Exp. Eye Res. (1992), 55, 51 l-520

A Role for

Endogenous Dopamine in Circadian Retinal Cone Movement

CHRISTOPHER

A. McCORMACK””

BETH

AND

Regulation

of

BURNSIDEb

aDepartment of Optometry and Vision Sciences, University of Wales College of Cardiff, PO Box 905, Cardiff CFl 3XF, U.K. and b Department of Molecular and Cell Biology, Division of Cell and Developmental Biology, University of California, Berkeley, CA 94720, U.S.A. (Received

Rockville

16 September

1991 and accepted

in revised

form 77 February

1992)

Cone movements in the retina of the Midas cichlid (Cichlusoma citrinellum) take placein responseboth to light andendogenouscircadiansignals.In the normal light/dark cycle (LD)conemyoidsare long at night (SO-55 ,um), begin to contract before expected dawn, and with light onset contract to their fully contractedpositions(5 km) which are retainedthroughout the day. In continuousdarkness(DD) cone myoidsare fully elongateat night, but undergopre-dawn contractionsto partially contractedpositions which they retain throughout expectedday (20-25 pm). To investigate the mechanismsby which circadian signalsmodulate cone myoid movementsin teleostretinas, we have testedthe effectson circadian cone movements of optic nerve section, intraocular injection of dopamine agonists or antagonists,and intraocular injection of melatonin. We report here that both light-inducedand circadian-drivenconemyoid movementscan occur in the absenceof efferent input from higher centres: both are retained with full amplitudeafter optic nerve sectionin vivo. Intraocular injection studiessuggestthat circadian regulation of cone myoid movement is mediatedlocally within the eye by dopamineacting via a dopaminergicD,-receptor. Cone myoid contraction can be inducedat midnight in LD or DD animalsby intraocular injection of dopamineor the D,-receptoragonistLY171555. The partially contracted conesof DD animalsat expectedmid-day can beinducedto fully contract by intraocular injection of dopamineor the D,-receptoragonist,or to elongate by intraocular injection of the dopamineD,-antagonistsulpiride.Furthermore,the pre-dawnconemyoid contraction observedin both LD and DD animalsin responseto circadian signalscan be completely blockedin DD animalsby intraocular injection of the D,-antagonistsulpirideshortly beforethe time of expectedlight onset. In contrast, circadian cone myoid movementswere unaffected by intraocular injection of the D,-receptoragonistSCH23390,or the D,-receptorantagonistSKF38393.In addition, we report that intraocularly injectedmelatoninhad no effect on conepositionwhen injectedin the light at mid-day,in darknessat midnight or in darknessjust beforeexpectedlight onsetat dawn. However, both melatonin and iodomelatonininduced cone myoid contraction (the light-adaptive movement) when injectedin darknessat expectedmid-day in DD animals.This paradoxicalresult is not consistentwith observationsfrom other speciesin which melatonin inducesdark-adaptivephotoreceptorresponses. Our optic nerve sectionobservationsindicatethat circadianregulation of conemyoid movementdoes not require efferent control. Instead they suggestthat circadian cone movementis regulated locally within the eye.Our resultsfrom intraocular injection of dopamineagonistsand antagonistssuggestthat this localcircadian regulationis mediatedby endogenousdopamine,acting via D,-receptors.We find no evidencefor a role for melatoninin producing dark-adaptiveretinomotor movementsin fish retina. Key words : teleosts; cones: retinomotor movements; circadian phase: efferent control ; dopamine: dopaminereceptorsubtypes; optic nerve section.

1. Introduction Our laboratory has been trying to understand the mechanism of regulation of teleost retinomotor movements. In darkness at night cone myoids elongate, rod myoids contract, and the screening melanin pigment granules of the retinal pigment epithelium (RPE) aggregate into the RPE cell body. In the light during the day, movements are reversed: cone myoids

contract, rod myoids elongate and the retinal pigment epithelial cell pigment granules disperse into the apical processes of the RPE cell. Previous studies have shown that retinomotor movements take place in response to both light and

endogenous circadian signals (cf. Besharse, 1982 ; McCormack and Burnside, 199 1). Under conditions of constant darkness cones elongate at expected dusk and contract just before expected dawn to assume partially contracted positions during expected day (McCormack and Burnside, 1991). The importance of endogenous circadian signal(s) in the regulation of cone retinomotor movements is emphasized by the fact that even in normal light/dark cycles initiation of cone contractions anticipates normal light onset at dawn. In a previous study we have shown that circadian control of retinomotor movement is particularly strong in the Midas cichlid (Cichhsoma citrinellum)

: (McCormack

and Burnside,

1991). For

this reason we have extended our studies of the *

For

mechanism

correspondence.

