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PubMed1987

NMR spectroscopy as an investigative technique in physiology.

Relating physiological variables on an organ system level to metabolic function within the intracellular environment has been exceedingly difficult because of a paucity of techniques. Most of the tools at our command necessitate either the removal or destruction of tissues before measurements can be made. Recently, NMR spectroscopy has been applied to several important questions relating organ system and cellular physiology. NMR has the distinct advantage of being noninvasive and nondestructive, allowing the investigator to make repetitive measurements of intracellular variables while manipulating experimental variables that are important on the organ system level. In this review we shall present several examples of such NMR investigations so that the reader will gain some appreciation of the potential of this relatively new technique. Cellular acid-base homeostatic mechanisms, high-energy phosphate metabolism, and regulation of anaerobic glycolysis will be discussed for such diverse cellular populations as mammalian brain, mammalian heart muscle, salamander skeletal muscle, amphibian skin, and invertebrate muscle. In addition, the role of phosphomonoesters and phosphodiesters in lipid metabolism for several tissues in different species will be evaluated.

Animals↗
PubMed1994

NBQX suppresses inhibitory glycine currents in retinal ganglion cells.

The quinoxaline NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline) is a potent non-NMDA receptor antagonist, which appears to be relatively free of antagonistic action at the glycine binding site of the NMDA receptor. However, we report here that at 50 microM, NBQX significantly attenuated the inhibitory currents induced by the exogenous application of 100 microM glycine as observed using whole-cell recordings from ganglion cells in a slice preparation of the tiger salamander retina. In contrast, NBQX had no effect on GABA-mediated inhibition. This observation suggests that care should be taken when attributing the action of NBQX solely to its antagonism of non-NMDA glutamate receptors, particularly when higher concentrations are used.

Ambystoma↗
PubMed1998

Actin cytoskeleton regulates ion channel activity in retinal neurons.

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.

Actins↗
PubMed1999

Glutamate receptors differ in rod- and cone-dominated off-center bipolar cells.

The axon morphology of off-center bipolar cells in salamander retinal slices was correlated with light responses and dendritic glutamate responses of these cells under voltage clamp. Cells with telodendria ramifying exclusively in the distal one-quarter of the inner plexiform layer were rod dominated, whereas cells with more centrally ramifying telodendria were cone dominated. The glutamate responses of the centrally ramifying cells displayed more apparent desensitization and a smaller underlying single channel conductance (1.2+/-0.3 pS) than did the distally ramifying cells (2.8+/-0.4 pS), although the responses for both classes of cells were strongly enhanced by cyclothiazide. These results suggest that different subtypes of AMPA receptors are present on rod- and cone-dominated cells, and that these may differ with respect to desensitization kinetics.

Ambystoma↗
PubMed1993

Residual cornea and the degenerate eye of the cryptophthalmic Typhlotriton spelaeus.

The cave-dwelling Typhlotriton spelaeus larvas live in daylight. The larvas undergo a metamorphosis when the thyroxin level (T3 and T4) increases. They leave aquatic life for terrestrial and subterranean aphotic life. The larval eyes are normal and show good vision response to tests. On the contrary, in the oldest larva and the adult, the eyes are small with poor vision or no vision at all. The lens can disappear but in any case the eyelids grow over the eye. The retinal degeneration takes place during and after metamorphosis. The admitted statement that "after metamorphosis . . . the developing lids close over the eye and invade the cornea" is not confirmed concerning the invasion, because it is now evident that the eyelids separate spatially from the residual cornea. Consequently, the fibroblasts of the eyelids are not able to invade the corneal stroma. In fact, the periocular tissues do not invade the cornea but invade the eyelids' tissues. We are able to confirm that the development of the larval eyes is complete and apparently normal. After metamorphosis, the eyes decrease in size. The cornea "sinks" with the eye into orbit tissues. The eyelids cover the eye and are themselves replaced by a supraocular skin. This fits the description of the human cryptophthalmia. We also demonstrate that even in the degenerate eye the residual cornea retains the main structure of a normal salamander cornea. The preservation of the endothelium and of the Descemet membrane is exceptional in blind cave-dwelling vertebrates because in the other degenerate corneas the edema of the stroma depends on the spatial disjunction of the underepithelial stroma and the supraendothelial stroma. Perhaps, the very advanced development of the eye and the late start of the degenerative processes could explain the relative preservation of the cornea. Thus, the arguments put forward tend to prove that the thyroxinic metamorphosis sets off the growth and fusion of the eyelids but that the eye degeneration is related to hereditary processes.

