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Chloride-sensitive receptive field mechanisms in the isolated retina-eye cup of the rabbit
Brain Research, 90 (1975) 329-334
329
~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Chloride-sensitive receptive field mechanisms in the isolated retina-eye cup of the rabbit
ROBERT F. MILLER AND RAMON DACHEUX Neurosensory Laboratory, Department of Physiology, State University of New York at Bt(ffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Accepted February 27th, 1975)
Removing chloride ions from the external environment of the perfused rabbit retina results in rapidly occurring changes in the light induced discharge pattern of the retinal ganglion cells 7. Recordings of mass ganglion cell activity from the surface of the retina show that the discharge which follows the onset of a diffuse light stimulus is abolished in a chloride-free (c-f) environment but the off-discharge is enhanced and prolonged. This observation suggests that a c-f environment selectively affects specific retinal pathways. In the present study we have examined the effects of a c-f medium on the receptive field properties of different types of ganglion cells using single unit recordings and local, center and surround light stimulation. This study is an analysis of 55 ganglion cells recorded from the periphery of the rabbit retina, including 19 on-center cells, 28 off-center cells and 8 on-off cells. Details of these methods have been previously described 2,7. The experiments were carried out on an isolated, perfused retina-eye cup preparation. The eye of an anesthetized rabbit (albino or pigmented) was excised, the cornea, lens and iris were removed, and the eye cup was then everted over a suitably shaped Teflon hemisphere. The hemisphere was mounted in a perfusion chamber which permitted rapid and well dispersed fluid flow over the vitreal surface of the retina. The perfusate was a horse serum-Ringer solution aerated with 95% Oz and 5% COs. Sulfate was used as a chloride substitute in most experiments with sucrose added to maintain osmotic balance. However, identical results were obtained by substituting propionate or methyl sulfate for chloride. Light stimulation was provided by spots of white light focused on the retina over the tip of a glass insulated tungsten electrode 6 which had been advanced to the retinal surface under microscopic observation. Small (0.175 mm diameter) and large (3.5 ram) spots were obtained from two independently shuttered tungsten iodine lamps. The spots could be easily changed in position by a micrometer drive which controlled the aperture system. Intensity was adjusted to give a vigorous discharge in response to center stimulation. Without exception, on-center cells became rapidly unresponsive to light stimulation in the c-f environment. Fig. 1 (upper two horizontal rows) illustrates an on-
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Fig. 1. C-f effects on an on-center (upper two horizontal rows), off-center (middle two horizontal rows) and on-off cell (lower two horizontal rows)• Left hand drawing indicates by the absence of shading whether small or large spot stimulus was used. Left hand vertical column shows discharge pattern observed in a control environment, middle columns shows responses in a c-f environment after steady state conditions were observed, right hand column shows discharge pattern after returning to normal medium. All responses are photographic reproductions of impulse activity displayed on storage oscilloscope. 200 rnsec calibration applies to all traces except 2rid lowest horizontal row. Negative down. Light energy constant for all stimuli ~ 1.6 7; 10 -~ W/sq.cm.
center cell which responded to a centrally located small spot with a phasic discharge at °on' (upper trace, left column). In response to a large spot stimulus (lower trace, left column) it showed an on- as well as an off-discharge. The off-discharge, resulting from stimulation o f the antagonistic surround, had a longer latency than the on-discharge (68 vs. 46 msec). In the c-f perfusate (middle column) the cell did not respond to either small or large spot stimulation (i.e., on- as well as off-discharges were abolished). This effect was completely reversed on returning to the normal medium, as illustrated in the right hand column. The on-center cells usually became light insensitive within 3 rain after initiating the c-f perfusate; an approximately equal time was required before light induced activity was evident after returning to the control environment. Eight o f the 19 oncenter cells were tonic, responding with a sustained discharge during the 0.5-1 sec light stimulus; l I were phasic, as is the cell illustrated in Fig. 1. Eleven cells showed the surround discharge to the large spot stimulus, while in 8, off-activity was not evoked until the center was selectively adapted by steady illumination with the small
