620 Reports Effects of Apomorphine on the Rabbit Elec

Invest. Ophthalmol. Vis. Sci. 620 Reports October 1981

to the ones described in Jagadeesh et al.,5 with Effects of apomorphine on the rabbit elec- variations in the way the drug was injected and in data processing. A brief summary indicating the troretinogram. J. M. JAGADEESH AND R. appropriate variations is given below. SANCHEZ. The ERG recording system consisted of an Experiments were conducted in both albino and pig- LSI-11/03 computer (Digital Equipment Corp.) mented rabbits to determine the effects of apomorphine with 32K words of memory, a HAWK disk drive on the electroretinogram (ERG). Injection of apomor- (Control Data Corp.) and an AD AC Model 600- phine (0.1 to 1.5 mg/kg) into the left carotid artery pro- LSI-11, 12-bit data-acquisition module. The light duced dose-related decreases in the b-wave, predomi- stimulus was from a Grass Model PS22 pho- nantly in the b-wave amplitude, and also increases in tostimulator. A Grass Model PS16 preamplifier the c-wave amplitude. No significant changes were ob- and a Tektronix Model AM502 amplifier were served in the a-wave amplitude or in the attendant used to amplify the ERG signal. The overall gain latencies. The ERG changes apparently were not related to systemic drug effects. The effects of the drug were was set to 5000, and AC coupling was used with a similar for both albino and pigmented rabbits. Apomor- bandwidth of 0.2 to 2.0 kHz. A Burian-Allen elec- phine, a dopamine agonist, has the opposite effect on the trode was used to record ERG. The data were b-wave of the ERG when compared with the effect of processed on a PDP-11/34 computer (Digital chlorpromazine, a dopamine antagonist. Involvement of Equipment Corp.) equipped with VT-11 refresh dopamine receptors is not unexpected, since the retina is graphics scope and Tektronix Model 4662 plotter. rich in dopamine, especially the inner plexifonn layer. The general anesthetics used were 100 mg/ml (INVEST OPHTHALMOL VIS SCI 21:620-625, 1981.) ketamine (Ketalar; Parke, Davis & Co.) and 20 Extensive research has been conducted on the mg/ml xylazine (Rompun; Haver-Lockhart Labora- synthesis, storage, release, uptake, and metabo- tories). Proparacaine (Alcaine) and tropicamide lism of dopamine (DA) during the past two de- (both manufactured by Alcon Laboratories) were cades. Since the discovery of its presence in the used as local anesthetic and to dilate the pupil, brain in the 1950s, DA has been identified in vari- respectively. ous organs, including the retina, and this catechol- The rabbit was dark-adapted for about 20 min amine has now been accepted as a neurotransmit- and then anesthetized by an intramuscular injec- ter in its own right. It has been demonstrated1"3 tion of 50 mg/kg ketamine and 10 mg/kg xylazine. that the retinas of several species contain large With the rabbit's eyes completely covered with concentrations of DA. Among these, the rabbit black tape, an incision was made into the rabbit's retina has one of the largest concentrations of DA4 neck to expose the arteries. A canula with a T was and is thus a good model to study receptor effects. inserted into the left carotid artery. The animal Understanding of the DA receptors is perhaps a was then placed in a metal box with its head inside precursor to the understanding of dopaminergic a globe, set up with flash for a ganzfeld system. systems, and hence the study of DA receptors has The animal was kept anesthetized by a multidose received the greatest attention in recent years. administration of a mixture of 4 ml of keta- Such studies invariably include experiments utiliz- mine + 4 ml of xylazine + 12 ml of 0.5% saline ing specific-acting agonists and antagonists. In our solution. No data were collected for the first 30 to previous study5 intravenous injection of chlor- 45 min after the rabbit was placed in the metal promazine markedly increased the b-wave of the box. Three intensity levels corresponding to 1-1, electroretinogram (ERG). If, indeed, chlorpro- 4, and 16 of the PS22 photostimulator were used at mazine was acting on the DA receptors in the ret- random. One ERG was recorded every minute. ina, we expected that such a DA antagonist would Normal ERGs were recorded for about 20 min, have an effect opposite to that of an agonist. Apo- and then apomorphine was injected through the morphine has been extensively studied as a spe- left carotid artery via the canula. Drug doses rang- cific-acting DA agonist in the central nervous sys- ing from 0.1 through 2 mg/kg were used. The ex- tem. In order to establish DA agonist/antagonist periments were conducted on both albino and pig- drug effects on the ERG, we conducted detailed mented rabbits. In a few experiments, the animals experiments concerning the effect of apormor- were light-adapted with high illumination (100 w phine on rabbit ERG, and the results are reported incandescent light in addition to normal fluores- here. cent room light) for about 10 min, and then the Methods. The methods used were very similar ERGs were recorded through the dark-adaptation

