Permanent Alterations in Muscarinic Receptors and Pupil Size Produced by Chronic Atropinization in Kittens

No. 2 Reports 239 Permanent Alterations in Muscarinic Receptors and Pupil Size Produced by Chronic Atropinization in Kittens Earl L. Smith III,* Dianna A. Redburn,f Ronald 5. Harwerrh,* and Gregory W. Maguire* Chronic mydriasis was induced in six kittens (four monocular, two binocular) and two adult cats (both monocular) by the daily topical application of atropine. Both the kittens and the adult cats were atropinized for a 13-week period with the treatment regimen beginning at the time of eye opening for the kittens. Pupil size measurements, obtained 1 year after the atropinization were discontinued, revealed that, although the pupils of the adult cats were normal, the pupils of the kittens' treated eyes were consistently smaller than pupils in control eyes. The status of the muscarinic receptors in the kittens' irides was investigated using 3 H-QNB binding assays. In comparison with iris muscle homogenates from the control eyes, those from the treated eyes demonstrated an eightfold increase in the number of receptor binding sites. The results indicate that pupil size can be altered permanently by chronic mydriasis initiated early in the life of a kitten and that the permanent change in pupil size may result, in part, from a type of permanent supersensitivity response in the muscle following chronic blockade of muscarinic transmission by atropine. Invest Ophthalmol Vis Sci 25:239-243, 1984 Postdilation miosis is a reduction in pupillary size relative to pretreatment dimensions or nontreated control pupils following recovery from the primary action of a mydriatic agent. In humans, postdilation miosis has been observed following the topical administration of certain sympathomimetic agents 12 (eg, phenylephrine and hydroxyamphetamine) and has been reported 2 to persist for as long as 7 days after treatment. To our knowledge, the phenomenon of postdilation miosis has not been associated with the use of parasympatholytic agents in humans. However, in a recent study 3 on the effects of chronic atropinization on visual acuity in kittens, it was noted that following recovery from mydriasis (approximately 3 wks posttreatment) the pupils of the kittens' treated eyes were consistently smaller than the pupils in the nontreated eyes. If confirmed, this observation would not only be clinically important, it would also provide insight into the physiology of the iris musculature. Moreover, since chronic atropinization is an experimental procedure sometimes used for depriving an eye of a clear retinal image, it would be potentially important in the interpretation of certain experimental results. Therefore, one of the purposes of the present study was to verify and document the presence of postdilation miosis in chronically atropinized kittens. In this respect, since the original observation was in kittens, it was important to determine if the phenomenon was reversible and if it represented a developmental age-dependent process. A second purpose of the present study was to examine the mechanism by which an alteration in the tone of the iris muscles is maintained over extended periods of time following chronic atropinization. This phenomenon could result from a variety of changes in the iris including simple atrophy of certain iris muscles, an up-regulation of parasympathetic neuronal activity (ie, an increase in acetylcholine release from each nerve terminal or an increase in the number of terminals as seen in sprouting), or an up-regulation in muscle response (ie, an increase in muscarinic receptors). The in vitro binding of the irreversible muscarinic ligand, 3 H-quinuclidinyl benzylate (QNB), which has been widely used as a measure of the number and affinity of muscarinic receptors in neuronal and muscle preparations, provides a way to examine the status of muscarinic receptors in the iris (for a review see reference 4). We therefore analyzed the QNB binding activity of iris from control and chronically atropinized eyes. Our results suggest that the number of muscarinic receptors is greatly increased in the treated iris and, therefore, may represent one of the mechanisms by which pupillary size is permanently altered after chronic atropinization in kittens. Materials and Methods. Eight kittens that were born in an isolated colony and 10 additional cats that were obtained as adults were used in the study. Six of the kittens were treated with a 1% atropine sulfate solution (four monocularly, right eye; two binocularly) on a daily basis from the time of eye opening («8-9 days of age) until 14 wks of age. Two of the adult cats had atropine topically applied to the right eye on a daily basis for 13 wks. In order to maintain a maximal mydriasis, it was necessary to instill atropine into the experimental animals' eyes three times each day. Two of the kittens that had coloration patterns similar to their binocularly treated littermates and the remaining eight adult cats were used as nontreated controls. Throughout the experiment, all of the animals were housed in a normally illuminated environment (12- hr light/12-hr dark cycle). The sizes of the animals' pupils were measured from individual photographs. To help minimize distractions, the animals were held 150 cm from the camera at one end of a 50 X 50 X 150 cm white rectangular box. The interior of the box was illuminated uniformly by

