Regional expression of the homeobox gene NKX2-1 defines pallidal and interneuronal populations in the basal ganglia of amphibians.

The distribution of gene expression domains during development constitutes a novel tool for the identification of distinct brain regions. This is particularly useful in the brain of amphibians where cell migration is very limited and most neurons organize in a periventricular layer. Here we report the expression pattern of NKX2-1 protein in the developing Xenopus telencephalon. In mammals, the Nkx2-1 gene is expressed in distinct subpallial regions such as the septum, the medial ganglionic eminence and preoptic region. The results of the present study demonstrate that the expression of NKX2-1 delineates the pallidal anlage and its derivatives in amphibians, as in mammals and birds. In addition, double-labeling immunohistochemistry and the combination of tracing experiments with NKX2-1 immunohistochemistry demonstrate that the amphibian striatum contains interneurons, which express NKX2-1 and produce, among other possible neurotransmitters, nitric oxide and acetylcholine. In sum, the results of the present study strengthen the notion that similar developmental programs exist during basal ganglia development in all tetrapods.

Origin and development of descending catecholaminergic pathways to the spinal cord in amphibians.

The origin and development of the supraspinal catecholaminergic (CA) innervation of the spinal cord was studied in representative species of the three amphibian orders (Anura: Xenopus laevis and Rana perezi; Urodela: Pleurodeles waltl; Gymnophiona: Dermophis mexicanus). Using retrograde dextran amine tracing in combination with tyrosine hydroxylase (TH)-immunohistochemistry, we showed that only four brain centers contribute to the CA innervation of the adult spinal cord: (1) the ventrolateral component of the posterior tubercle, (2) the periventricular nucleus of the zona incerta, (3) the locus coeruleus, and (4) the nucleus of the solitary tract (except for gymnophionans). The pattern observed is largely similar in all amphibian species studied. The development of the CA innervation of the spinal cord was studied with in vitro double labeling methods in Xenopus laevis tadpoles. At stage 40/41, the first CA neurons projecting to the spinal cord were found to originate in the posterior tubercle. At stage 43, spinal projections were found from the periventricular nucleus of the zona incerta and the locus coeruleus, whereas spinal projections from the nucleus of the solitary tract were not observed before stage 53. These results demonstrate a temporal sequence in the appearance of the CA cell groups projecting to the anuran spinal cord, organized along a rostrocaudal gradient.

Early development of NADPH diaphorase-expressing neurons in the brain of the urodele amphibian Pleurodeles waltl.

The distribution of nitrergic cells was studied in the brain of the urodele amphibian Pleurodeles waltl during embryonic and larval stages by means of NADPH-diaphorase histochemistry. The first positive neurons were observed at embryonic stage 30 in the ventrolateral area of the caudal rhombencephalon. Subsequently (stage 33b), weakly reactive cells appeared in the isthmic tegmentum, the mesencephalic tegmentum, the hypothalamus, and the nucleus of the solitary tract. At initial larval stages (34-38), two new groups of NADPH-diaphorase-positive cells appeared in the caudal telencephalon (the amygdaloid region) and in the middle reticular nucleus. During the beginning of the active larval life (stages 39-42), reactive cells were found in the granule cell layer of the olfactory bulb and in the telencephalic pallium. As in the adult, the nitrergic cells in the central nervous system are widely distributed during early development, pointing to important roles of nitric oxide through ontogenesis. The sequence of appearance of nitrergic cells suggests an early involvement in reticulospinal control most likely related to locomotor behavior.

Development of NADPH-diaphorase/nitric oxide synthase in the brain of the urodele amphibian Pleurodeles waltl.

