JOURNAL OF THE OPTICAL SOCIETY OF AMERICA

Standing Potentials of the Frog's Eye*

ERNEST DZENDOLETHunter Laboratory of Psychology, Brown University, Providence, Rhode Islandt

(Received October 12, 1959)

With the in-place frog's eye, the potential difference between the center of the corneal surface and therest of the cornea reached a maximum of about -15 mv at the corneoscleral junction. This large a cornealpotential difference may be the immediate source of the potential presumably utilized in the electro-oculo-gram, rather than the cornea-to-fundus potential. A slightly injured section of the cornea and also theaqueous humor had a potential of approximately +15 mv with reference to the corneal center. These twopotentials appeared to be separated by an insulating layer, presumably the interface between the cornealepithelium and Bowman's membrane. The potential difference between the corneal center and the frontinterior part of the lens was about -33 mv, and about -47 mv for the back. The vitreous side of the retinawas about -2 mv. Within the retina, transient steps of about -50 mv occurred. These were not the sameshape or in the same order from frog to frog, or from one place in the same retina to another, except for one.This was a step of -40 to -60 mv which was presumably Brindley's R membrane.

INTRODUCTION

THE results of early investigations of the standingT potentials in excised animal eyes, usually offrogs, were primarily qualitative. Their quantitativeaspect was summarized by Kohlrausch,l who reportedthat freshly prepared frog's eyes gave a standing po-tential generally not greater than 10 mv, the corneabeing positive with respect to the fundus.

Surface of the Eye

Measurements of the standing potential over theentire excised eyeball were cited by Kohlrausch asbeing carried out by DeHaas and by Westerlund. Theseinvestigators used the center of the cornea as the refer-ence point, and measured the potential at six equallyspaced points between the corneal center and theentrance of the optic nerve into the eyeball. Theirresults were essentially the same. The first point, at thecorneoscleral junction, gave a value of about -4 mv.In the region of the ora serrata, the potential wentalmost to zero, and remained at this value up to theoptic nerve.

Interior of the Eye

In the interior of the eye, Brindley,2 using a micro-pipette and entering the excised eyeball through a holeat the back, found that there was no change in potentialwhen moving the pipette through the vitreous body.A corneal reference was used. Brindley also found thatthe back part of the lens was at a mean potential of-74 mv with respect to the vitreous body. Movementof the electrode completely through the interior of thelens changed the potential by about -10 mv. Brindley's

* This investigation was supported under a contract betweenBrown University and the Office of Naval Research.

t Present address: Aerospace Medical Laboratory, Wright AirDevelopment Center, Wright-Patterson Air Force Base, Ohio.

1 A. Kohlrausch in Handbuch der normalen und pathalogischlenPhysiologie, edited by A. Bethe (Springer-Verlag, Berlin, 1931),Vol. XII'2, p. 1394.

2 G. S. Brindley, Brit. J. Ophthalmol. 40, 385 (1956).

discovery of the lens potential has been confirmed byAndree,3 who gave a value of - 73.7 mv for the interiorof the lens with respect to the exterior when an excisedfrog lens is placed in Ringer's solution.

Within the Retina

Tomita4 reported a potential of about -20 mvwithin the retina, but attributed this drop to injuriesof the intraretinal cells caused by the probe electrode.Ottoson and Svaetichin5 apparently did not find thissame pattern. Brindley,6 however, did confirm Tomita'sreport. Brindley reported values of -10 to -30 mv,ranging from -2 to -41 mv. The hypothetical struc-ture causing this drop was designated the "R membrane"because of the drop itself, and also because this part ofthe retina behaved as though it were a high resistancein parallel with a large capacitance. Brindley also men-tioned that other changes were found as the probemoved through the retina. These did not follow a clearpattern that could be identified in each experiment.Brindley believed that the R membrane was identicalwith the external limiting membrane.

