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Dynamics of dark adaptation in the cephalopod retina
Part II: Papers DYNAMICS
OF DARK ADAPTATION CEPHALOPOD RETINA
IN THE
G. DUNCANand P. B. PYEiSENT Biophysics Section. School of Biological Sciences, University of East Angtia. Norwich NR4 7TJ. England Key W&s--retina:
cephalopod:
dark adaptation:
photoreceptor.
Pynsent and Duncan (1977) have shown that the signals recorded at the proximal (y) and distal (V,) ends of Sepia/u photoreceptors are quite different in waveform. They later suggested (Duncan and Pynsent, 1979) that the differences can be explained if it is assumed that there is a primary light-stimulated sodium conductance increase at the distal (rhodopsinbearing) membranes and that the resulting current flow activates voltage-sensitive potassium channels in the membranes of the proximal segment. The basic waveform of the response at either end of the cell is also assumed to be modified by the time constant of the cell membranes and by the relatively large resistance of the extracellular space (Pynsent and Duncan, unpublished). The extracellular responses (V,) are relatively large in the cephalopods, and Weeks and Duncan (1974) have previously investigated the dynamics of dark adaptation of the extracellular responses using a paired flash technique. They found, as expected, that recovery of sensitivity to the second flash was prolonged as the intensity of the stimuli increased. They further showed that at a constant stimulus level, reduction in the external calcium concentration also prolonged the dark adaptation rate. This latter observation contrasts with several reports of similar studies with other invertebrate photoreceptors where reducing the external calcium was found to speed up the recovery of sensitivity (Baumann, 1974; Stieve and Hamani, 1976; Stieve, 1976). As there are in fact several ways of monitoring dark adaptation, even by the simple paired flash technique, and as the extracellular signals may not give a true representation of the electrical events at the membrane levei, we have rigorously examined. the double-flash technique using simultaneously recorded intracellular and extracellular signals. The perfusion chamber was an adaptation of the cooling stage used previously for non-perfused studies (Duncan and Pynsent. 1979). The normal perfusing solution was that used by Clark and Duncan (1978) and the stimulating and recording systems have been described in full elsewhere (Duncan and Pynsent. 1979). Essentially, the paired flashes were produced from a stroboscope with a flash duration of 10psec and the resulting signals were recorded on a d.c. tape recorder. They were then either displayed directly on
a storage oscilloscope screen, or digitised and processed on a computer. Figure 1 gives an example of a typical computer output from a double-flash experiment. The responses have all been scaled to the same height so that differences in waveform between y, V, and V, can be seen more clearly. The greatest difference in the shape of the response to the first flash appears to occur at the highest intensities where there is a pronounced sag phase in the v records comparable with the sag phase in vertebrate intracellular records (Baylor, Hodgkin and Lamb, 1974: Cervetto, Pasino and Torre, 1977). The V, responses are relatively flat over the saturating region, while the V, records show a biphasic response. With the flash separation of 250msec chosen for this series of experiments, there appears to be little decrease in amplitude of the second flash response at low intensities, but there is a considerable loss of sensitivity at high intensities. However, the extent of the adaptation depends on the method of measurement. Cervetto er al. (1977) used the trough between the responses as a reference point [Fig. 2(a)] and with their method there is little difference between the response ratios until an intensity of 100 photons pm-’ is reached. However, the y ratio is consistently below the others throughout the range. Duncan and Croghan (1973) simply measured the heights of the peaks above the baseline [Fig. 2(b)]. In their case, there is a consistent difference between the x and V, records at all intensities, and again the y records show the greatest adaptation. Weeks and Duncan (1974) attempted to obtain a “difference signal” as another criterion for adaptation. A single flash was applied and then after a suitable period, to ensure complete dark adaptation, the double flash stimulus was used. The baseline for the second amplitude was then measured from the point where the response would have been if the second flash had not been applied [Fig. 2(c)]. Using this method, there is little difference in the response ratios of K, V, and V,, throughout the range, except at the lowest intensities where, in fact, the measuring error is greatest. The dark adaptation rare can be measured by varying the separation between the two stimuli, and Fig. 3. shows that the differences between the V, and V, waveforms are most pronounced at short time inter-
359
360
G. DLWCAYand P. B. F’YNSENT
361
Cephalopod dark adaptation
I
PEAK RATIOS (Vi) SOIllS
100
200
1.9
0.60
0.75
0.91
0.65
0.76
0.94
9.6
0.39
0.68
0.90
0.40
0.72
0.88
(0 um2,
Fig. 3. Simultaneous me~urements of intracellular (q) and extracellular (K:.) responses to a series of double flashes of intensity 1.9 and 9.6 photons pm-‘, respectively. The time of the first flash corresponds to the origin of the response and the marker for the second flash is given below each response. A single flash served as the reference waveform for the flashes containing 9.6 photons pm-‘. while the first of two flashes spaced 4OOmsec apart served as the reference for the lower intensity series. The time marker represents 100 msec. The peak ratios for the three flash separations used in both experiments (SO. 100 and 2OOmsec) were calculated by the method of Weeks and Duncan (197-U.
