in photoreceptors, glia and extracellular space in

J. Physiol. (1985), 362, pp. 41.5-435 415With 11 text-figuresPrinted in greatt Britain

CHANGES IN SODIUM ACTIVITY DURING LIGHT STIMULATIONIN PHOTORECEPTORS, GLIA AND EXTRACELLULAR

SPACE IN DRONE RETINA

BY J. A. COLES AND R. K. ORKAND*From the Experimental Ophthalmology Laboratory, 22, rue Alcide-Jentzer, 1211,Geneva 4, Switzerland and the Department of Physiology, Geneva University

(Received 14 September 1984)

SUMMARY

1. Ion-selective micro-electrodes were used to measure Na+ activity, aNa, in thetwo types of cell, photoreceptors and glial cells, and in the extracellular space, insuperfused slices of the retina of the honey-bee drone, Apis mellifera3. Movementsof Na+ were induced by light stimulation, or by increasing [K+] in the superfusate.

2. In the dark. aNa in the photoreceptors was 10 mM (S.E. of the mean = 1 mM);in the glial cells it was higher: 37 + 2 mm. We estimate that in this preparation about2 of the free Na+ in the tissue is in the glial cells.

3. Stimulation with a train of light flashes, 1 s-1 for 90 s caused aNa in thephotoreceptors to increase by 16+ 2 mm. K+ activity, aK, decreased by 21 + 3 mM.

4. During the standard train of light flashes, aNa in glial cells decreased by only1 5 + 0 3 mm, much less than the increase in aK (7 + 2 mM). One possible interpretationof this result is that most of the increase in aK is due to K+ uptake by a mechanismother than Na+-K+ exchange.

5. In extracellular fluid, stimulation caused aNa to fall to a relatively steady valuein about 10 s. Unlike aK, there was no tendency for aNa to return to the base lineduring the remainder of the 90 s stimulation. The fall in aNa was 14 + 1 mM: a greaterfall is prevented by extracellular electric currents and a decrease in extracellularvolume.

6. When [K+] in the superfusate was increased from 7-5 to 18 mm, aNa decreasedin the glial cells but not in the photoreceptors.

7. In this tissue, stimulation causes changes in aNa in the neurones that might belarge enough to modify the biochemistry of the cells. But in the glia, the fractionalchanges are small.

INTRODUCTION

The drone retina is a nervous tissue in which it has been possible to use ion-selectivemicro-electrodes to measure K+ activity (aK) in the three compartments that makeup nearly all the volume: the photoreceptors, the outer pigment cells, which are glial

* Permanent address: Department of Physiology and Pharmacology, School of Dental Medicine,and Institute of Neurological Sciences, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

J. A. COLES AND R. K. ORKAND

cells, and the extracellular space. When the photoreceptors are stimulated by a flashof light there is an entry of Na+ (Coles & Orkand, 1982) and an efflux of K+(Tsacopoulos, Orkand, Coles, Levy & Poitry, 1983). When the stimulation is a trainof light flashes, 1 s-1, then the intracellular aK, a]K, falls by about one-quarter in 90 s,

mainly because of efflux of K+ into the extracellular space (Coles & Tsacopoulos,1979). From there, most of this K+ appears to pass into the glial cells, either by netuptake (Coles & Orkand, 1983) or in association with current loops through the glialsyncytium and the extracellular clefts ('spatial buffering': Gardner-Medwin, Coles& Tsacopoulos, 1981; Coles & Tsacopoulos, 1981). We now report measurements ofresting Na+ activity (aNa) in the three compartments and the changes induced bylight stimulation, and attempt to relate these changes to the K+ movements. Someof the findings have been published in abstracts (Orkand, Coles & Munoz, 1983; Coles,Orkand & Munoz, 1983).

METHODS

The experiments were done on 500-900 /tm thick slices of the retina of the honey-bee drone, Apismelliferad prepared, superfused and stimulated with light as already described (Coles & Orkand,1983). The compositions of the main solutions used for superfusion or for calibrating ion-selectiveelectrodes after each successful recording are given in Table 1. In solutions with high [K+] andconstant [Cl-], part of the NaCl was replaced by an equivalent amount of KCI. The solution with18 mM-K+ and constant [K+] [C1-] included (in mg ion 1-): Na, 210; Ca, 3-2; Mg, 10; Cl, 96-1;glucuronate, 145-1; gluconate, 3-2; HCO3, 10. (aca was within 20% of that in the normal Ringersolution; the measured osmolarity was 445 mosm compared to 431 mosm for the normal Ringersolution.) The superfusates were bubbled with 95% 02, 5%C02 which gave a pH of 7 1. For Na+and K+ we have taken the activity coefficient in the Ringer solution to be that in a solution ofNaCI or KC1 of equal ionic strength (0 70, Staples, 1971). For Ca2+ we have followed a conventionsimilar to that of Tsien & Rink (1980) and given the free concentration on the assumption thatthe activity coefficient in the cytoplasm is equal to that in the Ringer solution.

