Vision Ref. Vol. 34, No. 6, pp. 829-837, 1994 Copyright 0 1994 Ekvicr Science Ltd

Printed in Great Britain. All rights reserved 0042-6989{94 WJO + 0.00

The Effects of Acetazolamide in Albino Rabbits, Pigmented Rabbits, and Humans J. VERNON ODOM,* T. MICHAEL NORK,* BARBARA M. SCHROEDER,* SHERI A. CAVENDER,* STEVEN VAN SLYCKEN,? MONIQUE LEYSt

Received I2 hnuury 1993; in revised form 30 August 1993

In three separate experiments albino rabbits, pigmented rabbits, and hmnans were tested foamy administration of acetazolamide and witbout acetazolamide. In all three experiments, we recorded electroretiwgrams (ERGS) a&r dark adapted and light adapted conditions and measured tbe b-wave amplihdes. Dark adapted ERG b-wave amplitudes were increased following admiaistration of Adelaide as compared to control coaditioas, in albino rabbits, ante rabbits aad humans. Ligbt adapted b-wave amplitudes sbowed 80 statistically signifhnt changes as a function of acetazolamide admhistration sltbougb in all three experiments tbere was a trend toward ligbt adapted Q-wave amplitude rednction following administration of acetazolamide. In tbe buman experiments, ERG a-wave amplitudes were also measured. Light adapted e-wave ~~~ were redaced following admiahtration of acetazolamide. In tbe human experiments, severaI behavioral tests were performed, includiag L’Antbony de&orated D-15, Farnsworth-Mansell 100 hue, Cogan-Gunkel cbromatograpb, Nagel aaomaloscope, Goldmanro-Weekers dark adaptometry. Tbere were no consistent cbaages in tbe bmnan dark lactic guilds or color damnation, blob several -es approached significance.

Acetazolamide Rabbits Human Electroretinogram Color discrimination Dark adaptometry

INTRODUCTION

Carbonic anhydrase (CA) is found in many ocular cells, notably those of the ciliary epithelia, the retinal pigment epithelium, most cones and Miiller’s cells (Wistrand, Schenholm & Lonnerholm, 1986; Nork, McCormick, Chas & Odom, 1990; Musser & Rosen, 1973). Carbonic anhydrase inhibitors (CAI), such as acetazolamide (Diamox), play an important role in the treatment of several ocular diseases. Inhibition of CA activity reduces intraocular pressure (Becker, 1954; Maren, 1984) and macular edema (Fishman, Gilbert, Fiscella, Kimura & Jampol, 1989; Cox, Hay & Bird, 1988).

The electroretinogram (ERG) b-wave is primarily attributed to the activity of Miiller’s cells (Miller & Dowling, 1970; Wen & Oakley, 1988). The presence of carbonic anhydrase in Miiller’s cells suggests that inhibition of CA by a CA1 such as acetazolamide should alter the ERG b-wave. Two previous investigators have examined the effect of CA inhibitors, one finding an increase (Stanescu & Michiels, 1975) and the other a decrease (Broeders, Palmer & Dawson, 1988) of b-wave amplitude with administration of a CAI.

We observed previously that the effects of acetazo- lamide on ERG b-waves were dependent on the adap-

*Department of Ophthalmology, West Virginia University Health Sciences Center, PO Box 9193, Morgantown, WV 26506, U.S.A.

tDepartment of Ophthalmology, University Hospital, 185 De Pinte- laan, 9000 Ghent, Belgium.

tation level; b-wave amplitudes were increased in the dark but not in the light (Odom, Weinstein, Nork, Zavage, Brown & Hobson, 1989). A trivial explanation of the difference between the b-wave increase in the dark but absence of b-wave increase in the light might be that albino rabbits were tested in our exper- iments while pigmented rabbits were tested by Broeders et al. (1988). In the present set of experiments we extended the previous results to pigmented rabbits and humans to make more direct comparisons with previous results.

