Expression of mt1 melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT–PCR study across the diurnal cycle

doi:10.1016/S0169-328X(99)00222-3

Molecular Brain Research

Volume 72, Issue 2, 1 October 1999, Pages 176-182

https://doi.org/10.1016/S0169-328X(99)00222-3 Get rights and content

Abstract

Melatonin acts on specific receptors in the suprachiasmatic nuclei (SCN) to phase-dependently regulate the phase of the circadian clock. How the gating of melatonin's effect is restricted to particular times of day is not known, but may be related to temporal differences in receptor availability. In the present study, we used a competitive reverse transcription–polymerase chain reaction (RT–PCR) method to determine if the expression of mt₁ melatonin receptor subtype mRNA in rat SCN varied across the 12:12 light–dark (LD) cycle. Measurement of core body temperature using radiotelemetry confirmed that the male Wistar rats used exhibited a robust diurnal rhythm. mt₁ receptor mRNA was readily detected in reduced SCN slices at all times of day. However, there was no significant variation in the amount of mt₁ mRNA with time of day. Expression of MT₂ melatonin receptor subtype mRNA in reduced SCN slices was confirmed by nested PCR. These results indicate that changes in the level of mt₁ mRNA do not underlie the diurnal and/or circadian variation in the response of the SCN circadian clock to the phase-resetting effects of melatonin.

Introduction

In mammals, the synthesis and release of the pineal hormone melatonin is regulated by the circadian clock located in the suprachiasmatic nucleus (SCN) of the hypothalamus [10]. The environmental light–dark (LD) cycle synchronizes or entrains this intrinsic circadian SCN clock to the 24 h day (see Ref. [9]for review). Melatonin in turn can phase-dependently regulate the phase of the circadian clock via specific, high affinity melatonin receptors in the SCN 1, 13. In vitro autoradiography has detected melatonin binding sites in the SCN of adult rodents, and two G-protein coupled melatonin receptor subtypes have been cloned (mt₁ and MT₂) 17, 18. While expression of mt₁ subtype mRNA in rat SCN was readily detected by in situ hybridization [17], MT₂ mRNA was not detected [18], although it could be identified by reverse transcription–polymerase chain reaction (RT–PCR) in retina and brain. Recent work using in situ hybridization has detected MT₂ mRNA in mouse SCN [5].

The melatonin receptor subtype involved in the phase resetting action has not been specifically examined in the rat. However, in the mouse, targeted disruption of the mt₁ subtype blocked the acute inhibitory effects of melatonin on SCN firing, but did not prevent the phase-shifting effect of melatonin, even though these animals had no detectable high affinity melatonin binding sites in SCN [12]. Pharmacological studies using MT₂ selective receptor antagonists has implicated a role for this subtype in the phase-shifting effect in this species [5]. In the Siberian hamster, however, it is clear that the MT₂ subtype has no role as this species does not express a functional MT₂ receptor because of two nonsense mutations within the coding region of the gene, yet melatonin does phase advance the circadian rhythm in SCN neuronal firing rate [25].

The mechanisms responsible for the temporal gating of the effects of melatonin on SCN physiology are unknown, but are possibly linked to temporal differences in the availability of melatonin receptors and/or the coupling of these receptors to the second messenger systems such as protein kinase C (PKC) 14, 15. Several studies have indicated that the level of [ $^{125} I$ ]iodomelatonin binding sites in the rodent SCN may vary across the diurnal cycle 6, 11, 22. The maximal difference in density is modest in all studies (50–67%), and there are some differences in the timing of maximal and minimal density. A recent study [15]describes a dramatic change in the level of expression of the mt₁ receptor subtype mRNA in the rat SCN across the LD cycle which may drive the diurnal change in receptor density. In the present study, we used a competitive, quantitative RT–PCR procedure to determine whether the mRNA for the mt₁ subtype of melatonin receptor shows significant differences in the level of expression in the rat SCN across the day–night cycle.

