Pineal Content Research Articles

Embryonic Development of Avian Pineal Secretory Activity-A Lesson from the Goose Pineal Organs in Superfusion Culture.

The embryonic ontogeny of pineal secretory activity in birds has been investigated almost exclusively in chickens. This study aimed to characterize this process in domestic geese. The pineal organs of embryos aged 18–28 days were incubated in superfusion culture under different light conditions for 4–5 days and treated with norepinephrine (NE). Melatonin (MLT) was measured by radioimmunoassay and other indoles by HPLC with fluorescence detection. Additionally, pineal organs were collected from embryos at 14–28 days of age and used to measure catecholamines by HPLC with electrochemical detection. MLT secretion increased with embryo age, most intensively between the 22nd and 24th days of life. The daily changes in MLT secretion under the 12 L:12D cycle occurred on the first day of culture, starting from an embryonic age of 24 days. MLT secretion was controlled by the light-dark cycle in all age groups studied. However, exposure to light during the scotophase did not alter the secretion of MLT. The endogenous oscillator expressed its activity in regulating MLT secretion in the pineal organs of embryos aged 24 days and older but could not generate a rhythm after one cycle. The rhythm of 5-hydroxytryptophan release during the first day of culture was found in the pineal organs of all embryos, while the rhythmic release of N-acetylserotonin and 5-methoxyindole acetic acid started at the age of 24 days. The proportion of released indoles changed with embryo age. NE caused a decrease in MLT secretion and provoked an increase in serotonin release. Incubation of the pineal organs induced the development of MLT secretory machinery and its diurnal rhythmicity. The pineal content of catecholamines increased prominently at the end of embryonic development.

Effects of Streptozotocin-Induced Diabetes on the Pineal Gland in the Domestic Pig.

Several observations from experiments in rodents and human patients suggest that diabetes affects pineal gland function, including melatonin secretion; however, the accumulated data are not consistent. The aim of the present study was to determine the effects of streptozotocin-induced diabetes on the pineal gland in the domestic pig, a species widely used as a model in various biomedical studies. The study was performed on 10 juvenile pigs, which were divided into two groups: control and diabetic. Diabetes was evoked by administration of streptozotocin (150 mg/kg of body weight). After six weeks, the animals were euthanized between 12.00 and 14.00, and the pineal glands were removed and divided into two equal parts, which were used for biochemical analyses and for preparation of explants for the superfusion culture. The pineal contents (per 100 μg protein) of serotonin, 5-hydroxyindole acetic acid, 5-hydroxytryptophol, 5-methoxyindole acetic acid, 5-methoxytryptophol, and 5-methoxytryptamine were significantly lower in diabetic pigs than in control pigs. In contrast, the level of N-acetylserotonin was significantly higher in diabetic animals. No significant differences were found in the level of melatonin between control and experimental pigs. The amounts of 3,4-dihydroxyphenylalanine, dopamine, norepinephrine, and 3,4-dihydroxyphenylacetic acid were significantly lower in the pineal glands of diabetic animals. The level of vanillylmandelic acid was higher in diabetic pigs. No differences were observed in the level of basal and NE-stimulated release of N-acetylserotonin or melatonin between the pineal explants prepared from control and experimental animals. In vitro treatment with insulin was ineffective. In conclusion, streptozotocin-induced diabetes affects both indole metabolism and adrenergic neurotransmission in the pig pineal gland.

Human chorionic gonadotropin elevation is not an intracranial germ cell tumor signature

