EVIDENCE FOR A NONRETINAL PATHWAY OF LIGHT TO THEPINEAL GLAND OF NEWBORN RATS

BY MARK ZWEIG,* SOLOMON H. SNYDERJ AND JULIUS AXELRODNATIONAL INSTITUTE OF MENTAL HEALTH, NATIONAL INSTITUTES OF HEALTH,

BETHESDA, MARYLAND

Communicated by Seymour S. Kety, June 14, 1966

The pineal gland of rats is markedly affected by exposure of the animals to vary-ing periods of light and darkness. After maintenance of adult rats in continuouslighting, there is a significant decrease in pineal gland weight' and in the enzymaticcapacity of the pineal gland to synthesize melatonin,2 and an increase in the ac-tivity of the serotonin-synthesizing enzyme, 5-hydroxytryptophan decarboxylase,in the pineal gland.3-5 These effects of light exposure are abolished by bilateralorbital enucleation.3-7

Circadian, or 24-hr rhythms, have been demonstrated in the content of serotoninin the rat pineal gland8 and in the activity of the melatonin-forming enzyme.9The pineal content of serotonin is maximal at about 1 P.M.(lights on from 5 A.M.to 7 P.M.), and declines rapidly after the lights are turned off to reach a trough atabout 11 p.M.8 We found'0 11 that this rhythm appears to be endogenous, since itpersists in blinded animals and in rats kept in continuous darkness for up to 2 weeks.However, the nocturnal decline in pineal serotonin content can be prevented ifillumination is extended an additional 4 hr to 11 p.M.8 11 Additional light exposuredoes not prevent the nocturnal decline in pineal serotonin in adult rats subjectedto bilateral orbital enucleation, indicating that this effect required intact retinae."

Intact retinae appear to be required for lighting information to influence mostendocrine'2' 13 and circadian systems in mammals."'14 However, Ganong et al."5have found that measurable amounts of light can penetrate the skull to the brainsof mammals, without the intervention of the eyes. Other workers have obtainedevidence suggesting that light can directly affect hypothalamic neurons-in the duck'6and rat.'7The present work was undertaken to study the development of the circadian

rhythm in pineal gland serotonin content in newborn rats and its control by en-vironmental lighting. Experiments were designed to examine the possibility thatlighting information might influence the pineal gland by an extraretinal route innewborn rats.Methods.-Sprague-Dawley rats, both male and female, of varying ages were

maintained with their mothers under diurnal lighting conditions in clear plasticcages at a constant temperature of 250C for at least 3 days prior to experimentaltreatment. An overhead fluorescent lamp provided about 110-150 ft-c of illumi-nation at the level of the cages. Unless otherwise noted, lights were kept on from 5A.M. to 7 P.M. daily. Rats were killed by anesthesia with chloroform at 1 P.M.and 11 P.M. Pineal glands were removed immediately, placed on paper towels im-pregnated with cold, isotonic saline, and weighed on a 25-mg Roller Smith balance.Two pineal glands were used for serotonin assay. After weighing, the pineal

glands were homogenized in 0.5 ml of ice-cold 0.01 N hydrochloric acid with aconical ground-glass homogenizer and frozen. Serotonin assays were performed onthe following day by the method of Snyder et al. 18

515

516 PHYSIOLOGY: ZWE1G, SNYDER, AND AXELROD PROC. N. A. S.

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FIG. 1.-Twenty-four-hr serotonin rhythmin pineal glands of 6-day-old and 12S-day-old FIG. 2.-Persistence of a 24-hr rhythm inrats and the effect of additional lighting. pineal serotonin in blinded rats after addi-Each group contained 16 rats. Vertical bars tional lighting. Each group contained 12-16show the magnitude of the standard error of rats. Vertical bars show the magnitulde ofthe mean. the standard error of the mean.

Complete bilateral orbital enucleation was carried out under light ether anes-thesia.