240 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1984 Vol. 25 Fig. 1. Photographs taken 1 year posttreatment of a monocularly treated adult (A), a monocularly treated kitten (B), and a binocularly treated kitten (C, right) and its paired nontreated littermate (C, left). variable intensity incandescent light sources positioned above the camera. The outlines of the pupils were traced from enlarged projections of the photographic negatives, and the horizontal dimensions of the pupils were measured to the nearest 0.5 mm with a handheld magnifier. A correction factor to compensate for the magnification of the camera and enlarger was derived from photographs of a calibrated scale positioned in the same plane as the animals' eyes. For a given background level of illumination, measurements for each pupil were obtained from a minimum of three photographs. For the treated animals, estimates of pupillary size were made periodically during a 1-year recovery period beginning 1 month after the atropine regimen was discontinued. At the end of the 1 -year recovery period, the treated kittens were sacrificed by decapitation. The eyes were removed and hemisected. Muscles of the iris and ciliary body were obtained by gently exerting pressure on the attachments along the outer circumference of the iris with a blunt probe inserted through an incision in the center of the cornea. Iris and ciliary muscles were separated with scissors. Each tissue sample was frozen overnight at -20 C. By a modified filtration assay described by Yamamura and Snyder,5 3H-QNB binding was determined. Frozen pieces of tissue from the irides of the treated and nontreated eyes of the atropinized kittens were thawed, placed in 0.1 M Na-K phosphate buffer (ph 7.4) and homogenized for 30 sec with a Brinkman

No. 2 Reports 241 polytron at setting 6. The tissue suspensions then were diluted with Na-K phosphate buffer (0.05 M,finalconcentration) to a final volume containing 0.3-0.5 mg protein/ml for the 3 H-QNB assay. Fifty microliters of 3 H-QNB solution (final concentrations were 0.25 to 5.0 nm; New England Nuclear, Boston, MA) and 1 ml of phosphate buffer were placed in each assay tube. Twenty microliters of 10 fim atropine (final concentration 0.1 /xm; Sigma Chemical Co., St. Louis, MO) were placed in selected assay tubes to determine nonspecific binding. One milliliter of tissue suspension was added to each assay tube for a 1-hr incubation period at room temperature (25 C). Incubation was terminated by filtration on GF/C filters (Whatman, England) followed by three 5-ml washes with cold buffer. The filters were removed, placed in vials, and allowed to dry. A toluene base counting solution containing Triton X-100 was added, and the filters were counted on a Searle Mark III liquid scintillationcounter. Results. Pupil Size: There were no obvious behavioral differences between the treated and nontreated kittens, either during or after the treatment period. Casual inspection of the atropinized animals revealed that the responsiveness of the treated pupils to changes in illumination (both direct and consensual) increased gradually after the atropinization had been discontinued and that all of the treated pupils responded in a normal manner within 3 wks of the termination of the atropine treatment. Measurements of pupil size obtained 1 month posttreatment indicated that the pupils of the atropinized adult cats were essentially equal in size (ie, the differences in pupil size between the right and left eyes were within the range of size differences for the 8 nontreated controls). However, the differences in pupil size for all four monocularly atropinized kittens were outside the range for normals, the pupils of the treated eyes being consistently smaller than the pupils of the nontreated eyes. In addition, the pupils of the two binocularly treated kittens were essentially equal in size but smaller than the pupils of their paired nontreated littermates. The normal pupil sizes observed in the atropinized adult cats and the postdilation miosis demonstrated by the treated eyes of the kittens remained unchanged over a 1-year recovery period. The relative miotic nature of the kittens' treated pupils is illustrated in Figure 1, where photographs taken 1 year posttreatment of a monocularly treated adult (A), a monocularly treated kitten (B), and a binocularly treated kitten and its paired nontreated littermate (C) are shown. The ratios of pupil sizes (right eye/left eye) obtained for a moderate level of illumination (2.2 log lux) at the end of the 1- year recovery period are illustrated for each animal in Figure 2A. For the nontreated controls, the difference in the horizontal dimensions of the pupils never exceeded 6.0% and, as can be seen, the size ratios for both the monocularly treated adults and the binocularly treated kittens fall within the range of differences for the controls (dashed line). In contrast, the horizontal dimensions of the treated pupils of the monocularly atropinized kittens were between 12% and 27% smaller than those for the nontreated pupils. Similarly, both the pupils of the two binocularly treated kittens were A. 10.0 0.9 d 6.0 Q. 0.8 0.7 8.0-2.0- NORMAL MONOCULARLY BINOCULARLY MONOCULARLY CONTROLS TREATED TREATED TREATED ADULTS KITTENS KITTENS REARING CONDITION -1.0 0 1.0 2.0 LOG LUMINANCE (LUX) 3.0 Fig. 2A. Ratio of pupil sizes (right eye/left eye) obtained at a moderate background illuminance (2.2 log lux) plotted as a function of the rearing condition for each animal (filled circles). The open squares represent the ratios of the pupils of the binocularly treated kittens compared to their paired nontreated controls. B. Horizontal dimensions of the treated (filled circles) and nontreated pupils (open circles) of a monocularly atropinized kitten plotted as a function of background illuminance.