APPARATUS

The apparatus consisted of: (1) an optical systemfor stimulating the frog eye; (2) three mechanicalsystems, one for positioning the head and eye of thefrog, another for placement and movement of the re-cording or probe micropipette, and the last for place-ment of the reference micropipettes; (3) an electronicsystem for amplifying and displaying the electricalresponses of the eye, and (4) a photographic system forrecording the displayed responses. The stimulation ap-paratus was not used in this series of experiments.

3 G. Andree, Pflger's Arch. ges. Physiol. 267, 109 (1957).T. Tomita, Japan J. Physiol. 1, 110 (1950-51).D. Ottoson and G. Svaetichin, Cold Spring Harbor Symp.

Quant. Biol. 17, 165 (1952).6 G. S. Brindley, J. Physiol. 134, 339 (1956).

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ERNEST DZENDOLET

FIG. 1. The interior of the sheet-metal box with (A) the frogholder, (B) the ball-and-socket joint supporting it, (C) the lathecross feed which gives a degree of freedom perpendicular to thetwo of the probe micropipette manipulator, (D) the referencecalomel half cell, and (E) the air-driven probe micropipettemanipulator.

The MacNichol and Wagner7 high-input-impedancedc preamplifier circuit was used. For convenience inmaking adjustments of the preamplifier, ten-turn pre-cision potentiometers were used in place of standardpotentiometers for the calibrating, bucking-voltage,and zero controls.

Standing potentials were measured by the bucking-voltage method, using one of the auxiliary circuits ofthe MacNichol and Wagner preamplifier. In thismethod, a voltage of polarity opposite to the one to bemeasured was placed in series with it, between thereference electrode and ground. This bucking- orcounter-voltage was adjusted by a calibrated poten-tiometer so that it brought the one to be measured tozero. The voltage to do this was then read from thepotentiometer and recorded.

The preamplifier output was led into a dc amplifierbuilt according to Seely's 5 specifications. The 6AU7 tubeoriginally called for is obsolete, and was replaced bythe 6SL7. This change in tubes necessitated only achange in the negative voltage supply from -200 to-67 v. Wire-wound, noninductive 1% resistors wereused throughout both amplifiers for stability ofoperation.

The output of the dc amplifier was led to the dcamplifier section of a DuMont 304A oscilloscope. Thedisplayed signals were photographed with a GrassC4C Kymograph Camera, using Eastman Kodak Lina-graph paper.

The probe micropipette manipulator is made bymodifying a 10-in. Hite-Set made by the Brown and

7 E. F. MacNichol, Jr., and H1. G. Wagner, Research Report,Project NM 000 019.03.01, Naval Medical Research Institute,National Naval Medical Center, Bethesda, Maryland, 12, 97(1954).

8 S. Seely, Electron-Tube Circuits (McGraw-Hill Book Company,Inc., New York, 1950) p. 116.

Sharpe Mfg. Co. One without the measuring blockswas obtained. The vertical slot which contained theblocks allowed an extension of aluminum and plasticto be mounted easily on the set. This extension heldboth the micropipette and the reference half-cell. Thepipette itself was held in a vertical slot in the plasticwith a rubber band. Since the Hite-Set allowed onlyvertical movement, the entire manipulator was mountedon a lathe cross feed which was bolted to the steel plateforming the floor of the metal box used as the experi-mental area.

Vertical movement of the Hite-Set was controlledby an air-drive arrangement patterned after the typedeveloped by B6k6sy.Y

The reference micropipette manipulators were madeby combining the two degrees of freedom of a micro-scope mechanical stage with the one degree of freedomof a Harvard Apparatus Company Adjusting Clamp.One armof the stage was fastened to the adjustingclamp which was mounted, in turn, on a rod fixed to aplate on the cross feed on which the frog holder wasattached. As with the probe manipulator, the micro-pipette was held in a slot in a plastic block mounted onthe end of one of the stage arms.

