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PubMed1984

Location and function of voltage-sensitive conductances in retinal rods of the salamander, Ambystoma tigrinum.

The functional role and spatial location of voltage-sensitive conductances that modify the light-evoked electrical signals were studied in retinal rods of the tiger salamander. An isolated rod was drawn into a suction electrode for recording membrane current and impaled with an intracellular electrode for recording membrane potential and passing current. A bright flash gave a rapid initial hyperpolarization that relaxed to a smaller plateau. Simultaneously the dark current of the outer segment was shut off with the time course of a rounded step function. This characteristic difference between the wave forms demonstrates that the voltage relaxation does not result from reopening of light-sensitive channels. The voltage relaxation in (2) did not require light or interruption of the dark current, as the wave form was duplicated by suddenly switching off a depolarizing current injected during steady saturating light. This is explained if the relaxation depends purely on voltage-sensitive conductances. The voltage response to a dim flash reached its peak value before the current response. The voltage wave form was predicted assuming that the recorded photo-current drove a linear high-pass filter with parameters derived from analysis of the voltage response to injection of a current step. When the intracellular voltage was changed by current injection the slope resistance of the outer segment slowly declined to a lower level, indicating that the outer segment contains a voltage-sensitive conductance. When a current step was injected in bright steady light, the current recorded from the outer segment consisted of a capacity component proportional to dV/dt and a small extracellular leakage current but no detectable ionic current. This supports other evidence indicating that light-sensitive channels comprise the main or exclusive ionic conductance of the outer segment. The behaviour in (5) is explained if the light-sensitive channels themselves are slowly opened by hyperpolarization and closed by depolarization. Analysis of the current-injection experiments suggests that most of the high-pass filtering in a rod results from the action of voltage-sensitive conductances located in the inner segment. Addition of 10 mM-CsCl to the Ringer solution abolished the relaxation in the voltage response to a bright flash but left intact the high-pass filtering of small signals. This would be explained by a selective block of one of two sets of voltage-sensitive channels in the inner segment or by a voltage-sensitive block of one kind of channel.

Ambystoma↗
PubMed1984

The calcium current in inner segments of rods from the salamander (Ambystoma tigrinum) retina.

Solitary rod inner segments were isolated from salamander retinae. Their Ca current was studied with the 'whole-cell, gigaseal' technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The soluble constituents of the cytoplasm exchanged with the solution in the pipette. The external solution could be changed during continuous perfusion. Membrane voltage was controlled with a voltage clamp. After permeant ions other than Ca were replaced with impermeant ions (i.e. tetraethylammonium as a cation, and aspartate or methanesulphonate as an anion), an inward current remained. It activated at approximately -40 mV, reached a maximum at approximately 0 mV, and decreased as the membrane was further depolarized. The size of the current increased when Ba was substituted for external Ca. The current was blocked when Ca was replaced with Co. The voltage at which the current was half-maximum shifted from approximately -22 to -31 mV during the initial 3 min of an experiment. The maximum amplitude of the current continuously declined during the entire course of an experiment. The time course for activation of the Ca current following a step of depolarization could be described by the sum of two exponentials. The time constant of the slower exponential was voltage dependent. Deactivation following repolarization could also be described by the sum of two exponentials. Both time constants for deactivation were independent of voltage (between -30 and 0 mV) and faster than the slower time constant for activation. When the internal Ca concentration was buffered by 10 mM-EGTA, the Ca current did not inactivate during several seconds of maintained depolarization. When the concentration of EGTA was reduced to 0.1 mM, the Ca current declined and the membrane conductance decreased during several seconds of maintained depolarization. This inactivation was incomplete and only occurred after a substantial quantity of Ca entered. Following repolarization the Ca conductance recovered from inactivation. In contrast, the continuous decline observed during the course of an experiment (item 3) was not reversible. The difference suggests that inactivation and the decline are distinct processes.

Ambystoma↗
PubMed1986

Electrical properties of the light-sensitive conductance of rods of the salamander Ambystoma tigrinum.

