Characterization of the Day-Night Variation of Retinal Melatonin Content in the Chick

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1 Characterzaton of the Day-Nght Varaton of Retnal Melatonn Content n the Chck Steven M. Repperr and Stephen M. Sagar By montorng two tme ponts (one at md-lght and the other at md-dark), the day-nght varaton of melatonn content n the retna of 19-day-old chcks was characterzed. Melatonn was detected n the retna plus attached pgment epthelum by a specfc radommunoassay, and ts dentty was verfed by hgh performance lqud chromatography wth electrochemcal detecton. Melatonn content n the posteror pole of the eye showed a fvefold day-nght varaton, wth hgh levels durng the dark perod of durnal lghtng. Lght exposure durng the dark perod lowered the normally hgh nghttme value; mantenance of darkness durng the normal lght perod dd not alter the low melatonn values typcal of md-lght. Pneal melatonn content responded smlarly to the above lghtng manpulatons. Nether pnealectomy nor optc nerve transecton had an effect on retna-pgment epthelum melatonn or ts lght-dark rhythm. We next examned the relatve contrbutons of retnal and pneal melatonn to blood levels. Pnealectomy reduced the normally hgh md-dark plasma melatonn value by 80%. The addton of blateral enucleaton reduced the md-dark value by another 9% of control values. The day-nght varaton of retna-pgment epthelum melatonn was frst evdent n the embryo 2 days pror to hatchng and perssted through adulthood. It was concluded that the chck retna from the latter stages of embryonc development s capable of rhythmcally syntheszng melatonn; that retnal melatonn content dsplays a photcally controlled crcadan rhythm n phase wth, but ndependent of, the pneal gland; and that the retnal rhythm s not regulated by afferent optc nerve fbers. The pneal gland s the major source of plasma melatonn n the ntact chck, wth at most a small contrbuton from the retna. Invest Ophthalmol Vs Sc 24: , 1983 It s well establshed that the major source of melatonn for homootherms s the pneal gland. 19 Ths organ syntheszes melatonn from serotonn by use of two enzymes, N-acetyltransferase (NAT) and hydroxyndole-o-methyl transferase (HIOMT), wth NAT controllng the well-descrbed daly oscllatons of pneal melatonn producton n rats 10 and chckens. 1 The pneal melatonn rhythm, n turn, s reflected accurately n blood, urne, and cerebrospnal flud, wth hgh levels occurrng at nght. 19 Interestngly, the retnae of a number of vertebrate speces have been known for some tme to contan the enzymatc machnery necessary for melatonn bosynthess. 715 A resurgence of nterest n ths retnal capablty has led to the recent fndngs of large daly rhythms n NAT actvty 4 and melatonn content 8 n the chck retna. From the Neuroendocrne Research Laboratory, Chldren's and Neurology Servces, Massachusetts General Hosptal and Harvard Medcal School, Boston, Massachusetts. Supported by PHS grant HD and a postdoctoral fellowshp from the Natonal Eye Insttute. Steven M. Reppert s a research fellow of the Charles A. Kng Trust, Boston, Massachusetts. Submtted for publcaton July 27, Reprnt requests: Steven M. Reppert, Chldren's Servce, Massachusetts General Hosptal, Boston, MA Our nterests n melatonn physology and the potental endocrne functons of the retna led us to characterze the retnal rhythm of melatonn content n the chck by examnng ts response to alteratons n envronmental lghtng, by delneatng the nfluence of the pneal gland and of retnal optc nerve afferents n the generaton of the eye rhythm, and by studyng the ontogeny (embryo through adult) of the day-nght varaton n retnal melatonn content. An mportant am of our studes was to assess the relatve contrbutons of retnal and pneal melatonn to crculatng levels of the hormone. Materals and Methods Anmals Zero day fertle eggs and 12-day-old Whte Leghorn Chcks {Gallus domestcus) were purchased from Spafas, Inc. (Norwch, CT). Eggs were kept n a warmed (37 C), humdfed ncubator equpped wth an automated lghtng system; ntensty of llumnaton at the md-porton of the ncubator was 600 lux. Chcks were communally housed n a temperature-controlled brooder that was quartered n a lght-controlled room; ntensty of llumnaton was 400 to 600 lux at the food and water bns. Blnd chcks (see $ 1.15 Assocaton for Research n Vson and Ophthalmology 294 Downloaded From: on

