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New molecular targets for the geroprotective activity of AEDG (epitalon) and KE (vilon) peptides were verified via confocal laser scanning microscopy. It was shown that the aging of pineal and thymic cells in vitro led to a decrease in the staining of MitoTracker Red mitochondria and that there is a compensatory increase in the synthesis of L7A ribosomal protein. AEDG peptide increased the area of MitoTracker Red mitochondrial staining by 1.5 times and decreased by 22% the expression of ribosomal protein L7A in cultures of human pineal-gland cells with aging. KE peptide increased the area of MitoTracker Red mitochondrial staining by 1.5 times and decreased by 15% the expression of ribosomal protein L7A in cultures of human thymic cells with aging. The area of MitoTracker Red mitochondrial staining decreased and the compensatory expression of L7A ribosomal protein increased with aging of the pineal gland and thymic cells. Presumably, AEDG and KE peptides have a tissue-specific effect that normalizes the functions of mitochondria and ribosomes of pinealocytes and thymocytes.

Objective. To study the cellular composition of the human epiphysis. Materials and methods. An immunohistochemical method using cell-specific markers selectively detecting astroglial, endothelial, nerve, and mast cells was employed to study the epiphysis in seven males aged 16–68 years. Antibodies to glial fibrillary acidic protein (GFAP), vimentin, low molecular weight neurofilament proteins (clone 2F11), and mast cell tryptase were used. Results. Immunohistochemical reactions for GFAP in the human epiphysis identified a large number of astroglial processes but few astroglial cell bodies. Cells had relatively small primary processes which were significantly thicker than those of stellate astrocytes in other parts of the brain. Astrocyte processes formed dense plexuses around blood vessels and many concretions. Many cell processes in the stroma and particularly the parenchyma and blood vessel endothelial cells were vimentin-immunoreactive. Coexpression of GFAP and vimentin was not seen in any of the structures studied. Low molecular weight neurofilament proteins were detected in occasional pinealocytes and their processes. Mast cells immunohistochemically labeled for tryptase were detected in all the epiphyseal formations studied (most frequently in the stroma) Conclusions: 1) GFAP-immunopositive astrocytes in the human epiphysis differed in terms of their morphological features from typical stellate astrocytes in other parts of the brain, such that pineal GFAP-immunopositive astrocytes could be placed in a separate subgroup of astrocytes; 2) human epiphyseal astrocytes did not simultaneously contain GFAP and vimentin, in contrast to pineal astrocytes in other mammals; 3) mast cells are a consistent component of the human epiphysis – obligatory in the stroma and facultative in the parenchyma; 4) human epiphyseal pinealocytes express neuron-specific neurofilament protein, which is evidence supporting their neuron-like nature; 5) the localization of neuron-like endocrine cells and significant quantities of immunocompetent mast cells in the human epiphysis define this endocrine organ as an important component of the unified neuroimmunoendocrine system of the body.

Ethanol has chronobiological effects, which are associated with inhibition of melatonin synthesis and secretion and disruption of the sleep-wake cycle. Ethanol is known to cause sleep fragmentation due to frequent awakenings, prolong wakefulness, and reduce the duration of the slow sleep phase. At the same time, changes in the morphology of the pineal gland under chronic exposure to ethanol remain poorly studied. Of particular interest are changes in the basic morphometric parameters of pinealocytes, because they are a marker of the functional state of the pineal gland. The aim of the study was to morphometrically study the features of morphological changes in rat pinealocytes in the physiological norm and in chronic ethanol intoxication. To achieve this goal, we used 20 laboratory male rats: a control group and an experimental group. The control group was under standard vivarium conditions. For the experimental group, alcohol intoxication was modeled by injecting a 40% ethanol solution at the rate of 12 mg/kg of body weight intragastric 4 times a day. The morphometric parameters of pinealocytes were studied on day 30 from the start of the study. According to the results of morphometric measurements, a significant increase in the parameters of light pinealocytes and a decrease in the parameters of dark cells were established. It was determined that the average values of the cytoplasm area of light pinealocytes increase by 54.55% (p

The pineal gland, one of the three equivalent avian biological clock structures, is also the site of intensive neurosteroid synthesis (7α-hydroxypregnenolone and allopregnanolone). Pineal neurosteroid biosynthesis involves six enzymes: cytochrome P450 side-chain cleavage - Cyp11a1 encoded, cytochrome P4507α - Cyp7b1, 3β-hydroxysteroid dehydrogenase - Hsd3b2, 5α-reductase - Srd5a1, 3α-hydroxysteroid dehydrogenase - Akr1d1, and 5β-reductase - Srd5a3. Regulation of neurosteroid biosynthesis is not fully understood; although it is known that the E4BP4 transcription factor induces activation of biosynthetic cholesterol genes, which are the targets for SREBP (element-binding protein transcription factor). SREBP principal activity in the pineal gland is suppression and inhibition of the Period2 canonical clock gene, suggesting our hypothesis that genes encoding enzymes involved in neurosteroidogenesis are under circadian clock control and are the Clock Control Genes (CCGs). Therefore, through investigation of daily changes in Cyp11a1, Cyp7b1, Hsd3b2, Akr1d1, Srd5a1, and Srd5a3, pineal genes were tested in vivo and in vitro, in cultured pinealocytes. Experiments were carried out on pineal glands taken from 16-day-old chickens in vivo or using in vitro cultures of pinealocytes collected from 16-day-old animals. Both the birds in the in vivo experiments and the pinealocytes were kept under controlled light conditions (LD 12:12) or in constant darkness (DD). Subsequently, materials were prepared for RT-qPCR analysis. Results revealed that three of the six tested genes: Cyp11a1, Cyp7b1, and Srd5a3 demonstrated significant 24-hour variation in in vivo and in vitro. Findings of this study confirm that these genes could be under clock control and satisfy many of the requirements to be identified as CCGs.

A common, yet often neglectable, feature of neonatal hypoxic-ischemic brain damage (HIBD) is circadian rhythm disorders resulted from pineal gland dysfunction. Our previous work demonstrated that miRNAs play an important role in regulating key circadian genes in the pineal gland post HIBD [5,21]. In current study, we sought out to extend our investigation by profiling expression changes of pineal long non-coding RNAs (lncRNAs) upon neonatal HIBD using RNA-Seq. After validating lncRNA changes, we showed that one lncRNA: TCONS_00044595 is highly enriched in the pineal gland and exhibits a circadian expression pattern. Next, we performed bioinformatic analysis to predict the lncRNA-miRNA regulatory network and identified 168 miRNAs that potentially targetlncRNA TCONS_00044595. We further validated the bona fide interaction between one candidate miRNA: miR-182, a known factor to regulate pineal Clock expression, and lncRNA TCONS_00044595. Finally, we showed that suppression of lncRNA TCONS_00044595 alleviated the CLOCK activation both in the cultured pinealocytes under OGD conditions and in the pineal gland post HIBD in vivo. Our study thus shed light into novel mechanisms of pathophysiology of pineal dysfunction post neonatal HIBD.

Mazur Karol, Machaj Dominik, Mazur Dominika, Asztabska Aneta, Płaczek Alicja. The use of melatonin in the treatment of jet lag– clinical review. Journal of Education, Health and Sport. 2020;10(5):175-179. eISSN 2391-8306. DOIhttp://dx.doi.org/10.12775/JEHS.2020.10.05.018 https://apcz.umk.pl/czasopisma/index.php/JEHS/article/view/JEHS.2020.10.05.018 https://zenodo.org/record/3856458 The journal has had 5 points in Ministry of Science and Higher Education parametric evaluation. § 8. 2) and § 12. 1. 2) 22.02.2019. © The Authors 2020; This article is published with open access at Licensee Open Journal Systems of Nicolaus Copernicus University in Torun, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author (s) and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non commercial license Share alike. (http://creativecommons.org/licenses/by-nc-sa/4.0/) which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 05.05.2020. Revised: 10.05.2020. Accepted: 26.05.2020. The use of melatonin in the treatment of jet lag – clinical review Karol Mazur, Dominik Machaj, Dominika Mazur, Aneta Asztabska, Alicja Płaczek Karol Mazur, mazurkarol79@gmail.com, Faculty of Medicine, Medical University of Lublin, Chodźki Street 19, 20-093 Lublin, Poland Dominik Machaj, dominik5a4@tlen.pl, Faculty of Medicine, Medical University of Lublin, Chodźki Street 19, 20-093 Lublin, Poland Dominika Mazur, dominika.hul20@gmail.com, Medical Faculty, University of Rzeszow, Pigonia Street 6, 35-310 Rzeszow, Poland Aneta Asztabska, aneta.asztabska@gmail.com, Medical Faculty, University of Rzeszow, Pigonia Street 6, 35-310 Rzeszow, Poland Alicja Płaczek, alicja60@poczta.onet.pl, Medical Faculty, University of Rzeszow, Pigonia Street 6, 35-310 Rzeszow, Poland Summary: Jet lag is a circadian rhythm disorder, that occurs as a result of air travel across multiple time zones. Jet lag symptoms include: anxiety, constipation, diarrhea, confusion, dehydration, headache, irritability, nausea, indigestion, difficulty concentrating, sweating, coordination problems, dizziness, daytime sleepiness, malaise, and memory loss. Melatonin, is a hormone that regulates the circadian rhythm by acting on MT1 and MT2 melatonin receptors. It is produced by pinealocytes in the pineal gland and released directly into the blood. Main therapeutic application of this medication is re-entraining disturbed circadian rhythms. All the mentioned studies proved that melatonin is effective in the treatment of jet lag. None of the mentioned above trials didn’t report any serious side effects caused by this medication therefore occasional short-term use of melatonin seems to be safe.Most people should start treatment of jet lag with taking 2-3 mg melatonin, and if necessary increase the dose to 5 mg.Melatonin is the most effective in alleviating symptoms of jet lag when it is given at bedtime on the day of the flight and in the next few days after arrival.Further research is needed to find out the optimal dosage of melatonin for treatment of jet lag, timing of its administration, time of initiation and duration of the treatment or possible side effects of this medication. Key words: melatonin, jet lag, circadian rhythm

