Margarita Muñoz

TOPOGRAPHICAL DISTRIBUTION OF NADPH-DIAPHORASE ACTIVITY IN THE CENTRAL NERVOUS SYSTEM OF THE FROG, RANA PEREZI

The distribution of NADPH‐diaphorase (ND) activity was histochemically investigated in the brain of the frog Rana perezi. This technique provides a highly selective labeling of neurons and tracts. In the telencephalon, labeled cells are present in the olfactory bulb, pallial regions, septal area, nucleus of the diagonal band, striatum, and amygdala. Positive neurons surround the preoptic and infundibular recesses of the third ventricle. The magnocellular and suprachiasmatic hypothalamic nuclei contain stained cells. Numerous neurons are present in the anterior, lateral anterior, central, and lateral posteroventral thalamic nuclei. Positive terminal fields are organized in the same thalamic areas but most conspicuously in the visual recipient plexus of Bellonci, corpus geniculatum of the thalamus, and the superficial ventral thalamic nucleus. Labeled fibers and cell groups are observed in the pretectal area, the mesencephalic optic tectum, and the torus semicircularis. The nuclei of the mesencephalic tegmentum contain abundant labeled cells and a conspicuous cell population is localized medial and caudal to the isthmic nucleus. Numerous cells in the rhombencephalon are distributed in the octaval area, raphe nucleus, reticular nuclei, sensory trigeminal nuclei, nucleus of the solitary tract, and, at the obex levels, the dorsal column nucleus. Positive fibers are abundant in the superior olivary nucleus, the descending trigeminal, and the solitary tracts. In the spinal cord, a large population of intensely labeled neurons is present in all fields of the gray matter throughout its rostrocaudal extent. Several sensory pathways were heavily stained including part of the olfactory, visual, auditory, and somatosensory pathways. The distribution of ND‐positive cells did not correspond to any single known neurotransmitter or neuroactive molecule system. In particular, abundant codistribution of ND and catecholamines is found in the anuran brain. Double labeling techniques have revealed restricted colocalization in the same neurons and only in the posterior tubercle and locus coeruleus. If ND is in amphibians a selective marker for neurons containing nitric oxide synthase, as generally proposed, with this method the neurons that may synthesize nitric oxide would be identified. This study provides evidence that nitric oxide may be involved in novel tasks, primarily related to forebrain functions, that are already present in amphibians.

Nitric oxide synthase in the brain of a urodele amphibian (Pleurodeles waltl) and its relation to catecholaminergic neuronal structures

The neuronal structures with NADPH-diaphorase activity and nitric oxide synthase (NOS) immunoreactivity have been studied in the brain of the urodele amphibian Pleurodeles waltl by means of histochemical and immunocytochemical techniques. Both approaches resulted in the selective labeling of the same neurons and fiber tracts in the brain, except for the primary olfactory fibers that did not stain for NOS but were positive for NADPH-diaphorase. NOS-containing neurons were found in the olfactory bulbs, pallial regions, septum, caudal striatum, amygdala and preoptic area. Only a few diencephalic cells were labeled in the posterior tubercle and ventral hypothalamus. In the brainstem, abundant cells were labeled in the tectum, mesencephalic tegmentum and isthmic region. The most conspicuous cell population was found in the isthmic-pretrigeminal region. Particularly well stained cells were distributed throughout the rhombencephalon in areas related to the descending trigeminal tract, solitary tract, raphe nucleus and the mid-caudal reticular formation. In the cervical spinal cord, NOS-containing cells were present in the dorsal, intermediate and ventral grey fields. Cells in the preoptic, postotic and dorsal root ganglia were also labeled. Double labeling techniques revealed an extensive codistribution of neurons with NOS and catecholamines in the urodele brain but actual colocalization in the same cells was never observed. The organization of the central systems in urodeles with NOS appears to share many features not only with other anamniotes but also with amniotes.

Spinal ascending pathways in amphibians: Cells of origin and main targets

As part of a research program on the evolution of somatosensory systems in vertebrates, the various components of ascending spinal projections were studied with in vivo and in vitro tract‐tracing techniques in representative species of amphibians (the large green frog, Rana perezi, the clawed toad, Xenopus laevis and the ribbed newt, Pleurodeles waltl). Three main ascending sensory channels, each with largely separate targets, were demonstrated:

Localization of NADPH diaphorase/nitric oxide synthase and choline acetyltransferase in the spinal cord of the frog, Rana perezi.

