Relationships between neuronal cell adhesion molecule and LHRH neurons in the urodele brain: a developmental immunohistochemical study.

Polysialic acid (PSA), a homopolymer attached to neural cell adhesion molecule (NCAM) is considered a major hallmark of vertebrate cell migration. We studied the distribution of PSA-NCAM by immunohistochemistry, during brain development, in two urodele amphibians, Pleurodeles waltl and the neotenic newt Ambystoma mexicanum. In both species a gradual increase of immunolabelling was observed throughout the brain from developmental stage 30 to stage 52. At the onset of metamorphosis, some differences became evident: in Pleurodeles immunostaining was gradually restricted to the olfactory system while in Ambystoma, PSA-NCAM maintained a more extended distribution (for example throughout the telencephalic walls) suggesting, for the brain of this latter species, a rather preserved neuronal plasticity. The aim of the present work was to correlate the above described PSA-NCAM-immunoreactivity (IR) with the distribution of luteinizing hormone-releasing hormone (LH-RH) containing neurons, which represent a well known example of neural elements migrating from the olfactory placode. LHRH-IR, undetectable till stage 30, was later found together with PSA-NCAM-IR in both the olfactory system and septo-hypothalamic areas. Such observations further support a role of PSA in providing a migration route toward the establishment of a part, at least, of the urodele LHRH system. The possible functional meaning of the LHRH-containing neurons localized between dorsal and ventral thalamus of Ambystoma, never reported before in this area, almost devoid of PSA-NCAM-IR, is discussed.

Immunolocalization of aromatic L-amino acid decarboxylase, tyrosine hydroxylase, dopamine, and serotonin in the forebrain of Ambystoma mexicanum.

To improve basic knowledge about the neurochemical organization of the urodele brain, and to study discrepancies in the localization of monoaminergic markers, we immunohistochemically charted the distribution of four such markers (tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine, and serotonin) in the axolotl (Ambystoma mexicanum) forebrain. Catecholaminergic and serotoninergic systems were found in similar locations to those seen in other Urodela. As seen in other vertebrates, the localization of the different monoaminergic markers reveals some inconsistencies. Cells that are exclusively tyrosine hydroxylase-immunoreactive are observed in the olfactory bulb, anterior olfactory nucleus/nucleus accumbens region, the epichiasmatic portion of the preoptic nucleus, and in the pars intercalaris thalami, whereas cells that are only labelled by aromatic L-amino acid decarboxylase are seen in the anterior olfactory nucleus/nucleus accumbens region, the bed nuclei of the anterior commissure, the posterior portion of the preoptic nucleus, the ventral hypothalamus, and the pars intercalaris thalami. The presence of cells solely serotonin (5-HT)-immunoreactive is suggested for the nucleus infundibularis dorsalis. Conversely, there were no areas that appeared to be exclusively immunoreactive for dopamine. Double-labelling for aromatic L-amino acid decarboxylase/tyrosine hydroxylase and aromatic L-amino acid decarboxylase/serotonin, together with cell counting, confirmed the existence of neurons that express only one monoaminergic marker in amphibian, supporting the hypothesis that these cells are universally present in the central nervous system of vertebrates.

Larval development of tectal efferents and afferents in Xenopus laevis (Amphibia Anura).

The development of tectal connections in Xenopus laevis had been investigated using the degeneration technique to demonstrate the efferent pathways and the retrograde HRP transport to label the afferent pathways. Bilateral tectal efferents were present as soon as the beginning of metamorphosis. Ascending efferents originated from the anterior tectal part terminate in the secondary visual thalamic centres whereas the descending efferents coming from the posterior tectal part reached the tegmentum and the medulla oblongata. At this same time, the optic tectum already received secondary visual afferents originating in the ipsilateral pretectum and non-visual afferents from the ipsilateral semicircular torus and tegmentum. Some sparse bilateral isthmotectal connections were also present. Later, efferent pathways showed an increasing number of fibres whereas the sites of origin of afferents became more diversified: the dorsal thalamus, the suprachiasmatic area, the tegmental nuclei and in the medulla oblongata, the reticular and octavolateral areas sent bilateral projections to the optic tectum. At the end of metamorphosis, we noted ipsilateral olivotectal fibres and reciprocal connections between the tectum and the area of the Vth nerve. These last findings and the presence of the following direct projections, not previously reported in Anurans: the reciprocal connections between the tectum and the semicircular torus or the octavolateral area, underline the implication of the optic tectum in the multisensory (visual, acoustic, vibratory) integration elicited during the larval behavior. Also, the relations between the optic tectum and the lateral line system are particularly examined in the discussion.

