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The Pineal Gland

The Pineal Gland

THE PINEAL

The function of this small near the center of the mammalian has long been a mystery. Recent studies indicate that it is a "biological clock" that regulates the activity of the

by Richard J. Wurtman and

Buried nearly in the center of the eal in young female rats was frequently in some way on interactions between the brain in any is a small followed by an enlargement of the glands and the the new white structure, shaped somewhat , but the microscopic appearance discipline of has like a pinecone, called the pineal body. of the ovaries did not change consistent- developed. In this organ is roughly a quarter ly, and replacement of the extirpated In recent years much attention has of an inch long and weighs about a pineal by transplantation seemed to centered on the problem of locating the tenth of a gram. The function of the have little or no physiological effect. nervous structures that participate in pineal body has never been clearly Most experimental could sur- the control of glandular function. It understood. Now that the role of the vive the loss of the pineal body with no has been known for some time that spe- gland in establishing the body's major change in appearance or function. cial types of organs would be needed to immunological defenses has been dem- In retrospect much of the difficulty "transduce" neural information into en- onstrated, the pineal has become per- early workers had in exploring and de- docrine information. Nervous is haps the last great mystery in the fining the glandular function of the specialized to receive and transmit in- of mammalian organs. This pineal arose from limitations in the formation directly from to cell; ac- mystery may be nearing a : traditional concept of an endocrine or- cording to the traditional view, glands studies conducted within the past few gan. Glands were once thought to be are controlled by substances in the years indicate that the pineal is an in- entirely dependent on substances in bloodstream and dispatch their mes- tricate and sensitive "biological clock," the bloodstream both for their own con- sages to target organs by the secretion converting cyclic nervous activity gen- trol and for their effects on the rest of of into the bloodstream. In erated by light in the environment into the body: glands secreted hormones into order to transmit information from the endocrine-that is, hormonal-informa- the and were themselves regu- nervous system to an endocrine organ tion. It is not yet certain what physio- lated by other hormones, which were a hypothetical "neuroendocrine trans- logical processes depend on the pineal delivered to them by the circulation. ducer" would require some of the spe- clock for cues, but the evidence at hand The secretory activity of a gland was cial characteristics of both neural and suggests that the pineal participates in thought to be maintained at a fairly con- endocrine tissue. It should respond to some way in the regulation of the go- stant level by homeostatic mechanisms: substances (called neurohumors) re- nads, or sex glands. as the level of a particular in leased locally from endings, and the bloodstream rose, the gland invari- it should contain the biochemical ma- A Fourth Neuroendocrine Transducer ably responded by decreasing its secre- chinery necessary for synthesizing a tion of that hormone; when the level of hormone and releasing it into the blood- Until quite recently most investi- the hormone fell, the gland increased stream. Three such neurosecretory sys- gators thought that the mammalian pin- its secretion. tems have so far been identified. They eal was simply a vestige of a primitive In the past two decades this concept are (1) the -posterior- light-sensing organ: the "third " of how the works has pituitary system, which secretes the found in certain cold-blooded verte- proved inadequate to explain several antidiuretic hormone and , a brates such as the frog. Other workers, kinds of glandular response, including hormone that causes the to con- noting the precocious sexual develop- changes in hormone secretion brought tract during labor; (2) the pituitary- ment of some young boys with pineal about by changes in the external en- releasing-factor system, also located in tumors, had proposed that in vironment and also regular cyclic the hypothalamus, which secretes poly- the pineal was a gland. When the stan- changes in the secretion of certain hor- that control the function of dard endocrine tests were applied to mones (for example, the hormones re- the , and (3) the adrenal determine the possible glandular func- sponsible for the and medulla, whose cells respond to a ner- tion of the pineal, however, the results the hormones that are produced vous input by releasing into varied so much from experiment to ex- on a daily cycle by the ). the bloodstream. periment that few positive conclusions Out of the realization that these and The advent of neuroendocrinology seemed justified. Removal of the pin- other endocrine responses must depend has provided a conceptual framework

50 PINEAL BODY

CORPUS CALLOSUM

CEREBELLUM

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PINEAL BODY

CEREBELLUM CORPUS CALLOSUM

TWO VIEWS of the brain reveal the central position of the made in this view to reveal the region immediately surrounding pineal body. Section at top is cut in the median sagittal plane and the pineal. In mammals the pineal is the only unpaired midline is viewed from the side. Section at bottom is cut in a horizontal organ in the brain. The name "pineal" comes from the organ's re- plane and is viewed from above; an additional excision has been semblance to a pinecone, the Latin equivalent of which is pinea.

