Hamster Pineal Gland Research Articles

Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Daytime Gating in the Syrian Hamster Pineal Gland

Melatonin synthesis in rodents is tightly regulated at the transcriptional level by stimulatory and inhibitory transcription factors. Among them, phosphorylated cAMP-related element binding protein (pCREB) and inducible cAMP early repressor (ICER), a strong inhibitor of cAMP-related element-driven genes, have an antagonistic action in activating/inhibiting the transcription of the Aa-nat gene, which is an important enzyme in melatonin synthesis. In the Syrian hamster, a rodent displaying a seasonal control of reproduction, melatonin synthesis is strongly gated to the second part of the night. Indeed, exogenous adrenergic stimulation is unable to stimulate Aa-nat gene transcription and melatonin synthesis during daytime. In the present study, we investigated whether ICER may be the cause of this daytime repression by comparing the dynamic of ICER and the adrenergic regulation of two genes whose expression is rapidly activated by cAMP-dependant mechanisms, c-fos and Icer. Adrenergic induction of c-fos and Icer expression was not possible during daytime, except at early day. ICER immunoreactivity was elevated throughout the daily cycle but reached the highest levels at early day, when gene expression can be induced by adrenergic agonists. Additionally, CREB phosphorylation was subjected to the same daily gating with an adrenergic induction occurring in the early but not in the late day. Taken together, our results indicate that the diurnal gating of pineal activity in the Syrian hamster is not caused by the repressor ICER and that it may occur at the level of noradrenergic receptor signalling.

Endogenous rhythmicity of Bmal1 and Rev‐erbα in the hamster pineal gland is not driven by norepinephrine

Pineal melatonin is synthesized with daily and seasonal rhythms following the hypothalamic clock-driven release of norepinephrine (NE). The pineal gland of rats and mice, like the biological clock, expresses a number of clock genes. However, the role of pineal clock elements in pineal physiology is still unknown. We examined the expression and regulation of several clock genes (Per1, Cry2, Bmal1 and Rev-erb alpha) under different lighting conditions or following adrenergic treatments in the Syrian hamster, a seasonal rodent. We found that Per1 and Cry2 genes were similarly regulated by the nocturnal release of NE: levels of Per1 and Cry2 mRNA displayed a nocturnal increase that was maintained after 2 days in constant darkness (DD) but abolished after 2 days under constant light (LL), a condition that suppresses endogenous NE release, or after an early night administration of the adrenergic antagonist propranolol. In contrast, Bmal1 and Rev-erb alpha exhibited a different pattern of expression and regulation. mRNA levels of both clock genes displayed a marked daily variation, maintained in DD, with higher values at midday for Bmal1 and at day/night transition for Rev-erb alpha. Remarkably, the daily variation of both Bmal1 and Rev-erb alpha mRNA was maintained in LL conditions and was not affected by propranolol. This study confirms the daily regulation of Per1 and Cry2 gene expression by NE in the pineal gland of rodents and shows for the first time that a second set of clock genes, Bmal1 and Rev-erb alpha are expressed with a circadian rhythm independent of the hypothalamic clock-driven noradrenergic signal.

Transcription factors may frame Aa-nat gene expression and melatonin synthesis at night in the Syrian hamster pineal gland.

