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PubMed2005

Glutamate-induced Ca2+ influx in third-order neurons of salamander retina is regulated by the actin cytoskeleton.

Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.

Actins↗
PubMed2005

Variability of alkaloids in the skin secretion of the European fire salamander (Salamandra salamadra terrestris).

The two major alkaloids, samandarine and samandarone, were identified in the skin secretion of individual specimens from two populations of the European fire salamander (Salamandra salamandra terrestris) by gas chromatography/mass spectrometry. High intraspecific variability in the ratio of both alkaloids was observed, but also in individual specimens over a period of 4 months suggesting separate metabolic pathways of the compounds. Alkaloid synthesis appears to take place also in liver, testes and ovaries, whereas the larvae of the salamanders are entirely free of alkaloids.

Alkaloids↗
PubMed2005

Characterization of displaced bipolar cells in the tiger salamander retina.

In immunocytochemical studies of the tiger salamander retina, 17% of neurons in the outer nuclear layer did not label for recoverin, a photoreceptor marker. Lucifer yellow injection showed a population of cells in the ONL to be displaced bipolar cells, with axon terminals that stratified exclusively in the OFF sublamina of the inner plexiform layer (IPL), and predominately within the cone-dominated region of the OFF sublamina. Glutamate generated a dendritic cationic conductance increase in all displaced bipolar cells tested, and typical cone-dominated bipolar cell light responses were observed among displaced cells that stratified in the central IPL. We conclude that displaced bipolar cells in the tiger salamander retina are entirely OFF-center cells, and predominately cone-dominated cells.

Animals↗
PubMed1995

Diffusion coefficient of the cyclic GMP analog 8-(fluoresceinyl)thioguanosine 3',5' cyclic monophosphate in the salamander rod outer segment.

Cyclic GMP (cGMP) is the intracellular messenger mediating phototransduction in retinal rods, with its longitudinal diffusion in the rod outer segment (ROS) likely to be a factor in determining light sensitivity. From the kinetics of cGMP-activated currents in the truncated ROS of the salamander (Ambystoma tigrinum), the cGMP diffusion coefficient was previously estimated to be approximately 60 x 10(-8) cm2 s-1. On the other hand, fluorescence measurements in intact salamander ROS using 8-(fluoresceinyl)thioguanosine 3',5'-cyclic monophosphate (Fl-cGMP) led to a diffusion coefficient for this compound of 1 x 10(-8) cm2 s-1; after corrections for differences in size and in binding to cellular components between cGMP and Fl-cGMP, this gave an upper limit of 11 x 10(-8) cm2 s-1 for the cGMP diffusion coefficient. To properly compare the two sets of measurements, we have examined the diffusion of Fl-cGMP in the truncated ROS. From the kinetics of Fl-cGMP-activated currents, we have obtained a diffusion coefficient of 3 x 10(-8) cm2 s-1 for this analog; the cGMP diffusion coefficient measured from the same truncated ROSs was approximately 80 x 10(-8) cm2 s-1. Thus, a factor of 27 appears appropriate for correcting differences in size and intracellular binding between cGMP and Fl-cGMP. Application of this correction factor to the Fl-cGMP diffusion coefficient measurements by Olson and Pugh (1993) gives a cGMP diffusion coefficient of approximately 30 x 10(-8) cm2 s-1, in reasonable agreement with the value measured from the truncated ROS.

Ambystoma↗
PubMed2002

Pheromonal activation of vomeronasal neurons in plethodontid salamanders.

Pheromones from the mental glands of male plethodontid salamanders increase sexual receptivity in conspecific females. The pheromone enters the vomeronasal organ during courtship to produce this effect. Vomeronasal neurons from female Plethodon shermani were examined following exposure to male pheromone or saline placed on the nares. Agmatine was used in conjunction with the pheromone to enable immunocytochemical visualization of chemosensory neurons that were activated by the pheromone. Olfactory neurons exposed to pheromone or saline, and vomeronasal neurons exposed to saline did not demonstrate significant labeling. A population of vomeronasal neurons was intensely labeled following exposure to the pheromone. This study suggests that a specific population of vomeronasal neurons in a female plethodontid salamander is responsible for transmitting pheromonal information to the brain to produce modifications in behavior.

Animals↗
PubMed1992

A double-label study demonstrating that all serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina express GABA-like immunoreactivity.

