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T D Lamb

Publications and source records attributed to T D Lamb.

At least 55 records · Page 3Linked to original sources
PubMed1988

External and internal actions in the response of salamander retinal rods to altered external calcium concentration.

1. The membrane current was recorded from retinal rods isolated from Ambystoma tigrinum using the suction pipette and whole-cell patch pipette techniques, while the concentration of calcium bathing the outer segment was rapidly reduced. 2. The increase in outer segment current induced by lowered external calcium in darkness could be resolved into two components, one as rapid as the time course of the solution change (as judged by the junction current) and the other somewhat slower. 3. Introduction of the calcium buffer BAPTA (1,2-bis(o-aminophenoxy)ethane-N ,N ,N' ,N'-tetraacetic acid) into the cell from a patch pipette led to a progressive slowing of the second component of current increase. 4. When several minutes had elapsed following rupture of the patch, to allow a substantial amount of BAPTA into the cell (ca. 10 mM in the patch pipette), the second component was slowed by a factor of about 20-fold, while the first component continued to have the same rapid time course as the solution change. 5. The rapid component is attributed to a direct effect of external calcium, Ca2+o, and the delayed component to an indirect effect mediated by a reduction in internal calcium, Ca2+i. 6. These results confirm that, in previous experiments in which BAPTA was introduced into photoreceptors, the internal calcium concentration was very significantly buffered. 7. When Ca2+o drops from 1 mM to less than 10(-8) M, the rapid external component corresponds to an increase in circulating current of 3- to 4-fold, and the internal component corresponds to an increase of at least 4- to 5-fold. However, the total current at late times is limited by electrical factors, so that the size of the internal effect is bound to be considerably greater.

Action Potentials↗
PubMed1988

Incorporation of analogues of GTP and GDP into rod photoreceptors isolated from the tiger salamander.

1. Analogues of GTP and GDP were introduced into isolated rod photoreceptors using the whole-cell patch clamp technique, while simultaneously recording the photocurrent with a suction pipette. After several minutes of whole-cell recording the patch pipette was disengaged, thus trapping the analogue inside the cell. 2. During the introduction of the hydrolysis-resistant GTP analogues guanosine-5'-O-(3-thio-triphosphate) (GTP-gamma-S) and guanylyl-imidodiphosphate (GMP-PNP) the dark current progressively declined, and the duration of responses to flashes of light which had previously been just-saturating increased slightly. The form of the rising phases of the responses to dim or bright flashes was little affected. 3. Following the incorporation of these GTP analogues the response to an intense flash was prolonged by a factor of up to 300, and the circulating current remained suppressed for up to 1 h. Ultimately the circulating current recovered and the duration of the flash response returned to near its control value. 4. Superfusion of the outer segment with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) during the extended period of saturation resulted in a rapid increase in the circulating current, suggesting that the analogues had their major effect on the duration of phosphodiesterase activation by light. 5. Introduction of the phosphorylation-resistant GDP analogue guanosine-5'-O-(2-thio-diphosphate) (GDP-beta-S) resulted in a decrease in light sensitivity and a reduction in the slope of the rising phase of the flash response. 6. The response to an intense flash was also prolonged in cells containing GDP-beta-S, recovery becoming progressively slower on successive presentations of the flash following the withdrawal of the patch pipette. This observation suggests that GDP-beta-S may be slowly converted within the cell to form a hydrolysis-resistant product. 7. These results indicate that the presence of a hydrolysis-resistant analogue of GTP within the cell causes light activation of the transduction mechanism for an extended period. Our interpretation of this finding is that hydrolysis of the bound guanosine nucleotide is necessary for the quenching of activated GTP-binding protein.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Sources of noise in photoreceptor transduction.

Photoreceptors are surprisingly noisy, and the properties of this noise are providing clues to the molecular mechanisms underlying phototransduction. Three distinct components of noise arise from excitation of the rhodopsin photopigment molecule: the first is activation by photons, and the second is thermal activation in darkness. The third component, induced following intense bleaching lights, probably reflects reversibility in the reactions that inactivate isomerized rhodopsin; in this way a small degree of reactivation of the excited form of rhodopsin is generated from the vast amounts of bleached photoproduct that exist in the photoreceptor following intense exposures. A fourth component of noise, the continuous component present in darkness, probably arises from stochastic fluctuations in the number of excited molecules of a biochemical intermediate, either transducin (G-protein) or phosphodiesterase. This fourth component of noise appears to be much more prominent in cones than in rods. A fifth (and smaller) noise component is caused by the random opening and closing of light-sensitive channels in the outer segment.