242 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1984 Vol. 25 Q Z o CD A. IRIS (CONTROL) 14-0 B =2.98 12 - K d=0.8nm r value =0.99 10-8 - O 6-4 - 2-10 " ^ O 8 - o 6-4 - 2 - B. IRIS (ATROPINE-TREATED) O o 1 2 3 B max= 25 K d=2.8nm r value = 0.98 N 0 2 4 6 8 10 12 14 16 18 20 22 24 26 BOUND (fmoles/mg protein) Fig. 3. Scatchard analysis of specific 3 H-QNB binding in control (A) and atropine-treated (B) cat iris. The tissue was incubated in 0.05 M Na-K phosphate buffer (ph 7.4, 1 hr) containing different concentrations of ligand, and specific binding was obtained by subtracting the amount of radioactivity not displaced by 0.1 fim atropine from the total 3 H-QNB bound. K<j's and B max s, which represent estimates of the affinity and number of muscarinic receptors, respectively, were determined by linear regression analysis. 27% and 30% smaller than the pupils of their paired nontreated littermates (open squares, Figure 2A). The relative miotic state of the kittens' treated pupils was independent of the level of background illumination. Figure 2B shows the size of the treated (filled circles) and nontreated pupils (open circles) of a monocularly atropinized kitten plotted as a function of the background illumination. At all levels of illumination considered, the treated pupil was smaller than the nontreated pupil. Although the absolute difference in pupil size decreases with increasing illuminance levels, the percentage difference remains relatively constant («22%) across the nearly 4 log unit range of illuminance. 3 H-QNB Binding Assay: A total often separate samples of iris (five treated and five control) were subjected to in vitro analysis of 3 H-QNB binding in the presence of 0.25 to 5 nm concentrations of the ligand. Binding at each concentration was determined using three different treated and control iris samples, each of which was assayed in triplicate. The scatchard analysis of the results shown in Figure 3 demonstrates a significant difference (slope, P < 0.001; intercept, P = 0.008) in the binding characteristics of control versus treated iris samples. The treated iris samples bound significantly more 3 H-QNB in the presence of the higher concentrations of the ligand (2-5 nm). The calculated B max (an estimate of the number of muscarinic receptors) for the treated irides was eightfold higher than that for controls, which suggests that the increased binding is due mainly to an increase in the number of 3 H-QNB binding sites. However, at lower concentrations of the ligand (0.25-1 nm), the treated iris samples bound significantly less than the control samples. Thus the calculated affinity (KJ of the receptors from the treated samples is approximately one-third that of the control samples. Discussion. The relative miotic state noted for all of the kittens' treated pupils confirmed our earlier observation of postdilation miosis in chronically atropinized kittens. 3 Moreover, since the sizes of the kittens' treated pupils had not returned to normal by the end of the 1-year recovery period, it seems reasonable to assume that these pupillary alterations are permanent. The absence of postdilation miosis in the monocularly atropinized adult cats suggests that the relative miotic state of the kittens' treated pupils reflects an age-dependent process. Thus, the results indicate that a "critical period" exists wherein external factors can have a lasting influence on pupil size. In this respect, Cynader 6 has reported that the pupils of kittens previously dark reared from birth until 4 months of age are miotic compared with pupils of normally reared control kittens. The permanance of the enhanced pupillary reactions of dark-reared kittens has not been investigated thoroughly; however, it was observed for at least 3 wks after the kittens were brought into the light. Because chronic mydriasis would presumably be common to both dark-reared and chronically atropinized kittens, it is tempting to suggest that the relative posttreatment miosis produced by these rearing procedures is caused by the same mechanism. The results of the 3 H-QNB binding analyses suggest at least one possible mechanism that might be responsible for the postdilation miosis observed in the atropinized kittens. The overwhelming difference in the binding properties of the treated and control iris samples was an eightfold increase in the B max calculations, which is an approximation of the number of

