Circadian organization in Japanese quali

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 232:557-566 (1984)

Circadian Organization in Japanese Quail HERBERT UNDERWOOD AND THOMAS SIOPES Department of Zoology (H. U.) and Department of Poultry Science (TS.), North Carolina State Uniuersity, Raleigh, North Carolina 27695

ABSTRACT Our recent studies have implicated both the eyes and pineal as major components of the circadian system of Japanese quail. We assessed the role of these organs by examining the effect of their removal on the circadian activity rhythm of quail exposed to either 24 hr light-dark (LD) cycles or to continuous darkness (DD). Removal of only the pineal had no effect on the activity rhythm of quail in either LD or DD. Blinding (by orbital enucleation) had a major effect under both LD and DD. One third of the blinded birds showed entrainment under LD although entrainment patterns were very variable, whereas two thirds of blinded birds were arrhythmic. All blinded plus pinealectomized birds were arrhythmic in LD as were all blinded and blinded plus pinealectomized birds in DD. Accordingly, effects of pinealectomy can be seen only when pinealectomy is combined with blinding. The fact that blinding disrupts circadian organization in both LD and DD indicates that the eyes must act as major components of the quail's circadian system. In view of the postulated role for melatonin, an indoleamine, in circadian systems, the eyes, pineal, and blood of quail were assayed for this compound. Robust daily rhythms in melatonin content were observed in all three tissues. The blood rhythm is due to secretion of melatonin into the vascular system by both the pineal and eyes. The ocular melatonin rhythm continued after sectioning of the optic nerve, was reentrainable to a shift in the phase of the LD cycle, and persisted for a t least 2 days in DD. These data suggest that the eyes play a major role within the circadian system and support the hypothesis that circadian pacemakers may reside within the eyes of quail. The results are discussed in view of the findings of others in both quail and other avian species. A general model for circadian organization in birds is presented in which the eyes, the pineal, and the suprachiasmatic nuclei of the hypothalamus comprise major elements of a multioscillator circadian system.

Rapid strides have been made in elucidat- ing the physiological mechanisms underly- ing the generation of circadian rhythms in birds since the pioneering studies of Gaston and Menaker ('68), who demonstrated that the pineal organ is an important component of the avian circadian system. Interest in pineal function has been heightened by studies, mainly in rodents, showing an important role of the pineal in photoperiodic phenom- ena and implicating a class of pineal compounds (the indoleamines) as potent regulators of photoperiodic systems (Reiter, '80). In addition to the pineal, recent studies have implicated at least two other areas that are important in circadian organization: 1) the suprachiasmatic nuclei (SCN) of the hypothalamus and 2) the eyes.

Here we review our recent studies on circadian organization in Japanese quail and then discuss these studies in light of the findings of others in both quail and other avian species. We summarize, in general terms, how the multioscillator avian circadian system may be structured.

ACTIVITY

We have examined the effects of pinealectomy and blinding on the circadian activity rhythm of Japanese quail (Coturnix coturnix

Address reprint requests to Herbert Underwood, Department of Zoology, North Carolina State University, Raleigh, NC 27695.

0 1984 ALAN R. IJSS, INC.

558 H. UNDERWOOD AND T. SIOPES

japonica) under both 24-hr light-dark cycles and under constant darkness. In addition, we have assessed the melatonin content of the pineal, eyes, and plasma under different lighting regimens and after various surgical manipulations.

Twenty-four-hr ID cycles The circadian locomotor activity rhythm is

commonly used as an assay for the state of the “biological clock” since it is easy to meas- ure and imposes no constraints upon the animal. We have measured activity rhythms of individual quail maintained in cages with “tilting” floors under 24-hr LD cycles and after sham-pinealectomy, pinealectomy, blinding (by bilateral orbital enucleation), and both blinding plus pinealectomy. Figure 1 gives representative examples of the activity patterns of quail subjected to these surgical manipulations. Note that neither sham pinealectomy nor pinealectomy seemed to affect the pattern of the entrained activity rhythm. The activity pattern of sham-pinealectomized or pinealectomized birds was, in all cases, similar to the activity patterns of intact (unoperated) quail. Blinding, however, had a profound effect on the activity pattern. Two thirds of the blinded birds were arrhythmic under LD cycles (not shown) whereas one third showed evidence of entrainment (Fig. 1). The entrained, blinded bird shown in Figure 1 shows a marked in- crease in nocturnal activity and increased variability in daily activity onsets and offsets as compared to either control or pinealectomized birds. Extraretinal control of circadian rhythms is a common feature of circadian systems in submammalian vertebrates (Un- derwood and Groos, ’82). However, all blinded plus pinealectomized quail tested were arrhythmic under LD (Fig. 1). Accordingly, both the pineal and the eyes are involved in the control of the activity rhythm in LD but effects of pinealectomy are only seen when the birds are also blinded.

