(Received 31 October 1962)

Transcription

1 156 J. Physiol. (1963), 167, pp With 8 text-figures Printed in Great Britain THE EFFECT OF SODIUM ION CONCENTRATION ON THE ELECTRORETINOGRAM OF THE ISOLATED RETINA OF THE FROG BY D. I. HAMASAKI* From the Physiological Laboratory, University of Cambridge (Received 31 October 1962) It has been well established that sodium ions are essential in the extracellular fluid of most excitable tissues for the generation of an action potential (Hodgkin, 1951, 1958). The action potential of the frog's muscle or nerve is completely abolished if all the sodium in the Ringer's fluid is replaced by sucrose or choline (Nastuk & Hodgkin, 1950; Huxley & Stampfli, 1951). Partial replacement of the sodium reduces the amplitude of the action potential, and this reduction is linearly related to the logarithm of the sodium concentration in the bathing fluid. Where contact with the tissue is immediate the changes in the action potential occur almost instantaneously (Huxley & Stampfli, 1951). Sodium ions have also been found to be essential for the generation of the receptor potential of the cat's Pacinian corpuscle (Diamond, Gray & Inman, 1958). They found that the amplitude of the receptor potential was reduced in a graded manner with the concentration of sodium ions in the perfusing media. The receptor potential was reduced to 10 % of its initial value in min in a sodium-free medium. The generation of the electroretinogram (e.r.g.) also requires sodium ions in the extracellular fluid. Furukawa & Hanawa (1955) found that the e.r.g. was completely abolished when the isolated retina of the toad was placed in isotonic glucose. They also reported that lithium-ringer's solution could not maintain the e.r.g. in the toad. Thus sodium ions have been found to be essential for maintaining excitability in a variety of tissues. The hypothesis is that during excitation there is a change in the permeability of the cell membrane, allowing ions to move down their electrochemical gradient. This would then mean that the amplitude and rate of rise of the potential change would be related to the concentration gradient of one or more of the ions. The object of this study was to determine more quantitatively how extracellular sodium * Present address: Bascom Palmer Eye Institute, University of Miami School of Medicine, 1638 N.W. 10th Avenue, Miami 36, Florida.

2 FROG ELECTRORETINOGRAM 157 deficiency affected the amplitude of the frog's e.r.g. The results show that sodium ions are essential for the generation of the e.r.g., and suggest that any procedure which affects the ratio of [Na]0: [Na]i changes the amplitude of the e.r.g. METHODS Preparation of the retina. All experiments were performed on the isolated retina of Rana temporaria. Although the experiments were carried out from March to July, the results presented here were obtained from frogs obtained from May to July. After pithing the dark-adapted frog, one eye was enucleated and the anterior segment was removed. The choroid and retina were peeled out in one piece by running the points of a blunt forceps along the sclera and breaking the main point of attachment around the optic nerve. When placed in Ringer's solution, the choroid remained attached to the retina mainly around the optic nerve, and when this was cut the retina could be cleanly separated from the choroid and pigment epithelium. The retina was spread flat on a piece of filter paper, usually with the receptor side downward. Electrode Retina Filter paper... r i...~~u Drain ~ = = Oxygen Fig. 1. Diagram of Perspex box. The capacity of the box is approximately 100 ml. The retina and the ifilter paper were then placed on a platform mn a Perspex box (Fig. 1). The bottom of this platformn was perforated, and the retina and ifilter paper made contact with the fluid in the bottom of the box through the holes. Approximately two-thirds of the retina was not blocked by the platform. The fluid level was maintained at the level of the filter paper by the proper adjustment of the upper drain. Oxygen was constantly bubbled through the solutions, and the solutions were cooled to l0-l5o C. Solutions. The frog's Ringer's solution used had (mm): NaCl 110, KCI 2-5, CaCl2 2-2, NaHCO3 6. Low-sodium Ringer was made by replacing the NaCl by equal molar concentrations of choline chloride, or lithium chloride, or by twice the molar concentration of sucrose. In these the 6 mme of NaHCO3 was still present and thus all but 5 % of the sodium was replaced. Solutions with specific fractions of sodiuim replacement were made by mixing the normal Ringer's solution with the low-sodium Ringer. Recording apparatus. Responses were recorded between a large Ag: AgCl electrode on the surface of the retina and the fluid in contact with the ifilter paper. The diameter of the wire was 0 17 mm and 1-2 mm was in contact with the retina. The photo-electric effect from the Ag: AgCl electrode was less than 10 tlv.