00144835/92/090511+

10 $08.00/O

of circadian regulation 0

1992

using this fish.

AcademicPressLimited

C. A. McCORMACK

512

Circadian rhythms in activity have been reported for several physiological processes in a variety of vertebrate species (cf. Aschoff, 1982). Several cyclic processes in the vertebrate retina exhibit light and circadian regulation, including opsin synthesis (Korenbrot and Fernald, 1989). outer segment disc assembly and shedding (LaVail, 19 76) and horizontal cell spinule formation (Weiler and Wagner, 1984). In the present study we wished to assess the importance of retinal efferents and the roles of dopamine and melatonin in circadian regulation of cone myoid movement in the Midas cichlid retina. Several sources of efferent innervation are known for fish retina (Ebbesson and Meyer, 1981). One well studied retinal efferent, the terminal nerve, which originates in the olfactory bulb is known to contact bipolar, amacrine and interplexiform cells in the goldfish retina (Ball, Stell and Tutton, 1989). Dopaminergic interplexiform cells have recently been shown to contact not only horizontal and bipolar cells but photoreceptor cell terminals in catfish retina, thus completing a possible terminal nerve to photoreceptor pathway (Wagner and Wulle, 1990). The importance of efferent control for retinomotor movement is unclear. Both Easter and Macy (19 78) and Dearry and Barlow (1987) reported that optic nerve section did not interfere with light regulation of retinomotor movements. Dearry and Barlow (198 7) also reported that some degree of circadian cone retinomotor movement was retained in green sunfish retina after optic nerve section. We decided to examine the role of efferents in more detail in the Midas cichlid since it exhibits much stronger and more consistent circadian retinomotor movements than the green sunfish. In addition, we wished to examine the possible role of dopamine in circadian regulation of cone movements in the Midas cichlid. Several reported observations suggest a role for dopamine in both light and circadian regulation of cone movement. In carp retinas dopamine content and release have been reported to show circadian rhythmicity with maximal release taking place in expected day (Kolbinger et al., 1990). Dopamine is known to act via D,-dopaminergic receptors to mimic the effect of light onset by inducing light-adaptive cone retinomotor movements in teleost fish (Dearry and Burnside. 1986a, b) and frog retinas (Pierce and Besharse, 1985). Observations with isolated cone cell fragments indicate that D,-receptors are located on cone inner/outer segments (Dearry and Burnside, 198 6a). D,-receptors have been identified in human, rabbit, rat, bovine and chicken retinas (Jelsema, 1985 ; McGonigle, Wax and Molinoff, 1988 ; Qu et al., 1988; Elena et al., 1989; Iuvone, 1989; Denis et al., 1990). Finally, dopamine is the primary catecholamine found in retina (cf. Negishi et al., 198 1 ), where its synthesis and release have been reported to be light stimulated in rabbit (Nowak and Zurawska, 1989). rat (Brainard and Morgan, 1987) and fish retina (Kirsch and Wagner, 1989 ; Dearry,

AND

B. BURNSIDE

1991; McCormack and Burnside, 1992). On the other hand, studies of dopamine effects on fish horizontal cells have suggested that dopamine acts via D,receptors to mimic the effects of prolonged darkness on these cells (Mange1 and Dowling, 1985. 1987: Yang, Tornqvist and Dawling, 1988). The basis for these discrepancies are not understood. That they might result from differentially regulated paracrine (D,) and synaptic (D,) dopaminergic pathways has been suggested (Besharse and Iuvone, 1992). To further clarify the role of dopamine in circadian regulation of cone retinomotor movements we have carried out intraocular injection studies with dopamine agonists and antagonists. Since both dopamine and the indoleamine melatonin have been proposed as local circadian modulators in the frog regina (Pierce and Besharse, 1985) we have also used intraocular injections to investigate the possible effects of melatonin on cone retinomotor movements in the Midas cichlid retina. In the frog, dopamine triggers light-adaptive cone retinomotor movements, whereas melatonin triggers dark-adaptive movement (Pierce and Besharse, 1985). Previous studies of melatonin effects on green sunfish retina failed to find any effect of melatonin in that species (Dearry and Burnside, 1986b). We report here that circadian cone myoid movements persist in the absence of efferent input to the retina. Observations from intraocular injection studies with dopamine agonists and antagonists suggest that endogenous dopamine, acting via dopaminergic D,receptors, plays a critical role in the circadian initiation of cone myoid contraction just before dawn and in the maintenance of partially contracted cones during expected day in constant darkness. Melatonin does not induce dark-adaptive retinomotor movements in the Midas cichlid.