Results.-Presence of the serotonin rhythm in pineal of newborn rats: Groups of6-day-old and 12-day-old rats kept under diurnal lighting conditions were killed at1 P.M. (8 hr of light) or 11 P.M. (4 hr of darkness). Some groups were transferredat 7 P.M. on the day of killing to a room in which illumination was extended anadditional 4 hr to 11 P.M., at which time they were killed. Pineal glands of rats inall groups were examined for serotonin content (Fig. 1). The serotonin concentra-tion in pineal glands of 6-day-old rats at 1 P.M. was about half that of 12-day-oldrats, confirming earlier results.'9 Negligible amounts of serotonin were detected inten pooled pineal glands from 1- and 3-day-old rats. In both 6- and 12-day-oldrats, pineal serotonin levels were 2-3 times higher at 1 P.M. than at 11 P.M. in dark-ness. Exposure to 4 additional hr of lighting prevented the nocturnal decline ofpineal serotonin content in both of these groups. The extent of the day-nightchanges in pineal serotonin and the effect of additional lighting were essentially thesame as observed earlier in adult rats."l These effects of lighting were found despitethe fact that the eyelids of all 6- and 12-day-old rats in this study were firmly shut.

Effect of blinding on the serotonin rhythm and on the response to additional lightingin pineals of 12-day-old rats: The presence of the pineal serotonin rhythm and theresponse to additional lighting in newborn rats whose eyelids have not yet openedcan be explained in several ways: light may penetrate the closed eyelids; lightinginformation might be communicated to the newborn rats by the mother. Alter-natively, lighting information could reach the newborn rat directly, affecting the

VOL. 56, 1966 PHYSIOLOGY: ZWEIG, SNYDER, AND AXELROD 517

pineal gland by a pathway that does not require the retinae. To investigate thesepossibilities, groups of rats were subjected to bilateral orbital enucleation when11 days old and were killed at 12 days of age at 1 P.M. and 11 P.M. along with normalcontrols. Some normal and blinded rats were exposed to 4 additional hr oflight (7-11 P.M.) on the day they were killed. It was found earlier" that additionallighting was unable to prevent the nocturnal decline in pineal serotonin contentin blinded adult rats. To examine the possibility that the influence of additionallighting in 12-day-old rats might be mediated by the mother, the mother of onegroup of blinded rats exposed to additional lighting was herself blinded by bilateralorbital enucleation on the day before killing. The circadian rhythm in pinealserotonin of blinded rats did not differ from that of the normal controls (Fig. 2).When the blinded animals were exposed to 4 additional hr of lighting, their pinealserotonin content at 11 P.M. was significantly (p < 0.001) higher than that of blindedrats in darkness at 11 P.M. Their pineal serotonin concentration was lower (p< 0.001) than that of normal controls exposed to 4 additional hr of lighting. Sero-tonin levels for the blinded group with a blinded mother were the same as for thecorresponding group with a normal mother.

Thus, unlike earlier findings in adult blinded rats," additional lighting clearlyhad an effect in partially preventing the nocturnal decline in pineal serotonin in12-day-old blinded rats. In addition, these results indicate that this effect is notmediated by the mother. Since additional lighting completely abolished the noc-turnal decline in pineal serotonin in newborn rats with intact eyes but closed eye-lids and blinding partially eliminated this effect, some lighting information mustenter the eyes through the closed eyelids to affect the pineal gland. However, thepartial abolition of the nocturnal decline in pineal serotonin in blinded 12-day-oldrats by additional lighting would indicate that some lighting information reaches thepineal bland by an extraretinal pathway.

Effect of blinding and hooding on the pineal serotonin rhythm and the response toadditional lighting in 12-day-old rats: In order to ascertain whether the presumedextraretinal pathway of light to the pineal gland in 12-day-old rats involved thehead, the following experiment was designed. Groups of 11-day-old rats wereblinded and removed from their mothers on the 12th day of age. Rats were killedat 1 P.M. or 11 P.M. or transferred on the day of killing to a room in which the lightswere on until 11 P.M., providing 4 additional hr of lighting. Rats exposed to addi-tional lighting were subdivided into hooded and nonhooded groups. Hooded ratswere clothed in a black corduroy hood which covered the head and extendeddown to the inferior border of the rib cage with holes for the forelimbs. Animalswere able to breathe and move about freely while wearing the hoods. The hoodsdid not permit the entry of any detectable light under the experimental conditions.Animals were hooded at 5 P.M. on the day they were killed.As previously observed, 4 additional hr of lighting partially eliminated the noc-

turnal decline in pmeal serotonin of the 12-day-old blinded rats (Fig. 3). Pinealserotonin values at 11 P.M. of hooded rats exposed to additional lighting did notdiffer from those of blinded rats at 11 P.M. in darkness, indicating that hooding com-pletely abolished the effect of additional lighting. These results show that lightinginformation can reach the pineal gland by a pathway that involves the head but notthe eyes.