331 spot. All on- and off-activity of the on-center cells disappeared in the c-f medium. The off-center cells and the on-off cells were not silenced in the c-f environment. The middle 2 horizontal rows of Fig. 1 illustrate the results typically obtained from an off-center cell. The small spot (upper trace, left column) produced only an off-discharge, while the large spot generated both on- and off-activity (lower trace, left column). The off-latency was shorter than the on-latency (57 msec ~,s. 84 msec); thus in both on- and off-center cells, surround excitation was slow compared to center excitation. The middle column shows the altered discharge in a c-f environment: both small and large spot stimulation produced an off-discharge but the surround (or 'on') discharge in response to the large spot was abolished. Though it is difficult to appreciate from the illustration, the off-discharge has a higher frequency and a longer latency (by about 30 msec) than in the control medium; the spikes are also larger. On returning to a normal perfusate (right column), the surround discharge recovered rapidly (1-3 rain) but when it first appeared, a longer latency was apparent ( I l l msec right column v s . 84 msec in control). A return to the normal discharge latency usually required several additional minutes but the time-course of this recovery was not systematically studied. Twenty-three of the 28 off-center cells had surround excitation to the large spot stimulus, only 5 required center adaptation to generate a surround discharge. All off-center cells were affected in the same way, and center adaptation would not elicit surround excitation in a c-f medium. The lower traces of Fig. 1 illustrate the c-f effects on an on-off cell. Such cells were characterized by an on- and an off-discharge of nearly equal latency to small spot (left column: on = 52 msec; off = 47 msec) as well as large spot (on = 4l msec; off -- 47 msec) light stimulation 1. These on-off cells lost the on-discharge while the off-discharge became more vigorous and appeared in the form of repetitive, brief, high frequency bursts of firing. Spontaneous mass ganglion cell activity recorded from the retinal surface was increased in a c-f environment. Single unit recordings demonstrated that this spontaneous activity is restricted to off-center and on-off cells. The on-center cells were completely silent in a c-f environment, suggesting that spontaneous activity changes were dependent on a functional connection with the receptors. It is also possible that a loss of chloride-dependent IPSPs at the ganglion cell level leaves off-center and on-off cells more 'excitable' in a c-f environment. In contrast to the consistent effects of the c-f medium on the on- and offdischarges, it had a variable and unpredictable effect on the spontaneous activity of these cells. While in most off-center cells spontaneous activity was increased, some lost their spontaneous firing while the off-response persisted (Fig. 2A). In other cells (Fig. 2B) spontaneous activity developed into an oscillatory bursting pattern. A burst had generally 2-3 spikes and recurred at a rate of 2/sec. A pause of firing was typically interposed between off-discharges and the oscillatory firing. Some cells (Fig. 2C) furthermore were as active in a c-f medium as in the control. Exposure to the c-f medium for more than 5-10 min showed in some cells an additional phenomenon of 'spontaneous' firing (Fig. 2C, lower trace). In this case spontaneous discharge frequency suddenly and periodically increased to rates as
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Fig. 2. A, B, and C: spontaneous activity changes in a c-f medium. In each pair of traces the upper one was obtained in control and the lower one in a c-f environment. All light stimuli were large spot. Note in A no on-discharge was evoked by large spot stimulus. This cell required center adaptation to see surround excitation. Lower trace shows loss of spontaneous activity and a decrease in duration of off-excitation. B: increase in spontaneous activity appearing in the form of a bursting pattens. with a silent period following off activity. C: little change in spontaneous activity (note different time scales) but appearance of spontaneous inactivation, immediately after which cell is unresponsive to light stimulation. In A, B, and C, lower trace in each pair recorded at 0.5 gain of upper trace. D: light induced off-discharge (arrow) summates with spontaneous burst to produce rapid inactivation response. E: inhibitory influence of light as well as off-excitation in a c-f medium. Same cell as C with gain same as C, upper trace. Light intensity: A, B, C, and E, 1.6 :~ 10 '~W sq.cm, D, 1.6 10-4W/sq.cm.
