Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Acute toxicity of copper to the larval stage of three species of ambystomatid salamanders.

Copper (Cu) appears to be consistently more toxic to anuran species relative to other vertebrate taxa. There are limited Cu toxicity data for salamanders; of the few studies conducted on salamanders, most examined Cu effects on the embryonic, but not the larval, stage. We performed acute toxicity experiments, to quantify LC50s, on Harrison stage 46 larvae (free swimming hatchlings with egg yolk completely absorbed) of three ambystomatid salamander species. Each LC50 experiment used exposure concentrations of 10, 20, 30, 40, 50, and 60 µg/L with 10 replicates per concentration each containing one larva. We found very high toxicity for all species compared to previously published research on the embryo stage. Specifically, the 4-d LC50s for Ambystoma tigrinum and A. opacum were 35.3 and 18.73 µg/L, respectively. The same Cu concentrations caused similar toxicity to A. talpoideum (LC50 = 47.88 µg/L), but exposures required up to 48 d to elicit the same level of mortality. A time-to-event analysis indicated that time to mortality was significantly affected by Cu concentration. Additionally, for A. talpoideum, we observed that elevated levels of Cu decreased growth rate. Comparisons with previously reported Cu toxicity for embryos suggest that, as with fish, Cu may be more toxic to larval salamander stages than for embryos. Further, our data suggest that Cu is an important environmental contaminant that deserves increased scrutiny on the potential for population-level effects where contamination has occurred in wetlands and streams inhabited by salamanders.

Bioaccumulation of methylmercury in wood frogs and spotted salamanders in Vermont vernal pools.

Mercury (Hg) has accumulated in forested landscapes in the Northeastern U.S., and hotspots with enhanced deposition have been identified throughout the region. Due to a variety of favorable landscape characteristics, including relatively high dissolved organic carbon (DOC), fluctuating water levels, and low pH and dissolved oxygen, vernal pools provide ideal conditions for the conversion of Hg to its more toxic and bioavailable form, methylmercury (MeHg). Yet little is known about the concentrations, speciation, and bioavailability of Hg in vernal pools, or its bioaccumulation in vernal pool fauna and potential export into terrestrial systems. We investigated the role of forest cover type on the bioaccumulation of MeHg in wood frog (Lithobates sylvatica) and spotted salamander (Ambystoma maculatum) eggs, larvae, and adults, and investigated relationships among MeHg and water chemistry (pH, DOC). Water samples from pools located in coniferous stands had greater concentrations of THg and MeHg compared to deciduous pool water, and showed significant positive correlation to DOC (r = 0.683, P < 0.001) and correlated negatively with pH (r = -0.613, P < 0.001). Methylmercury levels in amphibian embryos were similar between the two species (L. sylvatica mean = 5.4 ng/g dw; A. maculatum mean = 3.5 ng/g dw). Concentrations of MeHg increased substantially in larvae, and were significantly greater in A. maculatum (mean = 237.6 ng/g ± 18.5 SE) than L. sylvatica larvae (62.5 ng/g ± 5.7 SE). Forest cover type did not explain variation in MeHg concentration among amphibian embryos or larvae. Methylmercury levels in adult tissue samples were significantly greater in A. maculatum (mean = 79.9 ng/g ± 8.9 SE) compared to L. sylvatica (mean = 47.7 ng/g ± 9.7 SE). This research demonstrates that vernal pools are important hotspots where amphibians bioaccumulate MeHg, which may then be transferred to terrestrial ecosystems. The abundance of amphibian larvae suggests they could be important bioindicators for monitoring MeHg loading and bioavailability.

Natural tissue concentrations in adult Ambystoma maculatum and larval DNA damage from exposure to arsenic and chromium.