No. 2 Reporrs 243 muscarinic receptor sites. Such an increase in the number of receptors would lead to an enhanced response of the muscle to ACh released from the parasympathetic innervation. A decrease in affinity is also suggested by the binding data, which would appear to have an opposing effect on the muscle response characteristics. However, the decrease in receptor affinity is smaller (0.8 vs. 2.8 nm) than the increase in the number of receptors (2.9 vs. 25 fmoles/mg protein). Thus, the response of pharmacologically induced deprivation of acetylcholine input to the iris musculature is similar in several respects to the denervation supersensitivity response commonly observed in other tissues. The supersensitivity response of postsynaptic receptors to long-term deprivation of transmitter is usually characterized by an increase in receptors, particularly extrajunctional receptors that typically have a somewhat lower affinity than junctional receptors (for a review see reference 4). Therefore, our results of a dramatic increase in the number of receptors with a somewhat lower affinity may reflect a "classical" supersensitivity response induced by atropine-block of cholinergic input. The observation that the permanent effects of treatment are found only in developing kittens may suggest that other factors, such as axon sprouting, may also be involved in stabilizing the response. It should be noted that both the iris sphincter and dilator muscles of the cat, like those of numerous species, receive cholinergic innervation. 7 ' 8 However, the 3 H-QNB binding analysis used in the present study does not allow us to determine if chronic atropinization affects the muscarinic receptors in both the iris muscles equally, or whether it differentially affects the receptor populations in the sphincter and dilator muscles. Investigations are currently being conducted to localize within the iris the increase in receptor sites observed in the present study. Key words: atropine, postdilation miosis, kitten, pupil size, anisocoria, muscarinic receptors, 3 H-QNB Acknowledgment. The authors thank Cheryl Mitchell for her technical assistance with the QNB binding assays. From the College of Optometry, University of Houston, University Park,* and the Department of Neurobiology and Anatomy, University of Texas Medical School,! Houston, Texas. Supported by Research Grants EY03611, EY01139 and EY01655 from the National Eye Institute to E.L.S., R.S.H. and D.A.R., respectively. Submitted for publication: October 18,1982. Dr. Maguire's present address: Bascom Palmer Eye Institute, University of Miami, School of Medicine, Miami, Florida 33101. Reprint requests: E. L. Smith III, College of Optometry, University of Houston, University Park, Houston, TX 77004. References 1. Haddad NJ, Norma NJ, Moyer RN, and Riley, FC Jr: Mydriatic effect of phenylephrine hydrochloride. Am J Ophthalmol 70:729, 1970. 2. Gillum WN: Sympathetic stimulators and blocker. Ophthalmic Seminars 2:283, 1977. 3. Smith EL, Harwerth RS, and Maguire, GW: Effects of chronic atropinization on visual acuity in kittens. Behav Brain Res 7:307, 1983. 4. Heilbronn E and Bartfai T: Muscarinic acetylcholine receptor. Prog Neurobiol 11:171, 1978. 5. Yamamura HI and Snyder SH: Muscarinic cholinergic binding in rat brain. Proc Natl Acad Sci USA 71:1725, 1974. 6. Cynader M: Interocular alignment following visual deprivation in the cat. Invest Ophthalmol Vis Sci 18:726, 1979. 7. Laties AM and Jacobowitz D: A comparative study of the autonomic innervation of the eye in monkey, cat, and rabbit. Anat Rec 156:383, 1966. 8. Geltzer AI: Autonomic innervation of the cat iris. An electron microscopic study. Arch Ophthalmol 81:70, 1969.