A previous study localized serotonin-like immunoreactivity to amacrine cell populations in the larval tiger salamander retina. The present double-label immunocytochemical analysis of the tiger salamander retina was performed to determine if gamma-aminobutyric acid (GABA)-like immunoreactivity is expressed by serotonin-immunoreactive amacrine cells. More than 3,000 serotonin-amacrine cells were observed in double-label preparations, and all were found to express GABA-like immunoreactivity. This finding extends previous studies of serotonin-GABA coexistence in the retina by providing the first report of the co-localization of endogenous serotonin and GABA-like compounds in a retinal neuron.

Animals↗
PubMed1998

Postsynaptic responses of horizontal cells in the tiger salamander retina are mediated by AMPA-preferring receptors.

The postsynaptic responses of sign-preserving second-order retinal neurons (horizontal cells (HCs) and off-bipolar cells) are mediated by CNQX-sensitive AMPA/KA glutamate receptors. In this study we used receptor-specific allosteric regulators of desensitization and selected antagonists to determine the glutamate receptor subtypes in tiger salamander horizontal cells. Two approaches were employed in this study. The first was to measure postsynaptic currents induced by exogenously applied glutamate under voltage clamp conditions in living retinal slices; and the second was to record voltage responses controlled by endogenous glutamate released from photoreceptors in whole retinas. Application of 100 microM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 microgram ml-1 Co nA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 microM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 microM depolarized the HC dark membrane potential by about 5 mV and reduced the amplitudes of the voltage responses to dim lights, but enhanced the voltage responses to bright lights. Cyclothiazide had no effect on either the dark potential or the light responses of rods and cones. Con A at 300 microgram ml-1 had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 microM) hyperpolarized the HC dark membrane potential by about 55 mV and almost completely suppressed the light responses. We conclude from these results that the postsynaptic glutamate- and light-induced responses in the tiger salamander retinal horizontal cells are mediated by AMPA-preferring, and not kainate-preferring glutamate receptors. The functional roles of AMPA receptors and their desensitization kinetics in visual information processing are discussed.

Ambystoma↗
PubMed1999

Multiple types of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.

Spontaneous and light-evoked excitatory postsynaptic currents (sEPSCs and leEPSCs) in retinal ganglion cells of the larval tiger salamander were recorded under voltage clamp conditions from living retinal slices. sEPSCs were isolated from the spontaneous inhibitory postsynaptic currents (sIPSCs) by application of 100 M picrotoxin+1 microM strychnine. In addition to the previously reported sEPSCs [K. Matsui, N. Hosoi, M. Tachibana, Excitatory synaptic transmission in the inner retina: pair recordings of bipolar cells and neurons of the ganglion cell layer, J. Neurosci. 18 (1998) 4500-4510; W.R. Taylor, E. Chen, D.R. Copenhagen, Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells, J. Physiol. 486 (1995) 207-221] [which are equivalent to our fast AMPA receptor-mediated sEPSCs (fAMPAsEPSCs)], we found another type of AMPA receptor-mediated sEPSC with slower rise and decay time courses and larger peak amplitudes (sAMPAsEPSCs), and the NMDA receptor-mediated sEPSCs (NMDAsEPSCs) in ON-OFF ganglion cells. The frequency of all three types of sEPSCs is greatly reduced by cobalt (with zero calcium) and increased by hyperosmotic solution, suggesting that these events are mediated by calcium-dependent exocytosis of glutamatergic synaptic vesicles. The amplitude histograms of sEPSCs do not show multiple peaks, suggesting that larger events are not discrete multiples of elementary events, or quanta, of similar neurotransmitter contents, as in the neuromuscular junction [P. Fatt, B. Katz, Spontaneous subthreshold activity at motor nerve endings, J. Physiol. 117 (1952) 109-128]. The average I-V relations of the fAMPAsEPSCs and sAMPAsEPSCs were outward rectified with reversal potentials at -12.2 mV and -10.8 mV, and that of the NMDAsEPSCs was N-shaped with a reversal potential at -5.8 mV. The average conductance increase associated with a single fAMPAsEPSC, a single sAMPAsEPSC, and a single NMDAsEPSC were 163. 26+/-51.02 pS, 233.33+/-163.64 pS, and 37.5+/-50.0 pS at -110 mV; 241.67+/-22.92 pS, 444.90+/-469.94 pS, and 25.93+/-70.37 pS at -60 mV; and 440.48+/-183.33 pS, 1,192.68+/-651.22 pS, and 517.71+/-238. 24 pS at +30 mV, respectively. The average frequency of the three sEPSCs at +30 mV were 15 Hz, 3.7 Hz and 3.6 Hz, respectively. The rise time (time to peak) of fAMPAsEPSCs was 1.5+/-1.05 ms and the decay time could be fitted with a single exponential with an average time constant of 3.4+/-4.1 ms. The rise and decay time course of the sAMPAsEPSCs and NMDAsEPSCs were much slower and sawtooth-shaped, and each 'sawtooth' had time course and amplitude similar to those of individual fAMPAsEPSCs. We propose that each fAMPAsEPSC is mediated by single or synchronized multiples of glutamatergic synaptic vesicles from bipolar cells, and each sAMPAsEPSC or NMDAsEPSC is mediated by larger clusters of synaptic vesicles triggered by spontaneous calcium spikes in bipolar cell axon terminals [J. Burrone, L. Lagnado, Electrical resonance and calcium influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina, J. Physiol. 505 (1997) 571-584; D. Zenisek, G. Matthews, Calcium action potentials in retinal bipolar neurons, Vis. Neurosci. 15 (1998) 69-75].