Ion-8elective electrodes. For double-barrelled electrodes, one barrel of a theta glass micropipettewas silanized with trimethyldimethylaminosilane (Fluka, Buchs, Switzerland, see Munoz, Deyhimi& Coles, 1983) and the tip filled with the appropriate sensor. The rest of the barrel was filled withRinger solution. For intracellular Na+ we used the sensor of Steiner, Oehme, Ammann & Simon(1979) based on ligand ETH 227 (Fluka), and for Ca21 that of Oehme, Kessler & Simon (1976):the reference barrels were filled with 2M-KCI. For K+ we used a valinomycin sensor the compositionof which was: valinomycin (Calbiochem), 5 mg; K tetra-p-chlorophenylborate (KTpCPB, Fluka)2 mg; in 01 ml 2,6 dimethylnitrobenzene (Fluka). Alternatively, we used an ion-exchanger sensor

of 3% KTpCPB in 2,6 dimethylnitrobenzene (Coles & Orkand, 1983). The reference barrel was filledwith 0-1M-KC1 and 2M-Li acetate. For measurements of extracellular aNa the ligand ETH 227 hasthe disadvantage that it is sensitive to changes in aca. We therefore used a sensor, a gift from DrD. Ammann, the composition ofwhich was: ligand ETH 157, 10 mg; Na tetraphenylborate, 05 mg;

in 01 ml 2-nitrophenyloctyl ether. The structure of the ligand is given in Ammann, Morf, Anker,Meier, Pretsch & Simon (1983). We found no references to the previous use of this sensor inmicro-electrodes, so as a check on the results we also used the ETH 227 sensor and corrected forchanges in aca with the aid of a third barrel containing the Ca2+ sensor (Dietzel, Heinemann,Hofmeier & Lux, 1982). A pair of calibration curves was constructed for the Ca2+ and'Na+' barrelsin modified Ringer solutions containing constant [Na+] and different[Ca2+1 (1, 1-6, 4 and 16 mM).From these curves the contribution of the Ca2+ to VNa, could be determined for any Vca.

Triple-barrelled micro-electrodes were made from tubing with two parallel partitions (R and DScientific Glass Co., 15931 Batson Rd, Spencerville, MD 20868). The two outer channels were

silanized and the tips filled with sensor. The centre channel was used for the reference, and as forthe other extracellular measurements, it was filled with Ringer solution, or in some cases, with1 M-Tris/TrisHOC (pH7-1).

416

Na+ IN PHOTORECEPTORS AND GLIA

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Recording arrangements. Each active barrel was connected directly to the input of an operationalamplifier, either an Analog Devices AD 515L or a Burr Brown OP104. Frequent checks were madeto ensure that the input resistance was > 1014 Q and the bias current < 75 fA.

Light stimulation. Light from a xenon arc (XBO 150) was focused through glass optics toilluminate uniformly the whole of one retina. I.R. radiation was attenuated by 21 mm of 1% CuS04solution. The photon flux density at the retina, measured with a photodiode (OSD 100-1, Centronic,Croydon CR9 OBG), was 2 x 1010 photons 1um-1 s-1. The two stable states of the photopigment werein an equilibrium that was unchanged by the light stimulation. Unless otherwise stated, light flasheswere of 20 ms duration and experiments analogous to that of fig. 10 in Muri & Jones (1983) showthat photons will have been absorbed by in the order of 1 % of the pigment molecules (unpublishedmeasurements by the late R. B. Muri). In some cases, to avoid prolonged after-depolarizations(Baumann & Hadjilazaro, 1972) the light was attenuated by a factor of up to 4: the ion changesduring a 90s series of flashes were little affected by this. Values of ion activities in the dark weremeasured at least 6 min after the last stimulation, when light-induced changes were no longerapparent.

RESULTS

Na+ in the dark. As a double-barrelled Na+ electrode was advanced into the retinathe reference barrel recorded a series of stable membrane potentials from eitherphotoreceptors or glial cells, the latter being encountered more often near thecorneal end of the ommatidia, where they occupy a greater proportion of the volume(Perrelet, 1970). The cell was identified by its response to a 20 ms flash of light, as

shown in Fig. 1. Photoreceptors respond with a depolarizing receptor potential ofabout 50 mV amplitude with a spike on the rising phase (Baumann, 1968). Glial cellsrespond with a smaller and slower depolarization, with a time to peak of about 70 ms,which results from the K+ that is released from the photoreceptors and accumulatesin the extracellular space (Bertrand, 1974; Coles & Tsacopoulos, 1979). Measure-ments with fine, single-barrelled micro-electrodes have given mean values of theresting membrane potential, Vm, of -46 mV for glial cells in the cut head (Bertrand,1974) or -51 mV in vivo (Coles & Tsacopoulos, 1979). As will be described below,intracellular aNa (aNa) in a given slice of retina tended to change during the time (upto 5 h) over which recordings were made. We therefore considered first measurementsmade in the first hour after the dissection.As illustrated in Fig. 1 A and B, aNa in the glial cells appeared to be much higher

than that in the photoreceptors. In the six photoreceptors that gave receptorpotentials of more than 40 mV and had the most negative resting potentials (- 48-5to -52 mV) aNa was 10 1 mM; S.E. of the mean = 1-2 mm. In ten glial cells from ten

retinas, with Vm -45 to -57 mV, aNa was 45 7 + 34 mm. Additional brief impale-ments of glial cells showed, as expected in a syncytium, less variation from one glialcell to the next in any one retina than was found between retinas. There was no signi-ficant correlation between aka and Vm (r = 0 05).

