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PubMed1985

Synaptic transmission from rods to bipolar cells in the tiger salamander retina.

Synaptic transmission between rods and depolarizing bipolar cells (DBC) was studied by using simultaneous recording techniques in the living retinal slice preparation. Current injection into the rod elicited a sign-inverting, sustained voltage change in the DBC. Voltage "tails" after the termination of a bright flash were observed in dark-adapted rods and DBC but not in cones. These simultaneously recorded voltage tails were used to isolate the rod input from the cone input and to study the input-output relation of the rod-DBC synapse. Within the voltage range between 0 and -10 mV from the rod dark potential (-39 +/- 1.2 mV), the input-output relation of the rod-DBC was approximately linear, with an estimated gain of about 3.7.

Animals↗
PubMed1986

Gated currents generate single spike activity in amacrine cells of the tiger salamander retina.

Amacrine cells form the neural networks mediating the second level of lateral interactions in the vertebrate retina. Members of a prominent class of amacrine cells, found in most vertebrates, respond at both the onset and termination of steps of illumination with a single, large transient depolarization. We show here how specific relationships between membrane currents control this single spike activity. Using whole-cell patch clamp on living retinal slices, we studied the membrane currents in amacrine cells. The currents elicited by depolarizing voltage steps could be separated into three main ionic components: a transient inward voltage-gated sodium current, a relatively small sustained inward voltage-gated calcium current, and a calcium-dependent outward current. A specific relationship between the sodium and potassium current alone appears to preclude repetitive spike activity. Potassium current is activated at potentials positive to -20 mV, but the sodium inactivation, between -60 and -20 mV, does not intersect potassium activation. Therefore, a steady depolarizing current step elicits an initial spike but then the membrane cannot be sufficiently hyperpolarized by potassium current to remove sodium inactivation and the cell remains refractory.

Action Potentials↗
PubMed1987

Gated currents in isolated olfactory receptor neurons of the larval tiger salamander.

The electrical properties of enzymatically isolated olfactory receptor cells were studied with whole-cell patch clamp. Voltage-dependent currents could be separated into three ionic components: a transient inward sodium current, a sustained inward calcium current, and an outward potassium current. Three components of the outward current could be identified by their gating and kinetics: a calcium-dependent potassium current [IK(Ca)], a voltage-dependent potassium current [IK(V)], and a transient potassium current (Ia). Typical resting potentials were near -54 mV, and typical input resistance was 3-6 G omega. Thus, only 3 pA of injected current was required to depolarize the cell to spike threshold near -45 mV. The response to a current step consisted of either a single spike regardless of stimulus strength, or a train of less than 8 spikes, decrementing in amplitude and frequency over approximately equal to 250 msec. Thus, the receptor response cannot be finely graded with stimulus intensity.

Animals↗
PubMed1989

Gamma-aminobutyrate type B receptor modulation of L-type calcium channel current at bipolar cell terminals in the retina of the tiger salamander.

Bipolar-cell axon terminals receive direct synaptic input from amacrine-cell processes, suggesting a possible pathway for modulation of transmitter release. In retinal slices, bath-applied baclofen, a gamma-aminobutyrate type B (GABAB) receptor agonist, reduced a patch-clamp-recorded L-type calcium channel current in a population of bipolar cells with axon terminals that ramify along the midline of the inner plexiform layer. Lucifer yellow staining revealed that this current was found only in bipolar cells that retain axon terminals and their associated telodendria, suggesting that the current is generated at the terminal and also possibly modulated there. T-type calcium currents were found in all bipolar cells, including those without axon terminals, but were not modulated by baclofen. The baclofen-induced reduction of calcium current was enhanced by guanosine 5'-[gamma-thio]triphosphate and eliminated by guanosine 5'-[beta-thio]diphosphate added to the cytoplasm by the patch recording electrode, suggesting that the GABAB receptors act through a guanine nucleotide-binding regulatory protein (G protein). Baclofen also reduced an excitatory synaptic input to a population of amacrine cells with processes that ramify along the midline of the inner plexiform layer--cells probably postsynaptic to the bipolar terminals. This suggests that GABAB receptors modulate not only the calcium current but also transmitter release by a pathway involving G proteins and L-type calcium channels.

