permanent alterations in muscarinic receptors and pupil size

No. 2 Reports 239

Permanent Alterations in Muscarinic Receptors and Pupil SizeProduced 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 thedaily topical application of atropine. Both the kittens andthe adult cats were atropinized for a 13-week period withthe treatment regimen beginning at the time of eye openingfor the kittens. Pupil size measurements, obtained 1 yearafter the atropinization were discontinued, revealed that, al-though the pupils of the adult cats were normal, the pupilsof the kittens' treated eyes were consistently smaller thanpupils in control eyes. The status of the muscarinic receptorsin the kittens' irides was investigated using 3H-QNB bindingassays. In comparison with iris muscle homogenates fromthe control eyes, those from the treated eyes demonstratedan eightfold increase in the number of receptor binding sites.The results indicate that pupil size can be altered permanentlyby chronic mydriasis initiated early in the life of a kitten andthat the permanent change in pupil size may result, in part,from a type of permanent supersensitivity response in themuscle following chronic blockade of muscarinic transmissionby atropine. Invest Ophthalmol Vis Sci 25:239-243, 1984

Postdilation miosis is a reduction in pupillary sizerelative to pretreatment dimensions or nontreatedcontrol pupils following recovery from the primaryaction of a mydriatic agent. In humans, postdilationmiosis has been observed following the topical ad-ministration of certain sympathomimetic agents12 (eg,phenylephrine and hydroxyamphetamine) and hasbeen reported2 to persist for as long as 7 days aftertreatment.

To our knowledge, the phenomenon of postdilationmiosis has not been associated with the use of para-sympatholytic agents in humans. However, in a recentstudy3 on the effects of chronic atropinization on visualacuity in kittens, it was noted that following recoveryfrom mydriasis (approximately 3 wks posttreatment)the pupils of the kittens' treated eyes were consistentlysmaller than the pupils in the nontreated eyes. If con-firmed, this observation would not only be clinicallyimportant, it would also provide insight into the phys-iology of the iris musculature. Moreover, since chronicatropinization is an experimental procedure sometimesused for depriving an eye of a clear retinal image, itwould be potentially important in the interpretationof certain experimental results. Therefore, one of thepurposes of the present study was to verify and doc-ument the presence of postdilation miosis in chroni-cally atropinized kittens. In this respect, since the orig-inal observation was in kittens, it was important to

determine if the phenomenon was reversible and if itrepresented a developmental age-dependent process.

A second purpose of the present study was to ex-amine the mechanism by which an alteration in thetone of the iris muscles is maintained over extendedperiods of time following chronic atropinization. Thisphenomenon could result from a variety of changesin the iris including simple atrophy of certain iris mus-cles, an up-regulation of parasympathetic neuronal ac-tivity (ie, an increase in acetylcholine release from eachnerve terminal or an increase in the number of ter-minals as seen in sprouting), or an up-regulation inmuscle response (ie, an increase in muscarinic recep-tors). The in vitro binding of the irreversible muscarinicligand, 3H-quinuclidinyl benzylate (QNB), which hasbeen widely used as a measure of the number andaffinity of muscarinic receptors in neuronal and musclepreparations, provides a way to examine the status ofmuscarinic receptors in the iris (for a review see ref-erence 4). We therefore analyzed the QNB bindingactivity of iris from control and chronically atropinizedeyes. Our results suggest that the number of muscarinicreceptors is greatly increased in the treated iris and,therefore, may represent one of the mechanisms bywhich pupillary size is permanently altered afterchronic atropinization in kittens.

Materials and Methods. Eight kittens that were bornin an isolated colony and 10 additional cats that wereobtained as adults were used in the study. Six of thekittens were treated with a 1% atropine sulfate solution(four monocularly, right eye; two binocularly) on adaily basis from the time of eye opening («8-9 daysof age) until 14 wks of age. Two of the adult cats hadatropine topically applied to the right eye on a dailybasis for 13 wks. In order to maintain a maximal my-driasis, it was necessary to instill atropine into theexperimental animals' eyes three times each day. Twoof the kittens that had coloration patterns similar totheir binocularly treated littermates and the remainingeight adult cats were used as nontreated controls.Throughout the experiment, all of the animals werehoused in a normally illuminated environment (12-hr light/12-hr dark cycle).

