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PubMed1991

Response properties of cones from the retina of the tiger salamander.

1. Spectral sensitivity measurements using the suction electrode technique reveal three types of cone in the retina of the tiger salamander, showing maximum sensitivity at wavelengths 610 nm (red-sensitive cone), 444 nm (blue-sensitive cone) and below 400 nm (UV-sensitive cone). 2. The absolute sensitivities of red- and blue-sensitive cones to flashes of optimal wavelength are 0.022 and 0.33 pA photon-1 micron 2 respectively. 3. The time-to-peak of the dim flash response and the recovery of membrane current after a flash of any intensity are fastest in red-sensitive and slowest in blue-sensitive cones. 4. In blue- and UV-sensitive cones the flash response peaks progressively earlier as the flash strength is increased, as in rods. In red-sensitive cones, however, bright flash responses take longer to peak than dim flash responses. 5. In all three cone types, voltage clamping at -40 mV reduces the time-to-peak of the response to a bright flash, showing that the rising phase of the bright flash response is normally limited by the time constant of the cell. Under voltage clamp, all cones show a decrease in time-to-peak with increasing flash intensity. 6. Voltage clamping red-sensitive cones reveals two components of the rising phase of the response to a bright flash. Most of the current is rapidly suppressed by a bright flash, and represents the closure of light-sensitive channels. The residual current decays with a mean time constant of 20 ms, and is probably attributable to the decline of electrogenic Na(+)-Ca2+, K+ exchange. The amplitude of this exchange current suggests that the proportion of the dark current carried by calcium ions is greater in red-sensitive cones than in rods of the same species. 7. In UV-sensitive cones, a prominent oscillation of light-sensitive current is observed during the recovery from flashes of intermediate intensity. A similar, but slower and less prominent oscillation is usually seen in blue-sensitive cones. 8. When a red-sensitive cone is voltage clamped an oscillation similar to those in the other two cone types is revealed. An underswing of up to 2 pA is also observed after recovery from intermediate or bright flashes in the majority of red-sensitive cones, and voltage clamping increases the amplitude of this underswing.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1991

Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods.

1. Blockage and permeation of divalent cations through channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in membrane patches excised from retinal rods of the tiger salamander Ambystoma tigrinum by rapidly changing the ionic medium bathing the intracellular side of the excised membrane. 2. The Na+ current, observed when 110 mM-NaCl was present on both sides of the membrane patch, was reduced by the addition of 1 mM of the chloride salts of Ca2+, Mg2+, Sr2+, Ba2+ or Mn2+ to the bathing medium. The sequence of blocking potency at +60 mV was Mg2+ greater than Mn2+ approximately Ba2+ greater than Ca2+ greater than Sr2+, while at -60 mV it was Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ approximately Mg2+. For all divalent cations the blocking effect depended, in a complex way, on the membrane potential. 3. The blocking effect of Ca2+ and Mg2+ increased when the concentration of cyclic GMP was reduced from 100 to 5 microM. At -60 mV 1 mM-Ca2+ blocked about 34% of the Na+ current in the presence of 100 microM-cyclic GMP, while in the presence of 5 microM-cyclic GMP, 1 mM-Ca2+ blocked about 56% of the Na+ current. 4. When, in the presence of 100 microM-cyclic GMP, 110 mM-NaCl at the intracellular side was replaced by equiosmolar amounts of chloride salts of divalent cations (73.3 mM) a small outward current carried by divalent cations could be observed at large positive membrane potentials. At +60 mV the ratio between the current carried by Na+, Sr2+, Ca2+, Ba2+, Mg2+ and Mn2+ was 83.3:1.4:1:0.58:0.33:0.25. 5. In agreement with previous observations the dependence of the Na+ current on the concentration of cyclic GMP shows a clear co-operativity among cyclic GMP molecules.4+ cyclic GMP-gated channel in excised patches is similar to but not identical to the selectivity sequence of divalent cations through the channel in intact rods.

Animals↗
PubMed1991

Rapid charge movements and photosensitivity of visual pigments in salamander rods and cones.