51 that has been most helpful in charac- out of its "storage bin" in the lateral course of the next 50 years many other terizing the role of the . On ventricles of the brain. In the 17th children with pineal tumors and pre- the basis of recent studies conducted by century Ren6 Descartes embellished this cocious sexual development were de- the authors and their colleagues at the notion; he believed that the pineal scribed, as well as a smaller number of National Institute of Mental Health, as housed the seat of the rational soul. In patients whose pineal tumors were as- well as by investigators at other institu- his formulation the perceived the sociated with delayed sexual develop- tions, it now appears that the pineal is events of the real world and transmitted ment. Inexplicably almost all the cases not a gland in the traditional but what they saw to the pineal by way of of precocious were observed in is a fourth neuroendocrine transducer; "strings" in the brain [see illustration boys. it is a gland that converts a nervous in- below]. The pineal responded by allow- In a review of the literature on pin- put into a hormonal output. ing humors to pass down hollow tubes eal tumors published in 1954 Julian I. to the muscles, where they produced Kitay, then a fellow in at A Prophetic Formulation the appropriate responses. With the the Harvard Medical School, found that hindsight of 300 years of scientific de- most of the tumors associated with pre- The existence of the pineal body has velopment, we can admire this prophet- cocious puberty were not really pineal been known for at least 2,000 years. ic formulation of the pineal as a neuro- in origin but either were tumors of sup- , writing in the second century endocrine transducer! porting tissues or were (prim- A.D., quoted studies of earlier Greek In the late 19th and early 20th cen- itive tumors containing many types of anatomists who were impressed with the turies the pineal fell from its exalted cells). The tumors associated with de- fact that the pineal was perched atop metaphysical state. In 1898 Otto Heub- layed puberty, however, were in most the aqueduct of the and was ner, a German physician, published a cases true pineal tumors. He concluded a single structure rather than a paired case report of a young boy who had that the cases of one; he concluded that it served as a shown precocious puberty and was also resulted from reduced pineal function valve to regulate the flow of thought found to have a pineal tumor. In the due to disease of the surrounding tis- sue, whereas delayed sexual develop- ment in children with true pineal tumors was a consequence of increased pineal activity. The association of pineal tumors and sexual malfunction gave rise to hun- dreds of research projects designed to test the hypothesis that the pineal was a gland whose function was to inhibit the . Little appears to have re- sulted from these early efforts. Later in 1954 Kitay and Mark D. Altschule, director of at McLean Hospital in Waverly, Mass., reviewed the entire world literature on the pin- eal: some 1,800 references, about half of which dealt with the pineal- question. They concluded that of all the studies published only two or three had used enough experimental animals and adequate controls for their data to be analyzed statistically. These few papers suggested a relation between the pineal and the gonads but did little to characterize it. After puberty the human pineal is hardened by ; this change in the appearance of the pineal led many investigators to assume that the organ xwas without function and further served to discourage research in the field. (Actually calcification appears to be unrelated to the pineal functions we have measured.) As long ago as 1918 Nils Holmgren, a Swedish anatomist, had examined the pineal region of the frog and the dogfish with a light microscope. He was sur- SEAT OF THE RATIONAL SOUL was the function assigned to the human pineal (H) by Ren6 Descartes in his mechanistic theory of . According to Descartes, the eyes prised to find that the pineal contained perceived the events of the real world and transmitted what they saw to the pineal by way distinct sensory cells; they bore a of "strings" in the brain. The pineal responded by allowing humors to pass down marked resemblance to the cone cells of hollow tubes to the muscles, where they produced the appropriate responses. The size of the retina and were in contact with the pineal has been exaggerated in this wood engraving, which first appeared in 1677. nerve cells. On the basis of these obser-

