Distribution and movement of sodium and potassium in the toad retina

Exp. Eye Res. (1972) 13,258-28s

Distribution and Movement of Sodium and Potassium in the Toad Retina GEORGE DUNCAN AND FRANK I. WEEKS

University

School of Biological Sciertces, of East Anglia, Norwich, NOR MC, Englad (Received 24 January

1972, London)

The water content of the toad retina is 8746 and half of this is accessible to inulin. The intracellular ion concentrations of sodium and potassium are 48 and 98 mM/kg water respectively after a 1 hr incubation in the dark, and after incubation in Ringers solution containing ouabain they become 71 and 70 m&r/kg water respectively. When retinas are light adapted, there is a decrease in the 42K uptake and a small increase in the 22Na uptake. A short light flash also increases the rate of loss of potassium. These data suggest that the depolarizing light response dominates the changes in ion movements. Glutamate, aspartate and high potassium concentrations increase the rate of loss of potassium. In solutions containing 10 mM aspartate or glutamate the increased efflux rate occurs even when most of the sodium has been replaced by choline. From the increased rate of efflux in high potassium solutions, the ratio of the sodium and potassium permeabilities of the retinal cell membranes (P~,/PI() was estimated to be 1.3 x 10-2, and substituting this value in the Goldman potential equation together with the intracellular ion concentrations gave -80 mV aa the average resting potential of the retinal cells.

1. Introduction There have been many investigations of the electrical properties of the vertebrate retina and these have shown that the primary response to light is a hyperpolarization of both rods and cones. [See Tomita (1970) for an excellent review]. This graded hyperpolarization is passed on through synaptic transmission to the horizontal cells which also hyperpolarize, to the bipolar cells which are capable of giving both hyperpolarizing and depolarizing responses, and finally to cells that give a graded depolarization, together with regenerative action potentials, the amacrine and ganglion cells (Werblin and Dowling, 1969). Just as the electroretinogram (ERG) is a complex sum of all of these potentials (Brown, 1968) any light-stimulated ion movements that might be observed in the retina treated as a whole could be expected to be of a complex nature. In the few flux studies that have been carried out, Sekoguti (1960b) has found an increased rate of potassium efflux from illuminated amphibian retinas, while Buckser and Diamoncl (1966) have found an increased uptake of sodium in frog retinas under similar conditions suggesting that the depolarizing response predominates. Unfortunately, however, in both studies very high light intensities were used and so it cannot be excluded that the observed responses were unphysiological ones. We have therefore undertaken a study of the effect of low-level light intensities on ion movements in the amphibian retina. We have been further stimulated to undertake a reinvestigation of retinal ion movements by the recent findings of Sillman, Ito and Tomita (1969). They have shown that aspartate inhibits the PI, PI1 and proximal PI11 component,s of the ERG, leaving the photoreceptor component of PI11 intact. In the same way we hoped to isolate the light-stimulated ion movements from photoreceptor cells in the presence of aspartate.

279

RETINALIONMOVEMENTS

2. Materials and Methods After a 3 hr dark adaptation period, specimens of the common toad Bufo bufo were decapitated and their retinas were removed under dim red light (Kodak safelight filter 1A) and placed in a dish of Ringers solution (A). Th e composition of the various solutions used is given in Table I. Choline chloride was purified before use by washing in amyl alcohol and ether and sodium aspartate was prepared by neutralizing aspartie acid with sodium hydroxide. I

TABLE

Composition of soltiions

(mar)

Nat ‘1

KC1

Tris

C&Cl,

MgCI,

Glucose

Choline

A

100

B C D

-. -. 100

2.5 100 2.5 -

5 5” 5

2.5 2.5 2.5 2..5

1.5 1.5 1.5 1.5

5 5 5 5

100 -

by mixing

A and B in the required

Potassium rich solutions solutions was 7.5-&0.1.

Radioactive

were obtained

proportions.