The sizes of the animals' pupils were measured fromindividual photographs. To help minimize distractions,the animals were held 150 cm from the camera at oneend of a 50 X 50 X 150 cm white rectangular box.The interior of the box was illuminated uniformly by

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240 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1984 Vol. 25

Fig. 1. Photographs taken1 year posttreatment of amonocularly treated adult(A), a monocularly treatedkitten (B), and a binocularlytreated kitten (C, right) andits paired nontreated litter-mate (C, left).

variable intensity incandescent light sources positionedabove the camera. The outlines of the pupils weretraced from enlarged projections of the photographicnegatives, and the horizontal dimensions of the pupilswere measured to the nearest 0.5 mm with a hand-held magnifier. A correction factor to compensate forthe magnification of the camera and enlarger was de-rived from photographs of a calibrated scale positionedin the same plane as the animals' eyes. For a givenbackground level of illumination, measurements foreach pupil were obtained from a minimum of threephotographs. For the treated animals, estimates of pu-pillary size were made periodically during a 1-yearrecovery period beginning 1 month after the atropineregimen was discontinued.

At the end of the 1 -year recovery period, the treatedkittens were sacrificed by decapitation. The eyes wereremoved and hemisected. Muscles of the iris and ciliarybody were obtained by gently exerting pressure on theattachments along the outer circumference of the iriswith a blunt probe inserted through an incision in thecenter of the cornea. Iris and ciliary muscles were sep-arated with scissors. Each tissue sample was frozenovernight at -20°C.

By a modified filtration assay described by Yama-mura and Snyder,5 3H-QNB binding was determined.Frozen pieces of tissue from the irides of the treatedand nontreated eyes of the atropinized kittens werethawed, placed in 0.1 M Na-K phosphate buffer (pH7.4) and homogenized for 30 sec with a Brinkman


No. 2 Reports 241

polytron at setting 6. The tissue suspensions then werediluted with Na-K phosphate buffer (0.05 M, final con-centration) to a final volume containing 0.3-0.5 mgprotein/ml for the 3H-QNB assay. Fifty microliters of3H-QNB solution (final concentrations were 0.25 to5.0 nM; New England Nuclear, Boston, MA) and 1ml of phosphate buffer were placed in each assay tube.Twenty microliters of 10 fiM atropine (final concen-tration 0.1 /xM; Sigma Chemical Co., St. Louis, MO)were placed in selected assay tubes to determine non-specific binding. One milliliter of tissue suspensionwas added to each assay tube for a 1-hr incubationperiod at room temperature (25 °C). Incubation wasterminated by filtration on GF/C filters (Whatman,England) followed by three 5-ml washes with coldbuffer. The filters were removed, placed in vials, andallowed to dry. A toluene base counting solution con-taining Triton X-100 was added, and the filters werecounted on a Searle Mark III liquid scintillation-counter.

Results. Pupil Size: There were no obvious behav-ioral differences between the treated and nontreatedkittens, either during or after the treatment period.Casual inspection of the atropinized animals revealedthat the responsiveness of the treated pupils to changesin illumination (both direct and consensual) increasedgradually after the atropinization had been discontin-ued and that all of the treated pupils responded in anormal manner within 3 wks of the termination ofthe atropine treatment. Measurements of pupil sizeobtained 1 month posttreatment indicated that thepupils of the atropinized adult cats were essentially

equal in size (ie, the differences in pupil size betweenthe right and left eyes were within the range of sizedifferences for the 8 nontreated controls). However,the differences in pupil size for all four monocularlyatropinized kittens were outside the range for normals,the pupils of the treated eyes being consistently smallerthan the pupils of the nontreated eyes. In addition,the pupils of the two binocularly treated kittens wereessentially equal in size but smaller than the pupils oftheir paired nontreated littermates.