1. Photosensitivities of visual pigments were determined by measuring early receptor currents (ERCs) in voltage-clamped photoreceptors from larval salamanders. 2. As expected from previous work of others, the ERC elicited by a brief flash consisted of a rapid inward component followed by a larger and slower outward component. The magnitude of the outward component corresponded to the movement of about 0.18 electronic charge across the membrane per photoisomerization. 3. The time course of the ERC was independent of the flash intensity, the flash wavelength and the magnitude of the response. The outward component of the cone ERC declined about twice as rapidly as the outward component of the rod ERC.. 4. The amplitude of the ERC decreased as successive flashes bleached the cell's pigment. Using the proportional relation between the size of the ERC and the number of pigment molecules photoisomerized, photosensitivities of the native A2 pigments in rods, red-sensitive cones, blue-sensitive cones and UV-sensitive cones were determined. Calculated solution photosensitivities for rhodopsin, red-sensitive and blue-sensitive cone pigments were not significantly different and the average value for all three pigments at their respective absorption maxima was (7.3 +/- 1.6) x 10(-9) micron 2 molecule-1. A value of 44.0 x 10(-9) micron 2 molecule-1 was obtained in a single UV-sensitive cone. 5. Substitution of the native dehydroretinal chromophore in the red-sensitive cone pigment with 11-cis-retinal increased the solution photosensitivity to (9.6 +/- 0.62) x 10(-9) micron 2 molecule-1. 6. We conclude that cone pigments have large molecular absorption cross-sections and high quantum efficiencies of photoisomerization. These properties seem well suited for the receptive molecules of a highly sensitive, miniaturized transducer.

Animals↗
PubMed1991

Electrogenic uptake of sulphur-containing analogues of glutamate and aspartate by Müller cells from the salamander retina.

1. The effect of sulphur-containing analogues of glutamate and aspartate on the membrane current of glial cells was studied by whole-cell clamping Müller cells isolated from the salamander retina. 2. L-Cysteic acid (CA), L-cysteinesulphinic acid (CSA), L-homocysteic acid (HCA), L-homocysteinesulphinic acid (HCSA) and S-sulpho-L-cysteine (SC) all evoked an inward membrane current that was large at negative potentials, and was smaller (but did not reverse) at more positive potentials up to +30 mV. 3. Removal of external sodium ions abolished the amino acid-evoked currents. Whole-cell clamping with pipettes containing no potassium led to a rapid suppression of the currents, that did not occur when potassium was included in the pipette. 4. The dependence of the currents on sulphur-containing amino acid concentration obeyed first-order Michaelis-Menten kinetics. The current evoked by co-application of L-glutamate and a sulphur-containing analogue was smaller than the sum of the currents produced by glutamate alone and by the sulphur analogue alone. 5. These data are consistent with the sulphur amino acid-evoked current being caused by uptake on the electrogenic glutamate uptake carrier, which co-transports an excess of Na+ ions into the cell, and counter-transports one K+ ion out of the cell. 6. The apparent Km (Michaelis-Menten constant) values for activation of uptake by CA (6 microM) and by CSA (60 microM) are low enough for uptake on the glutamate uptake carrier to be a plausible mechanism for terminating the postulated neurotransmitter action of these agents. However, the apparent Km values for uptake of HCA (2.95 mM), HCSA (1.65 mM) and SC (greater than 1 mM) are much higher than the EC50 (half-maximal effective concentration) concentrations for these agents' activation of NMDA (N-methyl-D-aspartate) channels. 7. Comparing the concentrations of sulphur amino acids needed to activate NMDA channels with their rate of uptake suggests that their potency for causing excitotoxic damage should follow the sequence HCA greater than SC greater than HCSA greater than Glu greater than CSA greater than Asp greater than CA.

Amino Acids, Sulfur↗
PubMed1992

Effects of 2-amino-4-phosphonobutyric acid on cells in the distal layers of the tiger salamander's retina.