Constant conditions The activity rhythms of Japanese quail

were also examined under conditions of continuous darkness (DD) (Fig. 2). Japanese quail are notorious for the lack of precision in their free-running activity patterns. In fact, a significant percentage of intact (unoperated) quail show arrhythmic activity patterns under constant conditions. For example, in our experiments approximately 50% of intact and sham-pinealectomized quail were rhythmic in DD (Fig. 2) whereas the remaining appeared arrhythmic. Also,

following pinealectomy about half of the birds tested were rhythmic in DD (Fig. 2). There- fore, the percentage of rhythmic birds after pinealectomy is similar to that seen in sham- pinealectomized and unoperated quail. That is, neither sham-pinealectomy or pinealectomy affected the free-running behavior of quail. However, all blinded and blinded plus pinealectomized birds were arrhythmic in DD (Fig. 2). The lack of effect of pinealectomy, when performed alone, is consistent with the lack of effect of pinealectomy noted by Simpson and Follett (’81) in free-running Coturnix. It is also consistent with our LD findings insofar as pinealectomy, when performed alone, did not affect entrained activity patterns. However, either blinding or blinding combined with pinealectomy induced arrhythmicity in all birds exposed to DD. The fact that blinding induced arrhythmicity in all birds in DD is inconsistent with the hypothesis that the role of the eyes in circadian organization is simply a photo- sensory one. Removal of a photoreceptor (the eyes) might be expected to affect activity rhythms of birds exposed to LD cycles but the eyes must play a more “fundamental” role in circadian organization if their removal can affect activity rhythms in DD as well. In quail the eyes’ role in circadian organization seems to be more substantial than the pineal’s role since blinding, when performed alone, can disrupt both entrained and free-running activity rhythms whereas pinealectomy, when performed alone, cannot.

MELATONIN RHYTHMS

The pineals of all vertebrates manufacture a host of biochemicals. However, interest has focused on a class of compounds called indoleamines and, in particular, on N-acetyl 5- methoxytryptamine (melatonin). Melatonin is derived from serotonin via the conversion of serotonin to N-acetylserotonin by the enzyme N-acetyltransferase (NAT) followed by the conversion of N-acetylserotonin to melatonin by the enzyme hydroxyindole-0-meth- yltransferase (HIOMT). The melatonin content of all pineals examined to date shows a pronounced circadian rhythm with high melatonin levels occurring at night regard- less of the habits (nocturnal vs. diurnal) of the animal. It is widely held that the rhythm in NAT activity is responsible for the rhythm in melatonin content. Until recently it was assumed that the methoxyindoles, including melatonin, were unique pineal products since HIOMT activity was assumed to be localized to pineal tissue. Recently, however, several other sites of melatonin production have been

C

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CIRCADIAN ORGANIZATION IN QUAIL

PX

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Fig. 1. Activity patterns of quail subjected to several surgical manipulations. C, control (sham-pinealectomized); PX, pinealectomized; BLX, blinded (by orbital

enucleation). The light-dark cycles to which the birds were exposed are diagrammed at the top of the records.

identified, including the gut, the Harderian glands, and the eyes (Ralph, '81). For example, in the chick, the eyes exhibit a rhythm in melatonin content and NAT activity that is affected by environmental lighting in a fashion similar to that of the pineal (Hamm et al., '83; Binkley et al., '80; Reppert and Sagar, '83). A role for melatonin within circadian systems has been implicated since daily melatonin injections can entrain activ- i ty rhythms in birds (starlings) and mammals (rats) and continuous exogenous

melatonin administration under constant conditions can cause period changes in activity rhythms, or arrhythmicity, in several species of birds and in a mammal, the armadillo (Gwinner and Benzinger, '78; Redman et al., '83; Turek et al., '76; Ebihara and Kawa- mura, '81; Harlow et al., '82). In view of the potential importance of melatonin in circadian organization, we have measured the melatonin content of the pineal, eyes, and plasma of quail under several experimental conditions in order to address the following.