Arsenic (As) and chromium (Cr) are two contaminants that are detected in aquatic and terrestrial habitats. Using the spotted salamander, Ambystoma maculatum, to assess impacts from these contaminants may be advantageous as adults live and breed in such environments. Adult amphibians typically exhibit elevated tissue concentrations of contaminants present in their environment, while larval stages were found to exhibit increased sensitivity to pollutants. From January through March of 2015, during the spring breeding season, 5 adults and approximately 32 egg masses were collected from a local breeding site. Field levels of As and Cr ranged from 5.99 to 8.88 µg/L and 1.45 to 2 µg/L, respectively, while mean adult As tissue concentrations were 56.74 µg/g dry weight for heart, 0.92 µg/g for liver, and 1.21 µg/g for tail tissue. Mean tissue concentrations for Cr were 87.64 µg/g for heart, 1.47 µg/g for liver, and 6.92 µg/g for tail. Developing larvae that were collected from the field and exposed in a lab setting for 12 d to 0.2 or 20 mg/L of either As or Cr displayed little DNA damage attributed to As, but marked damage due to exposure to 20 mg/L Cr when assessed using the comet assay. Exposure to a mixture of either 0.25:0.1 or 25:10 mg/L As and Cr resulted in significant DNA damage at the lower concentration of 0.25:0.1 mg/L. As adult spotted salamanders were found to possess high concentrations of these contaminants in cardiac tissue, and larvae were shown to be susceptible to DNA damage from increased exposures, assessing impacts and potential declines of amphibian populations exposed to As and Cr is needed.

Developmental and interactive effects of arsenic and chromium to developing Ambystoma maculatum embryos: Toxicity, teratogenicity, and whole-body concentrations.

Anthropogenic activity has contributed to elevated environmental concentrations of arsenic (As) and chromium (Cr). The spotted salamander, Ambystoma maculatum, may be useful for identifying developmental effects produced by exposure to these contaminants as adults breed and larvae develop in water that may contain As or Cr. Three sample sets among 700 developing larvae were exposed to a range of As, Cr, or 2.5:1 mixture of As:Cr concentrations, respectively. From these 700 larvae, samples containing approximately 24 larvae showed different patterns of whole-body As and Cr from individual and mixture exposure. Whole-body As concentrations were 20.27 and 45.4 µg/g dry weight for larvae exposed to 20 mg/L As and 25:10 mg/L As:Cr, respectively, while whole-body Cr concentrations were 24.8 and 22 µg/g dry weight for larvae exposed to 20 mg/L Cr and 25:10 As:Cr, respectively. Observed malformations included edema, tail kinking, facial deformities, and abnormal bending. Twelve-day lethal concentrations for As and Cr in Ambystoma maculatum larvae were 261.17 mg/L and 71.93 mg/L, respectively, while 12-d effective concentrations to induce malformations were 158.82 and 26.05 mg/L, giving teratogenic indices of 1.64 and 2.76 for individual metal exposure. Exposure to a mixture of As and Cr resulted in a response addition and yielded lower lethal and effective concentration values with a teratogenic index of 2.78, indicating that these contaminants are developmentally toxic at lower concentrations when exposed as a mixture. Data demonstrate that As and Cr affect development of amphibian larvae, and that Ambystoma maculatum may be a useful indicator of environmental toxicity for these metals.

Sources of protons and a role for bicarbonate in inhibitory feedback from horizontal cells to cones in Ambystoma tigrinum retina.

KEY POINTS: In the vertebrate retina, photoreceptors influence the signalling of neighbouring photoreceptors through lateral-inhibitory interactions mediated by horizontal cells (HCs). These interactions create antagonistic centre-surround receptive fields important for detecting edges and generating chromatically opponent responses in colour vision. The mechanisms responsible for inhibitory feedback from HCs involve changes in synaptic cleft pH that modulate photoreceptor calcium currents. However, the sources of synaptic protons involved in feedback and the mechanisms for their removal from the cleft when HCs hyperpolarize to light remain unknown. Our results indicate that Na+ -H+ exchangers are the principal source of synaptic cleft protons involved in HC feedback but that synaptic cleft alkalization during light-evoked hyperpolarization of HCs also involves changes in bicarbonate transport across the HC membrane. In addition to delineating processes that establish lateral inhibition in the retina, these results contribute to other evidence showing the key role for pH in regulating synaptic signalling throughout the nervous system. ABSTRACT: Lateral-inhibitory feedback from horizontal cells (HCs) to photoreceptors involves changes in synaptic cleft pH accompanying light-evoked changes in HC membrane potential. We analysed HC to cone feedback by studying surround-evoked light responses of cones and by obtaining paired whole cell recordings from cones and HCs in salamander retina. We tested three potential sources for synaptic cleft protons: (1) generation by extracellular carbonic anhydrase (CA), (2) release from acidic synaptic vesicles and (3) Na+ /H+ exchangers (NHEs). Neither antagonizing extracellular CA nor blocking loading of protons into synaptic vesicles eliminated feedback. However, feedback was eliminated when extracellular Na+ was replaced with choline and significantly reduced by an NHE inhibitor, cariporide. Depriving NHEs of intracellular protons by buffering HC cytosol with a pH 9.2 pipette solution eliminated feedback, whereas alkalinizing the cone cytosol did not, suggesting that HCs are a major source for protons in feedback. We also examined mechanisms for changing synaptic cleft pH in response to changes in HC membrane potential. Increasing the trans-membrane proton gradient by lowering the extracellular pH from 7.8 to 7.4 to 7.1 strengthened feedback. While maintaining constant extracellular pH with 1 mm HEPES, removal of bicarbonate abolished feedback. Elevating intracellular bicarbonate levels within HCs prevented this loss of feedback. A bicarbonate transport inhibitor, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), also blocked feedback. Together, these results suggest that NHEs are the primary source of extracellular protons in HC feedback but that changes in cleft pH accompanying changes in HC membrane voltage also require bicarbonate flux across the HC membrane.