The light-sensitive conductance of isolated rods from the retina of the tiger salamander was studied using a voltage-clamp method. The membrane current of the outer segment was collected with a suction electrode while the internal voltage was measured and controlled with a pair of intracellular electrodes. Saturating light blocked the outer segment current at all potentials, the residual conductance usually becoming less than 20 pS. This suggests that light-sensitive channels comprise the main ionic conductance in the surface membrane of the outer segment. Current-voltage relations determined 10-40 ms after changing the voltage showed outward-going rectification, the outward current increasing e-fold for a depolarization of 11-14 mV. The reversal potential of the light-sensitive current was estimated as 5 +/- 4 mV. This is consistent with other evidence indicating that the channel is not exclusively permeable to Na. Applying steady light, lowering external Ca, or changing the intracellular voltage to a new steady level scaled the light-sensitive current without altering the reversal potential or the form of the rectification. This suggests that all three manipulations change the number of channels in the conducting state without changing the ionic concentration gradients or the mechanism of permeation through an 'open' channel. Hyperpolarizing voltage steps slowly increased the light-sensitive current and depolarizing steps reduced it. A gating variable Y expressing the fractional activation of the light-sensitive conductance in the steady state was derived from the ratio of the instantaneous and steady-state currents. Y declined at voltages positive to -100 mV and usually reached a minimum near 0 mV, with a secondary rise positive to 0 mV. Around the dark voltage Y changed e-fold in roughly 25 mV. The voltage-dependent gating in (6). appeared to involve two delays similar in magnitude to those of the four principal delays in the rod's response to a dim flash. Steady background light shortened the time-scale of gating and flash responses to a similar degree. Clamping the voltage at the dark level had little effect on the photocurrent evoked by a flash. The small, delayed effect actually observed is explained by the slow voltage-dependent gating of the light-sensitive conductance. Hyperpolarization had little effect on the kinetics of the response to a flash, but depolarization slowed the response, causing it to reach a larger, later peak. Depolarization also prolonged the blockage of the light-sensitive current after a saturating flash.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Incorporation of calcium buffers into salamander retinal rods: a rejection of the calcium hypothesis of phototransduction.

The suction-electrode technique was used to monitor the photocurrent of isolated retinal rods from the tiger salamander, by drawing in the light-sensitive outer segment, or sometimes the inner segment. Calcium buffers or other agents were then introduced into the rod cytoplasm by the 'whole-cell patch-clamp' technique. A patch pipette was sealed against the region of the rod protruding from the suction pipette (usually the inner segment), and the membrane patch was ruptured to obtain a whole-cell recording. Several lines of evidence indicated that the pipette contents diffused into the outer segment, and showed that the cell could be adequately voltage clamped. With only trace quantities of chelator in the patch pipette (to bind stray calcium), a gradual decline of the dark current and slowing of responses was usually observed over a period of 10-20 min after rupture of the patch. When the patch pipette contained no added calcium and 10 mM of the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (free Ca2+ ca. 10(-9) M) rupture of the patch led, over a period of a few minutes, to an increase in mean dark current, an increased duration of responses, a substantial increase in flash sensitivity, and a pronounced overshoot in the recovery phase, but with virtually no change in the rising phase of the response to bright flashes. Similar results were obtained when EGTA was used in place of BAPTA, and also in the few cases when successful rupture of the outer segment membrane was obtained. With the free calcium concentration in the patch pipette buffered to the higher level of 1 microM (with 10 mM-Ca2+/11 mM-BAPTA) the results were qualitatively similar to those obtained with BAPTA alone, except that the mean dark current did not increase. This is consistent with a resting free calcium concentration in darkness in the region of 1 microM. In the presence of bright steady illumination with BAPTA in the cell the suppression of outer segment current could be maintained for at least 15 min. Upon extinction of the light a very large current transient developed (similar to the overshoot with flashes) which was light suppressible. With backgrounds of moderate intensity the incorporation of buffer led to a gradual reduction of the residual current.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1986

Are Merkel cell-neurite reciprocal synapses involved in the initiation of tactile responses in salamander skin?