We have previously hypothesized that CA1 may have a direct effect on retinal cells. Because carbonic anhy- drase is absent in rods but is present in most cones and in the Miiller’s cells (Nork et al., 1990), we suggested (Odom ef al., 1989) that differences in the effects of CA1 activity on ERG b-wave amplitudes in the light and dark might be attributable to two different effects, one an alteration of Miiller’s cell activity and the other an alteration of cone function (and possibly cone-rod inter- actions) due to inhibition of CA activity in Miiller’s cells and cones.

If photoreceptor activity is altered by acetazolamide, there should be some evidence in measures of cone activity, such as, light adapted a-waves and color dis- crimination or measures of rod activity such as dark adapted a-waves and dark adapted threshoIds. There- fore, we measured dark adaptation, light adapted and dark adapted ERGS and color vision in humans.

829

830 J. VERNON ODOM er a/

METHODS

Albino and pigmented rabbits

Ten albino rabbits and four pigmented rabbits weigh- ing approx. 3 kg were subjects in the experiments. All procedures were consistent with the guidelines for the care and handling of animals of the Association for Research in Vision and Ophthalmology and were approved by the West Virginia University Animal Care and Use Committee. The rabbits were restrained in a cloth wrap. Topical cyclopentolate 1% was used to produce mydriasis and cycloplegia. The corneas were anesthetized with topical proparacaine to reduce any discomfort the animals might experience as a result of ERG electrode placement. Neither general anesthesia nor sedation was used in order to avoid possible drug interactions that might contaminate the results. Acetazo- lamide was injected in the vein of the right pinna. Each albino rabbit was tested under three conditions during three separate sessions on three separate days; in Session 1 acetazolamide was injected with a dosage of 20 mg/kg, in Session 2 an equal volume of normal saline was injected, in Session 3 no injection was given (i.e. normal conditions). Although three conditions were employed, the critical comparison is between injection of acetazo- lamide and injection of saline and not between injection of acetazolamide and no injection. The sequence of sessions was randomized.

Pigmented rabbits were tested under the two critical conditions only, i.e. injection of acetazolamide and injection of saline, in two separate sessions on two

separate days. The sequence of sessions was randomized across rabbits.

Recording procedures. ERG procedures similar to Odom et al. (1989) were employed. The active electrode was a cornea1 contact lens electrode. It was referenced to an electrode on the nasion, and a clip electrode attached to the left pinna served as ground. ERGS were amplified 10,000 times with a half amplitude bandwidth of 0.1-200 Hz using a Lifetech signal averager. Oscillatory potentials were recorded from the pigmented rabbits by averaging responses to four stimuli using filter settings of 100-300 Hz and an amplification of 50,000 times. The time from injection of acetazolamide or saline to the beginning of recording was typically 20 min. This time did not vary systematically between conditions.

Stimulation procedures. Three flash intensities were presented: 132, 1400, and 3150 lx (0.05, 0.5, and 1 .l cd/m*/sec) under dark adapted and light adapted conditions. Light adaptation was obtained by placing the rabbit for 5 min in a room with an ambient illumina- tion of 750 lx. Dark adaptation was obtained by adapt- ing to a luminance below the threshold of our light meter (i.e. < 1 lx) for 15 min. The order of stimulus presen- tation was as follows: light adapted eye one, dark adapted eye one, dark adapted eye two, light adapted eye two. Right and left eyes were randomly assigned as eye one or eye two. Stimulus intensity was always increased from lower to higher within an adaptation level. Each ERG recording was averaged as the stimulus was pre-

sented four times. Two averages were acquired for each stimulus condition. Flashes were presented once every 15 set under dark adapted conditions and once per set under light adapted conditions. Oscillatory potentials (OPs) were elicited from the pigmented rabbits by the highest intensity flash under both dark adapted and light adapted conditions. The time from injection to stimulus presentation did not vary systematically be- tween experimental conditions and therefore cannot account for any systematic differences observed across experimental conditions.

