So small and remote as to be easily overlooked in a rapid perusal of the cerebrum, the pineal gland has been a source of fascination for philosopher and physician alike since its discovery by HerophiIus in 300 H.C. Galen dubbed the gland konareion (conarium in Latin), meaning "shaped like a pinecone." Descartes believed that the pineal's central location uniquely qualified it to be the "seat of the soul." To those for whom the anatomic situation was too suggestive not to postulate a psychic link, the pineal was the "sphincter of the mind."
Evolution of Techniques to Study the Pineal
Science began to make inroads in pineal physiology with early anatomic studies of animals such as fish and amphibians, which also possess pineal glands but in more superficial locations adjacent or in close proximity to visual structures. The human pineal is buried deep within the brain, far from the surface where it might interact directly with sources of light. Therefore, its photoreceptor function was not appreciated until anatomists could trace pathways from the optic apparatus to the pineal and comparative anatomists determined that the organ monitoring environmental photic information was the same in lower animals and humans. Only then did awareness of the link between this gland and photoperiodic homeostasis begin to emerge.
For 4 decades, isolation and assay techniques for a long list of biosynthetic precursors, intermediaries, neurotransmitters and peptide hormones have overwhelmed an entire scientific journal devoted to pineal research. Although the volume of data renders impossible, for the moment, the assemblage of a comprehensive summary of pineal physiology. it clearly establishes the gland's central role in coordinating vertebrate physiology.
The pineal is studied at the chemical level by analysis of its products and of interactions between these and others isolated from organs throughout the body. Individual pineal cells from a variety of species are subjected to chemical and physical manipulations. The isolated pineals of subhuman species have been examined and purified homogenates used in a variety of culture and tissue experiments. In vivo experiments are most frequently done in rats and lower animals but also in primates. Human in vivo studies are becoming increasingly feasible with improved techniques for chemical analysis and imaging of the nervous system.
Much of what is known about pineal physiology is learned from manipulations of exposure of an organism to light - the major influence on pineal physiology. Comparison of physiologic parameters before and after pinealectomy provides clues to the organ's function.
For a variety of reasons, frequently ethical ones, relatively few studies of pineal physiology have been done on human beings. Much of the available information about how the pineal operates and what its functions are is derived from animal studies, cell cultures, and tissue preparations. The precise significance of many experimental findings for humans is nebulous. Many of the functions ascribed to the human pineal are based on extrapolations from in vitro and nonhuman data.
Phylogenetic Clues to Pineal Function
Comparisons among species provide important clues to the pineal's role in humans. Phylogenetically, the gland has been primarily a photoreceptor organ. Throughout vertebrate species it is closely related to the optic apparatus. Some species have cortical and medullary portions of the pineal: the significance of this is unknown.
Pineal volume does appear to correlate with variations in hormonal activity. Species with larger pineals (relative to total organism size) tend to be homoeothermic (vs. heterothermic) and to have diurnal physiologic cycles (vs. nocturnal). Tropical animals tend to have smaller pineals than those adapted to living in temperate climates, where seasonal variations in temperature and daily duration of sunlight make reproductive timing more critical.
The pineal is present in all vertebrates. In lower species its location is often more superficial and its association with photoreceptor function is more obvious. Through evolution. the pineal changed from a photoreceptor organ to one associated with an organism's interactions with the light-dark cycle.
Analogies between mammalian and amphibian photoreceptors (anatomic, chemical or functional) are not identities. Interesting and exciting theories about human pineal function are frequently extrapolated from animal research or even from in vitro tissue or cell culture preparations. Cautious appraisal of pineal data from nonhuman studies is warranted to avoid over- or underestimation of the gland's importance in humans.
The pineal arises as an evagination of the neuroepithelial roof of the third ventricle, between the habenula and the posterior commissure in the second month of gestation. Neuroepithelial cells become pinealocytes, whereas mesenchymal cells become the connective tissue strands, fibroblasts and vessels of the gland. The pineal develops from two lobes that fuse. The gland's orientation changes from vertical to horizontal. Innervation of the gland is completed in the prenatal period.
At birth there are two types of pinealocyte which are distinguishable to approximation 3 years of age. Type I pinealocytes are S-100-positive and neuron-specific enolase-negative; type II pinealocytes are enolase-positive. S-100-negative, and melatonin negative. At birth. type I cells predominate. but by 1 year of age, they are outnumbered by type II cells.
Secretory activity, specifically of the primary pineal product melatonin, varies throughout the course of development. Serum melatonin levels increase from the third postnatal month to preschool age. They then decline steadily from preschool age until sexual maturity. This pattern raises the interesting possibility that, contrary to previous beliefs, the gland's effects on development are not necessarily related to sexual maturation. The pineal reaches its adult size by age 4 years and undergoes no significant histologic changes from childhood to adulthood.
Regional Gross Anatomy
The anatomic location of the pineal with respect to nearby neural and vascular structures accounts for the difficulty in surgical access as well as for the neurological symptoms that may accompany mass lesions arising in the gland. From the physiologic perspective, the pineal is well situated to serve as the hub of a system of input, feedback and regulation among numerous diverse brain regions.
Also called the epithalamus, the pineal is located in the posterior most portion of the epithelial roof of the third ventricle. The posterior commissure is found posteroinferiorly. Anteriorly the gland is in continuity with the habenular trigone and the striae medullaris thalami. The posterior portion of the corpus callosum, the splenium overlies the pineal.
Although the gland is suspended in the pineal recess, a CSFfilled cistern contiguous with the quadrigeminal cistern, the gland itself is not bathed in spinal fluid but rather is surrounded by a pial layer. Because the organ is solid, the substances it produces are probably not primarily released directly into the third ventricle but rather into the vascular system.
Major vascular structures lying in proximity to the pineal or passing by it are the medial posterior choroidal arteries (the major feeders to the gland) and numerous perforators. Ample venous outflow seems to facilitate the distribution of synthesized substances. Drainage is into the vein of Galen via the internal cerebral veins: the veins of Rosenthal are situated just lateral and superior to the pineal. Behind the gland is the precentral vein of the cerebellum.
Microscopically the pineal appears as clusters of parenchymal cells enclosed by bands of connective tissue of variable thickness that consist of fibrous astrocytes and other cell types. The pineal is surrounded by a capsule and is composed of lobules with separating connective tissue septae. Astrocytes are shown to be abundant by glial fibrillary acidic protein (GFAP) staining and form a barrier between vessels and pinealocytes.
There are many different cell types in the gland. In addition to supporting, neuronal, and endothelial cells, the stroma is made up of pinealocytes, which themselves may be subdivided by histologic and electron microscopic, and increasingly by biochemical and functional, criteria.
Pinealocytes make up 90 percent of the parenchyma of the gland. In sub mammalian species, they are derived from photoreceptor cells. In vertebrates there are thought to be two types of pineal cells, light and dark, so named on the basis of their responsiveness or unresponsiveness to light input at the retina, The existence of these two types in humans remains controversial.
Parenchymal pineal cells are thought to belong to the system of amine precursor uptake and decarboxylation (APUD) cells. They stain positively for neuron-specific enolase.
Pinealocytes are characterized by their prominent nuclei and nucleoli. Each pinealocyte has several cytoplasmic processes, which terminate in club-shaped endings on perivascular spaces and which may provide a means for communication between pinealocytes.
Because it produces substances chemically related to neurotransmitters, contains synaptic vesicles, secretes its products in response to stimulation of receptors on its cell membrane and originates in the ectoderm, the pinealocyte can be considered a "paraneuronal" cell. Kappers considers the pineal "a true endocrine organ" because pinealocytes, although derived from neuroectoderm, are not neurons.
In lower animals, pinealocytes morphologically resemble retinal photoreceptor cells. Some of the pinealocytes in these species have processes that resemble axons and many of them immuno-stain positively for gamma-aminobutyric acid (GABA).
Glial cells, primarily fibrous astrocytes, appear to serve both supportive and metabolic roles in the pineal, as they do in all central nervous system tissue. Staining with S-100 suggests that microglial cells are also present.
The endothelial cells that make up the vascular supply to the pineal do not have the tight junctions characteristic of the bloodbrain barrier: the pineal is thus rightly considered to be a circumventricular organ. Although parasympathetic, commissural. and peptidergic fibers have been demonstrated in the pineal. the only fibers with known physiologic significance in the gland are general visceral afferent sympathetics originating in the superior cervical ganglion and reaching the pineal via the nervi conarii. These postganglionic sympathetic fibers receive descending input from hypothalamic nuclei, particularly the suprachiasmatic nucleus (SCN), which receives direct input from retinal ganglion cells. Unmyelinated fibers travel through connective tissue in discrete bundles. In some animals all the sympathetic input reaches the pineal as unmyelinated fibers travelling with venules and arterioles, which vascularize the gland. There is also questionable parasympathetic innervation, possibly arriving via the habenula and posterior commissure. Neurotransmitters reach pineal cells not across a synaptic cleft but by diffusion after release from varicosities some distance away. Other cell and tissue types found in the pineal include fibrous connective tissue, skeletal muscle and lymphocytes.