In salamander skin the Merkel cell-neurite complexes located near the base of the epidermis are the morphological correlates of the rapidly adapting touch receptors (Parducz, Leslie, Cooper, Turner & Diamond, 1977). The present electron microscopic studies revealed that these complexes contain reciprocal synapses polarized in the direction Merkel cell to neurite, and in the opposite direction, neurite to Merkel cell. The possible involvement of chemical transmission in the initiation of the mechanosensory response, was studied in vitro with the aid of a stable skin-nerve preparation in which single mechanoreceptors were activated under controlled conditions. Mechanosensitivity was measured with a calibrated prodder (tip diameter 10-30 micron) applied to random or selected points on the surface of the skin while the afferent impulse was recorded in the attached nerve twig. In some experiments the (tungsten) prodder was also used as a surface electrode, allowing the same mechanosensory axon to be excited mechanically (i.e. physiologically), and/or electrically. When applied at a single 'touch spot', suitably timed subthreshold mechanical and subthreshold electrical stimuli could summate to produce a single action potential. The temperature coefficient (Q10) between 5 and 15 degrees C for the latency of the afferent spike was small, in the range 1.3-2, whether it was evoked by mechanical or electrical stimulation. The latency following the mechanical stimulus, which included the transduction step, was longer than that following the electrical stimulus by 0.5-2.5 ms, and this additional delay was also relatively insensitive to temperature. In several cases removal of the epidermis with its Merkel cells (and presumably the most distal portions of the afferent nerve terminations) did not render the remaining skin totally insensitive to mechanical stimulation; however, the remaining receptive elements, though still rapidly adapting, generally had increased mechanosensory thresholds. The mechanosensitivity of the skin was unaffected by bath application of several aminergic (e.g. noradrenaline, 5-hydroxytryptamine, octopamine) and purinergic (e.g. ATP, quinacrine) compounds at concentrations in the range 0.2-2 mM. Removal of extracellular Ca2+ combined with elevation of extracellular Mg2+ (10-40 mM) had relatively little effect on the mechanosensitivity over periods of up to 1 h. In contrast, application of Co2+ (2-10 mM) produced a decrease or blockade of the mechanosensitivity that was not associated with any obvious alterations in the ultrastructure of the Merkel cell-neurite complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1987

The properties and function of inward rectification in rod photoreceptors of the tiger salamander.

1. Rod photoreceptors were isolated from the retinae of tiger salamanders and voltage clamped using the whole-cell patch-clamp technique. 2. Hyperpolarizing the cell to potentials more negative than -50 mV evoked an inward current termed Ih. 3. Ih did not turn on immediately following a hyperpolarizing step but showed a marked delay. The activation time course of Ih could be described by the sum of two exponential components of opposite polarity. 4. The steady-state chord-conductance was half activated at -67 mV. 5. The reversal potential of Ih was close to -30 mV in normal standard salt solution. Increasing the external potassium concentration tenfold shifted the reversal potential by +17 mV. 6. The conductance-voltage relation and the kinetic parameters were not affected by changes in the external potassium concentration. 7. When fully activated, the zero-current conductance underlying Ih depended on the square root of the concentration of external potassium. 8. The permeability ratio PNa/PK depended on the external potassium concentration. It was 0.2 at an external potassium concentration of 2.0 mM and 0.3 at an external potassium concentration of 10.0 mM. The interaction of potassium with Ih suggests that Ih is a multi-ion pore. 9. It is concluded that Ih differs from the inward rectifier that is found in egg cells, frog muscle and heart muscle. 10. The kinetics and voltage sensitivity of Ih suggest that it does not play a role in the dark resting state or in the response to dim flashes of light. Its properties indicate that it may have a major role in the response to bright flashes.

Action Potentials↗
PubMed1987

Measurement of sodium-calcium exchange in salamander rods.

1. Methods employing suction electrodes to measure the small inward currents associated with the exchange of internal Ca2+ for external Na+ in salamander rod outer segments are described. 2. The ratio of the integral of the exchange current to the integral of the Ca2+ current during the loading period averaged 0.37, which is consistent with 1 Ca2+ ion exchanging with 2.7 Na+ ions, in approximate agreement with Yau & Nakatani (1984b). 3. The transient pumping current observed when external Na+ was restored after a few seconds in isotonic Ca2+ with IBMX (3-isobutyl-1-methylxanthine) consisted of a phase with current at a constant saturated level followed by a phase in which current declined along a characteristic S-shaped curve that was much steeper than expected from the Michaelis equation. 4. The relation between Ca2+ load and pumping current was also steeper than a Michaelis relation. 5. In Ringer solution at 20 degrees C the saturated exchange current was about 20 pA and the value of charge at which the current was half-saturated was 1-5 pC corresponding to 6-30 X 10(6) Ca2+ ions per rod outer segment. 6. The Ca2+ exchange current after small loads declined along the same curve as that determined with medium loads but fell more slowly after large loads. 7. The exchange current at the beginning of the plateau of a strong flash response usually declined along the curve determined with small or medium Ca2+ loads. 8. There was evidence that the exchange current at the tip of the outer segment remained saturated for longer than at the base. 9. The time to pump out Ca2+ through the Na+-Ca2+ exchange system is largely responsible for the delay in the recovery of the light-sensitive current after a Ca2+ load. 10. A theoretical analysis of some of the observations in this and the succeeding paper is based on assumptions about the binding of Ca2+ by exchange sites and by cytoplasmic Ca2+ buffers.

