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PubMed1995

Erratic deposition of agrin during the formation of Xenopus neuromuscular junctions in culture.

In order to disclose the mechanism that regulate synapse development we compared the distributions of agrin, acetylcholine receptors (AChR) and a basal lamina heparan sulfate proteoglycan (HSPG) in sections and cultures prepared from Xenopus laevis and Ambystoma mexicanum embryos. While agrin, AChR and HSPG may accumulate almost synchronously at synapses in vivo, agrin deposition usually lagged well behind the other synaptic markers during development in culture, and was not detectable at many differentiated junctions. Agrin deposition at nerve-muscle contacts in culture also appeared to require the presence of other synaptic components. A similarly variable deposition occurred on noninnervated myocytes, where agrin again collected near sites of HSPG and AChR accumulation on some cells. Profuse agrin accretion occurred consistently, however, within the extracellular matrices of surrounding epithelial cells derived from both myotomes and neural tubes. In cocultures of Ambystoma neurons and Xenopus myocytes Ambystoma agrin collected at some chimeric neuromuscular junctions, but also accumulated on noninnervated myocytes and in the extracellular matrices of salamander neuroendothelial cells. Based upon these observations we conclude that (a) focal agrin deposition is not required for synaptic differentiation on Xenopus myocytes and (b) agrin may be one of several muscle basal lamina components that stem mainly from the secreted products of nearby epithelial cells.

Agrin↗
PubMed1996

Reduction of the current of injury leaving the amputation inhibits limb regeneration in the red spotted newt.

Immediately following amputation of the limb in salamanders, a strong, steady, and polarized flow of ionic current is produced by the injury. Current flows in a proximodistal direction within the limb stump and is associated with a fall in electrical potential of about 50 mV/mm near the stump's end. This current is electrogenically driven by the Na(+)-dependent, internally positive transcutaneous voltage of the intact skin of the limb stump. Reduction of this EMF, the skin's battery, by topical application of Na+ blocking agents leads to inhibition or disruption of normal limb regeneration. This suggests electrical factors are a critical control of limb regeneration. Here we test another means to reduce the injury current and its associated electrical field within the forelimb stump of red spotted newts. A fine (40 gauge), insulated, multistrand wire was inserted beneath the skin of the animal's back, with the uninsulated portion terminating either at the shoulder region or at the base of the tail. When this cathodal (negative) electrode is connected to a regulated current source, sufficient current was pulled into the stump end from an external anode (placed in the water the animal was immersed in) to markedly reduce or null the endogenous current for the first 8 days following amputation. The extent of limb regeneration in sham-treated and experimentally treated animals was determined 1 month following amputation at the elbow. Sham-treated animals regenerated normally, with most producing digits within this time. Limb regeneration was completely arrested, or caused to be strikingly hypomorphic, in half of the experimentally treated animals. This effect was independent of where the subcutaneous electrode was placed and suggests that electrical (physiological) factors are indeed a critical control of limb regeneration in urodeles.

Animals↗
PubMed2000

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo.

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.

Amputation, Surgical↗
PubMed2000

Suppressive actions of betaxolol on ionic currents in retinal ganglion cells may explain its neuroprotective effects.

Betaxolol, a beta 1-selective adrenoceptor antagonist, is widely used in the treatment of glaucoma. In addition to its ocular hypotensive effects, betaxolol has been suggested to act as a retinal neuroprotective agent (Osborne et al., 1997). To investigate possible mechanisms underlying the neuroprotective effects, we tested the actions of betaxolol on ion channels and calcium signaling in isolated retinal ganglion cells. Betaxolol (50 microM) reduced by about 20% the high-voltage-activated (HVA) Ca channel currents in ganglion cells isolated from tiger salamander retina. In contrast, the beta 1-adrenoceptor antagonists propranolol (10 microM) and timolol (50 microM) had no inhibitory actions on HVA Ca channel currents. The L-type Ca channel antagonist, nisoldipine, blocked the HVA Ca channel current partially and the remaining current was not inhibited by betaxolol. Outward current was inhibited in the presence of betaxolol. Both iberiotoxin (IBTX; 10 nM), a selective inhibitor of large-conductance Ca-activated K channels, and Cd2+ (100 microM), which suppresses Ca-activated K channels subsequent to its block of Ca channels, reduced outward current and the remaining current was not blocked significantly with betaxolol. In the presence of betaxolol, Na channel currents were reduced by about 20%, as were currents evoked by glutamate (10 mM) and GABA (1 mM). Current clamp recordings from isolated ganglion cells showed that betaxolol had several effects on excitability: spike height decreased, repetitive spike activity was suppressed, spike width increased and hyperpolarization following spikes was reduced. Calcium imaging in isolated rat retinal ganglion cells revealed that betaxolol inhibited glutamate-induced increases in [Ca2+]i. These results suggest that betaxolol has a diversity of suppressive actions on ganglion cell ion channels and that, as a consequence, one of the net actions of the drug is to reduce Ca2+ influx. The subsequent reduction in [Ca2+]i may contribute to the apparent neuroprotective actions of betaxolol in promoting ganglion cell survival following ischemic insult, as may occur in glaucoma and retinal disease.