Acervuli, corpora arenacea, or pineal calcifications, the familiar and important pineal landmarks seen on skull roentgenography and computed tomography (CT) of the head are still incompletely understood. Calcium accumulates along plasmalemma and intracellularly in pinealocytes. This may be the basis of calcium deposition, which occurs in an organic matrix produced by pinealocytes. The calcareous concretions grow along growth zones and are composed of calcium, magnesium and ammonium ions as well as calcium carbonate. The growth process is thought to be age- and sex-independent and is probably related to the gland's secretory activity.
Although the etiology of these concretions is not known, two theories have been proposed. In the first a carrier protein is thought to be released into intracellular vacuoles. In the second there is decreased drainage of tissue fluid from the gland. Acervuli are seen in 3 percent of pineals by age 1 year, in 7.1 percent by age 10, and in 33 percent by age 18.
The functional significance of pineal calcifications is unknown. Recent CT studies of pineal calcifications have attempted to correlate their presence with diseases such as schizophrenia.
Another incompletely understood phenomenon is the formation of benign pineal cysts. These mass lesions, which can reach proportions sufficient to result in clinical symptoms and signs, are thought to result either from degeneration of foci of gliosis within the gland or from sequestration of CSF during pineal development. Why this happens remains unknown. In a recent radiologic review, Golzarian et al. reported a 2,4 percent incidence in 500 consecutive magnetic resonance imaging studies. Pineal cysts can be found in 25 to -40 percent of subjects studied at autopsy.
What the Pineal Does: Cycles and Signals
Generation of Cycles and Periodicity
Human physiologic systems fluctuate in a cyclic manner, reflecting an internal awareness of diurnal and seasonal cycles. The pineal supplies both clock and calendar information to the organism.
Although the pattern of nocturnal melatonin production varies among species, the duration of night-time melatonin elevation is always proportional to the duration of night. Because the duration of day and night changes during the year, photoperiod information is information about time of year as well.
To survive the cyclic variations, terrestrial organisms need mechanisms not just for detecting changes in the environment but also for anticipating them. In the absence of an explicit environmental cue, organisms needed an internal signal of impending change to make timely, adaptive behavioural preparations.
The organism must respond appropriately to cycles and oscillations of variable frequency and amplitude. In addition to the lightdark cycle, there are cycles of reproduction, seasons and aging (the cycle from birth to death). Virtually all of the multifarious pineal physiologic functions can be understood in the context of vertebrate biological cycles.
Cyclic environmental stimuli affect the organism in various ways: physical, mechanical and chemical. Input must be converted from external physical to internal signals. Electromagnetic energy in both the visible and the nonvisible portion of the spectrum reaches the pineal through a well-described neural pathway. Other external factors with cyclic variations seem to influence the pineal through less clearly understood transduction mechanisms.
The pineal integrates the transduced environmental signal information and generates its own signals. Through the integrated production and release of various neurohormonal substances, the pineal brings order to the apparent chaos of the disparate signals it receives.
Although melatonin is most important, the pineal produces other substances that have signal-transducing effects. Once released, pineal hormones have effects on other biosynthetic pathways, which then become important for signal transduction. Understanding their effects is essential to understanding pineal physiology.
The effects of pineal signals occur at the level of cells, organs and organism behaviour. Such effects result in physiologic changes within the internal environment, which are themselves feedback signals to the pineal.
The effects of electromagnetic field interactions with biological systems are only beginning to be understood. Both light and non visible electromagnetic energy decrease the conversion of serotonin to melatonin. Although the mechanisms are incompletely described, the alterations in melatonin production due to light appear to be the same as those due to nonvisible electromagnetic field exposure.
Light has an effect on the level of melatonin production and release as well as on the rhythm of this production and release. In humans exposed to moderate and low light, there are differences not only in the level of salivary melatonin but also in the duration of the melatonin peak and time of offset.
The control of melatonin synthesis by light gives the pineal its essential photoperiodicity. Light has two different effects on the circadian rhythm of melatonin production and release: it acutely suppresses melatonin output according to the wavelength of light and the circadian phase and it entrains or phase-shifts the underlying cellular pacemaker. The pathways for these different but related effects may have the same origin.
Light causes configurational and chemical changes in the rods and cones of the retina. These changes are the basis of transduction of light energy into neural signals, which can be passed along neural pathways to the pineal. In addition, pineal cells themselves have photoreceptor properties demonstrated by recordings of responses to light stimulation of isolated pineal cells, which show electrical reactions to illumination.
Static magnetic fields consistently and reproducibly perturb circadian melatonin rhythm. The effects are reflected in alterations in levels of cyclic adenosine monophosphate (cAMP), N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and pineal and blood melatonin concentrations, all of which are decreased by magnetic field exposure. S-Hydroxytryptamine (serotonin) is increased as a consequence of decreased melatonin synthesis.
Although the mechanisms for the influence of nonvisible electromagnetic energy on melatonin formation are not known, the retina is thought to be the magnetoreceptor. Alterations in the retinal magnetoreceptor are transmitted to the suprachiasmatic nucleus.
Magnetic fields cause depression of melatonin levels in experimental rats. The rat pineal also responds to pulsed static magnetic fields with a decrease in NAT activity. Interestingly, the effect occurs during the middle or late dark phase but not during the light phase or early dark phase. The reason for differences in the response to magnetic field during different portions of the photoperiod is not known. In humans. 6-hydroxymelatonin excretion in urine is lower in users of magnetic field-emitting as opposed to conventional electric blankets.
Alterations in periods of sun and seasonal temperature changes require homeostatic and behavioural adaptations in warm-blooded animals. As coordinator of circadian seasonal responses, the pineal can affect mechanisms of temperature change anticipation, detection and response.
Elevation of temperature has been shown to increase the amplitude of the melatonin rhythm in cultured chick pineal cells. In a study in which melatonin levels were manipulated through administration of a beta-adrenergic antagonist, there was an inverse relationship between body core temperature and melatonin levels. The result suggests a possible clinical use for melatonin in conditions associated with loss of circadian body core temperature rhythm.
Other forms of high energy also affect pineal activity. Whole body irradiation with 14.35-Gy gamma rays was shown to increase pineal NAT activity in rats.
Input to the Pineal
The neural pathway through which light influences the pineal originates at the retina. Axons of certain retinal ganglion cells travel through the optic nerves, branching off to form a separate pathway, the retinohypothalamic tract to the suprachiasmatic nucleus (SCN). Some light information also reaches the SCN via the lateral geniculate body.
The biological clock appears to be in the SCN, which has a high density of melatonin receptors to receive feedback from the pineal. Some melatonin receptors that mediate seasonal reproductive behavioural changes are thought to reside in the anterior pituitary gland (pars tuberalis).
Light activation of the retinal ganglion cells inhibits the SCN, while it leaves the SCN active to stimulate the next station of the pathway, the paraventricular nucleus. Innervation to the SCN is bilateral, although predominantly from the contralateral retina. The nucleus is better studied in lower animals, in whom it is better defined; in humans it is more diffuse.
Efferent signals of the SCN are not fully understood. Some appear to be neural (those to the hypothalamus in particular), but some may occur through release of a diffusible substance into the CSF.
Axons of paraventricular neurons travel through the medial forebrain bundle to the intermediolateral area of the upper thoracic spinal cord. The intermediolateral axons are the presynaptic input to superior cervical ganglion cells, whose efferents travel initially with other sympathetic fibers along the carotid artery; ultimately, however, they form distinct fiber tracts, the bilateral nervi conarii, which synapse on pineal cells.
The superior cervical ganglion is the source of the postganglionic output to the pineal. Norepinephrine is released from postganglionic fibers, primarily during darkness. In darkness, the SCN is electrically inactive. In light the SCN is inhibited, which leads to decreased norepinephrine release. The norepinephrine then stimulates beta1 receptors, which, through induction of protein synthesis via the G protein-mediated cAMP second messenger system, increase production of melatonin.
Sympathetic fiber endings do not directly end on pinealocytes but rather are precapillary. The norepinephrine they release, reaches the pineal cells by diffusion. Although sympathetic innervation of the pineal has been proved only in lower species, cervical spine injury is a model for sympathetic denervation in humans. Following high cervical spinal cord injury, there is loss of the normal melatonin cycle and an increase in 24-h production of the hormone. In addition, stages 3 and 4 and rapid-eye-movement (REM) sleep, all of which can be induced experimentally by melatonin administration, are disrupted by cord transection.
