THE PINEAL GLANDThe function of this small organ near the center of th - PDF document

THE PINEAL GLANDThe function of this small organ near the center of the mammalianbrain has long been a mystery. Recent studies indicate that it isa "biological clock" that regulates the activity of the sex glandsby Richard J. Wurtman and Julius AxelrodBuried nearly in the center of thebrain in any mammal is a smallwhite structure, shaped somewhatlike a pinecone, called the pineal body.In man this organ is roughly a quarterof an inch long and weighs about atenth of a gram. The function of thepineal body has never been clearlyunderstood. Now that the role of thethymus gland in establishing the body'simmunological defenses has been dem-onstrated, the pineal has become per-haps the last great mystery in thephysiology of mammalian organs. Thismystery may be nearing a solution:studies conducted within the past fewyears indicate that the pineal is an in-tricate and sensitive "biological clock,"converting cyclic nervous activity gen-erated by light in the environment intoendocrine-that is, hormonal-informa-tion. It is not yet certain what physio-logical processes depend on the pinealclock for cues, but the evidence at handsuggests that the pineal participates insome way in the regulation of the go-nads, or sex glands.A Fourth Neuroendocrine TransducerUntil quite recently most investi-gators thought that the mammalian pin-eal was simply a vestige of a primitivelight-sensing organ: the "third eye"found in certain cold-blooded verte-brates such as the frog. Other workers,noting the precocious sexual develop-ment of some young boys with pinealtumors, had proposed that in mammalsthe pineal was a gland. When the stan-dard endocrine tests were applied todetermine the possible glandular func-tion of the pineal, however, the resultsvaried so much from experiment to ex-periment that few positive conclusionsseemed justified. Removal of the pin-eal in young female rats was frequentlyfollowed by an enlargement of theovaries, but the microscopic appearanceof the ovaries did not change consistent-ly, and replacement of the extirpatedpineal by transplantation seemed tohave little or no physiological effect.Most experimental animals could sur-vive the loss of the pineal body with nomajor change in appearance or function.In retrospect much of the difficultyearly workers had in exploring and de-fining the glandular function of thepineal arose from limitations in thetraditional concept of an endocrine or-gan. Glands were once thought to beentirely dependent on substances inthe bloodstream both for their own con-trol and for their effects on the rest ofthe body: glands secreted hormones intothe blood and were themselves regu-lated by other hormones, which weredelivered to them by the circulation.The secretory activity of a gland wasthought to be maintained at a fairly con-stant level by homeostatic mechanisms:as the level of a particular hormone inthe bloodstream rose, the gland invari-ably responded by decreasing its secre-tion of that hormone; when the level ofthe hormone fell, the gland increasedits secretion.In the past two decades this conceptof how the endocrine system works hasproved inadequate to explain severalkinds of glandular response, includingchanges in hormone secretion broughtabout by changes in the external en-vironment and also regular cyclicchanges in the secretion of certain hor-mones (for example, the hormones re-sponsible for the menstrual cycle andthe steroid hormones that are producedon a daily cycle by the adrenal gland).Out of the realization that these andother endocrine responses must dependin some way on interactions between theglands and the nervous system the newdiscipline of neuroendocrinology hasdeveloped.In recent years much attention hascentered on the problem of locating thenervous structures that participate inthe control of glandular function. Ithas been known for some time that spe-cial types of organs would be needed to"transduce" neural information into en-docrine information. Nervous tissue isspecialized to receive and transmit in-formation directly from cell to cell; ac-cording to the traditional view, glandsare controlled by substances in thebloodstream and dispatch their mes-sages to target organs by the secretionof hormones into the bloodstream. Inorder to transmit information from thenervous system to an endocrine organa hypothetical "neuroendocrine trans-ducer" would require some of the spe-cial characteristics of both neural andendocrine tissue. It should respond tosubstances (called neurohumors) re-leased locally from nerve endings, andit should contain the biochemical ma-chinery necessary for synthesizing ahormone and releasing it into the blood-stream. Three such neurosecretory sys-tems have so far been identified. Theyare (1) the hypothalamus-posterior-pituitary system, which secretes theantidiuretic hormone and oxytocin, ahormone that causes the uterus to con-tract during labor; (2) the pituitary-releasing-factor system, also located inthe hypothalamus, which secretes poly-peptides that control the function ofthe pituitary gland, and (3) the adrenalmedulla, whose cells respond to a ner-vous input by releasing adrenaline intothe bloodstream.The advent of neuroendocrinologyhas provided a conceptual framework50 PINEAL BODYCORPUS