The possibility of a spurious ion signal. The Na+ sensor based on ETH 227 issensitive not only to aNa but also to Ca2+ activity aca (Steiner et al. 1979). Althoughmost measurements of intracellular aca have given values so low that they wouldnot interfere with the measurement of aNa, the only published report on aca in a glialcell gives an unusually high value (a free concentration of 56 JiM, Burhle & Sonnhof,1983). We therefore used Ca2+-sensitive electrodes to compare aca in drone photo-

418


receptors and glial cells. Typical records are shown in Fig. 2A and B and illustratethe finding that the resting signal from the Ca2+ electrode was similar in the two celltypes. A flash caused a marked increase in the Ca2+ signal in photoreceptors (sixteencells; Fig. 2C), but there were no large or consistent changes in glia, even in responseto a train of flashes (eight cells, Fig. 2 D). For photoreceptors, the apparent free [Ca2+]

A B C

Photoreceptor Glial cell Calibration

or_FVref(mV)

-50 _L aNa =147 mm

-45 F | - l 26 mM

-75 _ 1 min

5-5 mM

Fig. 1. Comparison of aNa in photoreceptors and glial cells. Upper traces show thepotential (1V/ef) recorded by the reference barrel. Lower traces show the Na+ signal, i.e.the difference in potential (VNa) between the sensor and reference barrels. In A, the recordbegins with the electrode in a photoreceptor. A flash of light evoked a receptor potentialand a small deflexion of the Na+ signal. (On the oscilloscope screen it was observed thatthe spike on the receptor potential crossed the zero potential line, but the chart recorderwas unable to follow such a rapid event. The initial downward deflexion on the Na+response is an electrical artifact.) The electrode was then withdrawn to the bath. B is froma glial cell and shows the characteristic, slowly declining, potential response to a light flash.C is an electrode calibration. The estimated aNa in this photoreceptor was 6 mm and inthe glial cell 24 mm. The records were obtained in the order B, C, A.

was 32 ,uM, S.E. of mean = 0-6 aM, n = 11; for glial cells, free [Ca2+] was 3 0+ 0-7 /M,n = 7. The correlation between VE and free [Ca2+] was negligible in the photoreceptors(r = +0 13), but significant in the glia (r = +0 82). The lowest free [Ca2+] that wemeasured was 0-95 JuM in a glial cell with Vm = -64 mV. To test if changes in [Ca2+]in the micromolar range would affect measurements of aNa, we looked for a changein the signal from a Na+ electrode immersed in 10 mM-Na+ when [Ca2+] was increasedfrom 0 3 to 10 /tM. The observed change was less than 1 mV.We also did experiments in which [Na+] in the superfusate was reduced to 2 mm.

In the photoreceptors this causes a decrease in a'a that has been described previously(Coles & Orkand, 1982). In glial cells in low [Na+], the membrane hyperpolarized by

14-2

419

420 J. A. COLES AND R. K. ORKAND

2to 5 mV, aka fell exponentially (Fig. 3, note the logarithmic activity scale) andrecovered when superfusate [Na+] was restored. The mean values from five cells were:initial aka, 41 + 2 mm and time constant of fall, 10 +1 min. Fig. 3 shows the longestrecording during exposure to low [Na+], and in this case aNa fell to a minimum of14 mm. Since the electrode signal was still falling at this time, this figure sets an upperlimit for possible interference by unidentified ions other than Na+. These tests suggestthat in the superfused slices aNa in the glia was indeed markedly higher than in thephotoreceptors.

A B0

VM F(MV)

-500

vC, r1 min(mV) m

-130 -

Photoreceptors Glial cells00

5mV

(mVi |LL

5 mV L - > __1 mV[

Fig. 2. Recordings with Ca2+-sensitive micro-electrodes. In each pair of traces, the upperis the potential recorded by the reference barrel, and the lower is the difference in potentialbetween the two barrels. A, the record starts with the electrode in a photoreceptor. A lightflash evoked a receptor potential. The electrode was then withdrawn to the bath. B,withdrawal of another electrode (with a different callibration curve) from a glial cell. Aand B suggest that aca is similar in the two types of cell. C, Ca2+ signal from aphotoreceptor at a higher sensitivity. The second of the two light flashes was four timesweaker than the first. Although the receptor potential was only slightly smaller, the Ca2+signal was considerably reduced. D, recording from a glial cell at high sensitivity. A seriesof ninety light flashes, 1 s-', produced no detectable change in Vca (the initial downwarddeflexion can be attributed to an electrical artifact). The approximate base-line free Ca2+concentrations in the four records were estimated to be: 2-6, 095, 2-4 and 3-4 /M.

Changes in aka with time. A proportion of the photoreceptors that were impaledhad Vm -45 mV but gave only small receptor potentials (from 40 to 5 mV). Thesecells had a high aka of 20-30 mm. (Cells with similar electrical properties also hadhigh aca ) The proportion of cells with these characteristics varied from one slice toanother and increased with time. In the glial cells, the behaviour was different. Themean aNa measured between 120 and 280 min after the dissection was slightly lower


than in the first 60 min: 355 + 53 mm, n = 10. The membrane potentials alsochanged, becoming more negative (the mean for the sample was -57 mV). In tworecordings, made more than 160 min after the dissection, aia appeared to fall to verylow values: 15 and 6 mm.

aka changes with light stimulation and raised bath [K+]. We induced Na+ movementsacross cell membranes either by light stimulation with trains of light flashes at 1 s-1for 90 s or by changing bath [K+], much as in previous work on K+ movements (Coles& Tsacopoulos, 1979; Coles & Orkand, 1983).

5 min

Vm(mV)

-50 147

] 48 aNa(mm)

14

[Na+] (mm) 210 2 210

Fig. 3. Effect of reducing bath [Na+] on glial aNa. At the time indicated on the bottomline, all but 2 mm of the Na+ in the superfusate was replaced by choline. aNa fell and then,when the normal bath [Na+] was restored, recovered to the initial level. Note that theaNa scale is approximately logarithmic. The electrode tip was 135,m from the surfaceof the slice.

Photoreceptors. Fig. 4 shows a recording from a photoreceptor that was stimulatedwith a train of light flashes and then depolarized by raising bath [K+]. In fivephotoreceptors with Vm < -48-5 mV (from different retinas), aNa increased by16 + 2 mm with light stimulation. When bath [K+] was raised to 18 mm, photoreceptoraNa decreased or increased by less than about 0 5 mm as in Fig. 4 and in the eightother cases where the membrane potential showed a simple depolarization with noinflexions. In a few cells with 18 mM-K+, and in most cells with 28 mm-K+, thedepolarization had a complex time course and was accompanied by an increase inaka (not shown).