high as 1000/sec, while spike amplitude simultaneously decreased. Firing then abruptly ceased, and all spike generation was blocked for about 2 sec as evidenced by the disappearance of any off-activity to light stimulation. At times this 'inactivation' phenomenon was triggered by the superposition of spontaneous bursting and the off-discharge resulting from light stimulation (Fig. 2D). Intracellular recordings obtained from rabbit off-center ganglion cells show that the light activated decrease in spike activity is accompanied by a hyperpolarization of the cellL Fig. 2E shows that the ability of the off-mechanism to inhibit in light as well as excite in early dark was not impaired in a c-f medium (same cell as Fig. 2C). The inhibition is probably not the result of a light activated chloride-dependent IPSP, since these mechanisms would be abolished in a prolonged exposure to the c-f medium 8. Recordings from a few off-center cells were maintained for as long as 2 h
333 in a c-f environment and the persistence of inhibition was noted. It seems more likely that the inhibitory effect, as it exists in a c-f environment, results from the turning off of a dark mediated EPSP response (i.e., disfacilitation). Dark dependent excitatory influences are not unexpected in view of findings which suggest that receptors release a transmitter in the dark 3,10. The findings of this study show that despite apparent complex phenomenology, a c-f medium reduces the connection between receptors and ganglion cells to a single functional channel subserving the off-discharge of off-center and on-off ganglion cells. As was previously shown, light evoked horizontal cell activity is abolished in a c-f environment v, this readily explains the loss of the surround excitation in off-center cells. It is reasonable to suppose that the horizontal cell is also involved in the surround excitation for on-center cells. However, a selective loss of the 'on' bipolar would abolish both center and surround influences, since the bipolar cells themselves are antagonistically organized. Two types of bipolar cells, depolarizing and hyperpolarizing, have been described3, a and it is likely that each bipolar cell type subserves one of the two sets of channels (on-center and off-center). These findings suggest that the on-off cells are connected to both bipolar cell types in the same way as an on- or off-center cell is connected to one type. This arrangement would account for the similar on- and off-latencies of on-off cells. Thus the c-f induced changes in ganglion cell receptive field organization could be accounted for by the loss of the 'on' bipolar in addition to the horizontal cell. The mechanism by which a c-f environment effects the retinal network is presently unknown. One obvious possibility is that transmembrane chloride movements play a critical role in the electrogenesis of the chloride-sensitive neurons. An increase in chloride conductance can lead to a hyperpolarization or a depolarization depending on the relative equilibrium potential for chloride with respect to the membrane potential 9. Another possibility is that chloride ions play a less specific role and their removal could conceivably alter the intracellular concentration of other ions 4. Regardless of the mechanism, the c-f environment clearly offers a very promising experimental condition to identify which type of bipolar cell is connected to which type of ganglion cell. This question is presently being investigated with intracellular recording techniques in the mudpuppy retina which is similar to the rabbit in its sensitivity to the removal of chloride ions. We appreciate the helpful suggestions of W.K. Noell. Supported by N I H Grant EY-00844.
1 BARLOW, H. B., HILL, R. M., AND LEVICK, W. R., Retinal ganglion cells responding selectively to direction and speed of image motion in the rabbit, J. Physiol. (Lond.), 173 (1964) 377-407. 2 DACHEUX, R. F., DELMELLE, M., MILLER, R. F., AND NOLLE, W. K., Isolated rabbit retina preparation suitable for intra- and extracellular analysis, Fed. Proc., 32 (1973) 327. 3 DOWLING, J, E., AND RIPPS, H., Effect of magnesium on horizontal cell activity in the skate retina, Nature (Lond.), 242 (1973) 101-103. 4 HODGKIN, D. L., AND HOROW1CZ, P., The influence of potassium and chloride ions on the membrane potential of single muscle fibres, J. Physiol. (Lond.), 148 0959) 127-160.
334 5 KANEKO, A. R., Physiological and morphological identification of horizontal, bipolar anti ~tm~crine cells in gold fish retina, J. Physiol. (Lond), 207 (1970) 623-633. 6 LEvlcK, W. R., Another tungsten microelectrode, Med. biol. Engng, 10 (1972) 510--515. 7 M~LLZR, R. F., AND DACHEtJX, R. F., Information processing in the retina: importance of chloride ions, Science, 121 (1973) 266 ~268. 8 MO:rOKIZAWA, F., REUBEN, J. P., AND GRUNDFEST, H., Tonic permeability of the inhibitory postsynaptic membrane of lobster muscle fibers, J. gen. PhysioL, 54 (1969) 437-461. 9 OOMURA, Y., OOVOMA, H., AND SAWADA, M., lntracellular chloride concentration in the onchidium neurons, Proc. 24th int. Congr. PhysioL, Soc., 6 (1968) 330. 10 TRWONOV, YU. A., ANt) BYZOV, A. L., The response of the cells generating s-potential on the current passed through the eye cup of the turtle, BioJ~zika, 10 (1965) 673-680. 11 WERamN, F. S., AND DOWJ-XNG, J. E., Organization of the retina of the mudpuppy, Ne~Tttrtts maculosus. I!. Intracellular recording, J. Neurophysiol. 32 (1969) 339-355.
