Reports

Erhamburol* Changes the Color Coding of Carp RetinalGanglion Cells Reversibly

B. W. von Dijk and H. Spekreijse

The influence of ethambutol on retinal function was studiedby recording ganglion cell responses in isolated carp retinassuperfused with a Ringer solution containing different con-centrations of ethambutol (0 mg/liter, 10 mg/liter, 20 mg/liter, 30 mg/liter). The results indicate that ethambutol re-versibly affects color opponency, without changing the sen-sitivity of the underlying receptor processes. The amacrineand bipolar cells are the most likely candidates to be af-fected by ethambutol. Invest Ophthalmol Vis Sci 24:128-133, 1983

Visual function is affected in about 0.5 to 1.5% ofpatients treated with ethambutol, a commonly usedtuberculostatic drug.' These patients show nontypicalcolor vision defects. For example, in the Farnsworth(100 Hue) test errors are present along both the deu-teran and protan axes.2'3 The visual acuity of someof these patients decreases, although generally with-out visual field losses.4 After treatment is stopped,pretreatment vision is generally regained.5

The site where ethambutol affects the visual systemis not clear. Very high doses of 1300-1600 mg/kg/day (about 65X the therapeutic dose) cause demyelin-ization of the optic nerve in rhesus monkey (MacacaMulatto).6 Ophthalmoscopic examination has re-vealed toxic effects in the human retina.7

Zrenner and Kriiger3 have performed an extensivepsychophysical and electrophysiological study on twoaffected patients. Since both patients showed normalERGs and since the signals of all three cone typeswere present in the visual evoked potential (VEP),they concluded that these toxic effects do not manifestthemselves in a loss of a particular receptor mecha-nism. They attributed the visual defects caused byethambutol to a modification of color-opponentneural mechanisms.

If this modification of color-opponent mechanismsoccurs at the retinal level, then recording the ganglioncell activity changes caused by ethambutol will be amore direct approach than the ERG, which is deter-mined predominantly by receptors and Miiller cells.

In the present study the influence of ethambutol

* Myambutol is manufactured by Lederle, Division of AmericanCyanamid Co., Wayne, NJ.

on the ganglion cell responses was examined in iso-lated carp retina. Carp has three cone types, with ac-tion spectra peaking in the blue (around 450 nm), thegreen (around 530 nm), and the red (around 620 nm)regions of the visual spectrum.8 In teleost fish colorinteractions occur as distal as the horizontal cells,9"11and most ganglion cells show spatial as well as spectralopponency.1213 It will be demonstrated, that spectralopponency can be modified reversibly, when etham-butol doses in the range of 10-30 mg/liter are applied.

Materials and Methods. Normal carp (Cyprinuscarpio) of 600 to 800 g were enucleated in the dark.The retina was isolated and placed receptor side upin a preparation chamber filled with a standardRinger solution (NaCl: 120 mM; KC1: 3.5 mM; CaCl2:1.6 mM; NaHCO3: 22.6 mM; MgSO4: 1.6 mM; dex-trose: 10 mM; a mixture of 97.5% O2 and 2.5% CO2is pumped through this solution to maintain a pH of7.4). This solution is pumped through the chamberexchanging the medium with a time constant of .8minute. Temperature is kept at 17.5 C. After eachchange in the composition of the Ringer solutionfrom addition of ethambutol, the retina was kept inthe dark or at a constant level of illumination for atleast 20 min, before data collection was restarted.

A two-channel optical stimulator could projectspots and annuli of different diameters on the retinafrom underneath. Wavelengths in the range of 410-780 nm could be set in one beam with a monochro-mator (width at half amplitude 20 nm) and in theother beam with interference filters (width at halfamplitude 10 nm). The intensity of the 450W Xenonlamp could be attenuated with neutral density wedges.The narrow band light stimuli are modulated by shut-ters that interrupt the beams once per 2 sec with aduty cycle of 50%. To restrict rod intrusion and tomaintain a constant state of adaptation the entireretina is illuminated continuously by a 509 nmbackground light of low photopic intensity(5-1014Q-s-'-m-2).

