CONTROL OF THE CIRCADIAN RHYTHM IN SEROTONIN CONTENT OF THE RAT PINEAL GLAND BY SOLOMON H. SNYDER, MARK ZWEIG,* JULIUS AXELROD, AND JOSEF E. FISCHER LAEOEATOItY OF CUNICAL SCIENCE, NATIONAL INSTITUTJ!i OF MENTAL EEALTE, NATIONAL INSTITUTES OF HEALTH Cmnmu~~ by Sey~ut S. Ketg, December 3,1964 Circadian rhythms have been demonstrated in mammals for a variety of biological phenomena, including running activity, l rectal temperat,ure,2 phma eosinophil and corticosterone levels,2* * liver mitoses, and nucleic acid content.' Certain of these rhythms are considered endogenous since they persist in the absence of en- vironmental lighting. l* s Controlling centers within the organism for these rhythms have been sought without success. Thus &renal-dependent rhythms in epidermal mitosea and rectal temperature persist after hypophysectomy, although their am- plitude is diminished.`, 7 In the rat pineal gland, marked circadian rhythms have been observed in serotonin* and melatonin9 content and in the activity of the melatonin-forming enzyme.`O This study was undertaken to examine possible mechanisms controlling the cir- cadian rhythm in pineal serotonin content. MetJlods.-Sprague-Dawley female rate (150-180 gm) were maintained under diurnal lighting conditions in clear plastic cage at a constant temperature of 25'T. An overhead fluorescent lamp provided about llO-15Oft-c of illumina tion at the level of the cages. Unless otherwise notad, lights were kept on from 5 A.M. to 7 P.M. daily. Rate were killed by neck fracture at 1 P.M. and 11 P.M. In experiments involving constant light or dark environments, rate were kept in an isolated, soundproofed and air-conditioned room equipped with double-door light baffles. Pineal glands were removed immediately, placed on paper towels impregnated with cold isotonic saline, and weighed on a 25mg Roller-Smith balance. f: Single pineal glands were used for serotonin assay. After weighing, the pineal glands were homogenized in 0.5 ml of water with a tapered nylon rod in a l&ml glass-stoppered centrifuge tube and frozen. Serotonin assap were performed on the following day by a modification of the ninhydrin procedure of Vanable which is described elsewhere." In this procedure serotinin ww extracted from pineal gland homogenates, reacted with ninhjrdrin, and the resultant compound was meas@ flu&ometrically. Complete bilateral orbital enucleation was &&d-out under light ether anesthesia. Bilateral superior cervical ganglionectomy was performed under ether anesthesia. Superior cervical ganglia were decentralized in rata under ether anesthesia by removing a l-cm segment of cervical sympathetic chaii beginning 3 mm below the ganglion, with care to avoid diiturbiig the ganglion iteelf. Adrenalectomired, thyroidectomized, hypophysectomized, and oijphorectomized Sprague- Uawley female rats (150-180 gm) with matched control rats were obtained from Hormone Assay Laboratories. ** Results.-The egect of varying kghting conditions and blinding on the circadian rhythm in skotonin content of the rat pineal gland: Groups of rats kept under diurnal lighting conditions were killed at 1 P.M. (G hr of light) and 11 P.M. (4 hr of dark- ness), and their pineal glands were assayed for serotonin content. Serotonin levels were 2 to 3 timea higher in pineal glands of rats killed at 1 P.M. than in those of rats k&d at 11 P.M. (Table 1). These results essentially confirm the findings of Quay." Groups of rats which had been kept under diurnal lighting for 1 week were trans- ferred to constant light or dark environments. After 5 days these animals were killed at 1 P.M. and 11 P.M. and their pineal glands examined for serotonin (Table 301 302 PHYSIOWGY: SNYDER ET AL. Paoc. N. A. S. TABLE 1 1). The circadian changes in pineal sero- EFFECT OF CONTINUOUS LIQHT OR D-NESS ON THE CIIKXDIAN RH~RM IN SEE~TONIN tonin levels were abolished in rats kept CONTENT OF TEE PINEAL GLAND in constant light. At 11 P.M. the pineal Pined serotonin Group (mrg/mg f S.E.M.) serotonin levels of rats maintained in con- Diurnal light tinuous light were elevated to the 1 P.M. I P.M. 72 f 5.2 34 f 3.1* values found in diurnal lighting. In con- 11 P.M. Continuous light trast to the effect of continuous light ex- 1 P.M. 65 f 6.4 11 P.M. 62 f 7.2 posure, the circadian rhythm of pineal Constant darknes serotonin persisted unchanged in rats 1 P.M. 68 f 1.3 11 P.M. 32 