The frog holder is a hollow, plastic, rectangularparallelepiped without a top or front end. At the frontwas an approximation of a human "biting-board" ar-rangement. The frog's mouth was placed so that theupper jaw "bit" on a narrow sheet of plastic cementedto the rest of the plastic box. A second strip of plastic,with sponge rubber cemented to one side, was arrangedso as to screw down on the upper part of the frog's nose.The entire holder itself was mounted on a camera-tripod head, which is essentially a ball-and-socket joint.A separate arrangement also allowed 360° rotation ofthe tripod head about a vertical axis. The holder andtripod head were mounted on a lathe cross feed fastenedto the steel flooring plate. The horizontal movement ofthis cross feed was perpendicular to the one holdingthe probe micropipette. The manipulators and the frogholder were housed in a sheet-metal box with the pre-viously mentioned heavy steel plate for a floor. Thebox shielded the electrodes from the various electricfields present in our society, primarily the 60-cycle housecurrent. The box also minimized the amount of straylight influencing the frog's eye. This part of the ap-paratus is shown in Fig. 1.

The micropipettes were drawn from commerciallyavailable capillary tubing on a Livingstone type me-chanical puller. They were filled with approximately3 M KCl solution, and connected by small diameterplastic tubing to calomel half-cells, and then to theinput of the preamplifier. The tip diameters usedranged from about 1 to 8 y.

9 G. v. Bdkesy, Rev. Sci. Instr. 27, 690 (1956).

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PROCEDURE

Male Rana pipiens frogs were used, and all measure-ments were made on the in-place eye. The frogs wereimmobilized by using 0.05 cc of 3 mg/cc d-tubocurarinechloride injected into each thigh. The frog was placedin its holder in the manner indicated in the apparatussection. Measurements on the cornea were made bymerely lowering the electrodes into place. The openedeye preparation was made by cutting away most of thecornea with scissors. The iris was always left intact.The lens and vitreous body were removed togetherwith the aid of an ordinary medicine dropper. Whenthis procedure was carried out successfully, the entireretina showed an even, purplish color. Final values ofthe electrical potential differences given in the resultsare the means of five frogs unless otherwise noted.

RESULTS

Surface of the Eye

When measuring the potential difference between thecornea and the skin, a fluctuation of the potential withtime was observed. For example, the potential differ-ence of one animal went from + 13 to -2 mv in 22 min.The skin was therefore not used as the reference.Instead, the center or another part of the cornea wasmade the reference point.

Figure 2 shows a plot of the potential difference dis-tribution on the surface of the cornea, from the centerto the caudal edge of the cornea, i.e., toward the tailend, and also from the center to the dorsal edge, i.e.,toward the spine of the frog. The distance betweensuccessive electrode positions was measured along ahorizontal plane tangent to the center of the cornea,rather than along the curved surface of the corneaitself. This procedure was chosen for its methodologicalconvenience. It can be seen that the potential nearthe center of the cornea falls off slowly at first, andthen rapidly, and linearly farther away from thecorneal center until a final mean value of -12 to -16mv was reached. Measurements on two animals indi-cated that similar distributions were present bothcranially and ventrally, i.e., toward the nose andtoward the belly, respectively, so that for convenienceonly the caudal and dorsal values were plotted.

If the cornea was injured, as by lightly scrapingwith a knife without cutting all the way through, amean injury potential of + 18.4 mv appeared, rangingfrom +6.8 to +30.4 mv.

The potential of the conjunctiva was approximatelythe same value as the section of the cornea which itadjoined. When injured, it also gave a positive potentiallike the cornea.

Interior of the Eye

If a slit was made through the cornea, a mean po-tential of +15.4 mv was found for the same five frogson which the corneal injury potential was determined.

These values ranged from +6.8 to +27.0 mv. Therewas little or no change in potential as the probe elec-trode was moved through the aqueous humor.

The potential of the lens was obtained by firstmaking a small slit in the lens capsule, and then in-serting the probe micropipette. The mean potential atthe front of the lens was -22.6 mv, and at the back,-37.0 mv. The same five frogs used in the abovemeasurements were also used in these. In these meas-urements, the center of the cornea was not used as refer-ence, but a section of the cornea near the ventral borderof the skin and the cornea. The back of the lens wasreached from the front by driving the micropipettethrough the lens.