52 vations he suggested that the pineal might function as a photoreceptor, or "," in cold-blooded . In the past five years this hypothesis has finally been confirmed by electrophysi- ological studies: Eberhardt Dodt and his colleagues in Germany have shotwn that the frog pineal is a wavelength dis- criminator: it converts light energy of certain wavelengths into nervous im- pulses. In 1927 Carey P. McCord and Floyd P. Allen, working at Johns Hop- kins University, observed that if they made extracts of cattle pineals and add- ed them to the media in which tadpoles were swimming, the tadpoles' skin blanched, that is, became lighter in color. Such was the state of knowledge about the pineal as late as five or six years ago. It appeared to be a photo- in the frog, had something to do with sexual function in rats and in INNERVATION OF RAT PINEAL was the subject of a meticulous study by the Dutch (at least those with pineal tu- neuroanatomist Johannes Arians Kappers in 1961. He demonstrated that the pineal of the mors) and contained a factor (at least adult rat is extensively innervated by from the sympathetic nervous system. The in cattle) that blanched pigment cells in sympathetic nerves to the pineal originate in the neck in the superior cervical ganglion, tadpoles. enter the along the blood vessels and eventually penetrate the pineal at its blunt end (top ). Aberrant from the sometimes run up the pineal stalk The Discovery of AMelatonin from 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 , into which its secretions proba. Then in 1958 Aaron B. Lemer and bly pass. According to AriEns Kappers, the innervation of the human pineal is quite similar. his co-workers at the Yale University School of Medicine identified a unique compound, , in the pineal gland of cattle [see "Hormones and Skin Color," by Aaron B. Lerner; SCIENTIFIC ANIERICAN, July, 1961]. During the next four years at least half a dozen other major discoveries were made about the pineal by investigators representing many different disciplines and institu- tions. Lerner, a dermatologist and bio- chemist, was interested in identifying the substance in cattle pineal extracts that blanched frog skin. He and his col- leagues prepared and purified extracts from more than 200,000 cattle pineals and tested the ability of the extracts to alter the reflectivity of light by pieces of excised frog skin. After four years of effort they succeeded in isolating and identifying the blanching agent and found that it was a new kind of biologi- cal compound: a methoxylated indole, whose biological activity requires a methyl group (CH,) attached to *an oxygen atom [see illustration on next two pages]. Metloxylation had been noted pre- viously in mammalian tissue, but the products of this reaction had always ap- peared to lose their biological activity as a result. The new compound, named SYMPATHETIC NERVE terminates directly on a pineal cell, instead of on a blood melatonin for its effect on cells contain- vessel or smooth muscle cell, in this electron micrograph of a portion of a rat pineal made ing the pigment melanin, appeared to by David Wolfe of the Harvard Medical School. The nerve ending is characterized by dark lighten the skin by causing vesicles, or sacs, that contain neurohumors. Magnification is about 12,500 diameters.

53 H H DECARBOXYLATING '~C\ H H ACETYLATING - HO-C C-C-C-C-NH 2 l 11 11 ~ I ENZYME ENZYME H'C C C, C-O H'Cc/C\ C , N / H H

5-HYDROXYTRYPTOPHAN

SYNTHESIS OF MELATONIN in the rat pineal begins with the hydroxytryptophan by the enzyme 5-hydroxytryptophan decarbox- removal of a carboxyl (COOH) group from the 5- ylase. Serotonin, the product of this reaction, is then enzymatically the aggregation of melanin granules tonin, an whose molecular struc- sequently they found that all mamma- within the cells. It was effective in a ture is similar to melatonin and whose lian pineals shared this biochemical prop- concentration of only a trillionth of a function in nervous tissue is largely un- erty but that no tissue other than pineal gram per cubic centimeter of medium. known. Studies by other investigators could make melatonin. Extensive studies No influence of melatonin could be subsequently showed that the rat pineal of a variety of mammalian species have demonstrated on mammalian pigmenta- contains the highest concentration of confirmed this original observation that tion, nor could the substance actually be serotonin yet recorded in any tissue of only the pineal appears to have the identified in , in which it any species. ability to synthesize melatonin. (In am- exerted such a striking effect. It re- A year before the discovery of mela- phibians and some small amounts mained a biological enigma that the tonin one of the authors (Axelrod) and of melatonin are also manufactured by mammalian pineal should produce a his co-workers had identified a meth- the brain and the eye.) Other investiga- substance that appeared to have no bi- oxylating enzyme (catechol-O-methyl tors have found that the pineal contains ological activity in mammals but was a ) in a number of tissues. This all the biochemical machinery needed potent skin-lightening agent in amphib- enzyme acted on a variety of catechols to make melatonin from an amino acid ians, which were unable to produce it! (compounds with two adjacent hydroxyl, precursor, 5-hydroxytryptophan, which Both aspects of the foregoing enigma or OH, groups on a benzene ring) but it obtains from the bloodstream. It was have now been resolved. Subsequent showed essentially no activity with also found that circulating melatonin is research has shown that melatonin does respect to single-hydroxyl compounds rapidly metabolized in the to form in fact have a biological effect in mam- such as serotonin, the most likely pre- 6-hydroxymelatonin. mals and can be produced by amphib- cursor of melatonin. In 1959 Axelrod ians. Spurred by Lerner's discovery of and Herbert Weissbach studied cattle of the Pineal this new indole in the cattle pineal, pineal tissue to see if it might have the Nicholas J. Giarman, a pharmacologist at special enzymatic capacity to methox- While these investigations of the bi- the Yale School of Medicine, analyzed ylate hydroxyindoles. They incubated N- ochemical properties of the pineal were pineal extracts for their content of other acetylserotonin (melatonin without the in progress, important advances were biologically active compounds. He found methoxyl group) with pineal tissue and being made in the anatomy of the pin- that both cattle and human pineals con- a suitable methyl donor and observed eal by the Dutch neuroanatomist Jo- tained comparatively high levels of sero- that melatonin was indeed formed. Sub- hannes Ariens Kappers and by several

