Immunohistochemical localization of neuropeptide FF-like in the brain of the turtle: relation to catecholaminergic structures.

A previous study in the lizard Gekko gecko has revealed that neuropeptide FF (NPFF, a neuropeptide involved in nociception, cardiovascular regulation, and endocrine function) is widely distributed throughout the brain and spinal cord. Although the distribution of NPFF immunoreactivity shares many features with that found in other vertebrates, it was noted that Gekko shared more features with anamniotes in terms of number of cell groups, more elaborate networks of fibers, and lack of colocalization with catecholamines, than with mammals. To assess the primitive or derived character of these features, NPFF and tyrosine hydroxylase (TH) antibodies have been applied to the brain and spinal cord of the turtle, Pseudemys scripta elegans, which belongs to a different radiation of reptiles. As in Gekko, major NPFF-ir cell groups were found in the diagonal band nucleus of Broca and in the hypothalamus, whereas additional cells were identified in the anterior olfactory nucleus, lateral and dorsal cortices, dorsal ventricular ridge, and the intergeniculate leaflet formation. Notable differences are the presence of NPFF-ir cells in the medial cortex and striatum of Pseudemys, which are lacking in Gekko. On the other hand, no NPFF-ir cells could be detected in the septal region and dorsal horn of the spinal cord in Pseudemys. Double staining with NPFF and TH antibodies revealed an intimate relationship between NPFF-ir and TH-ir structures but colocalization could not be established. In conclusion, the distribution of NPFF in the brain of Pseudemys has corroborated previous results in Gekko, but also revealed some notable species differences.

Evolution of the basal ganglia in tetrapods: a new perspective based on recent studies in amphibians.

It has been postulated frequently that the fundamental organization of the basal ganglia (BG) in vertebrates arose with the appearance of amniotes during evolution. An alternative hypothesis, however, is that such a condition was already present in early anamniotic tetrapods and, therefore, characterizes the acquisition of the tetrapod phenotype rather than the anamniotic-amniotic transition. Re-examination of the BG organization in tetrapods in the light of recent findings in amphibians strongly supports the notion that elementary BG structures were present in the brain of ancestral tetrapods and that they were organized according to a general plan shared today by all extant tetrapods.

Basal ganglia organization in amphibians: evidence for a common pattern in tetrapods.

The results of recent studies investigating the connections and chemoarchitecture of the basal forebrain of amphibians provide strong evidence that tetrapod vertebrates share a common pattern of basal ganglia organization. This pattern includes the existence of dorsal and ventral striatopallidal systems, reciprocal connections between the striatopallidal complex and the diencephalic and mesencephalic basal plate (striato-nigral and nigro-striatal projections), and descending pathways from the striatopallidal system to the midbrain tectum and reticular formation. The connectional similarities are parallelled by similarities in the distribution of chemical markers of striatal and pallidal structures such as dopamine, substance P and enkephalin. Moreover, studies of development and expression of homeobox genes have given further support to the notion that both amniotic and anamniotic tetrapods have a common pattern of basal ganglia organization. A new nomenclature of basal forebrain structures in amphibians is proposed which reflects our current understanding of basal ganglia organization in this class of vertebrates.

Retinofugal pathways in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata.

Retinofugal pathways in the spotted dogfish Scyliorhinus canicula and the thornback ray Raja clavata were studied with reduced silver techniques following unilateral eye enucleations. Optic nerve axons decussate in the chiasma opticum, except for a small ipsilateral projection to the area preoptica. After crossing, retinal projections distribute to the area preoptica, the thalamus dorsalis pars lateralis, the thalamus ventralis pars lateralis, the corpus geniculatum laterale, the nucleus pretectalis, and the superficial layers of the tectum mesencephali. In Scyliorhinus most primary optic fibers terminate in the stratum medullare externum of the mesencephalic tectum, while in Raja the zona externa of the stratum cellulare externum receives the bulk of the retinal input. A basal optic tract could be identified in Raja, but not in Scyliorhinus. The retinofugal pathways of the two species studied are compared with those of other cartilaginous fishes and other anamniotes. It is concluded that the primary visual system in chondrichthyans resembles that of actinopterygians and amphibians. However, there is a striking difference in the way in which the primary optic fibers reach the tectal target areas. In elasmobranch fish the optic nerve fibers enter the tectum through the zona interna of the stratum cellulare externum and send their axons into the more superficial tectal layers, while in actinopterygians and amphibians the majority of the optic fibers enter the tectum through the superficial layer and distribute their axons to deeper tectal layers.