3 158 D. 1. HAMASAKI The responses were fed into a Tektronix 122 pre-amplifier set at a coupling time constant of 1 sec. The responses were displayed on a Tektronix 502 oscilloscope and permanent records were made on a penwriter (Southern Instruments, M942C). A camera shutter was used to deliver flashes of about 0 9 sec duration. The illumination at the retina was 12 lm/m2. Experimental procedure. An e.r.g. was recorded immediately after placing the retina in the Perspex box. The retina was allowed to become dark-adapted for min and another response recorded. If the e.r.g. was not larger than the initial one the retina was discarded. The Ringer's solution was then drained from the box and the test solution added. The box was rinsed out once before allowing the retina to equilibrate in the test solution. The exact length of time for equilibration and the times when the recordings were made depended on the experiment and will be presented with the results. The retina was then returned to Ringer's solution and e.r.g.s recorded at comparable times. RESULTS Replacement of sodium 95 % replacement of sodium. After recording an e.r.g. with the retina equilibrated in Ringer's solution, a solution was substituted in which 95 % of the sodium was replaced by choline. The retina was immersed for 1 min, the fluid drained to the level of the filter paper, and an e.r.g. recorded. Recordings were made every minute thereafter. Tracings from two experiments are shown in Fig. 2. After the 1 min immersion, the amplitude of the b-wave was reduced by approximately 20 %. With time the amplitude gradually declined and finally disappeared after 9 min. In other retinas it took no longer than 15 min for complete abolition of the e.r.g. During the early stages the amplitude and rate of rise of both the a- and b-wave can be seen to have decreased. When the retina was returned to Ringer's solution, the amplitude recovered to at least 85 % within 5 min. In the records shown, the amplitude returned to 100 % in 3 min. If the retina was only briefly rinsed with Ringer's solution (10-15 sec) the amplitude of the e.r.g. returned to over 50 % of its initial value. If the same experimental procedure was followed but with the retina oriented with the receptor side upward, the changes in the e.r.g. were quite different. Tracings from two experiments are shown in Fig. 3. After the 1 min immersion, the e.r.g. was almost completely abolished. With time the amplitude of the e.r.g. increased rather than decreased. After 5 min the retina was again immersed for 1 min in the 95 % choline-ringer, and the e.r.g. was then completely abolished. Returning the retina to Ringer's solution brought the e.r.g. back to at least 85 % of its initial level within 5 min. In the records shown the e.r.g. returned to 100 % in 3 min. It has been reported that substitution of lithium for sodium can maintain the normal activity of the frog's muscle and nerve (Overton, 1902; Huxley & Stampfli, 1951). However, Gallego & Lorente de No (1947)

4 FROG ELECTRORETINOGRAM 159 reported that although lithium substitution can maintain the action potential in the frog's nerve fibres, it does tend to depolarize the fibres after 2 hr _( Fig. 2. The effect of replacing 95 % of the sodium by choline. Tracings from two isolated frog retinas with the receptor side downward. Control recording at 0. Retina immersed for 1 min in choline-ringer and recordings made every minute thereafter. Recovery of the e.r.g. 3 min after returning to Ringer's solution at 3. Calibration, 200 tv and 200 msec. Unlike its action on the frog's muscle and nerve, lithium replacement of sodium abolished the e.r.g. When the experimental procedure described for choline was used, lithium was found to abolish the e.r.g. in min when the receptors were oriented downward (Fig. 4A). The changes in the shape of the e.r.g. were more variable with lithium, and the changes only partially reversible. The changes with the inverted retina were very similar to those seen with choline replacement (Fig. 4B). As described above, if a retina which has had its e.r.g. abolished by replacing the sodium by choline is rinsed briefly with normal Ringer's solution, the e.r.g. returns to over 50 % of its initial value. If the same procedure is carried out with lithium-ringer, there is no recovery of the e.r.g. Sucrose replacement also abolished the e.r.g. reversibly. Tetraethylammonium

5 160 D. I. HAMASAKI chloride replacement abolished the e.r.g. but the changes were not wholly reversible Fig. 3. The effect of replacing 95 % of the sodium by choline. Tracings from two isolated frog retinas with the receptor side upward. Control recording at 0. Retina immersed for 1 min in the choline-ringer and recordings made every minute thereafter. Retina was immersed again for 1 min between the fifth and sixth minutes. Recovery of the e.r.g. 3 min after returning to Ringer's solution. Calibration, 200,tV and 200 msec. Partial replacement of sodium. The effect of partial replacement of sodium was studied most extensively with choline replacement. After recording an e.r.g. with the retina stabilized in Ringer's solution, the retina was immersed for 5 min in the test solution. A recording was made every 5 min for min. The retina was then returned to Ringer's solution and the same procedure followed. On replacing 75 % of the sodium by choline the amplitude of the e.r.g. was reduced by 30 % (Fig. 5A). In addition, the rate of rise of both the a- and b-waves was reduced and the shape of the e.r.g. was similar to that during the early stages of 95 % replacement of sodium. The changes were always wholly or almost wholly reversible. In Fig. 6 the percentage decrease in the b-wave is plotted against the sodium concentration in the test solution. The averages and ranges for