vals. At the relatively
low intensity of 1.9 photons pm-*, the K records seem very much more adapted with a 50msec separation between the flashes as the peak height achieved remains constant. However, when adaptation is assessed by the method of Weeks and Duncan (1974) there is little difference between the x and V, ratios. At low intensities, the q response to the first gash recovers to the baseline much faster than v, (Fig. 3 and Duncan and Pynsent, 1979) and this effect amplifies P? in the V, records. The effects of the adapting flash on the waveform of the response to the second flash were investigated by computing the first derivative (V/set) of the signal (Duncan and Pynsent. 1979) and the rate of change of voltage of the second flash was found to be greatly reduced compared to the rate of change for the first response. The maximum rate of change of voltage (D) is an easily measured parameter and when the ratio of the rates is plotted against intensity, the relationship obtain for al1 three responses (Fig. 4) is remarkably similar to the relationship for the peak ratios obtained by the Weeks and Duncan (1974) method. Although the measurement of adaptation in terms of the potentials involved probably gives a good indication of the possible form of the output from the optic nerves (especialfy method b in Fig. 2), it can obscure info~ation concerning the underlying mechanisms of adaptation at the membrane level. For
example, information will be lost at high intensities as the voltage change obtainable in response to a single flash probably saturates before the maximum possible conductance change is achieved (Clark and Duncan, i978; Lisman and Brown, 1975). However, bearing this point in mind, it is stilt possible to obtain useful information concerning dark adaptation from
Fig. 4. Relationship between maximum rate ratio and intensity for flashes spaced 250msec apart (data from responses given in Fig. 1). D, and DI are the maximum rates of change of voltage for the first and second re-, sponses. respectively. 0, V, responses: (D. V,: 8. V,,. The units of intensity are photons pm-‘.
362
G.
DUNCAN
and P. 0, PYSSENT
the interpretation of double flash experiments. For example, a detailed examination of Fig 1 shows that, as far as Y, and V, are concerned, the maximum volta8e reached by the second response is the same as that reached after the first flash for most of the intensity range. However, this is not true for y where the maximum amplitude of the second flash is much lower than the first. This difference can most readiiy be explained in terms of a voltage-sensitive potassium conductanse in the membranes of the proximal end of the photoreceptor cell. At the point in tune when the second waveform starts off, the potassium conductance of the proximal segment will have been greatly increased by the depolarising current from the distal segment initiated by the first flash. The current from the second flash will therefore be much less effective in depolarising the proximal membrane potential. An active process in the proximal segment will accentuate differences between the three methods of measuring adaptation and it should be noted that if the photoreceptor cell were acting as a purely passive, linear system, then none of the three methods outlined would show any adaptation. At very low intensities, it does indeed appear to behave in this way (Clark, Pynsent and Duncan, unpublished). In general, extracellular responses are much more stable and reproducible than those obtained intracellularly using high resistance microelectrodes (Fig. 3). Such properties are invaluable in the study of dark adaptation rate that may require the presentation of several test pulses over a relatively long period of time. It is therefore interesting to find that using one method for assessing adaptation, the V;, V, and V, records give identical results and it is therefore safe to assume that Weeks and Duncan (1974) were correct in claiming that reducing extracellular calcium prolongs dark adaptation recovery in the cephalopod retina. As one of the main effects of reducing external calcium is to make the extracellular (Clark and Duncan, 1978) and intracellular (Pynsent. 1978) responses more sensitive to light, it is not surprising that recovery is prolonged relative to controls in normal solution.
