THE PINEAL GLAND The function of this small organ near the center of the mammalian brain has long been a mystery. Recent studies indicate that it is a “biological clock” that regulates the activity of the sex glands by Richard J. Wurtman and Julius Axelrod uried nearly in the center of the brain in any mammal is a small white structure, shaped somewhat like a pinecone, called the pineal body. In man this organ is roughly a quarter of an inch long and weighs about a tenth of a gram. The function of the pineal body has never been clearly understood. Now that the role of the thymus gland in establishing the body's immunological defenses has been dem- onstrated, the pineal has become per- haps the last great mystery in the physiology of mammalian organs. This mystery may be nearing a solution: studies conducted within the past few years indicate that the pineal is an in- tricate and sensitive “biological clock,” converting cyclic nervous activity gen- erated by light in the environment into endocrine—that is, hormonal—informa- tion. It is not yet certain what physio- logical processes depend on the pineal clock for cues, but the evidence at hand - suggests that the pineal participates in some way in the regulation of the go- nads, or sex glands. A Fourth Neuroendocrine Transducer Until quite recently most investi- gators thought that the mammalian pin- eal was simply a vestige of a primitive light-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 pineal tumors, had proposed that in mammals the pineal was a gland. When the stan- dard endocrine tests were applied to determine the possible glandular func- tion of the pineal, however, the results varied so much from experiment to ex- periment that few positive conclusions seemed justified. Removal of the pin- 50 eal in young female rats was frequently followed by an enlargement of the ovaries, but the microscopic appearance of the ovaries did not change consistent- ly, and replacement of the extirpated pineal by transplantation seemed to have little or no physiological effect. Most experimental animals could sur- vive the loss of the pineal body with no major change in appearance or function. In retrospect much of the difficulty early workers had in exploring and de- fining the glandular function of the pineal arose from limitations in the traditional concept of an endocrine or- gan. Glands were once thought to be entirely dependent on substances in the bloodstream both for their own con- trol and for their effects on the rest of the body: glands secreted hormones into the blood and were themselves regu- lated by other hormones, which were delivered to them by the circulation. The secretory activity of a gland was thought to be maintained at a fairly con- stant level by homeostatic mechanisms: as the level of a particular hormone in the bloodstream rose, the gland invari- ably responded by decreasing its secre- tion of that hormone; when the level of the hormone fell, the gland increased its secretion. In the past two decades this concept of how the endocrine system works has proved inadequate to explain several kinds of glandular response, including changes in hormone secretion brought about by changes in the external en- vironment and also regular cyclic changes in the secretion of certain hor- mones (for example, the hormones re- sponsible for the menstrual cycle and the steroid hormones that are produced on a daily cycle by the adrenal gland). Out of the realization that these and other endocrine responses must depend in some way on interactions between the glands and the nervous system the new discipline of neuroendocrinology has developed. In recent years much attention has centered on the problem of locating the hervous structures that participate in the control of glandular function. It has been known for some time that spe- cial types of organs would be needed to “transduce” neural information into en- docrine information. Nervous tissue is specialized to receive and transmit in- formation directly from cell to cell; ac- cording to the traditional view, glands are controlled by substances in the bloodstream and dispatch their mes- Sages to target organs by the secretion of hormones into the bloodstream. In order to transmit information from the nervous system to an endocrine organ a hypothetical “neuroendocrine trans- ducer” would require some of the spe- cial characteristics of both neural and endocrine tissue. It should respond to substances (called neurohumors) _re- leased locally from nerve endings, and it should contain the biochemical ma- chinery necessary for synthesizing a hormone and releasing it into the blood- stream. Three such neurosecretory sys- tems have so far been ideritified. They are (1) the hypothalamus-posterior- pituitary system, which secretes the antidiuretic hormone and oxytocin, a hormone that causes the