The pineal gland is known to play an important role in adaptogenesis. The hypothalamus is one of the main links of the stress-reactive system; it participates in the regulation of the involution of the whole body. Thus, the study of changes in these organs under stress and during aging is of considerable interest. The goal of this work is to study the apoptosis mechanisms of pinealocytes and neurosecretory cells of the hypothalamic suprachiasmatic nucleus in aging, under stress, and during pharmacological correction of involutional processes and stress response (antioxidant α-tocopherol acetate, immunomodulator Cycloferon). Young (10–12 months) and old (24–30 months) Wistar rats were used as the model organism. Age-related features of the dynamics of apoptosis of pinealocytes and neurosecretory cells of the hypothalamic suprachiasmatic nucleus were studied via TUNEL and immunohistochemistry, and the potential for the pharmacological correction of apoptotic processes were determined. An age-dependent increase in the level of apoptosis of cells of the suprachiasmatic nucleus and pineal gland in rats was revealed. Stress (immobilization) led to increased cell death, which was more significant in older animals. This indicates that the pineal gland and suprachiasmatic nucleus, traditionally regarded as regulators of circadian rhythms, are also actively involved in general adaptation processes. The studied drugs (α-tocopherol-acetate, Cycloferon, and their combination) have a pronounced antiapoptotic, cytoprotective effect under physiological conditions during aging and under nonspecific emotional stress (immobilization) in young and old animals. The regulatory effect is implemented via activation of the expression of the antiapoptotic protein Bcl-2 in neurosecretory cells of the suprachiasmatic nucleus and pinealocytes.

In modern society increase of digitalization associated with grown exceed level of light at night – a new type of pollution. Presence of light at night inhibited endogenous melatonin synthesis by pineal gland, that influence on circadian system work cycles, so organism oftenbroken regime of wake/sleep, meals, physical activity. Also, a lack of melatonin in some certain time of dayand low melatonin concentration both, were shown take some intervention in diseases development through incorrect regulation of clock-depended genes expression. In connect with this, some latest clinical protocol in therapy or clinical trials of many different pathologies (for example, insomnia, metabolic syndrome, cardiovascular diseases, central nervous and immune system trouble, cancer, viral infection, etc.) include exogenous melatonin usage. As melatonin perform his function via endocrine and paracrine ways in variety types of cell, his application take place in wide range of doses and in different time of day (chronotherapeutic approach). Therefore, important to control state of circadian system central elements – pineal gland (main producer of endogenous melatonin) and suprachiasmatic nucleus (SCN) of hypothalamus (central pacemaker of circadian rhythm) in conditionsof exogenous melatonin treatment. Thus, the main goal of our research were analysis of rats pineal gland and hypothalamic SCN morpho-functional state after different time (morning, evening and continuously with drinking water) melatonin daily administration. Melatonin was administered by gavage for 7 weeks in dose 30 mg/kg 1 h before lights-off (M ZT11, evening), or 1 h after lights-on (M ZT01, morning), or continuously with drinking water during day-night period (MW). After melatonin use only in MW group pineal gland demonstrates changes in morphology (pinealocytes nucleus had mild basophilic color) and morphometric (increased cross-sectional area of the pinealocytes nucleus in compare with control group) analysis data. Besides, some similar changes were observed in SCN: the cross-sectional area of the SCN neurons nucleus grown in case of usage each of regime melatonin administration, while morphology characteristic remains without any alteration. In general, it suggesting about having by melatonin non-inhibiting features in context of circadian system feedback loop and supposing wide potential for melatonin use with absent huge side effect on central elements of above mentioned system.

In the current study, the prolonged light exposure at night on the function of pineal gland, melatonin production, pro-inflammatory response and progress of pregnancy in pregnant rats were investigated. A long term (entire gestation stage) of 24 h light exposure not only modify the morphologies of pinealocytes by decreasing their nucleus/cytoplasm ratio and mitochondrial numbers but also reduce the level of circulating melatonin. The biological consequences for this constant light exposure in pregnant rats were the elevated the pro-inflammatory response indicated by the increased production of IL-6 and finally, the termination of pregnancy compared to their controlled counterparts under the normal light/dark cycle. The result showed that the pregnancy was terminated at the early stage of embryo development. The report, for the first time, established a potential association among the pineal function, pro-inflammatory reaction and pregnant progress under the influence of light exposure. This observation has a high relevant to the rise in human infertility since humans have overexposed to the light at night with the increased light pollution globally.

The pineal gland is a naturally calcifying endocrine organ which secretes the sleep-promoting hormone melatonin. Age-related changes of the pineal have been observed, including decreased pinealocyte numbers, increased calcification, and a reduction in melatonin production. Since fluoride is attracted to calcium within the pineal gland, this study sought to examine the effects of a fluoride-free diet on the morphology of the pineal gland of aged male rats (26 months old). All animals had previously been raised on standard fluoridated food and drinking water. These control animals were compared to other animals that were placed on a fluoride-free diet ("fluoride flush") for 4 or 8 weeks. At 4 weeks, pineal glands from fluoride-free animals showed a 96% increase in supporting cell numbers and at 8 weeks a 73% increase in the number of pinealocytes compared to control animals. In contrast, the number of pinealocytes and supporting cells in animals given an initial 4-week fluoride flush followed by a return to fluoridated drinking water (1.2 ppm NaF) for 4 weeks were not different from control animals. Our findings therefore demonstrate that a fluoride-free diet encouraged pinealocyte proliferation and pineal gland growth in aged animals and fluoride treatment inhibited gland growth. These findings suggest that dietary fluoride may be detrimental to the pineal gland.

This study investigated whether and how fetal malnutrition would influence endogenous melatonin synthesis, and whether such effect of fetal malnutrition would transmit to the next generation. We enrolled 2466 participants and 1313 of their offspring. The urine 6-hydroxymelatonin sulfate and serum melatonin rhythm were measured. Methylation microarray detection and bioinformatics analysis were performed to identify hub methylated sites. Additionally, rat experiment was performed to elucidate mechanisms. The participants with fetal malnutrition had lower 6-hydroxymelatonin sulfate (16.59 ± 10.12 μg/24 hours vs 24.29 ± 11.99 μg/24 hours, P .001) and arear under curve of melatonin rhythm (67.11 ± 8.16 pg/mL vs 77.11 ± 8.04 pg/mL, P .001). We identified 961 differentially methylated sites, in which the hub methylated sites were locating on protein kinase C alpha (PRKCA) and cAMP response element-binding protein (CREB1) promoters, mediating the association of fetal malnutrition with impaired melatonin secretion. However, such effects were not observed in the offspring (all P .05). Impaired histomorphology of pineal, decreased melatonin in serum, pineal, and pinealocyte were also found in the in vivo and in vitro experiments (P .05 for the differences of the indicators). Hypermethylation of 10 CpG sites on the PRKCA promoter and 8 CpG sites on the CREB1 promoter were identified (all P .05), which down-regulated PRKCA and CREB1 expressions, leading to decreased expression of AANAT, and then resulting in the impaired melatonin synthesis. Collectively, fetal malnutrition can impair melatonin synthesis through hypermethylation of PRKCA and CREB1 promoters, and such effects cannot be transmitted to the next generation.