1-Methyl-3-isobutylxanthine↗
PubMed1987

The effect of ions on sodium-calcium exchange in salamander rods.

1. The influence of external cations on the rate at which a Ca2+ load was eliminated in exchange for external Na+ was studied by measuring the inward current associated with Na+-Ca2+ exchange in salamander rods. 2. In Ringer solution the exchange current saturated at a well-defined level of about 20 pA at 20 degrees C. 3. The saturation level of exchange current, j(sat), was increased by lowering the external concentrations of H+, Ca2+, Mg2+ and K+; it was decreased by raising the external concentration of these ions or by lowering [Na+]O. 4. J(sat) varied approximately as [Na+]O2.4 between 35 and 110 mM-Na+. 5. The inhibitory constants for external Ca2+ and Mg2+ were about 1 and 4 mM, respectively. 6. An acid pH decreased j(sat) and an alkaline one increased it; the shape of the relation between current and pH suggests that one inhibitory proton combines between pH 8 and 10 and a pair combine between pH 6 and 7. 7. Removing K+, Mg2+, and Ca2+, and increasing the pH from 7.5 to 10 increased the measured exchange current from 20 to ca. 100 pA. 8. The integral of the Na+-Ca2+ exchange current varied with the Ca2+ load but was largely independent of external ionic changes in spite of large changes in j(sat). The apparent Na+-Ca2+ exchange ratio remained at a little under 3 over a wide range of conditions. 9. The constancy of the integral of the exchange current was brought about by reciprocal variations of the amplitude and duration of the current transient. Records in different solutions could usually be matched by scaling amplitude and time by reciprocal factors. 10. Increasing Nai+ by allowing large light-sensitive currents to flow in low-Ca2+ solutions affected the Na+-Ca2+ exchange transient in a different way from lowering [Na+]o or raising [Ca2+]o, etc. In an Na+-rich rod there was little reduction in j(sat) but the response was prolonged and larger Ca2+ loads were needed to reach saturation. Analysis in terms of a simple model indicated that a substantial Na+ load might reduce the apparent affinity of the internal pumping sites for Ca2+ by a factor of 10. 11. An attempt is made to relate these findings to a model of Na+-Ca2+ exchange.

Action Potentials↗
PubMed1987

Kinetics and components of the flash photocurrent of isolated retinal rods of the larval salamander, Ambystoma tigrinum.

1. Membrane currents initiated by intense, 20 microseconds flashes (photocurrents) were recorded from isolated salamander rods by combined extracellular suction electrodes and intracellular tight-seal electrodes either in current or voltage clamp mode. The magnitudes (mean +/- 2 S.E.M.) of the maximal photoresponses recorded by the suction and by the intracellular electrode respectively were 40 +/- 5 pA (n = 18) and 35 +/- 7 mV (n = 8) for current clamp at zero current; 43 +/- 9 pA and 66 +/- 13 (n = 11) pA for voltage clamp at the zero-current holding potential, -24 +/- 3 mV. 2. Photocurrents initiated by flashes isomerizing 0.1% or more of the outer segment's rhodopsin achieved a saturated velocity and were 95% complete in less than 50 ms. The effect of incrementing flash intensity above 0.1% isomerization can be described as a translation of the photocurrent along the time axis towards the origin. Within the interval 0-50 ms the latter two-thirds of the velocity-saturated photocurrent is well described as a single-exponential decay. The decay was much faster in voltage clamp (2.8 +/- 1.2 ms, n = 11) than in current clamp mode (17 +/- 5 ms, n = 17). 3. The initial third of the velocity-saturated photocurrent, occurring over the interval from the flash to the onset of exponential decay, followed about the same time course in current and voltage clamp. The time interval occupied by this initial 'latent' phase decreased with increasing flash intensity and attained an apparent minimum of about 7 ms in response to flashes isomerizing 10% or more of the rhodopsin at ca. 22 degrees C. 4. The hypothesis that the decay of outer segment light-sensitive membrane current is the same in current and voltage clamp was supported by an analysis of the difference between outer segment currents measured successively in the two recording modes. First, the tail of the difference current decayed exponentially with a time constant approximately equal to R x C, where R and C are independently estimated slope resistance and capacitance of the rod. Secondly, the integral of the difference current, when divided by outer segment capacitance, closely approximated the hyperpolarizing light response measured under current clamp. Thus, displacement current accounted for the difference between photocurrents measured in current and voltage clamp.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1988

The ionic selectivity of the light-sensitive current in isolated rods of the tiger salamander.