6 FROG ELECTRORETINOGRAM 161 five retinas are plotted for 15 and 30 min equilibration. With 85 % replacement the e.r.g. is completely abolished within 15 min. The changes with 50 and 75 % replacement were almost the same at 15 and 30 min. A B 0~~~~~~~~~~ ~~~~~~~~~ _ 6-5 Fig. 4. The effect of replacing 95 % of the sodium by lithium. Tracings from two retinas; (A) receptor side downward, and (B) receptor side upward. Retinas immersed for 1 min in the lithium-ringer and recordings made every minute thereafter. The retina was immersed again for 1 min between the fifth and sixth minutes in record B. Recovery of the e.r.g. in Ringer's solution after 5 min. Calibration, 200,uV and 200 msec. The interrupted line in Fig. 6 was derived from Table 4 of Nastuk & Hodgkin (1950), and represents the percentage decrease in the intracellularly recorded action potential of the frog's sartorius muscle fibre in various concentrations of sodium. The solid line was derived from Fig. 4 of Huxley & Stampfli (1951), and represents the change in the action potential of a single myelinated nerve fibre of the frog in various concentrations of sodium. 11 Pbysiol 167

7 162 D. I. HAMASAKI The effect of replacing 75 % of the sodium by lithium is shown in Fig. 5B, and the results from eight retinas are plotted in Fig. 7. The decrease in the amplitude of the e.r.g. was much greater with lithium substitution than with choline, and the changes with lithium were only partially reversible. A B I Fig. 5. The effect of replacing 75 % sodium by choline (A) and by lithium (B). 1, Control recordings. 2, Responses after 15 min equilibration for choline and 10 min for lithium. 3, Recovery of e.r.g. in normal Ringer's solution. Calibration, 200 uv and 200 msec. Partial replacement by sucrose gave unexpected results. For a 50 % replacement the amplitude of the e.r.g. increased to 133 % of its initial value (average of three retinas). That this was not due to any specific action of sucrose was demonstrated by diluting the Ringer's solution to 50 % with distilled water. This hypotonic solution also gave b-waves which were larger by 131 % (three retinas). I

8 FROG ELECTRORETINOGRAM 163.-t. a- 0 0 C V [Na] in test solution (mm) Fig. 6. Reduction in action potential in various concentrations of sodium in test solution. Averages and ranges for five retinas are shown for 15 and 30 min equilibration Nastuk & Hodgkin (1950) muscle; - Huxley & St&mpfli (195-1) nerve. 0 = e.r.g. at 15 min; x at 30 min. of ' 40 4 L: a /* 0(2)' /0 /e / / / I / 0(4) / / /* I / I A(2) [Na] in test solution (mm) Fig. 7. Reduction in the e.r.g. with choline (-) and lithium (---) replacement. The numbers in brackets represent the numbers of retinas tested at that con. centration. 11-2

9 164 D. I. HAMASAKI Potassium-free Ringer's solution The results so far have shown that the amplitude and the rate of rise of the e.r.g. are related to the concentration of sodium in the external fluid or to the ratio of [Na]0: [Na]i. Then it should be expected that altering the intracellular sodium concentration will also change the e.r.g. in a predictable way. In the squid giant nerve fibre, Hodgkin & Keynes (1956) found that micro-injection of sodium reduced the amplitude of the action potential by approximately the amount expected by the Nernst equation. 0 10t 30 L:40 -.E 50 _\. Ct I I I I J I I Potassium-free Ringer Time (min) Normal Ringer's solution Fig. 8. The reduction in the e.r.g. in potassium-free Ringer. Recordings were made every 5 min in the potassium-free Ringer, and the retina was immersed between alternate recordings. It has been reported that the efflux of sodium from the cell is linked to the influx of potassium (Keynes, 1954, frog muscle; Ussing, Kruh0ffer, Hess-Thaysen & Thorny, 1960, frog skin). Desmedt (1953) found that the frog's sartorius muscle soaked in Ringer's solution with only 0-2 mm potassium increased its intracellular sodium, and the active membrane potential or the overshoot dropped from 33 to 13 mv. However, the overall action potential was little changed, because of the concurrent increase in the membrane potential. These changes were seen within 6 hr when the muscle was soaked in potassium-free Ringer's solution. The effect of potassium-free Ringer's solution on the e.r.g. is shown in Fig. 8. Recordings were made every 5 min in the potassium-free Ringer, and the retina was immersed in fresh potassium-free Ringer between