Melatonin is a hormone majorly secreted by the pineal gland and contributes to a various type of physiological functions in mammals. The melatonin production is tightly limited to the AANAT level, yet the most known molecular mechanisms underlying AANAT gene transcription is limited in the pinealocyte. Here, we find that c-Fos and cAMP-response element-binding protein (CREB) decreases and increases the AANAT transcriptional activity in renal tubular epithelial cell, respectively. Notably, c-Fos knockdown significantly upregulates melatonin levels in renal tubular cells. Functional results indicate that AANAT expression is decreased by c-Fos and resulted in enhancement of cell damage in albumin-injury cell model. We further find an inverse correlation between c-Fos and AANAT levels in renal tubular cells from experimental membranous nephropathy (MN) samples and clinical MN specimens. Our finding provides the molecular basis of c-Fos in transcriptionally downregulating expression of AANAT and melatonin, and elucidate the protective role of AANAT in preventing renal tubular cells death in albumin-injury cell model and MN progression.

The pineal gland secretes melatonin principally at night. Regulated by norepinephrine released from sympathetic nerve terminals, adrenergic receptors on pinealocytes activate aralkylamine N-acetyltransferase that converts 5-hydroxytryptamine (5-HT, serotonin) to N-acetylserotonin, the precursor of melatonin. Previous studies from our group and others reveal significant constitutive secretion of 5-HT from pinealocytes. Here, using mass spectrometry, we demonstrated that the 5-HT is secreted primarily via a decynium-22–sensitive equilibrative plasma membrane monoamine transporter instead of by typical exocytotic quantal secretion. Activation of the endogenous 5-HT receptors on pinealocytes evoked an intracellular Ca(2+) rise that was blocked by RS-102221, an antagonist of 5-HT(2C) receptors. Applied 5-HT did not evoke melatonin secretion by itself, but it did potentiate melatonin secretion evoked by submaximal norepinephrine. In addition, RS-102221 reduced the norepinephrine-induced melatonin secretion in strips of pineal gland, even when no exogenous 5-HT was added, suggesting that the 5-HT that is constitutively released from pinealocytes accumulates enough in the tissue to act as an autocrine feedback signal sensitizing melatonin release.

International audience; Melatonin (MLT) is a biological modulator of circadian and seasonal rhythms and reproduction. The photoperiodic information is detected by retinal photoreceptors and transmitted through nerve transmissions to the pineal gland, where MLT is synthesized and secreted at night into the blood. MLT interacts with two G protein-coupled receptors, MT 1 and MT 2 . The aim of our work was to provide evidence for the presence of MLT receptors in the ovine pineal gland and define their involvement on melatonin secretion. For the first time, we identified the expression of MLT receptors with the specific 2-[ 125 I]-MLT agonistic radioligand in ovin pinealocytes. The values of Kd and Bmax are 2.24 ± 1.1 nM and 20 ± 6.8 fmol/mg. MLT receptors are functional and inhibit cAMP production and activate ERK1/2 through pertussis toxin-sensitive G i/o proteins. The MLT receptor antagonist/ inverse agonist luzindole increased cAMP production (189 ± 30%) and MLT secretion (866 ± 13%). The effect of luzindole on MLT secretion was additive with the effect of well-described activators of this pathway such as the β-adrenergic agonist isoproterenol and the α-adrenergic agonist phenylephrine. Co-incubation of all three compounds increased MLT secretion by 1236 ± 199%. These results suggest that MLT receptors are involved in the negative regulation of the synthesis of its own ligand in pinealocytes. While adrenergic receptors promote MLT secretion, MLT receptors mitigate this effect to limit the quantity of MLT secreted by the pineal gland.

Melatonin is the primary circadian output signal from the brain and is mainly synthesized in pinealocytes. The rhythm and secretion of melatonin are under the control of an endogenous oscillator located in the SCN or the master biological clock. Disruptions in circadian rhythms by shift work, aging, or light at night are associated with bone loss and increased fracture risk. Restoration of nocturnal melatonin peaks to normal levels or therapeutic levels through timed melatonin supplementation has been demonstrated to provide bone-protective actions in various models. Melatonin is a unique molecule with diverse molecular actions targeting melatonin receptors located on the plasma membrane or mitochondria or acting independently of receptors through its actions as an antioxidant or free radical scavenger to stimulate osteoblastogenesis, inhibit osteoclastogenesis, and improve bone density. Its additional actions on entraining circadian rhythms and improving quality of life in an aging population coupled with its safety profile make it an ideal therapeutic candidate for protecting against bone loss in susceptible populations. The intent of this review is to provide a focused discussion on bone loss and disorders of the bone as it relates to melatonin and conditions that modify melatonin levels with the hope that future therapies include those that include melatonin and correct those factors that modify melatonin levels like circadian disruption.

Homeobox genes encode transcription factors controlling development; however, a number of homeobox genes are expressed postnatally specifically in melatonin-producing pinealocytes of the pineal gland and photoreceptors of the retina along with transcripts devoted to melatonin synthesis and phototransduction. Homeobox genes regulate melatonin synthesis in pinealocytes, but some homeobox genes also seem to be involved in regulation of retinal phototransduction. Due to the lack of photoreceptor models, we here introduce the rat pinealocyte culture as an in vitro model for studying retinal phototransduction. Systematic qPCR analyses were performed on the rat retina and pineal gland in 24 hour in vivo series and on primary cultures of rat pinealocytes: All homeobox genes and melatonin synthesis components, as well as nine out of ten phototransduction genes, were readily detectable in all three experimental settings, confirming molecular similarity between cultured pinealocytes and in vivo retinal tissue. 24 hours circadian expression was mostly confined to transcripts in the pineal gland, including a novel rhythm in arrestin (Sag). Individual knockdown of the homeobox genes orthodenticle homeobox 2 (Otx2), cone-rod homeobox (Crx) and LIM homeobox 4 (Lhx4) in pinealocyte culture using siRNA resulted in specific downregulation of transcripts representing all levels of phototransduction; thus, all phototransduction genes studied in culture were affected by one or several siRNA treatments. Histological colocalization of homeobox and phototransduction transcripts in the rat retinal photoreceptor was confirmed by RNAscope in situ hybridization, thus suggesting that homeobox gene-encoded transcription factors control postnatal expression of phototransduction genes in the retinal photoreceptor.

Melatonin is a highly conserved molecule that regulates day/night rhythms; it is associated with sleep improvement, reactive oxygen species (ROS) scavenging, anti-aging effects, and seasonal and circadian rhythms and has been a hot topic of research for decades. Using single-cell RNA sequencing, a recent study describes a single-cell transcriptome atlas for the rat pineal gland. Based on a more comprehensive analysis of the retrieved data (Mays et al., PLoS One, 2018, 13, e0205883), results from the current study unveiled the underappreciated gene regulatory network behind different cell populations in the pineal gland. More importantly, our study here characterized, for the first time, the day/night activation of autophagy flux in the rat pineal gland, indicating a potential role of autophagy in regulating melatonin synthesis in the rat pineal gland. These findings emphasized a hypothetical role of day/night autophagy in linking the biological clock with melatonin synthesis. Furthermore, ultrastructure analysis of pinealocytes provided fascinating insights into differences in their intracellular structure between daytime and nighttime. In addition, we also provide a preliminary description of cell-cell communication in the rat pineal gland. In summary, the current study unveils the day/night regulation of autophagy in the rat pineal gland, raising a potential role of autophagy in day/night-regulated melatonin synthesis.

Background and objectives: The pineal gland is a photoneuroendocrine organ in the midline of the brain, responsible primarily for melatonin synthesis. It is composed mainly of pinealocytes and glial tissue. This study examined human postmortem pineal glands to microscopically assess age-related changes using digital techniques, and offers a perspective on evolutionary tendencies compared to the past. Materials and Methods: A retrospective autopsy study has been performed on 72 pediatric and adult autopsy cases. The glands have been processed for histological analysis and immunohistochemical staining with glial fibrillary acidic protein (GFAP). Slides were assessed under polarized light and digitally scanned. Morphometric data were obtained using CaseViewer and ImageJ. Results: Thirty-three females and 39 males were included in the study, grouped under three age groups: 0–25, 46–65, and 66–96 years of age. The peak gland volume was found within the 46–65 age group, the overall mean volume was 519 mm3, the main architectural types were lobular and insular, and the mean percentage of pineal calcification was 15% of the gland, peaking within the 66–96 age group, with a predominantly globular shape. Glial cysts were found in 20.8% of cases. The intensity of GFAP stain was maximal in the pediatric age group, but the extent of glial tissue was much larger in elderly patients. Discussion: The degenerative process of the pineal gland can be quantified by measuring normal parenchyma, calcifications, glial tissue, and glial cysts. Morphometric differences have been observed and compared to a similar studies performed in the published literature. The current study, unfortunately, lacks a 26–45 age group. Digital techniques seemed to offer a more exact analysis, but returned similar results to studies performed over 40 years ago, therefore offering important information on evolutionary tendencies. Conclusions: Increase in glial tissue, calcifications, and glial cysts have a defining role as age-related changes in the pineal gland.

