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Electrical properties of cones isolated from the tiger salamander retina
0042.6’W
RESEARCH
Y I 08 I ?:%04502.00 0 Pcrgdmon Pror Ltd
NOTE
ELECTRICAL PROPERTIES OF CONES ISOLATED FROM THE TIGER SALAMANDER RETINA R. L. KONGand F. S. WERBLIN Electronics Research Laboratory. University of California. Berkeley. CA 94720. U.S.A. (Received 22 August
1980: in recised form 4
The voltage response in the cone to a step of illumination is typically not square, but shows an initial hyperpolarizing peak, followed by a less hyperpolarized plateau. What factors contribute to the transient nature of the cone response? Normann and Werblin (1974) suggested that some of the sag in the response was due to adaptation in the photochemical transducer itself. Others (Baylor et al., 1971; O’Bryan. 1973; Gerschenfeld and Piccolino, 1980) have implicated feedback from horizontal cells as a factor contributing to the response transient. Recent studies in rods have shown that a voltage-sensitive current in the photoreceptor membrane itself can dramatically shape the response (Bader er al., 1979; Attwell and Wilson, 1981). In order to sort out the factors that act to shape the cone response, we have physically isolated individual cones from the retina and examined the light response. Isolation eliminates coupling to adjacent receptors as well as feedback from horizontal ceils, and allows one to measure the effects of adaptation and voltage-sensitive currents alone. Retinal slices, 100 to 2OOc(m thick, were obtained from larvae of the tiger salamander by the method of Werblin (1978). These slices were superfused in an oxygenated Ringer’s solution, described previously by Marshall and Werblin (1978), at approx. ZO’C. As a
1980)
result of the slicing procedure. individual cones were often freed from the retinal network. These isolated cones were readily identified and impaled with two separate microelectrodes under visual control. Electrical measurements were performed by injecting current steps of 1 sec. duration in increasing increments of 0.1 nA, alternating polarity with each presentation. through one electrode, while monitoring voltage with the second electrode. Photoresponses could be elicited in the slice preparation for up to two hours in the Ringer’s solution. The light responses measured in a cone in the network and in an isolated cone are compared in Fig. 1. The records show responses to test flashes of different intensities of white light. increasing in 0.5 log unit steps from top to bottom. In both cells the dark potential level was - 3.5mV. The responses in the two records are similar, but there are two important differences; (1) the isolated cones were typically more .noisey: (2) the response in cones in the network were typically more transient. exhibiting prominent peak and plateau components. A significant observation, in both sets of recordings, as well as those taken from the eyecup (unpublished), was that the transient property of the response did not appear in records of maximal stimulus intensity. Both the isolated and
INTACT
T
December
ISOLATED
12 mV 0.5 set
0.5 set
Fig. 1. Comparison of photoresponses in an isolated cone and a cone in the intact retinal network. The ambient membrane potential in both cones was -35 mV. Stimulus timing is indicated by the bar below the response traces. Stimulus intensities were incremented in 0.5 log unit steps. The maximum stimulus intensity was 6 x 10s photons KC-’ pm-‘. effective quanta at 575 nm. Both cones displayed photoresponses of similar amplitude and wave-form. Note that the maximum light responses in both cones are square. 1329
1330
Research Note
NORMAL
MEDIUM
15mV
Fig. 2. I-V measurements in an isolated cone in normal medium. (a) Responses to depolarizing and hyperpolarizing current steps of 0.1 nA increments are shown. Hyperpolarizing responses show timedependent, peak-plateau waveforms. The magnitude of the responses to depolarizing current steps are smaller than the responses to hyperpolariring currents. The ambient potential is -35 mV. The onset of current injection is shown by an upward deflection of the bottom trace. (6) Current-voltage curve derived from the responses shown in part (a). Voltage measurements were taken at both the peaks (solid line) and plateaus (broken line) of the responses and are plotted as absolute membrane potential on the ordinate. Outward rectification is seen to occur at potentials positive to the ambient level.