The pH of all

solutions

22Nasolutions were prepared by adding 02 ml of stock solution (Code SKSl, RadiochemicalCentre, Amersham) to 15 ml of solution A and 42K solutions were prepared by adding O-5 ml of stock solution (PES. IP, Amersham.) to 30 ml of solution D to give a final potassiumconcentration of 2.5 my. 14C-Inulinsolutionswere obtained by dissolving 0.1 mg isotope (ICN, California) in 5 ml solution A. Water a& catiolz measurements Retinas were blotted on a glassmicroscopeslide to remove adhering water, placed in smallpreweighedpolyethylene tubes and weighedto ho.1 mg. After drying overnight in an oven t,he tubes were reweighed to obtain the dry weight and water content. 10 ml of deionized water were then added and the sodium and potassiumcontents of the leached retinas were determined after 48 hr using an Unicam SP900flame photometer (Unicam Instruments, Cambridge). Standardsusedwere in the range O-l-0.01 mM and contained equimolar amountsof sodiumand potassium. Radiomivity

metcswrements

(a) Sodium and potassiuminfluxes. After the retina had beenremoved from the eye, it wa.splaced in an oxygenated radioactive solution for periodsranging from 30-250 min. In caseswherethe tracer uptake wasto be determined, the retina wasblotted on a glassslide and transferred to a weighing tube. After drying, 10 ml deionized water were added as before and 1 ml was pipetted into a scintillation vial. 11 ml deionized water were then added and the radioactivity assayedby monitoring the Cerenkov radiation (Haberer, 1966)in a Packard Tricarb Counter (Model 574). 20 ~1 of loading solution were alsodried down and the sameprocedure carried out. When effluxes were performed, the radioactive retina was briefly washedin inactive solution and then transferred to the first of a seriesof scintillation bottles each containing 2 ml of inactive solution A or 2 ml of the test solution. The retina remained for 1 min in

280

GEORGE

DUNCAN

AND

FRANK

I. WEEKS

the first two bottles and then for 2 min in the rema,ining bottles, until the retina reached the final bottle where it remained. 10 ml deionized water were then added and the Mtles assayed by eerenkov counting as before. The radioactivity in the retina at the beginning of the efflux was found by adding the counts in each of the bott,les to those in the retina at, the end of the experiment. Subtracting counts in bottle I from t,he total gave the activity in the retina at the end of 1 min; subtracting the counts in bottle 2 gave the activity after 2 min. In this way, the activity in the retina could be found at known times during the efflux. (b) Inulin influx. The retinas were loaded for 1 hr in the radioactive solution, by which time the uptake was complete (see also Ames and Hastings, 1956) anti were then blotted, weighed and dried as before. In this case, after adding 10 ml deionized water, 1 ml was removed and added to a scintillation bottle containing 10 ml scintillation fluid (Duncan, 1970). The radioactivity was assayed on a Tricarb and flame-photomet,ry determinations carried out on the solution remaining. Illuminuhon

Except when otherwise stated, all the experiments were carried out under dim red light. In one set of experiments the retinas were subject to continuous illumination from a fluorescent source some distance away (0.1 lux in illuminance units) and in experiments where shorter light pulses were delivered, the light from a tungsten lamp was led to the retina via a bundle of optical fibres. In one series of experiments the retinas were illuminated with short pulses of light (100 lux, lasting 10psec) from a stroboscope (Dawes Transistor Strobotorch). 3. Results The toad retina has the relatively high water content of 87&l% (18) and this is in good agreement with the value obtained by Ames and Hastings (1956) for the rabbit retina. Using our blotting procedure, half of this water is accessible to radioactive inulin and so presumably extracellular. When the inulin space is taken into account, the intracellular concentration of sodium and potassium after a 1 hr incubation

260 240

/ I

X’

X

200

/

I

20

I

40

I

60 Time

I

60

1

100

X Dark n Contiwous light A Dark+ouabain . Ltght i-ouabain I I

120

140

Grid

FIG. 1. *2K!Influx into light and dark adapted retinas. Dark uptake in Solution -4 ( X-X - x ). Dark -t ouabain (A-A). Retinas exposed to continuous light of intensity 0.1 lux (m-m). Light and ouabain (0-0). The uptake was calculated in terms of m&r/kg dry weight from the activity in the retina (in counts/min/kg dry weight) and the specific activity of the loading solution (in counts/min/mole).