Pineal melatonin synthesis is stimulated at night following an increase in arylalkylamine-N-acetyltransferase (AA-NAT) activity. Depending on the species, two mechanisms of enzyme activation have been described: a cAMP/phospho-cAMP response element-binding protein-dependent stimulation of Aa-nat gene transcription in the rat, presumed to occur in all rodents, or a posttranslational regulation of AA-NAT protein in ongulates. The present data obtained in the Syrian hamster indicate another route of AA-NAT regulation. Elevated nocturnal levels of Aa-nat mRNA were strongly suppressed following light exposure or adrenergic antagonist administration, demonstrating the involvement of norepinephrine in the stimulation of melatonin synthesis. However, administration of adrenergic agonists during the day did not increase Aa-nat mRNA unless a protein synthesis inhibitor was given during the previous night. This indicates that an inhibitory protein, synthesized at night, prevents melatonin synthesis during the day. By contrast, a protein synthesis inhibitor given at the beginning of the night markedly reduced Aa-nat mRNA, suggesting that a stimulatory protein (transcription factor?) is necessary for Aa-nat gene transcription at night. Noteworthy, hamsters raised in long photoperiod were responsive to adrenergic agonist injection only in the first hour after light onset, a response that may be important in this photoperiodic species in which the melatonin peak extends into the morning hours in a short photoperiod.

Mechanisms regulating the marked seasonal variation in melatonin synthesis in the European hamster pineal gland.

Like many wild species, the European hamster (Cricetus cricetus) adapts to the marked seasonal changes in its environment, namely by hibernation and inhibition of sexual activity in winter. These annual functions are driven by the variation in the environmental factors (light, temperature) that are transmitted to the body through large variations in the duration and amplitude of the nocturnal melatonin rhythm. Here we report that the seasonal variation in melatonin synthesis is mainly driven by arylalkylamine N-acetyltransferase gene transcription and enzyme activation. This, however, does not exclude participation of hydroxyindole-O-methyltransferase, which may relay environmental temperature information. The in vivo experiments show that norepinephrine stimulates melatonin synthesis, this effect being gated at night. The possibility that the variation in pineal metabolism depends on a seasonal change in the suprachiasmatic nuclei clock circadian activity that is transmitted by norepinephrine is discussed.

Expression and regulation of Icer mRNA in the Syrian hamster pineal gland

Inducible-cAMP early repressor (ICER) is a potent inhibitor of CRE (cAMP-related element)-driven gene transcription. In the rat pineal gland, it has been proposed to be part of the mechanisms involved in the shutting down of the transcription of the gene coding for arylalkylamine N-acetyltransferase (AA-NAT, the melatonin rhythm-generating enzyme). In this study, we report that ICER is expressed in the pineal gland of the photoperiodic rodent Syrian hamster although with some difference compared to the rat. In the Syrian hamster pineal, Icer mRNA levels, low at daytime, displayed a 20-fold increase during the night. Nighttime administration of a β-adrenergic antagonist, propranolol, significantly reduced Icer mRNA levels although daytime administration of a β-adrenergic agonist, isoproterenol, was unable to raise the low amount of Icer mRNA. These observations indicate that Icer mRNA expression is induced by the clock-driven norepinephrine release and further suggest that this stimulation is restricted to nighttime, as already observed for Aa-nat gene transcription. Furthermore, we found that the daily profile of Icer mRNA displayed photoperiodic variation with a lengthening of the nocturnal peak in short versus long photoperiod. These data indicate that ICER may be involved in both daily and seasonal regulation of melatonin synthesis in the Syrian hamster.

Role of extracellular calcium on the regulation of melatonin synthesis in the Syrian hamster pineal gland.

The role of extracellular calcium on the induction of cyclic AMP production, N-acetyltransferase (NAT) activity and melatonin synthesis, induced by forskolin, was investigated in Syrian hamster pineal glands. Pineals were removed from hamsters killed either in the first half of the normal dark period or late in the dark period. Forskolin immediately increased NAT activity and melatonin levels only when the glands were collected late in the dark period, while in the first half of the dark phase, hamster pineals responded to forskolin with an increase of NAT activity and melatonin production only after 6 hr of incubation. The absence of calcium prevented the induction of melatonin synthesis by forskolin only when glands were collected early in the dark phase and incubated for 6 hr. In the second half of the normal dark period removal of calcium markedly decreased NAT activity and melatonin levels in glands incubated with forskolin for either 4 or 6 hr. However, the absence of extracellular calcium had no significant effect on the induction of cyclic AMP production by forskolin in pineals collected either early in the dark period or late in the dark phase. These data indicate that at least part of the action of extracellular calcium is indirect and that it affects steps in the induction of melatonin synthesis beyond the accumulation of cAMP.