Humans

Six human subjects gave their informed consent and participated in the experiments. Each subject was tested with and without administration of acetazolamide. The sequence of administration was randomized. Three of the subjects were tested first after ingestion of acetazo- lamide and then, several days later, they were retested without acetazolamide. The order was reversed for the remaining three subjects. Three subjects were male and three female. Their ages ranged from 26 to 41 yr. The dose of acetazolamide was 500 mg (8 mg/kg on average) taken orally, 2 hr prior to one session. The 2 hr wait was decided upon because acetazolamide reaches its peak plasma levels 2 hr after oral administration and has a half-life of 46 hr (Maren, 1984). Consequently, testing would begin when acetazolamide was likely to have its maximal effects and end before this effect had diminished significantly.

Tests included the L’Anthony desaturated D-15, Farnsworth-Munsell 100 hue, CoganGunkel chro- matograph, Nagel anomaloscope, Goldmann-Weekers dark adaptometry, and ERG. The ERG and anomalo- scope testing were performed monocularly using the right eye. All other testing was binocular. Color vision testing was performed first. Then patients were dark adapted for 15 min, bleached for 5 min with a light of 2000 apostilb, and light thresholds were measured during the next 15 min of dark adaptation. After dark adaptometry, pupils were dilated and a cornea1 ERG-jet electrode was placed on the right eye, referenced to an electrode at the ipsilateral outer canthus and grounded to the forehead. Full-field ERGS were elicited by flash intensities of 438, 1400, and 55001x (0.12, 0.5, and 1.9 cd/m*/sec) under dark and light adapted conditions. Single flash, dark adapted ERGS were recorded immedi- ately following Goldmann-Weekers dark adaptometry, about 20min after beginning dark adaptation, with a minimum of 15 set between subsequent flashes. Light adaptation was accomplished by increasing the back- ground intensity of the cupola to 6 lx and waiting 3 min before recording averaged ERGS elicited by 16 flashes presented once per sec.

RESULTS

Albino rabbits

Figure 1 presents the ERGS of an albino rabbit elicited by the brightest flash during dark and light adaptation

EFFECTS OF ACETAZOLAMIDE 831

Normal Saline Acetazolamide

I

FIGURE I. Albino rabbit ERGS. ERGS recorded from a single eye of one albino rabbit are presented. The solid and dashed waveforms represent two replications of the same condition. The upper set of waveforms represent light adapted responses and the lower set dark adapted responses. The responses elicited without any injection (normal) are on the left, those elicited following injection of physiological saline are in the center and those elicited following injection of acetazolamide am

on the right.

for the three conditions of drug administration. Figures 2 and 3 show the group mean ERG b-wave amplitudes and standard errors elicited by all flash intensities and drug administration conditions for light and dark adaptation respectively.

Analyses of variance (ANOVAs) of repeated measures for the 10 rabbits were calculated to compare the effects of drug condition and flash intensity under light and dark adaptation. Under light adapted conditions, only the effect of flash intensity was statistically significant (F = 444.86; d.f. = 2,18; P < 0.001). Light adapted ERG

A -.- Normal 0 -Saline x --- Acetazolamide

I I I

.05 .5 1.1

FLASH LUMINANCE (cd/m2 isec)

FIGURE 2. Albino rabbit dark adapted ERG b-waves. Dark adapted b-wave amplitudes are presented for each flash luminance and con- dition. Each point and its error bars represent the mean and standard error of the measurements made on 10 rabbits. For each rabbit four measurements were averaged for each condition (two replications for each of two eyes). Open triangles and dot-dashed lines represent the normal condition in which there was no injection; open circles and solid tines represent the saline condition in which physiological saline was injected i.v. and the xs and dashed lines represent the acetazo-

b-wave amplitude increased with increasing intensity, each higher level of intensity eliciting a greater amplitude b-wave (P < 0.05). Under dark adapted conditions, there was a statistically sign&ant effect of the drug condition (F = 10.8; d.f. = 2,18; P < 0.001) and of flash intensity (F = 283.03; d.f. = 2,18; P < 0.001) but not of their interaction (F = 2.34; d.f. = 4,36; P = 0.07). Stat- istically significant ANOVAs were followed by Scheffe post hoc tests of effects. Under dark adapted conditions, ERG b-wave amplitudes elicited following injection of acetazolamide were larger than those elicited following