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180 PIGMENTED RABBIT APOMORPHINE 1 mg/kg 19 min. BEFORE DRUG FLASH INTENSITY = 16 16 min. AFTER DRUG 3- 15 min. AFTER DRUG UJ Q • 51 min. AFTER DRUG 60

_l Q_ 0

-60 - 0 0.115 0.230 0.345 0.460 0.575 0.690 0.805 TIME (S) Fig. 1. ERG waveforms before and after intra-arterial injection of apomorphine. The ERGs were digitized with a 12-bit analog to digital converter and the sampling period was 0.5 msec.

process. A few experiments were also conducted ished. There was a slight reduction in the bj-wave with chlorpromazine by injecting the drug intra- amplitude. The reduction in b2-wave amplitude arterially to compare with our previous results was significantly greater than the reduction in using an intravenous injection. The blood pressure bj-wave amplitude in both types of animals and at was recorded on a Physiograph (Narco Bio-Sys- all dose levels. There was no measurable change in tems, Inc.) using the femoral artery. Experiments the a-wave amplitude. The c-wave amplitude in- were conducted on five albino and six pigmented creased after the injection of the drug. A very clear rabbits with drug and four albino and three pig- dose-amplitude relationship could not be estab- mented rabbits without drug. lished for the c-wave amplitude or latency. The An interactive program was used to calculate latencies of a-, br, b2-, and c-waves were the same the a-, b-, and c-wave amplitudes and latencies. before and after the drug injection. The results Each ERG was displayed on the VT-11 graphics were similar at all flash intensities except that the scope, and a flashing cursor was moved across the c-wave was absent at the lowest intensity. A series screen. When the operator judged that the cursor of ERGs taken before and after apomorphine was was located at the peak of interest, he typed an injected is shown in Fig. 1. The amplitudes of the "o"; the amplitude and the latency values at the br and b2-waves as a function of time before and cursor were stored on the disk. These disk files after the drug injection are shown in Fig. 2, A. were used to plot various graphs. The recovery of the ERG waveform was gradual Results. The nomenclature used in this report and dose-dependent. The reduction in the bj- and for different wavelets of the electroretinogram is b2-wave amplitudes was also dose-dependent. The given in the inset of Fig. 3. This nomenclature is maximum reduction for each dose was calculated very similar to the one given by Armington6 ex- and these values for different doses are given in cept that photopic (bp) and scotopic (bs) b-wave Table I. are labeled as bx- and b2-waves, respectively. Four Soon after the drug had been injected, there discrete doses (0.1, 0.6, 1.0, and 1.5 mg/kg) were was a small dip in the arterial blood pressure, and tested on both pigmented and albino rabbits. At it returned to predrug level in about 1 to 3 min, as doses higher than 2 mg/kg the recording was un- shown in Fig. 2, A. This transient change in blood stable. In all cases the b2-wave amplitude was re- pressure was observed in all experiments. There duced dramatically, irrespective of the type of ani- was also a gradual fall in blood pressure progress- mal. For large doses the b-2-wave almost van- ing toward the end of the experiment. We also

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500 A

UJ 400 cr Z) O) t C/) O O LJJ LU 300 -O O O O OOOOOO O O TO '1 Z) Q ^ 200 O CL 50 AP0M0RPHINE O.lmg/kg ALBINO RABBIT - 30 < 100 FLASH INTENSITY=I6 1 i il i i i i i i i i UJ 00 10 20 30 40 50 60 70 80 TIME(min)