The localization of nitrergic cells and fibers and cholinergic cells has been analyzed in the spinal cord of the anuran amphibian Rana perezi. Histochemistry for nicotinamide adenine dinucleotide phosphate–diaphorase and nitric oxide synthase immunohistochemistry revealed a concurrent pattern of labeled structures. A large population of nitrergic spinal neurons was found from the level of the obex to the filum terminale. They are abundant in the dorsal horn and intermediate gray matter, but also occur in territories of the ventral horn and, only occasionally, in somatic motoneurons. Numerous nitrergic fibers were present in the spinal white matter, particularly in the dorsal and dorsolateral funiculi. A special arrangement of nitrergic axons is present in Lissauers tract, where a collateral system is formed. Cholinergic cells, revealed by choline acetyltransferase immunohistochemistry, were observed throughout the spinal cord. The somatic motoneurons were the most conspicuously immunoreactive cells. A large population of cholinergic cells forms a discontinuous column in the intermediate gray, from the third spinal segment to lumbar segments. These cells were organized in a medially located or intercalated cell group, and a laterally located intermediolateral group. Numerous scattered cholinergic cells were present in the central zone of the ventral horn and were absent in the dorsal horn. Double‐labeling experiments revealed a high degree of codistribution of nitrergic and cholinergic cells, mainly in the intermediate gray, but colocalization of both markers in the same neurons was not found. This result contrasts with the situation found in mammals and raises the question of whether coexpression of both substances was acquired in spinal cord neurons through evolution only in amniotes or, even, only in mammals. J. Comp. Neurol. 419:451–470, 2000.

Distribution of adrenomedullin-like immunoreactivity in the central nervous system of the frog.

Adrenomedullin (AM) is a recently discovered peptide widely distributed in the mammalian brain. By using an antiserum specific for human AM, we have analyzed the localization of AM-like immunoreactivity in the brain and spinal cord of the anuran amphibian Rana perezi. Cell bodies immunoreactive (AMi) for AM were located in the dorsal, lateral and medial pallial regions, diagonal band of Broca, medial septum, and above and rostral to the anterior commissure. A large population of AMi neurons was located in the anterior preoptic area, suprachiasmatic nucleus and in the infundibular hypothalamus. The processes of these latter cells are part of the hypothalamo-hypophysial pathway to the neural and intermediate lobes. Labeled cells were observed in the pretectal region, posterior tubercle and the mesencephalic anteroventral tegmental nucleus. Strikingly, Purkinje cells in the cerebellum also showed AM immunoreactivity, albeit not all of these cells were equally stained. Additional cells were located in the parabrachial region, principal trigeminal sensory nucleus, reticular nuclei medius and inferior, and the intermediolateral gray of the spinal cord. Immunolabeled fibers were widespread throughout the brain and spinal cord of the frog. They were particularly abundant in the medial amygdala, hypothalamus, mesencephalic tectum, periventricular gray and spinal cord. The distribution pattern of AM-like immunoreactivity in the brain of the frog is very selective and does not correspond with the pattern observed for any other transmitter or neuroactive molecule. The wide distribution of this peptide strongly suggests that it may play a significant role in the multiple neuronal functions in the amphibian brain.

Evidence for an Anuran Homologue of the Mammalian Spinocervicothalamic System: An In Vitro Tract-tracing Study in Xenopus laevis

Evidence is presented for an anuran homologue of the mammalian spinocervicothalamic system. In vitro tract‐tracing experiments with biotinylated dextran amine in Xenopus laevis show that ascending spinal fibres from all levels of the spinal cord, passing via the dorsolateral funiculus, terminate in a cell area ventrolateral to the dorsal column nucleus. This cell area can be considered a possible homologue of the mammalian lateral cervical nucleus. After tracer applications to the ventral thalamus or to the torus semicircularis (both targets for somatosensory projections), the anuran lateral cervical nucleus was retrogradely labelled contralateral to the application sites. Tracer applications to the dorsolateral funiculus at the obex level and rostral spinal cord resulted in labelling of the cells of origin of the spinocervical tract. These were found, mainly ipsilaterally, in the ventral part of the dorsal horn, and were rather evenly distributed throughout the spinal cord. These data suggest the presence of an anuran homologue of the mammalian spinocervicothalamic system. A brief survey of the literature shows that such a system is much more common in vertebrates than previously thought.