Animals↗
PubMed1986

Role of calcium in regulating the cyclic GMP cascade of phototransduction in retinal rods.

Both cGMP and Ca2+ appear to be involved in the process of phototransduction in vertebrate rods, but their precise roles have been the subject of debate. To investigate the role of Ca2+ we have artificially increased the calcium buffering capacity of the rod by using a patch pipet to incorporate the calcium buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) into the rod cytoplasm. In the presence of buffer the Na+-Ca2+ exchange current became greatly slowed, suggesting that the cytoplasmic calcium concentration (Cai) had indeed been buffered substantially. Although the presence of buffer had negligible effect on the rising phase of the light response, it profoundly altered the later behavior. Responses to brief flashes became prolonged and exhibited an overshoot, apparently because the shut-off process was modified. The normal acceleration of time-to-peak with brighter flashes (an early sign of light adaptation) disappeared. Responses to steady adapting illumination took much longer than normal to settle to a steady level, although the final level represented a similar fractional suppression of current. With superimposed test flashes the presence of such adapting illumination caused a more rapid recovery, whereas the presence of calcium buffer slowed the recovery. The results are consistent with the idea that the rapid drop in Cai, which has recently been shown to accompany the light response, is involved in terminating the light response, and that Cai is thereby involved in setting the operating point and sensitivity of phototransduction. From comparison with other work we infer that Cai appears to act, at least in part, by means of control of cGMP phosphodiesterase activity.

3',5'-Cyclic-GMP Phosphodiesterases↗
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↗
PubMed1985

An inexpensive digital tape recorder suitable for neurophysiological signals.

Modifications are described which convert an inexpensive 'Digital Audio Processor' (Sony PCM-701ES), together with a video cassette recorder, into a high performance digital tape recorder, with two analog channels of 16 bit resolution and DC-20 kHz bandwidth. A further modification is described which optionally provides four additional 1-bit digital channels by sacrificing the least significant four bits of one analog channel. If required two additional high quality analog channels may be obtained by use of one of the new video cassette recorders (such as the Sony SL-HF100) which incorporate a pair of FM tracks.

Neurophysiology↗
PubMed1984

Effects of temperature changes on toad rod photocurrents.

Rod current responses were measured over the range 5-30 degrees C. Following a rapid decrease in temperature the amplitude of the dark current decreased without detectable delay (less than 3 s). Over a period of several minutes the amplitude of the dark current sometimes relaxed slightly towards its previous value. The rapid change cannot be accounted for simply by altered activity of the sodium pump and instead indicates that the conductance of the outer segment in darkness changes with temperature. Over the range 10-30 degrees C the amplitude of the dark current increased approximately linearly with temperature, and the straight line of best fit extrapolated to zero current at about 5 degrees C. The few points available below 10 degrees C indicated that the relationship flattened out, but this could not be investigated properly. The kinetics of responses to dim flashes accelerated with a Q10 of about 2.2, and were well described by an Arrhenius plot with an activation energy of 13.8 kcal mol-1 (HEPES Ringer solution). The time course of recovery of dark current following a saturating flash showed a similar temperature dependence to that of the dim flash kinetics. A simple explanation of the previous two findings is that the delays determining the time course of responses to both dim and bright flashes are largely determined by the fluidity of the disk membrane. The sensitivity to dim flashes had a broad peak at about 22 degrees C, decreasing at both lower and higher temperatures. The relative sensitivity to long wave-length light increased slightly with temperature. The sensitivity at 700 nm relative to that at 500 nm increased by 0.225 log10 units (1.68 times) upon a temperature increase from 11.5 to 29.3 degrees C (from approximately -5.0 log10 units to approximately -4.8 log10 units). This change appears to be approximately what would be expected theoretically.

Animals↗
PubMed1982

Local effects of bleaching in retinal rods of the toad.