In the present study, the ontogenesis of nitrergic neurons has been studied in the urodele amphibian Pleurodeles waltl by means of NADPH-diaphorase (NADPHd) histochemistry and neuronal nitric oxide synthase (NOS) immunohistochemistry. Embryonic and larval stages were studied. Except for the olfactory fibers and glomeruli, both methods were equally suitable to reveal nitrergic structures in the brain. The earliest positive neurons were observed in the inferior reticular nucleus (Ri) in the caudal rhombencephalon at embryonic stage 30. At stage 33b, weakly reactive cells appeared in the tegmentum of the mesencephalon and isthmus, in the ventral hypothalamus (VH), and in the proximity of the solitary tract (sol). At initial larval stages (stages 34-38), two new groups appeared in the caudal telencephalon (future amygdaloid complex (Am)) and in the middle reticular nucleus (Rm) of the rhombencephalon. During the active larval life (stages 39-55c) the nitrergic system developed progressively both in number of cells and fiber tracts. At stages 39-42 reactive cells were found in the inner granular layer (igl) of the olfactory bulb, the telencephalic pallium, the pretectal region, the optic tectum (OT) and retina. New populations of nitrergic cells appear during the second half of the larval period (stages 52-55). Rostrally, reactive cells were found in the telencephalic diagonal band (DB) nucleus, medial septum and in the thalamic eminence (TE), whereas caudally cells appeared in the raphe (Ra) and the descending trigeminal nucleus (Vd). The last changes occurred during the juvenile period (metamorphic climax), when cells of the spinal cord (sc) and the preoptic area became positive. The sequence of appearance of nitrergic cells revealed a first involvement of this system in reticulospinal control, likely influencing locomotor behavior. As development proceeds, cells in different sensory systems expressed progressively nitric oxide synthase in a pattern that shows many similarities with amniotes.

Comparative analysis of adrenomedullin-like immunoreactivity in the hypothalamus of amphibians.

Adrenomedullin (AM) is a novel neuropeptide with special significance in the mammalian hypothalamo-hypophysial axis. By using an antiserum specific for human AM, we have studied the localization of AM-like immunoreactive (AMi) cell bodies and fibers in the hypothalamus and hypophysis of the amphibians Rana perezi (anuran), Pleurodeles waltl (urodele), and Dermophis mexicanus (gymnophionan). Distinct AMi cell groups were found for each species. In the anuran, six cell groups were localized in the preoptic and infundibular regions, whereas only three and one were found in the urodele and gymnophionan, respectively. A comparative analysis of AMi cells and cells expressing arginine vasotocin (AVT), neuropeptide Y (NPY), and tyrosine hydroxylase (TH) revealed strong differences between species. Thus, colocalization of AVT/AM is most likely to occur in the preoptic magnocellular nucleus of urodeles and it is reflected by the intense AM immunoreactivity in the neural lobe of the hypophysis. Colocalization of NPY/AM seems to be possible in the suprachiasmatic nucleus of anurans. In the gymnophionan, cells containing AVT and NPY are distinct from AMi cells. Only in anurans, the ventral aspect of the suprachiasmatic nucleus possesses a small population of AMi cells that express also TH immunoreactivity and most likely also express NPY. The results strongly suggest that AM in amphibians plays an important regulatory role in the hypothalamo-hypophysial system, as has been demonstrated in mammals. On the other hand, substantial differences have been found between species with respect to the degree of colocalization with other chemical substances.

Descending supraspinal pathways in amphibians. I. A dextran amine tracing study of their cells of origin.

The present study is the first of a series on descending supraspinal pathways in amphibians in which hodologic and developmental aspects are studied. Representative species of anurans (the green frog, Rana perezi, and the clawed toad, Xenopus laevis), urodeles (the Iberian ribbed newt, Pleurodeles waltl), and gymnophionans (the Mexican caecilian, Dermophis mexicanus) have been used. By means of retrograde tracing with dextran amines, previous data in anurans were largely confirmed and extended, but the studies in P. waltl and D. mexicanus present the first detailed data on descending pathways to the spinal cord in urodeles and gymnophionans. In all three orders, extensive brainstem-spinal pathways were present with only minor representation of spinal projections originating in forebrain regions. In the rhombencephalon, spinal projections arise from the reticular formation, several parts of the octavolateral area, the locus coeruleus, the laterodorsal tegmental nucleus, the raphe nucleus, sensory nuclei (trigeminal sensory nuclei and the dorsal column nucleus), and the nucleus of the solitary tract. In all species studied, the cerebellar nucleus and scattered cerebellar cells innervate the spinal cord, predominantly contralaterally. Mesencephalic projections include modest tectospinal projections, torospinal projections, and extensive tegmentospinal projections. The tegmentospinal projections include projections from the nucleus of Edinger-Westphal, the red nucleus, and from anterodorsal, anteroventral, and posteroventral tegmental nuclei. In the forebrain, diencephalospinal projections originate in the ventral thalamus, posterior tubercle, the pretectal region, and the interstitial nucleus of the fasciculus longitudinalis medialis. The most rostrally located cells of origin of descending spinal pathways were found in the suprachiasmatic nucleus, the preoptic area and a subpallial region in the caudal telencephalic hemisphere, probably belonging to the amygdaloid complex. Our data are discussed in an evolutionary perspective.