The neuroendocrine effects of the superior cervical ganglion (SCG) can be demonstrated by ablation of the ganglion, which results in changes in prolactin release, changes in drinking behaviour, disruption of photoperiod reproductive control, and changes in thyroid and oxytocin activity.
The SCG is an important modulator at the pineal neuroeffector junction. Presynaptically, norepinephrine influences alpha- and beta-adrenergic receptors. Postsynaptic substances from the effector (pineal) cells, such as serotonin, provide feedback on presynaptic cells. Prostaglandin E2 exerts a negative influence on transmission.
The innervation of the pineal is probably almost exclusively sympathetic, although there is some evidence of parasympathetic innervation in some lower mammals. Parasympathetic fibers originate at the superior salivatory nucleus of the seventh cranial nerve, travel along the greater superficial petrosal nerve, and reach the pineal probably through the habenular and posterior commissures. The physiologic significance of acetylcholinergic parasympathetic input to the pineal is unknown.
Pineal substances are released by ependymal secretion or direct release of products of the endoplasmic reticulum after processing by the Golgi apparatus. This release, into pericellular and pericapillary spaces, is under the control of the sympathetic nervous system.
Immunohistochemical methods have been used to determine that histaminergic nerve fibers, originating from the posterior hypothalamus, project to the pineal complex of the rat. Histamine must therefore be considered a putative neurotransmitter contained in the central innervation of the pineal gland, but its function in pineal physiology has not been elucidated.
Although neural input of transduced environmental signals is important in setting the biological clock, pineal cells seem to have their own intrinsic rhythmicity, independent of external signals. Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, whose mechanism is not fully understood. A model with dissociated lizard pineal cells demonstrated circadian rhythms of melatonin secretion, in the absence of neural or humoral input. Blind humans with no retinas showed a slightly greater than 24-h cyclic variability in melatonin levels.
Pineal Output: Substrates for Pineal Effects
The neural pathways convey information about environmental electromagnetic energy. At the pineal, this information is transduced into physiologically effective signals through the release of various chemical compounds. Although modern assay techniques have detected numerous pineal products, the two most important groups are the indole amines melatonin and serotonin, and pineal peptides.
Melatonin synthesis and release follow a circadian rhythm with high nocturnal and low diurnal levels. Light of varying intensity, wavelength, and duration of exposure, and darkness influence melatonin production by affecting cAMP and norepinephrine control mechanisms.
Although influenced by cycles of light and darkness, melatonin has its own intrinsic circadian rhythm. Lewy and Newsome demonstrated cyclicity in melatonin levels in blind subjects.
Melatonin is only briefly stored in the pinealocyte. Because of its high lipophilicity, it rapidly crosses cell membranes and enters the blood stream and possibly the CSF, where levels of melatonin are much lower than but parallel to those in blood. Cisternal injection of melatonin leads to increases in hypothalamic cAMP. However, the significance of melatonin in CSF is not known.
Melatonin was initially described as a most potent substance in blanching amphibian melanocytes by causing aggregation of intracellular melanin granules. It also antagonizes the darkening effects of melanocyte-stimulating hormone.
Melatonin is a 232-dalton indole amine (a seven-carbon two-ring structure with an attaching -NH2), N-acetyl-5methoxytryptamine, which is synthesized in pinealocytes (as well as in the retina, red blood cells, hypothalamus, SCN, intestine and peripheral nerves) from tryptophan. The existence of extrapineal sites has been demonstrated in experiments in rats: loading with tryptophan caused increased melatonin levels in pinealectomized animals as well as those with intact glands.
Melatonin has a half-life of 10 to 40 min. In rats, 90 percent is cleared in one pass through the liver. In mice, 70 to 80 percent is converted into inactive metabolites by liver microsomal systems.
Melatonin synthesis from tryptophan is a two-stage process. After the circulating amino acid is taken up from blood by pinealocytes, tryptophan is first converted to serotonin by a hydroxylation step followed by a decarboxylation step. Serotonin is then converted to melatonin by three steps in which a series of enzymes successively add an acetyl, methyl and finally a hydroxyl group to the indole ring.
The rate-limiting step is the conversion of serotonin to N-acetylserotonin by the enzyme N-acetyltransferase (NAT), which is induced by darkness at the retina and converts serotonin to melatonin. NAT activity has its own rhythmicity and can be followed as a marker for melatonin. NAT activity decreases toward the end of the dark phase, suggesting that some inactivating substance may influence the enzyme. The next enzymatic reaction, conversion of N-acetyltryptophan to melatonin, is catalyzed by hydroxyindole-O-methyltransferase (HIOMT). Induction or suppression of this rate-limiting enzyme synthesis by cAMP is a potential site of melatonin synthesis regulation.
Once melatonin is produced in the pineal gland, it is quickly released into the vascular system. The rapid release of melatonin is generally believed to relate to its high lipophilicity, which allows it to pass readily through the membrane of the pinealocytes and the endothelial cells that line the capillaries. In addition to melatonin, two pineal peptides that can be used to follow melatonin effects are methoxytryptamine and methoxytryptophol.
Although melatonin is the most important compound produced by the pineal, serotonin, an intermediate product in the synthetic pathway, also displays periodicity and may itself be important as a homeostatic agent or as part of some "biological clock" mechanism.
The concentration of serotonin in the pineal is 250 times greater than that in any other region of the brain, and the concentration is higher in the brain than anywhere else in the body. At night the concentration is 10 to 20 mg/g of pineal tissue: during the day this rises to 60 to 90 mg/g. It is postulated that less serotonin is secreted at night because of its consumption as a precursor in melatonin synthesis.
Several peptides are also produced and released by the pineal and may participate in its functional activity. Some of these are exclusive to the pineal, whereas others are found elsewhere in the body. Several are putative neurotransmitters. It is extremely difficult to isolate peptides because so many are present in the pineal, but the number identified by immunocytochemical techniques continues to grow.
Many neuropeptides are produced at sympathetic synaptic endings and released at the pineal: they include vasopressin, oxytocin, somatostatin, α-melanocyte-stimulating hormone, endorphin, vasoactive intestinal peptide (VIP), substance P, luteinizing hormone-releasing hormone (LHRH), thyrotropin-releasing hormone, angiotensin II, adrenocorticotropic hormone (ACTH), neurophysins I and II and α-albumin (which is identical to GFAP). These substances may be released into the synaptic cleft or may enter the blood stream to act as hormones.
Several antigonadotropins were the first pineal peptides to be isolated. They act on the hypothalamus, affecting levels of prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Their mechanism of action seems to be an effect on catecholamine turnover, specifically by increasing dopamine synthesis.
The neurohypophyseal substances vasopressin and oxytocinlike peptides have also been isolated from the pineal: this has led to speculation of possible hypothalamic influences on pineal activity. These substances are thought to reach the pineal via extrahypothalamic fibers whose cell bodies are in the magnocellular portion of the hypothalamus. LHRH and VIP have also been immunocytochemically demonstrated in the pineal.
Arginine vasotocin (AVT), a nonapeptide differing from arginine vasopressin in only one amino acid, has been isolated from the pineal. AVT is found in lower animals and was initially isolated from bovine pineals, although it is also produced in the pituitary. In mammals it is produced by specialized pineal cells. AVT is thought to act through the serotonin pathway by interfering with serotonin release at postsynaptic receptor sites. It may also exert its effect through actions on the GABA-containing habenuloraphe pathway. The substance has actions like those of melatonin, but it is far more potent. It has sleep-inducing and anticonvulsant effects and can induce changes in the EEG and promote REM sleep.
Recoverin is a 26-kDa protein that binds to calcium and activates guanylate cyclase in retinal photoreceptors when, upon photoexcitation, the intracellular concentration of free calcium drops. It is found only in photosensitive cells and may be involved in photosignal transduction.
Pineal transduction of physical environmental signals is a function of the anatomic and chemical substrate of the gland as well as of physiologic mechanisms for signal generation and control. The anatomic location in relation to neural pathways, neurotransmitters and the receptors that pass and receive signals along the pathway, as well as the pathways for synthesis and release of hormonal products, are all important in transduction. Control and feedback mechanisms and mechanisms by which the pineal hormones interact with target cells underlie pineal's function as a link between an external environmental stimulus and internal end organ (homeostatic) responses.
Mechanisms of Action
At the molecular level, one of the proposed mechanisms of melatonin could be through binding to calmodulin. Studies have demonstrated a specific melatonin-binding site. Interactions with calmodulin would enable melatonin to participate in the modulation of many intracellular functions dependent on Ca2+, which could include cytoskeletal rearrangements and inhibition of calmodulin-dependent phosphodiesterase activity. Phylogenetic preservation of calmodulin and melatonin may reflect the participation of both in a fundamental physiologic mechanism of cellular regulation and synchronization.