RETINAL

ION

MOVEMENTS

281

period in the dark are 48*6 (18) and 98f5 (18) mM/kg water respectively. In this case the ratio of the intracellular volume to dry weight is 3.5 10.2 (18) cm3/g and this compares with a ratio of 3.8 cm3/g for the rabbit retina (-4mes and Hastings, 1956). In retinas that had been incubated for 1 hr in solution A containing 10” M ouabain (Sigma) the intracellular concentrations became 71&4 (8) and 7012 (8) mM/kg water respectively, while the intracellular to dry weight ratio increased to 4.2f0.2 (8), indicating that either the retinal cells swell when they gain sodium and lose potassium, or they lose some solid matter. Many tissues in fact swell in the presence of ouabain (Duncan and Croghan, 1969). In the bulk of the experiments performed the inulin space was not determined, and it was found, and indeed expected, that the most satisfactory way of expressing the potassium concentration was in terms of the dry weight, while the data for the sodium concentration had a smaller spread when expressed in terms of the total water. Under these circumstances, the total potassium content was 405&18 (18) m&i/kg dry weight, while the total sodium content was 95-&7 (18) mM/kg water. The former value is in good agreement with the value of 450 m&r/kg dry weight reported for the frog (Sekoguti, 1960a). TABLE

II

Tracer

Dark Dark Light Light

uptake and ouabain uptake and ouabain

sodium

84A.3 12017 9lzt4 110+5

content

(4) (4) (4) (4)

Retinas were sampled between t = 30 and t = 180 min and the data were pooled. The tracer sodium content was determined by dividing the activity in the retina (in counts mm-l) by the specific activity of the loading solution (in counts mini mole-r). As the water content of the retina is also known, the tracer uptake can be expressed in terms of mM/kg water. The light adapted retinas were exposed to continuous light of illuminance 0.1 lux.

The 42K influx graph (Fig. 1) shows that in the dark, after an incubation period of over 2 hr, only 60% of the total potassium has exchanged and furthermore that the uptake level is depressed when the retinas have been exposed to a steady, low level light intensity. As most of the potassium influx into animal cells is a passive process (Duncan and Croghan, 1969), this suggests that the force driving potassium into the cells has decreased in the light, perhaps as a result of a depolarization of the cell membranes which would follow a change in the sodium levels in the inhibited retina (Table 2). ,4s might be expected for an ion that is in high concentrations in the extracellular space, the initial influx of 22Na into the retina is both large and rapid (Fig. 2). This initial movement is followed by an extremely slow influx, which suggests that the bulk of the retinal cells are not very permeable to sodium ions. There is in fact little difference between the 22Na content at t = 30 and t = 180 min under any of the conditions studied and so only the mean of the results is given in Table 2. Ouabain, as might be expected, causes a significant increase in the 22Na content of the retina both in light and dark conditions, whereas low level illumination results in only a small increase in the 22Na content,. Much higher light intensities cause a much larger increase (Buckser and Diamond, 1966).

282

Time (mid

FIG. 2. 22Na influx

into dark

adapted

retinas.

The difference between the sodium and potassium ef0uxes (Fig. 3) is a result of their differing distributions and membrane permeabilities. Potassium is mainly an intracellular ion and so the initial rapid e%ux is small, whereas the initial efflux of 22Na is large. The rate constant of loss from the second compartment is relatively rapid in the ease of potassium, but slow for sodium. Ouabain had in fact little effect on the sodium e&x, suggesting that most of the sodium originated from extracellular regions whereas it caused a rapid increase in the potassium ef3ux from the cells (Table 3). The sodium efflux is in fact a matter for future study when much longer influx periods ( e 12 hr) and loading solutions of a higher specific activity will be used so that the relatively small slow intracellular compartment can be followed for long efflux periods.