Action Potentials↗
PubMed1996

Immunohisto- and immunocytochemical studies on the dynamics of TSH and GTH cells in normally metamorphosing, metamorphosed, and metamorphosis-arrested Hynobius retardatus.

The development and dynamics of TSH and GTH cells were studied immunohisto- and immunocytochemically in the salamander Hynobius retardatus, which had been reported to show neoteny in a specific environment and a precocious development and maturation of testis in goitrogen-treated larvae. Pituitary glands from normally metamorphosing and metamorphosed animals, and metamorphosis-arrested larvae which had been treated with goitrogens, were examined using anti-human TSH beta and anti-bullfrog LH beta sera. The immunoreactive TSH cells in the goitrogen-treated larvae began to exceed those in the controls in number at 40 days after hatching (stage 64). At the end of this experiment (220 days after hatching), the goitrogen-treated larvae had four times more immunoreactive TSH cells than the controls. The immunoreactive TSH cells in the goitrogen-treated larvae came to occupy nearly the whole area of the section of the pars distalis. Contrary to this, no significant differences were observed in the number of immunoreactive GTH cells between the goitrogen-treated larvae and the controls during their ontogeny. The number of immunoreactive GTH cells was much less than that of immunoreactive TSH cells. Immunocytochemistry at the electron microscopical level revealed many "thyroidectomy cells" with extraordinarily inflated ER and secretory granules which showed strong immunoreactivity with anti-TSH beta antibody only in the pars distalis of the goitrogen-treated larvae. These results suggest the possibility that TSH induces precocious testicular development and maturation in the goitrogen-treated larvae in H. retardatus.

Animals↗
PubMed2000

Spatio-temporal expression of TSH beta and FSH beta genes in normally metamorphosing, metamorphosed, and metamorphosis-arrested Hynobius retardatus.

Expression patterns of TSH beta and FSH beta genes were investigated in normally metamorphosing and metamorphosed individuals and in goitrogen-treated (TU-SPC), metamorphosis-arrested larvae of a Japanese salamander, Hynobius retardatus, a particular population of which had been reported to show neoteny. Cloned sequences for Hynobius TSH beta and FSH beta covered 66 and 56% of full-length genes, respectively, and both showed relatively high similarity to those of other vertebrates. TSH beta-expressing cells were observed at the ventro-caudal region of the pars distalis of controls, whereas an extraordinarily large number of cells which occupied the whole area of the pituitary glands strongly expressed TSH beta in TU-SPCs. In contrast, no FSH beta-expressing cells were observed in the pituitary glands of controls, whereas several positive cells were observed in the pituitary glands of TU-SPCs. Northern blot analyses revealed a rapid increase of TSH beta expression in the pituitary gland of TU-SPCs that finally reached the level of 20- to 30-fold as much as that of controls. Surprisingly, the expression of FSH beta also increased in the pituitary gland of TU-SPCs compared with that of controls. On the other hand, triiodothyronine administration to the control juveniles reduced the expression of TSH beta compared with saline-administrated controls, suggesting that thyroid hormone (TH) exerts a repressing effect on TSH beta gene. These results suggest that TSH beta overexpression in TU-SPCs must be caused by a deprivation of TH which plays a role in the negative feedback system and that FSH beta expression also might be enhanced by mechanisms similar to those of TSH beta, probably due to certain overlapping of the endocrinological regulation of TSH and FSH in H. retardatus.

Amino Acid Sequence↗
PubMed1996

A maximum-likelihood estimator of the genetic identity between polyploid species.