2 No. 3 RETINAL MELATONIN IN THE CHICK Repperr ond Sogor , 10 Md-lght Md-dark Lght at nght Dark durng day Pneal Eye reotaxc frame. All surgcal procedures were performed at 14 to 15 days of age. Pnealectomy was performed by the technque of Bnkley et al. 5 For sham operatons, the skn over the head was ncsed, the skull etched wth a trephne, and the ncson closed. Completeness of pnealectomy was corrfrmed on postmortem examnaton. Blndng was accomplshed by blateral orbtal enucleaton. Followng enucleaton, the vacant orbt was packed wth gel foam and eyelds were sutured closed. For optc nerve transecton, each optc nerve was vsualzed drectly by medal retracton of the globe through a ventrolateral ncson of the lower eyeld. The optc nerve was transected wth an rs scssors. The opposte eye, n whch the optc nerve was vsualzed but not cut, served as a control. Optc nerve transecton was confrmed on postmortem examnaton. 1 ^.3 s.15 0 fa Plasma Fg. 1. Photc regulaton of pneal, ocular, and plasma melatonn. At 19 days of age anmals were klled at md-lght or md-dark. Anmals were also klled at the subjectve md-dark tme wth lghts left on throughout the expermental dark perod or at the subjectve md-lght tme wth lghts left off throughout the expermental lght perod. Eye (mddle panel) refers to the posteror hemsphere of one eye. Data are presented as mean ± SE of sx to eght chcks n each group. "Surgcal Procedures") were housed wthn a fenced off porton of the communal brooder n order to protect them from sghted anmals. Purna Chck Starteena and water were avalable at all tmes. Unless otherwse specfed, the daly lght-dark cycle conssted of 12 hrs of lght per day (LD 12:12) wth lghts on from 0600 hrs EST. For experments nvolvng hens, anmals were reared by the anmal husbandry department of the Unversty of Connectcut at Storrs and housed there n the durnal lghtng cycle descrbed above. Surgcal Procedures Chcks were anesthetzed wth pentobarbtal (40 mgkg, IM), and the heads were secured n a rat ste- m Sample Collecton At the end of an experment, anmals were klled by decaptaton; kllng n darkness was accomplshed wth the ad of a low ntensty red lght (Kodak safe lght flter no. 2). Trunk blood was collected n tubes contanng 100 unts sodum heparn and centrfuged; the resultng plasma was decanted and stored at -20 C untl analyss. The pneal gland was removed rapdly, frozen on dry ce, and stored at -20 C. Eyes were removed from the skull and placed on ce. The globes were hemsected at the equator, and the vtreous was removed. For the experments depcted n Fgures 1 and 2, the posteror hemsphere was frozen on dry ce and stored at 20 C. For all subsequent experments, the retna plus attached pgment epthelum was dssected out and stored as above. We found that the eye could reman at 0 C for up to 3 hrs pror to dssecton wthout any sgnfcant change n melatonn concentraton. Melatonn Radommunoassay (RIA) Melatonn was measured usng the assay procedure of Rollag and Nswender 22 (antserum 1055) as modfed by Reppert et al. 20 At the tme of analyss, tssues were soncated for 5 sec n the followng volumes of PBS (0.01 M phosphate buffer wth 0.9% NaCl, ph 7.0): pneals, 120 A; retna pgment epthelum, 1 ml; and posteror pole, 2 ml. The pneal soncates were then dluted 1:40 n PBS. Melatonn from ether a 200- or 400-jl porton of each soncate was extracted nto 5 ml chloroform. The chloroform was washed, and melatonn quantfed as prevously descrbed. 20 For measurement of melatonn n chck plasma, an addtonal extracton step usng petroleum ether was used. 20 The assay procedure for plasma melatonn Downloaded From: on