Efferent tectal pathways in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata.

The efferent connections of the tectum mesencephali in the shark Scyliorhinus canicula and the ray Raja clavata have been studied by using the silver impregnation methods. of Nauta-Gygax ('54) and Fink-Heimer ('67). After a unilateral lesion made through all six tectal layers, three distinct pathways could be observed: 1) an ascending projection both ipsi- and contralateral to the pretectal area, the dorsomedial region of the thalamus, and the lateral geniculate body, 2) a commisural projection to the contralateral tectum and intercollicular nucleus, and 3) a descending projection to the rhombencephalic reticular formation. The last mentioned tract can be subdivided into (a) the ipsilateral tractus tectobulbaris ventralis and intermedius, giving off fibers to the intercollicular nucleus, the nucleus reticularis isthmi, and the medial and median reticular formation of the rhombencephalon and (b) the contralateral tractus tectobulbaris dorsalis, which connects the tectum with the contralateral medial reticular formation. Contrary to what has been found in other vertebrates there is no distinct segregation with respect to laterality of tectoreticular connections. Neither an ipsilateral projection to the nucleus isthmi nor a direct tectospinal pathway could be demonstrated with the techniques used.

The afferent connections of the tectum mesencephali in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata.

The afferent connections of the tectum mesencephali were studied in the spotted dogfish Scyliorhinus canicula and the thornback ray Raja clavata by means of the horseradish peroxidase (HRP) technique. Following unilateral injections in the tectum, labeled neurons could be identified in all main divisions of the brain and in the cervical spinal cord. Telencephalic neurons which project to the tectum mesencephali were observed in the caudal part of the pallium. Diencephalic projections to the tectum originate from the thalamus dorsalis pars medialis, the thalamus ventralis pars lateralis, the nucleus medius infundibuli, and the pretectal area. In Scyliorhinus labeled neurons could also be found in the corpus geniculatum laterale. Mesencephalic cells of origin of tectal afferent pathways were identified in the stratum cellulare externum of the contralateral tectum, in the nucleus tegmentalis lateralis, in the ventrolateral tegmentum, and in the nucleus ruber. Rhombencephalic cells projecting to the tectum could be identified in the nucleus cerebelli (only in Scyliorhinus), the nucleus vestibularis superior, the reticular formation, the nucleus funiculi lateralis, the nucleus tractus descendens nervi trigemini, and the nucleus dorsalis and intermedius areae octavolateralis. In addition a number of small-and medium-sized cells of the reticular formation were found labeled. Diffusely scattered labeled cells could be observed in the dorsal part of the cervical spinal cord. It is concluded that the tectal afferent connections in the chondrichthyans studied in general resemble those of other vertebrates, but that some striking differences exist. In particular, tectal afferents originating from the nucleus medius infundibuli, the nucleus cerebelli, and the nucleus dorsalis and intermedius areae octavolateralis have not been reported in other vertebrates.

Distribution of dopamine immunoreactivity in the forebrain and midbrain of the snake Python regius: a study with antibodies against dopamine.