CALLOSUMCEREBELLUMPINEAL BODYCORPUS CALLOSUMTWO VIEWS of the human brain reveal the central position of thepineal body. Section at top is cut in the median sagittal plane andis viewed from the side. Section at bottom is cut in a horizontalplane and is viewed from above; an additional excision has beenmade in this view to reveal the region immediately surroundingthe pineal. In mammals the pineal is the only unpaired midlineorgan in the brain. The name "pineal" comes from the organ's re-semblance to a pinecone, the Latin equivalent of which is pinea.51CEREBELLUM--,,1t11_ I/_,rlLvvLLm Vi that has been most helpful in charac-terizing the role of the pineal gland. Onthe basis of recent studies conducted bythe authors and their colleagues at theNational Institute of Mental Health, aswell as by investigators at other institu-tions, it now appears that the pineal isnot a gland in the traditional sense butis a fourth neuroendocrine transducer;it is a gland that converts a nervous in-put into a hormonal output.A Prophetic FormulationThe existence of the pineal body hasbeen known for at least 2,000 years.Galen, writing in the second centuryA.D., quoted studies of earlier Greekanatomists who were impressed with thefact that the pineal was perched atopthe aqueduct of the cerebrum and wasa single structure rather than a pairedone; he concluded that it served as avalve to regulate the flow of thoughtout of its "storage bin" in the lateralventricles of the brain. In the 17thcentury Ren6 Descartes embellished thisnotion; he believed that the pinealhoused the seat of the rational soul. Inhis formulation the eyes perceived theevents of the real world and transmittedwhat they saw to the pineal by way of"strings" in the brain [see illustrationbelow]. The pineal responded by allow-ing humors to pass down hollow tubesto the muscles, where they producedthe appropriate responses. With thehindsight of 300 years of scientific de-velopment, we can admire this prophet-ic formulation of the pineal as a neuro-endocrine transducer!In the late 19th and early 20th cen-turies the pineal fell from its exaltedmetaphysical state. In 1898 Otto Heub-ner, a German physician, published acase report of a young boy who hadshown precocious puberty and was alsofound to have a pineal tumor. In theSEAT OF THE RATIONAL SOUL was the function assigned to the human pineal (H) byRen6 Descartes in his mechanistic theory of perception. According to Descartes, the eyesperceived the events of the real world and transmitted what they saw to the pineal by wayof "strings" in the brain. The pineal responded by allowing animal humors to pass downhollow tubes to the muscles, where they produced the appropriate responses. The size ofthe pineal has been exaggerated in this wood engraving, which first appeared in 1677.52course of the next 50 years many otherchildren with pineal tumors and pre-cocious sexual development were de-scribed, as well as a smaller number ofpatients whose pineal tumors were as-sociated with delayed sexual develop-ment. Inexplicably almost all the casesof precocious puberty were observed inboys.In a review of the literature on pin-eal tumors published in 1954 Julian I.Kitay, then a fellow in endocrinology atthe Harvard Medical School, found thatmost of the tumors associated with pre-cocious puberty were not really pinealin origin but either were tumors of sup-porting tissues or were teratomas (prim-itive tumors containing many types ofcells). The tumors associated with de-layed puberty, however, were in mostcases true pineal tumors. He concludedthat the cases of precocious pubertyresulted from reduced pineal functiondue to disease of the surrounding tis-sue, whereas delayed sexual develop-ment in children with true pineal tumorswas a consequence of increased pinealactivity.The association of pineal tumors andsexual malfunction gave rise to hun-dreds of research projects designed totest the hypothesis that the pineal wasa gland whose function was to inhibitthe gonads. Little appears to have re-sulted from these early efforts. Laterin 1954 Kitay and Mark D. Altschule,director of internal medicine at McLeanHospital in Waverly, Mass., reviewedthe entire world literature on the pin-eal: some 1,800 references, about halfof which dealt with the pineal-gonadquestion. They concluded that of allthe studies published only two or threehad used enough experimental animalsand adequate controls for their data tobe analyzed statistically. These fewpapers suggested a relation between thepineal and the gonads but did little tocharacterize it. After puberty the humanpineal is hardened by calcification; thischange in the appearance of the pinealled many investigators to assume thatthe organ xwas without function andfurther served to discourage research inthe field. (Actually calcification appearsto be unrelated to the pineal functionswe have measured.)As long ago as 1918 Nils Holmgren,a Swedish anatomist, had examined thepineal region of the frog and the dogfishwith a light microscope. He was sur-prised to find that the pineal containeddistinct sensory cells; they bore amarked resemblance to the cone cells ofthe retina and were in contact withnerve cells. On the basis of these obser- vations he suggested that the pinealmight function as a photoreceptor, or"third eye," in cold-blooded vertebrates.In the