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PubMed2000

Microgravity and hypergravity effects on fertilization of the salamander Pleurodeles waltl (urodele amphibian).

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.

Animals↗
PubMed2005

Genetic exchange across a hybrid zone within the Iberian endemic golden-striped salamander, Chioglossa lusitanica.

The study of hybrid zones resulting from Pleistocene vicariance is central in examining the potential of genetically diverged evolutionary units either to introgress and merge or to proceed with further isolation. The hybrid zone between two mitochondrial lineages of Chioglossa lusitanica is located near the Mondego River in Central Portugal. We used mitochondrial and nuclear diagnostic markers to conduct a formal statistical analysis of the Chioglossa hybrid zone in the context of tension zone theory. Key results are: (i) cline centres are not coincident for all markers, with average widths of ca. 2-15 km; (ii) heterozygote deficit was not observed across loci near the transect centre; (iii) associations of parental allele combinations ('linkage disequilibrium'R) were not detected either across loci or across the transect. These observations suggest that the Chioglossa hybrid zone is not a tension zone with strong selection against hybrids but instead one shaped mostly by neutral mixing. The patterns uncovered suggest a complex history of populations over a small scale that may be common in southern Pleistocene refugia.

Animals↗
PubMed1999

C-terminal interactions modulate the affinity of GLAST glutamate transporters in salamander retinal glial cells.

1. Proteins that interact with the intracellular carboxy termini of neurotransmitter- and voltage-gated ion channels are known to control the subcellular localization of the channels, localize other proteins near those channels, and modulate channel activity. By contrast, little is known about the control of neurotransmitter transporter function by interacting proteins. 2. To competitively disrupt interactions of the C- and N-termini of the GLAST glutamate transporter with other proteins, we dialysed whole-cell patch-clamped retinal glia with peptides identical to the eight amino acids at the C- or N-termini of the transporter, and compared the effect on transporter-mediated currents with dialysis of scrambled versions of the same peptides. 3. Dialysis with the N-terminus peptide had no effect on the maximum glutamate-evoked current nor on the glutamate affinity of the transporter. Dialysis with the C-terminus peptide had no effect on the maximum current, but increased the affinity of the transporter for glutamate (compared with scrambled C-terminus peptide, and with N- and scrambled N-terminus peptides: Km decreased from 16 to 11 microM)). 4. These data suggest that disruption of an interaction between an intracellular protein and the last eight amino acids of the GLAST C-terminus, which have some similarity to the PDZ binding domain of ion channel C-termini, increases the glutamate affinity of GLAST. Thus, the interacting protein decreases the affinity of GLAST transporters. 5. Removing the GLAST C-terminus interaction increases the transporter current by 40 % at low glutamate concentrations. Thus, this interaction may significantly slow the removal of low concentrations of glutamate from the extracellular space, and affect the kinetics of retinal cell light responses.

ATP-Binding Cassette Transporters↗
PubMed2000

Modulation of the synaptic Ca2+ current in salamander photoreceptors by polyunsaturated fatty acids and retinoids.

Synaptic transmission between retinal photoreceptors and second-order neurones is controlled by an L-type Ca2+ conductance (gCa) in the photoreceptor inner segment. Modulation of this conductance therefore influences the flow of visual information to higher centres. Possible modulation of gCa by retinal factors was investigated using patch clamp and Ca2+ imaging. No significant modulation of gCa by retinal neurotransmitters nor by intracellular signalling pathways was found. gCa was inhibited by retinoids (all-trans retinal) and by polyunsaturated fatty acids (PUFAs) such as arachidonic acid and docosahexaenoic acid, which are known to be released in the retina by exposure to light. Some PUFAs tested are physiological substrates for the cyclo-oxygenase, lipoxygenase and epoxygenase pathways, but specific inhibitors of these pathways had no effect on the inhibition of gCa. Treatments designed to activate or inhibit G-protein-coupled pathways or protein kinases A and C similarly had no effect on the inhibition by PUFAs nor on gCa itself. Inhibitors of phosphatases 1 and 2A were also largely ineffective. The inhibition by PUFAs is, however, dependent on membrane potential, suggesting that it arises from a direct interaction of fatty acids with the Ca2+ channel. The effect was not use or frequency dependent, suggesting that the effect does not depend on channel gating state. Control by retinoids and by PUFAs may be an important mechanism by which the Ca2+ conductance, and consequently the transmission of the visual signal, is modulated at the first retinal synapse.