3 296 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE March 1983 Vol. 24 Sham M Pnx was valdated by demonstratng (1) parallel nhbton curves between seral dlutons of extracted nghttme plasma samples and the melatonn standard, and (2) quanttatve recovery of 125 pg, 250 pg, and 500 pg of melatonn from 1 -ml samples of pooled daytme plasma. The extracton effcency for all samples was greater than 95%; values reported are not corrected for the small loss durng extracton. The lmt of assay senstvty was 0.5 pgtube. The wthn and between assay coeffcents of varaton were 10 and 15%, respectvely. Chemcal Valdaton of Retnal Melatonn by Hgh- Performance Lqud Chromatography (HPLC) 0 Md-lght Md-dark Fg. 2. Effects of pnealectomy (Pnx) on the day-nght varaton of ocular melatonn. At 14 days of age, anmals were dvded nto two groups; one group was pnealectomzed and the other receved sham operatons. Fve days later anmals from each group were klled at md-lght or md-dark. At each tme pont, eght pnealectomzed anmals and four sham anmals were used. Data are presented as mean ± SE. 4r Radommunoassay analyss of retna-pgment epthelum melatonn was corroborated by HPLC wth electrochemcal detecton usng the method of Mefford and Barchas 1 ' wth modfcatons (Fg. 3). Pooled chloroform extracts of ocular tssue were dred under vacuum n darkness and redssolved n 0.1 ml HC1. After centrfugaton, alquots of the supernatant were njected nto a 0.4 X 30 cm mcron Bondapak C 8 reversed phase column (Waters Assoc, Mlford, MA) eluted wth a moble phase consstng of 0.1 M sodum acetate buffer (ph 4.7), 0.1 mm EDTA, and 25% methanol at a flow rate of 1.5 mlmn. A LC-4 electrochemcal detector (Boanalytcal Systems, West Lafayette, IN) wth a glassy carbon electrode was used at a potental of V. Peaks were dentfed by retenton tme and melatonn quantfed by peak heght. 1 MEL Expermental Protocols Two tme ponts (one at md-lght and the other at md-dark) were used to montor daly melatonn rhythms n the eye, pneal, and plasma. These tme ponts correspond to the nadr and peak, respectvely, of the daly melatonn rhythms n all three tssues n the chcken, as studed n durnal lghtng. 2 ' 814 The term ocular melatonn s used n the text to sgnfy melatonn content n the posteror hemsphere of the eye. Other expermental detals appear n the "Results" secton and the fgure legends. Statstcal Methods 0 L 20 O 20 to 0 TIME (mm) Fg. 3. Hgh performance lqud chromatograms of (a) 10 pmoles each of 5-hydroxytryptophan (5-HT), N-acetyl serotonn (NAS) and melatonn (MEL); b, pooled extracts of retna-pgment epthelum of chcks klled at md-dark; and c, pooled extracts of retna-pgment epthelum of chcks klled at md-lght. Melatonn concentratons calculated from the chromatograms were 1.3 ng retna at md-lght and 3.9 ngretna at md-dark. The same extracts analyzed by RIA gave values of 1.2 and 5.0 ngretna, respectvely. Statstcal analyses were performed usng the Mann- Whtney UWlcoxon nonparametrc test. Results Photc Regulaton of Pneal, Ocular, and Plasma Melatonn Day-nght varatons of melatonn concentratons n the pneal gland, eye, and plasma were examned n chcks exposed to durnal lghtng and klled at Downloaded From: on

4 No. 3 RETINAL MELATONIN IN THE CHICK Repperr and Sogor 297 ether md-lght or md-dark. In ths lghtng cycle, there are promnent, synchronous day-nght oscllatons n pneal, ocular, and plasma melatonn, wth hgh values at nght (Fg. 1); the magntudes of the nocturnal excursons are 17-fold n the pneal, fvefold n the eye, and tenfold n plasma. Lght suppresson of nocturnal melatonn producton was examned n the chck by kllng anmals at the md-dark tme wth lghts left on throughout the expermental dark perod. Ths manpulaton suppresses plasma and ocular melatonn levels such that they become smlar to normal md-lght values (Fg. 1). The treatment also lowers pneal melatonn levels, but not to normal mdday values. To determne whether melatonn levels can decrease wthout a lght cue, anmals were klled at the md-lght tme wth lghts left off throughout the expermental lght perod. Ths results n low ocular and plasma melatonn levels lke those normally found durng the lght porton of the day (Fg. 1). Pneal melatonn content s also reduced, compared to mddark levels, n ths envronment, but the resultng value s hgher than that usually found at md-lght. I ṣ 2 Sham H Pnx 7\ Pnx and Blnded Effects of Pnealectomy on Ocular and Plasma Melatonn Levels Pnealectomy does not alter the day-nght varaton n ocular melatonn content (Fg. 2). In contrast, pneal removal has a marked effect on the normally hgh md-dark plasma melatonn concentraton, lowerng t by 80% (Fg. 4). Pnealectomy does not, however, alter