Dear editor, For many years, in case of suspicion of a germ cell tumor (GCT), human chorionic gonadotropin (HCG), its beta subunit and alpha-fetoprotein (AFP) have been used as specific markers in the blood and/or cerebrospinal fluid (CSF) for clinical decision-making [4]. A positive test is an indication for treatment, without requiring histological confirmation [5]. We report a first description of CSF HCG elevation in a non-cystic suprasellar craniopharyngioma, contradicting this dogma. A 52-year-old male with a history of arterial hypertension and tinnitus complained of a decrease in left-side visual acuity over a few weeks, associated with polyuro-polydipsia syndrome. MRI examination revealed a suprasellar lesion of 16×18×16 mm, with a moderate hyper-T2 signal and an iso-T1 signal with homogeneous gadolinium enhancement. The lesion did not have a cystic component (see Fig. 1). Spinal MRI was negative, and the cerebral CT scan did not show any calcification of the tumor. Hormone analysis revealed a pituitary deficiency. Biological analysis of the CSF performed as part of the etiological assessment revealed an elevation of HCG to 10.8 IU/l (normal levels <5 IU/l) and of free beta-HCG to 1.1 ng/ml (normal levels <0.1 IU/l). Elevation of AFP was not detected in the blood or CSF. No abnormal cells were found in the CSF. No elevation of these markers was found in the blood. Given the many atypical features in our patient, we decided to perform a robot-assisted stereotaxic biopsy. The final diagnosis was papillary craniopharyngioma and excluded germinoma. Due to the high risk of surgical treatment, and after a multidisciplinary discussion, the tumor was treated by radiotherapy. Six months after that, the patient recovered his visual field loss, but the hypopituitarism remained. The MRI showed a volumetric reduction of the tumor and a decrease in contrast enhancement. This report points out two issues: First, some authors [2, 3, 6] have suggested that many cystic pineal, sellar or suprasellar lesions, including cystic craniopharyngioma, are able to produce theses markers through their wall (descriptions of HCG in benign pineal cyst contents and walls). This observation of a solid craniopharyngioma demonstrates for the first time that the tumor itself is able to secrete low levels of HCG and β-HCG that may be detectable in the CSF. CSF AFP elevation was also detected in two cases of craniopharyngiomas, one of which was proven to be a malignant form [2]. So far, no blood elevation of these markers has been reported in craniopharyngiomas. Second, therefore, physicians involved in brain management need to be aware that HCG or AFP CSF positivity in a patient with a suprasellar tumor is not a specific GCT signature and is not enough information for making therapeutic decisions. Our clinical case underlines the importance of multidisciplinary discussions and the necessity to perform a biopsy in patients showing isolated suprasellar lesions (bipolar pineal and suprasellar locations remain suggestive of germinoma), positive for CSF markers but with atypical P. Bourdillon (*) : E. Jouanneau Department of neurosurgery A, Hopital Neurologique et Neurochirurgical Pierre Wertheimer, Hospices Civils de Lyon, 59 bd Pinel, 69500 Bron, France e-mail: pierre.bourdillon@neurochirurgie.fr

Season-dependent Postembryonic Maturation of the Diurnal Rhythm of Melatonin Biosynthesis in the Chicken Pineal Gland

Previously, we have demonstrated that the timing of the nocturnal peak of activity of the pineal arylalkylamine-N-acetyltransferase – a key enzyme in the melatonin biosynthesis pathway – in 3-wk-old chickens kept from the day of hatch under controlled laboratory conditions (L:D 12:12) varies depending on the season of hatch (summer vs. winter). The present study was undertaken to answer the following questions: (1) are season-related differences seen in the level of transcription of genes encoding enzymes of the melatonin biosynthesis pathway? (2) Does the pineal content of the main precursor (serotonin) and the final product (melatonin) exhibit age- and season-related changes? (3) At which step in postembryonic development are these season-related variations in pineal gland function most pronounced? Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held on L:D 16:8, were kept from the day of hatch under L:D 12:12 conditions. At the age of 2, 9, or 16 d, chickens were sacrificed every 2 h over a 24-h period and their pineal glands, isolated under dim red light, were processed for the measurement of (i) the level of Aanat and Asmt (acetylserotonin O-methyltransferase) mRNAs encoding the two last enzymes involved in melatonin biosynthesis, (ii) the activity of these enzymes, and (iii) the pineal content of serotonin and melatonin. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The levels of Aanat mRNA, AANAT enzymatic activity, and the pineal melatonin content changed during postembryonic development in a manner that was dependent on the season of hatch. Furthermore, the diurnal profile of Asmt mRNA was elevated during the light phase. In “winter” birds, the pattern and amplitude of the diurnal rhythm of accumulation of this transcript did not change with age, while in “summer” birds it increased in an age-related way. In contrast, the enzymatic activity of hydroxyindole-O-methyltransferase (HIOMT; encoded by the Asmt gene) did not change rhythmically, although it increased with age in a season-related way. In “winter” chickens, the pineal serotonin content was low, regardless of age, and did not change rhythmically, whereas in “summer” individuals the serotonin rhythm was already well established by day 2, with the amplitude increasing with age. These results confirm the existence of a “seasonal memory” operating within the chicken pineal gland, although the mechanism(s) underlying this phenomenon have yet to be characterized.

