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Melatonin and rhythmic photoreceptor metabolism: melatonininduced cone elonption is blocked at high light intensity Mary E. Pierce and Joseph C. Besharse Department of Anatomy and Cell Biology, Emory UniversitySchool of Medicine, Atlanta. GA 30322 (U. S.A .) (Accepted 4 November 1986) Key words: Retinomotor movement; Cone; Melatonin; Light intensity

We have proposed a model for circadian regulation of cone position in Xenopus laevis that involves interaction of melatonin and dopamine as signals for darkness and light respectively. One problem, however, is that the effects of melatonin have not been detected in eye cups prepared from animals maintained on a cyclic light schedule. Since melatonin's effect would be expected to occur in low light intensity at night, we have investigated the relationship among melatonin, light intensity, and cone length. We report that melatonin mimics the effects of darkness and stimulates cone elongation in eye cups from cyclic light animals incubated at low but not at high light intensities. We have extensively utilized an in vitro eye cup preparation from the African clawed frog, Xenopus laevis, to investigate rhythmic photoreceptor metabolism including disc shedding 3-5, cone photoreceptor movements 3,26-3° and melatonin biosynthesis 6'7'1s-2l. The view is emerging that melatonin and dopamine may be integral components of a regulatory feedback system controlling rhythmic events in the retina 18'26. Melatonin may be a signal for darkness while dopamine may be a signal for light 28. In the retina, melatonin is synthesized and released at night 8A6,x9,2t,25. The rhythmic activity of serotonin N-acetyltransferase (NAT), a key enzyme in the synthesis of melatonin, exhibits both sustained oscillation with peak activity at night and entrainment in the Xenopus in vitro preparation 7. Dopamine is also synthesized rhythmically in some retinas, but peak synthetic activity occurs in the daytime 22'31' 32. Melatonin inhibits the release of dopamine in rabbit and chick retinas 14'15, and decreases levels of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid in Xenopus eye cups 3°. D o p a m i n e also regulates melatonin biosynthesis2°: it inhibits the nighttime rise in N A T activity through a mechanism involving D 2

receptors is. We have proposed that interactions between melatonin and dopamine are responsible for the regulation of cone photoreceptor position in Xenopus eye cups 28. At night, cone myoids elongate, while in the daytime they contract 1. In eye cups prepared from 4-day constant light-treated Xenopus. melatonin mimics the effects of darkness and stimulates cone elongation, perhaps by modulating the dopaminergic system and/or by directly affecting cones 2s. Conversely, dopamine mimics the effects of light; it induces cone contraction, blocks dark-induced elongation. and inhibits melatonin-induced elongation. The dopamine response ts mediated by a specific D 2 receptor 28 which is known to reduce levels of c A M P 23, a condition necessary for reactivated contraction in teleost detergent-lysed cone photoreceptors 9. Dopamine-induced contraction has been reported for an isolated fish cone photoreceptor inner/outer segment preparation, suggesting that d o p a m m e receptors are located on photoreceptors~°. One problem with the m e l a t o n i n - d o p a m i n e model has been the inability to detect an effect of melatonin at subjective night in eye cups prepared from cy-

Correspondence: M.E. Pierce, Department of Anatomy and Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, U.S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

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Fig. 1. Melatonin induces cone elongation. Eye cups were prepared from animals maintained on a 12-h light: 12-h dark schedule at the time of light offset and maintained in culture for 3 h at a light intensity of 7 x 10 -7 W / c m 2 (clear bar) or in darkness (hatched bar). Melatonin (0.5 pM) was added at the beginning of the incubation in light. Data are expressed as a percent change in cone length as compared to the light control (see ref. 26). Melatonin and darkness induce cone elongation (P < 0.001 compared to light control). clic light-treated animals 28. Its effects, although highly potent, have only been seen in eye cups prepared from constant light-treated animals. Our failure to detect an effect in cyclic light-treated animals, even though incubations in darkness clearly result in cone elongation, has raised questions regarding melatonin's normal role 28. In fact, a role for melatonin has been seriously questioned, based on the failure to detect melatonin-induced cone elongation in isolated retinas from cyclic light-treated green sunfish during subjective day. Since the effects of melatonin would normally be expected to occur at night in low light intensity when melatonin is normally synthesized, we wondered if the light intensities used to evaluate its effects experimentally might provide essentially non-permissive conditions for cone elongation. To evaluate this, we investigated the relationship among cone position, light intensity, and melatonin's effect. We report here that in eye cups prepared at the time of normal light offset from cyclic light-maintained Xenopus, melatonin-induced cone elongation occurs only at light intensities below those used in prior experiments. Experimental details have been published previously 2s. Briefly, Xenopus laevis (Fort Atkinson,