518 PHYSIOLOGY: ZWEIG, SNYDER, AND AXELROD PROC. N. A. S.

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FIG. 3.-Effect of hooding on the pineal FIG. 4.-Effect of blinding on the pinealgland response to additional lighting in gland response to additional lighting in 27-12-day-old blinded rats. Each group con- day-old rats. Groups contained 16 rats.tamed 16 rats. Vertical bars show the stand- Vertical bars indicate the standard error ofard error of the mean. the mean.

Pineal serotonin rhythm in- 7-day-old blinded rats: Groups of 24-day-old ratswhose eyelids were completely open were blinded by bilateral orbital enucleation.When they were 27 days old, animals were killed at 1 P.m. and 11 P.m. along withnormal controls and their pineal glands examined for serotonin (Fig. 4). On theday they were killed, some groups were transferred to a room in which the lightswere kept on until 11 P.m. when they were killed. In the control animals, 4 addi-tional hr of light completely prevented the nocturnal decline in pineal serotonin.Unlike findings in 12-day-old rats, additional lighting had no effect in blinded 27-day-old rats. These results indicate that in 27-day-old rats, the nonretinal path-way of light to the pineal gland is no longer operative.

Discussion.-Results of the present study indicate that the 24-hr rhythm in pinealserotonin content is present at 6 days of age, the earliest time at which serotoninis detectable in the rat pineal gland. Since blinding did not completely abolish theinfluence of lighting, it is not clear whether or not the pineal serotonin rhythm innewborn rats is "endogenous," i.e., persists in the absence of environmental cues.In the 27-day-old rats as well as the adult rat1' the serotonin rhythm has been foundto be endogenous, since a nocturnal fall in this amine occurs in blinded animals at11 P.M. It would appear that as early as the 6th day of life, lighting has a synchro-nizing influence on the pineal serotonin rhythm.The complete abolition and the partial loss, after blinding, of the response of

pineal serotonin to additional lighting suggests that some light penetrates the closedeyelids of these animals and is transmitted to the pineal gland.The most striking finding in this study is the demonstration that lighting informa-

tion can be conveyed to the pineal gland of the 12-day-old rat by a nonretinal routeinvolving the head. By 27 days of age, this nonretinal pathway does not appearto be functional. The possibility of nonretinal photoreception in mammals hasbeen observed by other workers. Benoit,'6 in numerous experiments, has shownthat the photosexual reflex in immature male ducks can take place after bilateral

VOL. 56, 1966 PHYSIOLOGY: ZWEIG, SNYDER, AND AXELROD 519

orbital enucleation. The response in blinded ducks can be accentuated by placinga quartz rod deep into the orbit at an area overlying the hypothalamus. As a re-sult of these experiments, Benoit has concluded that light can activate the photo-sexual reflex in ducks by a direct action on hypothalamic photoreceptors.

Lisk and Kannwischer'7 have obtained evidence for a direct effect of light onhypothalamic neurons of the adult rat. In their experiments, continuous light,impinging by means of a stereotaxically implanted glass rod, on the suprachiasmaticregion of enucleated rats resulted in a constant estrouslike vaginal cycle. Ifthe glass rods were covered by black masking tape, the effect of continuous lighton the estrous cycle was obliterated.The location of the presumed extraretinal photoreceptor described here is not

clear. In amphibians, the pineal complex itself has been shown to contain photo-receptive elements,20 and to transduce lighting information into nerve impulses.2'In the adult rat, however, histological studies have failed to reveal the presence ofphotoreceptive cells in the pineal gland.22 It is possible, however, that in the veryyoung rat there is a photoreceptive function for the pineal gland. It should benoted that the pineal gland of the rat has a superficial location, just beneath theskull at the junction of the sagittal and occipital sutures.Another possibility is that photoreception might take place via hypothalamic