140

S 2 120

x

5 100

% a 80

Y

480 I+

40 X --- Acetazolamide

1 I I .05 5 1.1

FLASH LUMINANCE (cd/m2/sec)

FIGURE 3. Albino rabbit light adapted ERG b-waves. Light adapted b-wave amplitudes are presented for each flash luminance and con- dition. Each point and its error bar represent the mean and standard error of the measurements made on 10 rabbits. For each rabbit four measurements were averaged for each condition (two replications for each of two eyes). Open triangles and dot-dashed lines represent the normal condition in which there was no injection, open circles and solid lines represent the saline condition in which physiological saline was injected i.v.; and the xs and dashed lines represent the acetazo-

lamide condition in which acetazolamide was injected iv. lamide condition in which acetazolamide was injected iv.

832 J. VERNON ODOM et al.

Saline Acetazolamide

Light adapted

Dark adapted

.,

_--

+

‘\ ‘,I- -

,/--, ,’ ‘.

-. ‘.

-. P I 100 pv

FIGURE 4. Pigmented rabbit ERGS. ERGS recorded from a single eye of one pigmented rabbit are presented. The sohd and dashed waveforms represent two replications of the same condition. The upper set of waveforms represent light adapted responses and the lower set dark adapted responses. The responses elicited following iv. injection of physiological saline are

on the left and those elicited following i.v. injection of acetazolamide are on the right.

an injection of saline for all flash intensities (P < 0.05). On average, the dark adapted b-wave amplitude recorded following injection of acetazolamide was 17.35% greater than that recorded following injection of saline. There were no statistically significant effects of acetazolamide on light adapted ERG b-wave ampli- tudes, although there was a nonsigni~~~t trend for acetazolamide to reduce the ERG b-wave amplitudes (see Broeders et al., 1988).

Pigmented rabbits

Figure 4 illustrates the ERGS of a pigmented rabbit elicited by the brightest flash during dark and light adaptation for the three conditions of drug adminis- tration. Figures 5 and 6 present the group mean ERG

1 200 ;

t

- Saline ---* Acetazolamide /

/ s /- s 180 -

FLAW L~NANCE (cd/m2 Isec)

FIGURE 5. Pigmented rabbit dark adepted ERG b-waves. Dark adapted b-wave amplitudes are presented for eaeh flash luminance and condition. Each point and its error bars represent the mean and standard error of the measurements recorded from four pigmented rabbits. For each rabbit four measurements were averaged for each condition (two replications for each of two eyes). Open circles and solid lines represent the condition in which physiological saline was injected i.v., and the xs and dashed tines represent the condition in which

acetazolamide was injected i.v.

b-wave amplitudes and standard errors elicited by all flash intensities and drug administration conditions for light and dark adaptation respectively.

For the four pigmented rabbits, ANOVAs of repeated measures were calculated to compare the effects of drug condition and response measure under light and dark adap~tion. Under light adapted conditions, only the effect of response measure was statistically significant (P < 0.001). Light adapted ERG b-wave amplitude in- creased with increasing intensity, each higher level of intensity eliciting a greater amptitude b-wave (P < 0.05). Under dark adapted conditions, there was a statistically significant effect of the drug condition (F = 17.988; d.f. = 1,7; P < 0.004) and of response measure (F = 41.05; d.f. = 3,21; P < 0.001) but not of their inter- action. Statistically significant ANOVAs were followed

90 0 - Saline X ---. Acetazolamide

T

10 I I t

.05 5 1.1

FLASH LUMINANCE (cd/& isec)

FIGURE 6. Pigmented rabbit light adapted ERG b-waves. Light adapted b-wave amplitudes are presented for each flask iuminance and condition. Rach point and its error bar represent the mean and standard error of the measurements recorded from four pigmented rabbits. For each rabbit four measurements were averaged for each condition (two replications for each of two eyes). Open circles and solid lines represent the condition in which physiological saline was injected i.v., and the xs and dashed lines represent the condition in which

acetazolamide was injected i.v.