past five years this hypothesis hasfinally been confirmed by electrophysi-ological studies: Eberhardt Dodt andhis colleagues in Germany have shotwnthat the frog pineal is a wavelength dis-criminator: it converts light energy ofcertain wavelengths into nervous im-pulses. In 1927 Carey P. McCord andFloyd P. Allen, working at Johns Hop-kins University, observed that if theymade extracts of cattle pineals and add-ed them to the media in which tadpoleswere swimming, the tadpoles' skinblanched, that is, became lighter incolor.Such was the state of knowledgeabout the pineal as late as five or sixyears ago. It appeared to be a photo-receptor in the frog, had something todo with sexual function in rats and inhumans (at least those with pineal tu-mors) and contained a factor (at leastin cattle) that blanched pigment cells intadpoles.The Discovery of AMelatoninThen in 1958 Aaron B. Lemer andhis co-workers at the Yale UniversitySchool of Medicine identified a uniquecompound, melatonin, in the pinealgland of cattle [see "Hormones and SkinColor," by Aaron B. Lerner; SCIENTIFICANIERICAN, July, 1961]. During the nextfour years at least half a dozen othermajor discoveries were made about thepineal by investigators representingmany different disciplines and institu-tions. Lerner, a dermatologist and bio-chemist, was interested in identifyingthe substance in cattle pineal extractsthat blanched frog skin. He and his col-leagues prepared and purified extractsfrom more than 200,000 cattle pinealsand tested the ability of the extracts toalter the reflectivity of light by piecesof excised frog skin. After four years ofeffort they succeeded in isolating andidentifying the blanching agent andfound that it was a new kind of biologi-cal compound: a methoxylated indole,whose biological activity requires amethyl group (CH,) attached to *anoxygen atom [see illustration on nexttwo pages].Metloxylation had been noted pre-viously in mammalian tissue, but theproducts of this reaction had always ap-peared to lose their biological activity asa result. The new compound, namedmelatonin for its effect on cells contain-ing the pigment melanin, appeared tolighten the amphibian skin by causingINNERVATION OF RAT PINEAL was the subject of a meticulous study by the Dutchneuroanatomist Johannes Arians Kappers in 1961. He demonstrated that the pineal of theadult rat is extensively innervated by nerves from the sympathetic nervous system. Thesympathetic nerves to the pineal originate in the neck in the superior cervical ganglion,enter the skull along the blood vessels and eventually penetrate the pineal at its blunt end(top ). Aberrant neurons from the central nervous system sometimes run up the pineal stalkfrom its base, but these generally turn and run back down the stalk again without synaps-ing. The pineal is surrounded by a network of great veins, into which its secretions proba.bly pass. According to AriEns Kappers, the innervation of the human pineal is quite similar.SYMPATHETIC NERVE terminates directly on a pineal cell, instead of on a bloodvessel or smooth muscle cell, in this electron micrograph of a portion of a rat pineal madeby David Wolfe of the Harvard Medical School. The nerve ending is characterized by darkvesicles, or sacs, that contain neurohumors. Magnification is about 12,500 diameters.53 Hl 11 11 ~ IH'C C C, C-OH5-HYDROXYTRYPTOPHANDECARBOXYLATINGENZYMESYNTHESIS OF MELATONIN in the rat pineal begins with theremoval of a carboxyl (COOH) group from the amino acid 5-H'~C\ H H-HO-C C-C-C-C-NH2H'Cc/C\N/C,Hthe aggregation of melanin granuleswithin the cells. It was effective in aconcentration of only a trillionth of agram per cubic centimeter of medium.No influence of melatonin could bedemonstrated on mammalian pigmenta-tion, nor could the substance actually beidentified in amphibians, in which itexerted such a striking effect. It re-mained a biological enigma that themammalian pineal should produce asubstance that appeared to have no bi-ological activity in mammals but was apotent skin-lightening agent in amphib-ians, which were unable to produce it!Both aspects of the foregoing enigmahave now been resolved. Subsequentresearch has shown that melatonin doesin fact have a biological effect in mam-mals and can be produced by amphib-ians. Spurred by Lerner's discovery ofthis new indole in the cattle pineal,Nicholas J. Giarman, a pharmacologist atthe Yale School of Medicine, analyzedpineal extracts for their content of otherbiologically active compounds. He foundthat both cattle and human pineals con-tained comparatively high levels of sero-tonin, an amine whose molecular struc-ture is similar to melatonin and whosefunction in nervous tissue is largely un-known. Studies by other investigatorssubsequently showed that the rat pinealcontains the highest concentration ofserotonin yet recorded in any tissue ofany species.A year before the discovery of mela-tonin one of the authors (Axelrod) andhis co-workers had identified a meth-oxylating enzyme (catechol-O-methyltransferase) in a number of tissues. Thisenzyme acted on a variety of catechols(compounds with two adjacent hydroxyl,or OH, groups on a benzene ring) butshowed essentially no activity withrespect to single-hydroxyl compoundssuch as serotonin, the most likely pre-cursor of melatonin. In 1959 Axelrodand Herbert Weissbach