Evolution of The Vertebrate Pineal Gland: The Aanat Hypothesis

The defining feature of the pineal gland is the capacity to function as a melatonin factory that operates on a ～24 h schedule, reflecting the unique synthetic capacities of the pinealocyte. Melatonin synthesis is typically elevated at night and serves to provide the organism with a signal of nighttime. Melatonin levels can be viewed as hands of the clock. Issues relating to the evolutionary events leading up to the immergence of this system have not received significant attention. When did melatonin synthesis appear in the evolutionary line leading to vertebrates? When did a distinct pineal gland first appear? What were the forces driving this evolutionary trend? As more knowledge has grown about the pinealocyte and the relationship it has to retinal photoreceptors, it has become possible to generate a plausible hypothesis to explain how the pineal gland and the melatonin rhythm evolved. At the heart of the hypothesis is the melatonin rhythm enzyme arylalkylamine N‐acetyltransferase (AANAT). The advances supporting the hypothesis will be reviewed here and expanded beyond the original foundation; the hypothesis and its implications will be addressed.     

Evolution of the vertebrate pineal gland: the AANAT hypothesis

The defining feature of the pineal gland is the capacity to function as a melatonin factory that operates on a approximately 24 h schedule, reflecting the unique synthetic capacities of the pinealocyte. Melatonin synthesis is typically elevated at night and serves to provide the organism with a signal of nighttime. Melatonin levels can be viewed as hands of the clock. Issues relating to the evolutionary events leading up to the immergence of this system have not received significant attention. When did melatonin synthesis appear in the evolutionary line leading to vertebrates? When did a distinct pineal gland first appear? What were the forces driving this evolutionary trend? As more knowledge has grown about the pinealocyte and the relationship it has to retinal photoreceptors, it has become possible to generate a plausible hypothesis to explain how the pineal gland and the melatonin rhythm evolved. At the heart of the hypothesis is the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (AANAT). The advances supporting the hypothesis will be reviewed here and expanded beyond the original foundation; the hypothesis and its implications will be addressed.     

The 2004 Aschoff/Pittendrigh lecture: Theory of the origin of the pineal gland--a tale of conflict and resolution

A theory is presented that explains the evolution of the pinealocyte from the common ancestral photoreceptor of both the pinealocyte and retinal photoreceptor. Central to the hypothesis is the previously unrecognized conflict between the two chemistries that define these cells-melatonin synthesis and retinoid recycling. At the core of the conflict is the formation of adducts composed of two molecules of retinaldehyde and one molecule of serotonin, analogous to formation in the retina of the toxic bis-retinyl ethanolamine (A2E). The hypothesis argues that early in chordate evolution, at a point before the genes required for melatonin synthesis were acquired, retinaldehyde--which is essential for photon capture--was depleted by reacting with naturally occurring arylalkylamines (tyramine, serotonin, tryptamine, phenylethylamine) and xenobiotic arylalkylamines. This generated toxic bis-retinyl arylalkylamines (A2AAs). The acquisition of arylalkylamine N-acetyltransferase (AANAT) prevented this by N-acetylating the arylalkylamines. Hydroxyindole-O-methyltransferase enhanced detoxification in the primitive photoreceptor by increasing the lipid solubility of serotonin and bis-retinyl serotonin. After the serotonin --> melatonin pathway was established, the next step leading toward the pinealocyte was the evolution of a daily rhythm in melatonin and the capacity to recognize it as a signal of darkness. The shift in melatonin from metabolic garbage to information developed a pressure to improve the reliability of the melatonin signal, which in turn led to higher levels of serotonin in the photodetector. This generated the conflict between serotonin and retinaldehyde, which was resolved by the cellular segregation of the two chemistries. The result, in primates, is a pineal gland that does not detect light and a retinal photodetector that does not make melatonin. High levels of AANAT in the latter tissue might serve the same function AANAT had when first acquired- prevention of A2AA formation.     