Proteomic analysis of fibroblastema formation in regenerating hind limbs of Xenopus laevis froglets and comparison to axolotl.

BACKGROUND: To gain insight into what differences might restrict the capacity for limb regeneration in Xenopus froglets, we used High Performance Liquid Chromatography (HPLC)/double mass spectrometry to characterize protein expression during fibroblastema formation in the amputated froglet hindlimb, and compared the results to those obtained previously for blastema formation in the axolotl limb. RESULTS: Comparison of the Xenopus fibroblastema and axolotl blastema revealed several similarities and significant differences in proteomic profiles. The most significant similarity was the strong parallel down regulation of muscle proteins and enzymes involved in carbohydrate metabolism. Regenerating Xenopus limbs differed significantly from axolotl regenerating limbs in several ways: deficiency in the inositol phosphate/diacylglycerol signaling pathway, down regulation of Wnt signaling, up regulation of extracellular matrix (ECM) proteins and proteins involved in chondrocyte differentiation, lack of expression of a key cell cycle protein, ecotropic viral integration site 5 (EVI5), that blocks mitosis in the axolotl, and the expression of several patterning proteins not seen in the axolotl that may dorsalize the fibroblastema. CONCLUSIONS: We have characterized global protein expression during fibroblastema formation after amputation of the Xenopus froglet hindlimb and identified several differences that lead to signaling deficiency, failure to retard mitosis, premature chondrocyte differentiation, and failure of dorsoventral axial asymmetry. These differences point to possible interventions to improve blastema formation and pattern formation in the froglet limb.

Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Sign-preserving and sign-inverting synaptic interactions between rod and cone photoreceptors in the dark-adapted retina.

We show that various types of rods and cones in the dark-adapted salamander retina are electrically coupled with linear and symmetrical junctional conductances G(j) (40-223 pS) and a rank order: Rod(C)-large single cone, rod-large single cone, rod-small single cone, rod-accessory double cone and rod-principal double cone. By systematically comparing the transjunctional current-voltage (I(j)-V(j)) relations and average G(j) values of the five types of rod-cone pairs recorded at day and night times, our results suggest that the differences in G(j) values among various types of rod-cone pairs are not caused by circadian differences, and the circadian-dependent changes in rod-cone coupling observed in the fish and rodent retinas are not present in the tiger salamander. In addition to rod-cone coupling, there is a sign-inverting, unidirectional rod→cone current I(RC), and the I(RC)-V(Cone) relations are linear, with a reversal potential near the chloride reversal potential E(Cl). I(RC) can be observed in rods and cones separated by at least 260 µm, and its waveform resembles that of the rod-elicited horizontal cell (HC) response I(HC). A glutamate transporter-associated chloride channel blocker TBOA suppresses I(RC) but not I(HC). These results suggest that I(RC) is largely mediated by HCs via a sign-inverting feedback chemical synapse associated with a chloride channel. I(RC) significantly reduced rod→cone coupling in the frequency range below 15 Hz, allowing better separation of rod and cone signals in the dark-adapted retina.

Lateral mobility of L-type calcium channels in synaptic terminals of retinal bipolar cells.