Glial cells. As illustrated in Fig. 5A, light stimulation caused only a small changein glial aNa: a fall that averaged about 4 o in 90 s. To analyse such small changes,we measured them with respect to the base line, allowing for any drift that waspresent. During light stimulation, extracellular K+ activity (ak) rises (Coles &Tsacopoulos, 1979) and aOa falls (see below). To see if these changes could reasonablybe the cause of the change in glial aka, we compared the effect of light stimulationwith that of superfusion with 18 mm-K+ Ringer solution. This concentration of K+was chosen because it depolarizes the glia about as much as does the light-induced

421


increase in a . Superfusion with this solution did indeed cause glial aka to fall(Fig. 5A, B and C). But in many cells, although the membrane depolarizations weresimilar with light stimulation and raised bath [K+], the changes in aka at the end of90 s (AaNa) were different. This difference depended on the depth of the recordingsite from the surface. In Fig. 5A, where the recording site was close (22 #sm) to

s~~~~~~~~~~~2min

(MV)_

-45 L(mM)I [

L r[K+]1 mM

Fig. 4. Changes in aNa in a photoreceptor. A train of light flashes evoked receptorpotentials that appear fused in the upper trace. The activity scale for aN8 (lower trace,left-hand scale) is approximately logarithmic. The sensitivity of the Na+ signal wasdoubled for the second part of the record (right-hand calibration bar). When [K+] in thesuperfusate was increased from 7-5 to 18 mm there was no detectable change in aNa. Theelectrode tip was 121 #am from the surface of the slice.

the surface of the slice, although the depolarization was slightly greater duringthe photostimulation than during the raised bath [K+], AaNa was greater with thelatter stimulus. Fig. 5B and C, shows examples of the effects of raised bath [K+] attwo depths in the same retina. As reported previously (Coles & Orkand, 1983),the membrane depolarizations began with almost the same, brief, delay after theswitching of the solution and were similar in amplitude and time course, near thesurface and deep in the slice. In contrast, at the deeper site, aNa began to fall onlyafter a marked delay. The changes in aNa 90 s after the onset of photostimulationor superfusion with raised bath [K+] are summarized in Fig. 6. With light stimulation(Fig. 6A), decreases in aNa were observed at all depths at which recordings weremade (down to 254 #tm), and within this range there was no obvious dependence ondepth. In one site no decrease was seen: this may have been partly because ofartifactual fluctuations in the electrode signal and partly because in this particularslice the proportion of photoreceptor cells that gave only small receptor potentialsappeared to be unusually large. The mean decrease in aNa with light stimulation inglial cells with Vm -50 mV was 1-5+0-3 mm, n = 10.

In Fig. 6B, AaNa, 90 s after switching to superfusate with 18 mM-K+, has beenplotted against depth. It is seen that the greatest changes in aNa were near the surfaceand at deep sites they were small, or even positive. In some superficial sites, aNa fellfirst rapidly, then after some tens of seconds, more slowly. In deep sites, the fall in

422

Na+ IN PHOTORECEPTORS AND GLIA 423

A

(mV)

50 -

aN8

(mM)

45_A

Light 18 mm90 s

B

v -30(MV) _40 -

a' 488Na

(mom) LH ~

18mM

C

VM -40 r(MV) _50 L_

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Fig. 5. A, the experiment of Fig. 4 was repeated with the electrode in a glial cell. Lightstimulation caused a small decrease in aNa, and increasing bath [K+] to 18 mm causeda larger decrease. The electrode tip was 22 sum from the surface. B and C, recordings fromglial cells at two depths in the same retina (A, 40 ,um; B, 151 sum). At the deeper site aNadoes not start to fall until about 90 s after the onset of the depolarization. In this recordthe base line for aNa is drifting upwards.

aNa always lagged behind the depolarization, as in Fig. 5C, and in a few cases therewere signs of an initial transient increase. We suggest, in the Appendix, that this iswhat might be expected from spatial buffering. When these spatial effects are takeninto account, it appears that the decrease in glial aNa induced by stimulation of thephotoreceptors could arise simply because the ionic composition of the extracellularfluid changes to resemble that of the 18 mM-K+ Ringer solution.

After the first 2 min, raised bath [K+] caused aNa to decrease steadily at a rate thatappeared to be independent of depth. Fig. 7A shows our longest recording, whichwas 15 min. In cells with Vm E -50 mV the mean fall in aNa after 5 min was3-5 + 06 mm, n = 7. The time constant of the decrease was 41 +3 min, n = 26. The


rate of decrease was greater when [K+] was 28 mm than when it was 18 mm (Fig. 7 B).Conversely, when bath [K+] was reduced to 2 mM, glial aNa rose steadily (in four cells;the longest exposure was 20 min). The results are summarized in Fig. 7C.The composition of the 18 mM-K+ solution used so far was similar to that in the

extracellular space during photostimulation in that apart from the increase in [K+],

A

-0 5i- --XJ3.50

A aNa 0(mm) 1

-1-5 0 0 0

-2-0 0

-20l

B~~~~~~~~~~~~~

+ 05

0

AaNa _1(mM)

-2-0

-2-5

-3-5

0 50 100 150z (Am)

200 250

Fig. 6. Change in glial aNa 90 s after the onset of light (A) or switching to 18 mM-bathK+ (B). The values are plotted as functions of the depth, z, of recording site.