The cAMP system is important in mediating the effects of the hormone. Prolonged melatonin exposure affects the sensitivity of the cAMP system and increases the production of cAMP. Melatonin also increases the sensitivity of the cAMP system to stimulation by substances such as forskolin through a mechanism not dependent on new protein synthesis.
Protein kinase C (PKC) prevents and reverses melatonininduced pigment aggregation. Presumably activation of PKC stimulates the intracellular machinery involved in the centrifugal translocation of pigment granules along microtubules.
Because of the high lipophilicity of the melatonin molecule, Reiter postulates that the most important mechanisms of melatonin action may occur not at the cell membrane but rather within the cell. He recently reviewed evidence that melatonin interacts with oxygen-centered free radicals, reducing their number by two mechanisms: stimulation of glutathione peroxidase, which breaks down hydroxyl radical and scavenging by the melatonin molecule itself, of free radicals. Melatonin binds within the nucleus, where it may have a role as protector of nearby DNA.
Melatonin action may be mediated through an effect on the microtubules, which are intracellular protein structures important in cellular movement, division and axon transport. There is evidence that melatonin causes ultrastructural changes in microtubules. An effect on microtubules like that of colchicine has been seen when the structures are allowed to recover following disruption by temperature elevation.
Melatonin causes a decrease in the protein content of hypothalamic microtubules. It has been shown to decrease axoplasmic transport in the sciatic and optic nerves.
The microtubule hypothesis for the action of melatonin is attractive because it provides a mechanism for melanin granule aggregation in melanocytes and consequent skin lightening. Aggregation of granules requires participation of microtubules.
The microtubule hypothesis has been challenged by investigators who administered melatonin to chick embryos and noted no changes in the central nervous system. Other mechanisms for melatonin action haw been postulated, including effects on cellular cyclic guanosine 3.5' -monophosphate (cGMP) and prostaglandins.
Melatonin has been found in the hypothalamus, midbrain, pituitary, peripheral nerves and gonads. Steroid receptors are found on pineal cells and it is postulated that melatonin may alter the number or affect intracellular processing of steroid-receptor complexes at that site. The physiologic significance of melatonin binding in multiple tissues is in many cases unknown. [125I] iodomelatonin binds with high specificity at several sites in the chicken spinal cord with diurnal variation. Although this suggests a direct melatonin effect on the cord, the actual significance of these binding sites is unknown.
Melatonin receptors are found in many organs throughout the body. In cardiac muscle, melatonin induces changes in calcium and magnesium ion-dependent ATPase activity. The presence of putative melatonin receptors in the guinea pig kidney supports hypotheses of melatonin-regulated renin secretion together with renal excretory functions via melatonin receptors. In many locations, the function of the pineal hormone remains unknown.
Control of and Feedback to the Pineal
Pineal activity can be controlled by affecting either the intrinsic cyclicity of pineal cells or by affecting rates of production and release of pineal products. Mechanisms by which intrinsic cellular rhythms would be reprogrammed remain to be worked out. Production and release of pineal products can be influenced at a genetic, enzymatic, or neural level. Although both show circadian oscillations, chick retina and pineal have slightly different NAT activity and melatonin content responses to constant light and darkness.
Reprogramming Cycles and Rhythmicity: Levels of melatonin can be regulated at many points along the synthetic and release pathways. Neurotransmitters, genes, and enzymes each have their own synthetic and production control pathways that affect melatonin production and release.
Factors known to influence melatonin level (besides electromagnetic radiation) include age, body weight and height, use of glasses, beta blockers, chlorpromazine, antidepressants and genetic variations. Melatonin production and release are affected by the change of seasons, phases of the menstrual cycle, puberty and aging.
Hormones such as testosterone, progesterone, prolactin, thyroxine, FSH, LH, and parathyroid hormone have all been shown to influence pineal activity as measured by the level of melatonin synthesis within the gland. They exert their effects either directly on pinealocytes or indirectly on sympathetic nerves in the pineal. Receptors for many hormones have been detected on pineal cells.
Genetic Control: The locus controlling pineal serotonin NAT has recently been localized to mouse chromosome 11. Studies of induction of messenger RNA (mRNA) transcription and other genetic changes will be important in the future definition of melatonin production and release control mechanisms. Protooncogenes may participate. Stressful stimuli induce a significant increase in the expression of c-fos mRNA in the pineal gland suggesting a possible mechanism by which such stimuli could influence pineal function.
Enzymatic Influences: Any factor that influences protein synthesis can potentially enhance or blunt melatonin's effects or even establish new cycles within the larger one regulated by light and darkness. Enzyme levels are determined by rates of protein synthesis but also by inhibitory and excitatory substances that may operate on NAT in the pineal. Alteration in receptor number and sensitivity is yet another mechanism by which cyclicity could be influenced.
The list of substances produced by the pineal all of which have their own control mechanisms susceptible to cyclic influences, includes such biologically active agents as tryptophan, histamine, and angiotensin I. Target tissue sensitivity to melatonin varies with the number and sensitivity of receptors there and the influence of other hormones. All these target tissue-resident factors are amenable to cyclic influences. A system of interacting regulating influences enables the organism to respond to a plethora of periodically variable challenges, of which light is just one.
Understanding the control of pineal melatonin production and release requires measurement of levels of melatonin and intermediate substances in its production pathway (such as serotonin) as well as activities of the various enzymes (adenyl cyclase, HIOMT, NAT) participating in its synthesis. The level of NAT is of special importance because this enzyme catalyzes the rate-limiting reaction in the synthetic pathway, the conversion of serotonin to melatonin. Its activity increases in darkness. Serotonin N-acetyltransferase (SNAT) is truly circadian (i.e., not dependent on light).
With the onset of darkness there is an increase in the firing rate of sympathetic neurons with endings on pineal cells. A large increase in pineal NAT activity is associated with increased local turnover of norepinephrine and the pineal takes up circulating dietary phenylalanine.
Electrical activity in the suprachiasmatic nucleus (and thus stimulation of norepinephrine release and ultimately of melatonin production and release) is shut off within 1s of exposure of the retina to light. The degree to which this activity is curtailed depends on the intensity of the light to which the retina is exposed, Light wavelength may be important in suppressing melatonin production. Blue light (wavelength of 500 to 520 nm) seems to be most effective and suggests that rhodopsin is an important participant in the effect.
Additional control is possible along the synthetic pathways of the substances that affect the melatonin pathway, the most important being norepinephrine, Phenylalanine is first converted to tyrosine, Tyrosine. in a rate-limiting step catalyzed by tyrosine hydroxylase is converted to 3,4-dihydroxyphenylalanine (DOPA). This reaction occurs 50 percent faster at night. DOPA is then converted to dopamine by DOPA decarboxylase. Finally, dopamine is converted to norepinephrine. This multienzymatic biosynthetic process is stimulated by darkness. The norepinephrine is then released, crosses through the synaptic space, and stimulates production and release of melatonin by pineal cells. Norepinephrine is deactivated by the well-described mechanisms of diffusion, reuptake and enzymatic degradation by monoamine oxidase and catechol-O-methyItransferase.
Levels of NAT, the enzyme that adds an -OCH3 to serotonin in the melatonin synthetic pathway, have been shown to increase following beta-adrenergic activation of cAMP activity at night. In light, NAT activity decreases. The enzyme may be regulated by a disulfide peptide, such as arginine vasopressin, somatomedin, or insulin in light, but the functional (teleological) significance of this is unclear.
Long-term ethanol administration has been shown to result in a significant decrease in NAT activity in the pineal. The significance of this finding is unclear.
NAT activity is variable during the light-dark cycle. Increases in the NAT conversion reaction require increases in NAT itself, which requires protein and therefore mRNA synthesis. These processes account for the time lag noted from onset of darkness to increase in melatonin level.
Data show that melatonin applied in the late light period advances the evening NAT rise during a short photoperiod only; during a longer photoperiod, the phase-advancing effect of melatonin may conflict with a phase-delaying effect of the end of a light period, and the effect of light exposure overrides that of melatonin.
Rhythmic control of melatonin production by modulation of NAT activity is mediated through activity of cAMP. cAMP is important for protein synthesis and maintenance of NAT in active form. cAMP concentration is higher in the pineal than in any other part of the brain. cAMP is a regulator of pineal melatonin production in the chick as evidenced by the fact that chemicals that raise cAMP (or analogue) levels also raise melatonin levels. Substances that lower cAMP lower melatonin. There is also a circadian pacemaker in pineal cells that regulates melatonin production independent of cAMP.
