Daily rhythm and regulation of clock gene expression in the rat pineal gland

doi:10.1016/j.molbrainres.2003.10.019

Molecular Brain Research

Volume 120, Issue 2, 5 January 2004, Pages 164-172

https://doi.org/10.1016/j.molbrainres.2003.10.019 Get rights and content

Abstract

Rhythms in pineal melatonin synthesis are controlled by the biological clock located in the suprachiasmatic nuclei. The endogenous clock oscillations rely upon genetic mechanisms involving clock genes coding for transcription factors working in negative and positive feedback loops. Most of these clock genes are expressed rhythmically in other tissues. Because of the peculiar role of the pineal gland in the photoneuroendocrine axis regulating biological rhythms, we studied whether clock genes are expressed in the rat pineal gland and how their expression is regulated.

Per1, Per3, Cry2 and Cry1 clock genes are expressed in the pineal gland and their transcription is increased during the night. Analysis of the regulation of these pineal clock genes indicates that they may be categorized into two groups. Expression of Per1 and Cry2 genes shows the following features: (1) the 24 h rhythm persists, although damped, in constant darkness ; (2) the nocturnal increase is abolished following light exposure or injection with a β-adrenergic antagonist; and (3) the expression during daytime is stimulated by an injection with a β-adrenergic agonist. In contrast, Per3 and Cry1 day and night mRNA levels are not responsive to adrenergic ligands (as previously reported for Per2) and daily expression of Per3 and Cry1 appears strongly damped or abolished in constant darkness.

These data show that the expression of Per1 and Cry2 in the rat pineal gland is regulated by the clock-driven changes in norepinephrine, in a similar manner to the melatonin rhythm-generating enzyme arylalkylamine N-acetyltransferase. The expression of Per3 and Cry1 displays a daily rhythm not regulated by norepinephrine, suggesting the involvement of another day/night regulated transmitter(s).

Introduction

Although the mammalian pineal gland has lost its capacity for endogenous rhythmicity in the course of evolution [12], [27], it still synthesizes and releases melatonin with daily and seasonal rhythms under the control of the master biological clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus [23]. The endogenous rhythmicity of the hypothalamic pacemaker relies upon genetic regulation involving a set of “clock” genes: three Period genes (Per1, Per2, Per3), two Cryptochrome genes (Cry1 and Cry2) and two other genes coding for transcription factors (Clock and Bmal1), all working in intercalating positive and negative feedback loops to produce 24-h oscillations [32], [35]. These clock transcription factors, besides acting on the transcription of their own genes, directly regulate the expression of other genes, such as the gene coding for vasopressin [19]. Such “clock-controlled genes” are considered as clock outputs that relay the timing information to other central and peripheral structures, among which is the pineal gland [7].

Most clock genes are expressed in structures outside of the SCN in central and peripheral structures [1], [4], [5], [45]. One of these structures is the pineal gland which expresses Per1, Per2, Clock and Bmal1 genes [14], [29], [39]. Because the pineal gland is part of a photoneuroendocrine system that includes the retina and the SCN, both of which contain an endogenous clock, it was remarkable to find that the pineal gland expresses clock genes.

The pineal gland synthesizes and releases melatonin at night upon the clock-driven noradrenergic input [2], [10], [23]. Melatonin is synthesized from serotonin following acetylation by the arylalkylamine N-acetyltransferase (AA-NAT), methylation by the hydroxyindole-O-methyltransferase (HIOMT) and then is immediately released into the bloodstream [20], [22]. In the rat pineal gland, the nocturnal release of norepinephrine induces a marked cAMP/phosphorylated cAMP-responsive element binding protein (P-CREB)-dependent increase in Aa-nat gene expression followed by a large rise in AA-NAT protein and enzyme activity [6], [26], [34], whereas HIOMT mRNA and activity are barely increased [33]. This result indicates that AA-NAT is the rate-limiting enzyme for the nocturnal synthesis of melatonin and the level of Aa-nat mRNA in the rat correlates with the adrenergic state of the gland [25], [36].

The finding that the rat Aa-nat gene promoter contains an E-box sequence (DNA binding site for the CLOCK/BMAL1 dimer) again raised the question of an endogenous clock in the mammalian pineal gland [9]. Transfection of rat pinealocytes with Clock and Bmal1-containing expression vectors, however, was unable to induce Aa-nat gene expression, although this was possible in cultured rat photoreceptors [9]. By contrast, the CLOCK/BMAL1 dimer is able to activate Per1 gene expression in transfected pinealocytes [15], indicating that the dimer is functioning in the pineal gland.