studied cattlepineal tissue to see if it might have thespecial enzymatic capacity to methox-ylate hydroxyindoles. They incubated N-acetylserotonin (melatonin without themethoxyl group) with pineal tissue anda suitable methyl donor and observedthat melatonin was indeed formed. Sub-sequently they found that all mamma-lian pineals shared this biochemical prop-erty but that no tissue other than pinealcould make melatonin. Extensive studiesof a variety of mammalian species haveconfirmed this original observation thatonly the pineal appears to have theability to synthesize melatonin. (In am-phibians and some birds small amountsof melatonin are also manufactured bythe brain and the eye.) Other investiga-tors have found that the pineal containsall the biochemical machinery neededto make melatonin from an amino acidprecursor, 5-hydroxytryptophan, whichit obtains from the bloodstream. It wasalso found that circulating melatonin israpidly metabolized in the liver to form6-hydroxymelatonin.Anatomy of the PinealWhile these investigations of the bi-ochemical properties of the pineal werein progress, important advances werebeing made in the anatomy of the pin-eal by the Dutch neuroanatomist Jo-hannes Ariens Kappers and by several0 1 2 3 4 5 6 7 8 9 10 11 12AGE OF RATS (WEEKS)-PLACEBO -MELATONINEFFECT OF MELATONIN on the estrus cycles of female rats is de-picted here. Rats that had been given daily injections of melatoninstarting in their fourth week of life developed a longer estrus cyclethan rats that had been similarly treated with a placebo. When themelatonin-treated animals were 10 weeks old, a placebo was substi-tuted for the melatonin and the estrus cycle returned to normal.54-z.,LU:wLUUry,LUU)Ic-U/)DU)LUizcr):aiCLUU)1008060402000 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30DAYS AFTER OPERATIONDARK LIGHTEFFECTS OF LIGHTING on the estrus cycles of three groups offemale rats are shown in the graphs on these two pages. The groups,each consisting of about 20 rats, were subjected respectively to asham operation (left), removal of their superior cervical ganglion(middle) and removal of their eyes (right). Each group was thenfurther subdivided, with about half being placed in constant lightACETYLATINGENZYMESEROTONINhydroxytryptophan by the enzyme 5-hydroxytryptophan decarbox-ylase. Serotonin, the product of this reaction, is then enzymatically10080604020I-zLUJZLU0ULUw(nU)KIaU)DU)LUzcnO-,,JKU)0 H OIC\ H H H I1HO-C C-C--C--N-C -CH3I II 1 H AH' C /C\ /C'HH HMETHOXYLATINGENZYMEH OICE H H H II-1 ;_ x v v v v --I 1 H HC C CH' % / \ /HH HN-ACETYLSEROTONINacetylated to form Nacetylserotonin. This compound in turn ismethoxylated by the enzyme hydroxyindole-O-methyl transferaseMELATONIN(HIOMT) to yield melatonin. In mammals HIOMT is found onlyin the pineal. Changes in basic molecule are indicated by color.American electron microscopists, in-cluding Douglas E. Kelly of the Uni-versity of Washington, Aaron Milofskyof the Yale School of Medicine andDavid Wolfe, then at the NationalInstitute of Neurologic Diseases andBlindness. In 1961 Ariens Kappers pub-lished a meticulous study of the nerveconnections in the rat pineal. He dem-onstrated clearly that although this or-gan originates in the brain in the de-velopment of the embryo, it loses allnerve connections with the brain soonafter birth. There is thus no anatomicalbasis for invoking "tracts from thebrain" as the pathway by which neuralinformation is delivered to the pineal.Ariens Kappers showed that insteadthe pineal of the adult rat is extensivelypenetrated by nerves from the sympa-thetic portion of the autonomic nervoussystem. The sympathetic nervous sys-tem is involuntary and is concernedwith adapting to rapid changes in theinternal and external environments; thesympathetic nerves to the pineal origi-nate in the superior cervical ganglionin the neck, enter the skull along thep-zZLDLoQ.(nIU)rU)LUJzc)10080604020n0 2 4 6 8DAYS AFTER OPERATION-DARK -LIGHTand the other half in constant darkness beginning one day aftertheir respective operations. Daily vaginal smears were taken on thefirst day and on the sixth through the 30th days after the operations.Results were plotted as the percentage of all the smears in a treat.ment group showing estrus phases each day. In general it wasfound that interference with the transmission of light informationblood vessels and eventually penetratethe pineal [see top illustration on page53]. Electron microscope studies latershowed that within the pineal manysympathetic nerve endings actually ter-minate directly on the pineal cells, in-stead of on blood vessels or smooth-muscle cells, as in most other organs[see bottom illustration on page 53].Among endocrine structures the orga-nization of nerves in the mammalianpineal appeared to be most analogous tothat of the adrenal medulla, one of thethree demonstrated neuroendocrinetransducers.Meanwhile electron microscope stud-ies by other workers on the pineal re-gions of frogs had confirmed many ofHolmgren's speculations. It was foundthat the pineal cells of amphibians con-tained light-sensitive elements that werepractically indistinguishable from thosefound in the cone cells of the retina,but that the pineal cells of mammalsdid not contain such elements. By 1962it could be stated with some assurancethat the mammalian pineal was notsimply