Molecular cloning of the arylalkylamine- N-acetyltransferase and daily variations of its mRNA expression in the Syrian hamster pineal gland

The arylalkylamine- N-acetyltransferase (AA-NAT) expressed in the vertebrate pineal gland catalyzes the N-acetylation of the serotonin into N-acetylserotonin and is considered to be the rate limiting enzyme of the pineal melatonin synthesis. Indeed, dramatic changes in its activity throughout the 24-h period drive the large day/night variations in plasma melatonin concentrations. Recently, AA-NAT was cloned in the rat pineal. In this species, AA-NAT mRNA variations were demonstrated to be responsible of the well known AA-NAT activity and plasma melatonin circadian fluctuations. In the Syrian hamster, the pineal melatonin secretion pattern is characterized by a late-night short-duration peak of melatonin synthesis. We investigated whether this typical pattern could be due to a late-night delayed pineal AA-NAT mRNA expression. The first part of our study was dedicated to the molecular cloning of a Syrian hamster AA-NAT cDNA. A PCR-generated clone of 1045 bp encoding the AA-NAT has been isolated and sequenced. In situ hybridization using an AA-NAT cRNA probe revealed that the AA-NAT mRNA expression undergoes strong daily fluctuations in the Syrian hamster pineal, with undetectable level in the second half of the light period and a dramatic increase at night. After lights off, the AA-NAT mRNA expression requires 6–7 h to reach its maximum expression. This result thus suggests that the transcription of the AA-NAT mRNA in the Syrian pineal gland determines the lag period in pineal responsiveness and melatonin synthesis to darkness.

RF exposure of biological systems in radial waveguides

Two radial waveguide exposure systems for multiple biological targets are described that were conceived, dimensioned, implemented, measured and dosimetrically analyzed. In the first case, a number of 120 hamsters was exposed with mobile communication signals at 383 and 900 MHz. The variation of the dissipated energy within the animals could be reduced to less than 30% though the objects were not restrained. Whole body specific absorption rates of 80 mW/kg were applied. The other system was used for exposing hamster's pineal glands in culture. The small size of the samples yields an even smaller variation of the exposure of 2.4%. This is a consequence of the excellent uniformity of the electromagnetic field distribution across the exposure region, which was achieved by a good rotational symmetry of the waveguide and a careful design of the feed antenna and the absorber/shortcut combination. The paper reports on the technical aspects of the RF exposure.

Possible involvement of neuropeptide Y in the seasonal control of hydroxyindole- O-methyltransferase activity in the pineal gland of the European hamster ( Cricetus cricetus)

Hydroxyindole- O-methyltransferase (HIOMT) catalyses the last step of all the 5-methoxyindoles synthesized in the pineal gland. The synthetic activity of this neuroendocrine structure is driven not only by noradrenaline but also by various neuropeptides. Recently we have established (1) that one of these neuropeptides, neuropeptide Y (NPY), stimulates specifically HIOMT activity in rat pinealocytes and (2) that the density of the NPY-immunoreactive (NPY-IR) fibers innervating the pineal gland of the European hamster ( Cricetus cricetus) displays seasonal variations with a large increase in the late autumn. These findings have led us to evaluate a possible seasonal control of NPY on the European hamster pineal gland. We thus compared the nycthemeral patterns of pineal HIOMT activity and 5-methoxytryptophol (5-ML) content and of circulating MEL levels in European hamsters when NPYergic innervation is low (end of October) and when it is the highest (mid-December). We report in this study that HIOMT activity is significantly increased by 80% in mid-December compared with end of October. This increase is correlated with the appearance of a nycthemeral rhythm of pineal 5-ML levels (with a fourfold increase occurring in early dawn and decreasing slowly towards the end of the day). These observations suggest that NPY could be an important neurotransmitter involved in the seasonal control of the biochemistry of the European hamster pineal gland via a stimulatory effect on HIOMT activity.