Ganglion cell responses were recorded with glass-coated Ptlr electrodes that penetrated the retina fromabove. In this condition the stimulus does not createa shadow of the electrode on the retina. Spikes were

0146-0404/83/0100/128/51.10 Association for Research in Vision and Ophthalmology

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spot annulus

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Fig. 1. Response vs logintensity curves recordedfrom a carp ganglion cellwith red, green and bluesensitive cone inputs. Leftcolumn response to spotstimulation (diameter: 0.30mm). Right column re-sponse to annular stimula-tion (inner diameter: 2.20mm; outer diameter: 3.30mm). Duty cycle of thestimuli: 800 msec on, 800msec off. Ethambutol con-centration was changed insteps of 10 mg/liter. FiguresI and J show the results ob-tained upon return to thestandard Ringer solution.After each concentratonchange, the retina was keptat least 20 min in the darkto settle to the new medium.The vertical axis is the dif-ference between the averagenumber of spikes recordedin successive ON phases andOFF phases of the stimulus.The horizontal axis is thelog attenuation of the stim-ulus light intensity. Twentystimulus cycles were aver-aged. Zero log units atten-uation corresponds with 9 10l6Q-s-'-m-2.

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shaped and played over a loudspeaker. The spikeswere counted during the ON phase and OFF phaseof the stimulus, and these numbers were recorded ona two-channel chart recorder. Criterion responsespectra were obtained by setting the intensity atten-uation in such a way that a just audible change in thespike discharge could be heard. Stable recordingsfrom isolated ganglion cell were possible for up to 11hours.

Results. Changes in the response properties of 14spectral opponent ganglion cells were recorded.Ethambutol had similar effects on all these units.Figures 1A and B show the number of spikes vs logintensity curves elicited by spot (left column) andannular (right column) stimulation of different wave-lengths. In this figure the average difference betweenthe number of spikes recorded during ON and OFTphases of the stimulus is plotted.

130 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1983 Vol. 24

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Fig. 2. Criterion responsespectra for a center red ON,green OFF (left column);surround red OFF green ON(right column) ganglion cell,when different concentra-tions of ethambutol areadded to the Ringer solu-tion. The center stimuluswas a spot with diameter0.15 mm; the surroundstimulus was an annuluswith inner diameter 1.80mm and outer diameter 3.30mm. Duty cycle of the stim-ulus was: 1 sec on, 1 sec off.The log attenuation thatyielded the criterion re-sponse is plotted against thewavelength of the stimulus.Thresholds were reproduc-able within 0.15 log units.

wavelength nm

Since this unit received input from all three conetypes (with different signs) the response vs log inten-sity curves have a rather complicated shape. One ofthe consequences of this is that a constant responsecriterion like a just noticeable change in the spikedischarge does not result in unambiguous spectralcharacteristics. For this reason the effect of etham-butol is shown in the form of response vs log intensitycurves. Data were recorded at least 20 min after thereplacement of the initial solution by another onewith a new ethambutol concentration. Inspection ofFigure 1 shows that application of ethambutol affectsthe responses to spot stimulation in the same way asto annular stimulation.

For increasing ethambutol concentrations the colorantagonism becomes less pronounced. For example,the responses obtained in the 10 mg/liter mediumstill have opposite signs in the short (ON response)and long (OFF response) wavelength regions of thevisible spectrum, whereas only OFF responses areobtained in the 30 mg/liter medium. This change incolor antagonism cannot be attributed to variationin absolute sensitivity of underlying receptor mech-anisms since for each wavelength the lowest intensityyielding a response is not affected.

After 11 hours of recording from the same unit thestandard Ringer solution was replaced, and the re-sponse vs log intensity curves depicted in Figures II

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Fig. 3. Spike density pro-files of the maintained ac-tivity from the unit of Fig-ure 2 upon adding 10 mg/liter ethambutol. The num-ber of spikes counted duringsuccessive (1 sec) time in-tervals is plotted vs time.The dashed line marks thestart of the concentrationchange, in the lower profilefrom 0 mg/liter to 10 mg/liter, in the upper profilefrom 10 mg/liter to 20 mg/liter. Replacement of theRinger solution occurs witha time constant of 0.8 min.

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and J were recorded. Comparison of these figures withFigures 1A and B shows that the unit had recoveredfully to the initial state.

The spontaneous activity from the same unit wasalso measured. No changes with concentration ofethambutol could be established.

Figure 2 shows criterion response spectra recordedfrom a ganglion cell with the most frequently en-countered spectral and spatial coding in carp retina:antagonistic coding for red and green in both centerand surround. The coding of this particular unit was:

center red on and green off (left column), surroundred off and green on (right column). As was the casefor the unit presented in Figure 1, the effect of etham-butol expresses itself on the interaction between thecone mechanisms and not on their absolute sensitiv-ities. This is most evident in the surround responsesof Figure 2.