C

BLX

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Fig. 2. Activity patterns of quail that were subjected to several surgical manipulations and exposed to contin-

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1) Are melatonin rhythms present in the pineal, eyes, or blood? Quail were entrained to LD 12:12 and groups of birds were then killed every 2 hr over a 24-hr period. The melatonin content of the pineal, eyes, and

blood was then measured using the radioim- munoassay of Rollag and Niswender ("76). Figure 3 shows that a robust rhythm in melatonin content occurs in all three tissues with high levels occurring at night and low

CIRCADIAN ORGANIZATION IN QUAIL 561

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Fig. 3. Melatonin rhythms in the pineal, eyes, and blood of quail entrained to LD 12:12.

levels occurring during the day. 2) What is the relative contribution of the

pineal and eyes to the plasma melatonin rhythm? Quail entrained to LD 12:12 were either left intact (controls), sham-pinealec-

tomized, pinealectomized, blinded (by orbital enucleation), or both pinealectomized and blinded; the ocular, pineal, and plasma levels of melatonin were subsequently assayed in groups of birds killed every 3 hr over a 24-hr period. Blinding did not obviously affect the pineal melatonin rhythm and pinealectomy did not affect the ocular melatonin rhythm (Underwood et al., '84). However, pinealectomy depressed the plasma melatonin rhythm by 54% as compared to controls and blinding depressed the rhythm by 33% as compared to controls (Fig. 4). After both pinealectomy plus blinding the plasma melatonin rhythm was virtually eliminated (Fig. 4). The small rhythm seen after pinealectomy plus blinding is due either to secretion from extrapineal and extraocular melatonj n- producing sites or, more likely, to small rem- nants of pineal tissue which may remain after pinealectomy. Therefore, in the intact bird on LD 12:12 the pineal's contribution to the plasma melatonin rhythm is about two- thirds and the eye contributes one-third.

3) What is responsible for the ocular melatonin rhythm?% The only known efferent innervation of the avian eye originates in the isthmo-optic nucleus of the midbrain and en- ters the eye via the optic nerves (O'Leary and Cowan, '82). In one experiment the optic nerves to the left eyes of quail entrained to LD 12:12 were sectioned (ONX) while the optic nerves to the right eyes of these birds were sham operated. The phase of the light cycle was shifted 180" and groups of these birds were killed 2 weeks later over a 24-hr period. Figure 5 shows that the melatonin rhythm persists after ONX and is capable of reentrainment to a shifted LD cycle. In a second experiment the endogenous nature of the ocular melatonin rhythm was explored. Birds were subjected to ONX (left eyes) or sham ONX (right eyes) while entrained to LD 12:12. The birds were subsequently placed into DD and the melatonin content of the eyes was assessed during the projected second day of DD. Figure 6 shows that the rhythm persisted in both the experimental and control eyes of these birds.

The profound effects of bilateral enucleation on the circadian activity rhythm (Figs. 1,2) are compatible with the hypothesis that blinding removes a major component of circadian organization such as removal of circadian oscillators from a multioscillator system. The demonstration of circadian rhythms in ocular melatonin content and the


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Fig. 4. Plasma melatonin levels in quail entrained to LD 12:12 after several surgical procedures. The dashed

lines represent the melatonin levels seen in the control birds.


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Fig. 5. Melatonin rhythms in the left (ONX, optic nerve sectioned) and right (Intact, sham operated) eyes of quail after reentrainment to a 180" shift in the phase of an entraining LD 12:12 cycle.

demonstration that these rhythms are unaf- fected by ONX in either LD or DD (Figs. 5, 6) offer some support for the existence of intraocular circadian clocks in birds. Recently, Besharse and Iuvone ('83) demonstrated a circadian rhythm in NAT activity in Xene pus eye cups maintained in culture. The in vitro NAT rhythm persisted in DD and was entrainable by LD cycles showing that the Xenopus eye is the locus of a circadian clock.

If intraocular clocks exist in birds they could be communicating with the rest of the system via either neural or hormonal routes (or both). However, melatonin can affect circadian rhythms, and ocular secretion of melatonin into the blood (Fig. 4) would offer a mechanism whereby such putative clocks could communicate with the rest of the system. Alternatively, if extraocular clocks are responsible for the ocular melatonin rhythm, they must be communicating with the eye either by a hormonal link or by a neural link other than the efferent innervation such as via sympathetic or parasympathetic nerves.