Pineal gland is one of the most important components of homeostasis - the supporting system of the body. It participates in the launch of stress responses, restriction of their development, prevention of adverse effects on the body. There was proved an impact of electromagnetic radiation on the epiphysis. However, morphological changes in the epiphysis under exposure to electromagnetic radiation of modern communication devices are studied not sufficiently. For the time present the population is daily exposed to electromagnetic radiation, including local irradiation on the brain. These date determined the task of this research - the study of the structure of rat pineal gland under the exposure to electromagnetic radiation from personal computers and mobile phones. These date determined the task of this research - the study of the structure of rat pineal gland under the exposure to electromagnetic radiation from personal computers and mobile phones. Performed transmission electron microscopy revealed signs of degeneration of dark and light pinealocytes. These signs were manifested in the development of a complex of general and specific morphological changes. There was revealed the appearance of signs of aging and depletion transmission electron microscopy both in light and dark pinealocytes. These signs were manifested in the accumulation of lipofuscin granules and electron-dense "brain sand", the disappearance of nucleoli, cytoplasm vacuolization and mitochondrial cristae enlightenment.

The suprachiasmatic nucleus (SCN) of the hypothalamus and the pineal gland (epiphysis) play an important role not only in the regulation of circadian rhythms but also in the implementation of adaptive responses, including those to various stress factors. Age-related morphofunctional changes in these brain structures, including those associated with increased oxidative stress, exert a significant effect on the organism as a whole. The aim of this work was to explore the dynamics and mechanism of apoptosis in pinealocytes and SCN neurosecretory cells as well as to determine the possibilities of pharmacological correcting this process by an antioxidant alpha-tocopherol and an immunomodulator cycloferon under physiological and immobilization stress conditions in young (2–4-month-old) and aged (30-month-old) Wistar rats. The preparations were administered perorally once a day for 14 days. While the apoptosis level increased with age both in the SCN and the pineal gland, administration of alpha-tocopherol, cycloferon and their combination led to abate this process. It was shown that stress-induced apoptosis in the SCN and the pineal gland proceeds via the p53-dependent pathway, while administration of alpha-tocopherol acetate, cycloferon and their combination decreases the apoptosis level in pinealocytes, suppressing p53 expression both in young and aged animals. In the SCN, no relationship was found between apoptosis and p53 expression levels after administration of the above preparations during stress, suggesting the involvement of different mechanisms.

Our previous study showed that the turkey pineal organ, in contrast to that of the chicken, is characterized by a follicular structure throughout the entire period of post-hatching life. Despite the preservation of the follicular organization, the histological structure of the pineal follicles in turkeys changes prominently with age. The present research was performed to investigate the cellular composition and organization of the follicle wall as well as the ultrastructure of parenchymal cells in the turkey pineal organ during the period of post-hatching development. Pineal organs were collected from female turkeys at 2 days, 2 weeks, 4 weeks, 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 56 weeks post-hatching. The organs were prepared for immunocytochemical studies using antibodies against N-acetylserotonin O-methyltransferase (ASMT), glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) and for ultrastructural examination. The results showed that regardless of age, the pineal follicle was formed by ASMT-immunopositive cells, among which rudimentary photoreceptor and secretory pinealocytes were identified. The second component of the follicle wall consisted of GFAP-immunopositive cells, as represented by ependymal-like and astrocyte-like cells. Rudimentary photoreceptor pinealocytes and ependymal-like cells formed the inner part of the follicle wall, while secretory pinealocytes and astrocyte-like cells created the outer part. Three forms of the pineal follicle structure characteristic of young (two days to ten weeks), young adult (20-30 weeks) and adult (40-56 weeks) turkeys were distinguished. These forms primarily differed in the relative dimensions of the inner and outer parts of the follicle wall. Ultrastructural studies showed prominent changes in the organization of rudimentary receptor pinealocytes during the investigated period of life. These cells developed until the age of 20 weeks, at which time they appeared as strongly elongated cells with a stratified, highly regular distribution of organelles. In adult turkeys, rudimentary receptor pinealocytes showed pronounced regressive changes; however, we never observed their transformation into cells of the secretory type. Secretory pinealocytes increased in number and size during the post-hatching period, which was especially pronounced after 20 weeks of age. The most prominent changes in the supporting cells included the intensification of GFAP-immunoreactivity due to the accumulation of filaments in the cytoplasm and the development of astrocyte-like cells. The increase in the number of secretory pinealocytes and astrocyte-like supporting cells resulted in the formation of two distinct parts of the follicle wall in the pineal organs of young adult and adult turkeys.

A local renin-angiotensin system (RAS) has been postulated in the pineal gland. In addition to angiotensin II (Ang II), other active metabolites have been described. In this study, we aimed to investigate a role for Ang IV in melatonin synthesis and the presence of its proposed (IRAP)/AT4 receptor (insulin-regulated aminopeptidase) in the pineal gland. The effect of Ang IV on melatonin synthesis was investigated in vitro using isolated pinealocytes. IRAP protein expression and activity were evaluated by Western blot and fluorimetry using Leu-4Me-β-naphthylamide as a substrate. Melatonin was analyzed by HPLC, calcium content by confocal microscopy and cAMP by immunoassay. Ang IV significantly augmented the NE-induced melatonin synthesis to a similar degree as that achieved by Ang II. This Ang IV effect in pinealocytes appears to be mediated by an increase in the intracellular calcium content but not by cAMP. The (IRAP)/AT4 expression and activity were identified in the pineal gland, which were significantly higher in membrane fractions than in soluble fractions. Ang IV significantly reduced IRAP activity in the pineal membrane fractions. The main findings of the present study are as follows: (1) Ang IV potentiates NE-stimulated melatonin production in pinealocytes, (2) the (IRAP)/AT4 receptor is present in the rat pineal gland, and (3) Ang IV inhibits IRAP activity and increases pinealocytes [Ca2+]i. We conclude that Ang IV is an important component of RAS and modulates melatonin synthesis in the rat pineal gland.

Melatonin, an indoleamine hormone produced and secreted at night by pinealocytes and extra-pineal cells, plays an important role in timing circadian rhythms (24-h internal clock) and regulating the sleep/wake cycle in humans. However, in recent years melatonin has gained much attention mainly because of its demonstrated powerful lipophilic antioxidant and free radical scavenging action. Melatonin has been proven to be twice as active as vitamin E, believed to be the most effective lipophilic antioxidant. Melatonin-induced signal transduction through melatonin receptors promotes the expression of antioxidant enzymes as well as inflammation-related genes. Melatonin also exerts an immunomodulatory action through the stimulation of high-affinity receptors expressed in immunocompetent cells. Here, we reviewed the efficacy, safety and side effects of melatonin supplementation in treating oxidative stress- and/or inflammation-related disorders, such as obesity, cardiovascular diseases, immune disorders, infectious diseases, cancer, neurodegenerative diseases, as well as osteoporosis and infertility.

It is well established that the adult mammalian pineal body (PB), with the exception of rodents, contains nerve cell bodies. Based on our previous results we have proposed that there is a pinealo-to-retinal neuronal connection in adult hamsters and in prebubertal rats. By the time the animals reached puberty, labeled cells in the PB were not observed in rats. In the present experiment, we provide light and electron microscopic immunohistochemical evidence that the labeled cells in the PB of prepubertal rats are neurons. Pinealocytes cannot transport neurotropic viruses. Virus labeled cells do not show S-antigen immunoreactivity typical for pinealocytes of six-day-old rats. Electron microscopic investigation confirmed the neuronal nature of virus labeled cells. These neurons, similarly to that of hamsters, also establish pinealo-to-retinal connections in prepubertal rats.