1. We studied the effects of 2-amino-4-phosphonobutyric acid (APB) on the response properties of rods, horizontal cells and bipolar cells in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. A concentration of 100 microM was found to be sufficient to elicit maximal effects. 2. Rods hyperpolarized slightly upon exposure to 100 microM-APB and their response amplitudes were slightly reduced. The amplitude of the cone-generated component of the rod's response to 700 nm light was not significantly affected by APB. 3. Horizontal cells hyperpolarized by 2-5 mV upon exposure to 100 microM-APB. The rod-driven component of the horizontal cell response increased in amplitude while the cone-driven component decreased in amplitude. APB thus causes an increase in voltage gain between rods and horizontal cells and a decrease in cone/horizontal cell gain. These findings can be explained in terms of an APB-induced reduction in transmitter release from the cones. 4. APB at a concentration of 100 microM caused an increase in the length constant of the horizontal cell syncytium. Our analysis shows this to be due primarily to a 50% reduction in the coupling impedance between the cells of the syncytium. 5. The effects of APB on off-centre bipolar cells were qualitatively similar to those on horizontal cells. APB increased the amplitudes of rod-driven responses and reduced those of cone-driven responses. The length constants, both of the receptive field centre and of the surround, were increased and the strength of the surround relative to the centre was reduced by about 20%. 6. APB abolished the depolarizing light responses of the receptive field centres of on-centre bipolar cells. A hyperpolarizing response remained whose spatial properties were similar to those of the receptive field surround. We believe this response to reflect a direct (feedforward) input to on-centre bipolar cells from horizontal cells.

Aminobutyrates↗
PubMed1992

Cyclic GMP-activated channels of salamander retinal rods: spatial distribution and variation of responsiveness.

1. Patch-clamp methods were used to investigate the areal density and spatial location of cyclic GMP-activated channels in the surface membrane of salamander rod outer segments. 2. The density of active channels (i.e. channels able to respond to cyclic GMP) in patches excised from outer segments was determined from the number of active channels, N, and the membrane area, A. N was estimated from the current induced by a saturating concentration of cyclic GMP, while A was estimated from the electrical capacitance of the patch. 3. In patches excised from forty-one isolated outer segments prepared in the light the active channel density varied over a remarkable range: 0.34-629 microns-2, with a mean of 166 microns-2. Density was not correlated with patch area in this or any of the conditions studied. 4. The spatial distribution of open channels on the outer segment of a transducing rod was measured by recording the local dark current at various positions with a loose-patch electrode. The apparent density of open channels varied by only about +/- 50% around the circumference of the outer segment and up and down its length. This indicates that the wide range of densities in excised patches did not result from sampling a non-uniform spatial distribution of channels. 5. Patches excised from sixteen dark-adapted whole cells with healthy appearances and saturating light responses of normal size had active channel densities of 1.1-200 microns-2, with a mean of 60 microns-2. Patches from twenty light-adapted whole cells had similar densities. Many densities from the whole cells were much lower than expected. This, and the wide variation in densities, suggests that obtaining a patch often lowered the density of active channels. The number of channels in a patch was quite stable from 1 s to 30 min after excision, ruling out progressive denaturation or adsorption of channels to the glass as a cause for this effect. 6. The mean active channel density in patches excised from whole cells was lower with calcium present in the external solution than with calcium absent (80 vs. 152 microns-2, n = 36 and 30 respectively). 7. We conclude that copies of the channel protein were present at a density of at least 650 microns-2 in the surface membrane of the outer segment and that the distribution of channels was fairly uniform on a 1 micron scale.(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation, Ocular↗
PubMed1992

Two types of glutamate receptors differentially excite amacrine cells in the tiger salamander retina.