Adaptation, Ocular↗
PubMedDate not supplied

Pigmentary system of the adult alpine salamander Salamandra atra aurorae (Trevisan, 1982).

The pigmentary system of skin from adult specimens of the amphibian urodele Salamandra atra aurorae was investigated by light microscope, electron microscope, and biochemical studies. Yellow (dorsum and head) and black (flank and belly) skin was tested. Three chromatophore types are present in yellow skin: xanthophores, iridophores, and melanophores. Xanthophores are located in the epidermis whereas iridophores and melanophores are found in the dermis. Xanthophores contain types I, II, and III pterinosomes. Some pterinosomes are very electron-dense. Black skin has a single type of chromatophore: the melanophores. Some melanophores are located in the epidermis. In contrast to the dermal melanophores, these present, in addition to typical melanosomes, organelles with different morphology and vesicles having a limiting membrane and containing little amorphous material. Both skin types present some pteridines and flavins, though they are qualitatively and quantitatively more abundant in yellow skin extracts.

Animals↗
PubMed1989

Genetic evidence for linkage with the Z and W sex chromosomes of two distinct couples of alleles controlling larval and postmetamorphic skin pigmentation in salamander.

In Pleurodeles waltl, progeny resulting from a cross between 2 individuals of the Z/W sexual genotype include 25% of W/W individuals, while those issued from crossing a Z/W neomale with a W/W thelygenous female include 50% of W/W individuals. W/W individuals can be identified through the peptidase-1 zymogram since, in P. waltl, this enzyme is controlled by codominant alleles which are linked to the sex chromosomes. In such progeny, we discovered 2 mutant phenotypes affecting larval and postmetamorphic skin pigmentation in W/W individuals. These phenotypes are described herein. The study of their inheritance in several offspring provides evidence that they are controlled by 2 distinct genes, the recessive mutant alleles of which are linked to the W sex chromosome; moreover, in thelygenous W/W females, the differential segment does not prevent the occurrence of meiotic recombinations between W sex chromosomes. Mutant skin pigmentary phenotypes are easily identified and constitute a tool for rapid, efficient selection of individuals of the W/W sexual genotype.

Alleles↗
PubMed1972

Calcium dependence of ciliary activity in the oviduct of the salamander Necturus.

1. Ciliary activity in the oviduct of the mud puppy Necturus maculosus was monitored by a photometric technique in normal, decalcified, and Triton X-extracted preparations to investigate the regulatory role of calcium ions.2. The frequency of ciliary beating in the isolated tissue ranged from 0 to 12 beats/sec. The frequency in any one group of cells underwent large cyclical variations with periods of 2 min or more.3. Frequency of beating reached a maximum and remained at a plateau upon addition of 1 mM caffeine. Beating temporarily ceased upon removal of the caffeine.4. Ionophoretic injection of calcium ions into an active cell bathed in Ringer solution produced an increased rate of beating. In cells rendered quiescent by prior decalcification with EGTA, injected calcium rapidly restored ciliary activity.5. Epithelia extracted in Triton X-100 were inactive until reactivated by addition of ATP and magnesium ions. The frequency of beating increased between 0.1 and 4.0 mM ATP in 1 mM magnesium, and between 0.2 and 2.5 mM magnesium in 1 mM ATP.6. The frequency of beating in the ATP-reactivated, Triton-extracted tissue was independent of the calcium ion concentration.7. Cells inactivated by decalcification were reactivated by injection of ATP or by extracellular ATP levels as low as 3 x 10(-8)M.8. It is concluded that the frequency of beating depends directly on the concentration of the available energy source, presumably ATP, and that an indirect dependence of beating frequency on calcium concentration in the living tissue results from rate-limiting effects of intracellular calcium on metabolic steps in pathways leading to ATP synthesis.

Adenosine Triphosphate↗
PubMed1973

Evidence that axoplasmic transport of trophic factors is involved in the regulation of peripheral nerve fields in salamanders.