10 FROG ELECTRORETINOGRAM 165 alternate recordings. Also shown are the e.r.g.s to show the change in their shape with increasing time. In 5 min the b-wave was reduced by about 10 %. With time the e.r.g. declined, until at 100 min it was only 30 % of its initial value. The average amplitude after min for six retinas was 32 % of the initial amplitude. The shape of the e.r.g. at 100 min was similar to the e.r.g. with partial sodium replacement and the e.r.g. during the early stages of 95 % replacement. On returning the retina to normal Ringer's solution the amplitude of the b-wave gradually increased, and at about 90 min was 75 % of its initial value. DISCUSSION The results of this study show that the frog's retina requires sodium ions in the extracellular fluid for the generation of its action potential, and thus does not differ from most excitable tissues. The complete absence of any potential change when all the sodium is replaced, suggests that if part of the e.r.g. is the receptor potential (Brown & Watanabe, 1962) then it too requires sodium for its generation. This is not unexpected from the findings of Diamond et al. (1958) on the Pacinian corpuscle. The much faster changes in the retina may indicate that the structures being affected are on or near the surface of the retina. The evidence below suggests that it is the receptors which are mainly being affected. The reduction of the e.r.g. when placed in the low-sodium Ringer was greatly dependent on the orientation of the retina. With the receptor side downward the amplitude was reduced by 20 % after the 1 min immersion, but with the receptor side upwardthe e.r.g. was almost completely abolished. The presence of the filter paper and the Perspex platform probably slows the exchange of sodium ions, and the elements generating the e.r.g. are affected the most when they are not covered by these physical barriers. This would then suggest that the elements must be near the receptor side of the retina. The difference in the course of the e.r.g.s following the 1 min immersion can be accounted for if the receptors are really the structures being affected. After the immersion, many of the sodium ions in the extracellular fluid around the receptors will have been replaced by choline. However, some sodium ions will diffuse from within the retina into this space, and some sodium will be pumped out of the receptors. With the receptors oriented downward the ions can diffuse through the filter paper into the large bath of choline-ringer in the bottom of the box. With the receptor side upward the sodium ions will be trapped in this space and thus will increase the concentration of sodium ions here. This would then result in a larger e.r.g. when next the retina was stimulated.

11 166 D. 1. HAMASAKI If this interpretation of the observations is correct, during excitation by light there must be a change in the permeability of the receptor membrane which will result in a movement of sodium ions (and perhaps other ions) into the receptors. The size of this receptor potential could then determine the amount of transmitter released at the bipolar junction and thus the potentials developed by these cells. With up to 75 % replacement of sodium by choline the reduction in the amplitude of the e.r.g. was linearly related to the logarithm of the sodium concentration in the test solution. This reduction agreed very closely with the reductions observed when the action potential is measured across the cell membrane. Thus the cells contributing to the e.r.g. generate action potentials which appear to be based on the sodium equilibrium potential. The findings with lithium agree with the report of Furukawa & Hanawa (1955). That the abolition of the e.r.g. was not due merely to a toxic action of the lithium was shown in the experiment where lithium-ringer was added to a retina which had its e.r.g. abolished by choline replacement. The quick rinse did not bring the e.r.g. partially back as it did with normal Ringer's solution. The greater reduction in the e.r.g. with partial lithium replacement was probably due to its depolarizing action. The increase in the amplitude of the e.r.g. with partial sucrose replacement or with dilution by distilled water was probably due to the reduction in the conductivity of the solution. This reduced conductivity would decrease the amount of short-circuiting around the edges of the retina as well as within the retina. This would then result in an increase in the recorded e.r.g. These findings with sucrose probably explain why Ottoson & Svaetichin (1953) found that isotonic glucose was better than Ringer's solution for moistening the retina. In their preparations there were enough sodium ions (and other ions) present in the choroid and sclera, and in the vitreous of their inverted preparation, to maintain the e.r.g. Adding isotonic glucose reduced the amount of short-circuiting in the retina which then gave larger e.r.g.s. The reduction in the amplitude of the e.r.g. when the retina was placed in a potassium-free Ringer was probably due to changes in both the sodium and potassium concentrations. The main change would probably be an increase in the intracellular sodium concentration, which would then reduce the amplitude of the e.r.g. By analogy with the findings of Desmedt (1953) there should be very little change in the amplitude of the e.r.g., since the over-all action potential of muscle fibres changes very little. However, in the single myelinated nerve fibres of the frog Huxley & Stampfli found that the resting membrane potential increased by only 3-5 mv in potassium-free Ringer. And in long term experiments such as

