From the Pineal Gland to the Central Clock in the Brain: Beginning of Studies of the Mammalian Biological Rhythms in the Institute of Physiology of the Czech Academy of Sciences.

The Institute of Physiology of the Czech Academy of Sciences (CAS) has been involved in the field of chronobiology, i.e., in research on temporal regulation of physiological processes, since 1970. The review describes the first 35 years of the research mostly on the effect of light and daylength, i.e., photoperiod, on entrainment or resetting of the pineal rhythm in melatonin production and of intrinsic rhythms in the central biological clock. This clock controls pineal and other circadian rhythms and is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. During the early chronobiological research, many original findings have been reported, e.g. on mechanisms of resetting of the pineal rhythm in melatonin production by short light pulses or by long exposures of animals to light at night, on modulation of the nocturnal melatonin production by the photoperiod or on the presence of high affinity melatonin binding sites in the SCN. The first evidence was given that the photoperiod modulates functional properties of the SCN and hence the SCN not only controls the daily programme of the organism but it may serve also as a calendar measuring the time of a year. During all the years, the chronobiological community has started to talk about "the Czech school of chronobiology". At present, the today´s Laboratory of Biological Rhythms of the Institute of Physiology CAS continues in the chronobiological research and the studies have been extended to the entire circadian timekeeping system in mammals with focus on its ontogenesis, entrainment mechanisms and circadian regulation of physiological functions. Key words: Pineal, Melatonin, AA-NAT rhythm, Light entrainment, Photoperiod, SCN clock.

Role of Melatonin in Viral, Bacterial and Parasitic Infections.

In all mammals, the circulating pool of MLTs is synthesized in the pineal gland during the night's darkness hours. Its main function is synchronizing the organism in the photoperiod. In contrast, extra-pineal MLT is synthesized in peripheral organs, does not follow any circadian rhythm or circulate, and plays a detoxifying and cytoprotective role. Circulating MLT may stimulate both innate and acquired immune responses through its circadian action and by activating high-affinity receptors on immunocompetent cells. Extra-pineal MLT may have antioxidant and anti-inflammatory effects that dampen the innate immune response. These two seemingly divergent roles may be considered to be two sides of the same coin. In fact, the integration of both circulating and extra-pineal MLT functions might generate a balanced and effective immune response against microbial pathogens. The studies described in this review investigated the effects of exogenous MLT in various models of infectious diseases using extremely different doses and treatment schedules. None of them evaluated the possibility of integrating the non-circadian anti-inflammatory effect with the circadian immunoenhancing action of MLT. As a consequence, in spite of the fact that most studies agree that MLT has a beneficial effect against infections, it seems difficult to draw any definite conclusion about its possible therapeutic use.

Contribution of the eye and of opn4xa function to circadian photoentrainment in the diurnal zebrafish.

The eye is instrumental for controlling circadian rhythms in mice and human. Here, we address the conservation of this function in the zebrafish, a diurnal vertebrate. Using lakritz (lak) mutant larvae, which lack retinal ganglion cells (RGCs), we show that while a functional eye contributes to masking, it is largely dispensable for the establishment of circadian rhythms of locomotor activity. Furthermore, the eye is dispensable for the induction of a phase delay following a pulse of white light at CT 16 but contributes to the induction of a phase advance upon a pulse of white light at CT21. Melanopsin photopigments are important mediators of photoentrainment, as shown in nocturnal mammals. One of the zebrafish melanopsin genes, opn4xa, is expressed in RGCs but also in photosensitive projection neurons in the pineal gland. Pineal opn4xa+ projection neurons function in a LIGHT ON manner in contrast to other projection neurons which function in a LIGHT OFF mode. We generated an opn4xa mutant in which the pineal LIGHT ON response is impaired. This mutation has no effect on masking and circadian rhythms of locomotor activity, or for the induction of phase shifts, but slightly modifies period length when larvae are subjected to constant light. Finally, analysis of opn4xa;lak double mutant larvae did not reveal redundancy between the function of the eye and opn4xa in the pineal for the control of phase shifts after light pulses. Our results support the idea that the eye is not the sole mediator of light influences on circadian rhythms of locomotor activity and highlight differences in the circadian system and photoentrainment of behaviour between different animal models.