1. Using the method of Hodgkin, McNaughton & Nunn (1985) for rapidly changing the extracellular medium, we analysed the effect of divalent cations on the photocurrent of isolated retinal rods of the tiger salamander. 2. When the extracellular NaCl was replaced by equiosmolar amounts of BaCl2, SrCl2, CaCl2, MgCl2 and MnCl2 the efficacy in carrying the photocurrent at early times was Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+ greater than Mn2+. At early times Ba2+ could carry a photocurrent similar to or larger than that carried by Na+. 3. The photocurrent carried by Ba2+ increased by about 50% when [Ca2+]o was reduced from 1 to 0.1 mM. In the presence of 0.1 mM-Ca2+ in the extracellular medium the photocurrent carried by Ba2+ saturated when [Ba2+]o was close to 50 mM and was half-activated at 15 mM [Ba2+]o. 4. The photocurrent which can be carried by Sr2+ is not larger than that carried by Ba2+ and does not saturate for [Sr2+]o up to 70 mM. 5. When extracellular Na+ is replaced by the impermeant organic ion choline it is possible to observe a transient photocurrent which is carried by Ca2+. This current has a maximal value of about 11 pA and has a half-activation constant of about 50 microM. 6. Movements of Mg2+ across the light-sensitive channel can be seen only when extracellular Ca2+ is reduced below 10 microM. Under these conditions the maximal photocurrent which can be carried by Mg2+ at early times is about 8 pA and has a half-activation of about 2 mM. Under normal conditions Mn2+ is hardly permeable through the light-sensitive channel. 7. It is concluded that the selectivity of the light-sensitive channel in the low ionic concentration range is Ca2+ greater than Sr2+ greater than Ba2+ greater than Mg2+ greater than Na+.

Action Potentials↗
PubMed1988

Control of light-sensitive current in salamander rods.

1. The exponential decline of light-sensitive current seen after switch from Na+ to Li+ in the presence of Ca2+ probably depends on the activity of the phosphodiesterase (PDE) which hydrolyses cyclic GMP. 2. This probability is supported by experiments with suction electrodes which show that in toad and salamander rods the rate constant, b, of the exponential decline of current was increased at least 10-fold by moderate light intensities and decreased about 10-fold by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of PDE. 3. The rate constant b is about 3 times more sensitive to weak lights or to IBMX than the membrane current. This may be explained by a feed-back involving calcium ions which tends to hold current constant, perhaps by calcium inhibition of guanylate cyclase. 4. The time course of b, which probably represents the changes in PDE activity, was measured by switching from Na+ to Li+ at various times after a flash. The results suggest that a moderate flash (140 Rh) increased b about 7 times in 0.5 s and that b then declined with a time constant of 1.5-2 s. 5. Extrapolated values of the parameter b suggest that strong flashes (5000-10,000 Rh) increased b from 1 s-1 in the dark to perhaps 60 s-1 and that b continued to increase with flash strength for several log units after the current had reached saturation. 6. The observations in 4 and 5 fit well with the idea that b is related to PDE activity and that changes in the latter are sufficient to account for the rising phase of the flash response. 7. After a flash the light-sensitive current recovers much more rapidly than the time constant b-1, a discrepancy which is explained if a light flash causes a delayed increase in guanylate cyclase activity. 8. The apparent delayed increase in cyclase activation is consistent with an inhibitory effect of [Ca2+]i which is reduced when calcium is pumped out during the plateau of the response. 9. Experiments in which pulses of IBMX were applied at different times during a flash response support the idea that a flash causes a delayed increase in the rate of supply of cyclic GMP. Quantitative analysis of these and other tests with IBMX gave rate constants similar to those obtained by the Na+----Li+ method.

1-Methyl-3-isobutylxanthine↗
PubMed1988

The modulation of the ionic selectivity of the light-sensitive current in isolated rods of the tiger salamander.