DISCUSSION

To date several sites have been identified as components of the avian circadian system: the pineal, the SCN, and the eyes.

Pineal Removal of the pineal from free-running

birds causes arrhythmicity in the locomotor

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Fig. 6. Melatonin rhythms in the left (ONX) and right (sham operated) eyes of quail 2 5 4 5 hr after being placed into DD following entrainment to LD 12:12.

activity rhythms of several species (house sparrow, Passer domesticus; white-crowned sparrow, Zonotrichia leucophrys gambelii; white-throated sparrow, Zonotrichia albicol- lis; Java sparrow, Padda oryziuora) (Gaston, '71; McMillan, '72: Ebihara and Kawamura, '81). Pinealectomy of free-running starlings (Sturnus uulgaris) usually causes changes in the period of the rhythm, and arrhythmicity is seen in only a few cases (Gwinner, '78). Pinealectomy is without effect on free-running activity rhythms of sighted Japanese quail (Simpson and Follett, '81) but, as demonstrated herein, effects of pinealectomy can be seen in blinded quail. Pinealectomy, when performed alone, was also without effect on the activity rhythm of sighted pigeons (Col- umba liuia) free-running in dim continuous light (LL) (Ebihara et al., '84). However, pinealectomized pigeons that were also blinded were arrhythmic after prolonged exposure to dim LL.

Perhaps the most thoroughly studied species with respect to the pineal's role in circadian organization is the house sparrow. These studies have shown 1) interruption of the nervous input or output from the pineal does not abolish rhythmicity in locomotor activity (Zimmerman and Menaker, '751, 2) trans- plantation of a pineal into the eye of a previously pinealectomized arrhythmic house sparrow reintroduces rhythmicity (Zimmer- man and Menaker, '79), and 3) the ieintro-


duced rhythm emerges rapidly and bears the phase of the donor bird (Zimmerman and Menaker, '79). The reintroduction of a rhythm in transplanted birds and the trans- fer of phase is strong evidence that the pineal can act as a circadian pacemaker. The fact that rhythmicity emerges rapidly in transplanted birds suggests that the pineal is coupled to the rest of the circadian system by a hormonal link.

Chicken pineals cultured in vitro continue to generate circadian rhythms in NAT activity and melatonin production, offering une- quivocal evidence that the avian pineal contains a circadian clock(s) (Deguchi, '79; Takahashi et al., '80; Binkley et al., '78). The chicken pineal is likely comprised of a num- ber of endogenous clocks since small frag- ments of pineal can still show rhythms in melatonin production and an NAT rhythm is seen in dispersed pineal cell cultures (Taka- hashi and Menaker, '84; Deguchi, '79). Rhythms in organ-cultured pineals are most persistent and robust in LD cycles but these rhythms will not persist for more than several cycles in DD. The dissipation of the rhythm in DD may represent a damping out of the oscillation(s) generating the rhythm or the uncoupling of the myriad clocks within the pineal. A robust rhythm in pineal melatonin production in an intact bird, therefore, may depend on a daily neural, hormonal, or photic input to the pineal. A recent study, for example, suggests that a daily neural signal through the sympathetic nerves to the pineal may be essential for maintaining a rhythm of melatonin production in the chicken pineal (Cassone and Menaker, '83).

SC" Bilateral lesions of the SCN of birds ex-

posed to constant conditions causes arrhythmicity in all species tested to date (Java sparrow, house sparrow, Japanese quail) (Ebihara and Kawamura, '81; Takahashi and Menaker, '82; Simpson and Follett, '81). After SCN lesions in Japanese quail the activity pattern typically "decayed" over several weeks, producing activity patterns in which circadian rhythmicity was either lost or barely discernible (Simpson and Follett, '81). In Java sparrows SCN lesions caused a rapid loss of rhythmicity but lesioned birds were rhythmic under LD cycles and could show positive phase angles to light onsets (Ebihara

and Kawamura, '81) SCN lesions also induced arrhythmicity in house sparrows in DD but in LD 12:12 the locomotor activity was rhythmic and similar to that of intact birds (Takahashi and Menaker, '82). Lesions of the SCN of mammals also abolish activity rhythms as well as a host of other overt circadian rhythms and in vitro studies of SCN tissue support the hypothesis that the SCN is the locus of a neural circadian pacemaker(s) (Rusak and Zucker, '79; Green and Gillette, '82).