In the chick pineal, cAMP appears to act downstream in the pathway that generates circadian rhythm. In experiments in which chemicals were added to raise cAMP to supersaturated levels, there was no continuous elevation of melatonin level. Rather, the cyclic rise and fall of melatonin continued. This indicates that the melatonin pacemaker is not cAMP. cAMP and the pacemaker act synergistically to regulate SNAT activity and the melatonin rhythm, with cAMP mediating acute effects and amplitude regulation.
Not surprisingly, rhodopson and retinoid activity in the retina is coordinated with activity of NAT and melatonin production. HIOMT catalyzes another step in the melatonin synthetic pathway and thus is a site for potential metabolic control. In rats, HIOMT peak enzyme activity occurs 5 h after the onset of darkness and is followed by two lesser bursts of activity preceding each change in the photoperiod. There is an approximately 18-fold increase in methylating capacity at night.
Neural Influences on Production and Release of Pineal Products
Beta-adrenergic receptors: Neurotransmitter receptors are an important site for control and modulation of pineal activity by the nervous system. Beta-adrenergic receptors are located outside of the blood-brain barrier, which supports the view of the pineal as a peripheral, rather than a central organ. Postganglionic cells from the superior cervical ganglion release norepinephrine: norepinephrine binds to beta1-adrenergic receptors on pinealocytes. which stimulate production of cAMP through a G proteinmediated mechanism. The increase in cAMP increases mRNA production, specifically of NAT.
The number of beta-adrenergic receptors increases at the end of the light period. At night, release of norepinephrine from terminals leads to a decrease in the number of receptors by the end of the dark phase. NAT activity increases further if the beta1 agonist isoproterenol is given at the end of the light phase. when receptors are more abundant, than if given at the end of the dark phase, when they are fewer. There is a great fluctuation in the level of beta-adrenergic receptors over 24 h.
The number of postsynaptic receptors can be increased by increasing the length of an animal's exposure to light or by surgically removing the cervical ganglion. Under such conditions the number of receptors on pineal cells increases and there is supersensitivity to adrenergic agents. Administration of isoproterenol is not associated with an increase in circulating melatonin in humans; perhaps some coneurotransmitter is required that is not stimulated by isoproterenol (e.g., GABA, histamine, DOPA). Administration of beta blockers blocks the night-time increase in pineal melatonin (alpha blockers have no effect).
Alpha-adrenergic receptors: Alpha-adrenergic receptors have not yet been identified in humans. In rats, alpha-adrenergic receptors of the pineal may be related to the regulation of phospholipid metabolism.
The activity of rat alpha-adrenergic receptors seems to be prostaglandin-mediated. Norepinephrine stimulates pineal production of prostaglandins and phosphatidyl inositol. The prostaglandinmediated change in the cell membrane and its components may be part of a neuroendocrine mechanism for signal transduction.
Alpha-adrenergic receptors that may respond to substances released by presynaptic axons whose neuronal cell bodies are located centrally in the brain are also present on pinealocytes. Stimulation of these is thought to induce changes in the phosphatidyl inositol pathway.
Adrenergic mechanisms of control: Adrenergic stimulation of the adult pineal gland increases cAMP and cGMP production by over 100-fold. Beta-adrenergic stimulation results in an increase in alpha-mediated cyclase activation, which is potentiated by alpha1-adrenergic-induced increases in intracellular calcium (Ca2+) and calcium-dependent protein kinase. Alpha-adrenergic receptors have a greater effect on the pineal during the course of development, whereas beta-adrenergic receptors are most important in the adult.
cAMP appears shortly after birth, cGMP after the second week of life. In a study that raised levels of intracellular calcium, cGMP did not appear until after the second week of life. However, if cells from before the second week were placed in same medium as cells from after second week, they would begin to make cGMP, suggesting the presence of a diffusible factor.
GABA-ergic receptors: Fifteen percent of pinealocytes are positive for GABA. Glutamic acid decarboxylase is found in the pineal gland as well as the GABA receptor complex, which includes the GABA-binding site, the benzodiazepine-binding site, and the chloride ionophore, GABA receptor-positive pinealocytes have neuron-like properties in that they release GABA in response to depolarizing stimuli. In the rat pineal, GABA is released following interaction of norepinephrine with alpha1 adrenoreceptors. Both A and B types of GABA receptors have been described in the pineal. Activation of A receptors interferes with norepinephrineinduced melatonin release. Activation of B receptors decreases norepinephrine release. Presynaptically, GABA increases maximal velocity but decreases the affinity of norepinephrine uptake. The pre- and postsynaptic effects of GABA in the pineal seem to be similar to those in other parts of the brain.
A relationship between melatonin and cortical benzodiazepine receptors is evidenced by the fact that melatonin administration maintains the concentration of cortical benzodiazepine receptors in pinealectomized and superior cervical ganglionectomized rats. Nicotine diminishes norepinephrine-stimulated melatonin accumulation, although it has no effect on melatonin production or release. A binding site for the excitatory neurotransmitter glutamate has recently been identified and characterized in the rat pineal. Glutamate may play a modulating role in pineal physiology.
Suprachiasmatic nucleus: Regulation of the suprachiasmatic biological clock is not fully understood. Administration of the pineal hormone melatonin to rats induces expression of Fos, the protein product of the c-fos proto-oncogene in the SCN. c-fos is activated only if the melatonin is given during the late phase of subjective day. Because melatonin administration late in the day advances the SCN biological clock, it must do so through a mechanism independent of c-fos.
Melatonin-receptor density in the rat in both the pars tuberalis and SCN increases in pinealectomized animals as well as those exposed to light. Administration of melatonin reverses this effect, indicating that melatonin itself regulates receptor density in these areas.
Melatonin-receptor levels are highly variable throughout the light-dark cycle. It is believed that receptor down-regulation may account for some of the different effects of the hormone at different phases in the cycle.
Ovarian hormones inhibit rat pineal melatonin production in the proestrous night. RU486 fails to block this effect, which suggests that estradiol is its mediator.
Pineal cells express a 3.5-kb mRNA that corresponds to the estradiol-17 beta receptor. At low concentrations, administered estradiol is inhibitory: at high concentrations it is stimulatory. These effects were modified, depending on when during the lightdark cycle the estradiol was administered.
Elevated levels of melatonin were found in association with pituitary tumors secreting either prolactin or growth hormone.
Effects on neural cells of melatonin: The effects of melatonin have been most studied in the hypothalamus. Melatonin has been shown to change protein synthesis, GABA content and neurohormone release, and to decrease cAMP accumulation as well as to alter excitatory responses. Melatonin has effects on neuronal activity and protein synthesis in several brain locations, including the hypothalamus, midbrain and pineal.
Melatonin and vasotocin have effects on spontaneous neuronal activity in certain areas of the brain. The effects of microiontophoretic application of melatonin and melatonin plus vasotocin on spontaneously active neurons of the caudate-putamen in shamoperated and pinealectomized rats were studied. Administration of melatonin alone to caudate-putamen cells caused inhibition of approximately three-quarters of neurons. Melatonin combined with vasotocin (another prominent pineal product) increased inhibition to 100 percent.
Ethanol Levels and Consumption, and Melatonin Production: Ethanol at usually consumed levels was shown to inhibit melatonin production in healthy volunteers. There was an associated increase in noradrenergic activity. The combined effects may be associated with disturbances of sleep and performance observed with this substance.
The pineal has been postulated to play a role in various other conditions, such as glaucoma, porphyria, hemochromatosis and endocrine disorders. Myelin formation and maintenance can be altered following pinealectomy: this is thought to be due to alterations in levels of long-chain fatty acids.
Endocrine effects of the pineal include influences on the thyroid and adrenals. The pineal has been demonstrated to have effects on growth, body temperature, blood pressure, motor activity and sleep. The effects of melatonin differ, depending on the point in the photoperiod at which it is given.
Melatonin binds to calmodulin with high affinity. This enables it to influence cellular activity within physiologic ranges. It may be through its interaction with calmodulin that melatonin affects many cellular rhythmic activities. Melatonin and calmodulin are both phylogenetically well-preserved molecules, which suggests that their interaction represents a primary mechanism for regulation and synchronization of cellular physiology.
Studies in rats have shown that pinealectomy induces hypertension that can be blocked by melatonin administration. This effect is thought to be related to stimulation of the renin-angiotensin system or perhaps to stimulation of central adrenergic receptors. Weight and volume of the pineal have been shown to be higher in aging hypertensives than in normotensives. Altered pineal sensitivity to norepinephrine and isoproterenol has been demonstrated in spontaneously hypertensive rats and is thought to be related to stimulation of PKC and intracellular calcium.