2. Materials

and Methods

Experimental Animals Adult Midas cichlids, Cichlasoma citrinellum (42500 g weight), were raised from fry and maintained either in outdoor ponds or indoor aquaria, with filtered aerated tap water at 24°C. Animals raised outdoors were transferred to indoor aquaria and entrained for at least 28 days prior to experimentation to an 11 hr light/l3 hr dark cycle with light onset at 0630 hr and offset at 1730 hr. This light cycle was chosen to match outdoor lighting conditions at the time of transfer and was then used throughout each part of the study. Indoor entraining light intensity was 1000 lx (General Electric, cool white fluorescent; PANLUX electronic 2 light meter. GOSSEN GMBH). Normal Circadian and Diurnal Cone Retinomotor Movements The time course

of cone retinomotor

movement

CIRCADIAN

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MOVEMENT

patterns in the normal light/dark cycle and in continuous darkness were determined as in McCormack and Burnside (1991). Briefly, cone position was analysed from pairs of eyes taken from a number of fish at selected time points in both the normal light/dark cycle and in continuous darkness. At all sample points, fish were killed by spinal cord section with pithing before eyes were enucleated and hemisected along the ora serrata to prepare eyecups. Cone Retinomotor Movement After Optic Nerve Section

Unilateral optic nerve section was performed 3 hr before light offset on adult fish of uniform size (150 g), following the protocol of Dearry and Barlow (1987). All fish used were anaesthetized with 0.1% tricane methane sulphonate (Sigma Chemical Co., St Louis, MO) before the optic nerve to the right eye of each animal was exposed and cut. Animals in which the ophthalmic artery was accidentally cut were killed immediately and their retinas excluded from the study. Animals in which there was no observable bleeding into the orbit were allowed to recover from surgery and were then used to assess cone myoid movements in cyclic light and in continuous darkness. Animals in which the optic nerve had been successfully cut displayed a dorsal light response. Control animals (sham operation) were anaesthetized and the optic nerve to their right eyes was exposed but left unsevered. Following optic nerve section, fish were killed throughout day and night phases of the LD and DD cycles by spinal cord section with pithing and eyecups were prepared from both the right (experimental) and left (control) eyes, for histological analysis as below. Modulation of Circadian Cone Myoid Movements

Intraocular injections were performed on adult fish of uniform size (150 g), according to Burnside et al. (1982) and killed 10, 30 or 60 min later as indicated. Drugs used were dopamine, sulpiride, melatonin and iodomelatonin (Sigma Chemical Co.), LY171555, SKF39393, and SCH23390, (RBI Research Biochemicals, MA, U.S.A.). Fish were injected in daylight or in darkness under infrared illumination ( > 880 nm). All drugs used were dissolved in buffer solution (modified Earle’s balanced salt solution containing ; 24 mM NaHCO,, 20 mM glucose, 3 mM Hepes, 1 mM ascorbic acid, 5 mM taurine, pH 7.4, with 0.01% DMSO at room temperature) at calculated final concentrations after injection as noted. For each injection, 7 ~1 of solution (average intraocular volume was approximately 150 ~1) was delivered to the centre of the vitreous using a Hamilton Microlitre syringe (Hamilton, Bonaduz 75 RN CH). In all cases right eyes received drug injections while left eyes received buffer and carrier (0.01 “/o DMSO) alone. Fish were killed in the light or under infrared illumination at 30 and