WI) were maintained in the laboratory for at least one month on a 12-h light (2 x 10 -4 W/cm2): 12-h dark schedule. Eye cups were prepared at the time of normal light offset by surgical removal of the cornea, iris, and lens. A defined culture medium supplemented with 35 mM NaHCO3, 100 p M ascorbic acid and gassed with 5% CO2/95% O2 (pH 7.4) was used during dissections and incubation. Melatonin (Sigma, St. Louis, MO) was added to culture medium just prior to use. Eye cups were incubated in a gassed incubation chamber on a rotary shaker for 3 h. The chamber was illuminated with white light from a tungsten-halogen lamp (Oriel Corp., Stamford, CN). Intensity was attenuated during the in vitro incubation by using calibrated neutral density filters (Oriel) and measured with an IL700 Research Radiometer (International Light, Newburyport, MA). Eye cups were fixed in a mixture of 1.65% glutaraldehyde and 1% OsO4 in 75 mM cacodylate buffer (pH 7.4), embedded in epoxy resins, and cone length was measured from light microscopic viewing of 1-pm plastic sections. Melatonin mimics the effects of darkness and induces cone elongation when eye cups from cyclic light-treated animals are incubated at a lower light intensity (Fig. 1). Eye cups were prepared at the time of normal light offset and when incubated for 3 h in light at 7 x 10-7 W/cm 2 cones remained contracted. Melatonin (0.5 pM) stimulated cone elongation in this light condition to lengths comparable to those achieved during a 3-h dark treatment. The fact that melatonin is a potent effector of elongation at a time when cones normally elongate and when melatonin synthesis in the retina is normally increasing supports our prior proposal 28 that melatonin or a related indoleamine may be a key component of the night-time signal leading to elongation. Fig. 2 is a composite of a series of experiments designed to determine the range of light intensities over which melatonin is effective. The solid line shows cone length in eye cups incubated without melatonin. Cone length was operationally defined as the distance from the outer limiting membrane to the base of the oil droplet found in the cone inner segment. Hence, in light, cone length is generally 10-15 pm. Cones elongate at intensities below 8 x 10-s W/cm z. This corresponds to a photon flux of about 2200 photons//~m2/s.

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Fig. 2. Melatonin induces cone elongation over a range of intensities. Eye cyps incubated without melatonin maintain their contracted lengths in light intensities at or above 7 × 10-s W/era2 (solid line). Melatonin (0.5/tM) causes cone to elongate at light intensities below 2 x 10-5 W/cm2 (dotted line). Cone length in the dark is shown at the far right of the figure. Actual cone length (urn) is plotted.

Cone position after dark treatment is shown at the far right of Fig. 2; total excursions are generally 25-30 ktm. The dotted line indicates cone position in eye cups incubated with 0.5/~M melatonin. Melatonin stimulated cone elongation at intensities of 2 × 10-5 W/cm 2 (5.5 × 105 photons//~m2/s) and lower, but not at the higher intensities examined. It is important to note that the effect of melatonin is overriden by light intensities which are much higher than those required to maintain cones in the light contracted state. It has been reported that melatonin does not stimulate cone movement in cyclic light-maintained teleost retinas u. However, the light intensity used in those experiments was even higher than our highest intensity. In the Atlantic salmon, Salmo salar, the response of cones to different light intensities has been measured in vivo2; cones elongate spontaneously at intensities lower than 3 × 10-3 W/cm 2. Our results suggest that to assess the role of melatonin in retinal circadian events it may be necessary to determine its effects over a range of light intensities. One possible explanation of why melatonin would be effective at low light intensity but not at high, is that as light intensity decreases more endogenous melatonin is released; the effect seen would simply be related to local melatonin concentration. If this were true, a higher concentration of melatonin would be expected to induce elongation at the higher inten-