The role of melatonin deficiency induced by pinealectomy on motor activity and anxiety responses in young adult, middle-aged and old rats.

BACKGROUND: Aging affects anxiety levels in rats while the pineal gland, via its hormone melatonin, could modulate their inherited life "clock." The present study aimed to explore the impact of plasma melatonin deficiency on anxiety responses and the possible involvement of the hypothalamic-pituitary-adrenocortical (HPA) axis and heat shock proteins (Hsp) 70 and 90 in the frontal cortex (FC) and the hippocampus in young adult, middle-aged and elderly rats with pinealectomy. RESULTS: Melatonin deficiency induced at different life stages did not affect the lifespan of rats. Pinealectomy abolished the circadian rhythm of motor activity, measured for 48 h in the actimeter, in young adult but not in middle-aged rats. Pinealectomy reduced the motor activity of the young adult rats during the dark phase and impaired the diurnal activity variations of old rats. The same generations (3- and 18 month-old rats with pinealectomy) had lower anxiety levels than the matched sham groups, measured in three tests: elevated-plus maze, light-dark test, and novelty-suppressed feeding test. While the activity of the HPA axis remained intact in young adult and middle-aged rats with melatonin deficiency, a high baseline corticosterone level and blunted stress-induced mechanism of its release were detected in the oldest rats. Age-associated reduced Hsp 70 and 90 levels in the FC but not in the hippocampus were detected. Pinealectomy diminished the expression of Hsp 70 in the FC of middle-aged rats compared to the matched sham rats. CONCLUSIONS: Our results suggest that while melatonin hormonal dysfunction impaired the motor activity in the actimeter and emotional behavior in young adult and elderly rats, the underlying pathogenic mechanism in these generations might be different and needs further verification.

The history of circadian rhythm research in Austria.

In view of the recent revival of interest in circadian biology and circadian epidemiology at the Medical University of Vienna, it seems appropriate to highlight the rich and pioneering history of circadian research in Austria. Among the forefathers of circadian research in Vienna are Otto Marburg (1874-1948), who discovered important elements of the pineal gland physiology, Robert Hofstätter (1883-1970), who used pineal gland extract in obstetrics/gynecology, and Paul Engel (1907-1997), who discovered that the pineal gland was controlled by light. More recently, Vera Lapin (1920-2007) showed that surgical removal of the pineal gland increased tumor growth, while Franz Waldhauser (*1946) investigated melatonin in conjunction with night work. Michael Kundi (*1950) and his team conducted among the first studies demonstrating differences in rhythms of night workers and early evidence for health impairments among them. Furthermore, Vienna-born Erhard Haus (1926-2013) pioneered the discovery of the role and importance of melatonin in relation to numerous diseases. This rich pioneering contribution of scientists in Vienna or with roots in Vienna is continued today by a new generation of chronobiologists, epidemiologists and clinicians in Vienna whose new insights contribute to the rapidly developing field of circadian rhythms research. Current topics and contributions relate to the impact of circadian rhythm disruption on health, and the application of chronotherapeutic approaches in clinical and preventive settings.

The Photoperiod-Driven Cyclical Secretion of Pineal Melatonin Regulates Seasonal Reproduction in Geese (Anser cygnoides).