Pineal arylalkylamine N-acetyltransferase (Aanat) gene expression as a target of inflammatory mediators in the chicken

Previously, we demonstrated that experimental peritonitis in chickens was attenuated by treatment with exogenous melatonin, while the developing inflammation decreased pineal AANAT activity. This suggested the existence of a bidirectional relationship between the activated immune system and pineal gland function. The aim of the present study was to identify the step(s) in the chicken pineal melatonin biosynthetic pathway that are affected by inflammation. Peritonitis was evoked by i.p. injection of thioglycollate solution, either 2h after the start, or 2h before the end of the light period, and the animals were sacrificed 4h later. The effect of inflammation on the expression of genes encoding enzymes participating in melatonin biosynthesis in the pineal gland, i.e. tryptophan hydroxylase 1 (Tph1), dopa decarboxylase (Ddc), arylalkylamine N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), was evaluated by qPCR. The pineal and serum melatonin concentration as well as the content of its precursors in the pineal gland were measured, along with the activity of the relevant biosynthetic enzymes. Developing peritonitis caused an increase in the pineal levels of the Tph1 mRNA during the night and the Asmt mRNA during the day, while nocturnal Aanat transcription was reduced. Both the pineal and serum melatonin level and the pineal content of N-acetylserotonin (NAS) were decreased during the night in birds with peritonitis. The amount and activity of pineal AANAT were significantly reduced, while the activity of HIOMT was increased under these experimental conditions. These results indicate that the observed decrease in MEL biosynthesis in chickens with developing inflammation is a result of transcriptional downregulation of the Aanat gene, followed by reduced synthesis and activity of the encoded enzyme.

Changes in plasma melatonin levels and pineal organ melatonin synthesis following acclimation of rainbow trout (Oncorhynchus mykiss) to different water salinities

Melatonin has been suggested to play a role in fish osmoregulation, and in salmonids has been related to the timing of adaptive mechanisms during smolting. It has been described that acclimation to different environmental salinities alters levels of circulating melatonin in a number of fish species, including rainbow trout. However, nothing is known regarding salinity effects on melatonin synthesis in the pineal organ, which is the main source of rhythmically produced and secreted melatonin in blood. In the present study we have evaluated, in rainbow trout, the effects of acclimation to different salinities on day and night plasma melatonin values and pineal organ melatonin synthesis. Groups of freshwater (FW)-adapted rainbow trout were placed in tanks with four different levels of water salinity (FW, 6, 12, 18 p.p.t.; parts per thousand) and maintained for 6 h or 5 days. Melatonin content in plasma and pineal organs, as well as the pineal content of serotonin (5-HT) and its main oxidative metabolite (5-hydroxyindole-3-acetic acid; 5-HIAA) were measured by high performance liquid chromatography. In addition, day-night changes in pineal organ arylalkylamine N-acetyltransferase (AANAT2) activity and aanat2 gene expression were studied. Plasma osmolalities were found to be higher in rainbow trout exposed to all salinity levels compared with the control FW groups. A salinity-dependent increase in melatonin content was found in both plasma and pineal organs. This effect was observed during the night, and was related to an increase in aanat2 mRNA abundance and AANAT2 enzyme activity, both of which also occurred during the day. Also, the levels of indoles (5-HT, 5-HIAA) in the pineal organ were negatively affected by increasing water salinity, which seems to be related to the higher recruitment of 5-HT as a substrate for the increased melatonin synthesis. A stimulatory effect of salinity on pineal aanat2 mRNA expression was also identified. These results indicate that increased external salinity promotes melatonin synthesis in the pineal organ of rainbow trout by enhancing synthesis of AANAT protein independently of its regulation by light. The possibility that pineal melatonin is a target for hormones involved in the response of fish to osmotic challenge is discussed, as well as the potential role of melatonin in the timing of osmoregulatory processes.