12 FROG ELECTRORETINOGRAM 167 the present one there should be a loss of potassium from the cells, which will tend to lower the membrane potential. This should be especially effective in the small retinal cells. Thus, the membrane potential of the retinal cells may not change very much in the potassium-free Ringer, and the decrease in the e.r.g. is probably mainly due to an increase in the intracellular sodium. Another possible explanation for the reduction of the e.r.g. may be that during excitation there is also a movement of potassium ions out of the cells, which tends to reduce the potential change developed by the movement of sodium ions into the cells (see Fatt & Katz, 1951, on muscle endplate potential). In the potassium-free Ringer there would be a steeper gradient for the efflux of potassium, which would cause a greater reduction in the potential change caused by the influx of sodium ions. SUMMARY 1. The e.r.g. of the isolated retina of the frog was completely abolished when 95 % of the sodium ions in the Ringer's solution was replaced by choline or sucrose. The changes were almost wholly reversible. 2. The reduction in the e.r.g. with partial replacement of the sodium by choline was linearly related to the logarithm of the sodium concentration in the test solution. The reduction in action potentials indicated that the amplitude of the e.r.g. was mainly determined by the sodium equilibrium potential. 3. Lithium could not substitute for sodium in the frog's retina. 4. The evidence suggests that the receptors were the structures mainly affected by the experimental procedures, and that during excitation there is a movement of sodium into the receptors. 5. When the retina was placed in potassium-free Ringer's solution, the amplitude of the e.r.g. decreased very slowly, falling to half in about 60 min; on replacing the retina in normal Ringer's solution the e.r.g. recovered at about the same slow speed. It is suggested that this effect of removing external potassium may depend mainly on a slow increase in intracellular sodium. The author wishes to acknowledge assistance by a Fellowship from U.S. Public Health Service, BF-14,692, from the National Institutes of Neurological Diseases and Blindness. REFERENCES BROWN, K. T. & WATANABE, K. (1962). Isolation and identification of a receptor potential from the pure cone fovea of the monkey retina. Nature, Lond., 193, DESMEDT, J. E. (1953). Electrical activity and intracellular sodium concentration in frog muscle. J. Physiol. 121, DIAMOND, J., GRAY, J. A. B. & INMAN, D. R. (1958). The relation between receptor potential and the concentration of sodium ions. J. Phy8iol. 142,

13 168 D. I. HAMASAKI FATT, P. & KATZ, B. (1951). An analysis of the end-plate potential recorded with an intracellular electrode. J. Physiol. 115, FURUKAWA, T. & HANAWA, I. (1955). Effects of some common cations on electroretinogram of the toad. Jap. J. Physiol. 5, GALTEGO, A. & LORENTE DE N6, R. (1947). On the effect of several monovalent ions upon frog nerve. J. cell. comp. Physiol. 29, HODGKIN, A. L. (1951). The ionic basis of electrical activity in nerve and muscle. Biol. Rev. 26, HODGKIN, A. L. (1958). Ionic movement and electrical activity in giant nerve fibres. Proc. Roy. Soc. B, 148, HODGKIN, A. L. & KEYNES, R. D. (1956). Experiments on the injection of substances into squid giant axons by means of a microsyringe. J. Physiol. 131, HUXLEY, A. F. & STXMPFLI, R. (1951). Effect of potassium and sodium on resting and action potentials of single myelinated nerve fibres. J. Physiol. 112, KEYNES, R. D. (1954). The ionic fluxes in frog muscle. Proc. Roy. Soc. B, 142, NASTUK, W. L. & HODGKIN, A. L. (1950). The electrical activity of single muscle fibres. J. cell. comp. Physiol. 35, OTTOSON, D. & SVAETICHIN, G. (1953). Electrophysiological investigations of the origin of the e.r.g. of the frog retina. Acta physiol. scand. 29, OVERTON, E. (1902). Beitrage zur allgemeinen Muskel- und Nervenphysiologie. Pflug. Arch. ges. Physiol. 92, USSING, H. H., KRUH0FFER, P., HESS-THAYSEN, J. & THORNY, N. A. (1960). The Alkali Metals in Biology. Berlin: Springer.