a vestige of the frog "third eye,"zLUcrLuJICLU)zcoLU100806040200since the "vestige" had undergoneprofound anatomical changes with ev-olution.The Melatonin HypothesisEven though the mammalian pinealno longer seemed to respond directly tolight, there now appeared good evi-dence that its function continued to berelated somehow to environmental light.In 1961 Virginia Fiske, working at Wel-lesley College, reported that the expo-sure of rats to continuous environmentalillumination for several weeks broughtabout a decrease in the weight of theirpineals. She had been interested instudying the mechanisms by which theexposure of rats to light for long periodsinduces changes in the function of theirgonads. (For example, continous lightincreased the weight of the ovaries andaccelerated the estrus cycle). At thesame time one of the authors (Wurt-man, then at the Harvard MedicalSchool), in collaboration with Altschuleand Willard Roth, was studying the con-ditions under which the administration0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30DAYS AFTER OPERATION-DARK -LIGHTto the pineal gland (either by blinding or by cutting the sympa-thetic nerves) also abolished most of the gonadal response to light.These findings supported the authors' melatonin hypothesis, whichholds that one mechanism whereby light is able to accelerate theestrus cycle in normal animals is by inhibiting the synthesis inthe pineal of melatonin, a compound that in turn inhibits estrus.55Ii ?~ I -:i l! -!t lv&#xU000;at-l-f-0Url 1 1~~~,..., 1 L.~~~~~~~~~~~~~~~~~~~~~~~i,10 12 14 16 18 20 22 24 26 28 30-------'YI of cattle pineal extracts decreased ovaryIREMOVAL OF PITUITARYweight and slowed the estrus cycle.We soon confirmed Mrs. Fiske's find-ings, and we were also able to showthat the exposure of female rats to con-Li-n1- lh- tar -he rLmInl of heirInIel ULhU a sll I llll u n11 I IIllpineals had similar, but not additive,effects on the weight of their ovaries.These experiments suggested that per-haps one way in which light stimulatesovary function in rats is by inhibitingBLINDING Ithe action ot an inhibitor ound in pin-pI l PYtrnrtz It nnw hPonmP rlmrinl tnidentify the gonad-inhibiting substancein pineal extracts and to see if its svn-thesis or its actions were modified byINTERRUPTION OF SYMPATHETIC NERVES OF PINEALenvironmental lighting.In 1962 we began to work togethernrx iclantr- 1o nt- i orl1l c1l-lcnrPMINIMUMI MAXIMUM-I DARK X== LIGHTRESPONSE OF MELATONIN-FORMING ENZYME hydroxyindole-O-methyl transferas(HIOMT) to continuous light or darkness is shown under four different circumstances. Ithe control, or normal, animal continuous darkness induces an increase in HIOMT activit3whereas exposing the animal to continuous light has the opposite effect. The ability of thpineal gland to respond to environmental lighting is unaffected by the removal of thpituitary gland but is abolished following blinding or sympathetic denervation of the pinealDINrAI R Y PIF l DFRMIS,OPTIC LOBECEREBRUMMEDULLA OBLONGATiPINEAL EYE is a primitive photoreceptive organ found in certain cold-blooded vertebratesuch as the frog. Frog's brain is shown from the side (top) and from above (bottom56present in pineal extracts. Our planwas to subject extracts of cattle pinealglands to successive purification stepsand test the purified material for itsability to block the induction by lightof an accelerated estrus cycle in therat. Before undertaking the complicatedand time-consuming procedure of isolat-ing the active substance in the pinealy, glands of cattle, we first tested a mix-e ture of all the constituents that hade already been identified in this tissue.l. The mixture was found to block theeffects of light on the estrus cycle.Next we tested melatonin alone, sinceit was apparently the only compoundproduced uniquely by the pineal. Toour good fortune we found that whenrats were given tiny doses (one to 10micrograms per day) of melatonin byinjection, starting before puberty andcontinuing for a month thereafter, theestrus cycle was slowed and the ovarieslost weight-just as though the animalshad been treated with pineal extracts.In later studies we found that this effectof melatonin was chemically specific: itwas simulated by neither N-acetylsero-tonin, the immediate precursor of me-latonin, nor 6-hydroxymelatonin, themajor product of its metabolism. More-over, it was possible to accelerate theestrus cycle by removal of the pinealand to block this response by the injec-tion of melatonin.On the basis of these studies, per-formed in collaboration with ElizabethChu of the National Cancer Institute,we postulated that melatonin was amammalian hormone, since it is pro-duced uniquely by a single gland (thepineal), is secreted into the blood-stream and has an effect on a distanttarget organ (the vagina and possiblyalso the ovaries). We were not able toes identify the precise site of action ofmelatonin in affecting the gonads. The_____ _I _ ·11····1_CONTROL slowing of the estrus cycle could be pro-duced by actions at any of several sitesin the neuroendocrine apparatus, in-cluding the brain, the pituitary, theovaries or the vagina itself. Whenmelatonin was labeled with radioactiveatoms and injected into cats, it wastaken up by all these organs and wasselectively concentrated by the ovaries.William M. MhcIsaac and his