Indoleamine Metabolism in the Pineal Gland of the Chinese Hamster,Cricetulus griseus

Day- and nighttime contents of pineal melatonin and other indoleamines were measured in the Chinese hamster,Cricetulus griseus.The hamsters were inbred in our colony and kept in LD 14:10. Absolute levels of pineal melatonin andN-acetylserotonin contents were extremely low, but showed a significant circadian rhythm with a minor peak during the light period. Pineal hydroxytryptophan and serotonin (5HT) contents were also found to be higher in the light period. The activity ofN-acetyltransferase in the pineal was higher in the light period than in the dark period, whereas there was no difference in hydroxyindole-O-methyltransferase activity between the light and dark periods. The uptake level of tryptophan into the pineal gland was higher in the light period than in the dark period. These results suggest that the 5HT synthesis pathway and/or tryptophan uptake may be involved in forming the circadian rhythm of 5HT content in the Chinese hamster pineal gland.

Daily time course of the contents in monoamines and their metabolites in the pineal gland of Syrian hamster

Twenty-four h changes in endogenous tyrosine hydroxylase (TH) activity, monoamines and their metabolites as well as N-acetyltransferase (NAT) activity and melatonin content were investigated in the Syrian hamster pineal gland. Both NAT activity and melatonin content exhibited an expected rise 8 h (0400 h) after darkness onset. Pineal dopamine (DA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) levels were highest during the dark phase reaching a peak at 0200 h, just prior to the rise in melatonin production. However no significant difference were found in both norepinephrine (NE) and TH activity levels during the 24 h period. However, in the present study, the rat pineal gland exhibited a clear day/night difference in its TH activity. These data suggest a possible role of pineal DA in the induction of melatonin synthesis in addition to its function as a NE precursor in the Syrian hamster pineal gland and also indicate that catecholamines may act on the hamster pineal in a different manner than in the rat.

Daily variation in the content of indoleamines, catecholamines and related compounds in the pineal gland of Syrian hamsters kept under long and short photoperiods.

This study examined the diurnal changes in the content of 5-hydroxytryptophan (5-HTP), serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), N-acetylserotonin (NAS), melatonin, 5-methoxytryptophol (5-ML), noradrenaline (NA), dopamine (DA), and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the pineal gland of Syrian hamsters kept under long (14L:10D) and short (10L:14D) photoperiods. The nocturnal increase in NAS and melatonin levels was dependent upon the prevailing photoperiod, with a prolonged duration when the night lengthened. In both photoperiods, NAS and melatonin contents increased several hours after the onset of darkness, and, in animals kept in short photoperiod, the levels of both compounds began to decrease before light onset. On the contrary, decreases were noted in 5-HT, 5-HIAA, and 5-ML contents during the night, which was directly proportional to the dark phase. 5-HTP levels did not show a rhythmic variation. Correlations between the mean values of 5-HT-related compounds showing daily rhythms were very high when group means were compared, but they decreased when values from individual animals were considered. In addition, when correlations were calculated on per-animal basis during the night phase, a weak negative correlation was found for 5-HT vs NAS and 5-HT vs melatonin, although the correlation of 5-HT with positively 5-HT-correlated compounds (5-HIAA and 5-ML) continued to be high. These results indicate that the nocturnal increase in the N-acetyl transferase activity is the major factor generating the rhythm of pineal 5-HT content, but that other photoperiod-dependent mechanisms (i.e., 5-HT synthesis or release) seem to be also implicated. On the other hand, this study shows that NA content in the Syrian hamster pineal gland does not exhibit daily variations, although marked nocturnal increases in the levels of DA and DOPAC were evident. These results suggest the existence of parallel daily alterations in pineal catecholamine synthesis and release, and suggest a role for DA in the pineal activation at night.