sity. Our data suggest that this is not the case. Increasing the concentration of melatonin to 200 ,uM does not stimulate movement at a light intensity of 2 x 10-4 W/cm 2. Another possibility more consistent with our observations is that melatonin interacts either directly or indirectly with another dark effector whose effect also depends on light intensity. When eye cups are incubated at I(V 3 W/cm 2, in medium containing both melatonin and the G A B A agonist muscimol, cones elongated, although neither drug alone was effective at the high light intensity >. Muscimol-induced cone elongation is also light-intensitydependent 27. In our earlier experiments using eye cups from animals maintained for 4 days in constant light, melatonin stimulated cone elongation even when incubated in light of a high intensity (10 -3 W/cm2) 28. The difference in sensitivity between cyclic and constant light animals may be due to a hypersensitization to melatonin or to a down regulation of the dopaminergic system in constant light conditions. Constant light treatment not only blocks circadian dark-adaptive photoreceptor movements 3, it also inhibits rhythmic N A T activity 21 and decreases levels of immunoreactive melatonin in the retina ~3,24. Furthermore, constant light conditions decrease the dopamine receptor number ~2'~3. It is possible that the constant light preparation is biased towards responding to dark-adaptive signals. It is not self-evident from our experiments what the relationship is between cone function and the light intensities below which cones either elongate in response to melatonin (2 x 10 -5 W/cm 2) or elongate spontaneously (8 x 10 -s W/cm2). Comparisons to other studies are complicated by differences in the way intensity measurements were reported, by the lack of knowledge of the duration of exposure to adapting lights, and by a tack of information about the time of day that experiments were conducted. Nonetheless, it seems likely that melatonin's effect is related to the adaptational state of the cone. For example, the intensity above which melatonin's effect is blocked in our experiments corresponds to a photon flux of about 5.5 x 105 photons/ktm2/s. In Rana pipiens a background white light of similar intensity used to adapt photoreceptors is approximately the threshold above which the intensity of a 630-nm test flash must be increased to elicit a threshold response

403 from cones17; that is, cones are measurably light-

ness as an activator of photoreceptor disc shedding in

adapted above this intensity. In summary, the results reported here demon-

Xenopus eye cups, suggesting that melatonin may

strate that m e l a t o n i n - i n d u c e d cone elongation in eye cups prepared from cyclic light-treated X e n o p u s laevis is blocked by a light intensity above 5 x 10-5 W/cm:. A t lower intensities, m el at o n i n stimulates

also play an important role in the dark-dependent process required for the initiation of disc shedding 5. Melatonin may be the h o r m o n a l analog for darkness in the retina, and its circadian synthesis and release may drive or time other rhythmic events in the retina.

cone elongation comparable to darkness. Melatonin stimulates m o v e m e n t at a time (light offset) when We thank Lou DeFelice for helpful discussions and

melatonin levels are thought to be increasing in the retina, supporting our hypothesis that m el at o n i n is an

Shirley Obie for typing this manuscript. This re-

integral part of the dark signal for cone elongation.

search was supported by a National Eye Institute

Furthermore, melatonin can be substituted for dark-

Grant EY02414.

1 Ali, M.A., Retinomotor responses. In M.A. Ali (Ed.), V/sion in Fishes: New Approaches in Research, Plenum Press, New York, 1975, pp. 313-355. 2 Ali, M.A., Histophysiological studies on the juvenile Atlantic salmon (Salmo salar) retina H. Responses to light intensities, wavelengths, temperatures and continuous light or dark, Can. J. Zool., 39 (1961) 511-526. 3 Besharse, J.C., The daily light-dark cycle and rhythmic metabolism in the photoreceptor-pigment epithelial complex. In N. Osborne and G. Chader, (Eds.), Progress in Retinal Research, Vol. 1, Pergamon Press, New York, 1982, pp. 81-124. 4 Besharse, J.C. and Dunis, D.A., Photoreceptor disc shedding in eye cups: relationship to bicarbonate and amino acids, Exp. Eye Res., 36 (1983) 567-580. 5 Besharse, J.C. and Dunis, D.A., Methoxyindoles and photoreceptor metabolism: activation of rod shedding, Science, 219 (1983) 1341-1343. 6 Besharse, J.C., Dunis, D.A. and Iuvone, P.M., Regulation and possible role of serotonin N-acetyltransferase in the retina, Fed. Proc., 43 (1984) 2704-2708. 7 Besharse, J.C. and Iuvone, P.M., Circadian clock in the Xenopus eye controlling retinal serotonin N-acetyltransferase, Nature (London), 305 (1983) 133-135. 8 Binkley, S., Reilly, K.B. and Hryshchyshyn, M., N-acetyltransferase in the chick retina. I. Circadian rhythms controlled by environmental lighting are similar to those in the pineal gland, J. Comp. Physiol., B. Biochem. System. Environ. Physiol., 139 (1980) 103-108. 9 Burnside, B., Smith, B., Nagata, M. and Porrello, K., Reactivation of contraction in detergent-lysed teleost retinal cones, J. Cell Biol., 92 (1982) 199-206. 10 Dearry, A. and Burnside, B., Dopaminergic regulation of cone retinomotor movement: I. Induction of cone contraction is mediated by D2 receptors, J. Neurochem., 46 (1986) 1006-1021. 11 Dearry, A. and Burnside, B., Dopaminergic regulation of cone retinomotor movements: II. Modulation by ~'-aminobutyric acid and serotonin, J. Neurochem., 46 (1986) 1022-1031. 12 DeMello, M.C.F., Ventura, A.L.M., Paes de Carvalho, R., Klein, W.L. and deMello, F.G., Regulation of dopamine and adenosine-dependent adenylate cyclase systems of chicken embryo retinal cells in culture, Proc. Natl. Acad.