MELATONIN CIRCADIAN RHYTHM IN THE RETINA OF MAMMALS

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood. (Chronobiology International, 17(5), 599–612, 2000)     

Cholinergic signaling in the rat pineal gland

Summary 1. Innervation of the mammalian pineal gland is mainly sympathetic. Pineal synthesis of melatonin and its levels in the circulation are thought to be under strict adrenergic control of serotoninN-acetyltransferase (NAT). In addition, several putative pineal neurotransmitters modulate melatonin synthesis and secretion.2. In this review, we summarize what is currently known on the pineal cholinergic system. Cholinergic signaling in the rat pineal gland is suggested based on the localization of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), as well as muscarinic and nicotinic ACh binding sites in the gland.3. A functional role of ACh may be regulation of pineal synaptic ribbon numbers and modulation of melatonin secretion, events possibly mediated by phosphoinositide (PI) hydrolysis and activation of protein kinase C via muscarinic ACh receptors (mAChRs).4. We also present previously unpublished data obtained using primary cultures of rat pinealocytes in an attempt to get more direct information on the effects of cholinergic stimulus on pinealocyte melatonin secretion. These studies revealed that the cholinergic effects on melatonin release are restricted mainly to intact pineal glands since they were not readily detected in primary pinealocyte cultures.     

Melatonin circadian rhythm in the retina of mammals

Melatonin has been traditionally considered to be derived principally from the pineal gland. However, several investigations have now demonstrated that melatonin synthesis occurs also in the retina (and in other organs as well) of several vertebrate classes, including mammals. As in the pineal, melatonin synthesis in the retina is elevated at night and reduced during the day. Since melatonin receptors are present in the retina and retinal melatonin does not contribute to the circulating levels, retinal melatonin probably acts locally as a neuromodulator. Melatonin synthesis in the retinas of mammals is under control of a circadian oscillator located within the retina itself, and circadian rhythms in melatonin synthesis and/or release have been described for several species of rodents. These rhythms are present in vivo, persist in vitro, are entrained by light, and are temperature compensated. The recent cloning of the gene responsible for the synthesis of the enzyme arylalkylamine N-acetyltransferase (the only enzyme unique to the melatonin synthetic pathway) will facilitate localizing the cellular site of melatonin synthesis in the retina and investigating the molecular mechanism responsible for the generation of retinal melatonin rhythmicity. Melatonin has been implicated in many retinal functions, and the levels of melatonin and dopamine appear to regulate several aspects of retinal physiology that relate to light and dark adaptation. In conclusion, it seems that retinal melatonin is involved in several functions, but its precise role is yet to be understood.     

Chick pineal melatonin synthesis: light and cyclic AMP control abundance of serotonin N-acetyltransferase protein

Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase [arylalkylamine N-acetyltransferase (AA-NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA-NAT mRNA levels. The major changes in AA-NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA-NAT protein levels and AA-NAT activity. Cyclic AMP might also regulate AA-NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA-NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA-NAT activity by altering the total amount of AA-NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA-NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP-induced changes in AA-NAT protein levels mediate rapid changes in chick pineal AA-NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA-NAT) within a photoreceptive cell.     

褪黑素和褪黑素受体对下丘脑-垂体-肾上腺轴作用的研究进展

松果体于儿童中期可发育至最高峰,普遍在7岁之后开始呈逐渐萎缩,并在成年后逐渐有钙盐沉着。褪黑素主要是由松果体进行合成和分泌所形成,存在较好的昼夜节律性,且通常是通过下丘脑的视交叉上核进行控制,并与环境中的光-暗呈现的周期改变存在密切关联。此外,褪黑素具有极其广泛的生物学作用,且其发挥作用的首站便是与特异性褪黑素受体相关结合,随后经由信号转导系统发挥相应的生物效应。褪黑素受体属于G蛋白耦联受体超家族重要成员之一,其主要是通过百日咳毒素敏感G蛋白的一致性G蛋白通路,减少环腺苷酸的急剧或(和)抑制毛喉菇素刺激的环腺苷酸升高,从而间接影响黑色素活动。下丘脑-垂体-肾上腺轴(HPA轴)是机体在发生应激反应过程中具有一定影响的系统,其所分泌的激素也会表现出昼夜节律性的改变,且此种改变与褪黑素的有关变化呈现出明显的相反性。提示了两者可能存在一定的相关,在机体免疫功能的调控中扮演着不同的角色。本文通过阐述褪黑素和褪黑素受体对HPA轴作用的最新研究进展,旨在明确三者存在的错综复杂的相互作用关系,继而为机体免疫功能调控的一系列疾病研究提供参考依据。     