1. Excitatory inputs to amacrine cells in the salamander retinal slice preparation were examined using whole-cell patch pipette voltage-clamp techniques. In strychnine (500 nM) and bicuculline (100 microM), two types of amacrine cell were easily distinguished by their light-evoked excitatory responses: transient and sustained. 2. In transient amacrine cells the current-voltage (I-V) relation for the peak light-evoked current was non-linear with a negative slope region between -50 and -70 mV. Responses reversed near +10 mV and were prolonged at more positive holding potentials. 3. In DL-2-amino-phosphonoheptanoate (AP7, 30 microM), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, both the negatively sloped region of the light I-V relation and the prolongation of the response at positive potentials were eliminated. In 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 2 microM), a selective non-NMDA receptor antagonist, light-evoked currents at the most hyperpolarized holding potentials were eliminated. At potentials positive to -85 mV the light-evoked currents lacked a fast onset. The light I-V relation in CNQX had a negative slope region between -35 and -80 mV. 4. With synaptic transmission blocked, kainate evoked responses in transient cells with a resultant I-V relation that was nearly linear, whereas glutamate and NMDA elicited responses with non-linear I-V relations. 5. Light-evoked currents in sustained amacrine cells had a nearly linear I-V relation and reversed near +10 mV. AP7 at a concentration of 30 microM did not affect the light-evoked currents in sustained cells, but 2 microM-CNQX eliminated all light-evoked currents in these cells. 6. With synaptic transmission blocked, sustained amacrine cells responded only to glutamate and kainate, not NMDA. The resultant I-V relations were linear. 7. We conclude that the light-evoked responses of transient amacrine cells are mediated by concomitant activation of both non-NMDA and NMDA receptors whereas the responses of sustained amacrine cells are mediated only by non-NMDA receptors. Furthermore, these data provide supportive evidence that the primary light-evoked excitatory neurotransmitter activating amacrine cells is glutamate.

2-Amino-5-phosphonovalerate↗
PubMed1992

Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander.

1. The ionic dependence of current through the 3',5'-cyclic guanosine monophosphate (cyclic GMP)-activated channels of salamander rods was studied in excised inside-out membrane patches from isolated outer segments. Voltage-clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP-induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman-Hodgkin-Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl- was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl- permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current-voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP-induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP-induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1-2 mM range. Block by Mg2+ was voltage dependent, suggesting that the binding site was within the channel's transmembrane electric field. Raising the Mg2+ concentration on the external surface of the patch increased the apparent dissociation constant of block by internal Mg2+, as expected if external and internal Mg2+ compete for the same binding site. 7. Block by internal Ca2+ had an opposite and weaker voltage dependence than block by internal Mg2+. 8. In symmetrical solutions containing both Na+ and Mg2+ the outward rectification was more pronounced than in solutions containing Na+ alone. In solutions thought to be close to physiological the outward patch current increased e-fold for a depolarization of 24-30 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Calcium homeostasis in the outer segments of retinal rods from the tiger salamander.

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

The permeability of the cGMP-activated channel to organic cations in retinal rods of the tiger salamander.

1. The permeability of the channel activated by guanosine 3',5'-cyclic monophosphate (cGMP) to many organic monovalent cations was determined by recording macroscopic currents in excised inside-out patches of plasma membrane from isolated retinal rod outer segments of the tiger salamander. 2. Current-voltage relations were measured when the NaCl of the bathing medium was replaced by salts of organic cations. Permeability ratios relative to Na+ ions were calculated with the Goldman-Hodgkin-Katz potential equation from the measured changes of reversal potentials. 3. Hydroxylammonium+, hydrazinium+ and methylammonium+, which are molecules of very similar shape and size, permeate the channel with very different permeability ratios: 5.92, 1.99 and 0.60 respectively. 4. Methylated and ethylated ammonium+ compounds were investigated. It was found that, not only methylammonium+, but also dimethylammonium+ and ethylammonium+ were permeant with permeability ratios of 0.6, 0.14 and 0.16 respectively. Trimethylammonium+, tetramethylammonium+, diethylammonium+, triethylammonium+, and tetraethylammonium+ were not permeant. 5. Guanidinium+ and its derivatives formamidinium+, aminoguanidinium+, acetamidinium+ and methylguanidinium+ were all permeant with permeability ratios 1.12, 1.00, 0.63, 0.36 and 0.33 respectively. 6. The cGMP-activated channel was found to be permeable to at least thirteen organic cations. Molecular models of the permeant cations indicate that the cross-section of the narrowest part of the pore must be at least as large as a rectangle of 0.38 x 0.5 nm dimensions.