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EFFECT OF MELATONIN on the estrus cycles of female rats is de- EFFECTS OF LIGHTING on the estrus cycles of three groups of picted here. Rats that had been given daily injections of melatonin female rats are shown in the graphs on these two pages. The groups, starting in their fourth week of developed a longer estrus cycle each consisting of about 20 rats, were subjected respectively to a than rats that had been similarly treated with a placebo. When the sham operation (left), removal of their superior cervical ganglion melatonin-treated animals were 10 weeks old, a placebo was substi- (middle) and removal of their eyes (right). Each group was then tuted for the melatonin and the estrus cycle returned to normal. further subdivided, with about half being placed in constant light

54 H O H O IC\ H H H I1 METHOXYLATING ICE H H HII 'Y -1 ;_ x v v v -v - HO-C C-C--C--N-C -CH 3 I II1 H ENZYME I 1 H H A C C C H' C /C\ /C'H H' % / \ /H H H H H

N-ACETYLSEROTONIN MELATONIN

acetylated to form Nacetylserotonin. This compound in turn is (HIOMT) to yield melatonin. In mammals HIOMT is found only methoxylated by the enzyme hydroxyindole-O-methyl transferase in the pineal. Changes in basic molecule are indicated by color.

American electron microscopists, in- blood vessels and eventually penetrate since the "vestige" had undergone cluding Douglas E. Kelly of the Uni- the pineal [see top illustration on page profound anatomical changes with ev- versity of Washington, Aaron Milofsky 53]. Electron microscope studies later olution. of the Yale School of Medicine and showed that within the pineal many David Wolfe, then at the National sympathetic nerve endings actually ter- The Melatonin Hypothesis Institute of Neurologic Diseases and minate directly on the pineal cells, in- Blindness. In 1961 Ariens Kappers pub- stead of on blood vessels or smooth- Even though the mammalian pineal lished a meticulous study of the nerve muscle cells, as in most other organs no longer seemed to respond directly to connections in the rat pineal. He dem- [see bottom illustration on page 53]. light, there now appeared good evi- onstrated clearly that although this or- Among endocrine structures the orga- dence that its function continued to be gan originates in the brain in the de- nization of nerves in the mammalian related somehow to environmental light. velopment of the , it loses all pineal appeared to be most analogous to In 1961 Virginia Fiske, working at Wel- nerve connections with the brain soon that of the , one of the lesley College, reported that the expo- after . There is thus no anatomical three demonstrated neuroendocrine sure of rats to continuous environmental basis for invoking "tracts from the transducers. illumination for several weeks brought brain" as the pathway by which neural Meanwhile electron microscope stud- about a decrease in the weight of their information is delivered to the pineal. ies by other workers on the pineal re- pineals. She had been interested in Ariens Kappers showed that instead gions of frogs had confirmed many of studying the mechanisms by which the the pineal of the adult rat is extensively Holmgren's speculations. It was found exposure of rats to light for long periods penetrated by nerves from the sympa- that the pineal cells of amphibians con- induces changes in the function of their thetic portion of the autonomic nervous tained light-sensitive elements that were gonads. (For example, continous light system. The sympathetic nervous sys- practically indistinguishable from those increased the weight of the ovaries and tem is involuntary and is concerned found in the cone cells of the retina, accelerated the estrus cycle). At the with adapting to rapid changes in the but that the pineal cells of mammals same time one of the authors (Wurt- internal and external environments; the did not contain such elements. By 1962 man, then at the Harvard Medical sympathetic nerves to the pineal origi- it could be stated with some assurance School), in collaboration with Altschule nate in the superior cervical ganglion that the mammalian pineal was not and Willard Roth, was studying the con- in the neck, enter the skull along the simply a vestige of the frog "third eye," ditions under which the administration

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and the other half in constant darkness beginning one day after to the pineal gland (either by blinding or by cutting the sympa- their respective operations. Daily vaginal smears were taken on the thetic nerves) also abolished most of the gonadal response to light. first day and on the sixth through the 30th days after the operations. These findings supported the authors' melatonin hypothesis, which Results were plotted as the percentage of all the smears in a treat. holds that one mechanism whereby light is able to accelerate the ment group showing estrus phases each day. In general it was estrus cycle in normal animals is by inhibiting the synthesis in found that interference with the transmission of light information the pineal of melatonin, a compound that in turn inhibits estrus.