&%nowvl&emenr~--We wish to thank Dr Peter C. Crog han for many helpful and stimulating discussions. Com-
puter facilities and support for PBP were made available through an SRC grant to Professor E. Rojas.
REFERENCES
3aumann F. (1974) Electrophysiological properties of the honey bee retina. In The CQ~~~~~~~~ Eye amf Vision Of Jnsrcrs (edited by Horridge G. A.). pp. 53-74. Clarendon press. Oxford. Baylor D. A., Hodgkin A. L. and Lamb T. D. (1974) The electrical response of turtle cones to flashes and steps of light. J. Ph~~iol. 242. 685-727. Cervetto L.. Pasino E. and Torre V. (1977) Electrical responses of rods in the retina of &fo marinu.5. J. Fb~s~o~. 267, 17-51. Clark R. 8. and Duncan G. (1978) TWO components of extracellular photoreceptor potentials in the cephalopod retina: differential effects of Na’. K” and Ca”. Biaph.m
Struct.
Mech.
4. 263-300.
Duncan G. and Croghan P. C. (1973) Electrical activity of the isolated cephalopod retina: an equivalent circuit model. Elcpl E,ru Res. 15. 4001-308. Duncan G. and Pynsent P. B. (1979) An analysis of the waveforms of photoreceptor potentials in the retina of the cephalopod Sepioln atlantica. J. Phpsid. In press. Lisman J. E. and Brown J. E. (1975) Light-induced changes of sensitivity in Limulus ventral photoreceptors. 1. gen. Phvsioi. 66, 473-488. Pynsent P. B. (1978) Photoresponses of the cephalopod retina. Ph.D. thesis. University of East Anglia. Pynsent P. B. and Duncan G. (1977) Reconstruction of photoreceptor membrane potentials from simultaneous intracellular and extracellular recordings. Nature 269, 257-259. Sieve H. (1976) On the influence of calcium ions on the properties of the arthropod photoreceptor cell membrane. Eioelectr. Bioenerg. 3, 151-157. Stieve H. and Hamani M. (1976) Light and dark adaptation of crayfish visual cells depending on extracellutar calcium concentration. Z. Naturforsch. 31, 324-327. Weeks F. I. and Duncan G. (1974) Photoreception by a cephalopod retina: response dynamics. Espl E~Y Rea 19. 483-509.
OF DARK ADAPTATION CEPHALOPOD RETINA
IN THE
G. DUNCANand P. B. PYEiSENT Biophysics Section. School of Biological Sciences, University of East Angtia. Norwich NR4 7TJ. England Key W&s--retina:
cephalopod:
dark adaptation:
photoreceptor.
Pynsent and Duncan (1977) have shown that the signals recorded at the proximal (y) and distal (V,) ends of Sepia/u photoreceptors are quite different in waveform. They later suggested (Duncan and Pynsent, 1979) that the differences can be explained if it is assumed that there is a primary light-stimulated sodium conductance increase at the distal (rhodopsinbearing) membranes and that the resulting current flow activates voltage-sensitive potassium channels in the membranes of the proximal segment. The basic waveform of the response at either end of the cell is also assumed to be modified by the time constant of the cell membranes and by the relatively large resistance of the extracellular space (Pynsent and Duncan, unpublished). The extracellular responses (V,) are relatively large in the cephalopods, and Weeks and Duncan (1974) have previously investigated the dynamics of dark adaptation of the extracellular responses using a paired flash technique. They found, as expected, that recovery of sensitivity to the second flash was prolonged as the intensity of the stimuli increased. They further showed that at a constant stimulus level, reduction in the external calcium concentration also prolonged the dark adaptation rate. This latter observation contrasts with several reports of similar studies with other invertebrate photoreceptors where reducing the external calcium was found to speed up the recovery of sensitivity (Baumann, 1974; Stieve and Hamani, 1976; Stieve, 1976). As there are in fact several ways of monitoring dark adaptation, even by the simple paired flash technique, and as the extracellular signals may not give a true representation of the electrical events at the membrane levei, we have rigorously examined. the double-flash technique using simultaneously recorded intracellular and extracellular signals. The perfusion chamber was an adaptation of the cooling stage used previously for non-perfused studies (Duncan and Pynsent. 1979). The normal perfusing solution was that used by Clark and Duncan (1978) and the stimulating and recording systems have been described in full elsewhere (Duncan and Pynsent. 1979). Essentially, the paired flashes were produced from a stroboscope with a flash duration of 10psec and the resulting signals were recorded on a d.c. tape recorder. They were then either displayed directly on