The role of the clock genes Per1, Per2, Clock and Bmal1 in the mammalian pineal physiology is therefore still unknown. Further experiments are needed to delineate whether the pineal contains other clockwork components and determine how these genes are regulated. In the present study, we report that the other clock genes, Per3, Cry1 and Cry2, are expressed in the rat pineal gland. In addition, we have analyzed, in parallel to that of Aa-nat, how expression of Per1, Per3, Cry1 and Cry2 coding genes are regulated by circadian, adrenergic and light components.

Section snippets

Animals

Male adult Wistar rats (250–350 g) were purchased from a local breeder (Faculté de Pharmacie, Strasbourg, France) and kept for at least 1 week in our animal facilities (12 h light/12 h dark (dim red light), with lights off at 20:00; 20±1 °C; food and water supplied ad libitum) before experimentation.

All experiments were performed in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC) with all efforts made to minimize animal suffering and to use the number of

Specific hybridization of rat pineal mRNA with Per1, Per3, Cry2 and Cr1 riboprobes

The specificity of the riboprobes was assessed on brain tissue sampled at 00:00. The labeling of the Per1, Per3, Cry2 and Cry1 riboprobe in the rat pineal was specific as determined by the difference between total (antisense) and non-specific (sense) hybridization, both being run in parallel (Fig. 1). In addition, the specificity of the antisense riboprobe binding to its molecular target was further assessed by the displacement of the antisense radio-riboprobe binding with a 20-fold excess of

Discussion

Soon after the discovery that the endogenous rhythmicity of the mammalian hypothalamic circadian clock relies upon self-sustained expression of clock genes ([32], [35] for reviews), it was reported that clock genes are also expressed in a circadian manner in both central and peripheral structures including the retina and the pineal gland [1], [4], [45]. Isolated retina from rodents maintain an endogenous rhythmicity in melatonin release and Per1 gene expression ([40], [41] for review). In most

Acknowledgements

Authors are very grateful to Dr. Leslie Krushel for English correction and to Dr. David Hazlerigg for helpful discussion. This study was supported by the Fondation Simone et Cino del Duca.