Ttme

(mid

FIG. 3. 42K and 2zNa effluxes from toad retinas. The difference between the effluxes being mainly an intracellular ion while the bulk of the sodium is extracellular.

is due to potassium

The relatively simple kinetics of potassium efflux (Fig. 3) suggest that the slow compartment originates from a homogeneous population of cells. However, closer inspection shows that this, while being approximately true is not strictly so. Figure 4 reveals that the rate of loss of potassium is dependent upon the length of incubation. The retina that has been loaded longer loses potassium less rapidly than a retina that has been incubated for only 30 min. This indicates that at least two cell populations exist in the retina. One population has a rapid turnover of isotopes, while the other loads up and effluxes more slowly. This also is responsible for the gradual decrease in rate constant observed during an e&x. However, comparisons can be made on the

RETINAL

ION

III

TABLE

__ -.~-

Potrtssium

concentration

0

124

in bathing

solution

25

50

Solution

C

Solution A + ouabain

0 Nn

------.

-.

Ratio of ratr cum&ants (k//k) Calculatc~d value for a ( x 102) Calculeted dE from k-‘/k (Kimizuka-Koketsu) Calculated dE from Goldman

2x3

MOVEMENTS

1.4

0+2

I.9

3.0

34

1.2

1.6

1.2

0-l

I4

.-

-23

+33

+5-t

-+6Z

-ts

+17

-30

$33

$51

-t-66

-7

+2r!

The ratio of rate constants is found for each retina by dividing the mean value in the test solution (I = 12 to 20 min) by the mean value in solution A (t = 6 to 10 min). a was calculated from equation 6. The Kimizuka-Koketsu PE was calculated from equation 5, and the Goldman dE from equation 4, assuming the value 1.3 x lo-% for a. The internal sodium and potassium concentrations used were 22 and 98 mix/kg water respectively, except in the case where ourtbain was applied, when they were 50 and 70 m#/kq water.

efiect of various substances and light regimes on test retinas have been subject to the same influx 3 hr and in order to describe the ion movements that in any 2 min efflux period, the rate of loss given by the equation

the retina provided the control and period. In our case this was usually mathematically, we have assumed of potassium ‘k’ is constant and is

dC+ -2 = - kt dt This equation can be integrated

to give

In C+(t,) -In

C+(t,)

= -k(t,

-tz)

(2)

where C:(t,) and Cc (t2) represents the internal activity, in this case of @K, in counts/ min, and t, and t, are the times at the beginning and end of the efflux period. The rate constant (k min-l) can be calculated from equation 2.

4

I2 Time

20

28

(min)

Fro. 4. The effect of load time on the rate of loss of potassium. The two graphs at least two cell populations in the retina with differing flux kinetics.

indicate

that

there

are

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GEORGE

DL7X;(‘A?C

AND

PRANK

1. WEEKS

Figure 5 gives the effect of a flash of low intensity light on the rate of loss of potassium from the toad retina. After a high rate of loss from the extracellular regions, a steady value of 2.8 x 10P2 min-r is achieved after 5 min. A 10 see flash after 9 nun results in an immediate increase in the rate of loss and a second flash after 21 ruin produces a further increase in the eflux rate. And indeed we found in every case that retinas exposed to a varied regime of light intensities always responded with an increase in potassium etllux, indicating that it is the retinal cells that depolarize in response to light that predominate in their contribution to the light stimulated potassium et&rx. 6x

4

i

x

\X

--x-xi\,

-X-x-X-x

2-

-px-

I IOsec

light

IO set light

Time

(mln)

FIG. 5. The effect of two separate

light flashes on the rate of loss of potassium. The light from a tungsten lamp had an &nninance of 5 lux and each flash lasted 10 sec. The light was switched on in the middle of a 2 min efflux period and the rate constant for the minute after switching on was calculated by assuming that the rate constant in the first minute (dark) was the same as in the preceding 2 min.