receptors as suggested by the work of Benoit"6 and of Lisk and Kannwischer.'7We have obtained evidence that the pathway by which lighting changes influencethe pineal gland in the adult rat involves the medial forebrain bundle as well as theeyes. The reduction of melatonin-synthesizing capacity in the pineal gland byconstant light exposure can be obliterated by lesions in the medial forebrain bun-dle.23 The circadian changes in pineal serotonin concentration are also abolishedby these lesions, suggesting that the central mechanisms controlling the pinealserotonin rhythm and central components of the pathway of light to the pinealgland may have the same or similar anatomic representation in the brain.The present demonstration of extraretinal photoreception in very young rats

would suggest that lighting information may be utilized in controlling physiologicalprocesses in mammals some time before visual function can be demonstrated.The pineal gland is an end-organ of the sympathetic nervous system24 and has

been implicated in gonadal regulation.7 The ability of lighting information toinfluence the pineal gland prior to the development of visual function would suggestthat photic control of autonomic and endocrine events in mammals may be sep-arate from and developmentally precede retinal photoreception.Summary.-A 24-hr rhythm has been found in serotonin content of the pineal

gland of newborn rats. Four additional hr of lighting can prevent the nocturnaldecline in pineal serotonin in 12-day-old rats whose eyelids are closed. There is apartial response to additional lighting in 12-day-old blinded rats. Hooding of 12-day-old rats completely abolishes the response to additional lighting. Additionallighting does not prevent the nocturnal decline in pineal serotonin in 27-day-oldblinded rats. These data indicate that in the very young rat, light can influence thepineal gland by an extraretinal pathway which involves the head.

* Medical student, Washington University School of Medicine, St. Louis, Missouri.t Present address: Henry Phipps Clinic, Johns Hopkins Hospital, Baltimore, Md. 21205.1 Fiske, V. M., K. Bryant, and J. Putnam, Endocrinology, 66, 489 (1960).

520 PHYSIOLOGY: ZWEIG, SNYDER, AND AXELROD PROC. N. A. S.

2Wurtman, R. J., J. Axelrod, and L. Phillips, Science, 142, 1071 (1963).3Snyder, S. H., J. Axelrod, J. Fischer, and R. J. Wurtman, Nature, 203, 981 (1964).4Snyder, S. H., and J. Axelrod, Federation Proc., 23, 206 (1964).5 Snyder, S. H., J. Axelrod, J. E. Fischer, and R. J. Wurtman, J. Pharmacol. Exptl. Therap.,

147, 371 (1965).'Wurtman, R. J., J. Axelrod, and J. E. Fischer, Science, 143, 1328 (1964).7Wurtman, R. J., J. Axelrod, E. W. Chu, and J. E. Fischer, Endocrinology, 75, 266 (1964).8 Quay, W. B., Gen. Comp. Endocrinol., 1, 3 (1963).9 Axelrod, J., R. J. Wurtman, and S. H. Snyder, J. Biol. Chem., 240, 949 (1965).10 Snyder, S. H., M. Zweig, and J. Axelrod, Life Sci., 3, 1175 (1964).11 Snyder, S. H., M. Zweig, J. Axelrod, and J. Fischer, these PROCEEDINGS, 53, 301 (1965).12 Browman, L. G., J. Exptl. Zool., 75, 375 (1937).Is Hollwich, F., Ann. N. Y. Acad. Sci., 117, 105 (1964).14Richter, C. P., Biological Clocks in Medicine and Psychiatry (Springfield, Illinois: Charles C

Thomas, 1965), p. 17.16 Ganong, W. F., M. D. Shepherd, J. R. Wall, E. Evanbrunt, and M. T. Clegg, Endocrinology,

72, 962 (1963)."Benoit, J., Ann. N. Y. Acad. Sci., 117, 204 (1964).17 Lisk, R. D., and L. R. Kannwischer, Science, 146, 272 (1964).18Snyder, S. H., J. Axelrod, and M. Zweig, Biochem. Pharmacol., 14, 831 (1965).19 Quay, W. B., and A. Halevy, Physiol. Zool., 35, 1 (1962).20 Oksche, A., Progr. Brain Res., 10, 3 (1965).21 Dodt, E., and M. Jacobson, J. Neurophysiol., 26, 752 (1963).22 Wolfe, D., Progr. Brain Res., 10, 332 (1965).23 Axelrod, J., S. H. Snyder, R. Y. Moore, and A. Heller, The Pharmacologist, 8, 187 (1966).24 Kappers, J. A., Jr., Z. Zellforsch., 52, 163 (1960).