there was a corresponding decrease in [Na+], and [Cl-] was constant (Coles & Orkand,1984). One way by which such a solution is expected to cause glial aNa to fall is simplyby the small reduction in extracellular [Na+] (see Fig. 3). A second way arises becausethe glial membranes are permeable to Cl- (Coles & Orkand, 1984). The 18 mM-K+superfusate causes entry of K+ and Cl- (Coles & Orkand, 1983, 1984) and thereforeof water, which will dilute the activity of intracellular Na+. If K+ and Cl- are inequilibrium across the membrane, then passive KCl entry is prevented if the productof the extracellular concentrations [K+] [Cl-] is maintained constant (e.g. Boyle &Conway, 1941). We were unable to do this in the slices of drone retina, first because

_

0

-- - - w - - - -

0

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0

* 0* 000 0 0

0

0

00

II III

-0-5

-10*0

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A

Vm -50F(mV) -60L

34 -

(mM)

[K+] 18 mmI

B

Vm -40 r5 min

(mV) [55[30-

(mm)25-

Light [K+] 18 mm 28 mm

C+0-8

0

daN8/dt(mM/min)

-1F

-20 10 20 30

[K@] (mM)

Fig. 7. A, the effect on glial aNa of prolonged superfusion with a Ringer solution con-

taining 18 mM-K+. The depth, z, of the recording site was 38 /sm. B, the effect of twodifferent values of bath [K+] on glial aNa. z = 107 ,um. C, summary of the effects ofdifferent concentrations of bath K+. The rate of change of aN., daN./dt, was measuredafter aNa had started to change steadily (up to 2 min after the change in solutions). Incases where aNa was changing with 7-5 mM-bath K+, then daNa/dt was measured relativeto this drifting base line, i.e. daNa/dt in 7-5 mM-K+ was defined as zero, which on averageis almost true. The number of measurements and the S.E. of the mean are indicated foreach point.

4

14

I I

425


[K+] is higher than in the bath and varies with depth (see Gardner-Medwin et al. 1981),and secondly because if bath [Cl-] is reduced, extracellular [Cl-] takes several hoursto reach the new value, presumably because the fairly large quantities that leave thecells diffuse only slowly to the bath (J. A. Coles & R. K. Orkand, in preparation). Forthese reasons, even if [K+] [Cl-] in the superfusate replacing the Ringer solution iskept constant, the product in the extracellular clefts will change, particularly during

TABLE 2. Intracellular aNa and aK

Photoreceptors Glial cells

Dark aNa (mM) 10-1+1-2(6) 37+2 (7)-485mV . Vm > -52mV -55mV> VM_ -59mV

Dark aK (mM)* 89+14 (3) 92+9 (5)-5OmV> Vm > -56mV -55 mV> Vm > -615mV

Light flashes for 90 s

AaNa(mM) +16+2(5) -1V5+0-3(10)-485mV _ Vm > -52mV -5OmV> Vm > -63-3mV

AaK (mM)* -21+2(7) +7-1+1-9(6)-45mV>Vm> -56mV -5OmV>Vm_-63mV

Bath [K+] increasedto 18 mMdaNa/dt (mM/min) > -024 (7)t -P12+02 (16)

-43mV> Vm . -485 mV -45mV > Vm _ -6OmVdaK/dt (mM/min)* 03+02 (6) +1-9+0-4 (8)

-48mV > Vm > -56mV -50mV > Vm . -59mV* From experiments described in Coles & Orkand (1983).t In four cells daNa/dt was > 0.Values are expressed as mean+ S.E. of mean; n in parentheses.

equilibration. Neverthless, experiments were made with a solution in which [K+] was18 mm, [Na+] was unchanged and [K+] [Cl-] was constant (see Methods forcomposition). In all of ten attempts to record the effect of switching to this solution,aNa began to fall. In the three cases in which a stable recording was obtained for 5 minof superfusion, aNa fell at a rate the mean value of which was indistinguishable fromthat with the standard 18 mM-K+ solution.

Extracellular space. When the electrode tip was at a site in the extracellular spacewhere its potential differed by less than 5 mV from that in the bath, and the electricalresponse to light was purely negative, we considered it to be in extracellular space.The Na+ sensor based on the ligand ETH 227 that was used for the intracellularmeasurements is affected by Ca2+ in the millimolar range found in the extracellularspace (Steiner et al. 1979; Dietzel et al. 1982), so electrodes based on ETH 157 wereused in most experiments. Compared to ETH 227, this ligand gives a sensor that isless sensitive to Ca2+ but more sensitive to K+ (Ammann et al. 1983), but the effectof changes in extracellular aK (a°k) was corrected for approximately by the increased[K+] in the calibration solution. We observed no deflexion when the [Ca2+] in theRinger solution was increased from 1-6 to 16 mm. We also made measurements usingETH 227 in electrodes with a third barrel containing Ca2+ sensor so that the aNa

426


measurement could be corrected for changes in aca (Fig. 8A and B). Results withboth kinds of electrodes are shown in Fig. 8C and D. When the electrodes were with-drawn to the bath after a recording the apparent VNa often showed a change. In fifteenrecordings the apparent aoa was less than in the bath, in ten it was greater and in

A B

VrefVref 5mV [5 mV L rV~a [ 1~1 2mim

2VN V[,//NII 2

14 mV [

5 mV [ [Ca2+] 1 6 6-0 [Na+] 200 210

D90s

10 mV r

10 aNr

AaN. 0-- 10mMI(MM) -10L

-2090 s 90 s

Fig. 8. A, extracellular recording with a triple-barrelled Na+/Ca2+ electrode. The top trace( Vr.e) is the potential between the reference barrel and the bath electrode. The middle trace(VNa) is the potential between the barrel containing the sensor based on ETH 227 andthe reference barrel; and the third trace (Vca) is the equivalent signal for the Ca2+ barrel.The filled rectangle indicates the time of stimulation with the standard train of flashes.The electrode tip was 126 ,sm from the surface. B, calibration of the Na+/Ca2+ electrode.First, the [Ca2+] and then the [Na+] of the Ringer solution were changed while the electrodewas in the preparation chamber. It is seen that the 'Na+' barrel responds to changes ineither [Na+] or [Ca2+], while the Ca2+ barrel is more selective. C, change in extracellularaNa. The line was calculated from the second and third traces in A. D, change in extra-cellular aNa. A double-barrelled electrode with the sensor based on ETH 157 had its tip104 ,sm from the surface.

four it was not detectably different. The greatest voltage deflexions were + 1-9 mVand - 2-7 mV. This implies that some slices gained Na+ during superfusion and otherslices lost Na+.