Comprehensive reviews dealing with the embryology, morphology, vascularization, innervation, and cytology of avian pineal glands are available. Among species of birds, the morphology and cytology of the pineal gland are remarkably varied. According to Collin, the unique cell type in the avian pineal, the secretory pinealocyte, is evolutionarily derived from a cone-like receptor cell found among some reptiles and anamniotes. The rhythmic variation in melatonin content of the pineal gland of chickens persists as a free-running, circadian rhythm for at least 2 weeks in continuous darkness, but it disappears within 2 weeks in constant light. The autotransplanted pineal gland of chickens apparently released greater amounts of melatonin into the blood at night than during the day, since by bioassay melatonin could be detected there only when sampled at night. The injury occasioned by the surgical removal of the pineal gland can affect the measured response in various ways, including increasing the opacity of the skull and underlying tissues.

The pineal gland (glandula pinealis) is neuroendocrine gland located at the epithalamus of the brain secreting melatonin. The aim of this study was to explore effects of prenatal hypoxia in rats at the age of 33 weeks on the occurrence of pineal gland calcification. Distribution and chemical composition of calcerous material by light, scanning and transmission electron microscopy was investigated. Melatonin concentrations in blood plasma by direct radioimmunoassay were measured. Rats were exposed to prenatal hypoxia for 12 h at day 20 of development and second group to prenatal hypoxia for 2x8 h at days 19 and 20 of development. Vacuoles of intracellular edema in the pineal samples after 12 h hypoxia were found. Their size ranges up to 30 µm. Some of them were filled with the flocculent and fibrous material. Samples of pineal glands after 2 x 8 h hypoxia revealed the pericellular edema of pinealocytes. The amount of calcium rich particles in 2 x 8 h hypoxia group was lower than in 12 h hypoxia group. Plasma melatonin levels did not differ between control and both hypoxia groups. We concluded that calcification is a process induced by osteoblasts and osteocytes with melatonin as a promotor and it is favored under hypoxic conditions.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

Motor-, behavior- and/or cognition-related symptoms are key hallmarks in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 pathology (FTLD-TDP), respectively. It has been reported that these patients also experience sleep disturbances, which might implicate a disturbed circadian rhythm of the sleep/wake cycle. It remains unknown, however, whether cells involved in the circadian sleep/wake cycle are affected by ALS- and FTLD-related neuropathological changes including phosphorylated TDP-43 (pTDP-43) aggregates and dipeptide repeat protein (DPR) inclusions resulting from the C9orf72 hexanucleotide repeat expansion. Immunohistochemistry for DPR and pTDP-43 pathology was performed in post-mortem hypothalamus and pineal gland tissue of patients with ALS and/or FTLD-TDP with and without the C9orf72 repeat expansion and healthy controls. Circadian sleep/wake-associated cells, including pinealocytes and hypothalamic neurons related to the suprachiasmatic nucleus (SCN), were microscopically assessed. We observed numerous DPR inclusions (poly(GA), poly(GP), poly(GR) and poly(PR)) in the pinealocytes and few poly(GA) inclusions in the SCN-related neurons in C9orf72-related ALS and/or FTLD-TDP cases. These circadian sleep/wake-associated cells, however, were devoid of pTDP-43 pathology both in C9orf72- and nonC9orf72-related ALS and/or FTLD-TDP cases. Our neuropathological findings show that pinealocytes and, to a lesser extent, SCN-related neurons are affected by DPR pathology. This may reflect an involvement of these cells in sleep/wake disturbances observed in ALS and/or FTLD-TDP patients.

The pineal gland structure and ultrastructure in the Northern (Blarina brevicauda) and Southern short-tailed shrew (Blarina carolinensis) are described by light and electron microscopy. Results observed were similar to other mammals of Insectivora described previously, specifically, the hedgehog (Erinaceus europaeus) and the Old World mole (Talpa europea). Two different types of pinealocytes were noticed by electron microscopy, in addition to relatively few glial cells. Granular vesicles were not noticed in abundance. The granular endoplasmic reticulum was observed and studded with vesicles. The golgi apparatus was well developed and appeared often. Synaptic ribbons were observed in several different formations consisting of ribbons and/or rods. The ciliary derivative, the rudimentary photoreceptor structures found in the pinealocytes of population I, was noticed in a 9 + 0 tubular pattern. Within these semifossorial shrews, the relationship between specific intracellular organelles and their function was discussed.

The avian circadian system is a complex of mutually coupled pacemakers residing in pineal gland, retina and suprachiasmatic nucleus. In this study, the self-regulation mechanism of pineal circadian rhythm was investigated by culturing chick primary pinealocytes exposed to red light (RL), green light (GL), blue light (BL), white light (WL) and constant darkness (DD), respectively. All illuminations were set up with a photoperiod of 12 light: 12 dark. The 24-h expression profiles of seven core clock genes (cBmal1/2, cClock, cCry1/2 and cPer2/3), cAanat and melatonin showed significant circadian oscillation in all groups, except for the loss of cCry1 rhythm in BL. Compared to WL, GL increased the amplitudes and mesors of positive elements (cClock and cBmal1/2) and reduced those of negative elements (cCry1/2 and cPer2/3), in contrast to RL. The temporal patterns of cAanatmRNA and melatonin secretion have always been consistent with the positive genes. Besides, GL advanced the acrophases of the positive elements, cAanat and melatonin, but RL and BL showed the opposite effect. Thereby, GL could promote the secretion of melatonin by enhancing the expressions of positive clock genes and repressing the expressions of negative clock genes.

The pineal gland is a neuroendocrine organ responsible for production of the nocturnal hormone melatonin. A specific set of homeobox gene-encoded transcription factors govern pineal development, and some are expressed in adulthood. The brain-specific homeobox gene (Bsx) falls into both categories. We here examined regulation and function of Bsx in the mature pineal gland of the rat. We report that Bsx is expressed from prenatal stages into adulthood, where Bsx transcripts are localized in the melatonin-synthesizing pinealocytes, as revealed by RNAscope in situ hybridization. Bsx transcripts were also detected in the adult human pineal gland. In the rat pineal gland, Bsx was found to exhibit a 10-fold circadian rhythm with a peak at night. By combining in vivo adrenergic stimulation and surgical denervation of the gland in the rat with in vitro stimulation and transcriptional inhibition in cultured pinealocytes, we show that rhythmic expression of Bsx is controlled at the transcriptional level by the sympathetic neural input to the gland acting via adrenergic stimulation with cyclic AMP as a second messenger. siRNA-mediated knockdown (>80% reduction) in pinealocyte cultures revealed Bsx to be a negative regulator of other pineal homeobox genes, including paired box 4 (Pax4), but no effect on genes encoding melatonin-synthesizing enzymes was detected. RNA sequencing analysis performed on siRNA-treated pinealocytes further revealed that downstream target genes of Bsx are mainly involved in developmental processes. Thus, rhythmic Bsx expression seems to govern other developmental regulators in the mature pineal gland.

The numerical density of secretory granules dense-core vesicles (DCV) in the cytoplasm of pinealocytes of the pineal gland was estimated by transmission electron microscopy in male white mice and Wistar rats. The 3-day biorhythm and lunaphase changes in the DCV content in the perikaryon and the processes of pinealocytes, which are manifested significantly in different seasons of the year, are established. The three-day biorhythm in adult male mice in comparison with younger male rats is not expressed uniformly in different phases of the moon. The in-phase manifestation of infradian biorhythms in different species of animals during the year with an unchanged daily photophase indicates the existence of common external synchronizers for mammals of these biorhythms that are not associated with the light/dark cycle.

Ample evidence indicates that in several mammalian species the pineal body contains neurons. In adult white albino rats neurons are not present in the pineal body; however, in perinatal rats many neurons were described. It was demonstrated that in adult mammalian species the pineal neurons contained some neuropeptides and neurotransmitters such as leu-enkephalin, met-enkephalin, substance-P, somatostatin and γ-aminobutiric acid. Oxytocin, vasopressin mRNAs and peptides were also demonstrated. No data are available on the chemical nature of the neurons in perinatal rats. In the present experiment we used immunohistochemistry to clarify this issue. After paraformaldehyde fixation frozen sections were prepared and stained for immunoreactivities of several neuropeptides and neurotransmitters. Dopamine β-hydroxylase (DBH), neuropeptide-Y (NPY), vesicular acetylcholine transporter (VAChT), vesicular glutamate transporter (VGluT) and calcitonin gene-related peptide (CGRP) antibodies were able to stain fibers. According to previous data these fibers may be sympathetic (DBH, NPY), parasympathetic (VAChT) or sensory (CGRP). VGluT antibody may stain pinealocytes as well. Some cells were immunoreactive for substance-P, oxytocin, vasopressin, leu-enkefalin and glutamic acid decarboxylase (GAD). These immnoreactivities showed colocalization with neuron-specific nuclear protein immunoreactivity indicating that these cells are neurons. Calbindin was observed in oval and elongated cells resembling pinealocytes. Based on the results obtained in adult mammals, the pineal neurons may be analogue to retinal ganglion cells, or they may function as interneurons in the retino-pinealo-retinal neuronal circuit or the peptidergic neurons may influence the pinealocytes in a paracrine manner.