Ambystoma↗
PubMed1993

Contribution of the ciliary cyclic nucleotide-gated conductance to olfactory transduction in the salamander.

1. Flash photolysis of caged cyclic nucleotides was used to examine the contribution of the ciliary cyclic nucleotide-gated conductance to olfactory transduction in the tiger salamander. Brief illumination of solitary olfactory receptor cells loaded with 100 microM caged cyclic AMP caused a large inward current (peak amplitude 355 +/- 200 pA; mean +/- S.D. for eleven cells) under whole-cell voltage clamp at -50 mV. 2. The photolysis response was initiated after a latency of 4-12 ms, whereas an odorant response of identical amplitude had a latency of several hundred milliseconds. The amplitudes of both responses exhibited almost identical voltage dependence between -50 and +25 mV, with both reversing near 0 mV. The time courses of the falling phases of odorant and photolysis responses also exhibited similar voltage dependence, both being prolonged at positive voltages. 3. Photolysis of caged cyclic GMP activated a current similar in amplitude and time course to that produced by photolysis of caged cyclic AMP. 4. When the flash was spatially limited to the cilia, the amplitude and duration of the photolysis response increased linearly with the length of the cilia illuminated (for cilia not longer than 30-40 microns) while the latency remained constant at 4-12 ms. The increase in duration was described semi-quantitatively by a model which incorporated diffusion and saturable hydrolysis of cyclic AMP. When the flash was limited to the soma or proximal dendrite, the response latency was proportional to the square of the distance between the illuminated region and the cilia. 5. Dialysis of cells with 500 microM cyclic AMP from a whole-cell electrode under voltage clamp activated a large transient inward current. Simultaneous suction electrode recording showed that this current originated almost entirely from the ciliary membrane. The density of cyclic nucleotide-gated channels was estimated to be 800-fold higher in the cilia than in the soma. 6. Summation of simultaneous odorant and photolysis responses was non-linear, the flash-induced current being enhanced during a small odorant response and attenuated during a large odorant response. Summation of two photolysis responses was similarly non-linear. The data were consistent with odorant stimuli and cyclic AMP both activating a common cyclic nucleotide-gated conductance with a Hill coefficient, n, of 2.0-4.4. For n = 2.5, the basal cyclic AMP concentration was estimated to be less than 20% of the K 1/2, which predicts a basal current of 5.8 pA, less than 2% of the maximum.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1993

The relation between stimulus and response in olfactory receptor cells of the tiger salamander.

1. Olfactory receptor cells were isolated from the adult tiger salamander Ambystoma tigrinum and the current in response to odorant stimuli was measured with the whole-cell voltage-clamp technique while odorants at known concentrations were rapidly applied for controlled exposure times. 2. Three odorants, cineole, isoamyl acetate and acetophenone, were first applied at 5 x 10(-4) M. Out of forty-nine cells tested, 53% responded to one odorant only, 22% to two odorants and 25% to all three odorants. 3. The amplitude of the current in response to a given odorant concentration was found to be dependent on the duration of the odorant stimulus and reached a saturating peak value at 1.2 s of stimulus duration. 4. The current measured at the peak of the response for odorant steps of 1.2 s as a function of odorant concentration was well described by the Hill equation for the three odorants with Hill coefficients higher than 1 and K1/2 (odorant concentration needed to activate half the maximal current) ranging from 3 x 10(-6) to 9 x 10(-5) M. 5. It is concluded that olfactory receptor cells are broadly tuned and have a low apparent affinity for odorants, integrate stimulus information over time, and have a narrow dynamic range.

Acetophenones↗
PubMed1994

Gating, selectivity and blockage of single channels activated by cyclic GMP in retinal rods of the tiger salamander.