Melatonin receptors are far more prevalent across cell types than had previously been suspected before radiolabeling assays were available. Although binding studies are a means of determining potential sites of melatonin action, their detection does not necessarily indicate the mechanism of action.
Binding of melatonin has been demonstrated in the anteroventral and anterodorsal nuclei of the rat, which suggests that some of the effects of melatonin are mediated via the limbic thalamus. These effects are thought to be due to interaction with specific, high-affinity melatonin receptors in the SCN and hypophyseal pars tuberalis, respectively. Receptor localization studies using 125I have shown melatonin receptors in the SCN and pars tuberalis of seasonally breeding species, including the rhesus monkey. The pars tuberalis receptors are absent in humans.
Melatonin Feedback onto SCN
Several studies have indicated that pineal melatonin feeds back on SCN rhythmicity to modulate circadian patterns of activity and other processes. However, the nature and system-level significance of this feedback are unknown. Recently published work indicates that although pinealectomy does not affect rat circadian rhythms in light-dark cycles or constant darkness, wheel-running activity rhythms are severely disrupted in constant light. These data suggest either that pineal feedback regulates the light sensitivity of the SCN or that it affects coupling among circadian oscillators within the SCN or between the SCN and its output.
Pineal (Melatonin) Influences on Hormones
Melatonin influences activity of many hormones and is, in turn, influences by them through feedback mechanisms. The interaction of melatonin and prolactin is of particular interest because it implicates several potential subsidiary control systems. Bright light at night leads to decreased prolactin levels paralleling those of melatonin.
Melatonin's effect on prolactin could be mediated through an effect on the SCN. perhaps mediated through an effect on the dopamine or endogenous opioid system. Total levels of prolactin secretion remained constant in light exposure experiments, which suggests that modulation occurs at the level of secretion rather than production. The interaction with prolactin may be part of the mechanism for melatonin to influence the reproductive system.
Studies in pinealectomized rats demonstrated a greater ACTH response to stress. This suggests that the pineal may suppress stress-reactive ACTH outflow.
There is a relationship between melatonin and the adrenal cortex that is dissociated from the hypothalamic-pituitary-adrenal axis. Administration of melatonin leads to an increase in adrenal corticosteroid levels.
Pineal influence over the neurohypophysis has been shown in experiments on pinealectomized animals. Exposure to constant light, while altering the patterns of neurohypophyseal activity in the pineal intact group, had little effect on the pinealectomized animals, indicating that the effect of light is mediated by the pineal.
Melatonin probably also acts through effects on the serotonergic system, which has an intrinsic component in the parvocellular nuclei of the hypothalamus as well as the system of raphe nuclear projections to the hypothalamus via the median forebrain bundle. Melatonin is not a competitive binder to serotonin receptors and therefore exerts its effect on cells of the serotonergic system by separate receptors. Melatonin and serotonin have antigonadotropic effects. The effect of melatonin on serotonin metabolism is controversial. Electrical stimulation of the pineal produces hypertension and tachycardia in the rat antagonized by serotonin receptor antagonism, bilateral vagotomy or spinal transection.
The emerging link between the immune system and the pineal may reflect an evolutionary connection between reproduction and recognition of self. Melatonin has an immunostimulatory effect, especially in states of immunodepression, including that induced by stress. The immune system link with melatonin suggests its possible use as a therapeutic agent in immunodeficiency as well as in cancer immunotherapy.
A principal target of melatonin is the thymus. The immunoenhancing effect seems to be due to T-helper cell-derived opioid peptides and lymphokines. Pituitary hormones may also be involved. Induction of these lymphokines by melatonin suggests the presence of specific binding sites on immune system cells. Melatonin production by the pineal is modulated by interleukin-2 (lL-2) and thymic hormones. The pineal gland might thus be viewed as the crux of a sophisticated immunoneuroendocrine network which functions as an unconscious, diffuse sensory organ. Administration of exogenous melatonin significantly enhances murine antibody-dependent cytotoxicity, whereas pinealectomy impairs it.
The presence of melatonin receptors on cells of the immune system does not necessarily implicate the pineal hormone as an immune regulator; however, a study on binding of 2iodomelatonin to human lymphocytes discovered that there were two types of receptors, differentiated by both the affinity of binding and the second messenger stimulated by binding. This is suggestive of a complex stimulation and regulatory mechanism.
Another study on diurnal rhythms of chick serum and granulocyte lysozyme found that rhythmicity abolished after pinealectomy could be restored by administering melatonin to the animals. This suggests that melatonin may have an influence on nonspecific immune mechanisms. Melatonin may be associated with multiple sclerosis through its effects on biological cycles and the immune system.
A study of IL-2 combined with melatonin in patients with solid neoplasms found a significant increase in the mean number of lymphocytes. This effect was not observed with melatonin alone, which suggests that IL-2 must be present for this immunostimulatory effect of melatonin to occur.
Melatonin promotes the growth of certain tumors. Although not itself known to be carcinogenic, the pineal hormone, acting through receptors linked to the cAMP and G protein second messenger systems, may participate in regulatory processes that become altered in preneoplastic states. Thus melatonin may act as an inductive factor where permissive intracellular conditions exist.
Pineal gland hyperplasia and elevated levels of melatonin have been demonstrated in association with disseminated melanoma. Melatonin receptors have been detected on certain types of breast tumor cells. There is an inverse relationship between melatonin level and level of estrogen receptors in patients with breast cancer.
An association between human breast cancer and levels of melatonin is suggested by the finding of depressed melatonin levels in the serum of patients with primary breast cancer. Experimental breast cancer cells have been successfully inhibited by melatonin, which suggests the possibility of using melatonin and other pinealderived substances as antineoplastics.
Levels of melatonin (but not those of prolactin and growth hormone) in 132 cancer patients were significantly higher than in 58 controls. Higher melatonin levels seemed to correlate with the stage of cancer. Interesting in the melatonin-neoplasia link is that levels of melatonin do not seem to be affected by surgical removal and that the rhythmicity of melatonin secretion is comparable to that in normals.
Several studies of the effect of melatonin on different tumor types have found it to be largely inhibitory. The mechanisms of this effect have been postulated to be mediated by effects on mitotic activity: immunocompetence, or secretion of growth hormone, somatomedin, ACTH or catecholamines. In one study, pinealectomy increased the growth of transplanted tumor cells in hamsters, whereas melatonin reversed this effect.
A study of tumor (erythroleukemia) cells in culture looked at the inhibition of cellular proliferation by pineal gland extracts. The gland had the most inhibitory activity in summer, whereas it was only weakly inhibitory or even excitatory in winter. This suggests seasonality of cancer occurrence.
Possible links between the pineal and neoplasia are being investigated intensively. Understanding the mechanisms whereby the pineal would induce or inhibit neoplasia might shed light onto the process of neoplastic transformation. Melatonin or another pineal substance could serve as a marker for neoplastic induction or progression.
Effects on the rhythmicity of melatonin secretion have been observed in cancer patients with breast, prostate and other neoplasms. The pineals of patients with cancer have been shown to be enlarged and degenerated, compared with age-matched controls.
Although an experimental model does not exist, numerous physiologic and epidemiologic factors are consistent with involvement of the pineal gland in the pathogenesis of endometrial carcinoma.
Pinealectomy inhibited leukemogenesis in a murine model, whereas melatonin promoted it. The melatonin appeared to work through an opioid mediator as an opioid antagonist blocked the melatonin effect.
Growth and Development
Mass lesions of the pineal region are associated with precocious puberty. However, the role of the pineal in puberty is still unclear. Melatonin levels are highest in both sexes between ages 1 and 5 and then decrease until the end of puberty. Melatonin levels have been shown to be the same for prepubertal and adult males as well as those undergoing precocious puberty. A study that measured melatonin levels in relation to stages of adrenarche found no relationship, indicating that pineal-puberty relationships are not mediated through an effect of melatonin on adrenarche.
A study of 57 normal children and 39 with disorders of onset of puberty found no correlation between nocturnal peak melatonin levels and those of testosterone, estradiol, or LH. This suggests that melatonin does not have a major inhibitory effect on the hypothalamic-pituitary-gonadal axis during childhood.
Evidence from other species, as well as the association between melatonin and dysmenorrhoea, gonadal growth and involution and reproduction. suggests a probable link between the pineal hormone and puberty. This remains to be fully elucidated by current experimental techniques.
The reason for the frequently observed association between pineal neoplasms and precocious puberty is not understood. Postulated mechanisms, other than an effect of melatonin, include the tumour's secretion of unknown substances that induce pubertal changes. Another possibility is modification of normal hormonal regulation mechanisms by pressure or by mechanical effects of a pineal region mass on nearby brain structures (such as the hypothalamus).