Action Potentials↗
PubMed2003

Corticosterone concentrations in free-living spotted salamanders (Ambystoma maculatum).

A non-lethal technique for drawing repeated blood samples from spotted salamanders (Ambystoma maculatum) was used to examine sex, seasonal, and capture technique differences in the physiological stress response under natural, undisturbed conditions. Baseline and stress-induced (30 min of handling and restraint) corticosterone (CORT) concentrations were measured at night during the spring migrations into and out of the breeding pond, as well as during the fall migration to over-wintering sites. Females had significantly higher CORT concentrations than males during the spring migration toward the breeding pond, but this difference was not present when animals emerged from the pond post-breeding or during the fall migration. CORT concentrations did not vary seasonally, but during the acute stress response, CORT concentrations nearly doubled during the spring inbound migration, exhibited a nearly significant increase during the spring outbound migration, and did not change in the fall. Allowing animals to sit overnight in the buckets in which they were captured elicited CORT concentrations that tended to be higher (although not statistically significant) than when animals were sampled on the night of capture. The ability to sample blood from spotted salamanders using a non-lethal technique offers an opportunity to better understand both amphibian physiology and ecology.

Ambystoma↗
PubMed2003

Targeted expression of androgen receptors in tooth-forming tissues of a neotropical salamander (Bolitoglossa schizodactyla) enables region-specific formation of dimorphic types of teeth in the male.

Bicuspid teeth are a morphological characteristic in adult metamorphosed urodeles. However, during the mating season the males of many species of plethodontid salamanders (Urodela, Plethodontidae) from northern America bear large monocuspid teeth on their jaws. Since androgen levels are elevated during the mating season, it is likely that the formation of monocuspid teeth is controlled by androgens. However, many plethodontid salamanders from southern Central America live in habitats which lack significant seasonal changes and therefore are no seasonal breeders. These males constantly bear monocuspid teeth, however their presence is restricted to the premaxillary bone, which forms the most frontal element of the upper jaw. Teeth of all other dentigerous bones are bicuspid. This study is based on an immunohistochemical localisation of androgen receptors and reveals a differential expression of androgen receptors in the three separate dental laminae of the upper jaw of Bolitoglossa schizodactyla (Urodela, Plethodontidae) from western Panama. Our findings suggest that the restriction of androgen receptor expression to the premaxillary dental lamina of the male and its absence in all other tooth-forming tissues leads to the differences in tooth morphogenesis. In conclusion, a differential and cell-specific expression of androgen receptors in urodeles-similar to the situation in mammalian species-allows a selective response of differentiating tissues to the hormonal status of the animal.

Animals↗
PubMed1987

Allometry of red cell oxygen binding and hematology in larvae of the salamander, Ambystoma tigrinum.