Based on a recently proposed method for estimating the genetic identity between polyploid species, maximum-likelihood estimators are given for the genetic identity between polyploid species, for the ancestral degree of homozygosity and their variances. As an example, the genetic identity, the ancestral degree of homozygosity and their variances were estimated from data sets of populations of two species, the weevil Polydrusus mollis and the salamander Ensatina complex. It was found that other indices of the genetic identity between species obviously underestimate genetic identity due to the pooling of differences caused by mutation and drift.

Animals↗
PubMed2000

High-definition mapping of neural activity using voltage-sensitive dyes.

The distribution of patterns of activity in different brain structures has been related to the encoding and processing of sensory information. Consequently, it is important to be able to image the distribution of these patterns to understand basic brain functions. The spatial resolution of voltage-sensitive dye (VSD) methods has recently been enhanced considerably by the use of video imaging techniques. The main factor that now hampers the resolution of VSD patterns is the inherent limitation of the optical systems. Unfortunately, the intrinsic characteristics of VSD images impose important limitations that restrict the use of general deconvolution techniques. To overcomes this problem, in this study an image restoration procedure has been implemented that takes into consideration the limiting characteristics of VSD signals. This technique is based on applying a set of imaging processing steps. First, the signal-to-noise (S/N) ratio of the images was improved to avoid an increase in the noise levels during the deconvolution procedures. For this purpose, a new filter technique was implemented that yielded better results than other methods currently used in optical imaging. Second, focal plane images were deconvolved using a modification of the well-known nearest-neighbor deconvolution algorithm. But to reduce the light exposure of the preparation and simplify image acquisition procedures, adjacent image planes were modeled according to the in-focus image planes and the empirical point spread function (PSF) profiles. Third, resulting focal plane responses were processed to reduce the contribution of optical responses that originate in distant image planes. This method was found to be satisfactory under simulated and real experimental conditions. By comparing the restored and unprocessed images, it was clearly demonstrated that this method can effectively remove the out-of-focus artifacts and produce focal plane images of better quality. Evaluations of the tissue optical properties allowed assessment of the maximum practical optical section thickness using this deconvolution technique in the optical system tested. Determination of the three-dimensional PSF permitted the correct application of deconvolution algorithms and the removal of the contaminating light arising from adjacent as well as distant optical planes. The implementation of this deconvolution approach in salamander olfactory bulb allowed the detailed study of the laminar distribution of voltage-sensitive changes across the bulb layer. It is concluded that (1) this deconvolution procedure is well suited to deconvolved low-contrast images and offers important advantages over other alternatives; (2) this method can be properly used only when the tissue optical properties are first determined; (3) high levels of light scattering in the tissue reduce the optical section capabilities of this technique as well as other deconvolution procedures; and (4) use of the highest numerical aperture in the objectives is advisable because this improves not only the light-collecting efficiency to detect poor-contrast images, but also the spatial frequency differences between adjacent image planes. Under this condition it is possible to overcome some of the limitations imposed by the light scattering/birefringence of the tissue.

Algorithms↗
PubMed1993

The relationship between the number of loci and the statistical support for the topology of UPGMA trees obtained from genetic distance data.

An analysis of the relationship between the number of loci utilized in an electrophoretic study of genetic relationships and the statistical support for the topology of UPGMA trees is reported for two published data sets. These are Highton and Larson (Syst. Zool.28:579-599, 1979), an analysis of the relationships of 28 species of plethodonine salamanders, and Hedges (Syst. Zool., 35:1-21, 1986), a similar study of 30 taxa of Holarctic hylid frogs. As the number of loci increases, the statistical support for the topology at each node in UPGMA trees was determined by both the bootstrap and jackknife methods. The results show that the bootstrap and jackknife probabilities supporting the topology at some nodes of UPGMA trees increase as the number of loci utilized in a study is increased, as expected for nodes that have groupings that reflect phylogenetic relationships. The pattern of increase varies and is especially rapid in the case of groups with no close relatives. At nodes that likely do not represent correct phylogenetic relationships, the bootstrap probabilities do not increase and often decline with the addition of more loci.

Algorithms↗
PubMed2001

Molecular systematics of Plethodon and Aneides (Caudata: Plethodontidae: Plethodontini): phylogenetic analysis of an old and rapid radiation.