PURPOSE: Efficient and precise release of glutamate from retinal bipolar cells is ensured by the positioning of L-type Ca(2+) channels close to release sites at the base of the synaptic ribbon. We investigated whether Ca(2+) channels at bipolar cell ribbon synapses are fixed in position or capable of moving in the membrane. METHODS: We tracked the movements of individual L-type Ca(2+) channels in bipolar cell terminals after labeling channels with quantum dots (QDs) attached to α(2)δ(4) accessory Ca(2+) channel subunits via intermediary antibodies. RESULTS: We found that individual Ca(2+) channels moved within a confined domain of 0.13-0.15 µm(2) in bipolar cell terminals, similar to ultrastructural estimates of the surface area of the active zone beneath the ribbon. Disruption of actin expanded the confinement domain indicating that cytoskeletal interactions help to confine channels at the synapse, but the relatively large diffusion coefficients of 0.3-0.45 µm(2)/s suggest that channels are not directly anchored to actin. Unlike photoreceptor synapses, removing membrane cholesterol did not change domain size, indicating that lipid rafts are not required to confine Ca(2+) channels at bipolar cell ribbon synapses. CONCLUSIONS: The ability of Ca(2+) channels to move within the presynaptic active zone suggests that regulating channel mobility may affect release from bipolar cell terminals.

The modulatory role of taurine in retinal ganglion cells.

Taurine (2-aminoethylsuphonic acid) is present in nearly all animal tissues, and is the most abundant free amino acid in muscle, heart, CNS, and retina. Although it is known to be a major cytoprotectant and essential for normal retinal development, its role in retinal neurotransmission and modulation is not well understood. We investigated the response of taurine in retinal ganglion cells, and its effect on synaptic transmission between ganglion cells and their presynaptic neurons. We find that taurine-elicited currents in ganglion cells could be fully blocked by both strychnine and SR95531, glycine and GABA(A) receptor antagonists, respectively. This suggests that taurine-activated receptors might share the antagonists with GABA and glycine receptors. The effect of taurine at micromolar concentrations can effectively suppress spontaneous vesicle release from the presynaptic neurons, but had limited effects on light-evoked synaptic signals in ganglion cells. We also describe a metabotropic effect of taurine in the suppression of light-evoked response in ganglion cells. Clearly, taurine acts in multiple ways to modulate synaptic signals in retinal output neurons, ganglion cells.

Taurine regulation of voltage-gated channels in retinal neurons.

Taurine activates not only Cl(-)-permeable ionotropic receptors but also receptors that mediate metabotropic responses. The metabotropic property of taurine was revealed in electrophysiological recordings obtained after fully blocking Cl(-)-permeable receptors with an inhibitory "cocktail" consisting of picrotoxin, SR95531, and strychnine. We found that taurine's metabotropic effects regulate voltage-gated channels in retinal neurons. After applying the inhibitory cocktail, taurine enhanced delayed outward rectifier K(+) channels preferentially in Off-bipolar cells, and the effect was completely blocked by the specific PKC inhibitor, GF109203X. Additionally, taurine also acted through a metabotropic pathway to suppress both L- and N-type Ca(2+) channels in retinal neurons, which were insensitive to the potent GABA(B) receptor inhibitor, CGP55845. This study reinforces our previous finding that taurine in physiological concentrations produces a multiplicity of metabotropic effects that precisely govern the integration of signals being transmitted from the retina to the brain.

Symbiosis maintenance in the facultative coral, Oculina arbuscula, relies on nitrogen cycling, cell cycle modulation, and immunity.

Symbiosis with unicellular algae in the family Symbiodiniaceae is common across tropical marine invertebrates. Reef-building corals offer a clear example of cellular dysfunction leading to a dysbiosis that disrupts entire ecosystems in a process termed coral bleaching. Due to their obligate symbiotic relationship, understanding the molecular underpinnings that sustain this symbiosis in tropical reef-building corals is challenging, as any aposymbiotic state is inherently coupled with severe physiological stress. Here, we leverage the subtropical, facultatively symbiotic and calcifying coral Oculina arbuscula to investigate gene expression differences between aposymbiotic and symbiotic branches within the same colonies under baseline conditions. We further compare gene ontology (GO) and KOG enrichment in gene expression patterns from O. arbuscula with prior work in the sea anemone Exaiptasia pallida (Aiptasia) and the salamander Ambystoma maculatum-both of which exhibit endophotosymbiosis with unicellular algae. We identify nitrogen cycling, cell cycle control, and immune responses as key pathways involved in the maintenance of symbiosis under baseline conditions. Understanding the mechanisms that sustain a healthy symbiosis between corals and Symbiodiniaceae algae is of urgent importance given the vulnerability of these partnerships to changing environmental conditions and their role in the continued functioning of critical and highly diverse marine ecosystems.