Organization of the serotoninergic system in the brain of two amphibian species, Ambystoma mexicanum (Urodela) and Typhlonectes compressicauda (Gymnophiona).

An immunocytochemical investigation was made of the distribution of serotonin (5-HT) in the brain of larval and adult Ambystoma mexicanum and adult Typhlonectes compressicauda. Immunoreactive perikarya can be identified in the caudal diencephalon (paraventricular organ and infundibular nucleus), in the ventral mesencephalon (interpeduncular nucleus) and in the raphe of the rhombencephalon. Immunopositive fibers and terminal arborizations are widely distributed, extending from the whole telencephalon to the spinal lemniscus area. However, the retinorecipient structures of the thalamus and mesencephalon are either very weakly innervated (Ambystoma) or completely immunonegative (Typhlonectes). The habenular system also exhibits very few 5-HT-positive structures. The major serotoninergic neuron clusters, in both Urodela and Gymnophiona, tend to gather, from the paraventricular organ to the raphe, on both sides of the sagittal plane, showing no tendency to "lateralization". A new interpretation of the limited development of the serotoninergic system in amphibians is given.

The connections of the anterior pallium in Pleurodeles waltl and Triturus carnifex: an HRP study.

In order to provide cues about the evolution of the telencephalon in tetrapods, the connections of the anterior pallium were studied in two adult Urodeles, Pleurodeles waltl and Triturus carnifex, by means of the HRP-tracing method. The staining of HRP-immunopositive cell bodies indicates that the pallial regions studied receive afferent projections from the main olfactory bulb and are reciprocally interconnected by intrapallial associative fiber systems. In the ventral hemispheric wall, HRP-immunoreactive perikarya are observed in the pars medialis of the amygdala and in the rostral and caudal striatum. Triturus exhibits a more complex pattern of pallial afferents, including interhemispheric connections and thalamic ascending projections that were not discovered in Pleurodeles. HRP-immunopositive fibers are observed in the dorsal and medial walls of the telencephalon, from the rostral part to the foraminal level. In Triturus, the dorsal fibers extend to the caudal part of the hemisphere. Another group of labelled fibers extends, throughout the lateral and ventral walls, to the most caudal part of the telencephalon, and, through the stria medullaris and the habenular commissure, crosses over to the controlateral hemisphere. These results allow us to specify the basic pattern of the pallial connections in Urodeles and to compare them with data previously obtained in other Amphibians.

Striatal afferents in the newt Triturus cristatus.

In order to investigate the neuronal populations projecting to the corpus striatum in the brain of a urodele, Triturus cristatus, horseradish peroxidase (HRP) retrograde labeling was used in parallel with anterograde degeneration, glyoxylic acid histofluorescence and behavioral testing. Striatal injections of HRP revealed that the main striatal afferent systems originate within the diencephalon, specifically in the dorsal thalamus and paraventricular organ of the hypothalamus. Several small groups of neurons in other diencephalic areas also participate in striatal innervation: proeminentia ventralis, amygdala, contralateral corpus striatum, preoptic area, posterior tuberal nucleus, locus coeruleus and raphe nuclei. Degeneration experiments after mechanical lesion of the paraventricular organ established the existence of a hypothalamostriatal projection. Degenerating axonal profiles were also found in many of the structures already identified as projecting to the striatum, suggesting that the paraventricular organ might influence the striatum not only directly but also indirectly through these other afferent systems. In the paraventricular organ, glyoxylic acid fluorescence histochemistry showed numerous monoamine neurons that corresponded in distribution and morphology to the retrogradely HRP-labeled neurons. Paraventricular-organ-lesioned males displayed a severe impairment of courtship behavior in the form of decreased tail beating and head stepping by the females. This suggests that the regulation of stereotyped hypermotricity might involve the monoamine component of the hypothalamo-striatal projection.

Habenular connections in the brain of the newt, Triturus cristatus carnifex Laurenti.

The connections of the habenular complex have been studied in the crested newt (Amphibia Urodela) by means of degeneration and HRP transport techniques. With the Fink-Heimer method, habenular efferents have been traced to the basal telencephalon, the pallium, the stria medullaris, the dorsal and ventral thalamus, the preoptic and anterior hypothalamus, the fasciculus retroflexus, the tegmentum and the interpeduncular neuropil. Anterograde transport of HRP by habenular neurons reveals fibers projections to the thalamus, the fasciculus retroflexus and the interpeduncular neuropil. After HRP injections in the habenulae, retrograde labelling of cells and fibres was observed, in the striatum, the posterior pole of the telencephalon, the thalamus, the preoptic area, the tegmentum and the raphe. The present results indicate that the habenular complex of the newt receives inputs of various sources (striatum, thalamus, hypothalamus, tegmentum) and is less directly involved in the olfactory functions.