55

------CONTROL of cattle pineal extracts decreased weight and slowed the estrus cycle. We soon confirmed Mrs. Fiske's find- ings, and we were also able to show that the exposure of female rats to con- Li-n1- lh- tar-he rLmInl of heir I InIelULhU a sll I llll u n11 I IIll REMOVAL OF PITUITARY pineals had similar, but not additive, effects on the weight of their ovaries. These experiments suggested that per- haps one way in which light stimulates ovary function in rats is by inhibiting the action ot an inhibitor ound in pin- BLINDING I pI l PYtrnrtz It nnw hPonmP rlmrinl tn identify the gonad-inhibiting substance in pineal extracts and to see if its svn- thesis or its actions were modified by environmental lighting. In 1962 we began to work together INTERRUPTION OF SYMPATHETIC NERVES OF PINEAL nrx iclantr- 1o nt-i orl1l c1l-lcnrP present in pineal extracts. Our plan was to subject extracts of cattle pineal glands to successive purification steps and test the purified material for its MINIMUMI MAXIMUM ability to block the induction by light of an accelerated estrus cycle in the -I DARK X== LIGHT rat. Before undertaking the complicated RESPONSE OF MELATONIN-FORMING ENZYME hydroxyindole-O-methyl transferas and time-consuming procedure of isolat- (HIOMT) to continuous light or darkness is shown under four different circumstances. I ing the active substance in the pineal the control, or normal, animal continuous darkness induces an increase in HIOMT activit3 y, glands of cattle, we first tested a mix- whereas exposing the animal to continuous light has the opposite effect. The ability of the ture of all the constituents that had pineal gland to respond to environmental lighting is unaffected by the removal of the already been identified in this tissue. pituitary gland but is abolished following blinding or sympathetic denervation of the pineall. The mixture was found to block the effects of light on the estrus cycle. Next we tested melatonin alone, since DINrAI R Y PIFl DFRMIS, it was apparently the only compound produced uniquely by the pineal. To our good fortune we found that when rats were given tiny doses (one to 10 micrograms per day) of melatonin by injection, starting before puberty and continuing for a month thereafter, the estrus cycle was slowed and the ovaries lost weight-just as though the animals had been treated with pineal extracts. In later studies we found that this effect of melatonin was chemically specific: it was simulated by neither N-acetylsero- tonin, the immediate precursor of me- latonin, nor 6-hydroxymelatonin, the OPTIC LOBE major product of its . More- over, it was possible to accelerate the estrus cycle by removal of the pineal and to block this response by the injec- tion of melatonin. On the basis of these studies, per- formed in collaboration with Elizabeth Chu of the National Institute, we postulated that melatonin was a mammalian hormone, since it is pro- duced uniquely by a single gland (the pineal), is secreted into the blood- stream and has an effect on a distant CEREBRUM MEDULLA OBLONGATi target organ (the and possibly also the ovaries). We were not able to PINEAL EYE is a primitive photoreceptive organ found in certain cold-blooded vertebratees identify the precise site of action of such as the frog. Frog's brain is shown from the side (top) and from above (bottom melatonin in affecting the gonads. The