The potential of the vitreous side of the retina wasmeasured after removal of the lens and vitreous. Thesetwo parts were most easily removed together. Themean potential for this was +12.9 mv, ranging from+2.4 to +17.3 mv. The reference, again, was a sectionof the cornea near the ventral border of the skin andthe cornea.

Within the Retina

When using the opened, in-place eye, in which mostof the cornea and all of the lens and vitreous bodywere removed, a fluid soon filled the vitreous cavity.The source of this fluid was unknown, and the rate offilling varied from frog to frog. After about 10 min, athin membrane was found to be present in the fluid, afew millimeters above the retina. The probe micro-pipette could not penetrate the membrane, so that themembrane had to' be either cut or removed. One tech-nique was to tear a slit in it with fine forceps, and passthe micropipette through the tear. The probe was thendriven automatically through the retina at a relativelyconstant rate using the air-drive system. Oscillographicrecordings of the results on one frog, at different pointson the retina are shown in Fig. 3. The electrode was

W

U 0

E -4._

, -12-

o -16a.

-200 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Distance from center of cornea in mm

FIG. 2. The electrical potential of the surface of the cornea,plotted in two mutually perpendicular directions from the centerof the cornea. The center was used as the reference point, bothelectrically and spatially. The plotted values are the means offive frogs.

I v - I

_~ Icentrodorsal

centroc ada

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ERNEST DZENDOLET

FIG. 3. Records from different points of the retina of one frogshowing the potential variations within the retina as the probemicropipette was driven at a constant speed through it. A negativedrop, R, roughly consistent both in shape and in order of occur-rence in all the records is presumably Brindley's R membrane. Adownward deflection in the record is negative, and the vitreousside of the retina was used as the electrical reference.

moving from the vitreous side toward the receptor sideof the retina. The reference was on the vitreous side.Large fluctuations of about -60 mv are seen to occur.They do not occur at the same approximate depth ofthe retina except for the last one in each record, labeledR. This is presumably Brindley's R membrane. No

attempt was made to measure the depth at which thesedrops occurred, or to identify the structures associatedwith the drops.

DISCUSSION

The distribution of potential over the corneal surfacehas apparently not been previously reported. Themagnitude of the potential difference between thecenter of the cornea and the corneoscleral junction wasfound to be about -15 mv in the present experiment.Previous measurements by DeHaas, and by Westerlundgave a value of about -4 mv. This difference may bedue to the fact that excised eyes were used before,whereas in-place eyes were used here.

This large a potential on the cornea, if it can also beshown to exist in humans, may be sufficient in itself toexplain the source of the potential used in the electro-oculogram or EOG. Generally, the explanation of theEOG is in terms of either the nasal, or the temporalelectrode being nearer the back part of the eye thanthe other electrode when the eye is rotated from itsforward-looking position. This, then, gives rise to apotential difference. The cornea was apparently thoughtto be an equipotential surface, and the source of thepotential difference measured in the EOG was thoughtto arise in the standing potential across the retina. Thepresent finding would merely modify the above ex-planation so that movement of the center of the corneatoward one electrode would make that electrode lessnegative than the other electrode. This explanationsays nothing concerning the source of the corneal po-tential distribution. It may be entirely due to thepotential across the retina, or it may be both a functionof the retina and the lens potential as Brindley2

suggested.The fact that the potential of an injured portion of

the cornea had a positive potential with respect to thecenter of the uninjured cornea suggests that an insu-lating layer is present. Since only a little scraping with-out cutting through the entire cornea is necessary toproduce the positive potential, it seems likely that thestructure in the cornea to which this insulating rolecan be assigned is either Bowman's membrane, or theinterface between it and the squamous epithelial layerof the cornea. The only other layer present in thecornea is Desemet's membrane and its interfaces. Thisis at the back of the cornea, and probably does not con-tribute much if any insulation, since the potential inthe injured cornea is only about +5 mv greater thanthat in the aqueous humor. Part of the reason for thisdifference in the two potentials may be a shuntingaction of the aqueous humor between the probe in theaqueous and the reference on the cornea. Since there islittle or no change in potential with depth in theaqueous humor after penetration of the cornea, the

10 E. Marg, A.M.A. Arch. Ophthalmol, (Chicago), 45, 169(1951).

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role of this membrane seems to be that of insulatingthe inside of the eye from the outside.