network cones shown here were strongly lightadapted which may account for the small maximum response amplitudes. In experiments where adaptation state was more carefully controlled, larger responses were obtained (up to 20 mV). The results in Fig. 1 suggest that some of the transient response may be due to properties of the network, since the responses in isolated cones appear less transient in nature. Also, these results are consistent with previous observations that isolated cones are more noisey than coupled cones (Lamb and Simon, 1976). To determine whether voltage-sensitive currents might contribute to the transient nature of the cone light response, the effects of voltage-sensitive currents alone were examined by measuring the voltage response to steps of injected current in an isolated cone. The results are shown in Fig. 2. Figure 2A shows that there is a time-dependent decay in potential from a peak to plateau level in response to hyperpolarizing current, a sign of a voltage-sensitive current. This decay is quite small for membrane hyperpolarizations of 10-20 mV from the dark potential level, the physiological light response range, but increases for greater hyperpolarizations. These properties of the voltage-sensitive current are incompatible with those of the light response transient. The effect of the voltage-sensitive current in the
physiological response range seems too small to account for the light response transient. Also. the light response transient disappears at maximal response levels, where the effects of the voltage-sensitive current should be growing. Thus. the voltage-sensitive current cannot contribute in a major way to the light response transient. The responses to injected currents are plotted as a function of the magnitude of the injected currents in Fig. 2B. This figure shows that the slope resistance for the isolated cone is about 150 MR at the dark potential level. For depolarizations beyond 10 mV from the dark level, the slope resistance decreases to near 25 MR, indicating the membrane is strongly outward rectifying. The normally strong outward rectification in the cone membrane makes depolarization and consequently, reversal of the light response extremely difficult. However, TEA blocks the outward rectification in cones, as shown previously for rods (Fain et al., 1978; Werblin, 1979). and increases the membrane slope resistance at positive potentials from 25 MR in normal medium to 85 MR in 2OmM TEA, as shown in Fig. 3. Furthermore. the outward rectification appears to be a separate process from the voltagesensitive current, since the voltage decay in responses to hyperpolarizing current steps persists in the presence of TEA. The use of TEA made it possible to
Research Note
TEA
25mV .15sec
(0)
(b)
Fig. 3. I-V measurements in 20 mM TEA. (a) The ambient potential was -25 mV. Both depolarizing and hyperpolarizing current steps elicited time-dependent responses. (b) Outward rectification is less severe in TEA as seen in the I-V curve obtained from part (a) (peak responses are plotted). Note that the membrane could be depolarized well beyond 0 mV.
depolarize the cone membrane sufficiently to measure reversal potentials for the light response near OmV. These measurements are similar to those of reversal potentials previously made in cones in the network (Baylor and Fuortes, 1970; Lasansky and Marchiafava, 1974) and are similar to reversal potentials measured in isolated rods (Werblin, 1978; Bader et al.. 1978; Attwell and Wilson, 1981). However, the intact cones examined by Baylor and Fuortes (1970) and Lasansky and Marchiafava (1974) seem unusual for two reasons: (1) they showed little outward rectification; (2) they were probably not coupled. We have never been able to reverse the light response in network cones of the salamander (even in 20mM TEA) for these reasons. These results suggest that the transient form of the cone response may be due, in part, to the properties of the network. since the response of cones in the network appears more transient in nature than that of isolated cones (Fig. 1). However, it is difficult to understand why the network would cease to operate at high light levels, where the responses of the cones become square. The voltage-sensitive current in the cone membrane. inferred from the measurements in Fig. 2. seems to play a minor role in the shaping of the cone response. The voltage decay due to this voltage-sensitive current increases with increasing hyperpolarization, but is practically non-existent at the potential levels of the normal light response. Thus,
it appears that a major contributor to the transient response in cones may be an adaptive process in the outer segments themselves. REFERENCES