56

______I _ ·11····1_ slowing of the estrus cycle could be pro- duced by actions at any of several sites in the neuroendocrine apparatus, in- cluding the brain, the pituitary, the ovaries or the vagina itself. When melatonin was labeled with radioactive atoms and injected into cats, it was taken up by all these organs and was selectively concentrated by the ovaries. William M. MhcIsaac and his col- leagues at the Cleveland Clinic have confinned the effects of melatonin on the estrus cycle and have identified an- other pineal methoxyindole-methoxy- tryptophol-that has similar effects. It appears likely that pineal extracts con- tain a family of hormones: the methoxy- indoles, all of which have in common the fact that they can be synthesized by the methoxylating enzyme found only in the mammalian pineal. We next set out to determine whether or not these effects of injected mela- tonin were physiological. Could the rat pineal synthesize melatonin and, if so, in what quantities? When rat pineal glands were examined for their ability to make melatonin, we were disap- pointed to find that the activity of the melatonin-forming enzyme (hydroxyin- RETINAL CONE CELL from the eye of an adult frog is shown in this electron micrograph dole-O-methyl transferase, or HIOMIT) made by Douglas E. Kelly of the University of Washington. The photoreceptive outer seg- in the rat was much lower than in most ment of the cell (top center) consists of a densely lamellated membrane. Parts of two larger rod photoreceptors can be seen on each side of cone. Magnification is about 13,000 diameters. other species; the maximum amount of melatonin that the rat could make was probably on the order of one microgram K- - - I : . .- ; I) per day. Our disappointment was soon 1 , '5~~~~~~~~ relieved, however, when we realized that the low activity of this enzyme made it likely that it was controlling the rate-limiting step in melatonin synthesis in the intact animal. Knowing that con- tinuous exposure to light decreased pineal weight, as well as the amount of ribonucleic acid (RNA) and in the pineal, we next explored what ef- fect illumination might have on HIOMIT activity and thus on melatonin synthesis. Since the rat pineal gland was so small (about a milligram in weight) and had so little enzymatic activity, it was necessary to devise extremely sensitive techniques to measure this activity. W\7hen rats were subjected to constant light for as short a period as a day or two, the rate of melatonin synthesis in their pineals fell to as little as a fifth that of animals kept in continuous dark- ness. Since this effect of illumination or its absence could be blocked by I agents that interfered with protein syn- thesis, it appeared that light was actu- ally influencing the rate of formation of the enzyme protein itself. PINEAL CONE CELL from the pineal eye of an adult frog is shown in this electron micro- How was information about the state graph made by Kelly at approximately the same magnification as the micrograph at top. of lighting being transmitted to the rat The lamellated outer segment of the pineal cell is practically indistinguishable from that of pineal? Three possible routes suggested the retinal photoreceptor. Part of the membrane has torn away from the cell (top left). 57

_ _1 _11_1 _1___ themselves. The first was that light pen- ing that blinded rats completely lost mone, perhaps by affecting the pituitary etrated the skull and acted directly on the capacity to respond to light with gland, and that this hormone secondar- the pineal; W. F. Ganong and his col- changed HIOMT activity; hence light ily influenced enzyme activity in the leagues at the University of California had to be perceived first by the retina pineal gland. This hypothesis was also at Berkeley had already shown that sig- and was not acting directly on the ruled out by demonstrating that the re- nificant quantities of light do penetrate pineal. moval of various endocrine organs, in- the of mammals. This hypothesis The second possibility was that light cluding the pituitary and the ovaries, was ruled out, however, by demonstrat- altered the level of a circulating hor- did not interfere with the response of pineal HIONIT to light. The third possibility was that infor- mation about lighting was transmitted to the pineal by nerves. Fortunately Arisns Kappers had just identified the nerve connections of the rat pineal as coming from the sympathetic nervous system. We found that if the sympa- thetic pathway to the pineal was inter- rupted by the removal of the superior cervical ganglion, the ability of mela- tonin-forming activity to be altered by light was completely lost. Thus it ap- peared that light was stimulating the retina and then information about this light was being transmitted to the pin- eal via sympathetic nerves. Within the LIGHT pineal the sympathetic nerves probably released neurohumors (noradrenaline or serotonin), which acted on pineal cells to induce (or block the induction of) HIONIT; this enzyme in turn regulated the synthesis of melatonin. SUPERIO Since one way light influences the gonads is by changing the amount of melatonin secreted from the pineal, we reasoned that the effects of light on the gonads might be blocked if the trans- mission of information about light to the pineal were interrupted. This could be accomplished by cutting the sympa- thetic nerves to the pineal-a procedure much less traumatic than the removal of the pineal itself. To test this hypothe- sis we placed groups of rats whose pin- eals had been denervated along with blinded and untreated animals in con- tinuous light or darkness for a month. Vaginal smears were checked daily for evidence of changes in the estrus cycle, and pineals were tested for melatonin- synthesizing ability at the end of the experiment. It was found that interrupt- ing the transmission of light information to the pineal (by cutting its sympathetic nerves-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 that SUGGESTED PATHWAY by which light influences the estrus cycle in the rat is depicted sympathetic nerves control enzyme syn- in this schematic diagram. Light stimuli impinge on the retinas and cause a change in the thesis in the pineal has provided, and neural 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 decrease should continue to provide, a useful tool in the activity of HIOMT and in the synthesis and release of melatonin. This decrease for studies in a number of other bio- in turn lessens the inhibiting effect of the circulating melatonin on the rate of the estrus logical disciplines. For example, study- cycle. The precise site of action of melatonin in influencing the gonads is unknown; the ing the changes in brain enzymes pro- slowing of the estrus cycle could be produced by actions at any one of several sites in the duced by environmental factors offers a neuroendocrine apparatus, including the brain, the pituitary, the ovaries and the vagina. useful method for tracing the anatomy 58