the normally low md-lght plasma melatonn level. Interestngly, a small but sgnfcant (P < 0.01) day-nght varaton n plasma melatonn levels, wth the hgh value at nght, s stll evdent n pnealectomzed chcks. Effects of Pnealectomy plus Blndng on Plasma Melatonn To examne the contrbuton of the retna to crculatng melatonn levels, blnd-pnealectomzed anmals were klled at ether md-lght or md-dark, and ther crculatng melatonn concentratons compared to those of pnealectomzed anmals. Beyond the reducton ncurred by pnealectomy alone (see precedng secton), blndng causes a further 9% reducton of the md-dark plasma melatonn value (Fg. 4). Ths procedure does not alter the mean md-lght plasma melatonn concentraton. Blnd-pnealectomzed chcks do not appear to exhbt a day-nght varaton n plasma melatonn levels. Effects of Optc Nerve Transecton on Retna- Pgment Epthelum Melatonn The role of optc nerve afferents to the retna n the generaton of the day-nght varaton n retna- 0 Md-lght Md-dark Fg. 4. Effects of pnealectomy (Pnx) and blndng plus pnealectomy on the day-nght varaton n plasma melatonn. At 14 days of age, groups of anmals receved one of three operatons: pnealxtomy, pnealectomy plus blndng, or sham pnealectomy. Fve days later, anmals from each group were klled at md-lght or md-dark. The data depcted represent a computaton of results from three separate experments. Data are presented as mean ± SE, and the number of anmals s depcted at the base of each bar. *P < 0.01; tp < pgment epthelum was also examned. As shown n Fgure 5, optc nerve transecton does not affect the eye rhythm, as studed under durnal lghtng condtons. Ontogeny of the Day-Nght Varaton n Retna- Pgment Epthelum Melatonn Developmentally, a sgnfcant (P < 0.01) day-nght varaton n retna-pgment epthelum melatonn content, wth hgh nghttme levels, s frst detected n the late embryo, 2 days pror to hatchng (Fg. 6). The ncrease n the magntude of the varaton durng maturaton results prmarly from an ncrease n the md-dark value. A smlar developmental pattern was found for pneal melatonn content (data not shown). Downloaded From: on

5 298 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE March 1983 Vol. 24 Optc Nerve Transecton pneal gland of each anmal, we have shown that the day-nght varatons n both organs are synchronous and regulated by envronmental lghtng n a smlar manner; hgh nghttme levels are suppressed by lght whereas the rhythms persst n darkness. We also dssected retna from pgment epthelum and found, as others have, 15 ' 8 that n the chck ocular melatonn and HIOMT are localzed to the retna (Sagar and Reppert, unpublshed data). The results of our pnealectomy experment show that the pneal gland s not the source of retnal melatonn snce ocular melatonn levels contnue to oscllate n anmals lackng pneals. Taken together, the fndngs to date ndcate that the chck retna rhythmcally syntheszes melatonn and that retnal melatonn content dsplays a photcally controlled crcadan rhythm n phase wth, but ndependent of, the pneal rhythm. The results of our developmental study suggest that melatonn s produced rhythmcally by the retna as early as the latter stages of embryonc development. Ths concdes wth the tme that day-nght varatons Md-lght Md-dark Fg. 5. Effects of optc nerve transecton on the day-nght varaton of retna-pgment epthelum melatonn. Optc nerve transecton (rght eye) and sham operaton (left eye) were performed n each anmal at 14 days of age. Fve days later anmals were klled at md-lght or md-dark. Data are the mean ± SE of sx to eght anmals n each group. Examnaton of retna-pgment epthelum melatonn content n hens showed that the day-nght varaton s promnently manfested n the adult anmal. Dscusson After Bnkley et al 4 dscovered a promnent daly rhythm n NAT actvty n the chck retna, Hamm and Menaker 8 showed that both NAT actvty and melatonn content exhbt synchronous daly retnal rhythms n ths speces and respond smlarly to manpulatons of the daly lght-dark cycle. They also showed that the retnal NAT rhythm perssts after pnealectomy. The results presented here confrm and extend those observatons. By montorng melatonn concentratons at two tme ponts n the posteror pole of the eye and n the ^S "7 - <b ( L- I 2 - s I 0 y 0 0 f I Md-lght Md-dark o A I - l -5 Brth Adult Age (d) Fg. 6. Developmental pattern of the day-nght varaton n retna-pgment epthelum melatonn. At 16 and 19 days of ncubaton, embryos were removed from ther eggs and klled at md-lght or md-dark. On the day of brth and at 2 and 19 days of age chcks were klled at md-lght or md-dark. The adult anmals were hens housed n durnal lghtng and klled at md-lght or md-dark. Data are the mean ± SE of fve to sx anmals n each group. *P < Downloaded From: on