col-leagues at the Cleveland Clinic haveconfinned the effects of melatonin onthe estrus cycle and have identified an-other pineal methoxyindole-methoxy-tryptophol-that has similar effects. Itappears likely that pineal extracts con-tain a family of hormones: the methoxy-indoles, all of which have in commonthe fact that they can be synthesized bythe methoxylating enzyme found onlyin the mammalian pineal.We next set out to determine whetheror not these effects of injected mela-tonin were physiological. Could the ratpineal synthesize melatonin and, if so,in what quantities? When rat pinealglands were examined for their abilityto make melatonin, we were disap-pointed to find that the activity of themelatonin-forming enzyme (hydroxyin-dole-O-methyl transferase, or HIOMIT)in the rat was much lower than in mostother species; the maximum amount ofmelatonin that the rat could make wasprobably on the order of one microgramper day. Our disappointment was soonrelieved, however, when we realizedthat the low activity of this enzymemade it likely that it was controlling therate-limiting step in melatonin synthesisin the intact animal. Knowing that con-tinuous exposure to light decreasedpineal weight, as well as the amount ofribonucleic acid (RNA) and protein inthe pineal, we next explored what ef-fect illumination might have on HIOMITactivity and thus on melatonin synthesis.Since the rat pineal gland was sosmall (about a milligram in weight) andhad so little enzymatic activity, it wasnecessary to devise extremely sensitivetechniques to measure this activity.W\7hen rats were subjected to constantlight for as short a period as a day ortwo, the rate of melatonin synthesis intheir pineals fell to as little as a fifththat of animals kept in continuous dark-ness. Since this effect of illuminationor its absence could be blocked byagents that interfered with protein syn-thesis, it appeared that light was actu-ally influencing the rate of formation ofthe enzyme protein itself.How was information about the stateof lighting being transmitted to the ratpineal? Three possible routes suggestedRETINAL CONE CELL from the eye of an adult frog is shown in this electron micrographmade by Douglas E. Kelly of the University of Washington. The photoreceptive outer seg-ment of the cell (top center) consists of a densely lamellated membrane. Parts of two largerrod photoreceptors can be seen on each side of cone. Magnification is about 13,000 diameters.K- --I : ..- ; I)1 , '5~~~~~~~~PINEAL CONE CELL from the pineal eye of an adult frog is shown in this electron micro-graph made by Kelly at approximately the same magnification as the micrograph at top.The lamellated outer segment of the pineal cell is practically indistinguishable from that ofthe retinal photoreceptor. Part of the membrane has torn away from the cell (top left).57 _ _1 _11_1 _1___I themselves. The first was that light pen-etrated the skull and acted directly onthe pineal; W. F. Ganong and his col-leagues at the University of Californiaat Berkeley had already shown that sig-nificant quantities of light do penetratethe skulls of mammals. This hypothesiswas ruled out, however, by demonstrat-LIGHTSUPERIOing that blinded rats completely lostthe capacity to respond to light withchanged HIOMT activity; hence lighthad to be perceived first by the retinaand was not acting directly on thepineal.The second possibility was that lightaltered the level of a circulating hor-SUGGESTED PATHWAY by which light influences the estrus cycle in the rat is depictedin this schematic diagram. Light stimuli impinge on the retinas and cause a change in theneural output of the superior cervical ganglion by way of an unknown route. This informa.tion is then carried by sympathetic nerves to the pineal gland, where it causes a decreasein the activity of HIOMT and in the synthesis and release of melatonin. This decreasein turn lessens the inhibiting effect of the circulating melatonin on the rate of the estruscycle. The precise site of action of melatonin in influencing the gonads is unknown; theslowing of the estrus cycle could be produced by actions at any one of several sites in theneuroendocrine apparatus, including the brain, the pituitary, the ovaries and the vagina.mone, perhaps by affecting the pituitarygland, and that this hormone secondar-ily influenced enzyme activity in thepineal gland. This hypothesis was alsoruled out by demonstrating that the re-moval of various endocrine organs, in-cluding the pituitary and the ovaries,did not interfere with the response ofpineal HIONIT to light.The third possibility was that infor-mation about lighting was transmittedto the pineal by nerves. FortunatelyArisns Kappers had just identified thenerve connections of the rat pineal ascoming from the sympathetic nervoussystem. We found that if the sympa-thetic pathway to the pineal was inter-rupted by the removal of the superiorcervical ganglion, the ability of mela-tonin-forming activity to be altered bylight was completely lost. Thus it ap-peared that light was stimulating theretina and then information about thislight was being transmitted to the pin-eal via sympathetic nerves. Within thepineal the sympathetic nerves probablyreleased neurohumors (noradrenaline orserotonin), which acted on pineal cellsto induce (or block the induction of)HIONIT; this enzyme