B 240 V V X Y Y v xx xxxx

i c 'E 180 \ o_ en LU c Q APOMOF PHINE lmg/kg 120 LU FLASH INTENSITY = I6 Q_ or 60 ^ 60 - _ / V y—"\ - 40 g 20 g n i i i i i 1 Q_ 0 25 150 00 50 75 100 125 LU TIME (min) cr Fig. 2. Amplitudes of bx- and b2-waves, blood pressure, and respiration rate of control and drug-treated rabbits. The arrows indicate the drug injection time. A, b2-wave amplitude (filled circles); bx-wave amplitude (open circles); arterial blood pressure (solid line). B, b2-wave amplitude (crosses) and respiration rate (dash-dotted line) of pigmented control (no injection) rabbit; b2-wave amplitude (full circles) and respiration rate (dashed line) of drug-treated rabbit.

noted such reduction in blood pressure in control and drug-treated animals (Fig. 2, B). The respira- animals. For low doses, even though the blood tion rate and the b2-wave amplitude measured pressure was below the predrug level, the b-wave during a control experiment with no injection are amplitudes returned to the predrug level. The also given in Fig. 2, B. Even though the frequency respiration rate fluctuated in both control animals of respiration fluctuated between 50 to 38 inspira-

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-Lb

400 r

^300 APOMORPHINE 0.6mg/kg FLASH INTENSITY = I6 m m9 ••• Id Q 200 250 Q_ 100 ALBINO RABBIT 200 1° 150 opo | Oopo 90 , I Y O 100 10 30 50 70 90 no 130 150 TIME (min

Fig. 3. Amplitudes of brwave (closed circles), b2-wave (triangles), and c-wave (open circles) of drug-treated rabbit. The arrow indicates drug injection time. Inset, How the various ERG peaks were measured.6

tions/min, the b2-wave remained constant for the Table I. Percent maximum reduction of br and control animal. However, in most cases there b2-wave amplitude from predrug values at seemed to be an increase in the respiration rate for different doses about 15 to 20 min after drug injection, and then either a gradual decrease or some fluctuation in Percent maximum reduction breathing rate was observed. Albino rabbit Pigmented rabbit Fig. 3 illustrates bx-, b2-, and c-wave amplitudes Dose as a function of time for an albino rabbit before and (mg/kg) b x-wave b 2-wave b rwave b 2-wave after the injection of 0.6 mg/kg apomorphine. It 0.1 11 28 18 35 may be noted that the c-wave amplitude returned 0.6 24 68 27 72 to the predrug level within 30 min, whereas the 1.0 14 43 20 63 1.5 21 45 28 60 recovery of br and b2-waves was rather slow. In the few experiments using intra-arterial injection of chlorpromazine, we found that the in- ERG.7 In addition, in a very systematic in vitro 8 crease in the b2-wave amplitude was greater than study using cats, Niemeyer has demonstrated the increase in the brwave. The highest increase that the b-wave of the ERG is affected by the flow occurred within 10 to 15 min after the drug injec- rate and oxygenation of the perfusate. Thus it is tion as compared to 30 to 50 min in the case of necessary to evaluate the systemic effects of intravenous injection. apomorphine that could indirectly cause the ob- Discussion. Our experiments demonstrated that served ERG changes. In all experiments, imme- intra-arterial injection of apomorphine caused a diately after the drug injection, there was a small significant reduction in the b2-wave amplitude, a dip in the arterial blood pressure. However, the small reduction in the bx-wave amplitude, and no effect was transient, lasting only 1 to 3 min. The change in the a-wave amplitude. Since these ob- observed blood pressure reduction of 5 to 7 mm servations were similar for both pigmented and Hg during the course of the drug experiment (Fig. albino rabbits, it can be concluded that pigmenta- 2, A) was not significantly different from the blood tion is not involved in the drug-induced ERG pressure change measured during control experi- changes. ments. Also, for low doses the ERG returned to Hypoxia can cause reduction in both photopic predrug levels although the blood pressure re- (x-wave) and scotopic b-waves (bj and b2) in human mained depressed. Thus it is not likely that blood