The distribution of dopamine (DA) immunoreactivity in the forebrain and midbrain of the ball python, Python regius, was studied by using recently developed antibodies against DA. In order to determine general and species-specific features of the DA system in reptiles, we have selected the ball python as a representative of a reptilian radiation that hitherto has not been the subject of (immuno)histochemical studies. Dopamine-containing cell bodies were found around the glomeruli and in the external plexiform layer of both the main and accessory olfactory bulb, but not in the telencephalon proper. In the diencephalon, DA cells were observed in several parts of the periventricular hypothalamic nucleus, in the periventricular organ, the ependymal wall of the infundibular recess, the lateral hypothalamic area, the magnocellular ventrolateral thalamic nucleus, and the pretectal posterodorsal nucleus. In the midbrain, DA cells were found in the ventral tegmental area, the substantia nigra, and the presumed reptilian homologue of the mammalian A8 cell group. Dopaminergic fibers and varicosities were observed throughout the whole brain, particularly in the telencephalon and diencephalon. The nucleus accumbens, striatum, olfactory tubercle, and nucleus of the accessory olfactory tract appear to have the most dense innervation, but the lateral septal nucleus, the dorsal ventricular ridge, and the nucleus sphericus also show numerous DA-containing fibers and varicosities. Except for the lateral cortex, cortical areas are not densely innervated by DA fibers. The DA system of the snake Python regius shares many features with that of lizards and turtles as determined with the same antibodies. The taxonomically close relationship between lizards and snakes, which together constitute the Squamata, is reflected in a similar distribution of DA fibers and varicosities to the dorsal ventricular ridge and the lateral cortex, and in the limited number of CSF-contacting DA neurons in the hypothalamus.

The secondary olfactory connections in two chondrichthians, the shark Scyliorhinus canicula and the ray Raja clavata.

The secondary olfactory connections in the shark Scyliorhinus canicula and the ray Raja clavata have been studied with reduced silver techniques. After transections through the pedunculus olfactorius in Scyliorhinus degenerating fibers could be traced to the telencephalic hemisphere. On entering the hemisphere these fibers subdivide into a small medial and a larger lateral olfactory tract. The medial tract distributes fibers to the lateral part of the ipsilateral pallium dorsale, pars superficialis and area periventricularis pallialis, but the majority of its fibers terminate in the submeningeal zone of the pallium. The medial olfactory tract also projects contralaterally to the submeningeal pallial zone via the commissura olfactoria inferior and to the stratum granulare bulbi olfactorii by way of the commissura olfactoria superior. The lateral olfactory tract distributes mainly to the pallium laterale and to the region superficial to the lateral part of the area superficialis basalis, though the striatum also receives some fibers. In Raja the secondary olfactory tract could not be subdivided into medial and lateral components and its projections seem to be restricted to the ipsilateral pallium laterale. A striking difference between Scyliorhinus and Raja is that in the latter no contralateral projections could be recognized nor a projection to the area superficialis basalis. When these results are compared with those reported in the literature for other cartilaginous fishes, it appears that the secondary olfactory connections of Scyliorhinus are more extensive than in other chondrichthians studied experimentally. In some cases of peduncle transection in Scyliorhinus the lateral part of the striatum was also involved in the lesion. In addition to the pattern of degeneration seen after olfactory peduncle lesions, degenerating fibers could be distinguished both in the stria medullaris and basal forebrain bundle. The former projects to habenular nucleus, whereas the latter distributes to the hypothalamus, ventral thalamus, and brainstem tegmentum.

Comparative aspects of the basal ganglia-tectal pathways in reptiles.

To determine how the basal ganglia in reptiles may influence visuomotor behavior, the connections from the basal ganglia to the tectum of the midbrain were studied in several species of reptiles. Immunohistochemical studies by means of antibodies against Leu-enkephalin (LENK) as well as experimental hodological studies with anterograde (PHA-L) and retrograde (HRP, Fluorogold, Cholera toxin) tracers were carried out. The results indicate that within the class of Reptilia, two different patterns occur: one in which information from the basal ganglia is relayed to the tectum via the substantia nigra as well as via a pretectal, enkephalinergic cell group, and another one in which only the ventral route, via the substantia nigra, is present. The former pattern is found in turtles, crocodiles, and the lacertid lizards Podarcis and Gallotia, and the latter pattern in the gekkonid lizards Gekko and Eublepharis, in Varanus, and in the snakes Python and Thamnophis. The presence or absence of the pretectal relay center is reflected in the laminar distribution of LENK immunoreactivity in the tectum. The apparent lack of a pretectal relay in nocturnal gekkonids and in snakes underlines the hypothesis (Reiner et al., '84: T.I.N.S. 7:320-325) that a de-emphasis of visual-basal ganglia mechanisms has occurred during the evolution of ancestral reptiles to modern mammals.