f 2.X* kept in continuous darkness. These rc+ *Differ from 1 P.M. values p < 0.001. sults indicate that this circadian rhythm Groups containing 10 rats were maintained in can be maintained in the absence of en- continuous liiht or dark environments 01` under diurnal light conditions. After 5 days, serotwi~ content of their piaeal glands wan m-red. vironmental lighting but can be sup- pressed by continuous light exposure. The effects of blinding on the serotonin rhythm in the pineal gland are shown in Table 2. Both eyes were removed from groups of rats which were then maintained under diurnal lighting conditions for a l- or 2-week period. The animals were then killed at 1 P.M. and 11 P.M. and their pineal glands assayed for serotonin. The serotonin rhythm was still present in both groups of blinded rats. To rule out the possible influence of extraretinal photoreceptors, blinded rats were kept in constant light, or dark environments for 1 week and their pineal glands assayed for serotonin content at 1 P.M. and 11 P.M. (Table 3). In contrast to the suppressive effect of constant light exposure in normal rats, the serotonin rhythm persisted in blinded rats kept in a constant light environment. To establish the rapidity with which continuous light abolishes the serotonin rhythm in the pineal gland, rats were placed in diurnal lighting conditions for 5 days. On the 6th day, groups of control and blinded rats were transferred to a room in which illumination was extended an additional 4 hr to 11 P.M. Pineal glands were removed at 1 P.M,,,and 11 P.M. on the 6th day and examined for serotonin con- tent (Table 4). There was no reduction at 1L P.M. in the serotonin levels of the pineals of mts which were exposed to additional illumination. Blinded rats showed a fall in pineal serotonin content %t lr P.M. whether or not the lights had been turned off at 7 P.M. These observations indicate that the circadian rhythm in pineal serotonin content can be extinguished by exposure to an additional 4 hr of TABLE 2 EFFECTOF BLINDINQ ON TEE CIBCADIAN TABLE 3 RHYTHM IN PINEALSIEROTONIN$JONTENT EFFECT OF CONTINUOUS LIGHT OR DARKNESS Pines1 serolonin ON THE CIRCADIAN RHYTHM IN PINEAL Treatment (mcg/mg 3~ S.E.M.) SEIWT~NIN OF BLINDED RATS None 1 P.M. 11 P.M. Bli;dyM( 1 week) . . 11 P.M. Blinded (2 weeks) 1 P.M. 11 P.M. 63 f 5.9 16 f 3.1* 55 4.8 f 20 f 2.8* 64 f 9.7 23 3.4" f Pineal serotonin Group (mrghg -fE S.E.M.) Continuous light 1 P.M. 63 i 7.4 11 P.M. 22 zt 2.5* Continuous darkness 1 P.M. 56 f 6.9 11 P.M. 23 f 2.2* *Differ from 1 P.&I. values p < 0.001. Pineal gLanda of group8 of 10 rata in diurnal lighting were examined for serotonin content 1 or 2 weeks after blinding. *Difier from 1 P.M. valuea p < 0.001. .4fter 4 days in diurnal lighting. groups of 10 rata were blinded and traderred to constrnt light or dark environments. Seven days l&x. their pine& went asayed for qerotonin. VOL. 53, 1965 PHYSIOLOGY: SNYDER ET AL. 303 light. Furthermore, the suppressive ef- TABLE 4 feet of additional light exposure requires EFFECT OF ONE ADDITIONAL PERIOD OF LIGHT intact retinae. ON THE CIRCADIAN RHYTHM IN PINEAL SEFWTONIN CONTENT Egect of the removal of endocrine glands 012 the circadian rhythm in the serotonin content of the rat pineal gland: Previous works indicated that the circadian rhythm of eosinophil count requires intact adrenal glands. To examine for possible influ- ences of endocrine organs on the circadian Pineal serotonin Group (mrg/mg rt S.E.M.) Control 1 P.M. 66 f 7.: i 11 P.M. (lights off) 23 * 4.1 L* 11 P.M. (lights on) 59 f 6.3 Blinded 1 P.M. 61 f 7.0 11 P.M. (lights off) 18 f 2.6* 11 P.M. (lights on) 13 f 2.8* rhythm in pineal serotonin, adrenalec- tomized, thyroidectomized, oijphorectom- ized, and hypophysectomized rats were *Differ from 1 P.Y. values p'< 0.001. After 5 days' exposure of rats to diurnal light- ing, acme groups were transferred at 9 A.Y. to s kept in diurnal lighting, killed at 1 P.M. room in which lights were not turned 05 at 7 P.Y. and the remainder kept in diurnal lighting condo- tions (lighta off st 7 P.M.). All groupa, contain- ing 6 to 10 rats, were killed 011 the day of transfer. and 11 P.M. 1 week after operation, and ' their pineal glands examined for serotonin content (Fig. 1). The removal of these glandular tissues had no effect on the circadian serotonin rhythm. The role of the sympathetic nervous system in