independently initiated. Secondary remodeling occurs in layer V and the barrels of some species.     

Ultrastructural analysis of glutamate-immunopositive synapses onto the rat jaw-closing motoneurons during postnatal development

The excitatory synapses on the jaw-closing (JC) motoneurons mediate the neuronal input that ensures smooth and rhythmic movements of the jaw. Recently, we have shown that the neurotransmitter phenotype of the inhibitory boutons onto JC motoneurons shifts from GABA to glycine, and new inhibitory synapses onto JC motoneurons are continuously formed during postnatal development (Paik et al. [2007] J. Comp. Neurol. 503:779–789). To test whether the developmental pattern of the excitatory synapses onto JC motoneurons differs from that of the inhibitory synapses, we studied the distribution of glutamate-immunopositive boutons onto the rat JC motoneurons during postnatal development by using a combination of retrograde labeling with horseradish peroxidase (HRP), postembedding immunogold staining, and quantitative ultrastructural analysis. The analysis of 175, 281, and 465 boutons contacting somata of JC motoneurons at postnatal days P2, P11, and P31, respectively, revealed that the number of glutamate-immunopositive (Glut(+)) boutons increased by 2.6 times from P2 to P11 and showed no significant change after that, whereas the length of apposition of these boutons increased continuously from P2 to P31, suggesting that the time course for the development of Glut(+) boutons differed from that for Glut(-) boutons, most of which were immunopositive for GABA and/or glycine. Our findings indicate that excitatory and inhibitory synapses onto JC motoneurons exhibit distinctly different developmental patterns that may be closely related to the maturation of the masticatory system.     

Comparative analysis of the vasotocinergic and mesotocinergic cells and fibers in the brain of two amphibians, the anuran Rana ridibunda and the urodele Pleurodeles waltlii.

To obtain more insight into the vasotocinergic and mesotocinergic systems of amphibians and the evolution of these neuropeptidergic systems in vertebrates in general, the distribution of vasotocin (AVT) and mesotocin (MST) was studied immunohistochemically in the brains of the anuran Rana ridibunda and the urodele Pleurodeles waltlii. In Rana, AVT-immunoreactive cell bodies are located in the nucleus accumbens, the dorsal striatum, the lateral and medial part of the amygdala, an area adjacent to the anterior commissure, the magnocellular preoptic nucleus, the hypothalamus, the mesencephalic tegmentum, and in an area adjacent to the solitary tract. In Pleurodeles, AVT-immunoreactive somata are confined to the medial amygdala, the preoptic area, and an area lateral to the presumed locus coeruleus. In both species, the distribution of MST-immunoreactive cell bodies is more restricted: in the frog, MST-immunoreactive somata are present in the medial amygdala and the preoptic area, whereas, in the urodele, cell bodies are found only in the preoptic area. Both in Rana and Pleurodeles, AVT- and MST-immunoreactive fibers are distributed throughout the brain and spinal cord. A major difference is that in Rana the number of MST-immunoreactive fibers is evidently higher than that of AVT-immunoreactive fibers, whereas the opposite is found in Pleurodeles. This holds, in particular, for the forebrain and the brainstem. The presence of several extrahypothalamic AVT-immunoreactive cell groups and the existence of well-developed extrahypothalamic networks of AVT- and MST-immunoreactive fibers are features that amphibians share with amniotes. However, this study has revealed that major differences exist not only between species of different classes of vertebrates, but also within a single class. In order to determine whether features of these neuropeptidergic systems are primitive or derived, a broad selection of species of each class of vertebrates is needed.     

Amphibian thalamic nuclear organization during larval development and in the adult frog Xenopus laevis: Genoarchitecture and hodological analysis

The early patterning of the thalamus during embryonic development defines rostral and caudal progenitor domains, which are conserved from fishes to mammals. However, the subsequent developmental mechanisms that lead to the adult thalamic configuration have only been investigated for mammals and other amniotes. In this study, we have analyzed in the anuran amphibian Xenopus laevis (an anamniote vertebrate), through larval and postmetamorphic development, the progressive regional expression of specific markers for the rostral (GABA, GAD67, Lhx1, and Nkx2.2) and caudal (Gbx2, VGlut2, Lhx2, Lhx9, and Sox2) domains. In addition, the regional distributions at different developmental stages of other markers such as calcium binding proteins and neuropeptides, helped the identification of thalamic nuclei. It was observed that the two embryonic domains were progressively specified and compartmentalized during premetamorphosis, and cell subpopulations characterized by particular gene expression combinations were located in periventricular, intermediate and superficial strata. During prometamorphosis, three dorsoventral tiers formed from the caudal domain and most pronuclei were defined, which were modified into the definitive nuclear configuration through the metamorphic climax. Mixed cell populations originated from the rostral and caudal domains constitute most of the final nuclei and allowed us to propose additional subdivisions in the adult thalamus, whose main afferent and efferent connections were assessed by tracing techniques under in vitro conditions. This study corroborates shared features of early gene expression patterns in the thalamus between Xenopus and mouse, however, the dynamic changes in gene expression observed at later stages in the amphibian support mechanisms different from those of mammals.     

Pattern of calbindin‐D28k and calretinin immunoreactivity in the brain of Xenopus laevis during embryonic and larval development

The present study represents a detailed spatiotemporal analysis of the localization of calbindin‐D28k (CB) and calretinin (CR) immunoreactive structures in the brain of Xenopus laevis throughout development, conducted with the aim to correlate the onset of the immunoreactivity with the development of compartmentalization of distinct subdivisions recently identified in the brain of adult amphibians and primarily highlighted when analyzed within a segmental paradigm. CR and CB are expressed early in the brain and showed a progressively increasing expression throughout development, although transient expression in some neuronal subpopulations was also noted. Common and distinct characteristics in Xenopus, as compared with reported features during development in the brain of mammals, were observed. The development of specific regions in the forebrain such as the olfactory bulbs, the components of the basal ganglia and the amygdaloid complex, the alar and basal hypothalamic regions, and the distinct diencephalic neuromeres could be analyzed on the basis of the distinct expression of CB and CR in subregions. Similarly, the compartments of the mesencephalon and the main rhombencephalic regions, including the cerebellum, were differently highlighted by their specific content in CB and CR throughout development. Our results show the usefulness of the analysis of the distribution of these proteins as a tool in neuroanatomy to interpret developmental aspects of many brain regions. J. Comp. Neurol. 521:79–108, 2013. © 2012 Wiley Periodicals, Inc.     

Claudin k is specifically expressed in cells that form myelin during development of the nervous system and regeneration of the optic nerve in adult zebrafish

The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.