Adrenoceptor Expression and Diurnal Rhythms of Melatonin and Its Precursors in the Pineal Gland of Type 2 Diabetic Goto-Kakizaki Rats

A decrease in the nighttime release of the pineal hormone melatonin is associated with aging and chronic diseases in animals an humans. Melatonin has a protective role in type 2 diabetes; however, its synthesis itself is affected in the disease. The aim of this study was to detect crucially impaired steps in the pineal melatonin synthesis of type 2 diabetic Goto-Kakizaki (GK) rats. Therefore, plasma melatonin concentrations and the pineal content of melatonin and its precursors (tryptophan, 5-hydroxytryptophan, serotonin, and N-acetylserotonin) were quantified in GK rats compared with Wistar rats (each group 8 and 50 wk old) in a diurnal manner (four animals per group and per time point). Additionally, the expression of pineal adrenoceptor subtype mRNA was investigated. We found that in diabetic GK rats, 1) inhibitory alpha-2-adrenoceptors are significantly more strongly expressed than in Wistar rats, 2) the formation of 5-hydroxytryptophan is crucially impaired, and 3) the pineal gland protein content is significantly reduced compared with that in Wistar rats. This is the first time that melatonin synthesis is examined in a type 2 diabetic rat model in a diurnal manner. The present data unveil several reasons for a reduced melatonin secretion in diabetic animals and present an important link in the interaction between melatonin and insulin.

Season-Related Differences in the Biosynthetic Activity of the Neonatal Chicken Pineal Gland

Influence of the season of hatch on the functional characteristics of the pineal gland was examined in neonatal Hi-Line male chickens. The pineal glands from 2-day-old birds hatched in and winter, and kept from the day of hatch in artificial lighting conditions (12L:12D), were isolated under dim red light in the middle of the day or night. The pineal glands were analyzed to characterize their melatonin biosynthetic activity: (1) expression of the Arylalkylamine- N-acetyltransferase and Hydroxyindole-O-methyltransferase genes, encoding the final two enzymes of the melatonin biosynthesis pathway; (2) the activity of AA-NAT and HIOMT; and (3) the content of the main substrates of this pathway, tryptophan (TRY) and serotonin (5-HT). Daily changes in pineal AA-NAT activity were observed in chickens hatched in both seasons, with a more pronounced nocturnal increase in summer. In contrast, the level of Aa-nat gene expression, although exhibited the same nocturnal/diurnal pattern in both seasons, was much lower in the summer. The activity of HIOMT was season- and daytime-independent. In winter chickens the pineal content of 5-HT was low and stable, while in summer birds it was correlated with levels of AA-NAT activity and Aa-nat gene expression. TRY content was very high and exhibited neither daily nor seasonal changes. The pineal gland of newly hatched chickens kept in controlled 12L:12D conditions exhibits daily variations in melatonin biosynthetic activity influenced by the season. This suggests a maternal effect on the perinatal/postnatal development of the circadian clock residing in the chicken pineal gland.

Pineal melatonin synthesis is decreased in type 2 diabetic Goto–Kakizaki rats

Aims It is not well understood why the amplitude of melatonin rhythms is reduced in diabetic animals and humans. This paper addresses the differences in the pineal melatonin synthesis of type 2 diabetic Goto–Kakizaki (GK) rats compared to non-diabetic Wistar rats (8 and 50 weeks old). Main methods Plasma melatonin concentrations and the pineal content of melatonin and its precursors (tryptophan, 5-hydroxytryptophan, serotonin, and N-acetylserotonin) were quantified at the middle of the day and night. Additionally, the expression of melatonin synthesizing enzymes, pineal noradrenaline content, and pineal protein content were considered, and the melatonin secreting capacity of pineal glands was studied in vitro. Key findings The pineal glands of diabetic GK rats have a different expression pattern of melatonin synthesizing enzymes. The amount of all precursors of melatonin is reduced in pineal glands of diabetic GK rats. The pineal glands of diabetic GK rats contain less noradrenaline, indicating a reduced stimulation of nighttime melatonin synthesis. The pineal glands of diabetic GK rats produce less melatonin in reaction to noradrenaline in vivo and in vitro. The pineal glands of diabetic GK rats contain less protein, probably a consequence of diabetic neuropathy. Significance This is the first time that melatonin synthesis is examined in a type 2 diabetic rat model. The present data unveiled several reasons for a reduced melatonin secretion in diabetic animals and presents an important link in the interaction between melatonin and insulin.