Cells of origin of pathways descending to the spinal cord in two chondrichthyans, the shark Scyliorhinus canicula and the ray Raja clavata.

The cells of origin of pathways descending to the spinal cord in the shark Scyliorhinus canicula and in the ray Raja clavata have been demonstrated by using the horseradish peroxidase (HRP) technique. Following HRP injections in the spinal cord of Scyliorhinus (fourth to sixth segment) and of Raja (15th to 20th segment) labeled neurons could be identified in the rhombencephalon, the mesencephalon, and in the diencephalon. Cells of origin of diencephalic nuclei, which project to the spinal cord, were observed in the nucleus periventricularis hypothalami and in the thalamus ventralis pars medialis which can in this respect be considered hypothalamic. Descending pathways from mesencephalic structures originate from the interstitial nucleus of the fasciculus longitudinalis medialis, the tectum mesencephali, the nucleus intercollicularis, the tectotegmental junction zone, and from diffusely arranged tegmental neurons. A contralateral rubrospinal pathway could be recognized in Raja, but not in Scyliorhinus. Rhombencephalic cells of origin of pathways descending to the spinal cord were found in all parts of the reticular formation, i.e., the nucleus raphes inferior, the nucleus reticularis inferior, medius, superior, and isthmi, in two vestibular nuclei, and in three nuclei, which have been tentatively indicated as nucleus B, F, and G. Furthermore cells of origin of descending pathways have been found in the nucleus tractus descendens nervi trigemini, in the nucleus funiculi lateralis, and in the nucleus tractus solitarii. The descending pathways of the two species studied have been compared with those of other vertebrates. It is concluded that the basic pattern in the organization of descending pathways to the spinal cord, as proposed by ten Donkelaar ('76) for terrestrial vertebrates, also holds for cartilaginous fishes.

Noradrenaline in the brain of the South African clawed frog Xenopus laevis: a study with antibodies against noradrenaline and dopamine-beta-hydroxylase.

To obtain insight into the noradrenergic system of amphibians, the distribution of noradrenaline was studied immunohistochemically with antibodies against noradrenaline (NA) and dopamine-beta-hydroxylase (DBH) in the brain of the South African clawed frog Xenopus laevis. Noradrenaline-containing cell bodies are found in the hypothalamic periventricular organ, the isthmic region, and in an area ventral and medial to the solitary tract. Noradrenaline-immunoreactive (NAi) fibers are widely, but not uniformly, distributed throughout the brain and spinal cord. In the telencephalon, dense plexuses of NAi fibers are present dorsomedial to the nucleus accumbens, in the nucleus of the diagonal band, the dorsolateral part of the striatum, the medial amygdala, and in an area that encompasses the lateral forebrain bundle. In the diencephalon, dense plexuses are found ventrolateral to the periventricular organ, in the posterior tubercle, and in the intermediate lobe of the hypophysis. Compared to the forebrain, the brainstem and spinal cord are less densely innervated by NAi fibers. The distribution of DBHi cell bodies and fibers resembles the pattern revealed with the NA antibodies. An exception is formed by the liquor contacting cells of the hypothalamic periventricular organ, which are immunonegative for the DBH antiserum. It is suggested that these cells accumulate rather than metabolize catecholamines. The present study combined with the results of a previous report in Xenopus on the distribution of dopamine (González, Tuinhof, Smeets, '93, Anat. Embryol. 187:193-201) offers the opportunity to differentiate between the two catecholamines. For example, it is now shown that both dopaminergic and noradrenergic fibers innervate the intermediate lobe of the hypophysis and that, therefore, both catecholamines are likely involved in background adaptation.