Pineal peptides and indoles seem to affect fertility by restricting reproductive function to an optimal time of the year, which improves survival of the species. The pineal converts photic and temperature cues into meaningful messages by which reproduction and nurturing are optimally timed.
The pineal effect on regulation of reproductive activity is mediated by melatonin levels, which increase and decrease during the lengthening and shortening of day-night periods as the seasons change. When days are longer, the period of melatonin production is prolonged, which may signal a season change to the animal, which could be a factor in controlling reproductive activity.
In many animals, maturation of the female reproductive organs is inhibited by the action of melatonin. During the reproductive phase, melatonin has important coordinating roles in regulating the timing of the LH surge and the production of progesterone. In 1963 Wurtman et al. first reported that melatonin given to female rats decreased ovarian weight and increased the frequency of estrus. This antigonadotropic effect has since been substantiated and may partly explain the effect of tumors that ablate the pineal: a positive gonadotropic effect and, frequently, precocious puberty.
After the identification of melatonin and the determination of its relationship to photoperiodicity, the most significant discovery about the hormone was of its effect on the reproductive organs and their function in mammals. Recent studies have confirmed that melatonin has effects on hypertrophy and atrophy of reproductive organs (e.g., ovaries) in experimental animals.
Ultrastructural changes in pineal cells have been observed in animals during different phases of the breeding cycle. There is a decline in levels of circulating melatonin with aging, suggesting that this chemical may be involved in the process of menopause. Increases in enzyme and cellular organelle activity during gestation have been documented.
The mediator of antigonadotropic activity of pineal extracts is unknown. Experiments with pineal extracts containing no melatonin or steroid fractions showed potent antigonadotropic effects in an ovine model. implicating an "inhibin-like" factor.
The pineal. presumably through melatonin, controls the timing of the LH surge, The LH surge in pinealectomized rats is variable. Administration of melatonin to pinealectomized animals shortly after a light-to-dark transition improved regulation of the timing of the LH surge. Administration of melatonin at other times in the light-dark cycle was ineffective. This shows that time of administration with respect to the light-dark cycle is important to melatonin effect.
Pineal effects on the menstrual cycle are probably mediated through effects on the pineal centres that produce and secrete gonadotropin-releasing hormone (GnRH) and other reproductionrelated hormones. The GnRH pulse generator can be inactivated by melatonin. Melatonin release is increased during the dark phase, which is longer during winter. Humans are not seasonal breeders, perhaps because of a defect in the retinopineal pathway. Melatonin, if it can be administered in a way that mimics the night-time amplitude and duration of long-day breeders, might be an effective contraceptive.
In one study, short photoperiods associated with increases in melatonin were associated in hamsters with loss of estrus. This could be restored by pinealectomy. Melatonin production and secretion patterns were altered in six studied amenorrheic women, reflecting a probable link between the pituitary-gonadal axis and the pineal.
Melatonin has been shown to decrease the release of GnRH in castrated animals (minks) but not in uncastrated controls. This implies that melatonin effects require testosterone feedback. Melatonin will cause testicular regression in pinealectomized hamsters only if delivered in a specific temporal pattern.
Exposure of hamster seminiferous tubule cells to melatonin caused regression and necrosis with increased incidence of aspermic tubules. This effect is absent if a much larger dose of melatonin is given over a prolonged period of time.
Melatonin levels in human milk exhibit a daily rhythm of high levels at night and undetectable levels during the day. These variations may communicate time-of-day information to breast-fed infants.
Melatonin may affect the behavioural as well as physiological aspects of reproduction, By using regulated. timed infusion of melatonin. it is possible to show which secretory profile is optimal to elicit certain seasonally appropriate behavioural and physiologic reproductive responses. In hamsters and sheep the duration of the melatonin infusion seems to be the crucial aspect of the signal.
The pineal is thought to be active in all stages of human life. The gland maintains its weight and ability to produce enzymes into the eighth decade. Circadian rhythms of pineal excretion are not affected by aging or senile dementia. However 24-h mean melatonin levels are half as great in the elderly as in the young. Melatonin levels in the CSF also decrease with age. Several reasons for these decreases have been suggested, including (1) changes in the release of the hormone, (2) an increase in its metabolism or excretion. (3) an increased sensitivity to light in the aged or (4) decreased or nonresponsive pineal beta-adrenergic receptors.
Melatonin and serotonin may be involved in the development of ischemic heart disease, Alzheimer's disease, tumor formation and other degenerative processes associated with aging. The favourable effects of dietary restriction on aging may also be related to melatonin.
Excitatory amino acids inhibit melatonin release. Melatonin is a potent nonenzymatic free radical scavenger. Increased release of excitatory amino acids may account for some of the effects of aging. The possible use of melatonin to slow down aging is supported by experiments demonstrating decreased hydroxyl radicalinduced oxidative damage in experimental rats.
Oral administration of melatonin to aging mice prolongs survival and preserves a youthful state. In one study, pineal glands were grafted from young mice to older mice thymuses. Thymus tissue remained youthful and T-cell function was preserved. (A diurnal rhythm in the activity of superoxide dismutase has been observed, the significance of which is not known.) Changes in the nightly melatonin peak provide a signal that informs the organism of its age. It has been hypothesized that this "durational signal" at the cellular level is the sleep-induced pCO2 changes in the blood.
Calcium availability is decreased in aging and is postulated to be the cause of decreased beta- and alpha-adrenergic responses leading to melatonin biosynthesis. A decreased response of short sympathetic neurons to applied melatonin has been demonstrated in rats. Increasing evidence that melatonin administered to rats slows the rate of aging has led to the proposed use of melatonin to inhibit aging due to free radicals.
Any discussion of melatonin's influences on human behavioural physiology must specify which effects result from direct administration of purified melatonin and which result from whole gland extract. In addition, many physiologic probabilities in animals are downgraded to mere possibilities in humans, pending confirmatory human investigation.
The actions of the pineal (and of melatonin) can be divided into two broad categories: behavioural and nonbehavioural. Behavioural effects include regulation and synchronization of biological activity into optimal cycles, such as for waking and sleeping, food seeking and reproduction. This activity is often tied to environmental light and dark variation, but there are intrinsic cycles as well. Persistence of locomotor circadian rhythms in the face of functional pinealectomy induced by bright light suggests the existence of a mechanism or mechanisms for cyclicity independent of the pineal. An interesting proposed behavioural link is that between melatonin-mediated changes in intracellular Ca2+ and infant colic.
A high dose of melatonin can produce ataxia, incoordination, muscle relaxation, ptosis, piloerection, muscle relaxation, extremity vasodilatation, and generalized decrease in movement. At very high doses, flexor reflexes are impaired, breathing becomes laboured, and body temperature decreases prior to death. Based on these findings, it has been postulated that melatonin plays a role in movement and therefore in movement disorders.
Studies of melatonin interaction with L-dopa in rats and detection of the hormone in the substantia nigra suggest a possible link to Parkinson's disease. Chronic oral melatonin administration ameliorates Parkinsonian tremor and rigidity.
A link between melatonin and amyotrophic lateral sclerosis (ALS) has also been proposed, based on the observed influences of melatonin on axonal transport in sciatic and other nerves. Melatonin is associated with the serotonergic transmission systems, and serotonin is believed to be involved in the pathogenesis of ALS.
Treatment with L-dopa is associated with dyskinesias thought to be due to striatal dopaminergic activity perturbations. In laboratory animals, the pineal hormone melatonin has been shown to regulate striatal dopaminergic activity and block L-dopa-induced dyskinesias. Sandyk et al. report a dramatic improvement in L-dopa-induced dyskinesias in a patient with Parkinson's disease treated with the application of external magnetic fields.
Both heat and cold increase locomotor activity in rats through a mechanism mediated by the pineal. Pinealectomy attenuates the response. It is thought that these and other non photic stimuli create stress from which pineal hormones promote escape through increased motor activity.
Wheel-running activity in hamsters is one of the experimental models used in correlating plasma melatonin levels with locomotor activity. Complex circadian melatonin rhythms have been associated with wheel-running activity.
Sleep- Wake Cycle and Epilepsy
Alpha rhythm appears around the time of puberty in the human brain. Thus it is a marker for psychosexual development, which is importantly influenced by the pineal. Administration of melatonin blocks alpha rhythm, which suggests that melatonin's progressive decline during childhood may enable the maturation of alpha rhythm. Sandyk proposes using alpha rhythm as a "neurophysiological marker" of pineal activity in disorders associated with alpha rhythm disturbances, such as autism. dyslexia, personality disorders, epilepsy, Tourette's syndrome and schizophrenia.