_____ 1_1 111-·1------of the nerve tracts involved. The obser- vation that the activity of at least one -C These part of the sympathetic nervous system Chemical "babies" will (the superior cervical ganglion) is affect- 6/ i .I _ build a new chemical world ed by environmental lighting raises the t - J- possibility that other regions of this ar---·--'"·-u'------·r""` ·, neural apparatus are affected similarly. i I'f If so, physiological studies of the effects Cl C, of light on other sympathetically inner- N.Q Hc3J cclnCH vated structures (for example the kid- C _/ I// neys and fat tissue) may be profitable. 2 DICYCLOPENTADIENEDIOXIDE We have also found that light influ- DICYCLOPENTADIENE HEXACHLOROCYCLOPENTADIENE ences the serotonin-forming enzyme in th nins_'l ln Ailt nntn nrth-r nran- s.11 c n ,t a ~l tosu u 111 UI z; ulct t 3 . . c -- In contrast to HIONIT. the activitv of , , \ C 0 this enzyme increases when rats are /C I Ho I C, 0 kept in constant light and decreases in C darkness. WlNhen rats are blinded or CHLORENDICANHYDRIDE ENDIC ANHYDRIDE METHENDIC ANHYDRIDE when the sympathetic nerves to the n--- - , -- ·- -- pineal are cut, the effect of light and \S _ \ % It )i i tUII 1f fi f/ / /' X/ darkness on the serotonin-forming en- I C-I_L. - Y ' - -. - - I - -·lr -Z PE$Lae;t zyme is also extinguished. Furthermore, This family of Velsicol dicyclopentadiene tems. We can encourage your interest by certain drugs that block the transmis- derived chemicals needs the benefit of providing sample quantities of these chem- sion of sympathetic nervous impulses your experience, imagination and chemi- icals and descriptive data. cal proficiency. Several of the growth areas VELSICOL CHEMICAL CORPORATION also abolish the effect of illumination on that are suggested for investigation include 341 East Ohio St., Chicago, III, 60611 this enzyme. The fact that lighting in- elastomers, plasticizers, fire retardant SA-75 fluences pineal weight and at least two chemicals, resins and epoxy curing sys- VELSICOLCHEMICAL CORPORATION Genlem."n:Plee tnd me dl. aend enzyme systems in this organ suggests For more information o ,: .m,,.,. _,,oo that it may regulate many additional, and testing samples. k ...CtRlIm[TO00100 £000T10M#1[ undiscovered biochemical events in the COMPANY pineal, via the sympathetic nervous ADDRESS Velsicol MC STATE system. ·.. the leading producerof dicyclopentadiene!.

INTERNATIONAL REPRESENTATIVE: VELSICOL INTERNATIONAL CORPORATION, C.A., P.O. BOX 1687, NASSAU. BAHAMAS Diurnal and Circadian _~ Rhythms .-- :..

w.R._ The pineal had been shown to re- spond and function under quite unusual conditions; for example, when an exper- NENEW imental animal was exposed to continu- ous light or darkness for several days. In , of course, animals that live intage in the temperate and tropical zones are BOOK rarely subjected to such conditions. It became important to determine if the pineal could also respond to naturally occurring changes in the environment. In nature the level of light exposure changes with both diurnal and annual cycles. Except in polar regions every 24-hour day includes a period of sun- The Celestron 10 The superior optical qualities of the light and a period of darkness; the ra- Schmidt type telescope, which combine tio of day to night varies with an an- the benefits of both lens and mirror nual rhythm that reaches its nadir at systems, are presented in the Celestron 10, a compound catadioptric system of the winter solstice and its zenith on the 150" e.f.l. This is an observatory quality first day of summer. Lighting cycles. telescope in large aperture, but its com- have been shown to be important in pact construction make it equally con- venient for your mountain-top excursions regulating several types of endocrine or permanent installation. Objects function: the increase in sunlight dur- viewed through the Celestron 10 appear ing the winter 1000 times brighter than to the unaided and spring triggers the eye, and it will resolve down to 0.4 sec- annual gonadal growth and breeding SCIENCE IN THE CAUSE OF MAN onds of arc. cycles in many birds and some mammals By GERARD PIEL Complete with pier. clock drive and set- that breed yearly, and the daily rhythm $1.65, now at your bookstore ting circles 1870.00. Write for details. of day and night synchronizes a variety RANDOM HOUSE of roughly daily rhythms in mammals, Celestron Pacific such as the cycle of adrenal-steroid se- 13214 Crenshaw Boulevard, cretion. Such rhythms are called cir- I _ S | Gardena, California