[Anatomy and development of the visual system of Pleurodeles poiretti]. / Anatomie et developpement du système visuel de Pleurodèles poiretti.

After a deposit of HRP crystals in one eye of larval and adult Pleurodeles and the PAP immunocytochemical procedure, the organization of the visual system was described at stages 36, 45; 52 and postmetamorphic (according to Gallien and Durocher, 1975) and in the adult. At hatching stage, the retinopreoptic and the accessory pathways differentiate, soon after the retino-thalamico-pretectal pathway appears. The tectum differentiates later and a true retinotectal bundle can be seen only after the end of metamorphosis. In the whole, the pattern of the normal system is very simple in Pleurodeles especially as regard the ipsilateral fibres which never reach the tectum. This rudimentary organization lead us to conclude that the Pleurodeles and the Protopterus visual patterns are very similar.

[Retinotopic projections of the trout (Salmo gairdneri)]. / Les projections rétinotopiques de la truite (Salmo gairdneri).

The retinotopic organization of the thalamic and tectal and visual centres was studied by Fink and Heimer method and radioautography. Quadratic lesions were achieved in the right retina of 21 adult trouts and 23 six month old specimens. Fourteen animals (adults) were kept alive for 20 to 25 days after operation, in running water at 13 degrees C and prepared for the Fink technique. In the remaining 7 adults and in the 23 six month specimens, we injected, 48 hours after the lesion, respectively, 2,5 microCi (in 20 microliters) and 0,5 microCi (in 1 microliter) of 14 C proline into the operated eye. The brains were prepared for radioautography. Results show that the thalamic pathways is not correlated with a peculiar quadrant. Nevertheless the temporo dorsal hemiretina sends the more numerous projections at this level. A topographic parallelism exists between retino-geniculate and retino-tectal projections. At the tectal level our data are somewhat at variance with those of electrophysiological mapping, since quadratic projections show a partial overlapping. In the optic tract, the fibres from every quadrant follow always the same pathway.

The visual system of the trout Salmo irideus Gibb. A degeneration and radioautographic study.

The organization of the primary visual system of the trout (Salmo irideus Gibb.) was studied by Fink-Heimer methods and radioautography. In addition to normal fiber, Nissl and Golgi staining techniques, we carried out total ablation of the retina on the right side on 16 animals which survived post-operatively at 13 degrees C for 9 to 31 days. The brains were cut at 35 micrometer and then stained by the Fink-Heimer techniques. Furthermore, we injected 20 microliter (2 microCi) of 14C proline into the right eye of 8 trouts which were then fixed between 24 and 72 hours after the injection, and then prepared for radioautography. The primary visual system projections are entirely contralateral. The optic tract is divided into three bundles which pass into tectal layers 2, 4 and 6. In the trout, a very obvious deep optic layer is present which extends throughout the length of the medial part of the tectum. In addition to the accessory optic tract and to a projection at the level of the preoptic area, the trout possesses a well-developed thalamic visual system composed of a ribboned geniculate nucleus and two medial thalamic visual centres fed by the retinothalamic pathways as well as a small retinal target located in the lateral thalamus. The pretectum receives only a few small sparse retinal projections. An interpretation of the trout visual system as the whole is discussed.

[Effects of ablation of the hindlimb on the organization of the ventral horn of the spinal cord in the lumbar region of green lizard embryos (Lacerta viridis Laur.)]. / Effets de l'ablation des membres postérieurs sur l'organisation des cornes ventrales de la moelle épinière, dans la région lombaire, chez les embryons de lézard vert (Lacerta viridis Laur.).

After extirpation of an hind limb in embryos of Lacerta viridis, numerous motor neuroblasts degenerate on the operated side, in the ventral horn of the lumbar spinal cord and the corresponding motor column is reduced or disappears. The lumbar spinal ganglia are affected and reduced on the operated side.

[Organization of the ventral branchial and lumbar horns of the spinal cord in embryos of snake-like and tetrapod reptiles]. / Organisation des cornes ventrales de la moelle épinière, dans les régions brachiale et lombiare, chez les embryons de Reptiles serpentiformes et de Reptiles à membres bien développés.

In embryos of Snake-like Reptiles (Anguis fragilis) and of Ophidians (Tropidonotus, Python), the lay-out of motor neuroblast in the ventral horns of the spinal cord, in the brachial and the lumbar regions differs from the one observed in the same regions of embryos of tetrapod Reptiles (Lacerta viridis).