329
~) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Chloride-sensitive receptive field mechanisms in the isolated retina-eye cup of the rabbit
ROBERT F. MILLER AND RAMON DACHEUX Neurosensory Laboratory, Department of Physiology, State University of New York at Bt(ffalo, Buffalo, N.Y. 14214 (U.S.A.)
(Accepted February 27th, 1975)
Removing chloride ions from the external environment of the perfused rabbit retina results in rapidly occurring changes in the light induced discharge pattern of the retinal ganglion cells 7. Recordings of mass ganglion cell activity from the surface of the retina show that the discharge which follows the onset of a diffuse light stimulus is abolished in a chloride-free (c-f) environment but the off-discharge is enhanced and prolonged. This observation suggests that a c-f environment selectively affects specific retinal pathways. In the present study we have examined the effects of a c-f medium on the receptive field properties of different types of ganglion cells using single unit recordings and local, center and surround light stimulation. This study is an analysis of 55 ganglion cells recorded from the periphery of the rabbit retina, including 19 on-center cells, 28 off-center cells and 8 on-off cells. Details of these methods have been previously described 2,7. The experiments were carried out on an isolated, perfused retina-eye cup preparation. The eye of an anesthetized rabbit (albino or pigmented) was excised, the cornea, lens and iris were removed, and the eye cup was then everted over a suitably shaped Teflon hemisphere. The hemisphere was mounted in a perfusion chamber which permitted rapid and well dispersed fluid flow over the vitreal surface of the retina. The perfusate was a horse serum-Ringer solution aerated with 95% Oz and 5% COs. Sulfate was used as a chloride substitute in most experiments with sucrose added to maintain osmotic balance. However, identical results were obtained by substituting propionate or methyl sulfate for chloride. Light stimulation was provided by spots of white light focused on the retina over the tip of a glass insulated tungsten electrode 6 which had been advanced to the retinal surface under microscopic observation. Small (0.175 mm diameter) and large (3.5 ram) spots were obtained from two independently shuttered tungsten iodine lamps. The spots could be easily changed in position by a micrometer drive which controlled the aperture system. Intensity was adjusted to give a vigorous discharge in response to center stimulation. Without exception, on-center cells became rapidly unresponsive to light stimulation in the c-f environment. Fig. 1 (upper two horizontal rows) illustrates an on-
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Fig. 1. C-f effects on an on-center (upper two horizontal rows), off-center (middle two horizontal rows) and on-off cell (lower two horizontal rows)• Left hand drawing indicates by the absence of shading whether small or large spot stimulus was used. Left hand vertical column shows discharge pattern observed in a control environment, middle columns shows responses in a c-f environment after steady state conditions were observed, right hand column shows discharge pattern after returning to normal medium. All responses are photographic reproductions of impulse activity displayed on storage oscilloscope. 200 rnsec calibration applies to all traces except 2rid lowest horizontal row. Negative down. Light energy constant for all stimuli ~ 1.6 7; 10 -~ W/sq.cm.
center cell which responded to a centrally located small spot with a phasic discharge at °on' (upper trace, left column). In response to a large spot stimulus (lower trace, left column) it showed an on- as well as an off-discharge. The off-discharge, resulting from stimulation o f the antagonistic surround, had a longer latency than the on-discharge (68 vs. 46 msec). In the c-f perfusate (middle column) the cell did not respond to either small or large spot stimulation (i.e., on- as well as off-discharges were abolished). This effect was completely reversed on returning to the normal medium, as illustrated in the right hand column. The on-center cells usually became light insensitive within 3 rain after initiating the c-f perfusate; an approximately equal time was required before light induced activity was evident after returning to the control environment. Eight o f the 19 oncenter cells were tonic, responding with a sustained discharge during the 0.5-1 sec light stimulus; l I were phasic, as is the cell illustrated in Fig. 1. Eleven cells showed the surround discharge to the large spot stimulus, while in 8, off-activity was not evoked until the center was selectively adapted by steady illumination with the small
