Description of a new octoploid frog species (Anura: Pipidae: Xenopus) from the Democratic Republic of the Congo, with a discussion of the biogeography of African clawed frogs in the Albertine Rift.

We describe a new octoploid species of African clawed frog (Xenopus) from the Lendu Plateau in the northern Albertine Rift of eastern Democratic Republic of the Congo. This species is the sister taxon of Xenopus vestitus (another octoploid), but is distinguished by a unique morphology, vocalization and molecular divergence in mitochondrial and autosomal DNA. Using a comprehensive genetic sample, we provide new information on the species ranges and intra-specific diversity of African clawed frogs from the Albertine Rift, including the details of a small range extension for the critically endangered Xenopus itombwensis and previously uncharacterized variation in Xenopus laevis. We also detail a new method for generating cytogenetic preparations in the field that can be stored at room temperature for up to 3 weeks. While extending our understanding of the extant diversity in the Albertine Rift, this new species highlights components of species diversity in ancestral African clawed frogs that are not represented by known extant descendants.

Auditory and vocal nuclei in the frog brain concentrate sex hormones.

Mate calling by South African clawed frogs, Xenopus laevis, is under the control of androgens. Autoradiographic studies demonstrate androgen-concentrating neurons in a motor nucleus that controls mate calling and a midbrain nucleus that is stimulated by sound. Hormone concentration by laryngeal motor neurons suggests that steroids regulate the final common path for vocal behavior. Modulation of auditory sensitivity by hormones could explain seasonal variations in behavioral responsiveness to conspecific vocalizations.

Saline culture of crops: a genetic approach.

Increasing salinity of soil and water threatens agriculture in arid and semiarid regions. By itself, the traditional engineering approach to the problem is no longer adequate. Genetic science offers the possibility of developing salt-tolerant crops, which, in conjunction with environmental manipulation, could improve agricultural production in saline regions and extend agriculture to previously unsuited regions.

Is song special?

Akutagawa and Konishi (2001)([this issue of Neuron]) describe the spatial and temporal pattern of SNAg (song system nuclear antigen) expression within a subset of song-associated forebrain nuclei of grass finches. The timing and estrogen inducibility of SNAg expression suggest that it may function in establishing neural connections key to vocal learning.

Generating sexually differentiated songs.

The past year has witnessed increased confusion as to the role of gonadal hormones in the development of neuroeffectors for sexually differentiated vocalizations in several species. Are sex differences in levels of circulating gonadal hormones robust enough to account for the full spectrum of male/female differences? Understanding how vocal behaviors are generated has improved, permitting greater insights into how differences in cell number and type contribute to male- and female-specific songs in frogs and birds.

Locations of androgen-concentrating cells in the brain of Xenopus laevis: autoradiography with 3H-dihydrotestosterone.

The distribution of hormone-concentrating cells in the brains of South African clawed frogs, Xenopus laevis, was examined autoradiographically after the administration of 3H-dihydrotestosterone. Hormone-accumulating cells were found in cranial nerve nucleus IX-X and adjacent smaller cells, a presumed medullary vestibular nucleus, a presumed sensory nucleus of cranial nerve V, dorsal tegmental area of the medulla, laminar nucleus of the torus semicircularis, ventral thalamus, and anterior pituitary. The pattern of dihydrotestosterone-labelled cells differs from previously reported results following testosterone or estradiol administration. Unlike these latter hormones, dihydrotestosterone does not accumulate in anterior preoptic or ventral infundibular nuclei. Both androgens but not estradiol label medullary motor neurons; limbic telencephalic nuclei appear to concentrate only estradiol. Hormone-concentrating brain nuclei in X. laevis have been implicated in neuro-endocrine regulation and the control of male and female reproductive behaviors.

Neurogenesis in the vocalization pathway of Xenopus laevis.

We examined possible contributions of neurogenesis to sex differences in the vocalization pathway of the South African clawed frog, Xenopus laevis. Birthdates of neurons were obtained from autoradiograms of animals receiving tritiated thymidine from gastrulation through 1 month after metamorphosis. Thymidine availability studies showed that 80% of the [3H]-thymidine injected into embryos and tadpoles was incorporated into the DNA of dividing cells within 3 hours. We observed 3 patterns of neurogenesis: late-short, a short burst of proliferation occurred late in development in the anterior preoptic area, the ventromedial nucleus of the thalamus, and the pretrigeminal nucleus of the dorsal tegmental area of the medulla; protracted-bimodal, a prolonged period of proliferation with an early and a late peak in the number of labeled cells occurred in the ventral striatum and in the ventrolateral and posterior nuclei of the thalamus; protracted-unimodal, a prolonged period of proliferation with a single early peak occurred in the inferior reticular formation and in the medial and lateral nucleus IX-X (containing laryngeal motor neurons). There were no differences between sexes in the number of tritiated thymidine labeled cells in any nucleus. The difference in nucleus IX-X neuron number in adults does not appear to result from sex differences in the proliferation of these cells during development. Since neurons in the vocalization pathway do not exhibit androgen receptors until after neurogenesis is complete, we also conclude that androgen probably does not regulate the genesis of these cells.