513

60 min post-injection (as noted), and eyecups were prepared and fixed for analyses of cone position. All fish used in this study were anaesthetized both before injection and again prior to killing with 0.1 y0 tricane methane sulphonate. Histological Analysis

For histological analyses of cone myoid length eyecups were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for > 12 hr. Cetylpyridium chloride (0.5%) was added to this fixative to enhance retinal-RPE adhesion. Following fixation, a central region was cut from the fundus of each retina and chopped into approximately 20 pm thick radial slices using a mechanical chopper designed by Frank Werblin (Werblin, 1978). Unstained slices were then examined in a light microscope equipped with an image analysis system (Optimax, Hollis, NH, U.S.A., Apple II microcomputer and Houston Instruments digitizing tablet), and Nomarski interference contrast optics. From each retina 30 representative cone myoid lengths were measured as the distance from the base of the cone ellipsoid to the outer limiting membrane. Date are presented as mean +s.D./s.E. as noted. For intraocular injection experiments data were compared by one-way analysis of variance (ANOVA). At all time points in all experiments, ‘ n’ refers to the number of fish examined. 3. Results Diurnal and Circadian Rhythms of ConeMovement

Cone movements under continuous dark (DD) and cyclic light (LD) conditions in the Midas cichlid retina have been reported previously (McCormack and Burnside, 1991). Briefly, they are as follows: in the normal light/dark cycle cone myoids are long (5 S60 pm) in the dark, begin to contract before dawn, contract to their maximally short position (5 pm) after dawn, remain short throughout the day and reelongate after light offset at dusk: in continuous darkness cone myoids begin to contract before expected dawn, contract to a partially contracted length of approximately 20-25 pm, maintain this length throughout expected day, and re-elongate after expected dusk. Optic Nerve Section Experiments

Optic nerve section had no effect on cone movement in the normal light/dark cycle (Fig. 1). Following optic nerve section or sham operation in the light 3 hr before light offset, cone myoids remained fully contracted in the light, elongated normally after light offset at dusk, and remained elongated during the night in darkness. The pre-dawn circadian initiation of cone myoid contraction occurred normally as did the

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1. Optic nerve section had no effect on cone movement in the normal light/dark cycle (ID) or in constant darkness (DD). Cone myoid lengths of optic nerve sectioned right eyes (0) and control left eyes (A) from the present study are superimposed on cone myoid lengths as previously described for the normal light/dark cycle and constant darkness as indicated by: (-) darkness; (---) light (McCormack and Burnside, 1991). In all cases animals were entrained to a light cycle with light onset at 0630 hr and light offset at 1730 hr. For continuous darkness lights were turned off at 1730 hr and left off for the remainder of the experiment. In all cases, cone myoid lengths from optic nerve sectioned, sham operated (S) and control eyes were not significantly different from one another or from previously reported control values (P < 0.05): n = 4 fish at all points: S.E.M. was always < 5 pm for all examples. (0) 10’ lights on in expected day: ONX, time of optic nerve section 1430 hr. FIG.

light-induced dawn.

full contraction

after light

onset at

Optic nerve section also had no effect on circadian cone movements in constant darkness. Following optic nerve section or sham operation in the light 3 hr before light offset in the light/dark cycle, fish were placed in darkness at dusk and then left in continuous darkness. In both operated and unoperated control fish, cone myoids exhibited the normal circadian partially contracted state during expected day. If lights were turned on during expected day cone myoids contracted fully to the normal light-adapted position.

Modulation of Circadian Cone Myoid Movements In order to ascertain whether dopamine or melatonin could influence circadian cone myoid movements, we tested the effects of dopamine and melatonin delivered by intraocular injection at various time points throughout the light/dark cycle and in continuous darkness. In addition, we tested the effects of D, (SCH23390 and SKF 38393) and D, (LY171555 and Sulpiride) type dopamine receptor agonists and

length 6013.5 pm), dopamine or the D,-agonist LY 17155 5 induced cone myoids to contract to 19.5 + 5 f 4.0 ,um and 26.5 f 1 ,um, respectively (Fig. 2). Final concentrations of agents were calculated to be approximately 10-6 M in the eye. Injected dopamine and D,-agonist were somewhat less effective than 10 min bright light (final cone myoid length 5.5 10.5 pm) at producing full contraction at this time. No significant effects on cone myoid length were produced by intraocular injection of the D,-antagonist sulpiride (1 O-4 M : 55 + 4.0 pm), the D,-agonist SCH23390 (104~: 57.512.3 pm), or the D,antagonist SKF38393 (104 M: 56.5k2.3 pm), as compared to cone myoid length prior to experimentation (To) at this time (P < 005). When injected just prior to initiation of the predawn circadian cone contraction, dopamine and the D,agonist LY 17 15 5 5 induced cone myoids to contract beyond the intermediate expected mid-day position (approximately