Effects of naphthalene, β-naphthoflavone and benzo( a)pyrene on the diurnal and nocturnal indoleamine metabolism and melatonin content in the pineal organ of rainbow trout, Oncorhynchus mykiss

Polycyclic aromatic hydrocarbons (PAHs) have deleterious effects on neuroendocrine systems in teleost fish affecting, among other processes, reproductive function or stress responses. The hormone melatonin, mainly produced in the pineal organ of vertebrates, is involved in the regulation of biological rhythms as well as other important functions, and may also act as an antioxidant molecule. The effects of environmental pollutants on the endocrine and metabolic activity of the pineal organ have been studied only in mammals. We here evaluate the effects of the PAHs naphthalene (NAP) and benzo( a)pyrene (BaP) and the flavonoid β-naphthoflavone (BNF) on the pineal organ of rainbow trout by quantifying the diurnal and nocturnal pineal content of some indoles and methoxyindoles, including melatonin. NAP mainly induced diurnal increases in the pineal content of melatonin and other methoxyindoles like 5-methoxytryptamine (5-MT), 5-methoxyindole-3-acetic acid (5-MIAA) or 5-methoxytryptophol (5-MTOL). Those increases did not occur at night, when even occasional decreases were observed compared with controls. NAP also induced some diurnal and nocturnal decreases in the levels of indolic compounds like serotonin (5-HT) and 5-hydroxyindole-3-acetic acid (5-HIAA), while pineal content of 5-hydroxytryptophan (5-HTP) was first decreased (few hours after injection) and then increased (few days after injection) during the day. BaP and BNF induced strong increases in diurnal levels of melatonin, whereas other pineal compounds were unaffected. It seems that an increase of the methylation capacity of the pineal organ takes place during the day, and a decrease occurs at night. Those effects could be mediated by changes in the activity of key enzymes involved in pineal melatonin biosynthesis, maybe as a result of the alteration of the cellular phototransduction mechanisms involved in the light-induced inhibition of melatonin synthesis in the pineal photoreceptor cells. These results demonstrate for the first time that environmental pollutants can disrupt the activity of the pineal organ of teleost fish. This disruption could be a threat for the survival of the animals in their natural environment, although the increases observed in melatonin levels could play a relevant role as a toxicity-protection factor.

Effect of Methylene Blue and Related Redox Dyes on Monoamine Oxidase Activity; Rat Pineal Content of N‐Acetylserotonin, Melatonin, and Related Indoles; and Righting Reflex in Melatonin‐Primed Frogs

The ability of methylene blue (MB) to inhibit the nitric oxide-induced stimulation of N-methyl-D-aspartate receptors has been suggested as a possible mechanism of MB's clinical antidepressant action. This study evaluated the alternative/additional mechanisms of the antidepressant effect of MB on biochemical and behavior levels. Selective inhibition of monoamine oxidase type A (MAO-A) is widely accepted as a major mechanism of the clinical antidepressant effect. MB and the related redox dyes toluidine blue O (TBO), thionine (TN), brilliant cresyl blue (BCB), and toluylene blue (TB) were reversible competitive inhibitors of both MAO-A and MAO-B and were highly selective toward MAO-A. TBO was the most potent inhibitor, followed by TN, BCB, MB, and TB. The dyes studied increased rat pineal N-acetylserotonin (NAS) and melatonin content, in accordance with our previous observations of the stimulating effect of selective inhibition of MAO-A on pineal melatonin biosynthesis. The redox dyes exerted antidepressant-like activity in frogs; that is, they suppressed the righting reflex in melatonin-primed frogs. This study's results indicate that selective inhibition of MAO-A might mediate the clinical antidepressant effect of MB through NAS stimulation and melatonin biosynthesis.

Bipedal ambulation induces experimental scoliosis in C57BL/6J mice with reduced plasma and pineal melatonin levels