Normally the presence of a strong red responseinhibits the other color mechanisms' contribution tothe spike discharge. For example, the green processin the surround (Fig. 2B) can only be found for stim-

132 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / January 1983 Vol. 24

ulus wavelengths up to 525 nm with the peak shiftedtowards shorter wavelengths. In the presence ofethambutol, however, this inhibition weakens: at 10mg/liter ON responses can be recorded up to 575 nm(Fig. 2D); at 20 mg/liter up to 625 nm (Fig. 2F). Sincethis change is achieved without significant changesin the sensitivities of the two receptor mechanisms,the functional site of the effect must be an active coloropponent mechanism.

This unit also recovered completely upon returnto the standard Ringer solution.

Figure 3 shows the spontaneous activity of the gan-glion cell of Figure 2 upon adding 10 mg/liter etham-butol. Directly following the ethambutol application(taking in account the 0.8 min time constant) dra-matic changes occur. At first the spontaneous activityis severely reduced. Next the spike frequency in-creases to about twice the initial rate. Finally the firingpattern slowly returns to the original rate, and afterabout 5 min the spontaneous activity is back to theoriginal mean level with, however, somewhat largervariations.

Discussion. The most prominent effect of etham-butol on ganglion cell function in the carp retina isthat it affects the color opponent characteristics of theganglion cell responses. These changes are not sec-ondary to a change of the spatial organization, norto a change in the absolute sensitivities of the un-derlying receptor mechanisms, nor to a change in themaintained discharge of the ganglion cells. The effectsof ethambutol on the color opponent interactions arereversible.

Several authors214"17 have suggested that etham-butol causes demyelinization of the optic nerve. How-ever, this is quite unlikely since affected patients oftenhave abnormal color vision without visual field oracuity losses that are generally associated with de-myelinization.

The results of this study clearly show that etham-butol affects visual function in the retina. Dick et al18have shown that in the mudpuppy retina ethanol en-hances ON responses of bipolars and the b-wave ofthe ERG, while suppressing OFF responses. Sinceethambutol is like ethanol, an alcohol, their actionon the retina might be related. However, in our ex-periments ethambutol did not show a selective actionon center or surround neither on ON or OFF mech-anisms. The changes monitored in ganglion cell re-sponses indicate that the most prominent effect ofethambutol is that inhibitive interactions betweenopponent color mechanisms (active interactions) areweakened. When ethambutol is applied it appears asif the receptor processes feed independently into theganglion cells, as such responses of all contributingmechanisms can be recorded in the yellow-green re-

gion of the spectrum. A similar change can also ac-count for the observed color deficiencies in affectedpatients. On the basis of our results it is possible tospeculate about the retinal cell types that are affectedby ETHAMBUTOL.

Since Zrenner and Kriiger3 did not find abnormalERGs and could measure signals from all three conetypes in the VEP it is unlikely that ETHAMBUTOLaffects the receptors. The present study confirms thissince no changes were found in the absolute sensitiv-ities of the cone mechanisms that underly the gan-glion cell responses.

Because no long-term changes in the ganglion cells'maintained activity was observed, it is unlikely thatthe site of action of ethambutol is proximal to theganglion cells' dendritic synapses. Toyoda and Ku-jiraoka19 have shown that in carp retina polarizationof any of the horizontal cell types influences the rel-ative sensitivity of the bipolar surround process andhence the center/surround balance. So it is likely thatalso at the ganglion cell level this balance will bechanged by polarization of the horizontal cells. How-ever, no changes were observed in the center/sur-round activity balance when ethambutol was applied.This indicates that ethambutol does not affect thehorizontal cells. This is supported by Zrenner andKriiger's observation that affected patients have nor-mal ERGs.

Our results, therefore, indicate that the most likelysite of ethambutol action is in the inner plexiformlayer and that the most likely candidates to be affectedby ethambutol are the amacrine and bipolar cells.Key words: acquired color vision defects, ganglion cell re-sponses, ethambutol, color coding, drug effects

From the Laboratory of Medical Physics, University of Am-sterdam, Amsterdam, The Netherlands, and the NetherlandsOphthalmic Research Institute, Wilhelmina Gasthuis, Amsterdam,The Netherlands. Supported partly by the Netherlands Organiza-tion for the Advancement of Pure Research (ZWO) through theFoundation of Biophysics. Submitted for publication December31, 1981. Reprint requests: Prof. H. Spekreijse, The NetherlandsOphthalmic Research Institute, PO Box 6411, 1005 Ek Amster-dam, The Netherlands.