Raising the potassium concentration in the medium bathing the retina and hence depolarizing the cell membranes is a well known means of abolishing the nervous component of the ERG (Granit, 1962). The results in Table 3 and in Fig. 6 show that raising the potassium concentration increasesthe rate of efHux, and at high potassium concentrations the very large increase in the dark rate (noise) would swamp the smaller light stimulated effect (signal). Replacing sodium in the Ringers solution by choline results in a small increasein the rate of lossof potassium.

“0

4I

8I

12I

16 I 20I Time

FIG. 6. The efflux of 42K from the an arbitrary value. Both Aspartate crease the rate of loss of potassium, efflux. Some recovery takes place in

24I 28I

32I

(mid

toad retina. The internal activity at zero time has been ascribed (10 mlu) and potassium rich solutions (25 m&r K+75 rnx Na) inwhile application of Solution D (zero K) reduces the potassium Solution A(R).

RETINAL

ION

MOVEMENTS

285

Aspartate applied to the retina isolates the distal PI11 component of the light response (Sillman, Ito and Tomita, 1969) and 10 mM sodium aspartate added to the Ringers solution causes a considerable increase in the dark efflux rate (Table 4 and Pig. 6). This increase in the “noise” level of the retina also occurred when the aspartate was applied in choline solutions indicating that the aspartate is probably directly increasing the potassium permeability. Glutamate (10 mM) had the same effect as aspartate, while GABA had no significant effect. Hanawa and Tateishi (1970) have previously shown that aspartate and glutamate have similar effects on the ERG, while GABA has no effect. TABLE

Efflux time

6-10 12-16 18-22 28-30

IV

Mean of rate Solution A

2.6 5.2 4.7 3.2

constant ( x Solution

10e2) C

2.8 5.2 4.9 3.7

Aepartate was present in both solutions during the efflux period t = 12 min to is more pronounced in Solution A. Each value is the mean of 3 experiments.

t=

22

min. Recovery

4. Discussion The toad retina has similar ion distribution to toad sciatic nerve (Shanes and Berman, 1955) and the relative distributions are in good agreement with those found by Ames and Hastings (1956) for the rabbit and by Duncan, De Pont and Bonting (1971), for the invertebrate Sepia. The data of the Ames group (Ames and Hastings, 1956 ; Ames, Tsukada and Nesbett 1967) show that the total internal osmolarity of cations in the retina is greater than the osmolarity of the bathing solution. In the toad retina, the same picture emerges, the sum of the internal Na +K is 146 mm, whereas the total sum of all cations in solution A is only 120 m&r. This means a 26 mM excess in the toad, and we compute from the data of Ames, Tsukada and Nesbett (1967) a 25 mM excess in the rabbit. As animal cells do not seem to have rigid cell walls to enable them to gain a higher internal osmotic pressure than the bathing solution, it seems that the cation excess must be complexed in some way so that its osmotic contribution is reduced. The cations could be in association with fixed negative charges either inside or outside the cell, perhaps with the mucopolysaccharides of the intercellular matrix. Such a cation excess mainly attributable to sodium also occurs in the cornea (Davson, 1949; Otori, 1967) and in amphibian skin (Aceves and Erlij, 1971). In calculations where internal ion concentrations are required (Table 3) it is assumed that the sodium excess is “sequestered” and not in free solution and so the internal “free” sodium in the dark is 22 mM and in cases where ouabain has been applied, it is 50 mM. The uptake data for both potassium and sodium reveal that a relatively large fraction of the ions are still unexchanged after 3 hr (Pigs 1 and 2). The influx of potassium into the cells is much more rapid than the sodium influx indicating that the

‘786

GEOl<~C:E

.I)I:~(‘AN

AND

PRANK

I. \VEEKS

retinal membranes are more permeable t’o potassium than sodium. This permeability ratio is characteristic of nerve cells, and a rough estimate of the permeability ratio can be derived from the potassium efflux data. (1964) equation, the membrane From the Goldman (1943) and Kimizuka-Koketsu potential E will be given by