1. We have compared the effects of partial denervation with those of colchicine-induced block of axoplasmic flow, on the peripheral fields of nerves innervating the hind limb of salamanders.2. Acute application of colchicine solution (0.03-0.10 M) to spinal nerve 16 results in a dose-dependent increase in skin and muscle fields of the adjacent nerves (15 and 17). The time course, magnitude and distribution of the response to 0.10 M colchicine is not distinguishable from that of the compensatory sprouting of nerves 15 and 17 that occurs after section of nerve 16.3. In contrast to the situation with nerve section, sprouting of adjacent nerves occurred after colchicine applications which produced no behavioural deficit, no change in the peripheral field of the treated nerve, and no interference with impulse conduction in it; nor was there subsequent degeneration in the nerve.4. The same concentrations of colchicine reduced the axoplasmic flow of catecholamines and cholinesterase; treated nerves contained fewer microtubules than untreated controls.5. A similar application of colchicine solution to nerve 15 prevented it from sprouting in response to the stimulus provided by section of nerve 16.6. We conclude that nerve terminals are continuously supplied by axoplasmic flow with a trophic factor concerned with the regulation of nerve fields. When the supply of this factor is reduced, adjacent nerves sprout and invade the territory of the treated nerve. In addition, the ability of nerves to sprout is itself dependent upon the maintenance of axoplasmic flow.

Action Potentials↗
PubMed1977

The distribution of acetylcholine receptors on muscle fibres of regenerating salamander limbs.

1. Muscle fibres developing during limb regeneration were examined for responsiveness to acetylcholine (ACh) applied iontophoretically along the membrane. 2. Fibres which were uninnervated as well as those with non-transmitting synapses had high over-all sensitivities, with only minor variations along their length. 3. Functionally innervated fibres in which depolarization did not yet elicit action potentials had high over-all sensitivities, even when the synaptic potentials ahd amplitudes of 40-50 mV. In these the membrane in the vicinity of synapses tended to have sensitivities above the background level. 4. Upon the appearance of action potentials, several weeks after fibre innervation, the responsiveness to ACh began to decline in synapse free regions of the membrane. In mature muscle the sensitivity to ACh is restricted to sites of synaptic contact.

Acetylcholine↗
PubMed1978

Synaptic transmission to the horizontal cells in the retina of the larval tiger salamander.

1. The receptive field diameter for most horizontal cells far exceeds the lateral spread of processes for any cell. Therefore horizontal cells probably receive synaptic input from neighbours as well as from the photoreceptors. The electrical effects of these two synaptic inputs were studied. 2. We have characterized the electrical properties of the horizontal cell inputs by determining the current-voltage curves in dark and light. These curves were compared with those obtained in the presence of Co2+ or Mg2 was nearly identical to the curve in the light. 4. The putative transmitter substances glutamate, aspartate and GABA depolarized the cells by increasing conductance. Current-voltage curves measured in the presence of these substances intersected the dark and light curves at +50 mV, the same level at which the dark and light curves intersect. 5. The light response of cells uith broad receptive fields, between 1.0 and 2.0 mm, showed little or no change in conductance associated with the light response. The input resistance was near 20 Momega, and the current-voltage curves intersected at an extrapolated potential level near 200 mV. 6. In the presence of ACh, electrical properties of the broad field cells reverted to those of the narrow field cells: the receptive field was reduced to 0.5 mm, the imput resistance increased, and the current-voltage curves intersected near +50 mV. Thus ACh appeared to interrupt synaptic input from neighbouring horizontal cells. 7. The results confirm the suggestion that horizontal cells receive a tonic excitatory input from the photoreceptors which is decreased by light. They show that horizontal cells receive an additional input from their neighbours, not associated with a measurable conductance change. The input from neighbours is selectively interrupted by ACh, but the nature of this synapse and of the cholinergic action is not known.

Acetylcholine↗
PubMed1978

Transmission along and between rods in the tiger salamander retina.