Attwell D. and Wilson M. (1981) Behavior of the rod network in the tiger salamander retina mediated by membrane properties of individual rods. J. Physiol. in press. Bader C. R., MacLeish P. R. and Schwartz E. A. (1978) Responses to light of solitary rod photoreceptors isolated from tiger salamander retina. Proc. Narn. Acnd. Sci. U.S.A. 75, 3507-3511. Bader C. R., MacLeish P. R. and Schwartz E. A. (1979) A voltage-clamp study of the light response in solitary rods of the tiger salamander. J. Physiol. 2%. l-26. Baylor D. A. and Fuortes M. G. F. (1970) Electrical responses of single cones in the retina of the turtle. J. Physiol. 207, 77-92. Baylor D. A., Fuortes M. G. F. and G’Bryan P. M. (1971) Receptive fields of cones in the retina of the turtle. J. Physiol. 214. 265-294. Fain G. L, Quandt F. N., Bastian B. L. and Gerschenfeld H. M. (1978) Contribution of &urn-sensitive conductance increase to the rod photoresponse. Norure 272, 467-469. Gerschenfeld H. M. and Piccolino M. (1980) Sustained feedback effects of L-horizontal cells on turtle cones. Proc. R. Sot. Lond. B. 206.439463. Lamb T. D. and Simon E. J. (1969) The relation between intercellular coupling and electrical noise in turtle photoreceptors. J. Physiol. 263, 257-286. Lasansky A. and Marchiafava P. L. (1974) Light-induced
1332
Research
resistance changes in retmal rods and cones of the tiger salamander. J. Physiol. 236. 171-191. Marshall L. M. and Werblin F. S. (1978) Synaptic transmission to the horizontal cells in the retina of the larval tiger salamander. J. Phpbl. 279, 311-346. Normann R. A. and Werblin F. S. (1974). Control of retinal sensitivity: 1. Light and dark adaptation of vertebrate rods and cones. J. gen. Physiol., 63. 37-61.
Note O’Bryan P. ht. (1973) Properties of the depolarizing synap tic potential evoked by peripheral illumination in cones of the turtle retina. J. Phpiol. 235, 107-223. Werblin F. S. (1978) Transmission along and between rods in the tiger salamander retina. J. Physiol. 280, 219170. Werblin F. S. (1979) Time- and voltage-dependent ionic components of the rod response. J. Physiol. 294. 6 13-626.
RESEARCH
Y I 08 I ?:%04502.00 0 Pcrgdmon Pror Ltd
NOTE
ELECTRICAL PROPERTIES OF CONES ISOLATED FROM THE TIGER SALAMANDER RETINA R. L. KONGand F. S. WERBLIN Electronics Research Laboratory. University of California. Berkeley. CA 94720. U.S.A. (Received 22 August
1980: in recised form 4
The voltage response in the cone to a step of illumination is typically not square, but shows an initial hyperpolarizing peak, followed by a less hyperpolarized plateau. What factors contribute to the transient nature of the cone response? Normann and Werblin (1974) suggested that some of the sag in the response was due to adaptation in the photochemical transducer itself. Others (Baylor et al., 1971; O’Bryan. 1973; Gerschenfeld and Piccolino, 1980) have implicated feedback from horizontal cells as a factor contributing to the response transient. Recent studies in rods have shown that a voltage-sensitive current in the photoreceptor membrane itself can dramatically shape the response (Bader er al., 1979; Attwell and Wilson, 1981). In order to sort out the factors that act to shape the cone response, we have physically isolated individual cones from the retina and examined the light response. Isolation eliminates coupling to adjacent receptors as well as feedback from horizontal ceils, and allows one to measure the effects of adaptation and voltage-sensitive currents alone. Retinal slices, 100 to 2OOc(m thick, were obtained from larvae of the tiger salamander by the method of Werblin (1978). These slices were superfused in an oxygenated Ringer’s solution, described previously by Marshall and Werblin (1978), at approx. ZO’C. As a
1980)
result of the slicing procedure. individual cones were often freed from the retinal network. These isolated cones were readily identified and impaled with two separate microelectrodes under visual control. Electrical measurements were performed by injecting current steps of 1 sec. duration in increasing increments of 0.1 nA, alternating polarity with each presentation. through one electrode, while monitoring voltage with the second electrode. Photoresponses could be elicited in the slice preparation for up to two hours in the Ringer’s solution. The light responses measured in a cone in the network and in an isolated cone are compared in Fig. 1. The records show responses to test flashes of different intensities of white light. increasing in 0.5 log unit steps from top to bottom. In both cells the dark potential level was - 3.5mV. The responses in the two records are similar, but there are two important differences; (1) the isolated cones were typically more .noisey: (2) the response in cones in the network were typically more transient. exhibiting prominent peak and plateau components. A significant observation, in both sets of recordings, as well as those taken from the eyecup (unpublished), was that the transient property of the response did not appear in records of maximal stimulus intensity. Both the isolated and