The photoperiod is the predominant environmental factor that governs seasonal reproduction in animals; however, the underlying molecular regulatory mechanism has yet to be fully elucidated. Herein, Yangzhou geese (Anser cygnoides) were selected at the spring equinox (SE), summer solstice (SS), autumn equinox (AE), and winter solstice (WS), and the regulation of seasonal reproduction via the light-driven cyclical secretion of pineal melatonin was investigated. We show that there were seasonal variations in the laying rate and GSI, while the ovarian area decreased 1.5-fold from the SS to the AE. Moreover, not only did the weight and volume of the pineal gland increase with a shortened photoperiod, but the secretory activity was also enhanced. Notably, tissue distribution further revealed seasonal oscillations in melatonin receptors (Mtnrs) in the pineal gland and the hypothalamus-pituitary-gonadal (HPG) axis. The immunohistochemical staining indicated higher Mtnr levels due to the shortened photoperiod. Furthermore, the upregulation of aralkylamine N-acetyltransferase (Aanat) was observed from the SS to the AE, concurrently resulting in a downregulation of the gonadotrophin-releasing hormone (GnRH) and gonadotropins (GtHs). This trend was also evident in the secretion of hormones. These data indicate that melatonin secretion during specific seasons is indicative of alterations in the photoperiod, thereby allowing for insight into the neuroendocrine regulation of reproduction via an intrinsic molecular depiction of external photoperiodic variations.

Potential therapeutic role of melatonin in hepatobiliary diseases: current evidence and clinical observations.

Melatonin (MEL) is produced and secreted by the pineal gland as well as the small intestine, liver, retina, lymphocytes, and melanocytes in the skin in both experimental animals as well as in humans. While pineal and retinas MEL is closely related to the light/dark cycle, the production of MEL by other so called extrapineal tissues is independent of such circadian rhythm. Among the primary mechanisms of action of MEL in humans, the most important are interaction of MEL with specific receptors (M1, M2, M3) and the MEL 'scavenging' activity against the formation of free oxygen metabolites as a result of MEL's ability to transfer free electrons and stimulation of the expression of redox reaction enzymes. In addition, MEL binds to intracellular proteins such as calmodulin, thereby affecting the course of cell cycle, and it has been shown to activate of nuclear receptors belonging to the retinoid orphan receptors/retinoid Z receptors (ROR/RZR) subfamily. MEL exerts regulatory effects on the master clock regulating diurnal rhythms. This updated review presents current view on the synthesis and metabolism of MEL and the growing body of experimental evidence transferable to the practical medicine supporting a pleiotropic molecule beneficial effects on the health including protection against various organ abnormalities, including internal organs such as the liver. Although the beneficial effects of MEL in various types of liver damage have been well documented in experimental studies, there are relatively few studies on liver dysfunction in humans. Considering the worldwide obesity pandemic often associated with the occurrence of steatohepatitis and cirrhosis, the beneficial effects of MEL in liver pathology should be proven in randomized trials involving patients presenting with hepatic disorders.

Effect of Melatonin on the Content of CD3low and CD3hi T Cells in the Thymus of Mice with Functional Pinealectomy.

Continuous lighting for 14 days (functional pinealectomy model) leads to a decrease in the relative number of CD3low and CD3hi T lymphocytes and the CD3low/CD3hi ratio in the thymus of C57BL/6 mice. Intragastric administration of melatonin in physiological doses (1 mg/kg body weight, 14 days) against the background of functional pinealectomy restores the percentage of CD3low and CD3hi thymocytes and CD3low/CD3hi ratio to the control values. Hence, prolonged continuous illumination inhibits the differentiation and maturation of young thymocytes into mature forms, while melatonin treatment helps to compensate the effects of functional pinealectomy triggering cell proliferation in the thymus from the earliest stages of proliferation and differentiation of T cells. Thus, melatonin has immunotropic properties and can be used for correction of the consequences of functional pinealectomy.

Special Issue on "Pleiotropic Benefits of Melatonin: From Basic Mechanisms to Disease".

Melatonin (N-acetyl-5-methoxytryptamine) is a multifunctional hormone that is naturally produced from tryptophan and released rhythmically throughout the night by the pineal gland to regulate sleep-wake cycles [...].

Melatonin in Reproductive Medicine: A Promising Therapeutic Target?