Very few studies have attempted to relate blood characteristics to body mass within, rather than between, species. Thus, respiratory and hematological properties of the blood of larval tiger salamanders (Ambystoma tigrinum) have been measured in animals ranging in body mass from approximately 2 to 112 g. This amphibian species was chosen because larvae of very different body mass may be of similar developmental stage, minimizing interference from ontogenetic factors. Mean corpuscular volume, mean corpuscular Hb, hematocrit, blood Hb concentration, blood O2 capacity and Hill's n were all positively correlated with body mass (P less than 0.005). Blood O2 affinity (P50) and Bohr shift were not significantly correlated with body mass (P greater than 0.20). The findings are discussed in the context of the general effect of body mass upon metabolic rate in vertebrates, as well as environmental and biological factors specific to larvae of the tiger salamander.

Animals↗
PubMed2001

Localization of neurotransmitters and calcium binding proteins to neurons of salamander and mudpuppy retinas.

We wished to identify the different types of retinal neurons on the basis of their content of neuroactive substances in both larval tiger salamander and mudpuppy retinas, favored species for electrophysiological investigation. Sections and wholemounts of retinas were labeled by immunocytochemical methods to demonstrate three calcium binding protein species and the common neurotransmitters, glycine, GABA and acetylcholine. Double immunostained sections and single labeled wholemount retinas were examined by confocal microscopy. Immunostaining patterns appeared to be the same in salamander and mudpuppy. Double and single cones, horizontal cells, some amacrine cells and ganglion cells were strongly calbindin-immunoreactive (IR). Calbindin-IR horizontal cells colocalized GABA. Many bipolar cells, horizontal cells, some amacrine cells and ganglion cells were strongly calretinin-IR. One type of horizontal cell and an infrequently occurring amacrine cell were parvalbumin-IR. Acetylcholine as visualized by ChAT-immunoreactivity was seen in a mirror-symmetric pair of amacrine cells that colocalized GABA and glycine. Glycine and GABA colocalized with calretinin, calbindin and occasionally with parvalbumin in amacrine cells.

Acetylcholine↗
PubMed1996

Synaptic inputs mediating bipolar cell responses in the tiger salamander retina.

Postsynaptic receptors in bipolar cells were studied by focal application of glutamate and GABA to the outer and inner plexiform layers (OPL and IPL) under visual guidance in living retinal slices of the tiger salamander. Two different types of conductance change could be elicited in bipolar cells by applying glutamate to the OPL. In off-center cells, which had axon telodendria ramifying in the distal 55% of the IPL, glutamate elicited a conductance increase associated with a reversal potential near -5 mV. In on-center cells, which had telodendria stratified in the proximal 45% of the IPL, glutamate caused a conductance decrease associated with a reversal potential near -11 mV. These observations suggest that glutamate gates relatively nonspecific cation channels at synapses between photoreceptors and bipolar cell dendrites. Application of glutamate to the IPL elicited no conductance change in Co2+ Ringer's solution, but in normal Ringer's it generated a conductance increase associated with a reversal potential near the chloride equilibrium potential (ECl). These findings are consistent with the notion that glutamate receptors exist in GABAergic and/or glycinergic amacrine cells, and that glutamate in the IPL depolarizes these cells, causing GABA and/or glycine release and the opening of chloride channels in bipolar cell axon terminals. In Co2+ Ringer's, application of GABA at the OPL elicited no conductance changes in bipolar cells, suggesting that GABA receptors do not exist on bipolar cell dendrites. Applied at the IPL, GABA elicited large conductance increases associated with a reversal potential near ECl. Implications of these results for the functional circuitry of the tiger salamander retina are discussed.

Ambystoma↗
PubMed1998

Localization and function of five glutamate transporters cloned from the salamander retina.

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.

ATP-Binding Cassette Transporters↗
PubMed1998

gamma-aminobutyric acid transporter-mediated current from bipolar cells in tiger salamander retinal slices.

About 10% of bipolar cells in salamander retina synthesize and take up gamma-aminobutyric acid (GABA), and may use GABA as a neurotransmitter. As GABA uptake is electrogenic, bipolar cells expressing GABA transporters (GATs) should give transport current (IGAT) to extracellular GABA. Using whole-cell patch recording, 28 bipolar cells responded to 30-200 microM GABA puffed to the axon terminals with a picrotoxin (PTX)-sensitive chloride current (ICI) only. Another three bipolar cells had, in addition to ICI, a PTX-resistant, sodium-dependent current that was completely and reversibly blocked by NO-711, an IGAT inhibitor, indicating that this component was an IGAT. This finding provides further support for a subset of GABAergic bipolar cells in the salamander retina.