References1. Pau H and Wahl M: Myambutol-Schadigung des Auges. Ber

Dtsch Ophthalmol Ges 72:176, 1972.2. Trusciewicz D: Farnsworth 100-Hue test in diagnosis of

ethambutol-induced damage to optic nerve. Ophthalmologica171:425, 1975.

3. Zrenner E and Kriiger CJ: Ethambutol mainly affects the func-tion of red/green opponent neurons. Doc Ophthalmol ProcSer 27:13, 1981.

4. Carr RE and Henkind P: Ocular manifestations of ethambutol.Toxic amblyopia after administration of an experimental an-tituberculous drug. Arch Ophthalmol 67:566, 1962.

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5. Pahlitzsch H and Tiburtius H: Augenuntersuchungen bei Be-handlung mit dem neuen Tuberkulostatikum Ethambutol-dihydrochlorid Lederle (Myambutol). Klin Monatsbl Augen-heilkd 154:228, 1969.

6. Schmidt IG: Central Nervous System effects of ethambutol inmonkeys. Ann NY Acad Sci 135:759, 1966.

7. Gross V, Eule H, and Hager G: Auswertung einer Toxizitats-studie bei intermittierender Ethambutol-Medikation. KJin.Monatsbl Augenheilkd 163:17, 1973.

8. Tomita T, Kaneko A, Murakami M, and Pautler EL: Spectralresponse curves of single cones in the carp. Vision Res 7:519,1967.

9. Svaetichin G: Spectral response curves from single cones. ActaPhysiol Scand Suppl 134:17, 1956.

10. Spekreijse H and Norton AL: The dynamic characteristics ofcolor-coded S-potentials. J Gen Physiol 56:1, 1970.

11. Stell WK, Lightfoot DO, Wheeler TG, and Leeper MF: Gold-fish retina: Functional polarization of cone-horizontal cell den-drites and synapses. Science 190:989, 1975.

12. Wagner HG, MacNicol EF Jr, and Wolbarsht ML: The re-

sponse properties of single ganglion cells in the goldfish retina.J Gen Physiol 43:45, 1960.

13. Spekreijse H, Mooy JEM, and Van den Berg TJTP: Photo-pigments and carp ganglion cell action spectra. Vision Res21:1601, 1981.

14. Weder W: Myambutolschaden des Sehnerves. Ber DtschOphthalmol Ges 72:172, 1972.

15. Stark N: Toxische Sehnervenschadigung durch Myambutol.Med Klin 67:913, 1972.

16. Orou F, Sideroff G, and Schabel F: Frequenzuntersuchungenvon Optikuserkrankungen im Rahmen der Myambutol-Be-handlung. KJin Monatsbl Augenheilkd 161:601, 1972.

17. Thaler A, Heilig P, Heiss WD, and Lessel MR: Toxische Scha-digung des Nervus opticus durch Ethambutol. Klin MonatsblAugenheilkd 165:660, 1974.

18. Dick E, Miller RF, and Dacheux RF: Neuronal origin of B-and D-waves in the I-type ERG. ARVO Abstracts. InvestOphthalmol Vis Sci 19(Suppl):34, 1979.

19. Toyoda J and Kujiraoka T: Analyses of bipolar cell responseselicited by polarization at horizontal cells. J Gen Physiol79:131, 1982.

Asteroid BodiesAn Ultrastructurol StudyHedva Miller, Benjamin Miller, Hanna Robinowitz, Shlomo Zonis, and Izhok Nir

An ultrastructural study of asteroid bodies in a vitreousaspirate from a patient suffering visual loss as a result ofasteroid hyalosis is presented. Under appropriate conditionsof fixation and high resolution transmission electron mi-croscopy, a lamellar arrangement with a periodicity of 4.6nm was observed. This lamellar arrangement is typical ofliquid crystalline phases of lipids in water. X-ray micro-analysis confirmed the presence of calcium and phosphorusin the asteroid bodies. We propose that the asteroid bodiesare not true crystals but rather liquid crystals of phospho-lipids in the vitreous humor. Invest Ophthalmol Vis Sci24:133-136, 1983

Asteroid bodies are seen as brilliant particles float-ing in an apparently normal vitreous body. They usu-ally appear unilaterally in the 60-80 age bracket.Their presence does not cause any impairment invision1 except in very rare instances.2

Even though the asteroid bodies have been quiteextensively investigated,1'3"5 their composition is stillnot fully elucidated, and there are various hypothesesconcerning their origin, organization and mode offormation.1-3'4

The asteroid bodies are known to be composed oflipids, calcium, and phosphorus.l>3