Acute toxicity and tissue distributions of malathion in Ambystoma tigrinum.

The kinetics of the bioaccumulation of malathion (O,O-dimethyl phosphorodithioate of diethyl mercaptosuccinate) and the biological impact of exposure for tiger salamanders, Ambystoma tigrinum, were assessed through exposure to soil surface contaminated with 50 microg/cm(2) or 100 microg/cm(2 )malathion and ingestion of an earthworm exposed to soil contaminated with 200 microg/cm(2) malathion. Malathion and malaoxon burdens in salamanders sampled at different times after exposure(s) were measured by gas chromatography in four tissue/organ subgroups: liver, epaxial muscle, pooled viscera (except the liver and brain), and pooled avisceral carcass (muscle, skin, and bone). The total tiger salamander xenobiotic burdens were calculated from these data. The malathion/malaoxon burden 1 day after exposure was greatest in the avisceral carcass and 2 days after exposure was greatest in the viscera. Bioconcentration and bioaccumulation factors remained less than unity throughout the experiment and did not support the hypothesis of bioaccumulation of malathion in the tiger salamander. Biological impact was assessed with a colorimetric brain cholinesterase microassay. Brain cholinesterase activities in salamanders exposed to malathion-contaminated soil (50 microg/cm(2) or 100 microg/cm(2 )malathion) were suppressed approximately 50-65% and 90%, respectively, compared to unexposed controls. The exposed animals did not exhibit overt clinical signs of malathion toxicosis.

Vasopressin-induced translocation and proteolysis of protein kinase Calpha in an amphibian brain: modulation by corticosterone.

In urodele amphibians, the hypothalamic neuropeptide arginine vasotocin and the adrenal steroid corticosterone interact to regulate reproductive behavior by actions in the brain. The present study investigated signal transduction pathways underlying acute effects of vasotocin and corticosterone, presumably mediated via "non-genomic" steroid action, in an amphibian brain. We used Western blot to examine the effects of corticosterone and the vasotocin receptor agonist arginine vasopressin, alone and in combination, on the subcellular localization and proteolytic processing of protein kinase C-alpha (PKCalpha) in tiger salamander brain tissue. Treatment of whole brain minces with vasopressin or vasotocin led to increases in PKCalpha in membrane fractions and concurrent decreases in PKCalpha in cytosolic fractions. Vasopressin or vasotocin treatment also induced the appearance in membrane and cytosolic fractions of a PKCalpha-immunoreactive band that corresponds to PKMalpha, the proteolytically generated, free catalytic subunit of PKCalpha. Treatment with corticosterone alone had no consistent effect on either PKCalpha or PKMalpha in either fraction. However, pretreatment with corticosterone reliably blocked vasopressin-induced increases in cytosolic PKMalpha. These data provide new information about the cellular mechanisms of action of vasopressin and corticosterone in the vertebrate brain and suggest a cellular mechanism by which the two hormones interact to regulate neuronal physiology and behavior.

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.

Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules.

We evaluated the dye 4',5'-dimethyl-5-(and -6-) carboxyfluorescein (Me2CF) for determining the intracellular pH(pHi) of isolated, perfused proximal tubules of the salamander. The intracellular absorbance spectrum, corrected for the intrinsic absorbance of the tubule, was obtained once per second. The dye was incorporated into tubule cells by exposing them to the membrane-permeable precursor 4',5'-dimethyl-5- (and -6-) carboxyfluorescein diacetate. The introduction of the dye had no significant effect on either pHi or cell voltage transients. Compared with dye contained in a cuvette, intracellular dye had a peak absorbance that was red-shifted by approximately 5 nm, and an apparent pK that was increased by approximately 0.3. These differences precluded an accurate calculation of pHi by the comparison of intracellular spectra with in vitro calibration spectra. However, when Me2CF was calibrated intracellularly, using the K-H exchanger nigericin to equalize external pH and pHi, the dye-derived, steady state pHi was within approximately 0.1 of the value obtained with pH-sensitive microelectrodes. Furthermore, when pHi was simultaneously measured with dye and microelectrodes during rapid pHi transients, the pHi time courses measured by the two methods were very similar. We conclude that the intracellular absorbance spectrum of Me2CF can be used to measure steady state pHi and rapid pHi transients reliably, provided the dye is calibrated intracellularly.

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander.

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.