1. The electrical pathways that couple the rods and that link the outer segments of the rods to the coupled network, were evaluated. Two separate micro-electrodes were inserted into the inner or outer segments of the same or neighbouring rods under visual control. Current was passed through one electrode, and the resulting potential recorded with the other. 2. The input resistance, measured at the inner or outer segment in a rod in the network, is strongly outward rectifying. It is typically near 40 Momega when the membrane is hyperpolarized 10 mV or more by extrinsic current, less than 10 Momega when the membrane is depolarized by 5 mV or more, and near 30 Momega at the no-current level. 3. When current is injected into the outer segment, the response in the inner segment is nearly identical with that at the outer segment, suggesting that the resistance coupling the segments is not high relative to the input resistance of the rod in the network. 4. Under voltage clamp the light response current for a rod in the network is of constant magnitude for potential levels between -80 and -20 mV. This suggests that there is little or no measurable light elicited conductance change associated with the response, possibly a consequence of coupling between rods. 5. The rod response increases with increasing diameter of a concentric test flash up to about 200 micron, or about 16 rod diameters. 6. When current is injected into one rod, the response in its immediate neighbours is between a quarter and one tenth that recorded in the injected rod for all potential levels in the injected rod. 7. The membrane time constant, measured in a rod in the network, is proportional to the voltage-dependent input resistance at 0.16 msec/Momega. With assumptions about the geometry of the rod network this represents a membrane capacitance of 1.5 muF/cm2. 8. The data can be approximated by a network model of square array. The model predicts that: the outer segment contributes less than half the current for the total rod response, the membrane resistance of an individual rod is greater than twice the measured input resistance for the rod in the network, near 60 Momega, and the coupling resistance for each arm of the network is about 4 times the individual rod resistance, near 240 Momega.

Animals↗
PubMed1978

Contacts between receptors and electrophysiologically identified neurones in the retina of the larval tiger salamander.

1. Following the intracellular recording of bipolar and horizontal cell responses, each unit was injected with horseradish peroxidase and a histochemical staining used to identify it at the level of the light and electron microscopes.2. Centre-depolarizing bipolar cells made contact with rods and cones at basal and ribbon junctions, the latter being fewer. Centre-hyperpolarizing bipolar cells made the same types of contacts with the receptors, but ribbon junctions predominated.3. It appears, therefore, that there is no fixed relationship between the sign of synaptic transmission from receptors to bipolar cells and the junctional features revealed by present methods for electron microscopy of tissue sections. Accordingly, the reason for the existence of more than one type of contact between receptors and bipolar cells remains to be determined.4. Light microscopy of the peroxidase-injected horizontal cells gave further support to the notion that type B responses are recorded from the cell body, and type A responses from the axon terminal, of a single type of horizontal cell. Electron microscopy showed that the processes (dendrites) originating from the cell body make ribbon and distal junctions with rods and cones, just as shown before for the axon terminals.5. As a result of these observations, it is possible to exclude one of two alternative hypotheses previously proposed to account for the properties of the surround responses recorded from the horizontal cell bodies.

Ambystoma↗
PubMed1982

The properties of single cones isolated from the tiger salamander retina.

1. The properties of isolated single cones were studied using the voltage-clamp technique, with two micro-electrodes inserted under visual control.2. Single cones had input resistances, when impaled with two electrodes, of up to 270 MOmega. This is probably lower than the true membrane resistance, because of damage by the impaling electrodes. The cone capacitance was about 85 pF.3. The cone membrane contains a time-dependent current, I(B), controlled by voltage, and a separate photosensitive current.4. The gated current, I(B), is an inward current with a reversal potential around -25 mV. It is activated by hyperpolarization over the range -30 to -80 mV, and at constant voltage obeys first order (exponential) kinetics. The gating time constant is typically 50 ms at the resting potential of -45 mV, rises to 170 ms at -70 mV, and decreases for further hyperpolarization.5. The spectral sensitivity curve of the cone light response peaks at 620 nm wave-length, and is narrower than the nomogram for vitamin A(2)-based pigments. The light responses of isolated cones are spectrally univariant.6. Voltage-clamped photocurrents were recorded at various membrane potentials, for light steps of various intensities. The photocurrent reversed at around -8 mV. The time course of the photocurrent, for a given intensity, was approximately independent of voltage (although its magnitude was voltage-dependent). The shape of the peak current-voltage relation of the light-sensitive current was independent of light intensity (although its magnitude was intensity-dependent).7. These results can be explained if: (a) light simply changes the number of photosensitive channels open, without altering the properties of an open channel; (b) the reactions controlling the production of internal transmitter, the binding of internal transmitter to the photosensitive channels, and the closing and opening of the channels are unaffected by the electric field in the cone membrane, even though at least some of these reactions take place in the membrane.8. I(B) plays only a small role in shaping the cone voltage response to light.

Ambystoma↗