The functional roles of microRNAs (miRNAs) have been studied in various diseases, including hypoxic-ischemic brain damage (HIBD). However, changes in the expression of miRNAs and the underlying mechanisms in the pineal gland during HIBD remain unknown. Based on the previous study by microRNA array, hundreds of miRNAs showed altered expression patterns in the pineal gland in a rat model of HIBD. MiR-375-3p was found to be significantly upregulated and abundant in the pineal gland. Further investigation in an in vitro HI model of pinealocytes showed that miRNA-375 exacerbated the damage to pineal function. After oxygen-glucose deprivation / reoxygenation (OGD/R), miR-375-3p expression increased, while aralkylamine N-acetyltransferase (AANAT) expression and melatonin (MT) secretion decreased. Overexpression of miRNA-375 in pinealocytes aggravated the influence of OGD/R on AANAT expression and MT secretion. Because miRNA-375 overexpression in pinealocytes induced decreased rasd1 mRNA and protein expression, rasd1 may mediate the effect of miR-375-3p on pineal function. Furthermore, miR-375-3p aggravated the cognitive impairment caused by HIBD in rats, as observed by Morris water maze test, and also affected emotion and circadian rhythm in HIBD-treated rats. Thus, miR-375-3p may be a key regulatory molecule in the pineal gland following HIBD, and targeting of miR-375-3p may represent a new strategy for the treatment of HIBD.

Melatonin is well known for its circadian production by the pineal gland, and there is a growing body of data showing that it is also produced by many other cells and organs, including immune cells. The chronobiotic role of pineal melatonin, as well as its protective effects in vitro and in vivo, have been extensively explored. However, the interaction between the chronobiotic and defence functions of endogenous melatonin has been little investigated. This review details the current knowledge regarding the coordinated shift in melatonin synthesis from the pineal gland (circadian and monitoring roles) to the regulation of acute immune responses via immune cell production and autocrine effects, producing systemic interactions termed the immune-pineal axis. An acute inflammatory response drives the transcription factor, NFκB, to switch melatonin synthesis from pinealocytes to macrophages/microglia and, upon acute inflammatory resolution, back to pinealocytes. The potential pathophysiological relevance of immune-pineal axis dysregulation is highlighted, with both research and clinical implications, across several medical conditions, including host/parasite interaction, neurodegenerative diseases and cancer. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.

Two structures, considered as secretory in nature, are present in the pinealocytes in of the domestic pig show the presence of two structures, which are considered as secretory in nature - the dense core vesicles (DCV) and the membrane bounded (dense) bodies (MBB). The latter are extremely numerous in pig pinealocytes (they occupy 6-20% of the cytoplasm), and the number of MBB changes under different physiological and experimental conditions. Norepinephrine is the main neurotransmitter that regulates the secretion of pineal melatonin. The present study was carried out to 1) clarify whether the DCV and their source - the Golgi apparatus (GA) - as well as the MBB are controlled by norepinephrine, 2) determine the effect of adrenergic stimulation on these structures, and 3) identify the receptors involved in the regulation of these structures. The studies were performed using a static organ culture of pig pineal explants. The explants were incubated in a control medium between 08:00 and 20:00 and in a medium with 10μM norepinephrine or alpha- or beta-adrenoceptor agonists between 20:00 and 08:00 on five consecutive days. The tissues were subsequently prepared for ultrastructural analysis. The results distinctly showed that the DCV, GA and MBB in pig pinealocytes are under adrenergic control. The stimulation of the beta-adrenoceptors resulted in an increase in the numerical density of the DCV and a decrease in the relative volume of the GA in the perikarya, while the incubation with agonists of the alpha1-adrenoceptors was ineffective. The relative volume of the MBB in the perikarya significantly decreased after treatment with both beta-agonists and alpha1-agonists, which suggested the involvement of two types of adrenoceptors in the regulation of these structures.

Melatonin (N-acetyl-5-methoxy tryptamine), following discovery from the extracts of bovine pineal gland, has been detected in the pineal as well as several extra-pineal tissues/organs of different vertebrates including fish. The unique feature of melatonin in the pineal gland is its rhythmic biosynthesis and release in blood in synchronization with the environmental light-–dark cycle. Accordingly, melatonin produced in the pineal of an animal living in a changing environment is implicated to the regulation of seasonal reproduction by acting as a hormone at one or more levels of hypothalamo-hypophyseal-gonadal axis. Additionally, melatonin is known to act as a potent free-radical scavenger or antioxidant to influence maturation of oocytes. However, possible relationship between extra-pineal melatonin and seasonality of reproduction in any animal remains enigmatic. Perhaps, carp is the only known animal in which temporal patterns of melatonin levels in the serum as well as in the extracts of pineal,...

Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 μm) or as unusually large pools of Cx43 ranging up to 4-7 μm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes.

Introduction: the pineal gland is a neuro-endocrine gland that secretes melatonin hormone whichregulates the circadian rhythm, psychiatric disorder and age changes resulting from exposure to differentlight wavelengths or darkness. Intensity, duration and wavelength of light control the activity of pinealgland.Aim of work: the purpose of this study was to monitor the morphological and electron microscopicchanges in the pinealocytes type I and II of albino rats exposed to either constant (24hrs/day) red light(long wavelength) or blue light (short wavelength) as well as confined to constant darkness as comparedto control group subjected to normal diurnal conditions.Material and methods: forty adult male albino rats were divided into four subgroups. Group I (GI):the rats were kept under normal diurnal conditions, group II (GII): the rats were confined to constantdarkness, group III (GIII): the rats were exposed to constant red light (long wavelength; 670nm) andgroup IV (GIV): the rats were subjected to constant blue light (short wavelength, 450nm); for four weekscontinuously.Results: morphological findings of pinealocytes type I (PI) were analyzed statistically and revealed anincrease in the nuclear number of PI in GII, decrease in the nuclear number of PI in GIV and nonsignificantdifference in nuclear number of PI in GIII as compared to the control group. In addition,rats of GIV reported a statistically significant increase in the amount of collagen fibers between looselypacked parenchymal cells. Electron microscopic results of the cytoplasm of PI in GII showed abundantcell organelles such as mitochondria, numerous ribosomes and multiple lipid droplets together withwell-developed Golgi bodies, scarce dense core vesicles (DCV), few cisternae of smooth endoplasmicreticulum (SER) and rough endoplasmic reticulum (RER). GIII demonstrated similar results to that ofGI. On the other hand, GIV showed scarce cytoplasmic organelles such as few mitochondria; mostlydistended with disturbed cristae, scattered lipid droplets with abundant DVC and numerous cisternaeof SER. Ultrastructurally, PI nuclei of GII exhibited infolded nuclear envelope with heterochromatickaryoplasm and prominent nucleoli while GIV revealed reduction of the nuclear chromatin with ruptureof nuclear envelope at certain sites. However, GIII nuclei demonstrated results similar to that of GI. Noobservable changes were detected by light and electron microscopy of PII in the different experimentalgroups apart from the presence of statistically significant increase in the nuclear number of PII in GII.Moreover, the PII cytoplasm showed scarce mitochondria in GI, II and RER in GIV. The glial cellsdemonstrated statistically significant increase in GIV but non-significant differences were noticed in theother groups as compared to GI.Conclusion: it could be concluded that the morphological and electron microscopic changes ofpinealocyte type I indicate decrease in the activity of pinealocytes type I on exposure to constant shortwavelength blue light and increase in the activity of PI confined to constant darkness. While the exposureto constant long wavelength red light demonstrates results similar to the control group. There were nochanges in the structure of pinealocytes type II in the different experimental groups, exposed to differentlight wavelengths or confined to darkness.

The aim of the present work was to use correlation analysis to identify the complex reaction of the pineal gland (PG) and immunocompetent organs in response to administration of melatonin (MT) in conditions of an abnormal light regime. Animals were kept for 14 days with natural or round-the-clock illumination (RTCI). Animals then received 0.9% sodium chloride solution or MT for seven days, after which they were decapitated and measurements were made of body, PG, thymus, and spleen weight. Thymus and spleen lymphocyte subpopulations were assessed by flow cytometry. PG lipofuscin content was determined in terms of autofluorescence intensity on frozen sections at wavelengths over the range 560–600 nm. The number of intraorgan relationships within the immune system was found to increase in response to MT regardless of light regime. Administration of MT to animals subjected to RTCI decreased the number of interorgan relationships, with the appearance of negative correlations between PG lipofuscin content and the cellular composition of the spleen. The most marked synchronizing and adaptogenic effects of MT were seen in RTCI conditions.