Animals↗
PubMed1990

Mitochondrial genotype of a unisexual salamander of hybrid origin is unrelated to either of its nuclear haplotypes.

We examined mitochondrial DNA (mtDNA), enzyme, and morphological variation among 17 unisexual Ambystoma of hybrid origin. Electrophoretic comparison of diagnostic enzymes indicates that these unisexuals are triploid with two nuclear genomes from the bisexual species Ambystoma laterale and one from Ambystoma jeffersonianum; however, according to restriction analysis, the mtDNAs of these specimens derive from a third species, Ambystoma texanum. This unusual situation is apparently due to a partially independent segregation event in an ancestor of these unisexuals. This situation highlights the potential importance of molecules with different inheritance patterns in elucidating complex cases of reticulate evolution.

Ambystoma↗
PubMed1960

Application of the electron microscope to the cytochemical peroxidase reaction in salamander leukocytes.

The present study has dealt with the localization by electron microscopy of the products of peroxidase reaction in neutrophil leukocytes in the subcapsular region of the livers of Triturus viridescens. Small pieces of liver tissue were fixed for 1 hour in buffered osmium tetroxide solution. After fixation they were divided into five groups: (a) Not treated with any reagent (control); (b) Treated for 4 minutes with the peroxidase reagent containing 0.3 per cent benzidine and 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol; (c) Treated for 4 minutes with 0.3 per cent benzidine solution in 50 per cent alcohol alone (control); (d) Treated for 4 minutes with 0.014 per cent (0.004 molar) hydrogen peroxide in 50 per cent alcohol alone (control); (e) Treated for 5 minutes with pure methanol, washed in water, and treated for 4 minutes with the peroxidase reagent (inhibition test). Each group was then dehydrated and embedded in either methacrylate or epoxy resin. In electron micrographs, the reaction products of peroxidase activity were evidenced in the form of dense materials localized in the specific granules in the cytoplasm of the neutrophil leukocytes. Neither mitochondria nor any other particles showed increases in density. The specific granules showed no change of density in the control and inhibition tests. Paraffin-embedded tissues of the above mentioned five groups, when examined with the light microscope, revealed that the brown granules denoting a positive reaction appeared only in leukocytes of the tissue treated with the peroxidase reagent. Although much further work is necessary before definitive and constant results are to be expected, the possibility that the electron microscope may be applicable to peroxidase cytochemistry in leukocytes has been suggested by the present study.

Animals↗
PubMed1979

Role of tropomyosin in actin filament formation in embryonic salamander heart cells.

Recessive mutant gene c in Ambystoma mexicanum embryos causes a failure of the heart to function even though initial heart development appears normal. An analysis of the constituent proteins of normal and mutant hearts by SDS-poly-acrylamide gel electrophoresis shows that actin (43,000 daltons) is present in almost normal amounts, while myosin heavy chain (200,000 daltons) is somewhat reduced in mutants. Both SDS-polyacrylamide gel electrophoresis and immunofluorescence studies reveal that tropomyosin is abundant in normal hearts, but very much reduced in mutants. Electron microscope studies of normal hearts show numerous well-organized myofibrils. Although mutant cardiomyocytes contain a few 60- and 150-A filaments, organized sacromeres are absent. Instead, amorphous proteinaceous collections are prominent. Previously reported heavy meromyosin (HMM)-binding experiments on glycerinated hearts demonstrate that most of the actin is contained within the amorphous collections in a nonfilamentous state, and the addition of HMM causes polymerization into F actin (Lemanski et al., 1976, J. Cell. Biol. 68:375-388). In the present study, glycerol-extracted hearts are incubated with tropomyosin, purified from rabbit or chicken skeletal muscle. This treatment causes the amorphous collections to disappear, and large numbers of distinct thin actin (60- to 80-A) filaments are seen in their place. Negative staining experiments corroborate this observation. These results suggest that the nonfilamentous actin located in the amorphous collections of mutant heart cells is induced to form into filaments with the addition of tropomyosin.