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.

Connections of the lobus inferior hypothalami of the clearnose skate Raja eglanteria (Chondrichthyes).

The afferent and efferent connections of the lobus inferior hypothalami of the clearnose skate were demonstrated by the anterograde and retrograde transport of horseradish peroxidase. The main source of afferents is from the midbrain tegmentum and telencephalon. The major midbrain input is from cells of the ipsilateral nucleus tegmentalis lateralis and the caudal tegmental area. Another prominent, mostly ipsilateral, projection arises from nucleus F of the isthmic region. A few labeled cells also occur in the nucleus interpeduncularis, nucleus raphes superior, and lateral reticular formation. Afferents from the telencephalon arise from cells of the area preoptica, area superficialis basalis, striatum, nucleus septalis lateralis, and area subpallialis 1. Of the pallial structures, the pallium mediale and anterior as well as posterior subdivisions of the pallium dorsale pars centralis appear to have strong projections to the inferior lobe. Efferent connections of the inferior lobe consist of ascending and descending pathways. Fibers of the main ascending efferent pathway course within the basal forebrain bundle and distribute to subpallial areas. The descending efferent pathways course within the tractus lobobulbaris and tractus lobocerebellaris. Of these, the former is traceable to the level of the facial motor nucleus, issuing fibers enroute to the midbrain tegmentum and to the lateral reticular formation. The lobocerebellar tract courses dorsolateral to the lobobulbar tract, and its fibers terminate within the ipsilateral granular ridge of the rostral pole of the cerebellar corpus. There appears to be a topological organization of the inferior lobe connections. In general, pallial areas project mainly to the lateral subdivision of the inferior lobe nucleus at midlobic levels, whereas connections with the brainstem arise from or terminate within the dorsal and intermediate subdivisions at midlobic as well as caudal levels. The widespread ascending and descending connections indicate that the hypothalamic inferior lobe of the clearnose skate is a major relay center between the telencephalon and brainstem.

Immunocytochemical analysis of the dopamine system in the forebrain and midbrain of Raja radiata: evidence for a substantia nigra and ventral tegmental area in cartilaginous fish.

The distribution of dopamine-containing cell somata and fibers in the forebrain and midbrain of a cartilaginous fish, Raja radiata, was investigated by means of antibodies directed against dopamine. Many small dopamine immunoreactive neurons are distributed throughout the telencephalon, including the olfactory bulbs. Within the diencephalon and particularly in the hypothalamus, i.e., in the nucleus preopticus, nucleus suprachiasmaticus, the paraventricular organ, lateral hypothalamic area, recessus mamillaris, and nucleus tuberculi posterioris, numerous cell somata stain for dopamine. In the mesencephalon, two distinct cell masses are found, which on the basis of their immunoreactivity for dopamine and their location, may be homologous to the substantia nigra and ventral tegmental area of other vertebrates. Dopamine immunoreactive fibers are found in the glomeruli of the olfactory bulbs, in ventral portions of the telencephalon, where a dense dopaminergic plexus innervates the area superficialis basalis and striatum, and in the diencephalon, where the inferior lobe is the most densely innervated structure. In the mesencephalon, the dopamine immunoreactive fibers are confined predominantly to the periventricular zone and lateral portions of the tectum. We conclude that much of the dopaminergic system in Raja radiata is strikingly similar to that seen in amniotes.

Distribution of noradrenaline immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko.