Homeobox genes generally encode transcription factors involved in regulating developmental processes. In the pineal gland, a brain structure devoted to nocturnal melatonin synthesis, a number of homeobox genes are also expressed postnatally; among these is the LIM homeobox 4 gene (Lhx4). We here report that Lhx4 is specifically expressed in the postnatal pineal gland of rats and humans. Circadian analyses revealed a four-fold rhythm in Lhx4 expression in the rat pineal gland, with rhythmic expression detectable from postnatal day 10. Pineal Lhx4 expression was confirmed to be positively driven by adrenergic signaling, as evidenced by in vivo modulation of Lhx4 expression by pharmacological (isoprenaline injection) and surgical (superior cervical ganglionectomy) interventions. In cultured pinealocytes, Lhx4 expression was upregulated by cyclic AMP, a second messenger of norepinephrine. By use of RNAscope technology, Lhx4 transcripts were found to be exclusively localized in melatonin-synthesizing pinealocytes. This prompted us to investigate the possible role of Lhx4 in regulation of melatonin-producing enzymes. By use of siRNA technology, we knocked down Lhx4 by 95% in cultured pinealocytes; this caused a reduction in transcripts encoding the melatonin-producing enzyme arylalkylamine N-acetyl transferase (Aanat). Screening the transcriptome of siRNA-treated pinealocytes by RNAseq revealed a significant impact of Lhx4 on the phototransduction pathway and on transcripts involved in development of the nervous system and photoreceptors. These data suggest that rhythmic expression of Lhx4 in the pineal gland is controlled via an adrenergic-cyclic AMP mechanism and that Lhx4 acts to promote nocturnal melatonin synthesis.

Smith-Magenis syndrome (SMS), linked to Retinoic Acid Induced (RAI1) haploinsufficiency, is a unique model of the inversion of circadian melatonin secretion. In this regard, this model is a formidable approach to better understand circadian melatonin secretion cycle disorders and the role of the RAI1 gene in this cycle. Sleep-wake cycle disorders in SMS include sleep maintenance disorders with a phase advance and intense sleepiness around noon. These disorders have been linked to a general disturbance of sleep-wake rhythm and coexist with inverted secretion of melatonin. The exact mechanism underlying the inversion of circadian melatonin secretion in SMS has rarely been discussed. We suggest three hypotheses that could account for the inversion of circadian melatonin secretion and discuss them. First, inversion of the circadian melatonin secretion rhythm could be linked to alterations in light signal transduction. Second, this inversion could imply global misalignment of the circadian system. Third, the inversion is not linked to a global circadian clock shift but rather to a specific impairment in the melatonin secretion pathway between the suprachiasmatic nuclei (SCN) and pinealocytes. The development of diurnal SMS animal models that produce melatonin appears to be an indispensable step to further understand the molecular basis of the circadian melatonin secretion rhythm.

The regulation of melatonin secretion in the avian pineal organ is highly complex and shows prominent interspecies differences. The aim of this study was to determine the roles of direct photoreception and the internal oscillator in the regulation of melatonin secretion in the pineal organ of the domestic turkey. The pineal organs were collected from 12-, 13- and 14-week-old female turkeys reared under a 12 L:12 D cycle with the photophase from 07.00 to 19.00, and were incubated in superfusion culture for 3&ndash, 6 days. The cultures were subjected to different light conditions including 12 L:12 D cycles with photophases between 07.00 and 19.00, 13.00 and 01.00 or 01.00 and 13.00, a reversed cycle 12 D:12 L, cycles with long (16 L:8 D) and short (8 L:16 D) photophases, and continuous darkness or illumination. The pineal organs were also exposed to light pulses of variable duration during incubation in darkness or to periods of darkness during the photophase. The secretion of melatonin was determined by direct radioimmunoassay. The turkey pineal organs secreted melatonin in a well-entrained diurnal rhythm with a very high amplitude. Direct photoreception as an independently acting mechanism was able to ensure quick and precise adaptation of the melatonin secretion rhythm to changes in light-dark conditions. The pineal organs secreted melatonin in circadian rhythms during incubation in continuous darkness or illumination. The endogenous oscillator of turkey pinealocytes was able to acquire and store information about the light-dark cycle and then to generate the circadian rhythm of melatonin secretion in continuous darkness according to the stored data. The obtained data suggest that the turkey pineal gland is highly autonomous in the generation and regulation of the melatonin secretion rhythm. They also demonstrate that the turkey pineal organ in superfusion culture is a valuable model for chronobiological studies, providing a highly precise clock and calendar. This system has several features which make it an attractive alternative to other avian pineal glands for circadian studies.

Melatonin, which is synthesized at night by the pineal gland, is present in the cerebrospinal fluid (CSF), but its entry site and its role in this compartment are not known. Using several approaches, we tested the hypothesis that melatonin enters the CSF through the pineal recess, an evagination of the third ventricle. CSF melatonin concentrations are higher near the pineal gland than in the anterior part of the third ventricle, and decrease markedly (80%) after sealing off the pineal recess. Moreover, ultrastructure and permeability analyses of the pineal-CSF interface showed that melatonin could reach the CSF either via delivery in situ by protruding pinealocytes that make direct contact with the CSF or via extracellular secretion and interstitial fluid draining into the ventricular lumen. These data indicate that melatonin in the CSF probably originates from a few pinealocytes of the basal part of the pineal gland neighbouring the pineal recess. Melatonin carried to the brain by the blood appears to be able to mediate the effects of photoperiod on reproduction, but it is unclear whether melatonin in CSF may fine-tune this response both in terms of timing and amplitude. It is critical to determine which pathway, blood or CSF, allows melatonin to reach its central targets more efficiently.

Homeobox genes encode transcription factors that regulate developmental processes; however, in the pineal gland, a neuroendocrine organ responsible for nocturnal melatonin synthesis, expression of the homeobox genes Otx2 (orthodenticle homeobox 2) and Crx (cone-rod homeobox) persists postnatally. We here show that OTX2 and CRX are exclusively present in melatonin-producing pinealocytes of the rat pineal gland. To understand the roles of Otx2 and Crx in the mature pineal gland, we used siRNA technology in cultured rat pinealocytes with the nocturnal situation mimicked by adding norepinephrine to the culture media. siRNA-induced knockdown of Otx2 was found to reduce expression levels of the enzymes involved in melatonin synthesis at both transcript and protein levels. Similar results were obtained when knocking down Crx. Knocking down Otx2 and Crx simultaneously produced an even larger reduction in both transcript and protein levels of the melatonin-producing enzymes and also reduced the levels of melatonin released to the culture media. These results suggest that Otx2 and Crx, both alone and in combination, act to control pineal melatonin synthesis.

Background: The study demonstrates, for the first time, the presence of substance P (SP) and calcitonin gene-related peptide (CGRP) in the nerve fibres supplying the pineal gland in the Arctic fox. Materials and methods: The expression and distribution pattern of the studied substances were examined by double-labelling immunofluorescence technique. Results: The SP-positive fibres enter into the pineal gland through the capsule as the nervi conarii. The fibres formed thick bundles in the capsule and connective tissue septa, from where they penetrated into the pineal parenchyma. Inside the parenchyma, the nerve fibres created basket-like structures surrounding clusters of pinealocytes. The density of intrapineal SP positive fibres was slightly higher in the distal and middle parts of the gland than in the proximal one. Double immunostaining with antibodies against SP and CGRP revealed that the vast majority of SP positive fibres were also CGRP positive. The fibres showing a positive reaction to SP and negative to CGRP were scattered within the whole gland. The fibres immunopositive to CGRP and immunonegative to SP were not observed. In the habenular and posterior commissural areas adjoining to the pineal gland the immunoreactive nerve fibres were not found. Moreover, no immunopositive cell bodies were observed in both the pineal gland and the commissural areas. Conclusions: These results reveal that SP and CGRP are involved in the innervation of pineal gland in carnivores. In turn we suggest that these peptides can regulate/modulate melatonin secretion.

