Elsevier

Brain Research

Volume 1203, 8 April 2008, Pages 89-96
Brain Research

Research Report
Daily oscillation of gene expression in the retina is phase-advanced with respect to the pineal gland

https://doi.org/10.1016/j.brainres.2008.01.073Get rights and content

Abstract

The photoreceptive retina and the non-photoreceptive pineal gland are components of the circadian and the melatonin forming system in mammals. To contribute to our understanding of the functional integrity of the circadian system and the melatonin forming system we have compared the daily oscillation of the two tissues under various seasonal lighting conditions. For this purpose, the 24-h profiles of the expression of the genes coding for arylalkylamine N-acetyltransferase (AA-NAT), nerve growth factor inducible gene-A (NGFI-A), nerve growth factor inducible gene-B (NGFI-B), retinoic acid related orphan receptor β (RORβ), dopamine D4 receptor, and period2 (Per2) have been simultaneously recorded in the retina and the pineal gland of rats under short day (light/dark 8:16) and long day (light/dark 16:8) conditions. We have found that the cyclical patterns of all genes are phase-advanced in the retina, often with a lengthened temporal interval under short day conditions. In both tissues, the AA-NAT gene expression represents an indication of the output of the relevant pacemakers. The temporal phasing in the AA-NAT transcript amount between the retina and the pineal gland is retained under constant darkness suggesting that the intrinsic self-cycling clock of the retina oscillates in a phase-advanced manner with respect to the self-cycling clock in the suprachiasmatic nucleus, which controls the pineal gland. We therefore conclude that daily rhythms in gene expression in the retina are phase-advanced with respect to the pineal gland, and that the same temporal relationship appears to be valid for the self-cycling clocks influencing the tissues.

Introduction

The retina and the pineal gland of mammals are components of the circadian system and display daily oscillations of biochemical, physiological, and morphological parameters. These changes are a consequence of oscillating gene expression in both tissues. Therefore, a subset of genes is under daily regulation in the retina (for review, see Iuvone et al., 2005) and the pineal gland (for review, see Karolczak et al., 2005). Among the daily regulated genes, the gene arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) has been investigated most intensively because of its importance for the formation of the hormone melatonin (for review, see Klein, 2007). In the retina, the daily AA-NAT rhythm not only occurs in vivo but is maintained in cultured retinas, persisting for at least 3 days in constant darkness (DD) indicating that AA-NAT rhythm generation is accomplished by an intrinsic self-cycling molecular oscillator (Tosini and Menaker, 1996, Tosini and Menaker, 1998, Sakamoto et al., 2000, Sakamoto et al., 2004). The retinal pacemaker (for review, see Ruan et al., 2006, Tosini and Fukuhara, 2002, Tosini and Fukuhara, 2003) appears to be located in photoreceptor cells (Tosini et al., 2007) and to target AA-NAT by the binding of clock gene products to an E-box in the AA-NAT promoter (for review, see Iuvone et al., 2005). In addition, AA-NAT gene transcription can be activated by the BMAL1/CLOCK heterodimer in cultured rat photoreceptors (Chen and Baler, 2000) suggesting that induction occurs primarily in photoreceptor cells. In the pineal gland, AA-NAT expression is driven by a circadian oscillator located in the suprachiasmatic nucleus (SCN), which in turn is entrained by the light/dark (L/D) cycle through neural inputs from the retina (for review, see Morin and Allen, 2006). The SCN sends signals to the pineal gland via the neurotransmitter noradrenalin, which, in the rat as in other rodents, induces nocturnal transcription of the aa-nat gene via β1- and α1-adrenoceptor stimulation and subsequent cAMP formation (for review, see Gupta et al., 2005, Simonneaux and Ribelayga, 2003). The functional integrity of the circadian system relies on the proper temporal phasing of its components (for review, see Morin and Allen, 2006), and thus, the temporal relationship between the retina and the pineal gland is of functional significance. Although the oscillations of each tissue have been investigated extensively (for review, see Iuvone et al., 2005, Karolczak et al., 2005), none of the studies has examined the temporal relationship between both systematically. Only two reports compare the oscillation of AA-NAT gene expression in the retina and the pineal gland. The findings described therein, however, do not provide a clear idea of the temporal relationship between AA-NAT transcription in both tissues, since the cyclical pattern in AA-NAT gene expression has been found to be phase-advanced in the retina under lighting conditions of LD 12:12 (Sakamoto and Ishida, 1998) but is synchronous in the retina and the pineal gland under extreme lighting regimes (LD 20:4 and LD 4:20) (Engel et al., 2004).

The aim of the present study has been to compare the oscillations of gene transcription in the retina and the pineal gland directly in order to contribute to our understanding of the relationship of these two compounds of the circadian and the melatonin forming system. For this purpose, we have first identified the genes under daily regulation in both the tissues and subsequently compared their oscillating patterns between the retina and the pineal gland of the same animals. Since the daily profiles of various genes in the retina and the pineal gland depend upon seasonal lighting conditions (Engel et al., 2004, Rohleder et al., 2006, Ribelayga et al., 1999, Spessert et al., 2006), the comparison between both tissues has been conducted under long days (LD 16:8) and short days (LD 8:16).

Section snippets

Identification of genes oscillating in the retina and the pineal gland

To compare oscillations of the retina and the pineal gland, we sought genes under daily regulation in both tissues. For this purpose, we compared the transcriptome of the retina and the pineal gland between ZT 18 and ZT 12, and between ZT 18 and ZT 6, under LD 12:12. To identify the genes in question, we first performed high density microarray analyses and subsequent real-time PCR of the retinal transcriptomes. Genes showing a more than two-fold change in transcript amount either between ZT 18

Discussion

In the present study, we have confirmed the daily oscillations of mRNA levels of the genes coding for aa-nat (Sakamoto and Ishida, 1998, Rohleder et al., 2006, Spessert et al., 2006, Borjigin et al., 1995, Sakamoto et al., 2002, Engel et al., 2005, Roseboom et al., 1996, Simonneaux et al., 2004), ngfi-a (Spessert et al., 2006, Carter, 1996, Gudehithlu et al., 1993), RORβ (Andre et al., 1998, Kamphuis et al., 2005, Baler et al., 1996), and Per2 (Rohleder et al., 2006, Engel et al., 2005,

Animals

Animal experimentation was carried out in accordance with the European Communities Council Directive (86/609/EEC). Adult male and female Sprague–Dawley rats (body weight: 150 to 180 g) were kept under standard laboratory conditions (illumination with fluorescent strip lights, 200 lux at cage level during the day and dim red light during the night; 20 ± 1 °C; water and food ad libitum) under various LD cycles (LD 12:12, LD 8:16, LD 16:8) for 3 weeks. When indicated, the rats were then kept for two

Acknowledgments

We thank Ms. U. Frederiksen and U. Göringer-Struwe for their excellent technical assistance. We also thank Ms. R. Dechau for secretarial help. The data contained in this study are included in theses presented by Sybille Zimmer, Oliver Rickes, and Nils Rohleder toward partial fulfillment of their degrees of medical doctor at the Johannes Gutenberg University, Mainz.

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