20-25 pm)

to the fully

contracted

cone myoid contraction should be taking place: at mid-day cone myoids should be fully contracted ; and at expected mid-day cone myoids should normally be

state (5.0) 1.0 pm and 5.5 + 1.0 pm, respectively), indistinguishable from that produced by 10 min light exposure delivered at this time (6.0 + 1.0 pm). Intraocular injection of the D,-antagonist sulpiride (lo6/104 M) just before dawn completely blocked the pre-dawn circadian cone contraction (myoid length after sulpiride = 60.0 + 4.0 ,um; myoid length prior to expected dawn = 59 +_2.0 pm: P < 0.05). Neither the D,-agonist nor the D,-antagonist had a significant effect on cone position, as compared to buffer, when injected at this time ; cones contracted to 3 5 + 0.9 ,um

partially

and 3 l_+ 2.5 pm, respectively

antagonists

at the same time points. Experiments

were

performed at four time points ; midnight, expected dawn, mid-day, and mid-expected day. These time points were chosen since: at midnight, cone myoids should be fully elongate; at expected dawn, circadian

contracted.

When injected at mid-dark

period (to cone myoid

(P < 0.05).

At mid-expected day in continuous

darkness, cone

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FIG. 2. Comparison of the effectsof light andintraocularly injecteddopamine,D,- and D,-receptoragonistsandantagonistson conepositionin DD animalsat midnight (A), expecteddawn (B). and expectedmid-day(C), and in LD animab at mid-day (D): D,-agonist LY1715S5 N- 10-6 M; D,-antagonist sulpirideN 10-6 M; RI-agonist SKF38393 < LO4 M: D,-antagonist SCH23390 z 104 M. Light, dopamine,and the Q-agonist LYI 71555 inducedconecontraction in dark maintainedanimals at all timestested.D,-agonistshad no effect. The Do-antagonistsulpirideblockedcircadianpre-dawn conemyoid con~action andinducede!o~gationin expectedday in DDanimals;it had no effecton LD animalsin the light. I?,-antagonistshad no effect. In all cases,To control representsconemyoid length at the time of injection. Circadianconecontraction at expecteddawn is seenin 60’ uninjectedand 60’ buffer injectedcontrols asindicated.At other time points 60’ uninjectedanimalsdo not differ from To controls and buffer-injectedcontrols.Midnight = 0030 hr; expecteddawn = 0500 hr; expectedmid-day = 1300 hr: n = It fish at ail control and 30’ buffer time points; n = 6 fish at 60’ buffer points: n = 6 fishat all dopaminetime points; and n = 4 fish at all agonist/antagonisttime points. Data are presentedas mean+ s.6.

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FIG. 3. Etrects of intraocularly injected melatonin on cone myoid length in DD at midnight (A), at expected dawn (B) and at expected mid-day (C), and in LD animals at mid-day (D). At midnight and expected dawn in DD animals or at mid-day in LD animals, melatonin had no effect. At expected mid-day in DD animals, melatonin and the metabolically stable analog 2iodomelatonin induced complete cone myoid contraction at mid-expected day, thus mimicking the effect of light. In all cases To control represents cone myoid length at the time of injection. Circadian cone contraction is seen in uninjected and buffer injected controls at expected dawn as explained for Fig 2. Light cycle as described for Fig 2 : n = 9 fish at all time points. Data are presented as mean & SE.

myoids are maintained in a partially contracted state (approximately 2 1 pm) by circadian signals (cf. McCormack and Burnside, 1991). Intraocular injection of dopamine (10-6~) or the D,-agonist LY171555 ( 10-6 M) at this time induced cone myoids to undergo complete contraction to lengths characteristic of daytime in the light (5.0 + 0.9 pm and 4.9 f 1.5 pm, respectively). In contrast, injection of the D,-antagonist sulpiride (lo-6 M) induced cone myoids to elongate toward but not to fully reach lengths characteristic of night-time in the dark (46.5 &2.0 pm). Again at this time neither the D,-agonist nor the D,-antagonist had any effect upon cone myoid position (2 5 f 2.5 pm and 24.9 f 1 pm, respectively : P < 0.05). These results suggest that the partially contracted cone myoid position in expected day is critically dependent on the local concentration of endogenously released dopamine, and that this dopamine influences cone position by interaction with D,receptors.