EFFECTS OF ACETAZOLAMIDE 833

Light adapted Dark adapted

Norma1

Acetaaolamide

FIGURE 7. Human ERGs. ERG waveforms are presented. The solid and dashed waveforms represent two different responses recorded from two different human subjects. The upper set of waveforms represent light adapted responses and the lower set dark adapted responses. The normal responses are on the left and those recorded following ingestion of acetazolamide are on

the right.

by t-tests for related samples. Under dark adapted ~nditions, ERG &-wave amp~tud~ elicited following injection of acetazolamide were larger than those elicited following injection of saline for all flash intensities (P < 0.05). On average, the dark adapted b-wave ampli- tude recorded following an injection of acetazolamide was 12% greater than that recorded following an injec- tion of saline. There was no statistically significant effect of acetazolamide on OP amplitudes. There were no statistically significant effects of acetazolamide on light adapted ERG b-wave amplitudes, although there was a nonsignificant trend for acetazolamide to reduce the ERG b-wave amp~tud~ (see Broeders et al., 1988).

Humans

Figure 7 presents a human subject’s ERG that was elicited by the brightest flash during dark and light adaptation for the three conditions of drug adminis- tration. Figures 8 and 9 show the group mean ERG a- and b-wave amplitudes and standard errors elicited by all flash intensities and drug administration con- ditions for light and dark adaptation respectively. t- Tests for related samples were used to compare b-wave amplitude in the various stimulus conditions, b-Wave amplitudes elicited by the highest intensity flash were greater following administration of acetazolamide than in the control condition (P < 0.03). Dark-adapted b- waves following administration of acetazolamide were 8% larger than in the control condition. There were no statistically significant effects of acetazolamide on light adapted ERG b-wave amp~tud~, although there was a nonsignificant trend for acetazolamide to reduce the ERG b-wave amplitudes (see Breeders et al., 1988).

An ANOVA of repeated measures with flash intensity and drug (accolade vs no a~~o~de) as factors was employed to examine the effects of acetazolamide on ERG u-wave amplitudes. In the light adapted

0 - Normal x - katazolamide

I I I

a12 0.5 1.Q FLASH LUMINANCE

~~2~)

FIGURE 8. Human dark adapted ERG b-waves. Dark adapted b-wave amplitudes are presented for each tlash I- and con- dition. Each point and its error bars represent the mean and standard error of the measurements recorded from six human subjects. Gmcr- ally only one response was recordexl for each condition from each subject. The xs and dashed lines represent the condition in which acetazolamide was ingest& prior to recording the EROS and the open circles and solid lines represent the condition in which EROS were

recorded normally without administration of acctazolamide.

834 J. VERNON ODOM et al.

220

T 2

0” 5 120

4

Y a 3

70

&I

20

---- Acetazolamide

t I I

0.12 0.5 1.9

FLASH LUMINANCE (cdlmalsec)

FIGURE 9. Human light adapted ERG b-waves. Light adapted b-wave amplitudes are presented for each flash luminance and con- dition. Each point and its error bar represent the mean and standard error of the measurements recorded from six human subjects. Gener- ally only one response was recorded for each condition from each subject. The xs and dashed lines represent the condition in which acetazolamide was ingested prior to recording the ERGs and the open circles and solid lines represent the condition in which ERGs were

recorded normally without administration of acetazolamide.

conditions, there was a statistically significant inter- action effect of flash intensity and acetazolamide admin- istration (F = 4.293; d.f. = 2, 10; P < OM), indicating

250 r o - Normal

200 -

150 -

190 -

50 -

01 0.12 0.5 1.9

FLASH LUMINANCE (cd/mVsec)

FIGURE 10. Human dark adapted ERG a-waves. Dark adapted a-wave amplitudes are presented for each flash luminance and con- dition. Each point and its error bars represent the mean and standard error of the measurements recorded from six human subjects. Gener- ally only one response was recorded for each condition from each subject. The xs and dashed lines represent the condition in which acetazolamide was ingested prior to recording the ERGS and the open circles and solid lines represent the condition in which ERGS were

recorded normally without administration of acetazolamide.