Photic and circadian regulation of melatonin production in the Mozambique tilapia Oreochromis mossambicus

Diverse circadian systems related to phylogeny and ecological adaptive strategies are proposed in teleosts. Recently, retinal photoreception was reported to be important for the circadian pacemaking activities of the Nile tilapia Oreochromis niloticus. We aimed to confirm the photic and circadian responsiveness of its close relative-the Mozambique tilapia O. mossambicus. Melatonin production in cannulated or ophthalmectomized fish and its secretion from cultured pineal glands were examined under several light regimes. Melatonin production in the cannulated tilapias was measured at 3-h intervals; it fluctuated daily, with a nocturnal increase and a diurnal decrease. Exposing the cannulated fish to several light intensities (1500-0.1 lx) and to natural light (0.1 and 0.3 lx) suppressed melatonin levels within 30 min. Static pineal gland culture under light-dark and reverse light-dark cycles revealed that melatonin synthesis increased during the dark periods. Rhythmic melatonin synthesis disappeared on pineal gland culture under constant dark and light conditions. After ophthalmectomy, plasma melatonin levels did not vary with light-dark cycles. These results suggest that (1) Mozambique tilapias possess strong photic responsiveness, (2) their pineal glands are sensitive to light but lack circadian pacemaker activity, and (3) they require lateral eyes for rhythmic melatonin secretion from the pineal gland.     

Urinary melatonin rhythms during sleep deprivation in depressed patients and normals

Melatonin excretion was measured in 8 hour urine aliquots for eight healthy controls and six depressed patients. Both groups had similar diurnal rhythms, with increased melatonin excretion during the night. When subjects were sleep deprived, remaining awake and active in continuous light from 7 a.m. one morning until 11 p.m. the following day, the diurnal rhythm in melatonin excretion remained unchanged. These data in man appear to be inconsistent with previous studies in rats showing rapid light-induced suppression of the nocturnal rise in pineal melatonin synthesis.     

Homeobox Genes in the Rodent Pineal Gland: Roles in Development and Phenotype Maintenance

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.     

Age-related morphometric changes in the pineal gland. A comparative study between C57BL/6J and CBA mice

Relatively little is known about the effects of melatonin on the aging of the pineal, the organ which is the main place for synthesis of this hormone. Using simple morphometric methods, some parameters of the pineal gland, such as total volume, number of pinealocytes and pinealocyte volume were estimated in two mice strains: normal CBA and melatonin-deficient C57BL/6J. Two age groups, 6 weeks and 10 months, were studied in order to evaluate possible differential age-related changes between both strains. Pineals of both strains have similar morphometric and morphological features at 6 weeks of age. This suggests that pineal development, which has already concluded at 6 weeks of age, is not affected by the absence of melatonin synthesis in the pinealocytes. Later on, CBA pineal showed an increase in size caused by cellular hypertrophy. In contrast, the C57BL/6J pineal volume decreased by loss of pinealocytes in the same period of time. Semithin sections analysed by light microscopy did not show that this cell death was evident in the C57BL/6J strain at any of the ages studied. Thus, a gradual loss of pinealocytes could be hypothesised in these pineals. These results suggest that pineal melatonin could have a role in the maintenance of pinealocyte viability and the increase of pineal size which takes place after development. The abnormal pattern observed in the C57BL/6J pineal should be taken into account in future studies on this gland.     