Descending supraspinal pathways in amphibians. II. Distribution and origin of the catecholaminergic innervation of the spinal cord.

Immunohistochemical studies with antibodies against tyrosine hydroxylase, dopamine, and noradrenaline have revealed that the spinal cord of anuran, urodele, and gymnophionan (apodan) amphibians is abundantly innervated by catecholaminergic (CA) fibers and terminals. Because intraspinal cells occur in all three orders of amphibians CA, it is unclear to what extent the CA innervation of the spinal cord is of supraspinal origin. In a previous study, we showed that many cell groups throughout the forebrain and brainstem project to the spinal cord of two anurans (the green frog, Rana perezi, and the clawed toad, Xenopus laevis), a urodele (the Iberian ribbed newt, Pleurodeles waltl), and a gymnophionan (the Mexican caecilian, Dermophis mexicanus). To determine the exact site of origin of the supraspinal CA innervation of the amphibian spinal cord, retrograde tracing techniques were combined with immunohistochemistry for tyrosine hydroxylase in the same sections. The double-labeling experiments demonstrated that four brain centers provide CA innervation to the amphibian spinal cord: 1.) the ventrolateral component of the posterior tubercle in the mammillary region, 2.) the periventricular nucleus of the zona incerta in the ventral thalamus, 3.) the locus coeruleus, and 4.) the nucleus of the solitary tract. This pattern holds for all three orders of amphibians, except for the CA projection from the nucleus of the solitary tract in gymnophionans. There are differences in the strength of the projections (based on the number of double-labeled cells), but in general, spinal functions in amphibians are controlled by CA innervation from brain centers that can easily be compared with their counterparts in amniotes. The organization of the CA input to the spinal cord of amphibians is largely similar to that described for mammals. Nevertheless, by using a segmental approach of the CNS, a remarkable difference was observed with respect to the diencephalic CA projections.

Cholinergic and catecholaminergic neurons relay striatal information to the optic tectum in amphibians.

In the amphibians Rana perezi and Xenopus laevis, the involvement of cholinergic and catecholaminergic neurons in the relay of basal ganglia inputs to the tectum was investigated. Tract-tracing experiments, in which anterograde tracers were applied to the basal ganglia and retrograde tracers to the optic tectum, were combined with immunohistochemistry for choline acetyltransferase and tyrosine hydroxylase. The results of these experiments suggest that dopaminergic neurons of the suprachiasmatic nucleus and pretectal region, noradrenergic cells of the locus coeruleus and the cholinergic neurons of the pedunculopontine and laterodorsal tegmental nuclei mediate at least part of the basal ganglia input to the tectum in anurans.

Localization of adrenomedullin-like immunoreactivity in the hypothalamo-hypophysial system of amphibians.

The presence of adrenomedullin-like immunoreactive (AMi) cell bodies and fibers in the hypothalamus and hypophysis of the amphibians Rana perezi (anuran) and Pleurodeles waltl (urodele) was examined by immunohistochemistry. A large population of AMi neurons was found in the suprachiasmatic nucleus of both species. Differently, AMi cells in the magnocellular nucleus of the preoptic area were only found in the urodele, whereas dispersed cells in the caudal infundibular region were exclusively present in the anuran. This different staining pattern is reflected in the hypophysis where the neural lobe is primarily immunoreactive in the urodele while the labeling in the intermediate lobe prevailed in the anuran. The results strongly suggest that, as is mammals, the AM in amphibians may play an important regulatory role in the hypothalamo-hypophysial system.