a storage oscilloscope screen, or digitised and processed on a computer. Figure 1 gives an example of a typical computer output from a double-flash experiment. The responses have all been scaled to the same height so that differences in waveform between y, V, and V, can be seen more clearly. The greatest difference in the shape of the response to the first flash appears to occur at the highest intensities where there is a pronounced sag phase in the v records comparable with the sag phase in vertebrate intracellular records (Baylor, Hodgkin and Lamb, 1974: Cervetto, Pasino and Torre, 1977). The V, responses are relatively flat over the saturating region, while the V, records show a biphasic response. With the flash separation of 250msec chosen for this series of experiments, there appears to be little decrease in amplitude of the second flash response at low intensities, but there is a considerable loss of sensitivity at high intensities. However, the extent of the adaptation depends on the method of measurement. Cervetto er al. (1977) used the trough between the responses as a reference point [Fig. 2(a)] and with their method there is little difference between the response ratios until an intensity of 100 photons pm-’ is reached. However, the y ratio is consistently below the others throughout the range. Duncan and Croghan (1973) simply measured the heights of the peaks above the baseline [Fig. 2(b)]. In their case, there is a consistent difference between the x and V, records at all intensities, and again the y records show the greatest adaptation. Weeks and Duncan (1974) attempted to obtain a “difference signal” as another criterion for adaptation. A single flash was applied and then after a suitable period, to ensure complete dark adaptation, the double flash stimulus was used. The baseline for the second amplitude was then measured from the point where the response would have been if the second flash had not been applied [Fig. 2(c)]. Using this method, there is little difference in the response ratios of K, V, and V,, throughout the range, except at the lowest intensities where, in fact, the measuring error is greatest. The dark adaptation rare can be measured by varying the separation between the two stimuli, and Fig. 3. shows that the differences between the V, and V, waveforms are most pronounced at short time inter-
359
360
G. DLWCAYand P. B. F’YNSENT
361
Cephalopod dark adaptation
I
PEAK RATIOS (Vi) SOIllS
100
200
1.9
0.60
0.75
0.91
0.65
0.76
0.94
9.6
0.39
0.68
0.90
0.40
0.72
0.88
(0 um2,
Fig. 3. Simultaneous me~urements of intracellular (q) and extracellular (K:.) responses to a series of double flashes of intensity 1.9 and 9.6 photons pm-‘, respectively. The time of the first flash corresponds to the origin of the response and the marker for the second flash is given below each response. A single flash served as the reference waveform for the flashes containing 9.6 photons pm-‘. while the first of two flashes spaced 4OOmsec apart served as the reference for the lower intensity series. The time marker represents 100 msec. The peak ratios for the three flash separations used in both experiments (SO. 100 and 2OOmsec) were calculated by the method of Weeks and Duncan (197-U.
vals. At the relatively
low intensity of 1.9 photons pm-*, the K records seem very much more adapted with a 50msec separation between the flashes as the peak height achieved remains constant. However, when adaptation is assessed by the method of Weeks and Duncan (1974) there is little difference between the x and V, ratios. At low intensities, the q response to the first gash recovers to the baseline much faster than v, (Fig. 3 and Duncan and Pynsent, 1979) and this effect amplifies P? in the V, records. The effects of the adapting flash on the waveform of the response to the second flash were investigated by computing the first derivative (V/set) of the signal (Duncan and Pynsent. 1979) and the rate of change of voltage of the second flash was found to be greatly reduced compared to the rate of change for the first response. The maximum rate of change of voltage (D) is an easily measured parameter and when the ratio of the rates is plotted against intensity, the relationship obtain for al1 three responses (Fig. 4) is remarkably similar to the relationship for the peak ratios obtained by the Weeks and Duncan (1974) method. Although the measurement of adaptation in terms of the potentials involved probably gives a good indication of the possible form of the output from the optic nerves (especialfy method b in Fig. 2), it can obscure info~ation concerning the underlying mechanisms of adaptation at the membrane level. For
example, information will be lost at high intensities as the voltage change obtainable in response to a single flash probably saturates before the maximum possible conductance change is achieved (Clark and Duncan, i978; Lisman and Brown, 1975). However, bearing this point in mind, it is stilt possible to obtain useful information concerning dark adaptation from
Fig. 4. Relationship between maximum rate ratio and intensity for flashes spaced 250msec apart (data from responses given in Fig. 1). D, and DI are the maximum rates of change of voltage for the first and second re-, sponses. respectively. 0, V, responses: (D. V,: 8. V,,. The units of intensity are photons pm-‘.