uterus to con- tract during labor; (2) the pituitary- releasing-factor system, also located in the hypothalamus, which secretes poly- peptides that control the function of the pituitary gland, and (3) the adrenal medulla, whose cells respond to a ner- vous input by releasing adrenaline into the bloodstream. The advent of neuroendocrinology has provided a conceptual framework PINEAL BODY CORPUS CALLOSUM CEREBELLUM \ \\ 1", . " PITUITARY GLAND ie oy PONS fy | MG MEDULLA OBLONGATA PINEAL BODY CEREBELLUM CORPUS CALLOSUM TWO VIEWS of the buman brain reveal the central position of the made in this view to reveal the region immediately surrounding pineal body. Section at top is ent in the median sagittal plane and the pincal. In mammals the pineal is the only unpaired midline is viewed from the side. Section at bottom is ent in » horizontal ergan in the brain. The neme “pineal” comes from the organ’s re- plane and is viewed from above; an additional excision has been semblance te a pinecone, the Latin equivalent of which is pinea, 5I that has been most helpful in charac- terizing the role of the pineal gland. On the basis of recent studies conducted by the authors and their colleagues at the National Institute of Mental Health, as well as by investigators at other institu- tions, it now appears that the pineal is not a gland in the traditional sense but is a fourth neuroendocrine transducer; it is a gland that converts a nervous in- put into a hormonal output. A Prophetic Formulation The existence of the pineal body has been known for at least 2,000 years. Galen, writing in the second century A.D., quoted studies of earlier Greek anatomists who were impressed with the fact that the pineal was perched atop the aqueduct of the cerebrum and was a single structure rather than a paired one; he concluded that it served as a valve to regulate the flow of thought out of its “storage bin” in the lateral ventricles of the brain. In the 17th century René Descartes embellished this notion; he believed that the pineal housed the seat of the rational soul. In his formulation the eyes perceived the events of the real world and transmitted what they saw to the pineal by way of “strings” in the brain [see illustration below]. The pineal responded by allow- ing humors to pass down hollow tubes to the muscles, where they produced the appropriate responses. With the hindsight 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 exalted metaphysical state. In 1898 Otto Heub- ner, a German physician, published a case report of a young boy who had shown precocious puberty and was also found to have a pineal tumor. In the SEAT OF THE RATIONAL SOUL was the function assigned to the human pineal (H) by René Descartes in his mechanistic theory of perception. According to Descartes, the eyes perceived the events of the real world and transmitted what they saw to the pineal by way of “strings” in the brain. The pineal responded by allowing animal hamors to pass down hollow tabes to the muscles, where they produced the appropriate responses. The sise of the pineal has been exaggerated in this wood engraving, which first appeared in 1677. §2 course of the next 50 years many other children with pineal tumors and pre- cocious sexual development were de- scribed, as well as a smaller number of patients whose pineal tumors were as- sociated with delayed sexual develop- ment. Inexplicably almost all the cases of precocious puberty were observed in boys. In a review of the literature on pin- eal tumors published in 1954 Julian I. Kitay, then a fellow in endocrinology at the Harvard Medical School, found that most of the tumors associated with pre- cocious puberty were not really pineal in origin but either were tumors of sup- porting tissues or were teratomas (prim- itive tumors containing many types of cells). The tumors associated with de- layed puberty, however, were in most cases true pineal tumors. He concluded that the cases of precocious puberty resulted from reduced pineal function 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 gonads. Little appears to have re- sulted from these early efforts, Later in 1954 Kitay and Mark D. Altschule, director of internal medicine 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-gonad 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 calcification; this change in the appearance of the pineal led many investigators to assume that the organ was 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 mi . He was sur- prised to find that the pineal contained distinct sensory cells; they bore a marked resemblance to the cone cells of the retina and were in contact with nerve cells. On the basis of these obser- vations he suggested that the pineal might function as a photoreceptor, or “third eye,” in cold-blooded vertebrates. In the past five years this hypothesis has finally been