331 spot. All on- and off-activity of the on-center cells disappeared in the c-f medium. The off-center cells and the on-off cells were not silenced in the c-f environment. The middle 2 horizontal rows of Fig. 1 illustrate the results typically obtained from an off-center cell. The small spot (upper trace, left column) produced only an off-discharge, while the large spot generated both on- and off-activity (lower trace, left column). The off-latency was shorter than the on-latency (57 msec ~,s. 84 msec); thus in both on- and off-center cells, surround excitation was slow compared to center excitation. The middle column shows the altered discharge in a c-f environment: both small and large spot stimulation produced an off-discharge but the surround (or 'on') discharge in response to the large spot was abolished. Though it is difficult to appreciate from the illustration, the off-discharge has a higher frequency and a longer latency (by about 30 msec) than in the control medium; the spikes are also larger. On returning to a normal perfusate (right column), the surround discharge recovered rapidly (1-3 rain) but when it first appeared, a longer latency was apparent ( I l l msec right column v s . 84 msec in control). A return to the normal discharge latency usually required several additional minutes but the time-course of this recovery was not systematically studied. Twenty-three of the 28 off-center cells had surround excitation to the large spot stimulus, only 5 required center adaptation to generate a surround discharge. All off-center cells were affected in the same way, and center adaptation would not elicit surround excitation in a c-f medium. The lower traces of Fig. 1 illustrate the c-f effects on an on-off cell. Such cells were characterized by an on- and an off-discharge of nearly equal latency to small spot (left column: on = 52 msec; off = 47 msec) as well as large spot (on = 4l msec; off -- 47 msec) light stimulation 1. These on-off cells lost the on-discharge while the off-discharge became more vigorous and appeared in the form of repetitive, brief, high frequency bursts of firing. Spontaneous mass ganglion cell activity recorded from the retinal surface was increased in a c-f environment. Single unit recordings demonstrated that this spontaneous activity is restricted to off-center and on-off cells. The on-center cells were completely silent in a c-f environment, suggesting that spontaneous activity changes were dependent on a functional connection with the receptors. It is also possible that a loss of chloride-dependent IPSPs at the ganglion cell level leaves off-center and on-off cells more 'excitable' in a c-f environment. In contrast to the consistent effects of the c-f medium on the on- and offdischarges, it had a variable and unpredictable effect on the spontaneous activity of these cells. While in most off-center cells spontaneous activity was increased, some lost their spontaneous firing while the off-response persisted (Fig. 2A). In other cells (Fig. 2B) spontaneous activity developed into an oscillatory bursting pattern. A burst had generally 2-3 spikes and recurred at a rate of 2/sec. A pause of firing was typically interposed between off-discharges and the oscillatory firing. Some cells (Fig. 2C) furthermore were as active in a c-f medium as in the control. Exposure to the c-f medium for more than 5-10 min showed in some cells an additional phenomenon of 'spontaneous' firing (Fig. 2C, lower trace). In this case spontaneous discharge frequency suddenly and periodically increased to rates as
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Fig. 2. A, B, and C: spontaneous activity changes in a c-f medium. In each pair of traces the upper one was obtained in control and the lower one in a c-f environment. All light stimuli were large spot. Note in A no on-discharge was evoked by large spot stimulus. This cell required center adaptation to see surround excitation. Lower trace shows loss of spontaneous activity and a decrease in duration of off-excitation. B: increase in spontaneous activity appearing in the form of a bursting pattens. with a silent period following off activity. C: little change in spontaneous activity (note different time scales) but appearance of spontaneous inactivation, immediately after which cell is unresponsive to light stimulation. In A, B, and C, lower trace in each pair recorded at 0.5 gain of upper trace. D: light induced off-discharge (arrow) summates with spontaneous burst to produce rapid inactivation response. E: inhibitory influence of light as well as off-excitation in a c-f medium. Same cell as C with gain same as C, upper trace. Light intensity: A, B, C, and E, 1.6 :~ 10 '~W sq.cm, D, 1.6 10-4W/sq.cm.