When injected in the light (1000 lx) at mid-day, neither dopamine (lo-6/ 104 M) nor any of the dopamine agonists or antagonists had any effect on cone myoid length (P < 0.05) cones remained fully contracted (e.g. 60’ sulpiride 4.5 f 0.9 ,um). Thus bright light appears to override any effect of dopamine antagonists on cone, position. This result is inconsistent with those of Dearry and Burnside (1986a), who report that sulpiride (lo-4 M) promoted darkadaptive cone elongation in isolated light-adapted green sunilsh retinas in which cones have first been induced to contract in response to ambient light (2000 lx approximately). Thus in Cichlids, unlike the green sunfish, bright ambient light appears to override any effects of dopamine antagonists on cone position. The effects of intraocular melatonin injections on cone positions in the Midas cichlid retina at midnight, expected dawn, expected day and normal day are illustrated in Fig. 3. Melatonin (IO4 M calculated final concentration) had no effect on cone myoid

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length at midnight (49 + 2 pm). at mid-day (5.9 4 3.9 pm) in normal cyclic light/dark animals, or at expected dawn in continuous darkness (31.7+2 pm) (P < 0.05). However, at expected mid-day in continuous darkness (DD), melatonin (104 M) induced complete cone myoid contraction, thus mimicking the effect of 10 min exposure to bright light at this time. Since this was a surprising result, inconsistent with findings in the frog (Pierce and Besharse, 1985), we investigated the effect of lower concentrations of melatonin and also the possibility that some metabolic product of melatonin was responsible for inducing cone contraction by injecting the metabolically stable melatonin analog 2-iodomelatonin. Both melatonin and 2-iodomelatonin at concentrations as low as lo-12 M were effective in producing cone myoid contraction when injected at mid-expected day (9.8 + 3.6 ym and 7.5 +_3.1 pm, respectively). 4. Discussion Our observations indicate that in the Midas cichlid retina both light-induced and circadian-driven cone myoid movements can occur in the absence of efferent input: however, both can be modulated by dopamine acting via a D,-type receptor. Optic nerve section had no effect on either light-induced or circadian-driven cone retinomotor movement in the cichlid. This finding is consistent with previous reports that optic nerve section failed to abolish light- (Easter and Macy, 1978) and circadian-driven (Dearry and Barlow, 198 7) retinomotor movements in other fish species. However, we did not see in the Midas cichlid the reduction in amplitude of cone elongation in expected night reported by Dearry and Barlow for the sunfish. Rather, circadian cone myoid movements continued unabated after optic nerve section in the Midas cichlid retina. We are as yet unable to rule out the possibility that a humoral stimulus regulates circadian-driven retinomotor movements. This possibility will be investigated in the future by in vitro studies. Our intraocular injection studies with dopamine agonists and antagonists indicate that dopamine acting via D,-receptors induces cone contraction at any point in the circadian cycle: thus, dopamine mimics the effect of light onset on cone movement. Intraocular injection of dopamine or the D,-agonist LYl71555 triggers cone myoid contraction under three different physiological states of the retina : (1) at midnight, when cone myoids are normally fully elongate : (2) at expected dawn, when cone myoids are beginning to contract in response to a circadian signal ; and (3) at expected mid-day when cone myoids are partially contracted. Interestingly, dopamine and the D,-agonist stimulate less extensive contraction than light at midnight but comparable extents of contraction at expected dawn or in expected day. This observation suggests that cones may be less responsive to dopamine in the middle of the night than they are