In addition to the induction of scoliosis in chickens by pinealectomy (PINX), we previously demonstrated that removal of the pineal gland also produces scoliosis in bipedal rats, which can be inhibited by melatonin treatment. Using C57BL/6J mice with genetically low circulating melatonin levels, the main objective of the present study was to investigate whether bipedal ambulation in C57BL/6J mice has the same effects on spinal deformity as those seen in pinealectomized bipedal rats. The present study consisted of two phases. The aim of the first experiment was to determine whether the C57BL/6J mouse strain actually exhibits depressed plasma concentrations and/or pineal contents of melatonin during both the light and the dark phase of the light:dark cycle. The aims of the second experiment were to evaluate; (i) whether bipedal ambulation alone in the C57BL/6J mouse induces scoliosis, and (ii) whether PINX with bipedal ambulation in another mouse strain, i.e. C3H/HeJ, which normally exhibits diurnal fluctuations in melatonin synthesis and secretion, has effects similar to those of bipedal ambulation alone in C57BL/6J mice. C3H/HeJ mice, serving as controls, showed significant increases in both plasma concentrations and pineal contents of melatonin during the dark phase when compared with the light phase. In contrast, there were no differences in either circulating levels or pineal contents of melatonin between the light and dark phases in C57BL/6J mice. Moreover, plasma melatonin levels were below the detection limit of the assay in both phases and pineal melatonin was < 10% of that in C3H/HeJ mice. Bipedal ambulation for 40 wk in C57BL/6J mice induced scoliosis at a rate of 64.3%, and two of nine scoliotic mice showed two sites of spinal deformity. This type of ambulation in C3H/HeJ mice resulted in scoliosis at a lower rate (25%), and affected animals had only a single scoliotic site. However, PINX combined with bipedal ambulation in C3H/HeJ mice produced scoliosis at a rate (70%) similar to that seen in C57BL/6J mice, and some double deformations were induced. These results confirm our previous observations in rats, and also support our hypothesis that melatonin as well as the bipedal ambulation appear to play a critical pathogenic role in scoliosis in experimental mammals.

Locally synthesized angiotensin modulates pineal melatonin generation.

We aimed to study the mechanisms and the significance of the influence exerted by the renin-angiotensin system (RAS) on the pineal melatonin production. Pineal melatonin and other indoles were determined by HPLC with electrochemical detection after angiotensin AT1-receptor blockade with Losartan in vivo or in cultured glands. N-acetyltransferase (NAT) activity was radiometricaly measured. To test the in vivo relevance of the local RAS, pineal melatonin and its indole precursors were determined in transgenic rats with inhibited production of angiotensinogen exclusively in astrocytes, TGR(ASrAOGEN). Tryptophan hydroxylase (TPH) and NAT mRNA levels were determined by real-time RT-PCR. Pineal melatonin content was significantly decreased by AT1-receptor blockade in vivo, in cultured glands and in TGR(ASrAOGEN) (35%, 32.4% and 17.5% from control, respectively). Losartan produced a significant decrease of pineal 5-hydroxytryptophan, serotonin, 5-hydroxyindole acetic acid and N-acetylserotonin in pineal cultures. Also, the pineal content of the precursor indoles in TGR(ASrAOGEN) rats was significantly lowered. The reduction of 5-hydroxytryptophan levels by 33-75% in both in vivo and in vitro studies suggests a decreased activity of TPH. Moreover, the TPH mRNA levels in TGR(ASrAOGEN) rats were significantly lower than control rats. On the other hand, NAT activity was unaffected by Losartan in pineal culture and its expression was not significantly different from control in TGR(ASrAOGEN) rats. Our results demonstrate that a local pineal RAS exerts a tonic modulation of indole synthesis by influencing the activity of TPH via AT1-receptors.

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice.

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.

Prenatal melatonin influences developmental changes of tachykinins in response to estradiol benzoate

The dev elopmental changes of hypothalamic, pituitary, striatum and pineal gland tachykinin concentrations, as well as the response to estradiol-benzoate (EB) administration, were studied in offspring of control and melatonin (MEL) treated mother rats. Female rats were studied throughout different phases of the sexual development: infantile, prepubertal and pubertal periods, in the four following groups; control-offspring+vehicle; control-offspring+EB; MEL-offspring+vehicle; MEL-offspring+EB. Hypothalamic NKA in control-offspring+vehicle was significantly increased only at 27 days of age and in control-offspring+EB at 27 days of age and during the infantile period. Hypothalamic SP levels increased similarly in control-offspring+EB during the infantile period but the EB influence was more pronounced with significantly increased concentrations at 32 days of age. Prenatal melatonin treatment produced major alterations in these patterns of postnatal development. In MEL-offspring+EB tachykinins concentrations in the hypothalamus during infantile and prepubertal periods did not increase, however at 37 days of age, they showed significantly higher values than in control-offspring+EB groups. The developmental pattern of pituitary NKA and SP concentrations in both; control-offspring+vehicle and control-offspring+EB groups, showed similar values from the infantile period to puberty, indicating that NKA and SP concentrations remained at similar levels independently of the sexual stage, only at 27 days of age in control-offspring+EB significantly increased values were found as compared to MEL-offspring+EB. Prenatal melatonin did not produce marked modifications, only significantly lower NKA and SP concentrations in MEL-offspring+EB group were observed at 25 days of age in comparison to control-offpring+EB group. Striatal NKA and SP concentrations showed a similar developmental pattern. In control-offspring, EB treatment produced NKA and SP decreased concentrations at the infantile period than in control-offspring+vehicle and significantly increased concentrations during the prepubertal period, then during the pubertal period NKA and SP concentrations decreased in control-group+EB. However, prenatal melatonin treatment reduced the levels of striatal NKA and SP during the prepubertal period after EB treatment and delayed until pubertal period the increase previously observed in control group during the prepubertal period. In MEL-offspring+vehicle group striatal concentrations of both tachykinins remained at low levels from infantile period until pubertal period. Prenatal melatonin and EB did not produce major alterations in SP pineal concentrations throughout sexual development. Plasma estradiol concentrations were significantly higher in the groups that received EB treatment than in those that received vehicle during prepubertal and juvenile periods in control-offspring+EB group and during the pubertal period in MEL-offspring+EB group. These data indicate that prenatal MEL treatment may influence NKA and SP developmental pattern from the infantile period until adulthood in the female rat.