The avian pineal gland, an independent circadian oscillator, receives external photic cues and translates them for the rhythmical synthesis of melatonin. Our previous study found that monochromatic green light could increase the secretion of melatonin and expression of CLOCK and BMAL1 in chick pinealocytes. This study further investigated the role of BMAL1 and CLOCK in monochromatic green light-induced melatonin secretion in chick pinealocytes using siRNAs interference and overexpression techniques. The results showed that si-BMAL1 destroyed the circadian rhythms of AANAT and melatonin, along with the disruption of the expression of all the seven clock genes, except CRY1. Furthermore, overexpression of BMAL1 also disturbed the circadian rhythms of AANAT and melatonin, in addition to causing arrhythmic expression of BMAL1 and CRY1/2, but had no effect on the circadian rhythms of CLOCK, BMAL2 and PER2/3. The knockdown or overexpression of CLOCK had no impact on the circadian rhythms of AANAT, melatonin, BMAL1 and PER2, but it significantly deregulated the circadian rhythms of CLOCK, BMAL2, CRY1/2 and PER3. These results suggested that BMAL1 rather than CLOCK plays a critical role in the regulation of monochromatic green light-induced melatonin rhythm synthesis in chicken pinealocytes. Moreover, both knockdown and overexpression of BMAL1 could change the expression levels of CRY2, it indicated CRY2 may be involved in the BMAL1 pathway by modulating the circadian rhythms of AANAT and melatonin.

MicroRNAs, including microRNA-7 (miR-7), are important modulators of numerous gene expressions and the related biological processes. Melatonin is a key hormone regulating daily and seasonal rhythms, in which a variety of positive and negative regulatory factors, such as norepinephrine (NE) and leptin, are involved. However, the interactions among these factors and the mechanisms remain to be elucidated. The aims of the present study were to identify the functions and the related mechanisms of miR-7 in regulating melatonin synthesis and secretion through in vitro and in vivo experiments in pineal gland of pigs, which is an important animal model for agricultural and biomedical studies. Our results firstly show that miR-7 is specifically expressed in porcine pinealocytes and negatively regulates melatonin synthesis. The further functional studies show that the dynamic expression levels of miR-7 are contrary to the melatonin levels throughout the day, and the forced inhibition of endogenous miR-7 in porcine pinealocytes sharply increases arylalkylamine N-acetyltransferase (AANAT) expression by 80.0% (P = 0.0031) and melatonin levels by 81.0% (P = 0.0421), whereas miR-7 over-expression down-regulates AANAT expression by 38.6% (P = 0.0004) and melatonin levels by 37.6% (P = 0.0212). In addition, the miR-7 expression is up-regulated by leptin through the JAK/STAT3 signaling pathway, and the in vivo intracerebroventricular injection of leptin increases miR-7 expression by 80.0% (P = 0.0044) in porcine pineal glands and reduces melatonin levels by 57.1% (P = 0.0060) compared with the controls. This functional inhibition of melatonin synthesis by miR-7 is accomplished by its binding to the 3'-UTR of Raf1. Further, our results demonstrate that the RAF1/MEK/ERK signaling pathway mediates NE-induced AANAT expression, whereas leptin attenuates NE's function through miR-7. Taken together, the results demonstrated that leptin activates the JAK/STAT3 signaling pathway to increase the expression of miR-7, which acts as a negative regulatory molecule inhibiting NE-activated RAF1/MEK/ERK signaling pathway by targeting Raf1, resulting in decreased AANAT expression and melatonin synthesis. These findings suggest that miR-7 is a novel negative regulator of melatonin synthesis and links leptin- and NE-mediated signaling pathways in porcine pineal glands, which will contribute to our understanding in the establishment of the biological rhythms resulting from melatonin.

Disorders of the serotonergic system are especially known to be present in the neurobiology of suicidal behavior. Studies investigating melatonin levels show that changes in pineal gland functions may also play a role in the pathogenesis of suicide. However, to our knowledge, there are no studies evaluating the activity of pinealocytes responsible for melatonin synthesis in suicide. This preliminary study aimed to investigate the relationship among pinealocyte, acetylserotonin O-methyltransferase (ASMT) immunoreactivity, and suicide. Samples of pineal gland, cerebrospinal fluid, blood, and urine were obtained from 21 suicide and 21 non-suicide cases on which medicolegal autopsies were performed. Expression of ASMT in human pineal gland was evaluated by immunohistochemical methods. A scoring system was used to define the anti-ASMT-positive staining in the sections. Enzyme-linked immunosorbent assays were employed to assess serum and cerebrospinal fluid melatonin levels and blood and urine noradrenaline levels. The ASMT-immunopositive pinealocyte count was observed to be lower in suicide cases compared to the non-suicide cases. With the exception of two cases (with moderate staining), all graded scores were 3 (strong staining) in non-suicide group, whereas scores were 1 (mild staining) or 2 (moderate staining) in the suicide group. Melatonin levels in the blood were lower among the suicide victims. These results support decreased pineal gland activity in suicide. However, further studies are needed to assess whether these changes are related to a psychiatric disorder. © The Author(s) 2018.

Circadian rhythm of pineal melatonin production is paced by the thalamus suprachiasmatic nucleus (SCN) depending on the lighting conditions via signal transduction to pinealocytes beta-receptors. Melatonin is a natural regulator of many physiological processes, and the decrease of its synthesis leads to various diseases, in particular, insomnia and metabolic disorders. It is known that administration of beta-blockers reduces melatonin production, but the data showing clinical significance of melatonin reduction associated with beta-blockers administration are still contradictory.The influence of long-term administration of beta-blockers to melatonin synthesis, sleep quality and vascular brain damage.The main study group included 114 patients, aged 47-83, with cardiovascular diseases, who were under a complex therapy with long-term administration of beta-blockers. The comparison group included 110 patients with cardiovascular diseases, similar in age and sex, who did not receive beta-blockers in their complex therapy. The circadian dynamics of melatonin synthesis was observed by excretion of 6-sulfatoxymelatonin (6-SM), the major metabolite of melatonin, in three urinary samples (day, evening, night). All the patients underwent night polysomnography to assess the severity of sleep disorders. The severity of vascular brain damage was assessed using magnetic resonance imaging.The analyses showed large variability in individual values of 6-SM circadian excretion of patients with cardiovascular diseases (from 0.9 to 133 μg/24h with a mid-point 16.8 μg/24h). A considerable decrease of 6-SM circadian excretion is detected in the group of patients taking beta-blockers comparing to those not Me [q 25; q 75]: 12.8 [6.2; 21.1] and 24.0 [12.5; 41.5] μg/24h, respectively (A low level of endogenous melatonin is a risk factor for development of sleep structure and quality disorders, vascular white matter brain damages with a higher risk for metabolic disorders. Long-term beta-blockers administration decrease endogenous melatonin synthesis to 50% increasing the risk for insomnia and vascular brain damage, mostly in patients with lower initial level of 6-SM circadian excretion.: melatonin, 6-sulfatoxymelatonin, beta-blockers, insomnia, vascular white matter brain damage, leptin, adiponectin.Оценка влияния длительного приема бета-блокаторов на синтез мелатонина, качество сна и сосудистое поражение головного мозга.Основную группу составили 114 пациентов, из них 103 мужчины, в возрасте 47—83 лет с сердечно-сосудистыми заболеваниями, комплексная терапия которых включала длительный прием бета-блокаторов. Группу сравнения составили 110 пациентов с сердечно-сосудистыми заболеваниями, сопоставимых по возрасту и полу, комплексная терапия которых не включала бета-блокаторы. Суточную динамику синтеза мелатонина определяли по экскреции основного метаболита мелатонина — 6-сульфатоксимелатонина (6-СОМТ) в трех порциях мочи (день, вечер, ночь). Для оценки выраженности нарушений сна всем пациентам проводили ночную полисомнографию. Выраженность сосудистого поражения головного мозга оценивали по результатам МРТ.Результаты анализа показали большой разброс в индивидуальных значениях суточной экскреции 6-СОМТ в группе пациентов с сердечно-сосудистыми заболеваниями (от 0,9 до 133 мкг/сут с медианой 16,8 мкг/сут). Выявлено значительное снижение суточной экскреции 6-СОМТ в группе пациентов, принимающих бета-блокаторы, по сравнению с пациентами, не принимающими бета-блокаторы, 12,8 [6,2; 21,1] и 24,0 [12,5; 41,5] мкг/сут соответственно (Низкий уровень суточной экскрецией 6-СОМТ является фактором риска развития нарушений структуры и качества сна, сосудистого поражения белого вещества головного мозга в сочетании с повышенным риском развития метаболических нарушений. Длительный прием бета-блокаторов снижает синтез мелатонина до 50%. что повышает риск развития инсомнии и сосудистого поражения головного мозга преимущественно у пациентов с более низким исходным уровнем суточной экскреции 6-СОМТ.