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at expected dawn or expected day when circadian signals are known to influence cone position, The basis for this differential sensitivity is not known: one possibility is that there may be circadian cycles in the numbers of dopamine D,-receptors on cones. Our findings are consistent with the results of several studies of dopamine effects on other retinal processes. In rabbit, rat, Xenopus and fish, retinal dopamine activity and release increase in response to light stimulation (Brainard and Morgan, 198 7; Godley and Wurtman, 1988 ; Boatright, Hoe1 and Iuvone, 1989; Kirsch and Wagner, 1989: Nowak and Zurawska, 1989; Witkovsky and Shi, 1990). Dopamine is thought to modulate many retinal activities in a light-adaptive fashion, including : cone myoid movements in other teleost fish (Dean-y and Burnside, 1986a; Kohler et al., 1990); retinal pigment epithelium pigment granule migration in teleost fish and bull frogs (Dearry and Burnside, 1989); cone myoid movements in bullfrog (Dean-y et al., 1990) and in Xenopus retinas (Pierce and Besharse, 198 5) ; horizontal cell spinule number and gap junction connexon density in fish (Weiler et al., 1988 ; Kohler and Weiler, 1990; Kohler et al., 1990); activity at horizontal cell synapses in Xenopus (Witkovsky, Stone and Besharse. 1988 ; Witkovsky and Shi, 1990) : and CAMP concentration in mouse photoreceptors (Cohen, 1989 ; Cohen and Blazynski, 1990). On the other hand, numerous studies have implicated dopamine in production of horizontal cell responses characteristic of prolonged darkness (Mangel and Dowling, 1985, 1987; Yang et al., 1988). Like the evidence for light-adaptive effects of dopamine above, evidence that dopamine acting via D,-receptors mimics the effect of prolonged darkness on horizontal cell physiology is quite compelling. The reasons for this baffling inconsistency are not yet clear. Besharse and Iuvone (1992) have suggested that both sets of observations might be accommodated by a model in which dopaminergic interplexiform cells release dopamine by two separate mechanisms (paracrine and synaptic) and that these two mechanisms exhibit differential regulation. Thus synaptic dopamine release onto horizontal cells to activate D,-receptors on these cells may be stimulated by prolonged darkness, while non-synaptic, paracrine dopamine release may be stimulated by light and circadian signals so that released dopamine diffuses to bind to D,-receptors on photoreceptors and trigger light adaptive retinomotor movements. Such a model is consistent with the observations that dopamine concentrations in the micromolar range are required to produce the D,mediated horizontal cell responses while nanomolar dopamine is sufficient to produce D,-mediated retinomotor movements in cones (cf. Besharse and Iuvone, 1992). Circadian fluctuations of endogenous retinal dopamine release in the nanomolar range could produce observed cycles of cone movement without ever

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reaching the threshold for activating horizontal cell responses. Thus, horizontal cells could be blind to circadian fluctuations in endogenous dopamine release in a range sufficient to regulate cone movement. Similarly, if local synaptic re-uptake mechanisms are sufficiently avid to prevent dopamine diffusion away from synaptic sites on horizontal cells, an increase in synaptic dopamine release in prolonged darkness might be undetectable by photoreceptors. Further, our intraocular injection observations implicate a role for endogenous dopamine acting via D,-dopaminergic receptors in circadian regulation of cone myoid length. The D,-antagonist sulpiride blocks circadian pre-dawn contraction and induces cone elongation in expected day, while D,-agonists and antagonists have no effect. These sulpiride results strongly suggest that D,-receptors are critical to circadian regulation of cone position and suggest that endogenous dopamine levels and/or the availability of cone D,-receptors dictate cone retinomotor position in constant darkness. This is consistent with previous reports in both fish and frogs that dopamine-induced retinomotor movements are effected via a D,-receptor mechanism (Dearry and Burnside, 1986a ; Pierce and Besharse, 1985). Kohler et al. (1990) report that dopamine release shows circadian cycles in the carp retina with maximal release during expected day. This finding is consistent with our mid-day injection results. However, Kohler et al. (1990) failed to detect any increase in dopamine before expected dawn, a finding inconsistent with our pre-dawn intraocular injection results. Our results suggest an increase in cone sensitivity to dopamine at expected dawn as compared to midnight. This increased sensitivity may trigger cone movements prior to detectable increases in dopamine release as measured by Kohler et al. ( 1990). The implication from our intraocular injection studies that circadian cone position is dictated by endogenous dopamine levels is inconsistent, however, with the results of studies in which retinal dopaminergic cells are ablated with 6-hydroxydopamine. Douglas, Wagner and Zaunreiter (199 1) have reported that both light-induced and circadian-driven cone movements continue in the retinas of the cichlid Aequidens p&her following 6-hydroxydopamine (6OHDA) induced ablation of dopamine producing cells. Pre-dawn injections of D,-antagonists fail to block circadian cone contraction in these 6-hydroxydopamine-lesioned fish. However, pre-dawn injections of D,-antagonists do block circadian cone contractions in normal control animals of the same species (Douglas and Wagner, pers. comm.). These two observations suggest that different physiological mechanisms are operative under acute vs. chronic deprivation of dopamine responses. The block of circadian cone contraction in normal fish by intraocular injection of D,-antagonists suggests (as does our study) that dopaminergic mechanisms are required for circadian cone contraction at expected dawn in normal animals.