in turn regulatedthe synthesis of melatonin.Since one way light influences thegonads is by changing the amount ofmelatonin secreted from the pineal, wereasoned that the effects of light on thegonads might be blocked if the trans-mission of information about light tothe pineal were interrupted. This couldbe accomplished by cutting the sympa-thetic nerves to the pineal-a proceduremuch less traumatic than the removalof the pineal itself. To test this hypothe-sis we placed groups of rats whose pin-eals had been denervated along withblinded and untreated animals in con-tinuous light or darkness for a month.Vaginal smears were checked daily forevidence of changes in the estrus cycle,and pineals were tested for melatonin-synthesizing ability at the end of theexperiment. It was found that interrupt-ing the transmission of light informationto the pineal (by cutting its sympatheticnerves-a procedure that does not inter-fere with the visual response to light)also abolished most of the gonadal re-sponse to light.Incidentally, the observation thatsympathetic nerves control enzyme syn-thesis in the pineal has provided, andshould continue to provide, a useful toolfor studies in a number of other bio-logical disciplines. For example, study-ing the changes in brain enzymes pro-duced by environmental factors offers auseful method for tracing the anatomy58_____ 1_1 111-·1------ of the nerve tracts involved. The obser-vation that the activity of at least onepart of the sympathetic nervous system(the superior cervical ganglion) is affect-ed by environmental lighting raises thepossibility that other regions of thisneural apparatus are affected similarly.If so, physiological studies of the effectsof light on other sympathetically inner-vated structures (for example the kid-neys and fat tissue) may be profitable.We have also found that light influ-ences the serotonin-forming enzyme inth nins_'l ln Ailt nntn nrth- r nran-s.11 ,t ~l su c n a to u 111 UI z; ulct t 3 .In contrast to HIONIT. the activitv ofthis enzyme increases when rats arekept in constant light and decreases indarkness. WlNhen rats are blinded orwhen the sympathetic nerves to thepineal are cut, the effect of light anddarkness on the serotonin-forming en-zyme is also extinguished. Furthermore,certain drugs that block the transmis-sion of sympathetic nervous impulsesalso abolish the effect of illumination onthis enzyme. The fact that lighting in-fluences pineal weight and at least twoenzyme systems in this organ suggeststhat it may regulate many additional,undiscovered biochemical events in thepineal, via the sympathetic nervoussystem.Diurnal and Circadian RhythmsThe pineal had been shown to re-spond and function under quite unusualconditions; for example, when an exper-imental animal was exposed to continu-ous light or darkness for several days.In nature, of course, animals that livein the temperate and tropical zones arerarely subjected to such conditions. Itbecame important to determine if thepineal could also respond to naturallyoccurring changes in the environment.In nature the level of light exposurechanges with both diurnal and annualcycles. Except in polar regions every24-hour day includes a period of sun-light and a period of darkness; the ra-tio of day to night varies with an an-nual rhythm that reaches its nadir atthe winter solstice and its zenith on thefirst day of summer. Lighting cycles.have been shown to be important in regulating several types of endocrinefunction: the increase in sunlight dur-ing the winter and spring triggers theannual gonadal growth and breedingcycles in many birds and some mammalsthat breed yearly, and the daily rhythmof day and night synchronizes a varietyof roughly daily rhythms in mammals,such as the cycle of adrenal-steroid se-cretion. Such rhythms are called cir--C6/ i .I_N.Q Hc 3J cclnCHDICYCLOPENTADIENE, ,C, 0CHLORENDIC ANHYDRIDEt -These Chemical "babies" willbuild a new chemical world2DICYCLOPENTADIENE DIOXIDE\ C 0CENDIC ANHYDRIDE\ % It )i i tU II 1fThis family of Velsicol dicyclopentadienederived chemicals needs the benefit ofyour experience, imagination and chemi-cal proficiency. Several of the growth areasthat are suggested for investigation includeelastomers, plasticizers, fire retardantchemicals, resins and epoxy curing sys-Cl C,C _/ I//HEXACHLOROCYCLOPENTADIENE.c --/C I Ho IMETHENDIC ANHYDRIDEfi f/ / /' X/tems. We can encourage your interest byproviding sample quantities of these chem-icals and descriptive data.VELSICOL CHEMICAL CORPORATION341 East Ohio St., Chicago, III, 60611SA-75VELSICOL CHEMICAL CORPORATIONGenlem."n: Plee tnd me dl. aendFor more information .m,,., o ,: ._ ,,oo...CtRlIm[TO00100 £000T10M#1[and testing samples. kCOMPANYADDRESSVelsicol MC STATE·.. the leading producer of dicyclopentadiene!. INTERNATIONAL REPRESENTATIVE: VELSICOL INTERNATIONAL CORPORATION, C.A., P.O. BOX 1687, NASSAU. BAHAMASNENEWintageBOOKSCIENCE IN THE CAUSE OF MANBy GERARD PIEL$1.65, now at your bookstoreRANDOM HOUSE59The Celestron 10The superior optical qualities of theSchmidt type telescope, which combinethe benefits of both lens and mirrorsystems, are presented in the Celestron10, a compound catadioptric system of150" e.f.l. This is an observatory qualitytelescope in large aperture, but its com-pact construction make it equally con-venient for your mountain-top excursionsor permanent installation. Objectsviewed through the Celestron 10 appear1000 times brighter than to the unaidedeye, and it will resolve down to 0.4 sec-onds of arc.Complete with pier. clock drive and set-ting circles 1870.00. Write for details.Celestron Pacific13214 Crenshaw Boulevard,Gardena, Californiaar---·--'"·-u'---------·r""` ·,J- n--- -, -- ·- --C- I L. -Y I_ ' --.