Melatonin rhythm onset in the adult siberian hamster: influence of photoperiod but not 60-Hz magnetic field exposure on melatonin content in the pineal gland and in circulation.

To determine the relationship between pineal melatonin production and its appearance in circulation, the rising phase of the pineal and serum melatonin rhythm was studied in the adult Siberian hamster. Melatonin concentrations increased in the pineal gland and in serum at 1.50 and 1.75 h, respectively, relative to lights off in long days (16 h of light/day) and at 2.00 and 2.75 h, respectively, in short days (10 h of light/day). Thus, a photoperiod-dependent melatonin rise in circulation lagged production by the pineal gland by 0.50 h--a delay of 0.75 h in short-day hamsters versus 0.25 h in long-day hamsters. Following initiation of this rise, concentrations that were typical of the nighttime peak were achieved within 2 h of melatonin rhythm onset, regardless of photoperiod. To determine whether clock control of the rising phase of the melatonin rhythm, in the absence of photoperiod cues, may be disrupted by perturbations in the ambient magnetic field, hamsters in constant darkness were acutely exposed to a 1-Gauss, 60-Hz magnetic field for 15 min or were daily exposed to this treatment for 14 or 21 days. Neither the melatonin rise in pineal content or circulation during subjective night was affected by acute or chronic magnetic field exposures; testes regression similarly occurred in sham and daily magnetic field-exposed hamsters in constant darkness. These findings indicate that magnetic field exposures are unlikely to serve as a zeitgeber for the circadian mechanism that controls onset of the melatonin rhythm; rather, photoperiod is a predominant cue that may differentially regulate the rising phase of melatonin production in the pineal gland and concentration in circulation.

Effect of various acute 60 Hz magnetic field exposures on the nocturnal melatonin rise in the adult Djungarian hamster.

Acute exposure to a 1 Gauss 60 Hz magnetic field for 15 min beginning 2 hr before darkness delays and blunts the nighttime melatonin rhythm in some but not all studies. To determine whether other exposure parameters (dose, mode, or time) influence the nocturnal melatonin rise, adult Djungarian hamsters reared in long days (16L:8D, lights off at 1000-1800 hr) were acutely exposed to a 60 Hz continuous magnetic field (15 min of 1 or 0.1 Gauss) beginning 4 hr before or 4 hr after lights off. Other hamsters were exposed to a 60 Hz intermittent magnetic field (15 or 60 min of a 1 Gauss field, 1 min on then 1 min off) between 1 and 2 hr before lights off. In sham-exposed controls, i.e., hamsters simultaneously placed in an adjacent coil system but without current, pineal and serum melatonin concentrations increased from a low baseline (1 hr after lights off) to concentrations that were typical of the nighttime peak by 3 hr after darkness. Acute exposure to the 0.1 or 1 Gauss continuous magnetic field for 15 min at either 4 hr before or 4 hr after lights off did not disrupt the nocturnal rise in pineal or serum melatonin. Similarly, onset of the melatonin rhythm was not suppressed by intermittent magnetic field exposures compared to that in sham controls. Thus, several magnetic field exposure paradigms failed to alter the rising phase of the melatonin rhythm in pineal gland content or in circulation. These findings indicate that the biological clock mechanism that mediates photoperiodic time measurement in this seasonally breeding rodent is resistant to a variety of acute continuous or intermittent magnetic field exposures.