1. Patches in the inside-out configuration were excised from the membrane of outer and inner segments of the larval tiger salamander, Ambystoma tigrinum. The current flowing through single channels opened by cyclic GMP was studied with the voltage clamp technique. 2. Amplitude histograms of current recordings from patches containing only one flickering channel, excised from the inner segment and in the presence of 100 microM cyclic GMP, could be fitted by a theoretical scheme in which the single channel conductance was at least 55 pS at +40 mV and at least 45 pS at -40 mV. The mean open time was no longer than the time constant of our recording system, about 35 microseconds. Similar results were obtained by analysis of the amplitude histograms of patches from the outer segment containing many channels, and in the presence of 1-5 microM cyclic GMP. 3. In membrane patches excised from the outer segment, reducing the temperature from 24 to 8 degrees C did not reduce the flickering, but changed the amplitude histograms of current fluctuations activated by 1 microM cyclic GMP in a way consistent with a decrease of 50% in the single channel conductance and a decrease of 50% in the open probability. 4. In the presence of 1 microM cyclic GMP at +60 mV, when Na+ was replaced by NH4+ or K+, brief outward current transients flowing through single channels were observed. When Na+ was replaced with Li+, Rb+ or Cs+, current transients were very small. 5. The shape of the power spectrum of current fluctuations induced by 1 microM cyclic GMP at +60 mV did not change when the permeating ion was Na+, K+ or NH4+. Analysis of the amplitude histogram did not show any effect of the tested monovalent cations on the open probability or on channel gating. At +60 mV, the estimated single channel currents were at least 4, 2.8 and 2 pA for NH4+, Na+ and K+ respectively. 6. The addition of 0.5 or 1 mM Ca2+ to the medium bathing the cytoplasmic side of the membrane greatly reduced the frequency of openings, but single channel activity could still be observed. The blocking effect of 1 mM Ca2+ on the channel activity induced by 2 microM cyclic GMP could be counterbalanced by increasing the cyclic GMP concentration. The addition of 0.5 or 1 mM Ca2+ did not change the shape of power spectra obtained at membrane voltages between -100 and +100 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1994

Bleached pigment activates transduction in isolated rods of the salamander retina.

1. We have used suction electrode recording together with rapid steps into Li+ solution and 0.5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady-state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10(-6) to 10(-7) times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization.

1-Methyl-3-isobutylxanthine↗
PubMed1995

The relationship between light-evoked synaptic excitation and spiking behaviour of salamander retinal ganglion cells.

1. Light-evoked input-output characteristics of ganglion cells in dark-adapted tiger salamander retina were studied in the slice preparation using patch-clamp techniques. Excitatory postsynaptic currents (EPSCs), isolated by blocking inhibitory inputs and evoked by a range of light stimulus intensities, were recorded under whole-cell voltage clamp. Spike responses, evoked by the same light intensities, were recorded extracellularly from the same cells using the cell-attached patch-clamp technique. 2. When N-methyl-D-aspartate (NMDA) receptor-mediated input was blocked by the competitive NMDA antagonist DL-2-amino-5-phosphonoheptanoate (AP7), light-evoked EPSC amplitude and peak firing rate were reduced at all light intensities. In both cases, the data obtained in the presence of AP7 scaled linearly to control data, indicating that NMDA and non-NMDA receptors are activated in the same proportions across the entire 2 log unit stimulus response range of these ganglion cells. 3. The relationship between light-evoked spike frequency and light-evoked EPSC amplitude was linear. The slope of the light-evoked synaptic current-spike frequency relationship was close to the slope of the injected current-spike frequency relationship, indicating that synaptic current and injected current drive spiking in a similar manner. The linearity of the synaptic current-spike frequency relationship was not compromised when NMDA input was blocked by AP7. 4. Light-evoked voltage responses, recorded under whole-cell current clamp, revealed that the average membrane potential during a spike response was depolarized only slightly with increased firing rate. Once the membrane potential surpassed spike threshold, it was maintained by the voltage-gated, spike-generating conductances at a depolarized plateau upon which action potentials were fired. The potential of this plateau varied only slightly with spike frequency. We conclude that the voltage control exerted by the spike-generating currents in ganglion cells prevents a substantial response-dependent decrease in the electrical driving force of the excitatory currents, obviating the need for the voltage-independent synaptic efficacy provided by the combination of NMDA and non-NMDA inputs.