Animals↗
PubMed2006

Physiological constraints on organismal response to global warming: Mechanistic insights from clinally varying populations and implications for assessing endangerment.

Recent syntheses indicate that global warming affects diverse biological processes, but also highlight the potential for some species to adapt behaviourally or evolutionarily to rapid climate change. Far less attention has addressed the alternative, that organisms lacking this ability may face extinction, a fate projected to befall one-quarter of global biodiversity. This conclusion is controversial, in part because there exist few mechanistic studies that show how climate change could precipitate extinction. We provide a concrete, mechanistic example of warming as a stressor of organisms that are closely adapted to cool climates from a comparative analysis of organismal tolerance among clinally varying populations along a natural thermal gradient. We found that two montane salamanders exhibit significant metabolic depression at temperatures within the natural thermal range experienced by low and middle elevation populations. Moreover, the magnitude of depression was inversely related to native elevation, suggesting that low elevation populations are already living near the limit of their physiological tolerances. If this finding generally applies to other montane specialists, the prognosis for biodiversity loss in typically diverse montane systems is sobering. We propose that indices of warming-induced stress tolerance may provide a critical new tool for quantitative assessments of endangerment due to anthropogenic climate change across diverse species.

Animals↗
PubMed2025

Blood capillary geometry helps to explain the link between genome size and metabolic rates.

In the past decades, several authors have investigated the possibility that genome size is correlated with metabolic rates, obtaining conflicting results. The main biological explanation among the supporters of this correlation was related to the nucleotypic effect of the genome size, which, determining the cellular volume and hence the surface area-to-volume ratio, influences cellular metabolism. In the present study, I tested a different hypothesis: genome size, influencing red blood cell (RBC) volume, is correlated with capillary density and diameter. These, in turn, are (directly and inversely) correlated with mass-specific metabolic rates, and this can explain the link between genome size and metabolic rates. I have found that these correlations are significant in vertebrates with nucleated RBCs (non-mammals), but not in species with enucleated RBCs (i.e. mammals). Although further research is needed (in particular to understand how to test the correlation between genome size and metabolic rates in ectotherms), my results show that genome size and metabolic rates act on each other through a physical constraint. Through enucleation, mammals (but also some fish and salamanders) removed this constraint.

Animals↗
PubMed1992

Variation across species in the size of the nuclear genome supports the junk-DNA explanation for the C-value paradox.

The amount of DNA in the nuclear genome (the DNA C-value) of eukaryotes varies at least 80,000-fold across species, and yet bears little or no relation to organismic complexity or to the number of protein-coding genes. This phenomenon is known as the C-value paradox. One explanation for the C-value paradox attributes the size of the nuclear genome to 'junk' (typically non-coding) genetic elements that accumulate until the costs to the organism of replicating excess DNA select against it. Across species, organisms that develop at a slower rate should tolerate more junk DNA. Alternatively, junk DNA may function as a nucleo-skeleton to maintain the volume of the nucleus at a size proportional to the volume of the cytoplasm in the cell. Across species, the DNA C-value is predicted to vary with the nuclear and cytoplasmic volumes of cells. Previous studies have not been able to distinguish between the skeletal-DNA and junk-DNA explanations for the C-value paradox. We report a study of DNA content in 24 salamander species which does. The size of the nuclear genome is correlated with developmental rate even after the effects of nuclear and cytoplasmic volume have been removed. However, genome size is not correlated with cytoplasmic volume after controlling for developmental rate. These results support the view that junk DNA accumulates in the nuclear genome until the costs of replicating it become too great, rather than that it functions as a nucleo-skeleton.