Actins↗
PubMed1994

Mechanism of Ba2+ block of M-like K channels of rod photoreceptors of tiger salamanders.

IKx is a voltage-dependent K+ current in the inner segment of rod photoreceptors that shows many similarities to M-current. The depression of IKx by external Ba2+ was studied with whole-cell voltage clamp. Ba2+ reduced the conductance and voltage sensitivity of IKx tail currents and shifted the voltage range over which they appeared to more positive potentials. These effects showed different sensitivities to Ba2+: conductance was the least sensitive (K0.5 = 7.6 mM), voltage dependence intermediate (K0.5 = 2.4 mM) and voltage sensitivity the most sensitive (K0.5 = 0.2 mM). Ca2+, Co2+, Mn2+, Sr2+, and Zn2+ did not have actions comparable to Ba2+ on the voltage dependence or the voltage sensitivity of IKx tail currents. In high K+ (100 mM), the voltage range of activation of IKx was shifted 20 mV negative, as was the tau-voltage relation. High K+ did not prevent the effect of Ba2+ on conductance, but abolished its ability to affect voltage dependence and voltage sensitivity. Ba2+ also altered the apparent time-course of activation and deactivation of IKx. Low Ba2+ (0.2 mM) slowed both deactivation and activation, with most effect on deactivation; at higher concentrations (1-25 mM), deactivation and activation time courses were equally affected, and at the highest concentrations, 5 and 25 mM Ba2+, the time course became faster than control. Rapid application of 5 mM Ba2+ suggested that the time dependent currents in Ba2+ reflect in part the slow voltage-dependent block and unblock of IKx channels by Ba2+. This blocking action of Ba2+ was steeply voltage-dependent with an apparent electrical distance of 1.07. Ba2+ appears to interact with IKx channels at multiple sites. A model which assumes that Ba2+ has a voltage-independent and a voltage-dependent blocking action on open or closed IKx channels reproduced many aspects of the data; the voltage-dependent component could account for both the Ba(2+)-induced shift in voltage dependence and reduction in voltage sensitivity of IKx tail currents.

Ambystoma↗
PubMed1998

Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Adaptation, Ocular↗
PubMed1989

Ionic channels of the inner segment of tiger salamander cone photoreceptors.

Cone photoreceptors were isolated enzymatically and their ionic currents studied by the whole-cell, gigaseal voltage-clamp technique. Five nonsynaptic currents were identified. A prominent, poorly selective cation current, Ih, activated after a delay during hyperpolarizations and then deactivated with a delay on return to potentials greater than -50 mV. An empirical model for Ih gating kinetics is developed with three open and two closed states. Depolarization elicits a small, voltage-gated calcium current (ICa). Block by nitrendipine, nickel, cadmium, and cobalt, increase of current with barium, lack of rapid inactivation, and relatively high threshold suggest an L-type Ca channel. No evidence was found for low-threshold Ca channels. An anion current ICl(Ca) was present after pulses that led to a significant inward ICa (but not IBa) and was not elicited when cobalt was present. Tails of ICl(Ca) were short (100 ms) after short depolarizations and were longer after longer depolarizations. Two TEA-sensitive K currents were also elicited by depolarizations. One, IK(Ca), was calcium sensitive. We looked for modulation of Ih, ICa, and ICl(Ca) by a number of neurotransmitters. No changes of Ih were seen, but ICa and ICl(Ca) were depressed in a few cones when GABA or adenosine were applied. We discuss how this modulation might contribute to the feedback effects of horizontal cells on cones when surrounding cones are illuminated.

Ambystoma↗
PubMed1990

Synaptic and voltage-gated currents in interplexiform cells of the tiger salamander retina.