Day- and nighttime content of monoamines and their metabolites in the pineal gland of rat and hamster

Day- and nighttime content of catecholamines, serotonin and their metabolites were measured in the pineal gland of Sprague-Dawley rats and Djungarian hamsters. In addition, monoamine turnover rates were determined in the hamster pineal gland following administration of α-methyl- p-tyrosine. Animals were decapitated in the middle of the light or dark period, respectively, and pineal tissue was analysed by high performance liquid chromatography with electrochemical detection. Pineals of both species exhibited day/night-differences in serotonin, 5-hydroxyindole-3-acetic acid and dopamine content. The hamster pineal gland further showed day/night differences in its content of epinephrine and 3-methyoxy-4-hydroxyphenylethyleneglycol. The dopamine turnover rate was augmented at night, while norepinephrine turnover was constant. Immunohistochemical incubations of pineal paraffine sections showed fibers and terminals stained by antisera to tyrosine-hydroxylase (TH) and dopamine-β-hydroxylase, and a few perikarya-like structures exhibiting TH immunoreactivity. The results support the view that dopamine, rather than only functioning as a norepinephrine precursor, is actively involved in the control of melatonin synthesis.

S‐Antigen and glial fibrillary acidic protein immunoreactivity in the in situ pineal gland of hamster and gerbil and in pineal grafts: Developmental expression of pinealocyte and glial markers

Postnatal development of S-Ag and GFAP immunoreactivity in the in situ pineal glands of golden hamsters and gerbils was examined using the avidin-biotin-peroxidase immunohistochemical technique. S-Ag was present in the gerbil pineal gland on the first postnatal day (P1), whereas it did not appear in the hamster pineal until P6. GFAP-immunoreactive astrocytes were first observed in the hamster pineal gland on P7 and in the gerbil pineal gland on P10. The number of S-Ag-immunoreactive pinealocytes and GFAP-immunoreactive astrocytes in the pineal glands of hamsters and gerbils increased with increasing age from P7 to 3 weeks. By 4 weeks, strong S-Ag and GFAP immunoreactivity was observed in both hamster and gerbil pineal glands. GFAP-immunoreactive stellate astrocytes were distributed evenly throughout the gerbil superficial pineal gland, but they were more often located in the peripheral region of the hamster superficial pineal. For the pineal grafts, pineal glands from neonatal (3-5 day old) hamsters were transplanted into the third cerebral ventricle (infundibular recess or posterior third ventricle) or beneath the renal capsule of adult male hamsters. S-Ag immunoreactivity appeared in the pineal grafts within 1 week following transplantation. By 4 weeks the pineal grafts showed strong S-Ag immunoreactivity which was maintained until at least 12 weeks after transplantation. The time course of glial cell maturation in the cerebroventricular pineal grafts is generally parallel to the hamster pineal gland in situ before 4 weeks. By 12 weeks, however, more astrocytes differentiated and developed GFAP-immunoreactivity in the pineal grafts than in the in situ pineals. These studies have described the postnatal development of S-Ag and GFAP immunoreactivity in in situ pineal glands and in neonatal pineal grafts.

Determination of 5-methoxyindoles in pineal gland and plasma samples by high-performance liquid chromatography with electrochemical detection

A liquid chromatographic analysis with electrochemical detection of 5-methoxytryptamine, 5-methoxytryptophol, 5-methoxyindoleacetic acid and melatonin is described. Optimal elution conditions were determined by studying several variables: pH, buffer salt, counter ion and organic modifier. Measurement of 5-methoxyindoles in the pineal gland and plasma of hamsters has been performed after extraction. This method is specific and sensitive, and enables detection of 5-methoxyindoles in a pool of two hamster pineal glands. This is also the first time that these three 5-methoxyindoles have been measured simultaneously in plasma.