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circulation changes induced the observed ERG generated by the photoreceptors and Miiller cells. changes. Hence, it is also likely that the c-wave change may In most cases the respiration rate increased after be mediated by the action of apomorphine in these drug injection. However, as can be seen from Fig. parts of the sensory retina. Histochemical studies 2, B, the b2-wave was quite constant in spite of the would likely elucidate specific sites of action of fact that the respiration rate fluctuated. Further- apomorphine in the retina. more, we could not correlate the changes in the breathing rate with the changes in the b-waves. We are grateful to Dr. P. N. Patil for his encourage- Thus it is highly improbable that the ERG altera- ment, guidance, and constructive criticism throughout this project. We are also grateful to Dr. T. D. Sokoloski tions after apomorphine were caused by changes for his careful reading of the manuscript and valuable in the breathing rate. suggestions. We appreciate Dr. Lawill and his col- DA is a prominent neurotransmitter in the ret- leagues (University of Kentucky) for sharing their expe- 9 ina, and Dowling and Ehinger have demon- rience in conducting ERG experiments and for many strated that a subset of amacrine cells synthesize useful hints. and degrade DA in rabbit retina. Also, Redburn From the College ol Pharmacy, The Ohio State Uni- and Kyles10 have shown that DA receptors are versity', Columbus. This research was supported in part concentrated in synaptosomal fractions of inner by National Eye Institute grant NO EY 1308-04. Submit- plexiform layer in the rabbit retina. In addition, it ' ted for publication Jan. 5, 1981. Reprint requests: Dr. has been suggested that DA functions as an inhibi- J. M. Jagadeesh, 500 W. 12th Ave., College of Phar- 11 tory transmitter in the retina. It was demon- macy, The Ohio State University, Columbus, Ohio strated that intravenous injection of chlorproma- 43210. zine, a DA antagonist, caused an increase in the Key words: ERG, a-wave, b-wave, c-wave, dopamine b-wave amplitude of the rabbit ERG.5 Thus it is highly probable that the reduction in the b2-wave amplitude observed in our experiments is caused REFERENCES by apomorphine acting as a DA agonist in the 1. Haggendal J and Malmfors T: Identification and cel- inner plexiform layer of the retina. lular localization of the catecholamines in the retina and the choroid of the rabbit. Acta Physiol Scand Dowling12 has postulated that amacrine cells are 64:58, 1965. involved in the light adaptation process, since 2. Laties AM and Jacobowitz D: Histochemical studies their synaptic contacts link bipolar, amacrine, and of monoamine-containing cells in the monkey ret- ganglion cells in both feed-forward and feed-back ina. J Histochem Cytochem 14:823, 1966. circuits. Our results showing similarity between 3. Vijay Sarthy P and Lam DMK: The uptake and re- 3 the growth of b2-wave during dark adaptation and lease of [ H] dopamine in the goldfish retina. J Neu- recovery from apomorphine, as well as the pre- rochem 32:1269, 1979. dominant effect of the drug on the b2-wave, indi- 4. Da Prada M: Dopamine content and synthesis in cate that apomorphine could be acting on the DA retina and N Accumbens Septi: pharmacological and receptors of amacrine cells. The results are thus light-induced modifications. Adv Biochem Psycho- compatible with Dowling's suggestion. pharmacol 16:311, 1977. 5. Jagadeesh JM, Lee HC, and Salazar-Bookaman M: In general, the pigment epithelium has been Influence of chlorpromazine on the rabbit electro- implicated in the generation of the c-wave. Since retinogram. INVEST OPHTHALMOL VIS SCI 19:1449, our results indicate an immediate increase in the 1980. c-wave amplitude, a direct influence of apomor- 6. Armington JC: The Electroretinogram. New York, phine on epithelial cells may be postulated. Also, 1974, Academic Press, Inc., p. 249. since the increase in the c-wave amplitude is com- 7. Brown JL, Hill JH, and Burke BE: The effect of pletely similar for both pigmented and albino rab- hypoxia on the human electroretinogram. Am J bits, the involvement of pigmentation may be ruled Ophthalmol 44:57, 1957. out. There is more recent evidence to suggest that 8. Niemeyer G: The function of the retina in the per- fused eye. Doc Ophthalmol 39:53, 1975. the c-wave may incorporate component voltages 9. Dowling JE and Ehinger B: Synaptic organization of from other sites in the retina, in addition to the 13 14 the dopaminergic neurons in the rabbit retina. J component from the pigment epithelium. ' Comp Neurol 180:203, 1978. These components include a cornea positive volt- 10. Redburn DA and Kyles CB: Localization of dopa- age generated synaptically in the inner retina and mine receptors within two synaptosome fractions of a cornea negative voltage considered to be signals rabbit and bovine retina. Exp Eye Res 30:699, 1980.