Auditory and lateral line inputs to the midbrain of an aquatic anuran: neuroanatomic studies in Xenopus laevis.

Computation of rate in auditory signals is essential to call recognition in anurans. This task is ascribed to a group of central nervous system nuclei in the dorsal midbrain or torus semicircularis, homologous to the inferior colliculus of mammals. We have mapped the connections of the subnuclei of the torus semicircularis in Xenopus laevis to determine which receive auditory and which receive lateral line information. Relative to terrestrial anurans, the torus of X. laevis is hypertrophied and occupies the entire caudal, dorsal midbrain. Auditory input to the torus, that arising directly from the dorsal medullary nucleus, is present only in the laminar nucleus. The principal and magnocellular nuclei receive their input from the lateral line nucleus of the medulla. All three nuclei of the torus also have reciprocal connections with the superior olive and the nucleus of the lateral lemniscus. Ascending efferents from all three nuclei of the torus innervate central and lateral thalamic nuclei, and all have a weak reciprocal connection with the posterior thalamus. The laminar and magnocellular nuclei have reciprocal connections with the ventral thalamus, and all three nuclei of the torus receive descending input from the anterior entopeduncular nucleus. The laminar and magnocellular nuclei also receive descending input from the preoptic area. Based on our identification of toral nuclei and these results we assign a major function for the detection of water-borne sounds to the laminar nucleus and a major function for the detection of near field disturbances in water pressure to the principal and magnocellular nuclei.

Projections of a telencephalic auditory nucleus-field L-in the canary.

Anterograde projections of a telencephalic auditory area - field L of the neostriatum - were traced in canaries, Serinus canarius. Field L was defined as the neostriatal projection of nucleus ovoidalis of the thalamus. Using amino acid autoradiography, two efferent projections of field L and adjacent neostriatum were observed: (1) a projection to the medial and ventral borders of nucleus hyperstriatum ventrale, pars caudale (HVc) and (2) a smaller projection to medial paleostriatum augmentatum (PA). When autoradiographic injection sites included neostriatum postero-ventral to field L, a projection to archistriatum outlining the anterior and ventral borders of the nucleus robustus archistriatalis (RA) resulted. Injection sites that included neostriatum antero-lateral to "L" gave rise to projections to the interior of HVc proper. Above background numbers of silver grains were consistently observed over caudal dorso-lateral portions of nucleus ovoidalis. Following lesion of field L and adjacent neostriatum, argyrophilic degeneration was traced to medial PA and to a shelf of neostriatum underlying HVc. All observed anterograde projections were ipsilateral. Two of the nuclei outlined by neostriatal projections in this study, HVc and RA, have important roles in the motor control of canary song (Nottebohm et al., '76). The development of song is dependent on auditory information. Auditory-vocal neural connections described here may be involved in song learning.

Connections of vocal control nuclei in the canary telencephalon.

Connections of two telencephalic vocal control nuclei, the hyperstriatum ventrale, pars caudale (HVc), and robust nucleus of the archistriatum (RA), were investigated in adult canaries. Methods used were transport of horseradish peroxidase and 3H-adenosine and silver staining of degenerating axons. Three nuclei project to HVc: medial nucleus magnocellularis of the anterior neostriatum (MAN), nucleus interfacialis (NIf) of midneostriatum, and nucleus uvaeformis (Uva) of the diecephalon. Uva also projects to NIf. NIf and Uva have not been described previously. HVc projects to area X of lobus parolfactorius, to RA, and to field Avalanche of hyperstriatum ventrale. Nucleus RA receives projections from HVc and from lateral MAN. All these projections are ipsilateral. No gross male/female differences were apparent in the projections to and from HVc. Uptake of HRP by cell somata in HVc following localized injections of this substance into RA or HVc suggests that HVc is composed of rostrocaudally organized clusters of cells, with little lateral communication between them.

Origin and identification of fibers in the cranial nerve IX-X complex of Xenopus laevis: Lucifer Yellow backfills in vitro.