RI’

K,+uNa,, Ki +aNa,

E=Yln

(when the chloride permeability is negligibly the membrane); a is the ratio of the sodium When the external potassium and sodium then the membrane potential will change to E,

= g

F The change in membrane potential

&Y-E

In h’,’ -__

low: or chloride is in equilibrium across and potassium permeabilities (PJP,). are changed to new values, Kb and Na&

E’. +M%’

K1 + crNai

will be given by

RT =A&! =pln--

K,’ -t aNa,,’ K, +aNa,

The change in membrane potential is also related to the ratio of the potassium rate contants in the Kimizuka-Koketsu (1964) flux equations

e%hrx

2RT AE = 7 In (U/k) and in the derivation of this equation it has been assumed that the potassium permeability does not change when the external potassium concentrations are changed. From equations (4) and (5)

a is the only unknown in this equation and the values calculated from the four experiments carried out are given in Table 3. The mean value for GCis 1.3 x 10-s which again suggests that the cell membranes are on average much more permeable to potassium than to sodium. This value of cc can now be used, together with the internal ion concentrations found in the inulin studies, to estimate the membrane potential using equation 3. The computed value is -80 mV while the range of values for vertebrate neurons is -70 to -80 mV (Aidley, 1971). The computed value using the internal ion concentrations after 1 hr application of ouabain ( 10w4M) is -58 mV; the resulting depolarization of -+22 mV from the resting potential is sufficient to drive potassium ions from the cell at an increased rate (Table 3) provided the change occurs rapidly enough. Experiments are now underway to find whether or not there is a rapid change in the sodium level in ouabain solutions. The increased efflux rate of potassium in choline is anomalous as the Goldman potential equation predicts a hyperpolarization of the membrane (Table 3). This anomaly, although unexplained, has previously been observed by Duncan (1969) in the toad lens and von Hagen and Horowitz (1967) in muscle. One interpretation of the increased 22Na uptake under continuous illumination

RETlNALIONMOVEMEN!l-S

257

(Table 2 and Buckser and Diamond, 1966) is that the depolarizing cells in the retina dominate the observed response. The depolarization would be provided by an increase in the sodium permeability of the cell membranes and the resulting sodium influx might also lead to an increase in the sodium content of the cells. Whether this increase in the sodium content is a result of fatigue, or whether it might play a part in light-adaptation processes can only be resolved by sodium influx techniques with a much greater time resolution than at present possible. The data presented above for the effect of light-adaptation on the tracer sodium and potassium levels in the retina differ from the flame-photometric data presented by Ames, Tsukada and Nesbett (1967) who found that photic stimulation had no effect on the cation content of rabbit retinas even when bleached. The small but significant increase in the potassium e&x rate constant following a light flash, similar to that found in the invertebrate retina (De Pont, Duncan and Bonting 1971) probably indicates activity in the depolarizing cells as it does in the receptor cells making up most of the response in the Sepia retina, and a fuller investigation of both responses is underway. Any possibility of isolating the photoreceptor response in aspartate solutions is unfortunately lost because of the great increase in the dark potassium efflux rate in aspartate (Table 4). This increase could be due either to depolarization of the cell membranes or an increase in potassium permeability. Curtis, Phillis and Watkins (1960) have concluded from electrophysiological studies on cat motoneurones that aspartate and glutamate depolarize the neuronal membrane directly and that no alteration in synaptic transmission is involved. They also suggest that neuronal permeability to all ions might be raised in the presence of aspartate. Depolarization could result from an aspartate-stimulated sodium permeability increase, or a stimulated sodium influx brought about by a coupling of sodium and aspartate fluxes [see Schultz and Curran (1970) for a review of coupling phenomena]. However, as the increased potassium efflux still occurs in choline solutions which contain only lo- 15 mM sodium (Table 4) it cannot be a result of coupling of the fluxes as this would be restricted in low sodium solutions, nor can it be a resuIt of an initial sodium permeability increase as the external sodium ion concentration has been lowered to below the internal value. This leaves the unexpected conclusions that either aspartate causes a direct increase in the potassium permeability of the retinal cell membranes or the membranes are as permeable to choline as they are to sodium in the presence of aspartate. Aspartate seems to have no effect on photoreceptor cell membranes, as the photoreceptor response is unaltered in t’he presence of aspartate both in the frog (Sillman, Ito and Tomita, 1969) and in the invertebrate, Sepiola atlanticus (Duncan and Weeks, unpublished data).