C.A.

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In the chronic absence of dopamine, as induced by 6OHDA ablation, it appears that other circadian signals can take over the role played by dopamine. A chronically dopamine-deprived retina may differ in other ways than in the absence of dopamine from normal retina. Nonetheless, these studies make it clear that dopamine is certainly not the sole mechanism by which circadian signals can control retinomotor movements. In the chronic absence of dopamine other mechanisms can suffice. As the indoleamine melatonin has been proposed as a possible modulator of circadian rhythmicities in the retina (cf. Besharse, 1982), we chose to assess its effects on cone myoid movements in the Midas cichlid retina. Intraocularly injected melatonin had no effect on cone myoid movements at mid-dark, at expected dawn or in normal day. However, when delivered at expected mid-day, melatonin ( < lo-12 M) induced cone myoid contraction. It would thus appear that at expected mid-day, melatonin was acting like a light signal. We have also tested the metabolically stable melatonin analog 2-iodomelatonin to examine the possibility that the cone myoid contraction was induced by melatonin metabolites which have been reported to have antagonistic effects (Clemens and Flaugh, 1986). Since the metabolically stable analog also induced contraction with a similar dose-response profile to melatonin, it seems likely that melatonin really does induce cone contraction in this fish. This finding is in conflict with evidence that melatonin had no effect on cone myoid movements in the green sunfish retina (Dearry and Burnside, 1986b) and with most of the published literature on the pharmacological effects of melatonin which suggest that melatonin acts like a dark signal (cf. Besharse, 1982). At this time we can offer no further explanation of our observed effects of intraocularly injected melatonin on cone myoid length except to note that there is significant evidence to indicate an interdependence of melatonin and dopamine as circadian modulators. Both melatonin and dopamine are involved in the circadian regulation of retinomotor movements in Xenopus where melatonin is known to act as a dark signal, acting through an inhibition of dopamine release (Pierce and Besharse, 1985). Melatonin content and NAT activity in the chick retina are D,dopamine receptor activated (Zawilska and Iuvone, 1989), while in rabbit retina melatonin may control dopamine release (Dubocovich, 198 5). Xenopus eyecups maintained in culture and supplied with serotonin display a circadian rhythm in melatonin production which may be phase shifted with dopamine (Iuvone, 1988; Cahill and Besharse, 1991). Such studies indicate that, in these systems at least, dopamine and melatonin may both be involved in the circadian regulation of retinal function. Furthermore, we see the paradoxical effect of injected melatonin only in expected day in constant darkness, i.e. under physiological conditions which the fish might never

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experience in nature. If there is indeed some interdependence of the dopamine and melatonin systems in the retina, perhaps abnormal darkness might affect one system differently from the other. Finally, others have noted that the time of application may be critical to the pharmacological effects observed with melatonin (Clemens and Flaugh, 1986). The results of this study indicate that circadian regulation of cone myoid movement in the Midas cichlid retina does not require efferent input. Our intraocular injection studies do not support a role for melatonin in circadian regulation of cone movement ; instead they suggest that endogenous dopamine, acting via D,-dopaminergic receptors, plays an important role in this circadian regulatory mechanism. Blocking D,-receptors prevents the circadian-induced core contraction that occurs at expected dawn. Acknowledgements This work has been supported by the NIH grants EY03 5 75 (B.B.) and Fogarty International Center 8FSTW042 17A (CMcC.).

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