1. By using the method of Hodgkin, McNaughton & Nunn (1985) for rapidly changing the extracellular medium, we analysed the effect of the organic compound IBMX (3-isobutyl-1-methylxanthine) on the movement of divalent cations through the light-sensitive channels of isolated retinal rods of the tiger salamander. 2. When the rod is treated with 0.5 mM-IBMX it is possible to observe photocurrents larger than 50 pA carried by Ba2+, Sr2+, Ca2+, Mg2+ and Mn2+. Under these conditions Ca2+, Mg2+ and Mn2+ carry photocurrents of similar amplitude, while Ba2+ and Sr2+ usually carry larger photocurrents. 3. The movement of Mn2+ through the light-sensitive channel, which is hardly detected under normal conditions, can also be observed after treating the rod for a few seconds with a solution containing 35 mM[Na+]o and 10(-7) M[Ca2+]o. Under these conditions the photocurrent carried by Mn2+ is fully saturated in the presence of 1 mM-extracellular Mn2+. 4. When the rod is pre-treated with an extracellular solution containing 0.5 mM-IBMX the maximal photocurrent which can be carried by 10 mM [Ca2+]o increases from about 10 pA to approximately 200 pA. In these conditions the half-activation of the Ca2+ current is between 1 and 10 mM, that is 20-50 times higher than in normal conditions (Menini, Rispoli & Torre, 1988). 5. When the rod is pre-treated with an extracellular solution containing 0.5 mM-IBMX the half-activation of the photocurrent which can be carried by Mg2+, Ba2+ and Sr2+ is equivalent to or greater than 10 mM. In the absence of pre-treatment with IBMX the half-activation of the photocurrent carried by Mg2+, Ba2+ and Sr2+ is less than 5 mM. 6. We conclude that the light-sensitive channel can exist in at least two distinct open states. The selectivity of the channel in the first open state is as described in a previous paper (Menini et al. 1988). Mn2+, which is hardly permeable through the light-sensitive channel in the first open state, can move through the light-sensitive channel in the second open state. Ca2+, Mg2+, Ba2+ and Sr2+ permeate more freely through the light-sensitive channel in the second open state, probably because the electrostatic interactions between these ions and the channel are less strong.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium.

1. Light adaptation has been studied in isolated red-sensitive cone photoreceptors of the salamander, using suction pipette recordings of circulating current. 2. In the presence of background illumination, the response to incremental dim flashes became desensitized according to the Weber-Fechner law. The recovery phase of the flash response was accelerated significantly, although the time-to-peak was reduced only slightly, and for dim backgrounds the rising phase was unaltered. 3. The role of cytoplasmic calcium concentration, Cai2+, in mediating cone adaptation was investigated by minimizing light-induced changes in Cai2+, either by incorporating calcium buffer into the cytoplasm or by exposing the outer segment to low-Ca2+, 0-Na+ solution. Both treatments appeared to slow dramatically or even to eliminate the onset of light adaptation in the cone. 4. When the low-Ca2+, 0-Na+ solution was presented in darkness, responses to subsequent illumination were affected in a characteristic manner: (i) the response-intensity relation was steepened and shifted to lower intensities, (ii) the response to a step of light could be predicted by integration and compression of the flash response, and (iii) the flash sensitivity declined steeply as a function of background intensity. 5. After extended exposure of the cone to bright backgrounds, the sensitivity in darkness failed to return to its original level. The flash response kinetics were faster and more biphasic than for dark-adapted responses or for responses desensitized to a comparable degree by exposure to steady background illumination. 6. The results indicate that, in cones isolated from the pigment epithelium, the primary factor influencing the adaptational state of the cell is the cytoplasmic concentration of free calcium, but that at high intensities the effects of pigment bleaching are likely to be significant.

Action Potentials↗
PubMed1990

Spatial organization of the bipolar cell's receptive field in the retina of the tiger salamander.