so

70

60

50

40

30

0 - Normal X--aAcetazolamide

FLASH LUMINANCE (cdhn2 kec)

FIGURE 11. Human light adapted ERG a-waves. Light adapted a-wave amplitudes are presented for each flash huninance and con- dition. Each ‘Roint and its error bar represent the mean and standard error of the measurements recorded from six human subjects. Gener- ally only one response. was recorded for each condition from each subject. The xs and dashed lines represent the condition in which acetazolamide was ingested prior to recording the ERGs and the open circles and solid lines represent the condition in which ERGS were

recorded normally without administration of acetazolamide.

that light adapted u-wave amplitude was reduced follow- ing administration of acetazolamide and that the re- duction was greater as gash intensity increased. The light adapted a-wave amplitude was reduced 14% at the highest Aash intensity. There were no statistically signifi- cant effect of acetazolamide on dark adapted ERG a-wave amplitudes, although there was a nonsignificant trend in the same direction as for the light adapted a -waves.

t-Tests of related samples were used to assess the effects of acetazolamide on dark adaptometry and on the various tests of color vision. The rod threshold after 15 min in the dark was 0.24 log apostilbs lower following administration of acetazolamide than without acetazo- lamide, but this was not statistically significant (P = 0.06). Differences in the time of the rod/cone break and cone threshold did not reach significance (P > 0.10). The color vision tests did not reveal any classic color vision losses. There was an insignificant increase in the total number of errors on the lOO-hue test following acetazolamide (P = 0.08).

DISCUSSION

The present study confirms and extends previous reports that administration of acetazolamide increases the amplitude of the dark adapted ERG b-wave but not that of the light adapted b-wave (Odom et ai., 1989). These results were confirmed in a larger sample of ahho rabbits, and extended to pigmented rabbits and humans.

EFFECTS OF ACET~OLAMI~E 835

Therefore, the results are not a consequence of albinism, but a more general characteristic of the role of CA and CA1 in retinal function.

Dark adapted ERG &-wave amplitudes were increased by acetazolamide relative to control conditions by 1419% in the albino rabbits, ll-13% in pigmented rabbits and 58% in humans; the percentage of increase tended to increase as flash intensity increased. The differences between the rabbits and humans may be a function of the relatively smaller dose given humans as compared to rabbits (8 vs 20mg/kg). Under light adapted conditions no statistically si~ifi~nt change in b-wave amplitude was observed in any of the three groups tested; however, in each experiment there was a nonsi~ficant trend for reduced b-wave amplitudes in the light adapted conditions.

Previous experiments have examined the effects of CAIs on ERG b-waves with conflicting results. Human b-wave amplitudes increased a few minutes after an intravenous injection of acetazolamide (Stanescus & Michiels, 1975), but ERG b-waves recorded from pig- mented rabbits decreased following intraocular injec- tions of methazolamide (Broeders et al., 1988). Our previous (Odom et al., 1989) investigation using albino rabbits observed CA1 induced ERG b-wave increases only in the dark. The present study extended those results to pigmented rabbits and humans. Therefore, it appears that the discrepancies in previous studies are primarily the consequence of varying levels of dark adaptation and stimulus intensity used in the studies and are probably not related to species differences. However, differences in the CA1 employed and its concentration in the retina also cannot be excluded as factors con- tributing to the differences in results across experiments.

We have previously hypothesized that the above effects can be accounted for by the hy~thesi~ng a direct effect of CA1 on the cells in the retina which contain CA (Odom et al., 1989). For this to be a plausible hypoth- esis, acetazolamide must cross the blood-brain barrier into the retina and its concentration at the times corre- sponding to our experiments must be large enough to inhibit a sui-hcient amount of CA in order to create the observed effects. Below we shall briefly summarize what is known about CA and CAI activity in the retina and consider several alternative hypotheses,

CA is an enzyme which catalyzes the combination of carbon dioxide and water to form carbonic acid and the reversal of this process. Interestingly, the process occurs even in the absence of CA. Therefore, many tissues where CA is observed, red blood cells and the renal cortex, for example, have far more CA than is required to catalyze the reaction. In the tissues where it has been observed, 99% of the enzyme must be inhibited before physiological effects are observed (Maren, 1984). These processes have never been directly studied in the retina and given the high metabolic rate of the retina it is unclear that the same excess of enzyme is present.