Section snippets

Animals

Male Wistar rats (inbred King's College London strain) were housed under a LD schedule of 12 h of light and 12 h of dark (L on 07:00 h) with food and water available ad libitum, and room temperature of 21±1°C. To monitor the physiological and behavioural rhythms of the animals, a representative sample of rats (23 out of 36 animals) were anaesthetized with halothane and a telemetry probe (VM-FH disc model, ∼4.0 g; Mini-Mitter, OR, USA) implanted in the i.p. cavity. All animals were then housed

Quantitation of mt₁ receptor subtype expression in SCN

Using the mt₁ receptor subtype primers a single PCR product of the expected size (360 bp) was amplified from 34 of the 36 SCN cDNA samples. This product was shown by sequencing to be derived from authentic mt₁ receptor mRNA. No PCR product was amplified from SCN samples when the RT step was omitted (data not shown). A competitor DNA (280 bp) having the same primer annealing sequences as mt₁ cDNA was prepared by low stringency PCR, and was shown to amplify with very similar efficiency (94%

Discussion

In this study, we found that the expression of mt₁ receptor mRNA was readily detectable in isolated rat SCN tissue. When examined across the LD cycle, no significant differences in the level of expression of mt₁ receptor mRNA in the SCN could be detected. The absence of a significant rhythm in the expression of the mt₁ receptor mRNA in the rat SCN suggests that either it is not regulated by the circadian clock or that diurnal changes occur only in selective regions of the SCN which are

Acknowledgements

We are grateful to Dr. Jiri Vanecek, Institute of Physiology, Prague for providing the mt₁ receptor primers. This work was supported through a postgraduate studentship from the Royal Thai Government to S.A., a Tempus exchange studentship to K.D., and a MRC project grant to H.D.P. (G9622937). We thank Dr. Clive Coen for the use of the telemetry equipment and Dr. Anne Stirland for her comments on the manuscript.

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Agomelatine can act through melatonin receptors to synchronize circadian rhythms [57], and the combination of effects on melatonin and 5-HT2C receptors has been proposed to mediate its therapeutic effects [58,59]. Both 5-HT2C and melatonin receptors are found in the SCN [18–23] and a 5-HT2C agonist can induce expression of c-Fos and the circadian clock genes Per1 and Per2 in the SCN [60]. Thus, the dual actions of agomelatine acting through these different receptor types may contribute to its antidepressant properties.
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These results are consistent with the hypothesis that agomelatine acts directly on the SCN via both agonist effects at melatonergic receptors and antagonist effects at 5-HT_2C receptors, which parallel its mechanisms of action as an antidepressant.
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Citation Excerpt :
Two PCR-based investigations on the day/night expression of MT1 mRNA were recently conducted but led to conflicting results [21,38]. One paper reported a dramatic increase in mRNA expression during the daytime [21], while the second study could not detect any significant daily variation [38]. The competitive PCR procedure used by Sugden et al. [38] is, incontestably, more reliable than the semi-quantitative estimation of MT1 mRNA transcription expressed relative to β-actin mRNA expression performed by Neu and Niles [21] especially since housekeeping gene expression cannot be assumed to remain perfectly constant throughout the 24-h period.
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¹: Present address: School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK.

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Research reportExpression of mt1 melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT–PCR study across the diurnal cycle

Abstract

Introduction

Section snippets

Animals

Quantitation of mt1 receptor subtype expression in SCN

Discussion

Acknowledgements

Mol. Brain Res.

Brain Res.

Neuron

Brain Res.

Mol. Brain Res.

Neuron

Neuron

J. Biol. Chem.

Mol. Cell Endocrinol.

Melatonin and circadian control in mammals

Experientia

Maximizing sensitivity and specificity of PCR by pre-amplification heating

Nucleic Acids Res.

A comprehensive set of sequence analysis programs for VAX and CONVEX systems

Nucleic Acids Res.

Research report
Expression of mt₁ melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT–PCR study across the diurnal cycle

Quantitation of mt₁ receptor subtype expression in SCN