[Architecture of the visual system of Discoglossus pictus (Oth)]. / Architecture du systéme visuel de Discoglossus pictus (Oth).

The retinofugal pathways of an "archaic" Anura Discogossus pictus were examinated with Fink-Heimer method following ablation of the retina and after survival times of 5--105 days at 14 degrees C. A description of the diencephalic and mensencephalic architectonics has been done in order to replace the visual system into the general pattern. The scheme of primary optic pathways and centers is similar in Discoglossus and Rana. However, some retinofugal fibers reach the posterolateral part of the ipsilateral tectum opticum. Beside this study, a Golgi study has shown the link which exists between the degeneration territories (neuropils) and the periventricular cell-masses by the way of the dendritic trees of multipolar, bipolar and pear-shaped neurons. An hypothesis about the notion of visual center is formulated. In the tectum, the distribution of degenerations allows to understand the existence of two visual behaviors: avoidance and prey-catching. The signification of the ipsilateral retinotectal pathway is discussed.

[A new visual pathway in an anour amphibian, Discoglossus pictus]. / Une nouvelle voiè visuelle chez un amphibien anoure, discoglossus pictus

The retino-tectai projections of the Anura Discoglossus pictus were examined on seventeen adult specimens unilaterally enucleated. After a postoperative survival period of 9 to 105 days, the degenerating fibers and terminals were shown by Nauta and electromicroscopic techniques. This work points out the existence of a direct ipsilateral retino-tectal pathway in Anura. Typical terminal degenerations were seen in the postero-medial part of the ipsilateral tectum. The implications of this new anatomical pathway in binocular vision is discussed.

[Architecture of the pathways and primary optical centers in trout (Salmo irideus Gibb.)]. / Sur l'architecture des voies et centres optiques primaires de la Truite (Salmo irideus Gibb.)

The retinal projections of the Trout were examined on fifteen adult specimens (eighteen months) unilaterally enucleated. After a post-operative survival period of six to 31 days, the degenerating fibers were stained with Fink-Heimer technique. The optic tract is entirely crossed. At the rostral end of the thalamus a first fascicle diverges which runs to aventral thalamic center and to a dorsal thalamic center. The latter extends caudally as a pretectal center. The second fascicle is a retinogeniculo-tectal pathway which borders the geniculate body and enters the external tectal layer. The corpus geniculatum is large, well laminated and contains terminal degeneration. The main part of the optic tract includes three fascicles. Each of them sends fibers to all three tectal layers.

[Primary visual centers in Protopterus dolloi Boulenger]. / Les centers visuels primaries de Protopterus dolloi Boulenger

Modifications of the Nauta's method (Fink-Heimer, Ebbesson-Heimer) were used to study the optic centers and pathways of rotoperus dolloi following unilateral enucleation. After ablation, animals were kept at 25degrees C from 6 to 51 days. We saw optimum degenerescences from 10 to 13 days of postoperatory life. Retinofugal fibers are completely crossed. The projections to necleus preopticus and hypothalamus seem to be bi-lateral. The mean primary optic centers are: the thalamic neuropil which is may be homologous with the Bellonci's neuropil in Amphilbia; the pretectal and tectal neuropils; the neuropil of the basal optic root. Results obtained in Protopterus can be compared with our knowledges in Amphibia.

[Development of the telencephalon of Salmo irideus Gib]. / Développement du télencéphale de Salmo irideus Gib

A developmental study of the Telencephalon of the trout (Salmo irideus) has been done. The stages of fixation were 18 days after fecondation, hatching; 5 days, 1, 2, and 3 months, and 1 year after hatching. The different cell-masses are summarized in table 1. In the young trout, eversion is not important. Just, 2 olfactory bulbs are evaginated. In front of the commissura anterior, we can see: the Nucleus ventroventralis and the Nucleus ventrodorsalis, on the one hand; a voluminous dorsal area which includes: the Nucleus dorsolateralis, the Nucleus dorsomedialis, the Nucleus dorsocentralis and the dorso and ventro-lateral groups, on the other hand. The different Nuclei of the dorsal area are differentiated from a primordial territory which is the area intermedius at the hatching stage. On the hemispheric wall at the level of the tela, we can see the Nucleus teniae. Behind the plan of the commissura anterior, the Nucleus posterior is already seeing at the end of the first month after the hatching. A Golgi-Cox study showed some aspects of different kinds of neurons, and an important neuropil at the level of the Nucleus dorsocentralis too.