6 No. 3 RETINAL MELATONIN IN THE CHICK Repperr and Sagar 299 n pneal NAT actvty 3 and n pneal melatonn content are frst detectable n the chck embryo. Thus, t seems that the rhythmc producton of melatonn by the retna and pneal gland exhbt smlar developmental patterns. An nterestng queston arses as to the ste of the endogenous oscllator that generates the retnal rhythm. It does not resde n the pneal gland snce the retnal rhythm perssts after pnealectomy. Another possblty s that the rhythm mght be generated by neural nput from the bran. One way that neural nformaton could reach the retna s va optc nerve afferents, whch are prevalent n avan speces. 21 Ths does not appear to be the case, however, snce optc nerve transecton does not affect the eye rhythm n durnal lghtng. Other possble neural nputs to the retna that could be consdered n ths regard, such as sympathetc fbers, requre further evaluaton. It s worth notng that Bnkley and coworkers 6 have shown by usng patch experments and montorng retnal NAT actvty that wthn an ndvdual chck the two retnae can respond to lght ndependently of each other, suggestng that each eye contans an endogenous mechansm for rhythm generaton. A major focus of our study was to examne the contrbuton of retnal melatonn to crculatng concentratons of the substance. Such a contrbuton seemed possble snce melatonn readly passes through cell membranes and many bologc barrers, ncludng the blood-bran barrer and the placenta 18 ; ths dffusblty s due to the nonpolar, lpophlc nature of ths small molecule (mol. wt. 232). Also, based on studes wth the rat pneal gland, melatonn s not stored n secretory vescles, but nstead dffuses rapdly nto blood upon producton We examned the contrbuton of retnal melatonn to blood levels by frst showng that our RIA system detects a large day-nght varaton n plasma melatonn concentratons n the ntact chck (Fg. 1). The varaton that we found (mean md-day value of 31 pgml; mean md-dark value of 322 pgml) s n good agreement wth those reported by three dfferent laboratores (range of md-lght values 10 to 50 pgml; range of md-dark values 210 to 350 pgml) where boassay 17 and two other melatonn RIA systems were used. 912 We next showed that the pneal gland s the major source of crculatng melatonn concentratons at nght, snce the ncrease at md-dark s nearly abolshed n pnealectomzed anmals; ths agan s consstent wth an earler report. 14 We also found that retnal melatonn contrbutes at best a small, albet statstcally sgnfcant, amount of melatonn to the md-dark crculatng levels. Furthermore, anmals lackng pneals exhbt a detectable day-nght varaton n blood melatonn, wth the hgher levels at nght. Whether ths small varaton actually represents a daly rhythm of retnal orgn or s of physologc sgnfcance s not possble to determne from the two tme ponts examned n our experment. Based on the bophyscal propertes of melatonn dscussed above, the apparently small retnal contrbuton to crculatng levels of the hormone was unexpected. If one estmates from Fgure 1 the amount of melatonn produced by each eye, assumng that melatonn levels reflect producton rates n the retna, as suggested by the parallel rses and falls of NAT actvty and melatonn content n the retna, 8 then we would have expected both retnae together to be responsble for roughly 50% of the hgh nghttme crculatng melatonn concentratons. That ths s not the case s dffcult to explan. A possble explanaton s that retnal melatonn mght be metabolzed locally wthn the eye. Snce our results suggest that the contrbuton of retnal melatonn to blood s qute small, the most lkely functon of retnally derved melatonn s n the regulaton of rhythmc phenomena wthn the eye tself. There are three processes n the retna and pgment epthelum of varous speces known to dsplay crcadan rhythms: pgment mgraton, the elongaton and contracton of the nner segment of photoreceptors (retnomotor movements), and rod outer segment dsk sheddng There s some evdence n nonavan speces that exogenously admnstered melatonn can nfluence each of these processes. Pgment mgraton n mammals 13 and retnomotor movements n amphbans 16 can be nfluenced by melatonn. Moreover, rod outer segment sheddng n rats can be modulated by systemcally admnstered melatonn. 