The distribution of noradrenaline (NA) immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko was studied by means of recently developed antibodies against NA. Noradrenaline-containing cell bodies are found in the hypothalamic periventricular organ and ependymal wall of the infundibular recess of the diencephalon. They are also present in the locus coeruleus and the nucleus of the solitary tract of the brainstem. Noradrenaline-immunoreactive (NAi) fibers and varicosities are widely, but not uniformly, distributed throughout the forebrain and midbrain. In the telencephalon, dense plexuses of NAi fibers are found in the bed nucleus of the medial forebrain bundle, the vertical limb of the nucleus of the diagonal band of Broca, and the caudoventral part of the septal region. The diencephalon, the periventricular preoptic area, the supraoptic nucleus, and, in particular, the medial habenular nucleus are densely innervated by NAi fibers, whereas in the midbrain NAi plexuses are found in the ventral tegmental area, the substantia nigra and its dorsolateral extension (RA8), and in an area ventral to the nucleus interpeduncularis, pars ventralis. Moderately dense plexuses of NAi fibers are found in the small-celled medial cortex, the dorsal cortex, and the midbrain tectum. The remaining forebrain and midbrain areas are generally not or only sparsely innervated by NAi fibers. The distribution of NAi cell bodies and fibers resembles the pattern revealed with antibodies against dopamine-beta-hydroxylase (DBH). A remarkable exception is that the cells in the hypothalamic periventricular organ and ependymal wall of the infundibular recess are immunonegative for DBH. Possible explanations for this discrepancy are discussed. The present study on the distribution of NA immunoreactivity in the brain of Gekko gecko combined with the results of a previous report on the distribution of dopamine in the same species (Smeets et al., '86b) offer the opportunity to differentiate between the two catecholamines in the brain of this vertebrate.

Distribution of serotonin immunoreactivity in the forebrain and midbrain of the lizard Gekko gecko.

The distribution of serotonin (5-hydroxytryptamine, 5-HT) in the forebrain and midbrain of the lizard Gekko gecko was studied by means of antibodies against serotonin. In the diencephalon, serotonin-immunoreactive (5-HTi) cell bodies were found in the hypothalamic periventricular organ and the ependymal wall of the infundibular recess. In the midbrain, 5-HTi cells were observed in the nucleus raphes superior and the lateral portion of the nucleus reticularis superior. In addition, 5-HTi cell bodies were found lateral to the ventral interpeduncular nucleus and around the ventral aspect of the medial longitudinal fasciculus. Serotonin-immunoreactive fibers and varicosities are present throughout the forebrain and the midbrain, but particularly in the nucleus accumbens, the septal area, the dorsal cortex, the dorsal thalamus, the lateral geniculate body, the ventromedial hypothalamic nucleus, the pretectal nucleus, and the basal optic nucleus. The medial habenular nucleus contains a dense 5-HTi plexus that shows a patchlike pattern. A laminar organization of 5-HTi fibers and varicosities is present in the midbrain tectum. When compared with data obtained in other vertebrates, the present study has confirmed that in the phylogenetic series fishes-amphibians-reptiles-birds-mammals there appears to be (1) a gradual decrease in the number of cerebrospinal-fluid-contacting serotoninergic cells in the hypothalamic periventricular layer and (2) a remarkable increase in number of serotoninergic cells in the midbrain tegmentum. As in mammals, a strong serotoninergic innervation of structures related to sensory, in particular visual, pathways could be recognized.

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.

Development of catecholamine systems in the brain of the lizard Gallotia galloti.

For a better insight into general and derived traits of developmental aspects of catecholaminergic (CA) systems in amniotes, we have studied the development of these systems in the brain of a lizard, Gallotia galloti, with tyrosine hydroxylase (TH)- and dopamine (DA) immunohistochemical techniques. Two main groups of TH-immunoreactive (THi) perikarya appear very early in development: one group in the midbrain which gives rise to the future ventral tegmental area, substantia nigra and retrorubral cell groups, and another group in the tuberomammillary hypothalamus. Somewhat later in development, TH/DA-immunoreactive cells are observed in the thalamus, rostrodorsal hypothalamus and spinal cord, and, with another delay, in the suprachiasmatic nucleus, the periventricular organ, and the pretectal posterodorsal nucleus. CA cell groups that appear rather late in development include the cells in the olfactory bulb, the locus coeruleus and the caudal brainstem. As expected, the development of immunoreactive fibers stays behind that of the cell bodies, but reaches the adult-like pattern just prior to hatching. The present study revealed considerable variation in the relation between the state of cytodifferentiation and first expression of TH/DA immunoreactivity between CA cell groups. Catecholamine cells in the midbrain and tuberomammillary hypothalamus are still migrating, immature (absence of dendrites) and express only TH immunoreactivity at the time of first detection. Cells which appear at later developmental stages lie already further away from the ventricle, possess two or more dendritic processes, and generally express both TH- and DA immunoreactivity.