The closely related salamander genera Plethodon and Aneides (Plethodontidae) differ in morphology, behavior, and ecology. Although the systematics of these taxa has been the focus of much study, many details remain unresolved. To generate an hypothesis for the relationships among these taxa, I sequenced a segment of the mitochondrial protein-coding gene ND4 and portions of mitochondrial tRNAs. Taxa sampled were 5 species of Aneides, 7 species of western Plethodon, and 13 species of eastern Plethodon. Ensatina eschscholtzii was used as the outgroup. Phylogenetic analyses using maximum-parsimony, neighbor-joining, and maximum-likelihood consistently recovered some relationships. The eastern species of Plethodon are a robust, well-supported clade. Sister taxon relationships of P. elongatus and P. stormi, of P. dunni and P. vehiculum, and of A. hardii and the three west coast species of Aneides were also consistently resolved with good support. The monophyly of Aneides was only weakly supported in some analyses and there is no evidence for the monophyly of Plethodon or of the western species of Plethodon. Excluding the relatively distant outgroup, down-weighting saturated substitutions, and analyzing conserved data partitions did not yield additional resolution or support among the lineages of western Plethodon and Aneides. These results are consistent either with saturation of sequences, due to the age of the lineages, or with relatively rapid radiation. An old, rapid radiation is consistent with the results of previous studies. An analysis of current taxonomy within the phylogenetic framework presented here retains Aneides and recognizes Plethodon as a metataxon (indicated with an asterisk, Plethodon*).

Animals↗
PubMed1975

Phylogeny of functional humoral transplantation immunity: comparative studies in amphibians and rodents.

Remarkably comparable observations from parallel experiments in salamanders and mice utilizing three related model systems (implant-induced immunomanipulation; passive transfer; and putative B cell suppression) argue directly that functional humoral transplantation immunity is highly developed at the phylogenetic level of Amphibia and that it plays a major role in regulating graft survival in these species (Fig. 4). Although it is still conjectural whether such humoral immunity and weak H-antigens evolved concurrently, the argument that enhancing atibodies evolved exclusively in viviparous species to protect the fetus from potential rejection by the maternal immune system no longer seems tenable (1).

Animals↗
PubMed1990

Calretinin and other CaBPs in the nervous system.

At least three CaBPs are abundant in various types of nerve cells: calbindin-D28, calretinin, and parvalbumin. The sequence of chick calretinin, from cDNA clones, is 60% homologous to that of chick calbindin. The genomic calretinin gene has also been partially sequenced. Calretinin is a protein of 29-30 kilodaltons. Antisera have been raised against beta-galactosidase-calretinin fusion proteins, and used to compare the distribution of calretinin with that of calbindin by two-colour immunofluorescence. Some sections have also been stained for parvalbumin. In chick brain and retina, the three proteins are largely in different neurons. Calbindin and calretinin are particularly abundant in some sensory nuclei, and co-expression is more common in peripheral sensory neurons. In rat brain, and in retinae of rat, cat, and salamander, some of the expression patterns are conserved, but some are not. In the chick embryonic retina, some cells show a transient phase of calbindin immunoreactivity during development.

Amino Acid Sequence↗
PubMed1993

Cyclic AMP-gated cation channels of olfactory receptor neurons.

Odor-induced electrical activity in vertebrate olfactory receptor neurons is, at least in part, the result of the direct cyclic AMP-dependent activation of a nonselective cation channel. Single-channel recordings from extraciliary regions of isolated salamander olfactory receptor neurons have greatly improved our knowledge about distinctive properties of the cAMP-gated channel such as channel kinetics, modulation through divalent cations, and pharmacology. Because of the central role of these channels in the transduction cascade, these efforts have led to a better understanding of the physiology of olfactory transduction.

1-Methyl-3-isobutylxanthine↗
PubMed1994

The comparison of morphological and molecular data in phylogenetic systematics.

Analytical methods facilitating the use of molecular and morphological characters as complementary sources of phylogenetic information are explored. Separation of phylogenetically useful information from misleading patterns of character variation is most effective when the methods of "taxonomic congruence" and "character congruence" are used together. Statistical approaches for implementing both methods are described and illustrated with an example from the phylogeny of salamanders.

Animals↗
PubMed1983

Merkel cell distribution in the epidermis as determined by quinacrine fluorescence.