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.

Some connections of the area octavolateralis pf Pleurodeles waltlii. A study with horseradish peroxidase under in vitro conditions

Horseradish peroxidase application in exsanguinated, pre-perfused newts, Pleurodeles waltlii, appeared to be very useful to study the connections of the area octavolateralis. Efferents from the octavolateral area course via bilateral lemnisci to the tectum and a presumptive torus semicircularis in the midbrain. Additional projections to the oculomotor nuclei, cerebellum, reticular formation, contralateral octavolateral area, and spinal cord were observed. The area octavolateralis receives inputs from the cerebellum and the contralateral octavolateral area. It is concluded that the octavolateral second order projections in Pleurodeles waltlii resemble in many respects those reported for other vertebrates that possess a complete octavolateralis system.

Anuran olfactory bulb organization : Embryology, neurochemistry and hodology

In Xenopus laevis, we analyzed the origin of the projection neurons and interneurons in the developing olfactory bulbs by studying the expression patterns of the genes x-Eomes, x-Lhx5, x-Dll3 and x-Pax6. Olfactory bulb interneurons were characterized by using four conserved molecular markers for distinct subpopulations: gamma-aminobutyric acid, calretinin, calbindin, and tyrosine hydroxylase. Immunohistochemistry was combined with tract-tracing experiments to demonstrate the projection neurons and the interneurons of the olfactory bulbs simultaneously. Taken together, the results showed: (1) the pallial nature of the olfactory bulb and its projection neurons in Xenopus, like in mammals with comparable central projection areas, (2) the subpallial origin of the interneurons that, most likely, follow migratory pathways comparable to those described for mammals, (3) the different interneuron types possess neurochemical characteristics similar to mammals. Therefore, the present results show that the origin, chemoarchitecture and central connections of the olfactory bulbs are highly conserved in evolution.

Distribution of somatostatin-like immunoreactivity in the brain of the caecilian Dermophis mexicanus (Amphibia: Gymnophiona): comparative aspects in amphibians.

The organization of the somatostatin‐like‐immunoreactive (SOM‐ir) structures in the brain of anuran and urodele amphibians has been well documented, and significant differences were noted between the two amphibian orders. However, comparable data are not available for the third order of amphibians, the gymnophionans (caecilians). In the present study, we analyzed the anatomical distribution of SOM‐ir cells and fibers in the brain of the gymnophionan Dermophis mexicanus. In addition, because of its known relationship with catecholamines in other vertebrates, double immunostaining for SOM and tyrosine hydroxylase was used to investigate this situation in the gymnophionan. Abundant SOM‐ir cell bodies and fibers were widely distributed throughout the brain. In the telencephalon, pallial and subpallial cells were labeled, being most numerous in the medial pallium and amygdaloid region. Most of the SOM‐ir neurons were found in the preoptic area and hypothalamus and showed a clear projection to the median eminence. Less conspicuously, SOM‐ir structures were found in the thalamus, tectum, tegmentum, and reticular formation. Both SOM‐ir cells and fibers were demonstrated in the spinal cord. The double‐immunohistofluoresce technique revealed that catecholaminergic neurons and SOM‐ir cells are largely intermingled in many brain regions but form totally separated populations. Many differences were found between the distribution of SOM‐ir structures in Dermophis and that in anurans or urodeles. Some features were shared only with anurans, such as the abundant pallial SOM‐ir cells, whereas others were common only to urodeles, such as the organization of the hypothalamohypophysial SOM‐ir system. In addition, some characteristics were found only in Dermophis, such as the localization of the SOM‐ir spinal cells and the lack of colocalization of catecholamines and SOM throughout the brain. Therefore, any conclusions concerning the SOM system in amphibians are incomplete without considering evidence for gymnophionans. J. Comp. Neurol. 501:413–430, 2007.