Acetazolamide does not appear to enter the retina or have any direct physiological effects in the retina in the first minutes after i.v. injection in cats (Yamamoto &

Steinberg, 1992b). However, 2 hr following injection acetazolamide is present in the retina and in larger concentration than in the retinal pigment epithelium (Goren, Newell & O’Toole, 1961). The time-course of this accumulation and its functional consequences are unknown. Consequently, long term direct effects of acetazolamide on retinal physiology cannot be excluded (Yamamoto & Steinberg, 1992b; Oakley dz Wen, 1989).

Because of the uncertainty of direct effects of CA1 on retinal cells, it has been more common to assume that the effects of CA1 have indirect effects on retinal cells. Generally, these hypotheses assume that the altered pH induced by CA1 diffuses into retina tissue and alters the function of retinal cells. The source of this altered pH may be either from the blood or the retinal pigment epithelium.

Stanescu and Michiels (1975) suggested that the in- crease in b-wave amplitude following administration of acetazolamide might be accounted for by its effects on pH. Acetazolamide lowers arterial pH by altering the level of COZ in the blood (Gotah, Meyer 8z Tomita, 1966). ERG b-waves are affected by pH (Linsenmeier, Mines & Steinberg, 1983); therefore, increased b-wave amplitudes might be accounted for solely on the basis of altered extracellular pH (Stanescu & Michiels, 1975). However, three lines of evidence argue against this interpretation. First, we know of no evidence that arterial pH is different in dark or light adaptation. Second, the pH change required to affect the b-wave is probably greater than that obtained in this study (Linsenmeier et al., 1983). In isolated retinal prep- arations, acetazolamide increases ERG b-wave ampli- tude, even when altered extracellular pH is insolent to account for the increase (Wen & Oakley, 1988). Third, extracellular pH in the retina is only loosely related to plasma pH (Yamamoto & Steinberg, 1992a).

An alternative explanation based on pH is to assume that the acetazolamide has a direct effect on the retinal pigment epithelium, altering extracelluar pH which then is diffused through out the retina, affecting the function of retinal cells, notably the Miiller’s cells. Yamamoto and Steinberg (1992b) have presented evidence that retinal pH changes following administration of acetazo- lamide begin near the retinal pigment epithelium. They suggested that these pH changes diffuse throughout the retina with time ahering retinal function. However, they could not exclude direct retinal effects of acetazolamide which have been observed in other preparations (Oakley & Wen, 1989) accounting for the later pH changes.

One might also assume a direct effect of acetazolamide and other CA1 on Mtiller’s cell activity (Broeders et al.,

1988). Miiller’s cell activity at about the level of the bipolar cell determines ERG b-wave amplitude (Miller & Dowling, 1970) and CA is located in the Miiller’s cells (Wistrand et al., 1986).

One of the difficulties of using a single process to explain the effects of CA1 on ERG b-waves whether that single process is altered pH which originates in the plasma or in the retinal pigment epithelium or a direct effect of acetazolamide on the Mullet’s cell is that two

836 J. VERNON ODOM et al.

effects are observed, an increase in b-wave amplitude in the dark and no change (present study) or a decrease (Broeders et al., 1988) in the light. Therefore, one must assume that the single mechanism has either a greater effect in the dark adapted state or two effects one in the dark and one in the light. While such possibilities cannot be excluded, we suggest a two process explanation which assumes direct effects on the Miiller’s cell and photoreceptors.