the control of the circadian rhythm of serotonin content in the pineal gland: The major, if not the sole, innervation of the rat pineal gland is derived from sympathetic fibers whose cell bodies are located in the superior cervical ganglia. l8 It has been shown in this laboratory that the ac- tivities of hydroxyindole-O-methyl transferase, the melatonin-synthesizing enzyme," and 5-hydroxytryptophan decarboxylase15 in the pineal gland are affected by its sympathetic innervation. The influence of the sympathetic nerves on the circadian rhythm of serotonin in the pineal gland was examined in the following experiments. Groups of rats whose superior cervical ganglia had been bilaterally extirpated were kept in diurnal lighting for 6 days after the operation. These rats were examined for pineal serotonin content in the usual manner at 1 P.M. and 11 P.M. (Table 5); The circadian rhythm in pineal serotonin was completely abolished in ganglion- ectomized rats. The 1 P.M. values were lowered and the 11 P.M. values were ele- vated. These results differ. somewhat from the suppressive action of constant change in 1 P.M. levels. These obser- vations would suggest that constant light exposure and gangiionectomy dif- fer in the mechanisms of their aboli- tion of the pineal circadian serotopin rhythm. There are several possible explana- tions for the suppressive action. of superior cervical ganglionectomy : the - ; circadian rhythm may be intrinsic to the pineal gland but requires intact FIG. L-Lack of effect of removal of glands on the circadian serotonin rhythm in the rat pineal sympathetic nerves for its expression; gland. Each group contained 10 rats. Vertical bars show the magnitude of the standard error of the controlling mechanism for the the man. light exposure fhich resulted in an a w llPY elevation of 11 P.M. values with no r; i 304 PHYSIOLOGY: SNYDER ET AL. PROC. N. A. S. TABLE 5 EFFECT OF S~~PEEIOB CERVICAL GANQLIONECT~MY ON TEF, CIBCUJIAN RHYTHM IN ENRmyTu SBFUWONIN TABLE 6 Em OF DECFJN TRALIZATION ON TEE fkG4DIAN yNI&NNEAL &6ROTONIN Pined ssrotonin Group (mro/mg f S.E.M.) 11 P:Y: Pines1 ._erotanin (mpg/me 3~ S.E.M.) ShiSIIl-oprated 1 P.M. 66 i 6.7 45 zt 2.1t 42 f 3.3t 11 P.M. Decentralized 1 P.M. 11 P.M. (lights off) 11 P.M. (lighta on) 18 i 2.1* 42 f 4.0t 44 f 3.2t 43 f 2.8t +Diere from sbam-opcwated 1 P.M. v&w *Differ from abarn-operated 1 P.Y. value p < 0.001. p < 0.001. t Di5er from &am-operated 1 P.Y. and 11 P.M. vsluea p < 0.001. Rata were kept in diurnal Ii after operation and were kill&n the 7th day. tie for 6 days Eaah group contsined 12-14 rata. t Di5er from both ahrm-operatad 1 P.Y. and 11 P.Y. valued p < 0.001. All rata were examined for pine81 w?otonin 6 daym after operation. One up of decentralized rata wan exposed to 4 hr o additional illumina- T tion before pines1 gland removal. rhythm could be localized in the superior cervical ganglia; the control might be neural but local&d elsewhere in the body and communicated to the pineal gland via preganglionic fibers. To examine this latter possibiity, the preganglionic nerves to the superior cer- vical ganglia were severed bilaterally in groups of rats which were then maintained in diurnal lighting for 6 days before assaying their pineals for serotonin (Table 6). This treatment abolished the circadian rhythm in pineal serotonin in the same manner as did ganglionectomy. One group of decentralized rats was transferred, on the day they were killed, to a room in which the lights were not turned off at 7 P.M., and were killed at 11 P.M. Pineal serotonin levels for this group were the same as for decentralized groups under diurnal lighting. These results indicate that the serotonin rhythm is extrinsic to the pineal gland and communicated from the central nervous system ta this organ via preganglionic sympathetic fibers to the superior cervical ganglia. Discussion .