Eyes In only two avian species, the pigeon and

the Japanese quail, have the role of the eyes in circadian organization been examined. As discussed previously, the eyes play a major role in circadian organization in Japanese quail and bilateral enucleation has a major impact on entrained activity rhythms and can even completely disrupt entrainment in many cases. All blinded quail are arrhythmic in DD. Whereas pinealectomy a- lone has no discernible effect either in LD or DD in quail, pinealectomy effects can be seen if pinealectomy is combined with blinding. In the pigeon neither pinealectomy, blinding, nor pinealectomy plus blinding seemed to affect entrained activity rhythms and neither blinding nor pinealectomy, when performed alone, abolished rhythmicity in dim LL (Ebi- hara et al., '84). However, pigeons that had been both blinded and pinealectomized were arrhythmic in prolonged dim LL. Therefore, the pigeon is similar to the quail insofar as combining pinealectomy and blinding has greater effects than either performed alone. Also, in both species the eyes are importantly involved in circadian organization but greater effects of blinding are noted in the quail since blinding alone causes a major disruption or arrhythmicity in LD, and arrhythmicity in DD, whereas in the pigeon blinding alone does not have such effects.

There are two possible routes by which the eyes could communicate with the rest of the circadian system: hormonal andlor neural. A hormonal output involving melatonin is an attractive possibility for several reasons. First, exogenous administration of melatonin can affect circadian systems in a vari- ety of vertebrates (Ebihara and Kawamura, '81; Turek et al., '76; Gwinner and Benzin- ger, '78). Second, only the pineal and eyes of quail secrete melatonin into the blood (Un-


derwood et al., ’84) and these are the organs whose removal affects the circadian rhythm of activity. Third, if the premise that the pineal of birds communicates with the rest of the circadian system via the cyclic release of melatonin is valid (Cassone and Menaker, ’ 83 , then it must logically follow that there must also be some role for the ocular secretion of melatonin that occurs in quail. A neural output for the eye, however, is at least as attractive a possibility since there is a direct neural pathway that originates in the eye and terminates on the SCN, a region of the hypothalamus that may well act as a central neural clock (Takahashi and Men- aker, ’82; Ebihara and Kawamura, ’81; Simpson and Follett, ’81). Clearly, much needs to be learned concerning the role of the eye of quail in circadian organization including whether or not a circadian pacemaker actually resides within the eye and how the eye and the rest of the system are linked together.

Circadian organization of different species of birds appears to involve the same components (pineal, SCN, and eyes) but the relative importance of these components varies between species. It seems likely that all of these components may exhibit autonomous rhythmicity and, in fact, each site may in turn be comprised of a population of interacting circadian clocks (cells). Clearly, in order to invest structure to the system these different (presumably oscillatory) components must be able to interact with one another either in a mutual or hierarchical fashion. The different species-dependent effects of pinealectomy or blinding observed would reflect the amount of residual integrity of the multioscillatory system after it has been deprived of one of its major components. For example, after pinealectomy the strength of mutual coupling between (and possibly within) putative extrapineal oscillators (SCN, eyes?) may vary between species. If coupling is too weak to keep them synchronized in the absence of the pineal then the oscillators would drift out of phase or become damped, producing an arrhythmic activity pattern in constant conditions (i.e., house sparrow, Java sparrow). If sufficient mutual coupling remains, the pinealectomized birds will remain rhythmic in constant conditions but show period changes (starlings). If the extrapineal oscillators are strongly coupled, no effects of pinealectomy may be seen (pigeon, Japanese quail). This kind of model would also explain why com-

bining pinealectomy with blinding may be more effective in disrupting circadian organization in some species (i.e., Japanese quail, pigeons) than either pinealectomy or blinding alone.

Clearly much remains to be learned about the nature of the interactions between these components of the circadian system. In fact, it is likely that the avian circadian system is more “complex” and contains more components than we can as yet appreciate.

ACKNOWLEDGMENTS

This research was supported by NSF grant PCM-8116880 (to H.U.) and by an NICHHD research career development award 1 KO4 HD00276-01 (to H.U.).

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Circadian organization in Japanese quali