1. The spatial properties of rods, horizontal cells and bipolar cells were studied by intracellular recording in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. Low stimulus intensities were used in order to keep cell responses close to, or within, their linear intensity/response range. 2. Spatial properties of bipolar cell receptive fields, measured while perfusing with normal Ringer solution, were compared with those measured during exposure to agents that eliminated the bipolar cells' receptive field surround (RFS). In this way, the spatial properties of the receptive field centre (RFC) and those of the RFS could be characterized independently. 3. To a good approximation, the contribution to the horizontal cell's response of unit area of its receptive field declined exponentially with distance from the centre of the receptive field. The (apparent) length constant describing this decay was 200 microns. The one-dimensional length constant of the horizontal cell syncytium was thus 248 microns. The variation of response amplitude with the radius of a centred circular stimulus was consistent with this finding. 4. This was true also of the RFCs of bipolar cells. The one-dimensional length constant of the RFC of off-centre bipolar cells averaged 124 microns. That of the RFC of on-centre cells averaged 62 microns though values were more variable, the RFCs of some on-centre cells being comparable to those of off-centre cells. These values were independent of the class of photoreceptor driving the bipolar cell. 5. The large size of the RFCs of off-centre cells and many on-centre cells cannot by explained by light scatter within the retina or by voltage spread within the rod syncytium. We proposed that off-centre cells are tightly coupled in a syncytium. On-centre cells, on average, are less tightly coupled. 6. The spatial properties of the bipolar cell's RFS were consistent with the notion that the RFS represents a convolution of the horizontal cell's receptive field and the bipolar cell's RFC. 7. The spatial properties of bipolar cell receptive fields were reconstructed from the measured properties of their RFCs and the measured properties of horizontal cell receptive fields. Under the conditions of our experiments, the bipolar cell's response could be described by a linear difference between a component generated by the RFC and a component generated by the RFS. 8. The spatial filtering characteristics of the bipolar cells were calculated from our data.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1990

Currents carried by monovalent cations through cyclic GMP-activated channels in excised patches from salamander rods.

1. Ionic selectivity and affinity for monovalent cations of channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in excised inside-out patches of plasma membrane from retinal rods of the tiger salamander. Channels were activated by addition of cyclic GMP to the medium bathing the cytoplasmic side of the membrane. The ionic solution at the cytoplasmic side was rapidly changed using the method of Nunn (1987 a). 2. Permeability ratios were calculated with the Goldman-Hodgkin-Katz potential equation from reversal potential measurements for alkali monovalent cations in bi-ionic conditions. The permeability sequence was: Li+:Na+:K+:Rb+:Cs+ = 1.14:1:0.98:0.84:0.58. 3. The selectivity sequence obtained from macroscopic current measurements in bi-ionic conditions at +100 mV was: Na+:K+:Rb+:Li+:Cs+ = 1:1:0.67:0.36:0.25. 4. The organic cations tetramethylammonium (TMA+), choline and tetraethylammonium (TEA+) were not permeant through the cyclic GMP-activated channels and caused a reduction of the Na+ inward current. At -100 mV the current ratio for inward current was 1:0.75:0.58:0.2 in the presence, at the cytoplasmic side, of 110 mM-Na+, TMA+, choline or TEA+ respectively. 5. The concentration dependence of the macroscopic current and the reversal potential was studied by changing the internal concentration of Na+ or K+ or Li+ from 5 mM to 500 mM. The permeability ratios were nearly constant regardless of the permeant ion concentration. 6. The current as a function of internal ion activity could be described by a Michaelis-Menten relation with a half-saturating activity, Km, at +90 mV equal to 249, 203 and 160 mM for Na+, K+ and Li+ respectively. The ratio of the extrapolated saturating current Imax at +90 mV was 1:0.86:0.26 for Na+, K+ and Li+ respectively. 7. The outward currents and the reversal potentials measured in different mixtures of Na+ and Li+ were monotonic function of the mole fraction. 8. These results can be explained by assuming that, at least in a narrow region, the cyclic GMP-activated channel is a one-ion channel, possibly with other poorly voltage-dependent binding sites in a large inner vestibule.

Animals↗
PubMed1990

Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.

1. Müller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole-cell voltage clamp. External D-aspartate, L-aspartate and L-glutamate each induced a membrane current. D-Glutamate, kainate, quisqualate and N-methyl-D-aspartate were more than 100x less effective than L-aspartate. Kynurenic acid had no effect on the current produced by L-glutamate, L-aspartate or D-aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl- concentrations demonstrated that these ions are not required for transport. 3. The shape of the current-voltage relation did not depend on the external amino acid concentration. The relationship between D-aspartate concentration and current amplitude can be described by the binding of D-aspartate to a single site with an apparent affinity constant of 20 microM. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA-induced current had a Q10 between 8 and 18 degrees C of 1.95. The Q10 is consistent with a rate constant for influx of 10(4) s-1 (at -70 mV and 20 degrees C). The maximum rate of influx was measured following a concentration jump produced by the photolysis of 'caged' L-glutamate. The onset of the observed current was limited by the 1.3 ms resolution of the recording system. Hence, the rate constant for influx must be faster than 10(3) s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambystoma↗
PubMed1990

Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells.