During light stimulation, aOa fell to a comparatively steady value in about 10 s,and showed no tendency to return to the base line during the remaining 80 s ofstimulation. The average decrease in aoa was 14+1 mm (twenty-four measurementsin seventeen retinas). The failure to return towards the base line during thestimulation is in striking contrast to the behaviour of a0, as is illustrated by the

recording in Fig. 9 made with a triple-barrelled electrode that measured aNa and aK


simultaneously. Note that aO is on a non-linear scale. For aO, the mean change atthe peak was 105+ 1I1 mm, and at the end of the 90 s it was 5-9+0-6 mm (n = 10).The results from extracellular space and also photoreceptors and glial cells aresummarized schematically in Fig. 10.

Vref Ir1(mV)-

-10 _

20

aK(mM) 10 X

5-25

3

IN[

20 mm_

90S

Fig. 9. Simultaneous measurement of extracellular aK and aNa. The upper trace is thepotential recorded by the reference barrel of a triple-barrelled electrode. Stimulation withthe standard ninety flashes (filled rectangle at bottom) evoked negative-going deflexionswhich appear fused. One active barrel contained K+ ion exchanger: aK (middle trace) roseto a peak then fell to a plateau during the stimulation. The electrode responded rapidlyenough to follow at least part of the pulsatile changes in aK associated with the light flashesand these are the main cause of the broadening of the trace. The other active barrelcontained the Na+ sensor based on ETH 157. During stimulation aNa fell to a steady value.At the end of the stimulation the decrease in aNa was 24-6 mm while the increase in aKwas only 9-3 mm. Because of its high resistance, the Na+ barrel was not rapid enough tofollow the field potentials and there was a subtraction artifact: despite filtering with atime constant of 03 s some of the artifact remains and is the cause of the widening ofthe trace. The electrode tip was 128 ,um from the surface of the slice.

DISCUSSION

Dark aNa in glial cells. For the cells accepted for inclusion in Table 2 on the basisof their membrane potentials, aNa was more than three times higher in glial cells thanin photoreceptors. It is unlikely that the electrode was responding to a substance otherthan Na+ because, as illustrated by Fig. 2 B, the only known and plausible interferingion, Ca2+, gave an electrode response as low as in the photoreceptors. Measurements


150

Extracellular space

100 _

aNa(mM)

40 Glial cells

Photoreceptors10 _

90s

Fig. 10. Schematic summary of the light-induced changes in aNa. The mean measuredvalues in the dark and after 90 s of stimulation have been indicated and curves joiningthem sketched in.

with an electron microprobe show that the Na+ concentration in the glia is sufficientto account for an aNa as high as that measured (Coles & Rick, 1985).We considered the possibility that aNa in the superfused slice was higher than in

the intact retina. In this connexion, we saw no sign that the cutting of the glialsyncytium during the preparation of the slice permanently changed the propertiesof the glial membranes. Apart from the new measurements with Ca2+-sensitiveelectrodes, previous work on slices under identical conditions showed that glial aKwas at least as high as in the photoreceptors and stable for more than 4 h (Coles &Orkand, 1983). Damage to the cells might be expected both to depolarize membranepotentials and to increase aNa, but provided Vm < -45 mV, no significant correlationwas found between glial aNa and Vm in measurements made during the first hour.We tentatively conclude that, after being cut, the syncytial membrane reseals withits original properties.

Glial aNa tended to fall over the 4-5 h during which measurements were made inany one slice. Glial aNa also changed when bath [K+] was changed; and from the graphof the results (Fig. 7 C), it can be seen that if a° differed by only 1 mm from the valuenecessary to maintain aNa constant, then aNa would fall fast enough to account forthe observed average rate in the slices. aO in the drone retina in vivo was found byColes & Tsacopoulos (1979) to be 7-7 mm. The Ringer solution in the present work hadan aK of 5-25 mm, but aK in the extracellular clefts was found to be higher than this,as in Fig. 9. A contributing factor would have been those photoreceptors that gainedNa+ over the lifetime of the slice, and, presumably, lost K+ to the extracellular clefts.


Hence, glial aNa might have been driven up or down by the balance of the effectsof the non-uniform aO. Measured aOa usually was apparently either greater or lessthan in the bath: this could have depended on whether entry of Na+ into dyingphotoreceptors was under- or over-compensated by extrusion of Na+ from the glia.This scheme will also explain the commonly observed drift in base-line glial aNa (asin Fig. 5C).To summarize, we conclude that, in the slices, aNa was higher in the glia than in

the photoreceptors; we argue that this is true in vivo; and we speculate that glial aNain the slices tended to fall slowly because aO was higher than in vivo.