2-Amino-5-phosphonovalerate↗
PubMed1996

Role of cytoplasmic calcium concentration in the bleaching adaptation of salamander cone photoreceptors.

1. In order to study the possible involvement of Ca2+ in the bleaching adaptation of cones isolated from the retina of the salamander Ambystoma tigrinum, changes in cytoplasmic calcium concentration ([Ca2+]i were opposed by exposing the outer segment to a low-Ca(2+)-O Na+ solution designed to minimize Ca2+ fluxes across the outer segment membrane. 2. When a cone was exposed in normal Ringer solution to bright light bleaching a significant fraction of the photopigment, the circulating current was initially suppressed completely and then recovered to a maintained value less than the value in darkness before the bleach. When the outer segment of the cone was stepped to low-Ca(2+)-O Na+ solution before the bleach was delivered, the circulating current recovered more slowly or (for large bleaches) remained completely suppressed for the duration of the solution exposure. 3. If, during the period for which the current was suppressed in low-Ca(2+)-O Na+ solution, the cone outer segment was exposed to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the circulating current was restored. The dim flash response recorded under these conditions exhibited kinetics and integration times similar to those recorded in low-Ca(2+)-O Na+ solution in darkness before the bleach. If, instead, the outer segment was returned to Ringer solution after the bleach, thereby allowing [Ca2+]i to fall from its dark-adapted level to the appropriate bleach-adapted level, the kinetics of the response in low-Ca(2+)-O Na+ solution were greatly accelerated, and the integration time considerably reduced. This was true regardless of whether or not the low-Ca(2+)-O Na+ solution included IBMX. 4. The role of Ca2+ in bleaching adaptation appeared to resemble its role in background adaptation, since in both cases exposure to low-Ca(2+)-O Na+ solution suppressed the acceleration of response kinetics. Responses recorded from cones in low-Ca(2+)-O Na+ solution were nearly identical in waveform and sensitivity during background light or after bleaches, provided that IBMX was used to restore sufficient photocurrent so that responses to flashes could be recorded, and sensitivity was corrected for loss in quantum catch. 5. These results indicate that the fall in [Ca2+]i in cones after a bleach is necessary both for the acceleration of the flash response and the adaptational decrease in sensitivity, as is the case for adaptation by background light.

1-Methyl-3-isobutylxanthine↗
PubMed1996

Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2-4 microM. 3. An average cytoplasmic Ca2+ concentration of 2-4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter.

Acetates↗
PubMed1996

Kinetics of desensitization induced by saturating flashes in toad and salamander rods.

1. The suction pipette technique was used to examine the effect of a conditioning pre-flash on the saturation time (tsat) of a bright test flash (intensity 10,000-250,000 isomerizations) delivered to intact salamander or toad rod outer segments. The conditioning flash was delivered 0-60 s before the test flash; its intensity was typically between six and sixty times dimmer than the test flash, and it was sufficient by itself to fully saturate the photocurrent. 2. A saturating pre-flash delivered before a saturating test flash reduced the tsat of the test flash. This was equivalent to a reduction in phototransduction gain (psi). 3. The pre-flash had little effect on tau zero the time constant of decay of the rate-limiting species in photoresponse inactivation (activated rhodopsin or the activated G-protein-phosphodiesterase complex). 4. The tsat declined exponentially as the separation time between a fixed intensity pre-flash and test flash was increased. The time constant (tau p) of decline in tsat was approximately 2.4s. The maximum reduction in tsat corresponded to a reduction in the apparent gain of phototransduction to approximately 0.10 of its original level. This exponential decline is consistent with a [Ca2+]i-mediated effect. 5. We conclude that the rate-limiting step in response inactivation and the step responsible for light-induced gain reduction constitute separate and distinct steps of the phototransduction cascade.

Animals↗