The normal pupil sizes observed in the atropinizedadult cats and the postdilation miosis demonstratedby the treated eyes of the kittens remained unchangedover a 1-year recovery period. The relative miotic na-ture of the kittens' treated pupils is illustrated in Figure1, where photographs taken 1 year posttreatment of amonocularly treated adult (A), a monocularly treatedkitten (B), and a binocularly treated kitten and itspaired nontreated littermate (C) are shown. The ratiosof pupil sizes (right eye/left eye) obtained for a moderatelevel of illumination (2.2 log lux) at the end of the 1-year recovery period are illustrated for each animal inFigure 2A. For the nontreated controls, the differencein the horizontal dimensions of the pupils never ex-ceeded 6.0% and, as can be seen, the size ratios forboth the monocularly treated adults and the binocularlytreated kittens fall within the range of differences forthe controls (dashed line). In contrast, the horizontaldimensions of the treated pupils of the monocularlyatropinized kittens were between 12% and 27% smallerthan those for the nontreated pupils. Similarly, boththe pupils of the two binocularly treated kittens were

A.

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8.0-

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2.0-

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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 therearing condition for each animal (filled circles). The open squares represent the ratios of the pupils of the binocularly treated kittens comparedto their paired nontreated controls. B. Horizontal dimensions of the treated (filled circles) and nontreated pupils (open circles) of a monocularlyatropinized kitten plotted as a function of background illuminance.


242 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1984 Vol. 25

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Fig. 3. Scatchard analysis of specific 3H-QNB binding in control(A) and atropine-treated (B) cat iris. The tissue was incubated in0.05 M Na-K phosphate buffer (pH 7.4, 1 hr) containing differentconcentrations of ligand, and specific binding was obtained by sub-tracting the amount of radioactivity not displaced by 0.1 fiM atropinefrom the total 3H-QNB bound. K<j's and Bmaxs, which representestimates of the affinity and number of muscarinic receptors, re-spectively, were determined by linear regression analysis.

27% and 30% smaller than the pupils of their pairednontreated littermates (open squares, Figure 2A).

The relative miotic state of the kittens' treated pupilswas independent of the level of background illumi-nation. Figure 2B shows the size of the treated (filledcircles) and nontreated pupils (open circles) of a mon-ocularly atropinized kitten plotted as a function of thebackground illumination. At all levels of illuminationconsidered, the treated pupil was smaller than the non-treated pupil. Although the absolute difference in pupilsize decreases with increasing illuminance levels, thepercentage difference remains relatively constant(«22%) across the nearly 4 log unit range of illumi-nance.

3H-QNB Binding Assay: A total often separate sam-ples of iris (five treated and five control) were subjectedto in vitro analysis of 3H-QNB binding in the presenceof 0.25 to 5 nM concentrations of the ligand. Bindingat each concentration was determined using three dif-ferent treated and control iris samples, each of whichwas assayed in triplicate. The scatchard analysis of theresults shown in Figure 3 demonstrates a significantdifference (slope, P < 0.001; intercept, P = 0.008) inthe binding characteristics of control versus treated irissamples. The treated iris samples bound significantlymore 3H-QNB in the presence of the higher concen-trations of the ligand (2-5 nM). The calculated Bmax

(an estimate of the number of muscarinic receptors)for the treated irides was eightfold higher than that forcontrols, which suggests that the increased binding isdue mainly to an increase in the number of 3H-QNBbinding sites. However, at lower concentrations of theligand (0.25-1 nM), the treated iris samples boundsignificantly less than the control samples. Thus thecalculated affinity (KJ of the receptors from the treatedsamples is approximately one-third that of the controlsamples.