Melatonin, mainly released from the pineal gland, also produced in the reproductive organs and cells, plays important roles in rhythms of the sleep-wake cycle, retardation of ageing processes, and antioxidant/anti-inflammatory functions. As a key mediator in reproductive systems, melatonin is participated in the reproductive process via regulating gamete and embryo development and influences reproductive diseases and pregnancy outcomes. The underlying mechanisms include epigenetic and other regulations, which are interesting for exploring new targets in the prevention and treatment of reproductive diseases. This review discusses the relationship between melatonin and reproductive functions and dysfunction, as well as potential clinical applications of melatonin in reproductive medicine. Notably, Developmental Origins of Health and Diseases (DOHaD) is closely linked to reproduction, this article is the first to review the new progress in studies on the possible relationship between melatonin and DOHaD.

Pinealectomy in Rats.

The pinealectomy technique consists of the surgical removal of the superficial pineal gland. This procedure allows the ablation of circulating indoles produced by this gland. Withdrawal of systemic melatonin, a pineal hormone, affects animal circadian rhythms and induces several physiological changes that are the subject of many investigations. In this chapter, we describe the pinealectomy protocol adapted to rats. We describe the animal placement on the stereotaxic fixation system, and the procedure for the pineal gland removal and animal recovery from surgery.

Physiologic intracranial calcifications incidentally detected on cone beam computed tomography.

OBJECTIVES: This study aims to determine the incidence of physiologic intracranial calcifications on cone beam computed tomography (CBCT) and to contribute to the differentiation between physiologic and pathologic intracranial calcifications by sharing their characteristic appearances, with the largest number of patients in the literature and many different types of physiologic calcifications. STUDY DESIGN: CBCT images of 996 patients admitted to the clinic between 2018 and 2019 were scanned retrospectively. Petroclinoid (PCL) and interclinoid ligaments (ICL), pineal gland, coronoid plexus, falx cerebri, tentorium cerebelli, and other dural calcifications were evaluated in each radiograph. RESULTS: Physiologic calcification was present in 49.4% of the cases, and pineal gland calcification was the most common (47.6%) type. PCL calcification was detected in 22.1%, and it was mostly bilateral partial. There were complete ICL in 1.2%, coronoid plexus in 11.3%, falx cerebri in 6.3%, tentorium cerebelli in 1.1%, and other dural calcifications in 3.6%. Only PCL calcifications differed between sexes and were more common in males. PCL, pineal gland, and coronoid plexus calcifications were most detected in the third decade of the cases. CONCLUSIONS: Intracranial calcifications are detected incidentally in CBCT examinations. Some calcifications may indicate pathologic conditions, and patients may need to be referred for further evaluation.

Impact of Melatonin Deficit on Emotional Status and Oxidative Stress-Induced Changes in Sphingomyelin and Cholesterol Level in Young Adult, Mature, and Aged Rats.

The pineal gland regulates the aging process via the hormone melatonin. The present report aims to evaluate the effect of pinealectomy (pin) on behavioral and oxidative stress-induced alterations in cholesterol and sphingomyelin (SM) levels in young adult, mature and aging rats. Sham and pin rats aged 3, 14 and 18 months were tested in behavioral tests for motor activity, anxiety, and depression. The ELISA test explored oxidative stress parameters and SM in the hippocampus, while total cholesterol was measured in serum via a commercial autoanalyzer. Mature and aged sham rats showed low motor activity and increased anxiety compared to the youngest rats. Pinealectomy affected emotional responses, induced depressive-like behavior, and elevated cholesterol levels in the youngest rats. However, removal of the pineal gland enhanced oxidative stress by diminishing antioxidant capacity and increasing the MDA level, and decreased SM level in the hippocampus of 14-month-old rats. Our findings suggest that young adult rats are vulnerable to emotional disturbance and changes in cholesterol levels resulting from melatonin deficiency. In contrast, mature rats with pinealectomy are exposed to an oxidative stress-induced decrease in SM levels in the hippocampus.

Metabolic footprint in young, middle-aged and elderly rats with melatonin deficit.