The central projections of individual components of the IX-X nerve complex in the South African clawed frog, Xenopus laevis, were mapped by dye diffusion with Lucifer Yellow in an isolated brain preparation. The method reliably revealed fiber tracts, termination zones, and detailed cell morphology. In addition, motor neurons could be doubly labelled by retrograde transport of horseradish peroxidase from muscle targets followed by backfilling the appropriate nerves with Lucifer Yellow. The most anterior root associated with the nerve IX-X complex, root 1, is composed of lateral line afferents that terminate in the medial medulla. Root 2 contains sensory fibers that terminate in the nucleus tractus solitarii and axons of lateral line efferent neurons. Root 3 is composed of sensory and motor fibers, including a major somatosensory component that terminates in posterior medulla and anterior spinal cord, and axons from cranial nerve nucleus IX-X. The most posterior root of the IX-X nerve complex, root 4, contains axons of laryngeal motor neurons and of general visceral efferent neurons.

Autoradiographic localization of hormone-concentrating cells in the brain of an amphibian, Xenopus laevis. I. Testosterone.

Autoradiographic methods were used to investigate locations of hormone concentrating cells in the CNS of Xenopus laevis. Both male and female frog brains contained cells. Four major hormone uptake sites were identified: the anterior preoptic area, the ventral infundibular nucleus, a dorsal tegmental area of the medulla and a presumptive motor nucleus of cranial nerves IX-X. The distribution of labelled cells was very similar for male and female brains. Available information on these testosterone uptake sites in anurans indicates possible roles in gonadotropin regulation and reproductive behavior.

Autoradiographic localization of hormone-concentrating cells in the brain of an amphibian, Xenopus laevis. II. Estradiol.

Autoradiographic techniques for light microscopic examination of sex steroid retention were applied to the brains of male and female Xenopus laevis, and anuran amphibian, after 3H-estradiol administration. Estrogen was concentrated by cells in three telencephalic areas (the ventral striatum, the ventral-lateral septum and the amygdala), the anterior preoptic area, the ventral thalamus, the ventral infundibular nucleus, and in the torus semicircularis. The anterior preoptic area and the ventral infundibular nucleus contained the greatest number of labelled cells. The topography of estrogen-concentrating cells was the same in male and female brains. This fact and comparisons of 3H-estradiol with 3H-testosterone retention in Xenopus suggest that the sex steroid itself, and not the genetic sex of Xenopus determines the pattern of uptake by cells in the brain. The distribution of hormone-concentrating cells in Xenopus has many similarities to that found in birds and mammals. Preoptic, hypothalamic (tuberal), limbic forebrain and specific mesencephalic sites in all these forms contain labelled cells following radioactive sex steroid administration. Findings in Xenopus add to the argument for a phylogenetically stable system of hormone-concentrating nerve cells in limbic, hypothalamic and mesencephalic structures.

An improved method for mounting frozen-section specimens.

We describe an efficient method for embedding frozen-section specimens. Transparent tape is used to produce a well on the surface of the tissue holder ("chuck"). This well contains a quantity of nonmotile mounting media in which a tissue specimen can be easily oriented. After freezing, the tape is removed, leaving a relatively smooth surface.

The ontogeny of androgen receptors in the CNS of Xenopus laevis frogs.

Androgenic steroids have been implicated in the development of sex differences in Xenopus laevis frogs. In order to determine when neurons first acquire the ability to concentrate androgen, we prepared autoradiograms of CNS in developing frogs following injection of tritiated dihydrotestosterone (DHT). X. laevis tadpoles and juveniles from stage 60 to 2 months post-metamorphosis (PM) were injected with [3H]DHT. Brain and spinal cord autoradiograms from these animals were examined for the presence of labelled cells. The pattern of [3H]DHT labelling in stage-64 tadpoles and in PM juveniles was similar but not identical to that seen in adults. Heavily labelled cells were seen in the motor nucleus of cranial nerves IX and X, medullary reticular formation, a presumed sensory nucleus of cranial nerve V, pretrigeminal nucleus of the dorsal tegmental area of the medulla, laminar nucleus of the torus semicircularis, anterior pituitary, ventral thalamus and anterior spinal cord. The vestibular sensory nucleus of cranial nerve VIII was the only area that concentrates DHT in adults but did not contain labelled cells in young animals. No [3H]DHT-labelled cells were found in stage-60 tadpoles. The onset of androgen concentrating capability in X. laevis CNS thus probably occurs between stages 60 and 64.

[3H]-2-deoxyglucose autoradiography in a molluscan nervous system.

We have used [3H]2-deoxyglucose autoradiography to correlate the labeling of individual neurons with electrical activity within the central nervous system of a terrestrial mollusc, Limax maximus. In an electrically quiescent control preparation where a single neuron is impaled with a glass microelectrode but not stimulated, several somata are uniformly labeled at 3-5 times background. In preparations where a single cell is impaled and stimulated, one or more somata are heavily labeled with [3H]2-deoxyglucose at 10-50 times tissue background. This technique may be useful for surveying metabolically active neurons during spontaneous and driven electrical activity.