We have correlated the membrane properties and synaptic inputs of interplexiform cells (IPCs) with their morphology using whole-cell patch-clamp and Lucifer yellow staining in retinal slices. Three morphological types were identified: (a) a bistratified IPC with descending processes ramifying in both sublaminas a and b of the inner plexiform layer (IPL), and an ascending process that branched in the outer plexiform layer (OPL) and originated from the soma, (b) another bistratified IPC with descending processes ramifying in both sublaminas a and b, and an ascending process that branched in the OPL and originated directly from IPC processes in the IPL, and (c) a monostratified IPC with a descending process ramifying over large lateral extents within the most distal stratum of the IPL, and sending an ascending process to the OPL with little branching. Similar voltage-gated currents were measured in all three types including: (a) a transient inward sodium current, (b) an outward potassium current, and (c) an L-type calcium current. All cells generated multiple spikes with frequency increasing monotonically with the magnitude of injected current. The IPCs that send their descending processes into both sublaminas of the IPL (bistratified) receive excitatory synaptic inputs at both light ON and OFF that decay with a time constant of approximately 1.3 s. Slowly decaying excitation at both ON and OFF suggests that bistratified IPCs may spike continuously in the presence of a dynamic visual environment.

Animals↗
PubMed1991

Light and dark active phosphodiesterase regulation in salamander rods.

We studied the activation of 3',5'-cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE) by using a cell-permeant enzyme inhibitor. Rods of Ambystoma tigrinum held in a suction electrode were jumped into a stream of 3-isobutyl-1-methylxanthine (IBMX), 0.01-1 mM. Initial transient light-sensitive currents fit the notion that dark and light-activated forms of PDE contributed independently to metabolic activity and were equivalently inhibited by IBMX (apparent Ki 30 microns). Inhibition developed within 50 ms, producing a step decrease of enzyme velocity, which could be offset by activation with flashes or steps of light. The dark PDE activity was equivalent to light activation of enzyme by 1,000 isomerization rod-1s-1, sufficient to hydrolyze the free cGMP pool (1/e) in 0.6 s. Steady light activated PDE in linear proportion to isomerization rate, the range from darkness to current saturation amounting to a 10-fold increase. The conditions for simultaneous onset of inhibitor and illumination to produce no net change of membrane current defined the apparent lifetime of light-activated PDE, TPDE* = 0.9 s, which was independent of both background illumination and current over the range 0-3 x 10(5) isomerization s-1, from 50 to 0 pA. Adaptation was a function of current rather than isomerization: jumps with different proportions of IBMX concentration to steady light intensity produced equal currents, and followed the same course of adaptation in maintained light, despite a 10-fold difference of illumination. Judged from the delay between IBMX- and light-induced currents, the dominant feedback regulatory site comes after PDE on the signal path. The dark active PDE affects the hydrolytic flux and cytoplasmic diffusion of cGMP, as well as the proportional range of the cGMP activity signal in response to light.

1-Methyl-3-isobutylxanthine↗
PubMed2004

The influence of ecology and genetics on behavioral variation in salamander populations across the Eastern Continental Divide.

Understanding the unique contributions of ecology and history to the distribution of species within communities requires an integrative approach. The Eastern Continental Divide in southwestern Virginia separates river drainages that differ in species composition: the more aquatic, predatory Desmognathus quadramaculatus is present only in the New River drainage (which drains to the Gulf of Mexico), while Desmognathus monticola is present in both the New River drainage and the James River drainage (which drains to the Atlantic Ocean). We investigated natural distributions, behavioral variation in experimental mesocosms, population genetic, and phylogenetic implications of community structure. The presence of D. quadramaculatus increased the terrestriality of D. monticola in natural and experimental situations but to different degrees in allopatric and sympatric populations. Our ecological data suggest that the degree of terrestriality in D. monticola is a result of a balance between the optimal aquatic habitat and risks of predation. Our genetic analyses suggest that D. monticola has experienced a recent range expansion and has only a recent history of association with D. quadramaculatus in Virginia. This is surprising given the strong behavioral variation that exists in populations experiencing unique community compositions over a scale of meters. This study demonstrates the need to combine both ecology and genetics toward an understanding of the factors affecting species distributions, behavioral variation between populations, and patterns of genetic variation across a landscape.

Animals↗