--· -l r I --Z_~ .-- :.. w. R._I _ S | i I'f\S _PE$Lae;t cadian, from the Latin phrase meaning"about one day." Could the pineal re-spond to natural diurnal lighting shifts?If so, it might function to synchronizethe endocrine apparatus with theseshifts.In order to determine if normal light-ing rhythms influenced the pineal, wekept a large population of rats undercontrolled lighting conditions (lights onfrom 7:00 A.M. to 7:00 P.M.) for severalweeks and then tested their pineals formelatonin-forming ability at 6:00 A.M.,noon, 6:00 P.M. and midnight. In thefive hours after the onset of darkness(that is, by midnight) this enzymaticcapacity increased between two andthree times. Moreover, pineal weightalso changed significantly during thisperiod, again indicating that light wasaffecting many more compounds in thepineal than the single enzyme we weremeasuring.All circadian rhythms studied up tothis stage had in common the ability topersist for some weeks after animalswere deprived of environmental light-ing cues (by blinding or being placedin darkness). These rhythms no longershowed a period of precisely 24 hours,but they did fall in a range between 22and 26 hours and hence were thought tobe regulated by some internal mecha-nism not dependent on, but usually syn-chronized with, environmental lighting.Such endogenous, or internally regu-lated, circadian rhythms in rodentsinclude motor activity and rectal tem-perature, as well as the rhythm in ad-renal-steroid secretion. When we blind-ed rats or placed them in continuouslight or darkness, the pineal rhythm inmelatonin-forming activity was rapidlyextinguished. If instead of turning offthe lights at 7:00 P.m. illumination wascontinued for an additional five hoursand pineals were examined as usual atmidnight, the expected rise in melato-nin-forming activity was completelyblocked. This pineal rhythm in HIOMTactivity thus appears to be truly exoge-nous, or externally regulated, and is en-tirely dependent on shifts in environ-mental lighting. Hence this enzymerhythm may be more important in car-rying information about light to theglands than other circadian rhythmsthat do not depend on light for theirexistence.Recently Wilbur Quay of the Univer-sity of California at Berkeley has foundthat the content of serotonin in the ratpineal also undergoes marked circadianrhythms. The highest levels of thisamine are found in pineals at noon andthe lowest levels at midnight. Serotonincontent falls rapidly just at the timethat melatonin-forming activity is rising.In collaboration with Solomon Snyderwe studied the mechanism of the sero-tonin cycle. When rats are kept in con-tinuous light, the serotonin cycle is ex-tinguished. To our surprise, however,when rats are kept continuously in dark-ness or blinded, this rhythm persists,unlike the rhythm in the melatonin-forming ezyme. When the sympatheticnerves to the pineal are cut, the sero-tonin and HIONIT cycles are both sup-pressed. When the nerves from thecentral nervous system to the superiorcervical ganglion are interrupted, theserotonin rhythm is also abolished [seeillustration below]. Hence the serotoninrhythm in the pineal gland is similar tomost other circadian rhythms (and dif-fers from the HIONIT cycle) in that itis endogenous and depends on environ-mental light only as an external syn-chronizer. The mechanism that controlsthe serotonin rhythm appears to residewithin the central nervous system. Thepineal gland thus contains at least twodistinct biological clocks, one totallv de-pendent on environmental lighting andthe other originating within the brainbut cued by changes in lighting.At present little is known about whatorgans are dependent on the pinealclock for cues. The ability of melatoninto modify gonadal function suggests,but does not prove, that its secretionmay have something to do with the tim-ing of the estrus and menstrual cycles-two phenomena about whose mecha-nisms of control very little is known.One is tempted to argue teleologicallythat any control mechanism as compli-cated and sensitive as that found in themammalian pineal gland must havesome place in the economy of the body.M N M N M N M N M N M N. M N M N M N M N M N MBIOCHEMICAL RHYTHMS in the pineal gland of the rat wererecorded under various lighting and other conditions. Normallyboth the content of serotonin and the activity of the melatonin-forming enzyme (HIOMT) vary with a 24-hour cycle. The serotonincontent is greatest at noon (N), whereas the HIOMT activity isgreatest at midnight (M). The HIOMT cycle is completely depen-60dent on environmental lighting conditions: it disappears when ani-mals are kept in continuous light or darkness, or when they areblinded. The serotonin cycle persists in continuous darkness orafter blinding but can be abolished by keeping the rats in continu.ous light. Both cycles are depressed when the sympathetic nerves tothe pineal gland are cut (extreme right). Gray areas signify darkness.