The benefits of four weeks of melatonin treatment on circadian patterns in resistance-trained athletes

Exercise can induce circadian phase shifts depending on the duration, intensity and frequency. These modifications are of special meaning in athletes during training and competition. Melatonin, which is produced by the pineal gland in a circadian manner, behaves as an endogenous rhythms synchronizer, and it is used as a supplement to promote resynchronization of altered circadian rhythms. In this study, we tested the effect of melatonin administration on the circadian system in athletes. Two groups of athletes were treated with 100?mg?day^?1 of melatonin or placebo 30?min before bed for four weeks. Daily rhythm of salivary melatonin was measured before and after melatonin administration. Moreover, circadian variables, including wrist temperature (WT), motor activity and body position rhythmicity, were recorded during seven days before and seven days after melatonin or placebo treatment with the aid of specific sensors placed in the wrist and arm of each athlete. Before treatment, the athletes showed a phase-shift delay of the melatonin circadian rhythm, with an acrophase at 05:00?h. Exercise induced a phase advance of the melatonin rhythm, restoring its acrophase accordingly to the chronotype of the athletes. Melatonin, but not placebo treatment, changed daily waveforms of WT, activity and position. These changes included a one-hour phase advance in the WT rhythm before bedtime, with a longer nocturnal steady state and a smaller reduction when arising at morning than the placebo group. Melatonin, but not placebo, also reduced the nocturnal activity and the activity and position during lunch/nap time. Together, these data reflect the beneficial effect of melatonin to modulate the circadian components of the sleep–wake cycle, improving sleep efficiency.     

Gastrointestinal melatonin: cellular identification and biological role

Melatonin, a pineal hormone, because of its wide activity spectrum, is a subject of much current interest for biologists and physicians. It has been demonstrated that pineal gland is not an exclusive source of melatonin synthesis. Melatonin synthesis has been found in different sites of the organism, and a major source of extrapineal melatonin is the gastrointestinal tract. The role of melatonin in gastrointestinal functions is considered in the present review.     

Vitamin A deficiency reduces the responsiveness of pineal gland to light in Japanese quail (Coturnix japonica)

Synthesis of melatonin in pineal gland is under the control of light environment. The recent finding of the presence of rhodopsin-like photopigment (pinopsin) and retinal in the avian pinealocytes has led to a hypothesis that vitamin A is involved in photoresponses of the pineal gland. We have thus analyzed the effect of vitamin A deficiency on the regulatory system of melatonin synthesis in the pineal gland of Japanese quail. Depletion of vitamin A from Japanese quails was attained by feeding them with a vitamin A-free diet supplemented with retinoic acid. In the vitamin A-deficient birds, diurnal rhythm in melatonin production persisted such that the phase of the wave was similar to that seen in the control birds. However, the amplitude of the nighttime surge of pineal melatonin was damped by vitamin A deficiency. When the control birds were briefly exposed to light at night, pineal melatonin dropped to the daytime level. In contrast, only slight decrease was observed in the vitamin A-deficient quails. The light responsiveness was restored after feeding the vitamin A-deficient quails with the control diet for 1 week. These results indicate that vitamin A plays essential roles in maintaining sufficient responsiveness of the avian pineal gland to photic input.     

The ageing pineal gland and its physiological consequences.

Melatonin, the chief hormone of the pineal gland, is produced and secreted into the blood in a circadian manner with maximal production always occurring during the dark phase of the light:dark cycle. Whereas the 24h rhythm of melatonin production is very robust in young animals including humans, the cycle deteriorates during ageing. The rhythm of melatonin can be substantially preserved during ageing by restricting the food intake of experimental animals; this same treatment increases the life span of the animals. The exogenous administration of melatonin to non-food restricted animals also reportedly increases their survival. Moreover, melatonin has been shown to have immunoenhancing effects and oncostatic properties. The implication of these studies is that melatonin may have both direct and indirect beneficial effects in delaying ageing processes or it may retard the development of processes (e.g., immunodeficiency and tumor growth) which contribute to a reduced life span.     