The light-induced increase in aNa in photoreceptors. During light stimulation aNaincreases and aK falls (Coles & Tsacopoulos, 1979). In Table 2, it is seen that the meanof the decreases in aK was 5 mm greater than the mean of the increases in aNa,although the difference was scarcely significant (P - 0-21). Since other ions cross themembrane, such as Cl- (Fulpius & Baumann, 1966) and perhaps HCO3-, and theremay be water movements (Orkand, Dietzel & Coles, 1984), a discrepancy would notnecessarily contravene electroneutrality.Mechanismsfor the decreases in aNa in the glial cells. During the 90 s of the standard

light stimulation, at least two space-independent mechanisms will have contributedto the fall in glial aNa. First, the extracellular aNa fell by about 10 %. When bathaNa was experimentally reduced by 99% (Fig. 3), glial aNa fell with a time constantof 10 min, which would correspond to a fall of 5-7 mm in the first 90 s (if there wereno diffusion delay). Hence, during light stimulation, the fall in aoa might cause aiato fall by about 0-1 x 5-7 mm = 0-57 mm. The second mechanism is the swelling of theglial cells. K+ and Cl- enter the glia (Coles & Orkand, 1983, 1984) and will bring aboutan entry of water, a sign of which is the observed shrinkage of extracellular space(Orkand et al. 1984). Within the cell, this water will dilute all the ion concentrations,including [Na+]. A swelling of less than 3 %, together with the first mechanism, wouldbe sufficient to account for the observed fall in glial aNa.During prolonged exposure to raised bath [K+] the situation is different and there

is evidence for another process. The reduction in bath aNa with the 18 mm-K+ Ringersolution was only 5.0 %, but over periods of many minutes, glial aNa could fall muchmore than this, e.g. by 18 % in the recording in Fig. 7 A. It is therefore unlikely thatthe decrease in aOa made much contribution. Similarly, the contribution of swellingwas probably small after the first 2 min of a switch to 18 mM-K+ Ringer solution.This is because ac1 changes rapidly to follow changes in Vm and then appears to remainconstant (J. A. Coles & R. K. Orkand, in preparation). Consequently, entry of KClwould no longer contribute to increasing the osmotic content of the glia. Hence, someother mechanism is needed. This might be a Na+/K+ exchangepump that is stimulatedby the increase in ao.The initial time course of the change of glial aNa was complicated by the

redistribution of Na+ in the glial syncytium by the spatial buffering currents(Gardner-Medwin et al. 1981; Coles & Orkand, 1983). In the regions of highextracellular [K+], where current enters the syncytium, there will have been de-pletion of Na+, and conversely, in the regions where K+ leaves the syncytium, therewill have been an accumulation (the transport number effect of Barry & Hope, 1969;see also Fig. 9 in Coles & Orkand, 1983). When bath [K+] was raised, this mechanism

430


92-

aNa, aK(mM)

37 -

-a 0 +aFig. I1. Model geometry for analyzing the effect of spatial buffer fluxes on intracellularaNa and aK. An idealized glial syncytium forms a slab extending from x = -a (deep inthe slice of retina) to x = +a (the surface of the slice). The membranes are permeable onlyto K+, and within the syncytium K+ and Na' are the only mobile ions. Initially, K+ andNa+ are uniformly distributed through the syncytium (dashed lines). At time t = 0, aK isincreased at the outer face of the membrane at x = -a and causes a constant current ofdensity J to flow through the syncytium. This leads to activity gradients: , aK;-*---, aNa. The extracellular clefts and the return current loops through them are notrepresented.

will have contributed to the fall in aNa near the surface and will have transportedNa+ into the deeper parts of the syncytium. The calculations in the Appendix suggestthat this interpretation is quantitatively plausible. Comparison of Fig. 6A and Bsuggests that when spatial effects are taken into account, the fall in glial aNa duringlight stimulation might well be accounted for simply by the rise in aO and the fall0~~~~~~~~~~~~~~in a.

Homoeostasis of aoa and aK. At the onset of photostimulation, the main processesthat tend to limit changes of ao and of aO are quite different. Because aOa is high,the shrinking of the extracellular space provides most of the Na+ that enters thephotoreceptors, without causing large fractional changes in aNa. In contrast, this sameshrinkage tends to aggravate the rise in aK due to the efflux from the photoreceptors;it is the glia that tend to prevent aO from rising. The shrinking also causes aOa toincrease (Orkand et al. 1984) and presumably aOg does too. Hence the observeddecrease in the sum (aa+aO) will tend to reduce changes in osmolarity. Na+ willalso be carried to the extracellular space in the active region by the extracellularcurrents associated with spatial buffering (Gardner-Medwin et al. 1981). The sameprocesses will account for the changes in aO and aoa that have been observed inmammalian brain (Dietzel et al. 1982).

APPENDIX

Theoretical contribution of spatial buffering to AaNa in the glial syncytiumThe aim is to estimate the changes in glial aNa that could be expected to result

from spatial buffer currents set up by light stimulation. As shown in Fig. 11, we

431


represent part of the glial syncytium as an infinite plane slab of thickness 2a that isinterposed between the centre of the zone of active photoreceptors, and the bath. Theextracellular clefts that run through the slab between the membranes at + a are notrepresented. At t = 0, [K+] is increased in the extracellular spaces adjoining themembrane at x = -a in such a way that a constant current ofdensity J flows throughthe slab. In crossing the membranes, J is carried solely by K+, and within thecytoplasm it is carried by K+ and also Na+ but there are no mobile anions. It isassumed that there is no water flow, and possible effects of cell membranes withinthe slab are ignored.

SymbolsJ: total current density imposed as a constant for t . 0.CNa(X, t), CK(X, t): concentrations of Na+ and K+.

CNa(x, t)+CK(x, t) = constant = CT. (1)

C°Na: concentration at t . 0.gnNa' UK: mobility coefficients.DNa, DK: diffusion coefficients.V(x, t): electrical potential.R, T and F have their usual meaning.