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

In the present study, "synaptic" ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of "synaptic" ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for "synaptic" spherules, indicating different roles for these two structures.

MRNA transcription determines the lag period for the induction of pineal melatonin synthesis in the Syrian hamster pineal gland

The nocturnal pattern of Syrian hamster pineal melatonin synthesis is characterized by a 6-8 h lag period, followed by a late-night, short-duration peak in both N-acetyltransferase (NAT) activity and melatonin content. Administration of cycloheximide (20 mg/kg body weight) given either at the time of lights out or 4 h into the dark phase to Syrian hamsters blocked the nocturnal increase in both pineal NAT activity and melatonin content. Actinomycin D (5 mg/kg body weight) prevented the nocturnal increase in both constituents only when it was administered at darkness onset, being significantly less effective when injected after 4 h of dark exposure. Reinduction of melatonin production by isoproterenol (2 mg/kg body weight) administration to acutely light-exposed animals during late darkness was prevented by cycloheximide, but not by actinomycin D administration. The results suggest that whereas Syrian hamster pineal melatonin production requires protein synthesis both early and late in the dark phase, the transcription of a putative NAT-related mRNA, which occurs only during the early night, seems to determine the lag period in melatonin synthesis and pineal responsiveness to beta-adrenergic receptor agonist stimulation.

Effects of Stimulation of the Superior Cervical Ganglia and Local Application of Noradrenaline on Electrical Activity of the Syrian Hamster Pineal Gland

Abstract The effects of electrical stimulation of either or both superior cervical ganglia and the effects of local application of L-noradrenaline on the spontaneous electrical activity of hamster pineal cells were evaluated. Extracellular recordings from pineals of anaesthetized hamsters revealed that the spontaneous electrical activity was mainly regular with interspike intervals attributed between 12 to 20 ms during the daytime and mainly irregular with interspike intervals of 1 to 250 ms during the night. Following stimulation of the superior cervical ganglia, either unilaterally or bilaterally, or local application of noradrenaline, the responses of these pineal cells fell into three major categories: A) non-responsive, B) excited, and C) inhibited. There was no relationship between the magnitude or form of response and the source of stimulus i.e. the right superior cervical ganglia or the left superior cervical ganglia. Almost all inhibited responses from electrical stimulation of the superior cervical ganglia could be correlated with inhibited responses from the local application of noradrenaline whereas excited responses could not. In general, these results suggest that the spontaneous electrical activity of some pineal cells is influenced by inputs from the superior cervical ganglia and that the inhibitory input is likely to be mediated through the release of noradrenaline. The excitatory input from the superior cervical ganglia is probably mediated by another neurotransmitter. The heterogeneity of responses suggests that different receptors or different cell types may be involved.

Treatment with forskolin for 8 hours during the day increases melatonin synthesis in the Syrian hamster pineal gland in organ culture: The long lag period is required for RNA synthesis

The exposure of organ-cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in melatonin levels when glands were collected in the second half of the dark period and incubated for 4 h. However, when glands were collected at the beginning of the dark period and incubated with the same drug, a significant increase in melatonin content was observed only after 6-8 h. Likewise, when glands were collected at the beginning of the light period, forskolin stimulated melatonin synthesis only after 6-8 h of incubation with the drug. These results support the existence of a relatively long lag period necessary for the induction of melatonin production in the Syrian hamster pineal gland. Experiments with actinomycin D indicate that RNA synthesis occurs during the lag period; thus, actinomycin D blocks the induction of melatonin synthesis by forskolin in glands collected at the beginning of the dark period and incubated for 8 h. In contrast, when pineal glands were collected from hamsters killed in the second half of the dark period, and incubated with forskolin for either 4 or 8 h, actinomycin D was unable to block the induction of melatonin production. These data suggest that RNA, presumably messenger RNA, which is necessary for increasing hamster pineal melatonin synthesis, is synthesized and accumulates during the first half of the night.