Immunocytochemical and circadian biochemical analysis of neuroactive amino acids in the pineal gland of the rat: effect of superior cervical ganglionectomy

Summary Semiquantitative immunocytochemistry by immuno-gold techniques revealed differences in the spatial distribution of glutamate, glutamine, and taurine within the pineal gland, with greatest labeling over pinealocytes, glia, and endothelia, respectively. At the subcellular level, glutamate labeling tended to be highest over pinealocyte synaptic ribbons and mitochondria, and lowest over lipid inclusions. Pineal levels of glutamate, glutamine and taurine, as measured by high performance liquid chromatography, did not vary over a light: dark cycle. Superior cervical sympathetic denervation, which abolishes pineal melatonin synthesis, resulted in a nearly 50% reduction in pineal glutamate levels, but had no effect on levels of glutamine and taurine. Other amino acids (alanine, arginine, aspartate, serine) were reduced by 23%–33% following sympathectomy. These data suggest an important role for glutamate in pinealocyte function(s) possibly related to the noradrenergic innervation of the gland.     

Bidirectional communication between the pineal gland and the immune system

The pineal gland is a vertebrate neuroendocrine organ converting environmental photoperiodic information into a biochemical message (melatonin) that subsequently regulates the activity of numerous target tissues after its release into the bloodstream. A phylogenetically conserved feature is increased melatonin synthesis during darkness, even though there are differences between mammals and birds in the regulation of rhythmic pinealocyte function. Membrane-bound melatonin receptors are found in many peripheral organs, including lymphoid glands and immune cells, from which melatonin receptor genes have been characterized and cloned. The expression of melatonin receptor genes within the immune system shows species and organ specificity. The pineal gland, via the rhythmical synthesis and release of melatonin, influences the development and function of the immune system, although the postreceptor signal transduction system is poorly understood. Circulating messages produced by activated immune cells are reciprocally perceived by the pineal gland and provide feedback for the regulation of pineal function. The pineal gland and the immune system are, therefore, reciprocally linked by bidirectional communication.     

The circadian timing system and reproduction in mammals.

Circadian systems in a wide variety of organisms all appear to include three basic components: 1) biological oscillators that maintain a self-sustained circadian periodicity in the absence of environmental time cues; 2) input pathways that convey environmental information, especially light cues, that can entrain the circadian oscillations to local time; and 3) output pathways that drive overt circadian rhythms, such as the rhythms of locomotor activity and a variety of endocrine rhythms. In mammals, the circadian system is employed in the regulation of reproductive physiology and behavior in two very important ways. 1) In some species, there is a strong circadian component in the timing of ovulation and reproductive behavior, ensuring that these events will occur at a time when the animal is most likely to encounter a potential mate. 2) Many mammals exhibit seasonal reproductive rhythms that are largely under photoperiod regulation; in these species, the circadian system and the pineal gland are crucial components of the mechanism that is used to measure day length. The rhythm of pineal melatonin secretion is driven by a neural pathway that includes the circadian oscillator(s) in the suprachiasmatic nuclei. Melatonin is secreted at night in all mammals, and the duration of each nocturnal episode of melatonin secretion is inversely related to day length. The pineal melatonin rhythm appears to serve as an internal signal that represents day length and that is capable of regulating a variety of seasonal variations in physiology and behavior.     

Melatonin the "light of night" in human biology and adolescent idiopathic scoliosis

Melatonin "the light of night" is secreted from the pineal gland principally at night. The hormone is involved in sleep regulation, as well as in a number of other cyclical bodily activities and circadian rhythm in humans. Melatonin is exclusively involved in signalling the 'time of day' and 'time of year' (hence considered to help both clock and calendar functions) to all tissues and is thus considered to be the body's chronological pacemaker or 'Zeitgeber'. The last decades melatonin has been used as a therapeutic chemical in a large spectrum of diseases, mainly in sleep disturbances and tumours and may play a role in the biologic regulation of mood, affective disorders, cardiovascular system, reproduction and aging. There are few papers regarding melatonin and its role in adolescent idiopathic scoliosis (AIS). Melatonin may play a role in the pathogenesis of scoliosis (neuroendocrine hypothesis) but at present, the data available cannot clearly support this hypothesis. Uncertainties and doubts still surround the role of melatonin in human physiology and pathophysiology and future research is needed.