Basal ganglia organization in amphibians: development of striatal and nucleus accumbens connections with emphasis on the catecholaminergic inputs.

To broaden our insight into the organization of the basal ganglia of amphibians, the development of the connections of the striatum and the nucleus accumbens was studied by means of tract-tracing techniques based on the transport of biotinylated dextran amines. In a number of experiments, these techniques were combined with tyrosine hydroxylase immunohistochemistry to identify the sources of catecholaminergic inputs to the striatum and the nucleus accumbens. Already at late embryonic stages, the basal telencephalon receives inputs from cells located in the amygdala, the thalamus, the suprachiasmatic nucleus, the raphe nucleus, and the rhombencephalic reticular formation. At these stages, the rostral part of the posterior tubercle seems to be the only source of the dopaminergic input to the basal telencephalon. During premetamorphosis, not only a differentiation between connections of the striatum and the nucleus accumbens could be made, but new sources of inputs were also detected in the mesencephalic and isthmic tegmentum, the parabrachial nucleus, and the nucleus of the solitary tract. Double-labeling experiments revealed that, at these stages, in addition to the posterior tubercle, cells within the mesencephalic tegmentum, the locus coeruleus, and the solitary tract nucleus contribute to the catecholaminergic innervation of the basal forebrain. During prometamorphic stages, a gradual increase occurs in the number of cells that project to the basal telencephalon. At the beginning of the metamorphic climax, the organization of the basal ganglia afferents largely resembles the pattern observed in juveniles and adults. Remarkably, during larval stages, the cells that contribute to the dopaminergic innervation of the basal forebrain show a rostrocaudal gradient in time of appearance. Moreover, the dopaminergic fibers reach the striatum earlier than the nucleus accumbens, and they precede markedly the development of the efferent connections of both brain structures. These developmental aspects are easily correlated with the situation in amniotes; therefore, the notion that amphibians share an essentially similar pattern of basal ganglia organization with other tetrapods is further strengthened.

Distribution of choline acetyltransferase immunoreactivity in the brain of anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians.

Because our knowledge of cholinergic systems in the brains of amphibians is limited, the present study aimed to provide detailed information on the distribution of cholinergic cell bodies and fibers as revealed by immunohistochemistry with antibodies directed against the enzyme choline acetyltransferase (ChAT). To determine general and derived features of the cholinergic systems within the class of Amphibia, both anuran (Rana perezi, Xenopus laevis) and urodele (Pleurodeles waltl) amphibians were studied. Distinct groups of ChAT-immunoreactive cell bodies were observed in the basal telencephalon, hypothalamus, habenula, isthmic nucleus, isthmic reticular formation, cranial nerve motor nuclei, and spinal cord. Prominent plexuses of cholinergic fibers were found in the olfactory bulb, pallium, basal telencephalon, ventral thalamus, tectum, and nucleus interpeduncularis. Comparison of these results with those obtained in other vertebrates, including a segmental approach to correlate cell populations, reveals that the cholinergic systems in amphibians share many features with amniotes. Thus, cholinergic pedunculopontine and laterodorsal tegmental nuclei could be identified in the amphibian brain. The finding of weakly immunoreactive cells in the striatum of Rana, which is in contrast with the condition found in Xenopus, Pleurodeles, and other anamniotes studied so far, has revived the notion that basal ganglia organization is more preserved during evolution than previously thought.