1. Cells in the ganglion cell layer of salamander retinal slices were voltage clamped using patch pipettes. Light elicited transient excitatory postsynaptic currents (EPSCs) in on-off ganglion cells and sustained EPSCs in on ganglion cells. Light-evoked inhibitory postsynaptic currents in these cells could be blocked by 100 microM-bicuculline methobromide and 500 nM-strychnine. 2. In the presence of external Cd2+, at a concentration that blocked light-evoked synaptic inputs, N-methyl-D-aspartate (NMDA) and the non-NMDA-receptor agonists, quisqualate and kainate, gated conductances in both on-off and on ganglion cells. The current-voltage (I-V) curve for the conductance elicited by NMDA had a negative slope between -40 and -70 mV and a reversal potential near 0 mV. The I-V curves for the non-NMDA-receptor-mediated conductances were nearly linear and also had reversal potentials near 0 mV. 3. I-V curves were measured at an early time point near the peak of transient EPSCs and at a later time point during the decay phase of the responses. The late I-V curve had a negative slope below -40 mV. The early I-V curve had a positive slope over the entire voltage range but the slope was greater at positive than at negative potentials. The evoked current reversed near 0 mV at both time points. 4. The region of negative slope of the late I-V curve was eliminated when Mg2+ was removed from the external saline. A slowly decaying component of transient EPSCs was eliminated in 20 microM-DL-2-amino-7-phosphonoheptanoate (AP7), an NMDA-receptor antagonist. 5. Application of 1 microM-6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA-receptor antagonist at this concentration, blocked a fast component of transient EPSCs. 6. Our results demonstrate that the synaptic inputs to on-off ganglion cells have two components: a slower NMDA-receptor-mediated component having a time-to-peak of 110 +/- 45 ms and an e-fold decay time of 209 +/- 35 ms at -31 mV (mean +/- S.D., n = 5), and a faster non-NMDA-receptor-mediated component having a time-to-peak of 28 +/- 10 ms and an e-fold decay time of 43 +/- 20 ms at -31 mV (n = 8). 7. A similar analysis of sustained EPSCs of on ganglion cells showed that these currents resulted from sustained activation of both NMDA and non-NMDA receptors.

Ambystoma↗
PubMed1990

The magnitude, time course and spatial distribution of current induced in salamander rods by cyclic guanine nucleotides.

1. Cyclic GMP was introduced into isolated salamander rods through a tight-seal electrode attached to the inner segment while the outer segment was held in a suction electrode; nucleotide-induced membrane current was recorded by both electrodes. After 3-15s of nucleotide exposure the cells were stimulated with intense, brief flashes, which suppressed 90-95% of the induced membrane current. 2. The magnitude of the induced light-sensitive current depended little on the pipette cyclic GMP concentration in the range 10-20 nM: the mean whole-cell current magnitude was 1256 +/- 160 pA (mean +/- 2 S.E.M., n = 41). 3. Experiments and analyses addressed hypotheses about the nature of the magnitude limitation on the induced current. It was shown that the spatial distribution of nucleotide, the residual series resistance of the whole-cell electrode, and the diminution of the ion gradients driving the induced current did not limit the current magnitude by more than 20%. 4. In contrast, the hypothesis that outer segment internal longitudinal resistance severely limits the magnitude of the cyclic GMP-induced current was supported by experiments in which various lengths of the outer segment were drawn into the suction electrode. These showed that the ratio of nucleotide-induced light-sensitive current collected by the suction electrode to that collected by a whole-cell electrode decreased steeply as a function of outer segment length excluded from the suction electrode, having an apparent space constant of 5-7 microns. 5. A cable model of the rod was developed and used to analyse the magnitude of the nucleotide-induced currents. The data are consistent with an outer segment longitudinal resistance of 1-4 M omega microns-1, and a maximum (space-clamped) light-sensitive current density of 313 pA microns-1, equivalent to a total induced current of 7200 pA (23 microns outer segment). 6. A diffusion model was developed and combined with the non-linear cable model to provide an account for the time course of the induced membrane currents. The results are consistent with an effective longitudinal diffusion coefficient of cyclic GMP in the outer segment of 3-10 microns2 s-1, and Hill coefficient of 2-3 for the cyclic GMP gating of the light-sensitive conductance. 7. 8-Bromo-cyclic GMP also caused the light-sensitive membrane current to increase to about the same magnitude as did cyclic GMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