362
G.
DUNCAN
and P. 0, PYSSENT
the interpretation of double flash experiments. For example, a detailed examination of Fig 1 shows that, as far as Y, and V, are concerned, the maximum volta8e reached by the second response is the same as that reached after the first flash for most of the intensity range. However, this is not true for y where the maximum amplitude of the second flash is much lower than the first. This difference can most readiiy be explained in terms of a voltage-sensitive potassium conductanse in the membranes of the proximal end of the photoreceptor cell. At the point in tune when the second waveform starts off, the potassium conductance of the proximal segment will have been greatly increased by the depolarising current from the distal segment initiated by the first flash. The current from the second flash will therefore be much less effective in depolarising the proximal membrane potential. An active process in the proximal segment will accentuate differences between the three methods of measuring adaptation and it should be noted that if the photoreceptor cell were acting as a purely passive, linear system, then none of the three methods outlined would show any adaptation. At very low intensities, it does indeed appear to behave in this way (Clark, Pynsent and Duncan, unpublished). In general, extracellular responses are much more stable and reproducible than those obtained intracellularly using high resistance microelectrodes (Fig. 3). Such properties are invaluable in the study of dark adaptation rate that may require the presentation of several test pulses over a relatively long period of time. It is therefore interesting to find that using one method for assessing adaptation, the V;, V, and V, records give identical results and it is therefore safe to assume that Weeks and Duncan (1974) were correct in claiming that reducing extracellular calcium prolongs dark adaptation recovery in the cephalopod retina. As one of the main effects of reducing external calcium is to make the extracellular (Clark and Duncan, 1978) and intracellular (Pynsent. 1978) responses more sensitive to light, it is not surprising that recovery is prolonged relative to controls in normal solution.
&%nowvl&emenr~--We wish to thank Dr Peter C. Crog han for many helpful and stimulating discussions. Com-
puter facilities and support for PBP were made available through an SRC grant to Professor E. Rojas.
REFERENCES
3aumann F. (1974) Electrophysiological properties of the honey bee retina. In The CQ~~~~~~~~ Eye amf Vision Of Jnsrcrs (edited by Horridge G. A.). pp. 53-74. Clarendon press. Oxford. Baylor D. A., Hodgkin A. L. and Lamb T. D. (1974) The electrical response of turtle cones to flashes and steps of light. J. Ph~~iol. 242. 685-727. Cervetto L.. Pasino E. and Torre V. (1977) Electrical responses of rods in the retina of &fo marinu.5. J. Fb~s~o~. 267, 17-51. Clark R. 8. and Duncan G. (1978) TWO components of extracellular photoreceptor potentials in the cephalopod retina: differential effects of Na’. K” and Ca”. Biaph.m
Struct.
Mech.
4. 263-300.
Duncan G. and Croghan P. C. (1973) Electrical activity of the isolated cephalopod retina: an equivalent circuit model. Elcpl E,ru Res. 15. 4001-308. Duncan G. and Pynsent P. B. (1979) An analysis of the waveforms of photoreceptor potentials in the retina of the cephalopod Sepioln atlantica. J. Phpsid. In press. Lisman J. E. and Brown J. E. (1975) Light-induced changes of sensitivity in Limulus ventral photoreceptors. 1. gen. Phvsioi. 66, 473-488. Pynsent P. B. (1978) Photoresponses of the cephalopod retina. Ph.D. thesis. University of East Anglia. Pynsent P. B. and Duncan G. (1977) Reconstruction of photoreceptor membrane potentials from simultaneous intracellular and extracellular recordings. Nature 269, 257-259. Sieve H. (1976) On the influence of calcium ions on the properties of the arthropod photoreceptor cell membrane. Eioelectr. Bioenerg. 3, 151-157. Stieve H. and Hamani M. (1976) Light and dark adaptation of crayfish visual cells depending on extracellutar calcium concentration. Z. Naturforsch. 31, 324-327. Weeks F. I. and Duncan G. (1974) Photoreception by a cephalopod retina: response dynamics. Espl E~Y Rea 19. 483-509.