Sci. U.S.A., 79 (1982) 5708-5712. 13 Dubocovich, M.L., Lucas, R.C. and Takahashi, J.S., Light-dependent regulation of dopamine receptors in mammalian retina, Brain Research, 335 (1985) 321-325. 14 Dubocovich, M.L., N-acetyltryptamine antagonizes the melatonin-induced inhibitionof [3H]dopaminerelease from retina, Eur. J. Pharmacol., 105 (1984) 193-194. 15 Dubocovich, M.L., Melatonin is a potent modulator of dopamine release in the retina, Nature (London), 306 (1983) 782-784. 16 Hamm, H.E. and Menaker, M., Retinal rhythms in chicks: circadian variation in melatonin and serotonin N-acetyltransferase activity, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 4998-5002. 17 Hood, D.C. and Hock, P.A., Light adaptation of the receptors: increment threshold functions for the frogs rods and cones, Vision Res., 15 (1975) 545-553. 18 Iuvone, P.M., Evidence for a D 2 dopamine receptor in frog retina that decreases cyclic AMP accumulation and serotonin N-acetyl transferase activity, Life Sci., 38 (1986) 331-342. 19 Iuvone, P.M., Rhythms of melatonin biosynthesis in retina: involvement of calcium, cyclic AMP and dopamine in the regulation of serotonin N-acetyltransferase. In D.W. Klein and P.J. O'Brien (Eds.), Pineal and Retina Relationships, Academic Press, New York, in press. 20 Iuvone, P.M. and Besharse, J.C., Dopamine receptor-mediated inhibition of serotonin N-acetyltransferase activity in retina, Brain Research, 369 (1986) 168-176. 21 Iuvone, P.M. and Besharse, J.C., Regulation of indoleamine N-acetyltransferase activity in the retina: effects of light and dark, protein synthesis inhibitors, and cyclic nucleotide analogues, Brain Research, 273 (1983) 111-119. 22 Iuvone, P.M., Galti, C.L., Garrison-Gund, C.K. and Neff, N.H., Light stimulates tyrosine hydroxylase activity and dopamine synthesis in retinal amacrine neurons, Science, 202 (1978) 901-902. 23 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (London), 277 (1979) 93-96. 24 Lucas, R.C., Takahashi, J.S. and Dubocovich, M.L., Effect of light and dark on retinal melatonin levels and dopamine receptor number, Soc. Neurosci. Abstr., 10 (1984) 328.11. 25 Pang, S.F., Shiu, S.Y.W. and Tse, S.F., Effect of photic

404 manipulation on the level of melatonin in the retinas of frogs (Rana tigrina regulosa), Gen. Comp. Endocrinol., 58 (1985) 464-470. 26 Pierce, M.E. and Besharse, J.C., Melatonin and dopamine interactions in the regulation of rhythmic photoreceptor metabolism. In D.W. Klein and P.J. O'Brien (Eds.), Pineal and Retina Relationships, Academic Press, New York, in press. 27 Pierce, M.E. and Besharse, J.C., Circadian regulation of retinomotor movements: II. The role of G A B A in the regulation of cone retinomotor movements, in preparation. 28 Pierce, M.E. and Besharse, J.C., Circadian regulation of retinomotor movements: I. Interaction of melatonin and dopamine in the control of cone length, J. Gen. Physiol., 86 (1985) 671-689.

29 Pierce, M.E. and Besharse, J.C., Melatonin and photorcceptor metabolism: interactions of melatonin, dopaminc, and GABA in the regulation of cone movement, Soc. Neurosci. Abstr., II (1985) 163.4. 30 Pierce, M.E., Iuvone, P.M. and Besharse, J.C., Melatonin and photoreceptor metabolism: regulation of cone retinomotor movement by melatonin and dopamine, Soc. Neurosci. Abstr., 10 (1984) 10.1. 31 Reme, C.E., Wirz-Justice, A. and DaPrada, M., Retinal rhythms in photoreceptors and dopamine synthetic rate: the effect of a monoamineoxidase inhibitor, Trans. Ophthal. Soc. U.K., 103 (1983)405-410. 32 Wirz-Justice, A., DaPrada, M. and Reme, C., Circadian rhythm in rat retinal dopamine level, Neurosci. Lett., 45 (1984) 21-25.