Biosynthesis of melatonin during early postnatal ontogeny in pineal gland of healthy rats and animals with hereditary retinal degeneration

The content of melatonin and its precursors in the pineal gland of healthy rats and rats with hereditary retinal degeneration is studied on days 20 and 40 of postnatal development. The measurements are performed between 02:30 and 03:00, when the content of melatonin is maximal. In December, the content of melatonin and N-acetylserotonin in 20-day-old rats with hereditary retinal degeneration is 1.9- and 3.2-fold higher than in healthy controls. The content of norepinephrine in the pineal gland of these rats is increased 1.8-fold. On postnatal day 40, no differences in the pineal content of indolamines are noted. An increase in the content of melatonin in 20-day-old rats with hereditary retinal degeneration in comparison with healthy controls is also observed in May. A decrease in the maximum nocturnal level of melatonin in May in comparison with December is shown both in rats with hereditary retinal degeneration (by 46%) and healthy controls (by 41%) only on postnatal day 40.

Afternoon injections of melatonin in the Djungarian hamster Phodopus sungorus: long lasting sex-specific effects and influence of acute treatment on the endogenous pineal melatonin rhythm.

The effects of exogenous melatonin on gonadal function and on the endogenous melatonin profiles of male and female Djungarian hamsters were investigated. Daily afternoon subcutaneous injections of melatonin (50 micrograms) in hamsters kept in long photoperiods (LD 16:8) led to gonadal inhibition in all the treated females (n = 13) but in only 2 of 13 males within the treatment period of 8 weeks (uteri: 244 +/- 11 mg in controls vs. 79 +/- 4 mg in treated hamsters, P < 0.001; ovaries: 13.6 +/- 0.6 mg in controls vs. 7.9 +/- 0.7 mg in treated hamsters, P < 0.001; testes: 1,021 +/- 54 mg in controls vs. 732 +/- 100 mg in treated hamsters, P < 0.05; and accessory glands: 641 +/- 38 mg in controls vs. 548 +/- 70 mg in treated hamsters, P > 0.05). These results indicate that there are some circumstances under which the gonadal responses of the reproductive organs of male and female Djungarian hamsters differ. Interestingly, pineal concentrations of melatonin were found to be significantly higher in males (P < 0.01), possibly indicating a more robust endogenous supply with the hormone, whereas serum melatonin levels were not significantly different between males and females. However, the subcutaneous injection of melatonin (5, 25, and 125 micrograms) exerted no acute effect on the endogenous, circadian melatonin-profile, independent of dosage and sex.

Mechanism by which 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) reduces circulating melatonin levels in the rat

We have previously shown that the prototype for halogenated aromatic hydrocarbons, 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD), diminishes serum melatonin concentration at the same dose in both the most TCDD-susceptible (Long-Evans, Turku AB; L-E) and the most TCDD-resistant (Han/Wistar, Kuopio; H/W) rat strain. The change developed within 24 h and persisted for at least 28 days after TCDD exposure; was independent of the time of day and was not associated with any morphological damage to the pineal gland. In the present study, we investigated the mechanism of this endocrine effect. Despite a 40–50% decrease in circulating melatonin levels, the pineal content of melatonin, serotonin and 5-hydroxyindole acetic acid remained unaltered and the rate-limiting enzyme of pineal melatonin biosynthesis, N-acetyltransferase, displayed only a relatively minor suppression in activity (30%) in TCDD-treated L-E rats. Likewise, TCDD did not influence the ability of pineal glands from L-E rats to synthesize and secrete melatonin in ex vivo or in vitro experiments. TCDD accelerated the disappearance of exogeneous melatonin from the serum in both rat strains. This enhancement probably did not originate in the liver, because liver perfusion studies revealed that even control rat livers were capable of total melatonin clearance in spite of the fact that the melatonin concentration far exceeded physiological levels. Urine excretion of the normal main metabolite of melatonin, 6-hydroxymelatoninsulfate, was reduced by TCDD treatment in both strains. This was accompanied by an altered HPLC pattern of metabolites, especially in H/W rats. We conclude that TCDD decreases serum melatonin levels in rats by enhancing the peripheral, evidently extrahepatic, metabolism of the hormone.