1. Suction electrode recordings were used to study the recovery of responsiveness in single toad rods after bleaching a small fraction (less than 5%) of the rhodopsin. 2. After a spatially uniform bleach that initially abolished the dark current over the entire length of the outer segment, the more proximal regions recovered faster than the more distal regions. For a time the most basal region was almost fully recovered while the tip remained fully saturated. 3. Such a gradient of responsiveness did not occur during uniform steady background illumination of dark-adapted cells. 4. The entire outer segment recovered uniformly after a longitudinally graded bleach that simulated the pattern produced by self-screening in the intact eye. 5. The recovery of the distal end of the outer segment was not affected by a bleach at the proximal end. This suggests that the differences in recovery rate reflect intrinsic local properties of the outer segment rather than longitudinal diffusion of a substance from the inner segment. 6. For at least the first 3 min after bleaching with a narrow transverse slit the reduction of responsiveness remained most pronounced in the bleached region, suggesting that this effect of bleaching does not spread extensively. 7. The increased noise induced by bleaching is shown to originate locally in the bleached region of outer segment. 8. When the tip was locally saturated after a bleach or during steady light, the current recorded from the tip was predominantly capacitive, resulting from intracellular voltage change. This indicates that when the dark current is abolished the outer segment plasma membrane has negligible leakage conductance.

Action Potentials↗
PubMed1981

Spatial spread of activation and background desensitization in toad rod outer segments.

1. The spread of activation and background desensitization in rods was studied by recording membrane current from single outer segments in pieces of isolated toad retina.2. Flash sensitivity changed slightly along the outer segment, falling by about 30% from base to tip.3. When only the distal half of an outer segment was in the recording pipette, illumination of the unrecorded part elicited little or no photocurrent at the recorded part, indicating that a photoisomerization does not cause activation of the entire outer segment.4. With diffuse illumination of an outer segment fully drawn into the pipette, the intensity-response relations at fixed times were invariant in form for most of the rising phase of the flash response and were considerably steeper than the Michaelis relation. The observed relation was consistent with a model in which a photoisomerization blocks all channels over a short region of the outer segment.5. With illumination restricted to a narrow transverse slit, the intensity-response relations at fixed times were much less steep, as would be expected for a very limited longitudinal spread of activation. An upper limit for the effective longitudinal diffusion coefficient of the internal transmitter was estimated to be about 3 x 10(-7) cm(2) sec(-1). This corresponds to a space constant for longitudinal spread of transmitter of about 3 mum at the time of the dim response peak.6. The time course of flash responses elicited with light positioned either on the edge or on the centre of the outer segment was very similar.7. Desensitization resulting from steady illumination by a transverse slit was also localized longitudinally. A linear desensitization parameter T, defined in Results, decayed approximately exponentially along the outer segment, on either side of the site of photoisomerization, with a space constant of about 6 mum.8. Transverse spread of desensitization was more effective than longitudinal spread.9. After turning off a dim diffuse background light, the decay of T was roughly exponential with a time constant of several seconds. From this, and the steady state space constant of 6 mum, it is estimated that the effective longitudinal diffusion coefficient for a ;desensitizing substance' would also be about 10(-7) cm(2) sec(-1).10. The restricted longitudinal spread of activation and desensitization may be explained by the barrier to diffusion presented by the stacked membranous disks in the outer segment. This baffling reduces the effective longitudinal diffusion coefficient to about 1/50 that of ordinary aqueous diffusion, but it does not significantly affect transverse spread.

Animals↗
PubMed1980

Spontaneous quantal events induced in toad rods by pigment bleaching.

Barlow proposed that absolute visual threshold is limited by photon-like noise events in the rod photoreceptors, and he later extended the idea to explain the elevation of threshold following bright bleaching lights in terms of increased noise in the photoreceptors. Rushton, on the other hand, has proposed that the threshold elevation during dark adaptation involves changes not within the photoreceptors themselves but rather in the gain of a subsequent 'pool'. I report here measurements of outer segment current in individual rod photoreceptors which demonstrate that spontaneous fluctuations occur at a greatly increased rate following bleaches of around 1%, and that these fluctuations have the form expected for random occurrences of the single photon events. This is consistent with Barlow's ideas but does not indicate whether a gain change subsequent to the receptors also occurs.

Action Potentials↗
PubMed1980

Spread of activation and desensitisation in rod outer segments.

Visual transduction in vertebrate photoreceptors is thought to involve a diffusible internal transmitter which links photon absorption in disk membranes to the conductance decrease in the plasma membrane of the outer segment. The desensitisation of the photoresponse which occurs during and after illumination may also involve a diffusible substance, because a photon absorbed in one disk can desensitise the cell's response to a subsequent photon absorbed in a different disk. Although both activation and desensitisation spread longitudinally from the site of photon absorption, neither spreads over the entire length of the outer segment. We report here experiments which indicate that, with dim illumination, both effects decline to be half-maximal in less than 8 micrometers from the site of light absorption.