A difficulty occurs in attempting to assign a con-tinuation of the insulating layer to a structure in thesclera. The same electrical sign change occurs on theinjured conjunctival portion of the frog eye, but thereis no structure there comparable to Bowman's mem-brane. Bowman's membrane ends right at the corneo-scleral junction. This suggests that the role of the in-sulator is taken over by the interface between the sub-conjunctival tissue and the conjunctiva, since thecorneal epithelium appears to be continuous with theconjunctiva. The insulator in the cornea is thus ap-parently the interface between Bowman's membraneand the corneal epithelium.

In the present experiment, the mean potential of thefront of the lens, with a small slit in it to allow entranceof the probe electrode, is - 22.6 mv. The reference forthis measurement was not the center of the cornea asit was for the other measurements, since a relativelylarge portion of the cornea was removed to allow work-ing space near the lens. This means that the referencewas at a more negative potential than the center of thecornea. From the data previously given on the poten-tial distribution over the cornea, the reference for thismeasurement was probably at a potential of about- 10 mv with respect to the center of the cornea. Thisvalue has to be added to the -22.6 mv. The final po-tential of the front part of the lens is thus about -33mv with respect to the center of the cornea. The sameis true for the back of the lens, which then has a cor-rected value of approximately -47 mv. The differenceof 14.4 mv between the front and the back of the lensis about the same as mentioned by Brindley.2 Theagreement on this value leads to the hypothesis thatthe disagreement in the absolute value of the lens po-tential, -74 mv for Brindley, and also for Andr6e, ascompared with the -30 mv here, may lie in the refer-ence points chosen in each case. The potential differenceof the inside of the lens with respect to the outside,since the outside of the lens is about +20 mv withrespect to the corneal center, is hence about -53 mv.In the same way, the back of the lens is about -67 mvwith respect to the outside front part of the lens.

The potential of the vitreous with reference to thecornea is measured after removal of a large part of thecornea, the entire lens, and the vitreous body. Underthese conditions, it is actually the vitreous side of theretina which is measured. Separation of the lens fromthe vitreous body is extremely difficult. The referencenecessarily has to be placed near the edge of the cornea.The potential under these conditions is a mean of

Reference

Fig. 4. Diagram showing the electrical potentials of the frog'seye with respect to the cornea. The relative magnitude is indi-cated by the size of the potential sign. Blank sections were notdirectly measured, but are assumed to be of the same sign andsimilar magnitude to the adjacent measured sections.

+ 12.9 mv. A correction must be made to find the valuewith respect to the center of the cornea. Since thecenter is about +15 mv more than the edge of thecornea where the reference is placed, the estimate ofthe vitreous side of the retina is approximately -2 mvwith respect to the center of the cornea.

The above data give the following picture of thestanding potentials of the eye. With reference to thecenter of the cornea, the corneal surface becomes pro-gressively negative toward its junction with the scleraor conjunctiva. Beneath the surface of the cornea, thepotential becomes positive, and remains so within theaqueous humor up to the lens. The interior of the lens,however, is negative. Approached from the front, withthe lens and body of the vitreous removed, the vitreousside of the retina is slightly negative with respect tothe cornea. These various potential differences aresummarized in Fig. 4. Within the retina, there are twoor three negative changes which are not reproduciblefrom frog to frog, and from one part of the retina toanother. One negative change, however, is. This ispresumably Brindley's R membrane.

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