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Attention deficit/hyperactivity disorder (ADHD) is frequently associated with comorbid aggression and sleep disturbances. The sleep/wake cycle is under the control of the circadian system which is moderated by clock genes. Clock genes can regulate the transcription of monoamine oxidase A, which is involved in the degradation of monoamines. Disturbances in monoamine interaction with clock genes in those with monoamine gene polymorphisms may regulate susceptibility of ADHD and comorbid aggression/sleep disturbances. While monoamines influence circadian rhythm and clock gene expression, circadian rhythm components modulate aggressive behavior, and altered clock genes expression have been associated with ADHD. We propose a mechanism by which circadian rhythm and clock gene expression may influence ADHD and comorbid aggression through the modulation of neurotransmitters. The role of clock genes in ADHD patients with comorbid aggression awaits further research; therefore we also indicate directions for future studies to help increase understanding of the underlying mechanisms in ADHD with comorbid aggression and sleep disturbances.
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The SCN possesses a set of proteins that generate self-sustained positive and negative transcriptional feedback loops with a free-running period of 24 h, called clock genes (Dardente and Cermakian, 2007; Duguay and Cermakian, 2009; Okamura et al., 2002). The expression of the clock genes has been characterized in almost all cell types in mammals, including in the pineal gland (Fukuhara et al., 2005; Namihira et al., 1999; Simonneaux et al., 2004; Wongchitrat et al., 2009; Wu et al., 2009), where it seems to have an important role, for example, in controlling the timing of Aanat mRNA expression (Fukuhara et al., 2005). The technique of pineal gland organ culture has been performed in a relative similar way since 70s (Parfitt et al., 1976).
Although the norepinephrine (NE) synchronization protocol was proved to be an important procedure for further modulating in vitro pineal melatonin synthesis, the maintenance of clock genes under the same conditions remained to be investigated. The aim of this study was to investigate the maintenance of the clock genes expression in pineal gland cultures under standard and NE-synchronized stimulation. The glands were separated into three experimental groups: Control, Standard (acute NE-stimulation), and NE-synchronized. The expression of Bmal1, Per2, Cry2, Rev-erbα, the clock controlled gene Dbp and Arylalkylamine-N-acetyltransferase were investigated, as well as melatonin content. No oscillations were observed in the expression of the investigated genes from the control group. Under Standard NE stimulation, the clock genes did not exhibit a rhythmic pattern of expression. However, in the NE-synchronized condition, a rhythmic expression pattern was observed in all cases. An enhancement in pineal gland responsiveness to NE stimulation, reflected in an advanced synthesis of melatonin was also observed. Our results reinforce our previous hypothesis that NE synchronization of pineal gland culture mimics the natural rhythmic release of NE in the gland, increasing melatonin synthesis and keeping the pineal circadian clock synchronized, ensuring the fine adjustments that are relied in the clockwork machinery.
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The photoreceptive teleost pineal is considered to be essential to the generation, synchronisation and maintenance of biological rhythms, primarily via melatonin release. The role of internal (circadian clock) and external (light) signals controlling melatonin production in the fish pineal differs between species, yet the reasons underpinning this remain largely unknown. Whilst in salmonids, pineal melatonin is apparently regulated directly by light, in all other studied teleosts, rhythmic melatonin production persists endogenously under the regulation of clock gene expression. To better understand the role of clocks in teleost pineals, this study aimed to characterise the expression of selected clock genes in vitro under different photoperiodic conditions in comparison to in vivo in both Atlantic salmon (Salmo salar) and in European seabass (Dicentrarchus labrax) (in vitro 12L:12D), a species known to display endogenous rhythmic melatonin synthesis. Results revealed no rhythmic clock gene (Clock, Period 1 & 2) expression in Atlantic salmon or European seabass (Clock and Period 1) pineal in vitro. However rhythmic expression of Cryptochrome 2 and Period 1 in the Atlantic salmon pineal was observed in vivo, which infers extra-pineal regulation of clocks in this species. No rhythmic arylalkylamine N-acetyltransferase 2 (Aanat2) expression was observed in the Atlantic salmon yet in the European seabass, circadian Aanat2 expression was observed. Subsequent in silico analysis of available Aanat2 genomic sequences reveals that Atlantic salmon Aanat2 promoter sequences do not contain similar regulatory architecture as present in European seabass, and previously described in other teleosts which alludes to a loss in functional connection in the pathway.
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Symptoms of opiate withdrawal include disturbances in circadian rhythms. We examined in male Wistar rats (n=48) the effects of a daily, mid-morning morphine injection (5–40 mg/kg, i.p.) and its withdrawal on 24-h wheel-running activity and on the expression of the clock protein, PERIOD2 (PER2), in the suprachiasmatic nucleus (SCN), oval nucleus of the bed nucleus of the stria terminalis (BNSTov), central amygdala (CEA), and dorsal striatum. Rats were killed over 2 days at 10, 22, 46, and 58 h after the last daily morphine injection at zeitgeber times (ZT) 1 or ZT13. Daily morphine injections and their withdrawal suppressed nighttime wheel running, but did not entrain any increase in activity in advance of the injection. Neither morphine injection nor its withdrawal affected PER2 expression in the SCN, whereas the normal daily peaks of PER2 in the BNSTov, CEA, and dorsal striatum were blunted both during morphine administration and its withdrawal. Treatment with a dopaminergic agonist (the D2/3 agonist, quinpirole, 1.0 mg/kg) or a noradrenergic agonist (alpha2 agonist, clonidine, 0.1 mg/kg) in morphine withdrawal did not restore normal PER2 patterns in each affected region; however, both quinpirole and clonidine themselves altered normal daily PER2 expression patterns in morphine-naive rats. These findings confirm and extend previous observations that opiates disrupt daily patterns of clock gene expression in the limbic forebrain. Furthermore, catecholaminergic drugs, which have been previously found to alleviate symptoms of opiate withdrawal, do not alleviate the effects of morphine withdrawal on PER2, but do modulate daily patterns of PER2 expression in saline controls.

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Research reportDaily rhythm and regulation of clock gene expression in the rat pineal gland

Abstract

Introduction

Section snippets

Animals

Specific hybridization of rat pineal mRNA with Per1, Per3, Cry2 and Cr1 riboprobes

Discussion

Acknowledgements

J. Biol. Chem.

Cell

Neuroscience

Mol. Brain Res.

Neurosci. Lett.

Prog. Neurobiol.

Neurosci. Lett.

Neuroscience

Cell

Cell

Brain Res.

Neurosci. Res.

Neurosci. Lett.

Brain Res.

Curr. Biol.

Circadian rhythms in isolated brain regions

J. Neurosci.

Stimulation of C14-melatonin synthesis from C14-tryptophan by noradrenaline in rat pineal in organ culture

Proc. Natl. Acad. Sci.

Clock genes in mammalian peripheral tissues

Cell Tissue Res.

Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland

Nature

Hypothalamic integration of central and peripheral clocks

Nat. Rev., Neurosci.

Pineal and retinal peptides and their receptors

Circadian clock system in the pineal gland

Mol. Neurobiol.

Research report
Daily rhythm and regulation of clock gene expression in the rat pineal gland

Stimulation of C¹⁴-melatonin synthesis from C¹⁴-tryptophan by noradrenaline in rat pineal in organ culture