Animals↗
PubMed1996

Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells.

Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

Ambystoma↗
PubMed1999

Spatial heterogeneity and function of voltage- and ligand-gated ion channels in retinal amacrine neurons.

The spatial distribution of ion channels within amacrine cells of the tiger salamander retina was studied using patch recording in the retinal slice preparation. By focally puffing kainate, GABA and glycine at amacrine cell processes in the inner plexiform layer, it was determined that the cell's glutamate receptors were located in a confined region of the processes near the soma, while glycine and GABA receptors were located throughout the processes. Likewise, similar techniques in conjunction with voltage steps demonstrated that voltage-gated sodium channels were located throughout the cell and were shown to generate sodium-dependent spikes, while only the processes contained voltage-gated calcium channels. These results suggest that this form of transient amacrine cell collects its excitatory synaptic inputs in a region confined to a central annular region near the soma, that the signal is actively propagated throughout its processes by voltage-gated sodium channels and that calcium-dependent neurotransmitter release of glycine from this neuron can occur throughout its processes. Thus, excitatory signals are collected in the processes near the soma, inhibitory signals throughout the processes and excitation is probably propagated throughout the processes of the amacrine cell.

Ambystoma↗
PubMed1981

Strategies for specifying form and pattern: adhesion-guided multicellular assembly.

We define a material pattern as a particular arrangement of material elements in space. We then make an effort to categorize the developmental strategies that underlie the emergence of multicellular patterns. These strategies are divided into three broad categories according to whether cell position influences or is influenced by cell fate. In that category of strategies in which cell fate influences cells to move to particular positions, we focus our attention upon morphogenetic and patterning phenomena that appear to be determined by adhesion-mediated interactions of cells with each other and with their surroundings. The differential adhesion hypothesis details how cellular adhesive properties can guide tissue movements and specify patterns of cell association. Motile, adhesive cells will naturally tend to group so as to maximize their adhesive interactions (minimize interfacial free energy). A homogeneous population of uniformly adhesive (isotropic) cells will tend toward spherical form. Cell surface adhesive anisotropies can determine other most-stable (equilibrium) configurations of the population, such as cell sheets, tubes and vesicles. Heterogeneous cell populations may preferentially either intermix or sort out, depending upon the balance of adhesive forces between like and unlike elements. The precise configuration adopted will depend upon the particular adhesive relationships that prevail. Both this end state and the approach toward it arise from the adhesive relationships among the interacting cells. Such morphogenetic phenomena as tissue spreading and the segregation of organ primordia are probably brought about in this way. We outline here some results of our recent experiments on the morphogenesis of the salamander pronephric duct. These illustrate the reality of emergent adhesion-generated tissue immiscibility as a cause of organ segregation and point toward a craniocaudally travelling adhesion gradient as the information that guides the migrating pronephric duct to the cloaca.

Ambystoma↗
PubMed1989

Mass extinctions among tetrapods and the quality of the fossil record.

The fossil record of tetrapods is very patchy because of the problems of preservation, in terrestrial sediments in particular, and because vertebrates are rarely very abundant. However, the fossil record of tetrapods has the advantages that it is easier to establish a phylogenetic taxonomy than for many invertebrate groups, and there is the potential for more detailed ecological analyses. The relative incompleteness of a fossil record may be assessed readily, and this can be used to test whether drops in overall diversity are related to mass extinctions or to gaps in our knowledge. Absolute incompleteness cannot be assessed directly, but a historical approach may offer clues to future improvements in our knowledge. One of the key problems facing palaeobiologists is paraphyly, the fact that many higher taxa in common use do not contain all of the descendants of the common ancestor. This may be overcome by cladistic analysis and the identification of monophyletic groups. The diversity of tetrapods increased from the Devonian to the Permian, remained roughly constant during the Mesozoic, and then began to increase in the late Cretaceous, and continued to do so during the Tertiary. The rapid radiation of 'modern' tetrapod groups--frogs, salamanders, lizards, snakes, turtles, crocodilians, birds and mammals--was hardly affected by the celebrated end-Cretaceous extinction event. Major mass extinctions among tetrapods took place in the early Permian, late Permian, early Triassic, late Triassic, late Cretaceous, early Oligocene and late Miocene. Many of these events appear to coincide with the major mass extinctions among marine invertebrates, but the tetrapod record is largely equivocal with regard to the theory of periodicity of mass extinctions.