INTACT
T
December
ISOLATED
12 mV 0.5 set
0.5 set
Fig. 1. Comparison of photoresponses in an isolated cone and a cone in the intact retinal network. The ambient membrane potential in both cones was -35 mV. Stimulus timing is indicated by the bar below the response traces. Stimulus intensities were incremented in 0.5 log unit steps. The maximum stimulus intensity was 6 x 10s photons KC-’ pm-‘. effective quanta at 575 nm. Both cones displayed photoresponses of similar amplitude and wave-form. Note that the maximum light responses in both cones are square. 1329
1330
Research Note
NORMAL
MEDIUM
15mV
Fig. 2. I-V measurements in an isolated cone in normal medium. (a) Responses to depolarizing and hyperpolarizing current steps of 0.1 nA increments are shown. Hyperpolarizing responses show timedependent, peak-plateau waveforms. The magnitude of the responses to depolarizing current steps are smaller than the responses to hyperpolariring currents. The ambient potential is -35 mV. The onset of current injection is shown by an upward deflection of the bottom trace. (6) Current-voltage curve derived from the responses shown in part (a). Voltage measurements were taken at both the peaks (solid line) and plateaus (broken line) of the responses and are plotted as absolute membrane potential on the ordinate. Outward rectification is seen to occur at potentials positive to the ambient level.
network cones shown here were strongly lightadapted which may account for the small maximum response amplitudes. In experiments where adaptation state was more carefully controlled, larger responses were obtained (up to 20 mV). The results in Fig. 1 suggest that some of the transient response may be due to properties of the network, since the responses in isolated cones appear less transient in nature. Also, these results are consistent with previous observations that isolated cones are more noisey than coupled cones (Lamb and Simon, 1976). To determine whether voltage-sensitive currents might contribute to the transient nature of the cone light response, the effects of voltage-sensitive currents alone were examined by measuring the voltage response to steps of injected current in an isolated cone. The results are shown in Fig. 2. Figure 2A shows that there is a time-dependent decay in potential from a peak to plateau level in response to hyperpolarizing current, a sign of a voltage-sensitive current. This decay is quite small for membrane hyperpolarizations of 10-20 mV from the dark potential level, the physiological light response range, but increases for greater hyperpolarizations. These properties of the voltage-sensitive current are incompatible with those of the light response transient. The effect of the voltage-sensitive current in the
physiological response range seems too small to account for the light response transient. Also. the light response transient disappears at maximal response levels, where the effects of the voltage-sensitive current should be growing. Thus. the voltage-sensitive current cannot contribute in a major way to the light response transient. The responses to injected currents are plotted as a function of the magnitude of the injected currents in Fig. 2B. This figure shows that the slope resistance for the isolated cone is about 150 MR at the dark potential level. For depolarizations beyond 10 mV from the dark level, the slope resistance decreases to near 25 MR, indicating the membrane is strongly outward rectifying. The normally strong outward rectification in the cone membrane makes depolarization and consequently, reversal of the light response extremely difficult. However, TEA blocks the outward rectification in cones, as shown previously for rods (Fain et al., 1978; Werblin, 1979). and increases the membrane slope resistance at positive potentials from 25 MR in normal medium to 85 MR in 2OmM TEA, as shown in Fig. 3. Furthermore. the outward rectification appears to be a separate process from the voltagesensitive current, since the voltage decay in responses to hyperpolarizing current steps persists in the presence of TEA. The use of TEA made it possible to
Research Note
TEA
25mV .15sec
(0)
(b)
Fig. 3. I-V measurements in 20 mM TEA. (a) The ambient potential was -25 mV. Both depolarizing and hyperpolarizing current steps elicited time-dependent responses. (b) Outward rectification is less severe in TEA as seen in the I-V curve obtained from part (a) (peak responses are plotted). Note that the membrane could be depolarized well beyond 0 mV.