Melatonin may act through a thermoregulatory mechanism by lowering core body temperature and thereby increasing sleep propensity. Melatonin has circadian cycling and hypnotic effects, which make it a good candidate as an adjuvant drug for sleep in conjunction with other drugs known to alter melatonin production, such as beta-blockers and benzodiazepines.
Developmental periods associated with increased melatonin are associated with decreased REM sleep: those with low melatonin are associated with increased REM sleep. Sandyk proposes that low melatonin is permissive for REM sleep. He further suggests that narcolepsy may be due to a maturational defect of the pineal gland in infancy.
Melatonin is important for maintaining sleep-wake cycles, and its absence or excess can cause irregularities in REM sleep. Melatonin has been shown to induce sleep and characteristic EEG rhythms in several species. Following intraperitoneal administration of melatonin, brain stem serotonin has increased, which is associated with sleep induction. In the elderly, decreased sleep is associated with decreased melatonin levels. Intensive research is under way to investigate the therapeutic use of melatonin or an analogue for patients with insomnia.
The extremely high levels of melatonin observed in patients suffering from narcolepsy and therefore with REM sleep deprivation have led some to suggest an etiologic link between hormone and sleep disorder. Melatonin has been proposed as a treatment for narcolepsy. but no clinical trials have been undertaken.
REM sleep onset is associated with increased melatonin levels, which may be due to increased norepinephrine release associated with the increased autonomic activity. Oral administration of melatonin in gelatine was associated with a decreased subjective feeling of jet lag in aeroplane travellers.
A more recent double-blind, placebo-controlled study of a small group of police officers working for seven consecutive night shifts found improved performance in various letter-target and visual search tests after melatonin was administered. Melatonin administration is also associated with prolonged time needed for animals to emerge from the effects of barbiturates and with decreased anxiety-inducing behaviours. It decreases alertness and increases sleepiness in animals.
Exogenous melatonin administration is associated with decreased epileptiform activity in animal models of epilepsy. Pinealectomy in these animals produces increased seizure activity. Antimelatonin antibodies have been shown to have an epileptogenic effect in rats. Evidence from humans supports the findings that melatonin increases sleep, decreases motor activity, and decreases paroxysmal EEG activity observed in epileptic patients.
Evidence that melatonin is anticonvulsive is that pinealectomy results in seizures in experimental animals. Recent studies with magnetoencephalography have shown an apparent proconvulsive effect of melatonin. This would be consistent with the observed five- to eight-fold higher levels of plasma melatonin observed at night than during the day. Seizures are also more frequent during pregnancy, when melatonin levels are higher. Anticonvulsants that decrease melatonin secretion, such as the benzodiazepines, may exert their antiepileptic activity by attenuating nocturnal melatonin secretion.
In rats the period associated with the development of enkephalin-induced seizures coincides with the peak of melatonin plasma levels at 3 weeks of age; therefore, it is proposed that melatonin mediates the anticonvulsant action of drugs effective for petit-mal (absence) epilepsy and that the pineal gland is implicated in the pathogenesis of this form of childhood epilepsy . In addition, a relationship between pineal calcification and the laterality of temporal lobe seizures has been proposed, based on the fact that the pineal receives predominant limbic input from the right temporal lobe, which itself has greater limbic and reticular input than the left temporal lobe.
The mechanisms for melatonin's sedative and antiepileptogenic effects remain obscure but may be due to interactions with the serotonergic and/or GABA-ergic neurotransmitter systems. Benzodiazepines have been demonstrated to have a stimulatory effect on acetyltransferase.
Artificial magnetic fields may attenuate seizure activity by altering the functioning of the pineal gland and melatonin levels. Humans kept under bright light during the night will show deterioration in parameters measuring performance in spite of decreases in their melatonin level; this suggests that these parameters worsen for other reasons.
Other Behavioural Effects
Nociceptive responses have been significantly diminished by functional pinealectomy (induced by a single long-light-duration day). Oral melatonin administration restored the pain response.
Aggression is decreased after pinealectomy and increased again when melatonin is administered. Ethanol preference does not seem to be affected by pinealectomy. Passive avoidance is increased with a pineal extract. Social isolation induces pineal hypertrophy. Stress leads to increased production of melatonin and tryptophan uptake by the pineal. A dramatic demonstration of the probable link between psychology and pineal physiology is the observed alteration in NAT activity observed in rat pups deprived of maternal contact.
Melatonin is known to decrease activity in the SCN and reticular formation. Melatonin may be involved in certain mental processes and has been implicated in mental illnesses, such as cyclic affective disorders.
The pineal and melatonin seem to interact with several psychiatric drugs, especially antidepressant and antipsychotic medications that operate on the beta-adrenergic system. Chlorpromazine and haloperidol inhibit HIOMT, the responsible enzyme. Chronic lithium administration suppresses a shift in the peak night-time melatonin concentration and decreases hormone levels.
In patients with no cortisol response to administration of dexamethasone, there may be a pineal factor that stimulates the production of corticotropin-releasing factor by inhibiting its action. Depressed patients with an abnormal dexamethasone suppression test result have lower-than-normal melatonin levels. Low melatonin levels have been suggested to indicate a genetic trait for depression. These abnormalities have been correlated with other changes in the hypothalamic-pituitary-adrenal axis. Patients with low melatonin levels also often have early escape on dexamethasone suppression testing.
Melatonin is increased in mania, which is consistent with a condition associated with increased sympathetic activity. There is a possible relationship between pineal hormonal activity and eating disorders, such as anorexia nervosa. It is unclear if the abnormalities are the cause or effect of the psychopathologic process.
Seasonal affective disorder (SAD), recently popularized in the media, is a condition associated with onset of depression during winter, with its longer periods of darkness. Exposure of SAD patients to several hours of bright light lowers circulating levels of melatonin and concomitantly elevates mood. Whether the two effects are coincidental or causally related is under investigation.
A patient with both SAD and cocaine addiction was described. SAD is related to disorders in circadian rhythms, and cocaine addiction may be related to disorders of melatonin. The two conditions may thus both be linked through the pineal.
Investigators of the causes of the sudden infant death syndrome (SIDS) have suggested that the pineal acts as the "masterswitch of life and death" through neurohormonal regulation and neural connections with the hypothalamus: it acts especially through its influence on hypothalamic noradrenaline levels that contribute to SIDS by influencing the function of the autonomic nervous system, specifically the control of such vital functions as breathing.
Although other infra- and ultradian cycles influence behaviour, the light-dark variation is the most blatantly regulatory cycle in humans. The sensitivity of melatonin production and release to environmental light was demonstrated in a study that followed serum melatonin levels in night shift workers in Antarctica. A period of night shift work requires readaptation of melatonin rhythms. This study shows that the time taken to adapt is longer (3 weeks) in winter than in summer (1 week). This has important implications for shift workers in temperate zones. A study of melatonin levels in a population living at a latitude of 70 degrees with almost continuous darkness in winter and almost continuous light in summer, showed the highest levels in January and the lowest in June.
Serotonin may contribute to an antidepressant effect of the pineal. Forced swimming, a test of immobility in rats (and an animal model for depression), was improved following the transplant to the frontal neocortex of pineal tissue.
Analysis of CT scans showed increased pineal calcification in premenopausal schizophrenic women. This suggests a link between pineal activity (presumably decreased in the presence of calcification) and schizophrenia.
Because of relationships between melatonin and physiologic processes associated with arousal, manipulations of melatonin levels through imposed magnetic fields have been proposed for disorders of sleep and for epilepsy.
Sandyk noting the seasonal and possibly cyclic occurrence of cluster headaches and to a lesser extent migraine headaches, believes that the pineal and melatonin may be involved in their pathogenesis. He cites successful treatment of a patient with migraine with picoTesla magnetic fields.
An important consideration in any therapeutic intervention with melatonin will be mode of delivery. An oral melatonin regimen in rats was found to be effective in re-establishing normal circadian levels of urinary 6-sulphatoxymelatonin in functionally pinealectomized rats.
The pineal enables the organism to coordinate its activities with cyclic variations in the physical environment. This is achieved primarily through links between the pineal and the photoreceptor organs as well as interrelationships between the gland and the information-processing neuroendocrine system. An interesting question for neurosurgeons to ponder is why lesions that destroy or increase pineal tissue seem to be associated more with mechanical effects on adjacent brain tissue and CSF circulation than with alterations in physiology. The pineal is a modulator, not a primary mover in the physiologic engine.
From the little structure that seemed to do nothing except give rise to difficult tumors, the pineal has emerged as a central organ in human physiology. Consistent with the evolving holistic picture of the organism, the pineal defies understanding if conceptually relegated to one system or function. The updated concept of the soul is that of a dynamically interacting totality in which the pineal may exert an important if not dominant role, entitling it to a share of the throne on which Descartes placed it.