During the preparation of the manuscript a report appeared [Sorbi, R. T. and Cavaggioni, A. (1971). Vision Res. 11, 9851 giving convincing evidence for a light stimulated decrease in potassium e&x, in complete contradiction to the data presented above. The only significant difference in methods, apart from a speciesdifference. is in the influx period. Sorbi and Cavaggioni loaded their frog retinas for only 30 min while we employed usually a 3 hr load period. In view of the evidence presented in Pig. 4 for the existence of at least two cell populations in the retina, it is tempting to conclude that cells which give a hyperpolarizing responsehave a rapid turnover of isotope, while cells which depolarize dominate the response after long in0ux periods.

We wish to thank Dr P. C. Croghan for many helpful discussions. F.I. W. wishes to thank the Nuffield Foundation for a Biological Bursarp and the Medical Research Council for a Research Studentship. REFERENCES Aceves, J. and Erlij, D. (1971). J. Physiol. 212, 195. Aidley, D. J. (1971). The PhysioZogy of Excitable Cells. Cambridge University Press, London. Ames, A. and Hastings, A. B. (1956). J. Neurophysiol. 19, 201. Ames, A., Tsukada, Y. and Nesbett, I?. B. (1967). J. Neurochem. 14, 145. Brown, K. T. (1968). Vision Res. 8, 633. Buckser, S. and Diamond, H. (1966). Biochem. Biophys. Res. Common. 23, 240. Curtis, D. R., Phillis, J. W. and Watkins, J. C. (1960). J. Physiol. 150, 656. Davson, H. (1949). Brit. J. OphthaZmoZ. 33, 175. De Pont, J. J. H. H. M., Duncan, G. and Bonting, S. L. (1971). Pjugers Arch. 322,278. Duncan, G. (1969). Exp. Eye Res. 8,413. Duncan, G. (1970). Exp. Eye Res. 9, 188. Duncan, G. and Croghan, P. C. (1969). Exp. Eye Res. 8,421. Duncan, G., De Pont, J. J. H. H. M. and Bonting, S. L. (1971). PjZugers Arch. 322, 264. Goldman, D. E. (1943). J. Gen. Physiol. 27, 37. Granit, R. (1962). in The Eye, Vol 2. (Ed. Davson H.). Academic Press, London. Haberer, K. (1966). in Packard Technical Bulletin No. 16, p. 1-14. (Packard Instrument Company, Illinois). von Hagen, S. and Horowitz, L. (1967). Am. J. Physiol. 213, 579. Hanawa, I. and Tateishi, T. (1970). Experientiu. 26, 1311. Kimizuka, H. and Koketsu, K. (1964). J. Theor. BioZ. 6, 290. Orti, T. (1967). Exp. Eye Res. 6, 356. Schultz, S. G. and Cm-ran, P. F. (1970). Physiol. Rev. 50,637. Sekoguti, Y. (1960a). Ann. Rep. Scient. work8 Fat. Sci. Osaka University 8, 75. Sekoguti, Y. (1960b). Ann. Rep. Scient. work<9 Fat. Sci. Osaka University 8, 83. Shanes, A. M. and Berman, M. D. (1955). J. Cell. Comp. PhysioZ. 45, 177. Sillman, A. J., Ito, H. and Tomita, T. (1969). Vision Res. 9, 1435. Tomita, T. (1970). Quart. Rev. Biophys. 3, 189. Werblin, F. S. and Dowling, J. E. (1969). J. Neurophysiol. 32, 339.