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11. Ehinger B: Biogenic monoamines as transmitters in from reaching the screen. Test stimuli were 1 sec the retina. In Transmitters in the Visual Process, duration blue (Ilford 621) flashes;th e steady back- Bonting SL, editor. New York, 1976, Pergaman ground beam was red (Wratten 29). Both back- Press, p. 145. ground and test-spot intensity were controlled by 12. Dowling JE: The site of visual adaptation. Science neutral density filters. Luminance measurements 155:273, 1967. were made" with a calibrated photodiode (UDT; 13. Marmor MF and Lurie M: Light-induced electrical responses of the retinal pigment epithelium. In The Model 11 A). The unattenuated test beams had a Retinal Pigment Epithelium, Marmor MF and Zinn luminance of 2.11 log mL, and the background KM, editors. Cambridge, Mass., 1979, Harvard had a luminance of 2.86 log mL. University Press, p. 226. After dark adaptation for3 0 min, the infant was 14. Rodieck RW: Components of the electroretino- held 50 cm in front of the dark rear projection gram—a reappraisal. Vision Res 12:773, 1972. screen by an adult (the holder). A second adult (the experimenter, positioned behind the screen) at- tracted the infant's gaze to the center of the screen Behavioral measurement of background by flickering a dim yellow flashlight (30 min arc adaptation in infants. RONALD M. HANSEN diameter). A third adult (the observer) watched AND ANNE B. FULTON. the child with an infrared viewer (FJW Industries; Thresholds for detecting blue test flashes in the dark- Model 3420). The observer, hidden in black cur- adapted condition and on steady red background fields tains adjacent to the screen, could view the infant were measured in 2- to 18-week-old human infants by a from above, below, or to the right or left of the two-alternative forced-choice preferential looking meth- screen. When the observer reported that the in- od. The results show that dark-adapted sensitivity infant was alert and looking at the center of the creases and background adaptation develops during the screen, the experimenter extinguished the fixation early postnatal weeks. Thus, the retinal mechanisms that light and presented a test spot. The observer faced underlie (1) detection of brief flashes and (2) neural pro- the infant and could not see the stimuli. Based on cessing in background adaptation appear to mature the infant's looking behavior, the observer re- postnatally. (INVEST OPHTHALMOL VIS SCI 21:625-629, 1981.) ported stimulus location on every trial. After each response, the experimenter told the observer Recently Powers et al.' showed that the scotopic whether the judgment was correct. We assume sensitivity of human infants increased postnatally. that the infant looked toward the flash if it was This demonstration, along with the finding that detected. Stimuli were repeated, when necessary, background adaptation in infant rats continued to to allow the observer to make a decision. develop after distal retinal morphogenesis was Initially the infant was shown stimuli that were 2 nearly complete2 (as in neonatal humans3), raised to 3 log units above the adult dark-adapted thresh- the possibility that scotopic background adaptation old. These stimuli were detected readily and en- also developed postnatally in human infants. To abled the observer to become familiar with the find out, we have modified the two-alternative infant's looking behavior. If the observer correctly forced-choice preferential looking method4 to mea- reported spot location on the first five trials (100% sure the effects of steady backgrounds on thresh- correct), test-spot intensity was reduced by 0.6 log olds for detecting flashes of light. Preliminary re- units.* Target intensity was reduced further until sults indicate that background adaptation in very the observer made an error. Then, ten trials were young infants is different from that of adults but run at that and each succeeding test-flash intensity. matures during the early postnatal weeks. Stimulus intensity was reduced in 0.3 log unit steps Methods. Three 500 W tungsten sources pro- jected test stimuli and backgrounds onto a 107 by 107 cm rear projection screen in a dark room. One *The probability of guessing stimulus location correctly is source produced a diffusely illuminated 110° di- 0.5 in a two-alternative forced-choice task. Assuming that ameter circular background field centered on the each trial was an independent event, the probability that the observer correctly guessed test-spot location on five screen. The other beams, one on the right and one trials in a row was 0.031 (from the binomial probability on the left, produced 10° diameter test spots lo- distribution with n = 5, r = 5, and p = 0.5). Thus it was cated 20° to the right and left of the center of the unlikely that the observer's percent correct was due to screen. The sources, lenses, and stops were guessing, but rather reflected the infant's visual per- mounted in light-tight boxes to prevent stray light formance.

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