The steady stateIn the steady state, the current carried by Na+ must be zero, and the distribution

of [Na+] is determined by the opposing effects of the gradients ofthe electric potentialand the concentration.

-uNa CNa dV/dx-DNa dCNa/dx = 0. (2)

K+ carry all the current, so

-TtK CK dV/dx-DK dCK/dx = J/F. (3)

Hence, using eqn. (1),

d V/dx =-(J/F-DK dCNa/dX)/{#K (CT-CNa)}1 (4)

Substituting (4) in (2):

/K DNa CT N dx +(/tNa DK-tK DNa) dCNa INa J/F. (5)

From Einstein's relationD/It = RT/F, (6)

the coefficient of the second term is zero.

0Na (X) = CNa (0) exp(/LNaTFo(0 KDN(aCTF)or, using (6),

CNa (x = CNa (°) exp (DCT-P (7)

432


It will be shown that the exponent < 1 in which case

CNa (X) CNa (°) DKC F (t o ). (8)

r+aSince I CNa (x, t) dx = constant with respect to t, then with the approximation of

-aeqn. (8), 0Na (0, t) = Coa. (9)

Hence,AC ~~~~~Co~aJX (0CNa (X, 00) CNa (X, 00)-CNa (O, ) -DCF (10)

Numerical values

CNa (0), CT: the values for the activities, from Table 2, are 37 mm and 129 mM.In eqn. (10) only the ratio of these quantities appears so there is no need to convertto concentrations.

J: from measurements of extracellular potential gradients, Gardner-Medwin et al.(1981) estimated that the spatial buffer current through the glia in the cut-head pre-paration had a density of 2-7 x 10-4 A cm-2 of tissue. The glia occupy about half thevolume, so, as a rough estimate, we double the figure and obtain J = 5-4 x 10-4 A cm-2.F= 97x 104C mol-i.DK: the diffusion of K+ has been studied in single cells (Hodgkin & Keynes, 1953;

Kushmerick & Podolsky, 1969) and in the syncytial tissue of the mammalian heart(Weidmann, 1966). It appears that in heart muscle the intercalated disks do notreduce the apparent DK by a factor of as much as 2 (Weingart, 1974). Coles & Orkand(1983) could not detect significant potential gradients in the glial syncytium of thedrone retina when one surface was depolarized, and injected Lucifer Yellow appearsto diffuse readily (R. K. Orkand & J. A. Coles, unpublished observation). We thereforeassume that DK in the glial syncytium is as large as in heart muscle: 8 x 10-6 cm2 s-(Weidmann, 1966).

x: we assume that the distance from the depth of maximum extracellular [K+] tothe surface of the slice is 200 ,tm (cf. Gardner-Medwin et al. 1981), i.e. a = 100 /Im.When these numbers are inserted in eqn. (10), the change in aNa at the extreme

ends of the model syncytium, after a steady state has been reached, is AaNa = 1-4 mM.This corresponds to a gradient that is about the same as that induced after 90 s byexposure of the slice to 18 mM-bath K+ (Fig. 6B). However, there are a number offactors of considerable uncertainty in the estimate of the component of AaNa dueto spatial buffering. The estimate of J depends on the value taken for the volumeof the extracellular space, which has been estimated for the drone retina only fromelectron micrographs (Coles & Tsacopoulos, 1979; Gardner-Medwin et al. 1981). Thedistribution of extracellular [K+] illustrated in Fig. 11 is unrealistic: there is actuallya continuous gradient, which will have the effect of reducing AaNa(a). Also, sinceneither ItNa nor DNa appear in eqn. (10) any other ion besides Na+ and K+ that iscapable of passing through the junctions between the cells will tend to redistributein the same way as Na+. Dye-injection experiments (R. K. Orkand & J. A. Coles,unpublished observation) suggest that ions as large as Lucifer Yellow CH can passreadily through these junctions. The presence of such additional mobile ions,

433


probably including Cl-, would reduce AaNa(a). Finally, the time necessary for thesteady-state distribution of glial aNa to be reached has not been evaluated, and mayhave been greater than the usual 90 s of stimulation. We now estimate this time.Time for the redistribution of Na+. The diffusion equation for Na+ was solved by

the method of Laplace transforms and the inverse transform evaluated approxi-mately. The result was that in the model system Na+ came halfway to its steady-state concentration 2-4 s after the imposition of J, so the steady-state was reachedwell within the usual 90 s of light stimulation. A much simpler argument suggeststhe same conclusion.The initial flux density of Na+ across the plane x = 0 is (assuming MNa = /K);

E CNaF CT'

The final excess of Na+ in the slab 0 < x < a is, by integration of eqn. (10),

1 COa Ja22 DK CTF

The time necessary for this quantity of Na+ to cross x = 0 at the rate of the initialflux is

T = a2/DKt 5 S << 90 S.

Mr J.-L. Munoz made most of the ion-sensitive electrodes, and Mrs J. Steiner and G. Champendalhelped type the manuscript. We thank Professor P. Jeanquartier for evaluating the inverse Laplacetransform referred to in the Appendix; Dr D. Ammann of Zurich for advice and gifts of neutralligand sensors and Drs I. Dietzel, A. R. Gardner-Medwin, G. Van de Werve and M. Tsacopoulosfor helpful criticism of the manuscript. Supported by NRSA Fellowship NS 06898, Grant NS 12253(R.K.0.) and Grant EY03504 (J.A.C.) from USPHS, Swiss NSF Grant 3.399.0.78, the GeorgesKernen Foundation and the Cloetta Foundation.

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in photoreceptors, glia and extracellular space in

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