The pineal gland is suggested to be an essential area involved in the programming of fertility, growth, aging, and death of mammals via the released hormone melatonin.The present study aimed to ascertain the effect of melatonin deficit on several physiological and metabolic parameters, closely associated with the aging process, at certain stages of ontogenesis. Sham and rats with pinealectomy, operated at ages 3, 14, and 18-months, respectively, were tested two months later. Sham rats demonstrated an age-related decline of muscle strength, exercise endurance, motor activity, food intake, calorimetric parameters, and impaired lipid profile. Pinealectomy reduced the maximal time to exhaustion and body weight gain while diminished motor activity, food intake, O2 consumption, CO2 production, and energy expenditure during the Dark phase in the youngest rat group. In addition, melatonin deficit elevated arterial blood pressure (systolic, diastolic, and mean arterial pressure) and increased serum glucose and triglyceride level in 3-month-old rats while decreased the liver enzyme activity in 14-month-old rats. In conclusion, the present study brought new insights confirming the complex impact of melatonin deficit on important physiological, metabolic and biochemical markers related to aging and demonstrated for the first time that the lack of melatonin hormone is harmful in young adult rats.

Autism Spectrum Disorder patients may be susceptible to COVID-19 disease due to deficiency in melatonin.

Patients with Autism Spectrum Disorder (ASD) may be particularly prone to develop COVID-19. An unusual extended course of COVID-19 disease illness has been reported in one ASD patient and a group of patients have COVID-19 disease in a neurodevelopmental facility. It has been widely reported that many of those with ASD have substantial sleep disorders with low levels of melatonin and various genetic alterations related to melatonin production have been found. Several lines of evidence point to a substantial role of melatonin in the body's innate defense system including acting as a scavenger, an antioxidant and modulating the immune system. We therefore hypothesize that melatonin deficiency may predispose those ASD patients who have low melatonin output to COVID-19 disease. Potential implications for treatment are discussed.

Roles of melatonin in the teleost ovary: A review of the current status.

Melatonin, the neurohormone mainly synthesized in and secreted from the pineal gland of vertebrates following a circadian rhythm, is an important factor regulating various physiological processes, including reproduction. Recent data indicate that melatonin is also synthesized in the ovary and that it acts directly at the level of the ovary to modulate ovarian physiology. In some teleosts, melatonin is reported to affect ovarian steroidogenesis. The direct action of melatonin on the ovary could be a possible factor promoting oocyte maturation in teleosts. A role for melatonin in follicle rupture during ovulation in the teleost medaka has recently emerged. In addition, melatonin is suggested to affect oocyte maturation by its antioxidant activity. However, the molecular mechanisms underlying these direct effects of melatonin are largely unknown.

Metabolism of Melatonin Synthesis-Related Indoles in the Turkey Pineal Organ and Its Modification by Monochromatic Light.

The metabolism of pineal indoles is closely related to alterations in the light and dark phases of a daily cycle. Recent research showed important interspecies differences in the pineal biochemistry, and a strong impact of monochromatic light on many physiological processes in birds. Therefore, the aims of study were to characterize the metabolism of melatonin-synthesis indoles in the pineal organ of the domestic turkey, and to determine the changes occurring in this metabolism under the influence of different wavelengths and intensities of light. For this purpose, 3-week-old turkeys were kept under 16 lx white light, or under blue, green, and red light with intensities of 16, 32, and 64 lx during the photophase, and after 7 d were sacrificed at 4 h intervals. The activities of melatonin-synthesizing enzymes and the contents of indoles were measured in the same pineal organ. The results revealed that the activities of tryptophan hydroxylase and arylalkylamine N-acetyltransferase, and the levels of all tryptophan derivatives had significant daily changes in birds kept under each light condition used. The profile of pineal indole metabolism in 4-week-old turkeys was characterized by high-amplitude rhythms in the activity of arylalkylamine N-acetyltransferase and the contents of N-acetylserotonin and melatonin, equal relative amounts of serotonin and 5-hydroxyindoleacetic acid, and higher content of melatonin than N-acetylserotonin. The monochromatic light significantly modified the pineal indole metabolism, and its effects were dependent on the color and intensity of light. Pronounced changes occurred in the level of serotonin synthesis and the daily rhythm course of melatonin synthesis.

Thymus-Pineal Gland Axis: Revisiting Its Role in Human Life and Ageing.