Discussion. The relative miotic state noted for allof the kittens' treated pupils confirmed our earlier ob-servation of postdilation miosis in chronically atro-pinized kittens.3 Moreover, since the sizes of the kittens'treated pupils had not returned to normal by the endof the 1-year recovery period, it seems reasonable toassume that these pupillary alterations are permanent.The absence of postdilation miosis in the monocularlyatropinized adult cats suggests that the relative mioticstate of the kittens' treated pupils reflects an age-de-pendent process. Thus, the results indicate that a "crit-ical period" exists wherein external factors can havea lasting influence on pupil size. In this respect, Cy-nader6 has reported that the pupils of kittens previouslydark reared from birth until 4 months of age are mioticcompared with pupils of normally reared control kit-tens. The permanance of the enhanced pupillary re-actions of dark-reared kittens has not been investigatedthoroughly; however, it was observed for at least 3 wksafter the kittens were brought into the light. Becausechronic mydriasis would presumably be common toboth dark-reared and chronically atropinized kittens,it is tempting to suggest that the relative posttreatmentmiosis produced by these rearing procedures is causedby the same mechanism.

The results of the 3H-QNB binding analyses suggestat least one possible mechanism that might be re-sponsible for the postdilation miosis observed in theatropinized kittens. The overwhelming difference inthe binding properties of the treated and control irissamples was an eightfold increase in the Bmax calcu-lations, which is an approximation of the number of


No. 2 Reporrs 243

muscarinic receptor sites. Such an increase in thenumber of receptors would lead to an enhanced re-sponse of the muscle to ACh released from the para-sympathetic innervation. A decrease in affinity is alsosuggested by the binding data, which would appear tohave an opposing effect on the muscle response char-acteristics. However, the decrease in receptor affinityis smaller (0.8 vs. 2.8 nM) than the increase in thenumber of receptors (2.9 vs. 25 fmoles/mg protein).Thus, the response of pharmacologically induced de-privation of acetylcholine input to the iris musculatureis similar in several respects to the denervation su-persensitivity response commonly observed in othertissues. The supersensitivity response of postsynapticreceptors to long-term deprivation of transmitter isusually characterized by an increase in receptors, par-ticularly extrajunctional receptors that typically havea somewhat lower affinity than junctional receptors(for a review see reference 4). Therefore, our resultsof a dramatic increase in the number of receptors witha somewhat lower affinity may reflect a "classical"supersensitivity response induced by atropine-block ofcholinergic input. The observation that the permanenteffects of treatment are found only in developing kittensmay 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 anddilator muscles of the cat, like those of numerous spe-cies, receive cholinergic innervation.7'8 However, the3H-QNB binding analysis used in the present studydoes not allow us to determine if chronic atropinizationaffects the muscarinic receptors in both the iris musclesequally, or whether it differentially affects the receptorpopulations in the sphincter and dilator muscles. In-vestigations are currently being conducted to localize

within the iris the increase in receptor sites observedin 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, UniversityPark,* and the Department of Neurobiology and Anatomy, Universityof Texas Medical School,! Houston, Texas. Supported by ResearchGrants EY03611, EY01139 and EY01655 from the National EyeInstitute to E.L.S., R.S.H. and D.A.R., respectively. Submitted forpublication: October 18,1982. Dr. Maguire's present address: BascomPalmer Eye Institute, University of Miami, School of Medicine,Miami, Florida 33101. Reprint requests: E. L. Smith III, College ofOptometry, University of Houston, University Park, Houston, TX77004.

References

1. Haddad NJ, Norma NJ, Moyer RN, and Riley, FC Jr: Mydriaticeffect of phenylephrine hydrochloride. Am J Ophthalmol 70:729,1970.

2. Gillum WN: Sympathetic stimulators and blocker. OphthalmicSeminars 2:283, 1977.

3. Smith EL, Harwerth RS, and Maguire, GW: Effects of chronicatropinization 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 bindingin rat brain. Proc Natl Acad Sci USA 71:1725, 1974.

6. Cynader M: Interocular alignment following visual deprivationin the cat. Invest Ophthalmol Vis Sci 18:726, 1979.

7. Laties AM and Jacobowitz D: A comparative study of the au-tonomic innervation of the eye in monkey, cat, and rabbit. AnatRec 156:383, 1966.

8. Geltzer AI: Autonomic innervation of the cat iris. An electronmicroscopic study. Arch Ophthalmol 81:70, 1969.


permanent alterations in muscarinic receptors and pupil size

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