23 The role of retnal melatonn n the regulaton of these mechansms n the chck deserves systematc study. Furthermore, the hypothess that n other speces ncludng mammals, the retna may be a target organ for melatonn should be tested. Key words: retna, pneal gland, melatonn, crcadan rhythms, chcks References 1. Bnkley S: Pneal gland borhythms: N-acetyltransferase n chckens and rats. Fed Proc 35:2347, Bnkley S, MacBrde SE, Klen DC, and Ralph CL: Pneal enzymes: Regulaton of avan melatonn synthess. Scence 181:273, Bnkley S and Geller EB: Pneal enzymes n chckens: Development of daly rhythmcty. Gen Comp Endocrnol 27:424, Bnkley S, Hryshchyshyn M, and Relly K: N-acetyltransferase actvty responds to envronmental lghtng n the eye as well as n the pneal gland. Nature 281:479, Bnkley S, Kluth E, and Menaker M: Pneal functon n sparrows: crcadan rhythms and body temperature. Scence 174:311, Bnkley S, Relly K, and Hernandez T: N-acetyltransferase n the chck retna. II. Interactons of the eyes and pneal gland Downloaded From: on

7 300 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE March 1983 Vol. 24 n response to lght. J Comp Physol 140:181, Cardnal DP and Rosner JM: Retnal localzaton of the hydroxyndole-o-methyl transferase (HIOMT) n the rat. Endocrnology 89:301, Hamm HE and Menaker M: Retnal rhythms n chcks: Crcadan varaton n melatonn and serotonn N-acetyltransferase actvty. Proc Natl Acad Sc USA 77:4948, Kennaway DJ, Frth RG, Phllpou G, Matthews CD, and Seamark RF: A specfc radommunoassay for melatonn n bologcal tssue and fluds and ts valdaton by gas chromatography-mass spectrometry. Endocrnology 101:119, Klen DC: The pneal gland: A model of neuroendocrne regulaton. Res Publ Assoc Res Nerv Ment Ds 56:303, Mefford IN and Barchas JD: Determnaton of tryptophan and metaboltes n rat bran and pneal tssue by reversed-phase hgh-performance lqud chromatography wth electrochemcal detecton. J Chromatogr 181:187, Pang SF, Brown GM, Grota LJ, Chambers JW, and Rodman RL: Determnaton of N-acetylserotonn and melatonn actvtes n the pneal gland, retna, harderan gland, bran and serum of rats and chckens. Neuroendocrnology 23:1, Pang SF and Yew DT: Pgment aggregaton by melatonn n the retnal pgment epthelum and chorod of gunea-pgs, Cava porcellus. Experenta 35:231, Pelham RW: A serum melatonn rhythm n chckens and ts abolton by pnealectomy. Endocrnology 96:543, Quay WB: Retnal and pneal hydroxyndole-o-methyl transferase actvty n vertebrates. Lfe Sc 4:983, Quay WB and McLoed RW: Melatonn and photc stmulaton of cone contracton n the retna of larval Xenopus laevs. Anat Rec 160:491, Ralph CL, Pelham RW, MacBrde SE, and Relly DP: Persstent rhythms of pneal and serum melatonn n cockerels n contnuous darkness. J Endocrnol 63:319, Reppert SM, Chez RA, Anderson A, and Klen DC: Maternalfetal transfer of melatonn n the non-human prmate. Pedatr Res 13:788, Reppert SM and Klen DC: Mammalan pneal gland: basc and clncal aspects. In The Endocrne Functons of the Bran. M Motta, edtor. New York, Raven Press, 1980, p Reppert SM, Perlow MJ, Tamarkn L, and Klen DC: A durnal melatonn rhythm n prmate cerebrospnal flud. Endocrnology 104:295, Rodeck RW: The Vertebrate Retna; Prncples of Structure and Functon. San Francsco, W. H. Freeman and Co, Rollag MD and Nswender GD: Radommunoassay of serum concentratons of melatonn n sheep exposed to dfferent lghtng regmens. Endocrnology 98:482, Whte MP and Fsher LJ: Melatonn effects on crcadan rod outer segment sheddng. Neuroscence Abstracts 6:344, Young RW: The daly rhythm of sheddng and degradaton of rod and cone outer segment membranes n the chck retna. Invest Ophthalmol Vs Sc 17:105, Downloaded From: on