-The circadian rhythm in ser&onin content of the pineal gland is probably'endogenous since it persi$s ip the absence of environmental lighting and when other environmental cues Isound, temperature) are kept constant. Unlike endogenous circadian rhythms of eosinophil count, rectal temperature, plasma cor- ticosterone levels, ahd running activity, l6 the endogenous serotonin rhythm is not influenced by the removal of the adrenal gland, nor is it tiected by oijphorectomy, hypophysectomy, or thyroidectomy. Exposure to additional light for as little as 4 hr will completely suppv the rhythm by preventing the nocturnal decline in pineal serotonin content. In contr&st to the almost immediate and complete aboli- tion of the pineal serotonin rhythm by a single additional period of light, other en- dogenous rhythms, such aa running activity in rodents,' are altered by continuous light exposure more gradually. As is the case with other circadian rhythms," daily changes in light exposure might act as an external synchronizer (Zeifgder) for the serotonin rhythm in the pineal gland. Preliminary reports"* l* had shown that superior cervical ganglionectomy abolished the circadian rhythm in pineal serotonin content, although blinding was without effect. Ii These data, coupled with the present experiments on decentraliza- tion, clearly demonstrate that the controlling mechanism for the pineal serotonin VOL. 53,1965 PHYSIOL0GY: SNYDER ET AL. 305 rhythm is extrinsic to this gland. Information regarding the control of the rhythm is probably transmitted from the central nervous system to the pineal gland via preganglionic fibers to the superior cervical ganglia where they synapse with post- ganglionic sympathetic nerves. Since the serotonin rhythm in the pineal gland, which is controlled by sympathetic centers in the central nervous system, closely resembles other endogenous circadian rhythms, it is possible that a similar central sympathetic mechanism may be involved in their control. The sympathetic nervous system has also been implicated in the regulation of the circadian rhythm in melatonin synthesis in the pineal gland.`0 However, unlike the serotonin rhythm, the circadian variations in melatonin synthesis are not en- dogenous, but are directly influenced by external lighting information which is com- municated to the pineal gland via the superior cervical ganglia.`O Sympathetic nerves are necessary for other actions of light on the pineal gland. Constant light exposure elevates the activity of 5hydroxytqyptophan decarboxylase, the ser- otonm-forming enzyme, in the rat pineal gland and results in a decrease in pineal gland weight.lgn m Removal of the superior cervical ganglia abolishes the effect of light on this enzymeI and on pineal weight." Summary.-The presence of a circadian rhythm in serotonin content of the rat pineal gland has been confirmed. This rhythm persists in blinded rats and in rats kept in constant darkness but is abolished by one additional period. of light.. The suppressive effects of light exposure act via the retinae. The rhythm is unaffected by hypophysectomy, thyroidectomy, adrenalectomy, or oophorectomy. Superior cervical ganglionectomy or decentralization of the superior cervical ganglia abolishes the pineal serotonin rhythm. * NIH Special Fellow, firs&year medical student, Washington University Medical School, St. Louis, MO. ' Johnson, M. S., J. ExpU. Zool., 82,315 (1939). 3 Halberg, F., A. Zander, M. W. Houglum, and H. R. Milhlemann, Am. J. Physzbl., 177,361 (1954). * Halberg, F., R. E. Peterson, and R. H. Silber, Endacrinol., 64,222 (1959). 4 Jardetsky, C. D., C. P. Barnum,,.and F. Halberg, Am. J. Phystil., 187, 608 (1956). 6 Halberg, F., M. B. Visscher, and J. J. Bittner, Am. J. Physiol., 174,109 (1953). 0 Zander, H. A., J. Waerhaug, and F. Halberg, J. Clin. Embcrinol. Metab., 14, 829 (1954). r Ferguson, D. J., M. B. Visscher, F. HalbergGand 4. M. Levy, Am. J. Physiol., 190, 235 (1957). 8 Quay, W. B., Gen. Comp. Enducrinol., 1, 3 (1963). 9 Quay, W. B., Proc. Sot. Exptl.`BioZ., 115, 710 (1964). IO Axelrod, J., R. J. Wurtrnan, and S. H. Snyder, J. Biol. Chem., in press. ii Vanable, J. W., Anal. Bioehcm., 6, 393 (1963). I* Snyder, S. H., J. Axelrod, and M. Zweig, Biochem. Pharmzwl., in press. la Kappem, J. A., Jr., 2. ZeUfor&h., 52, 163 (1960). 14 Wurtman, R. J., J. Axelrod, and J. Fischer, Seiace, 143,132s (1964). fi Snyder, S. H., J. Axelrod, J. Fischer, and R. J. Wurtman,`N&ure, 203, 4948 (1964). 16 Halberg, F., E. Halberg, C. P. Barnum, and J. J. Bittner, in Ph&periadi.sm anrl Related Phenolnerur in Plants and An&x&, ed. R. B. Withrow (Washington: AAAS, 1959), p. 803. 17 Snyder, S. H., M. Zweig, and J. Axelrod, Life Sciences, 3, 1175 ( 1964). `a Fiske, V. M., &x&x, 146,253 (1964). is Snyder, 5. II., and J. Axelrod, Federation Proc., 23,206 (1964). m Fiske, V. M., K. Bryant, and J. Putnam, Endacrinol., 66,489 (1960).