Adaptation, Physiological↗
PubMed1979

The membrane current of single rod outer segments.

1. Outer segments of individual rods in the retina of the toad, Bufo marinus, were drawn into a glass pipette to record the membrane current. 2. Light flashes evoked transient outward currents. The peak response amplitude was related to flash intensity by a Michaelis equation with half-saturating intensity about 1 photon mum-2. 3. The saturating response amplitude ranged up to 27 pA and corresponded closely to complete suppression of the steady inward current present in darkness. 4. For a given cell the saturating response amplitude varied linearly with the length of outer segment within the pipette. This is consistent with a uniform density of light-sensitive channels and negligible gradient of membrane potential along the outer segment. 5. Responses to bright flashes never showed the relaxation from an initial peak seen previously in intracellular voltage recordings, suggesting that the conductance change responsible for the relaxation does not occur in the outer segment. 6. Responses to local illumination of only the recorded outer segment were very similar to those obtained with diffuse light at the same intensity, indicating that peripheral rods made little contribution to the responses. 7. The spectral sensitivity of 'red' rods was consistent with a retinal1-based pigment with lambda max = 498 +/- 2 nm. 8. The kinetics of the response were consistent with four stages of delay affecting action of the internal transmitter. Responses were faster at the basal end of the outer segment than at the distal tip. 9. Background light reduced the sensitivity to a superposed dim test flash and shortened the time course of the response, indicating that adapting light modifies the kinetics and gain of the transduction mechanism within the outer segment. 10. Responses to dim lights exhibited pronounced fluctuations which are attributed in the succeeding paper (Baylor, Lamb & Yau, 1979) to the quantal nature of light.

Animals↗
PubMed1979

Responses of retinal rods to single photons.

1. A suction electrode was used to record the membrane current of single rod outer segments in pieces of toad retina. During dim illumination the membrane current showed pronounced fluctuations. 2. Amplitude histograms of responses to dim flashes of fixed intensity exhibited two discrete peaks, one at 0 pA and one near 1 pA, suggesting that the response was quantized. By setting a criterion amplitude level, flash responses could be classed as 'failures' (no response) or as 'successes' (at least one quantal event). 3. The variation of fraction of successes with flash intensity was consistent with the hypothesis that each quantal electrical event resulted from a single photoisomerization. 4. The quantal event had a mean amplitude of about 1 pA (5% of the standing dark current) and a standard deviation of 0.2 pA. Dispersion in the event amplitude prevented identification of histogram peaks corresponding to two or more photoisomerizations. 5. Individual quantal responses exhibited a smooth shape very similar to that of the average quantal response. This suggests that a single photoisomerization releases many particles of transmitter and that radial diffusion of internal transmitter is not a major source of delay in the light response. 6. The 'quantum efficiency' with which an absorbed photon generated an electrical event was measured as 0.5 +/- 0.1 (S.E. of mean, n = 4). This is slightly lower than the quantum efficiency of photoisomerization obtained previously for rhodopsin in solution. 7. At wavelengths between 420 and 700 nm the quantal event was invariant in size, although the cell's sensitivity varied over a range of 10(5). 8. The power spectrum of the fluctuations in dim steady light was predicted by assuming that a random series of quantal events occurred independently. 9. In brighter light the fluctuations were faster, and the response to an incremental flash was reduced in size and duration. The power spectrum could be predicted by assuming random superposition of events with the shape of the incremental flash response.

Animals↗
PubMed1978

Properties of the membrane current of rod outer segments.

The membrane current of single rod outer segments in pieces of isolated toad retina was recorded with a glass suction electrode. Light evoked a slow net outward photocurrent consisting of a reduction in the steady inward dark current. In very dim light, the photocurrent broke up into discrete shot effects with a rounded shape and an amplitude of about 1 pA. These events were shown to result from photoisomerization of single rhodopsin molecules. The current in darkness showed fluctuations consisting of (a) discrete events apparently resulting from thermal isomerization of rhodopsin molecules, and (b) smaller amplitude shot effects shaped by two of the four rate processes of the light response.

Animals↗