For years the thymus gland (TG) and the pineal gland (PG) have been subject of increasingly in-depth studies, but only recently a link that can associate the activities of the two organs has been identified. Considering, on the one hand, the well-known immune activity of thymus and, on the other, the increasingly emerging immunological roles of circadian oscillators and the rhythmically secreted main pineal product, melatonin, many studies aimed to analyse the possible existence of an interaction between these two systems. Moreover, data confirmed that the immune system is functionally associated with the nervous and endocrine systems determining an integrated dynamic network. In addition, recent researches showed a similar, characteristic involution process both in TG and PG. Since the second half of the 20th century, evidence led to the definition of an effectively interacting thymus-pineal axis (TG-PG axis), but much has to be done. In this sense, the aim of this review is to summarize what is actually known about this topic, focusing on the impact of the TG-PG axis on human life and ageing. We would like to give more emphasis to the implications of this dynamical interaction in a possible therapeutic strategy for human health. Moreover, we focused on all the products of TG and PG in order to collect what is known about the role of peptides other than melatonin. The results available today are often unclear and not linear. These peptides have not been well studied and defined over the years. In this review we hope to awake the interest of the scientific community in them and in their future pharmacological applications.

Season dependent effects of urban environment on circadian clock of tree sparrow (Passer montanus).

Great efforts have been made recently to understand the effect(s) of urban environments on the circadian and seasonal physiology of wild animals, but the mechanisms involved remain largely unknown. Most laboratory studies and a few studies on animals in the wild suggest alterations occur in the physiological functions of organisms in urban habitats. Here, we addressed the effects of the interaction of seasons and urban environments on clock gene expression in three tissues of tree sparrows (Passer montanus). Tree sparrows (N = 30 per site per time of year) were procured from rural and urban habitats during periods corresponding to their three physiological states, i.e., June (longest photoperiod; reproductive phase), September (equinox photoperiod; refractory phase), and December (shortest photoperiod; sensitive phase). Birds (N = 5 per time per site per month) were sampled at six time points; ZT1, ZT5, ZT9, ZT13, ZT17, and ZT21 (ZT0 = sunrise time) and clock gene expression in the hypothalamus, pineal gland, and retina was studied. Our results show that there is persistence of the circadian clock in both rural and urban birds throughout the year. In urban birds Bmal1, Npas2, Per2, and Cry1 acrophases were advanced, compared to rural birds, while Clock acrophase was delayed, depending on the tissue and time of year. This difference could be because of changes in the availability, duration, and intensity of sunlight during different times of the year and/or differential photoreceptor sensitivities, differential physiological states, or a combination of all these factors. These important results reveal, for the first time in any species, season-dependent effects of an urban environment on the molecular machinery of the circadian clock.

Melatonin and Autophagy in Aging-Related Neurodegenerative Diseases.

With aging, the nervous system gradually undergoes degeneration. Increased oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, and cell death are considered to be common pathophysiological mechanisms of various neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), organophosphate-induced delayed neuropathy (OPIDN), and amyotrophic lateral sclerosis (ALS). Autophagy is a cellular basic metabolic process that degrades the aggregated or misfolded proteins and abnormal organelles in cells. The abnormal regulation of neuronal autophagy is accompanied by the accumulation and deposition of irregular proteins, leading to changes in neuron homeostasis and neurodegeneration. Autophagy exhibits both a protective mechanism and a damage pathway related to programmed cell death. Because of its "double-edged sword", autophagy plays an important role in neurological damage and NDDs including AD, PD, HD, OPIDN, and ALS. Melatonin is a neuroendocrine hormone mainly synthesized in the pineal gland and exhibits a wide range of biological functions, such as sleep control, regulating circadian rhythm, immune enhancement, metabolism regulation, antioxidant, anti-aging, and anti-tumor effects. It can prevent cell death, reduce inflammation, block calcium channels, etc. In this review, we briefly discuss the neuroprotective role of melatonin against various NDDs via regulating autophagy, which could be a new field for future translational research and clinical studies to discover preventive or therapeutic agents for many NDDs.