depolarize the cone membrane sufficiently to measure reversal potentials for the light response near OmV. These measurements are similar to those of reversal potentials previously made in cones in the network (Baylor and Fuortes, 1970; Lasansky and Marchiafava, 1974) and are similar to reversal potentials measured in isolated rods (Werblin, 1978; Bader et al.. 1978; Attwell and Wilson, 1981). However, the intact cones examined by Baylor and Fuortes (1970) and Lasansky and Marchiafava (1974) seem unusual for two reasons: (1) they showed little outward rectification; (2) they were probably not coupled. We have never been able to reverse the light response in network cones of the salamander (even in 20mM TEA) for these reasons. These results suggest that the transient form of the cone response may be due, in part, to the properties of the network. since the response of cones in the network appears more transient in nature than that of isolated cones (Fig. 1). However, it is difficult to understand why the network would cease to operate at high light levels, where the responses of the cones become square. The voltage-sensitive current in the cone membrane. inferred from the measurements in Fig. 2. seems to play a minor role in the shaping of the cone response. The voltage decay due to this voltage-sensitive current increases with increasing hyperpolarization, but is practically non-existent at the potential levels of the normal light response. Thus,
it appears that a major contributor to the transient response in cones may be an adaptive process in the outer segments themselves. REFERENCES
Attwell D. and Wilson M. (1981) Behavior of the rod network in the tiger salamander retina mediated by membrane properties of individual rods. J. Physiol. in press. Bader C. R., MacLeish P. R. and Schwartz E. A. (1978) Responses to light of solitary rod photoreceptors isolated from tiger salamander retina. Proc. Narn. Acnd. Sci. U.S.A. 75, 3507-3511. Bader C. R., MacLeish P. R. and Schwartz E. A. (1979) A voltage-clamp study of the light response in solitary rods of the tiger salamander. J. Physiol. 2%. l-26. Baylor D. A. and Fuortes M. G. F. (1970) Electrical responses of single cones in the retina of the turtle. J. Physiol. 207, 77-92. Baylor D. A., Fuortes M. G. F. and G’Bryan P. M. (1971) Receptive fields of cones in the retina of the turtle. J. Physiol. 214. 265-294. Fain G. L, Quandt F. N., Bastian B. L. and Gerschenfeld H. M. (1978) Contribution of &urn-sensitive conductance increase to the rod photoresponse. Norure 272, 467-469. Gerschenfeld H. M. and Piccolino M. (1980) Sustained feedback effects of L-horizontal cells on turtle cones. Proc. R. Sot. Lond. B. 206.439463. Lamb T. D. and Simon E. J. (1969) The relation between intercellular coupling and electrical noise in turtle photoreceptors. J. Physiol. 263, 257-286. Lasansky A. and Marchiafava P. L. (1974) Light-induced
1332
Research
resistance changes in retmal rods and cones of the tiger salamander. J. Physiol. 236. 171-191. Marshall L. M. and Werblin F. S. (1978) Synaptic transmission to the horizontal cells in the retina of the larval tiger salamander. J. Phpbl. 279, 311-346. Normann R. A. and Werblin F. S. (1974). Control of retinal sensitivity: 1. Light and dark adaptation of vertebrate rods and cones. J. gen. Physiol., 63. 37-61.
Note O’Bryan P. ht. (1973) Properties of the depolarizing synap tic potential evoked by peripheral illumination in cones of the turtle retina. J. Phpiol. 235, 107-223. Werblin F. S. (1978) Transmission along and between rods in the tiger salamander retina. J. Physiol. 280, 219170. Werblin F. S. (1979) Time- and voltage-dependent ionic components of the rod response. J. Physiol. 294. 6 13-626.