The Merkel cell distribution in the basal epidermis of amphibian and mammalian skin was visualized in whole mounts by means of quinacrine fluorescence. In most cases only the isolated epidermis was viewed following dermal-epidermal separation. Tadpole tentacles contained numerous quinacrine fluorescent cells (QFC) 25-40 microns apart. Groups of 2-4 QFC were found around the gland openings in frog epidermis but not in salamander epidermis where the QFC were irregularly scattered 40-100 microns apart. In the rat, ring-like clusters of a few to 200 or more QFC were distributed across the basal epidermis of trunk skin (at touch domes or Haarscheiben), eyelid, ear, nose, and whisker pad. The ridged (glabrous) skin of the nose and footpad contained numerous QFC that appeared to follow the contours of the epidermal ridges. The isolated external root sheath of rat vibrissae contained an upper cylindrical cuff of several hundred QFC; enzymatic dissociation of these sheaths produced individually isolated as well as small clusters of fluorescent and non-fluorescent cells. Electron-microscopic examination of several of these cells confirmed that the fluorescent ones are Merkel cells, identified by the presence of characteristic dense-cored granules; in contrast, the non-fluorescent cells lack this ultrastructural feature.

Ambystoma↗
PubMed1993

Pit organs in axolotls: a second class of lateral line neuromasts.

The lateral line system of axolotls (Ambystoma mexicanum) consists of mechanoreceptive neuromasts and electroreceptive ampullary organs. All neuromasts in salamanders are located superficially and are organized into lines that are homologous to canal neuromasts in fishes. Ampullary organs are confined to the head and generally are located adjacent to the lines of superficial neuromasts. Axolotls, however, also possess a third class of receptors; these form restricted patches on the head and are possibly homologous to the superficial pit organs in fishes. In order to test this hypothesis the morphology of the suspected pit organs was examined with scanning electron microscopy, and a number of their physiological properties were determined. Pit organs are approximately half the size of neuromasts and have fewer hair cells, although these hair cells do possess kinocilia and stereocilia like those of neuromasts. Pit organs also possess cupulae and exhibit a pattern of innervation identical to that of neuromasts. Pit organs and neuromasts also exhibit similar rates of spontaneous activity, are excited by weak water currents but not weak electric stimuli, and are not inhibited by magnesium ions. Pit organs appear to have slightly lower rates of spontaneous discharge than neuromasts, however, and have slightly lower displacement thresholds to low frequency wave stimuli. These data support the contention that the pit organs of axolotls constitute a second class of neuromasts homologous to the pit organs of fishes.

Ambystoma mexicanum↗
PubMed1982

Ultrastructure of gut-associated lymphoid tissue (GALT) in the amphibian urodele, Pleurodeles waltlii.

The ultrastructure of gut-associated lymphoid tissue (GALT) has been studied in the salamander, Pleurodeles waltlii. Lymphoid accumulations appear as true infiltrates scattered throughout the lamina propria cell elements. The most important components of these infiltrates are small and medium sized lymphocytes, and, in lesser amounts, developing and mature plasma cells, macrophages and granulocytes. Migrating lymphoid cells massively invade the intestinal epithelium inducing noticeable modifications, such as the disappearance of the basement membrane and decreased numbers of mucous cells. Thus, in its organization and cell composition, the GALT of P. waltlii appears to represent a primitive phylogenetic precursor of the mammalian "intestinal-immunologic" barrier.

Animals↗
PubMed1984

Ultrastructure and morphometry of the stomach muscle of Amphiuma tridactylum.

An ultrastructural and stereological examination was performed on stomach smooth muscle of the salamander Amphiuma. This tissue has very large cells, ranging up to 12 X 1500 micron when relaxed. The extracellular space is 31% of the tissue volume, and the tissue contains 84.6% water. These values are similar to those of other amphibian and mammalian gastrointestinal smooth muscle. The cells possess the usual smooth muscle organelles. Thick, thin and intermediate filaments are present, along with membrane-associated and cytoplasmic dense regions. There is a well-developed sarcoplasmic reticulum and many microtubules. Caveolae are found in rows along the cellular surface; the caveolae increase the cellular surface area by about 70%. The ratio mean volume: surface area of the cells is 1.26 micron. This tissue appears to be typical of gastrointestinal smooth muscle, with the exception of the very large size of the cells.

Animals↗