Animals↗
PubMed2003

Regulative germ cell specification in axolotl embryos: a primitive trait conserved in the mammalian lineage.

How germ cells are specified in the embryos of animals has been a mystery for decades. Unlike most developmental processes, which are highly conserved, embryos specify germ cells in very different ways. Curiously, in mouse embryos germ cells are specified by extracellular signals; they are not autonomously specified by maternal germ cell determinants (germ plasm), as are the germ cells in most animal model systems. We have developed the axolotl (Ambystoma mexicanum), a salamander, as an experimental system, because classic experiments have shown that the germ cells in this species are induced by extracellular signals in the absence of germ plasm. Here, we provide evidence that the germ cells in axolotls arise from naive mesoderm in response to simple inducing agents. In addition, by analysing the sequences of axolotl germ-cell-specific genes, we provide evidence that mice and urodele amphibians share a common mechanism of germ cell development that is ancestral to tetrapods. Our results imply that germ plasm, as found in species such as frogs and teleosts, is the result of convergent evolution. We discuss the evolutionary implications of our findings.

Ambystoma↗
PubMed2005

Deciphering amphibian diversity through DNA barcoding: chances and challenges.

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecific cox1 divergences of 7-14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences, cox1 sequences were able to correctly identify species including disparate geographic variants. The main problems with cox1 barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complement cox1.

Amphibians↗
PubMed1977

The effect of the divalent cation ionophore A23187 on amphibian melanophores and iridophores.

Calcium ions are required in the medium for the full darkening action of melanocyte-stimulating hormone, but not for its second messenger, cyclic adenosine monophosphate, on isolated frog skin. Thus, the possible effect of the divalent cation ionophore A23187 was studied, using the skin of Rana pipiens. It is a potent darkening agent over the range of 1-2 micrometer, as measured by reflectance change and microscopic observation. During the darkening response of A23187, melanosome dispersion takes place in both dermal and epidermal melanophores, as well as aggregation of iridophore reflecting platelets. The darkening is slowly reversible in Ringer's solution, thus it is not due to toxicity. The darkening is partially dependent on the presence of calcium in the medium under certain conditions, suggesting that the response involves both the uptake and release of calcium ions. Cytochalasin B reversibly inhibits the darkening produced by A23187. Since this drug is known to bring about the breakdown of microfilaments, its inhibitory action is in accord with the concept that the ionophore is stimulating microfilament function. A23187 administered to tissue cultured embryonic salamander (Ambystoma maculatum) melanophores produced an irreversible rounding up of the cells, suggesting a toxic effect. The results with frog skin are interpreted as supporting the concept that the action of melanocyte-stimulating hormone involves the production of pigment granule movements as a result of the interaction of calcium ions with intracellular microfilaments and possible also the breakdown of microtubules. The mobilization of calcium ions could be brought about by cyclic adenosine monophosphate.

Animals↗
PubMed2002

The evolution of development: two portraits of skull ossification in pipoid frogs.