Because CA is located in 9 1% of human cones but not in the rods (Nork et al., 1990), the second effect of CA1 may be attributable to differential effects of CA1 on the activity of rods and cones. Evidence of this second effect is provided by changes in human ERG a-wave amplitudes. In humans, where ERG u-waves could be measured reliably, light adapted u-wave amplitudes were reduced about 14% following administration of acetazo- lamide compared to the control condition, but the a- to b-wave ratio remained about the same; 0.4 as compared to 0.42 respectively.

Let us provide a more detailed description of this suggestion. One may account for the electrophysiologi- cal results, increased dark adapted b-wave amplitudes and reduced light adapted a-wave amplitudes by assum- ing the operation of two processes: a direct effect of acetazolamide on the Mtiller’s cells which increases b-wave amplitudes and an effect on the cones which reduces a-waves and indirectly reduces b-wave ampli- tudes. In the dark adapted state, the effect on the Miiller’s cells would be the predominant effect present, because the cones would be minimally stimulated. How- ever, the cone effect would increase with increasing flash intensity as suggested by our nonsignificant a-wave results. Consequently, the b-wave amplitude would in- crease. In the light adapted state, the direct effect on the Miiller’s cells would act to increase b-wave amplitude and the effect on the cones would act to reduce u-wave amplitude. If photoreceptor activity is reduced there should also be a reduction in b-wave amplitude. Because one measures b-wave amplitude as the sum of a- and b-wave amplitudes, these two effects would tend to counterbalance one another, so that there would be no change (or a slight decrease) in the light adapted b-wave amplitude. The reduction of light adapted u-wave ampli- tudes in humans and trend for reduced light adapted b-waves supports the suggestion that there is an effect of CA1 on cones.

If 91% of the cones have CA (Nork et al., 1990) and if cones are affected by CAI as indicated by the reduced amplitudes of photopic human u-waves, we would pre- dict that alteration of CA1 would affect cone function and possibly cone-rod interactions. One potential ad- vantage of studying human subjects is that the behav- ioral effects of the drug can be tested easily. However, we did not observe any clear axis in the error scores, indicative of a color vision defect following adminis- tration of acetazolamide on any of the color vision tests at our disposal, i.e. the 100 hue, Lanthony desaturated D-15, Cogan-Gunkel chromograph or Nagel anomalo- scope. Similarly, dark adaptometry was not altered in a

consistent manner following administration of acetazo- lamide. We cannot exclude the possibility that by using a larger sample or more sophisticated analysis methods, we might have detected changes in human behavioral thresholds as several effects approached statistical significance. The absence of these effects may be inter- preted as evidence against our suggestion of altered cone function.

We suggest that our psychophysical tests did not adequately test our present hypothesis. The tests and scoring procedures were selected before we determined that the cones containing CA were the “red” and “green” cones and had completely framed the current hypothesis. They were selected to determine changes in rod or cone function. Dark adaptometry dark adapted thresholds should, in fact, have shown little or no change and most clinical tests of cone function are tests of color discrimination which are designed to detect either an absolute defect or a relative defect in a single cone type. We are suggesting a relatively minor alteration in two cone types which equal in the two types. It is not clear how such a change would affect color discrimination. Consideration of these technical factors may be useful for further experiments. For example, our procedure using the Nagel anomaloscope was to measure the mid-match point. No effort was made to measure the range of acceptable matches or the luminance of the yellow light which gave the best match. However, if both the “red” and “green” sensitive cones are affected equally, it is not clear that one would expect a change in the Nagel mid-match point, although the range over which an acceptable could be accepted might be expected to increase and the sensitivity might decrease resulting in a need for an increase in the luminance of the yellow light required to make the best match.

Therefore, detecting the changes in cone function which we suggest exist will require either different or more extensive testing than we performed. A more effective approach to detect changes in the cone activity as a result of CAIs may be to employ tests which specifically measure cone sensitivity. Results using these alternative strategies do show altered cone sensitivity in normal human subjects who take a one time oral dose of acetazolamide (Leys, van Slycken, Nork & Odom, 1990). A related strategy would be to measure the spectral sensitivity functions of each cone type.

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