Animals↗
PubMed1999

Anatomical and electrophysiological evidence for GABAergic bipolar cells in tiger salamander retina.

Our previous work showed that about 12% of bipolar cells in salamander retina synthesize and take up gamma-aminobutyric acid (GABA), are GABA transporter (GAT)-immunoreactive, and respond with a GAT current to extracellularly applied GABA, suggesting that these bipolar cells use GABA, in addition to glutamate, as a neurotransmitter. Further support for this idea was obtained in this study by use of immunogold electron microscopy and whole-cell patch clamp electrophysiology. Ultrastructural analysis showed that amacrine cell and ganglion cell processes were postsynaptic to GABA-immunoreactive synapses made by bipolar cell axon terminals. Whole-cell recordings were obtained from amacrine and ganglion cells in response to activation of bipolar cells by puffing KCl at their dendrites in the outer plexiform layer. Inhibitory postsynaptic currents were observed in several third order neurons, even after blocking the excitatory postsynaptic responses, generated in the inner plexiform layer, with a combined application of NMDA and non-NMDA receptor antagonists, AP-5 and CNQX. These ultrastructural and electrophysiological data support our previous neurochemical results, and suggest that the retinal through-information pathway in salamander includes both inhibitory GABAergic as well as excitatory glutamatergic synaptic mechanisms.

Action Potentials↗
PubMed2003

Complete nucleotide sequence of the mitochondrial genome of a salamander, Mertensiella luschani.

The complete nucleotide sequence (16,650 bp) of the mitochondrial genome of the salamander Mertensiella luschani (Caudata, Amphibia) was determined. This molecule conforms to the consensus vertebrate mitochondrial gene order. However, it is characterized by a long non-coding intervening sequence with two 124-bp repeats between the tRNA(Thr) and tRNA(Pro) genes. The new sequence data were used to reconstruct a phylogeny of jawed vertebrates. Phylogenetic analyses of all mitochondrial protein-coding genes at the amino acid level recovered a robust vertebrate tree in which lungfishes are the closest living relatives of tetrapods, salamanders and frogs are grouped together to the exclusion of caecilians (the Batrachia hypothesis) in a monophyletic amphibian clade, turtles show diapsid affinities and are placed as sister group of crocodiles+birds, and the marsupials are grouped together with monotremes and basal to placental mammals. The deduced phylogeny was used to characterize the molecular evolution of vertebrate mitochondrial proteins. Amino acid frequencies were analyzed across the main lineages of jawed vertebrates, and leucine and cysteine were found to be the most and least abundant amino acids in mitochondrial proteins, respectively. Patterns of amino acid replacements were conserved among vertebrates. Overall, cartilaginous fishes showed the least variation in amino acid frequencies and replacements. Constancy of rates of evolution among the main lineages of jawed vertebrates was rejected.

Amino Acid Sequence↗
PubMedDate not supplied

Altered behaviors in male mice, male quail, and salamander larvae following early exposures to the estrogenic pesticide methoxychlor.

Numerous publications show that methoxychlor (MXC), in use today as a DDT substitute, is a reproductive toxicant; it produces deleterious effects on the structure and function of the reproductive organs in exposed species. Exposure of mice (33 mg/kg body weight) to purified (95%) MXC at the time of implantation, or injection, of 5 mg MXC into freshly laid quail eggs prior to artificial incubation, altered sexual arousal and sexual behavior in adult males of both species. When placed near a plastic partition with an estrus female behind it, the MXC-exposed male mice showed no sexual arousal, spent less time near the partition, and exhibited lower testosterone levels. Similarly, adult quail males that were exposed to MXC during incubation showed a lack of sexual interest and copulatory behavior when presented with a receptive female. Some males showed a longer latency period before mounting the female, while others did not show any sexual interest at all. Exposure of salamander embryos to purified MXC at or above 0.3 microM (0.1 mg/l) induced precocial hatching of embryos and reduced the startle response and the distance traveled in response to startle. Exposing hatched larvae to MXC for 3 days also resulted in a blunted startle response. Due to the blunted startle response and decreased avoidance travel, the exposed salamander larvae appear more susceptible to predation and these effects may contribute to amphibian population declines. The results of these studies indicate that developmental exposures to environmental chemicals with hormonal activities produce undesirable behaviors that may affect population dynamics and survivability of exposed species.

Animals↗