high as 1000/sec, while spike amplitude simultaneously decreased. Firing then abruptly ceased, and all spike generation was blocked for about 2 sec as evidenced by the disappearance of any off-activity to light stimulation. At times this 'inactivation' phenomenon was triggered by the superposition of spontaneous bursting and the off-discharge resulting from light stimulation (Fig. 2D). Intracellular recordings obtained from rabbit off-center ganglion cells show that the light activated decrease in spike activity is accompanied by a hyperpolarization of the cellL Fig. 2E shows that the ability of the off-mechanism to inhibit in light as well as excite in early dark was not impaired in a c-f medium (same cell as Fig. 2C). The inhibition is probably not the result of a light activated chloride-dependent IPSP, since these mechanisms would be abolished in a prolonged exposure to the c-f medium 8. Recordings from a few off-center cells were maintained for as long as 2 h
333 in a c-f environment and the persistence of inhibition was noted. It seems more likely that the inhibitory effect, as it exists in a c-f environment, results from the turning off of a dark mediated EPSP response (i.e., disfacilitation). Dark dependent excitatory influences are not unexpected in view of findings which suggest that receptors release a transmitter in the dark 3,10. The findings of this study show that despite apparent complex phenomenology, a c-f medium reduces the connection between receptors and ganglion cells to a single functional channel subserving the off-discharge of off-center and on-off ganglion cells. As was previously shown, light evoked horizontal cell activity is abolished in a c-f environment v, this readily explains the loss of the surround excitation in off-center cells. It is reasonable to suppose that the horizontal cell is also involved in the surround excitation for on-center cells. However, a selective loss of the 'on' bipolar would abolish both center and surround influences, since the bipolar cells themselves are antagonistically organized. Two types of bipolar cells, depolarizing and hyperpolarizing, have been described3, a and it is likely that each bipolar cell type subserves one of the two sets of channels (on-center and off-center). These findings suggest that the on-off cells are connected to both bipolar cell types in the same way as an on- or off-center cell is connected to one type. This arrangement would account for the similar on- and off-latencies of on-off cells. Thus the c-f induced changes in ganglion cell receptive field organization could be accounted for by the loss of the 'on' bipolar in addition to the horizontal cell. The mechanism by which a c-f environment effects the retinal network is presently unknown. One obvious possibility is that transmembrane chloride movements play a critical role in the electrogenesis of the chloride-sensitive neurons. An increase in chloride conductance can lead to a hyperpolarization or a depolarization depending on the relative equilibrium potential for chloride with respect to the membrane potential 9. Another possibility is that chloride ions play a less specific role and their removal could conceivably alter the intracellular concentration of other ions 4. Regardless of the mechanism, the c-f environment clearly offers a very promising experimental condition to identify which type of bipolar cell is connected to which type of ganglion cell. This question is presently being investigated with intracellular recording techniques in the mudpuppy retina which is similar to the rabbit in its sensitivity to the removal of chloride ions. We appreciate the helpful suggestions of W.K. Noell. Supported by N I H Grant EY-00844.
1 BARLOW, H. B., HILL, R. M., AND LEVICK, W. R., Retinal ganglion cells responding selectively to direction and speed of image motion in the rabbit, J. Physiol. (Lond.), 173 (1964) 377-407. 2 DACHEUX, R. F., DELMELLE, M., MILLER, R. F., AND NOLLE, W. K., Isolated rabbit retina preparation suitable for intra- and extracellular analysis, Fed. Proc., 32 (1973) 327. 3 DOWLING, J, E., AND RIPPS, H., Effect of magnesium on horizontal cell activity in the skate retina, Nature (Lond.), 242 (1973) 101-103. 4 HODGKIN, D. L., AND HOROW1CZ, P., The influence of potassium and chloride ions on the membrane potential of single muscle fibres, J. Physiol. (Lond.), 148 0959) 127-160.
334 5 KANEKO, A. R., Physiological and morphological identification of horizontal, bipolar anti ~tm~crine cells in gold fish retina, J. Physiol. (Lond), 207 (1970) 623-633. 6 LEvlcK, W. R., Another tungsten microelectrode, Med. biol. Engng, 10 (1972) 510--515. 7 M~LLZR, R. F., AND DACHEtJX, R. F., Information processing in the retina: importance of chloride ions, Science, 121 (1973) 266 ~268. 8 MO:rOKIZAWA, F., REUBEN, J. P., AND GRUNDFEST, H., Tonic permeability of the inhibitory postsynaptic membrane of lobster muscle fibers, J. gen. PhysioL, 54 (1969) 437-461. 9 OOMURA, Y., OOVOMA, H., AND SAWADA, M., lntracellular chloride concentration in the onchidium neurons, Proc. 24th int. Congr. PhysioL, Soc., 6 (1968) 330. 10 TRWONOV, YU. A., ANt) BYZOV, A. L., The response of the cells generating s-potential on the current passed through the eye cup of the turtle, BioJ~zika, 10 (1965) 673-680. 11 WERamN, F. S., AND DOWJ-XNG, J. E., Organization of the retina of the mudpuppy, Ne~Tttrtts maculosus. I!. Intracellular recording, J. Neurophysiol. 32 (1969) 339-355.