confirmed by electrophysi- ological studies: Eberhardt Dodt and his colleagues in Germany have shown 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- receptor in the frog, had something to do with sexual function in rats and in humans (at least those with pineal tu- mors) and contained a factor (at Jeast in cattle) that blanched pigment cells in tadpoles. The Discovery of Melatonin Then in 1958 Aaron B. Lerner and his co-workers at the Yale University School of Medicine identified a unique compound, melatonin, in the pineal gland of cattle [see “Hormones and Skin Color,” by Aaron B, Lerner; ScrentiFic AMERICAN, 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 (CH3) attached to an oxygen atom [see illustration on next two pages]. Methoxylation 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 melatonin for its effect on cells contain- ing the pigment melanin, appeared to lighten the amphibian skin by causing Tien sig geigbit At Ll ladle lee : PERINEAL BODY NN? CU, CORPUS CALLOSUM S | ll i HoH ENZYME WAN Any H H SEROTONIN hydroxytryptophan by the enzyme S-hydroxytryptophan decarbox- ylase. Serotonin, the product of this reaction, is then enzymatically the aggregation of melanin granules within the cells. It was effective in a concentration of only a trillionth of a gram per cubic centimeter of medium. No influence of melatonin could be demonstrated on mammalian pigmenta- tion, nor could the substance actually be identified in amphibians, in which it exerted such a striking effect. It re- mained a biological enigma that the mammalian pineal should produce a substance that appeared to have no bi- ological activity in mammals but was a potent skin-lightening agent in amphib- ians, which were unable to produce it! Both aspects of the foregoing enigma have now been resolved. Subsequent research has shown that melatonin does in fact have a biological effect in mam- mals and can be produced by amphib- ians. Spurred by Lemer’s discovery of this new indole in the cattle pineal, Nicholas J. Giarman, a pharmacologist at the Yale School of Medicine, analyzed pineal extracts for their content of other biologically active compounds. He found that both cattle and human pineals con- tained comparatively high levels of sero- tonin, an amine whose molecular struc- ture is similar to melatonin and whose function in nervous tissue is largely un- known. Studies by other investigators subsequently showed that the rat pineal contains the highest concentration of serotonin yet recorded in any tissue of any species. A year before the discovery of mela- tonin one of the authors (Axelrod) and his co-workers had identified a meth- oxylating enzyme (catechol-O-methyl transferase) in a number of tissues. This enzyme acted on a variety of catechols (compounds with two adjacent hydroxyl, or OH, groups on a benzene ring) but showed essentially no activity with respect to single-hydroxy] compounds such as serotonin, the most likely pre- cursor of melatonin. In 1959 Axelrod and Herbert Weissbach studied cattle pineal tissue to see if it might have the special enzymatic capacity to methox- ylate hydroxyindoles. They incubated N- acetylserotonin (melatonin without the methoxyl group) with pineal tissue and a suitable methyl donor and observed that melatonin was indeed formed. Sub- 100 sequently they found that all mamma- lian pineals shared this biochemical prop- erty but that no tissue other than pineal could make melatonin. Extensive studies of a variety of mammalian species have confirmed this original observation that only the pineal appears to have the ability to synthesize melatonin. (In am- phibians and some birds small amounts of melatonin are also manufactured by the brain and the eye.) Other investiga- tors have found that the pineal contains all the biochemical machinery needed to make melatonin from an amino acid precursor, 5-hydroxytryptophan, which it obtains from the bloodstream. It was also found that circulating melatonin is rapidly metabolized in the liver to form 6-hydroxymelatonin. Anatomy of the Pineal While these investigations of the bi- ochemical properties of the pineal were in progress, important advances were being made in the anatomy of the pin- eal by the Dutch neuroanatomist Jo- hannes Ariéns Kappers and by several 100 AGE OF RATS (WEEKS) —— PLACEBO == MELATONIN EFFECT OF MELATONIN on the estrus cycles of female rats is de- picted here. Rats that had been given daily injections of melatonin starting in their fourth week of life developed a longer estrus cycle than rats that had been similarly treated with a placebo. When the melatonin-treated animals were 10 weeks old, a placebo wae substi- tated for the melatonin and the estrus cycle retarned to normal. 54 c e a q o ey ANU w 8 w XN a t a \ I ” ” a Bey Bot MW Lf f \ o YY 3 40 3 40 : x E \ aw 20 START KN > 20 J Z TREATMENT 7 = tf oe