Development creates morphology, and the study of developmental processes has repeatedly shed light on patterns of morphological evolution. However, development itself evolves as well, often concomitantly with changes in life history or in morphology. In this paper, two approaches are used to examine the evolution of skull development in pipoid frogs. Pipoids have highly unusual morphologies and life histories compared to other frogs, and their development also proves to be remarkable. First, a phylogenetic examination of skull bone ossification sequences reveals that jaw ossification occurs significantly earlier in pipoids than in other frogs; this represents a reversal to the primitive vertebrate condition. Early jaw ossification in pipoids is hypothesized to result from the absence of certain larval specializations possessed by other frogs, combined with unusual larval feeding behaviors. Second, thin-plate spline morphometric studies of ontogenetic shape change reveal important differences between pipoid skull development and that of other frogs. In the course of frog evolution, there has been a shift away from salamander-like patterns of ontogenetic shape change. The pipoids represent the culmination of this trend, and their morphologies are highly derived in numerous respects. This study represents the first detailed examination of the evolution of skull development in a diverse vertebrate clade within a phylogenetic framework. It is also the first study to examine ossification sequences across vertebrates, and the first to use thin-plate spline morphometrics to quantitatively describe ontogenetic trajectories.

Animals↗
PubMed2006

Flexible architecture of inducible morphological plasticity.

1. Predator-induced morphological defences are produced in response to an emergent predator regime. In natural systems, prey organisms usually experience temporal shifting of the composition of the predator assemblage and of the intensity of predation risk from each predator species. Although, a repetitive morphological change in response to a sequential shift of the predator regime such as alteration of the predator species or diminution of the predation risk may be adaptive, such flexible inducible morphological defences are not ubiquitous. 2. We experimentally addressed whether a flexible inducible morphological defence is accomplished in response to serial changes in the predation regime, using a model prey species which adopt different defensive morphological phenotypes in response to different predator species. Rana pirica (Matsui) tadpoles increased body depth and tail depth against the predatory larval salamander Hynobius retardatus (Dunn); on the other hand, they only increased tail depth against the predatory larval dragonfly Aeshna nigroflava (Martin). 3. Rana pirica tadpoles with the predator-specific phenotypes were subjected to removal or exchange of the predator species. After removal of the predator species, tadpoles with each predator-specific phenotype changed their phenotype to the nondefensive basic one, suggesting that both predator-specific phenotypes are costly to maintain. After an exchange of the predator species, tadpoles with each predator-specific phenotype reciprocally, flexibly shifted their phenotype to the now more suitable predator-specific one only by modifying their body part. The partial modification can effectively reduce time and energy expenditures involved in repetitive morphological changes, and therefore suggest that the costs of the flexible morphological changes are reduced.

Adaptation, Physiological↗
PubMed2004

Multiple paternity in an aggregate breeding amphibian: the effect of reproductive skew on estimates of male reproductive success.

Aggregate, or explosive, breeding is widespread among vertebrates and likely increases the probability of multiple paternity. We assessed paternity in seven field-collected clutches of the explosively breeding spotted salamander (Ambystoma maculatum) using 10 microsatellite loci to determine the frequency of multiple paternity and the number of males contributing to a female's clutch. Using the Minimum Method of allele counts, multiple paternity was evident in 70% of these egg masses. Simple allele counts underestimate the number of contributing males because this method cannot distinguish multiple fathers with common or similar alleles. Therefore, we used computer simulations to estimate from the offspring genotypes the most likely number of contributing fathers given the distributions of allele frequencies in this population. We determined that two to eight males may contribute to A. maculatum clutches; therefore, multiple paternity is a common strategy in this aggregate breeding species. In aggregate mating systems competition for mates can be intense, thus differential reproductive success (reproductive skew) among males contributing to a female's clutch could be a probable outcome. We use our data to evaluate the potential effect of reproductive skew on estimates of the number of contributing males. We simulated varying scenarios of differential male reproductive success, ranging from equal contribution to high reproductive skew among contributing sires in multiply sired clutches. Our data suggest that even intermediate levels of reproductive skew decrease confidence substantially in estimates of the number of contributing sires when parental genotypes are unknown.

Ambystoma↗