59 cadian, from the Latin phrase meaning include motor activity and rectal tem- ness or blinded, this rhythm persists, "about one day." Could the pineal re- perature, as well as the rhythm in ad- unlike the rhythm in the melatonin- spond to natural diurnal lighting shifts? renal-steroid secretion. When we blind- forming ezyme. When the sympathetic If so, it might function to synchronize ed rats or placed them in continuous nerves to the pineal are cut, the sero- the endocrine apparatus with these light or darkness, the pineal rhythm in tonin and HIONIT cycles are both sup- shifts. melatonin-forming activity was rapidly pressed. When the nerves from the In order to determine if normal light- extinguished. If instead of turning off central nervous system to the superior ing rhythms influenced the pineal, we the lights at 7:00 P.m. illumination was cervical ganglion are interrupted, the kept a large population of rats under continued for an additional five hours serotonin rhythm is also abolished [see controlled lighting conditions (lights on and pineals were examined as usual at illustration below]. Hence the serotonin from 7:00 A.M. to 7:00 P.M.) for several midnight, the expected rise in melato- rhythm in the pineal gland is similar to weeks and then tested their pineals for nin-forming activity was completely most other circadian rhythms (and dif- melatonin-forming ability at 6:00 A.M., blocked. This pineal rhythm in HIOMT fers from the HIONIT cycle) in that it noon, 6:00 P.M. and midnight. In the activity thus appears to be truly exoge- is endogenous and depends on environ- five hours after the onset of darkness nous, or externally regulated, and is en- mental light only as an external syn- (that is, by midnight) this enzymatic tirely dependent on shifts in environ- chronizer. The mechanism that controls capacity increased between two and mental lighting. Hence this enzyme the serotonin rhythm appears to reside three times. Moreover, pineal weight rhythm may be more important in car- within the central nervous system. The also changed significantly during this rying information about light to the pineal gland thus contains at least two period, again indicating that light was glands than other circadian rhythms distinct biological clocks, one totallv de- affecting many more compounds in the that do not depend on light for their pendent on environmental lighting and pineal than the single enzyme we were existence. the other originating within the brain measuring. Recently Wilbur Quay of the Univer- but cued by changes in lighting. All circadian rhythms studied up to sity of California at Berkeley has found At present little is known about what this stage had in common the ability to that the content of serotonin in the rat organs are dependent on the pineal persist for some weeks after animals pineal also undergoes marked for cues. The ability of melatonin were deprived of environmental light- rhythms. The highest levels of this to modify gonadal function suggests, ing cues (by blinding or being placed amine are found in pineals at noon and but does not prove, that its secretion in darkness). These rhythms no longer the lowest levels at midnight. Serotonin may have something to do with the tim- showed a period of precisely 24 hours, content falls rapidly just at the time ing of the estrus and menstrual cycles- but they did fall in a range between 22 that melatonin-forming activity is rising. two phenomena about whose mecha- and 26 hours and hence were thought to In collaboration with Solomon Snyder nisms of control very little is known. be regulated by some internal mecha- we studied the mechanism of the sero- One is tempted to argue teleologically nism not dependent on, but usually syn- tonin cycle. When rats are kept in con- that any control mechanism as compli- chronized with, environmental lighting. tinuous light, the serotonin cycle is ex- cated and sensitive as that found in the Such endogenous, or internally regu- tinguished. To our surprise, however, mammalian pineal gland must have lated, circadian rhythms in rodents when rats are kept continuously in dark- some 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 M

BIOCHEMICAL RHYTHMS in the pineal gland of the rat were dent on environmental lighting conditions: it disappears when ani- recorded under various lighting and other conditions. Normally mals are kept in continuous light or darkness, or when they are both the content of serotonin and the activity of the melatonin- blinded. The serotonin cycle persists in continuous darkness or forming enzyme (HIOMT) vary with a 24-hour cycle. The serotonin after blinding but can be abolished by keeping the rats in continu. content is greatest at noon (N), whereas the HIOMT activity is ous light. Both cycles are depressed when the sympathetic nerves to greatest at midnight (M). The HIOMT cycle is completely depen- the pineal gland are cut (extreme right). Gray areas signify darkness.

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