Melatonin Receptor mRNA Expression Research Articles

Melatonin injection and red light irradiation affect the antioxidant response and cell damage in disk abalone (Haliotis discus hannai) exposed to high water temperatures.

The effects of red light-emitting diode (LED) light irradiation (630 nm, 0.5 W/m2) and melatonin (10-8 and 10-7 M) on oxidative stress and physiological responses in abalones exposed to high temperatures (28°C) were investigated. Changes in messenger RNA (mRNA) expressions of melatonin receptor (MT-R), heat shock protein 70 (HSP70), and antioxidant enzymes, as well as alterations in H2O2 levels in the hemolymph, were examined. The results revealed that high-temperature-stressed abalones treated with melatonin injections or exposed to red LED light showed a significant increase in MT-R mRNA expression, while HSP70 mRNA expression decreased. Notably, HSP70 mRNA expression levels in the red LED light-irradiated group were similar to those in the group injected with 10-8 M melatonin after 24 h exposure. Abalones treated with melatonin at 20°C or irradiated with red LED light exhibited decreased H2O2 levels and reduced antioxidant enzyme mRNA expression compared with those of the control group. However, the high-temperature environment induced oxidative stress in abalones, leading to increased antioxidant enzyme mRNA expression compared with that under 20°C conditions. Moreover, abalones exposed to high-temperature stress exhibited hepatopancreatic DNA damage, which was attenuated by melatonin treatment or red LED light irradiation. Hence, red LED light reduces oxidative stress, boosts antioxidant enzymes, and alleviates DNA damage in high-temperature-stressed abalones, akin to 10-8 M melatonin treatment. Therefore, considering the practical challenges of continuous melatonin administration to abalones, utilizing red LED light emerges as a practical, effective alternative to protect abalones from oxidative stress compared to 10-8 M melatonin treatment.

Photoperiodic modulation of melatonin receptor and immune genes in migratory redheaded bunting

The transcriptional regulation of innate immune function across annual life history states (LHS) remains obscure in avian migrants. We, therefore, investigated this in a migratory passerine songbird, redheaded bunting (Emberiza bruniceps), which exhibits long-distance vernal migration from India to Central Asia. We exposed the birds (N = 10) to differential photoperiodic conditions to induce a non-migratory (NM), pre-migratory (PM), migratory (MIG), and refractory (REF) state, and performed gene expression assays of melatonin receptors (MEL1A and MEL1B), and innate immunity-linked genes (IL1B, IL6, TLR4, and NFKB) in spleen and blood. We found a significant reduction in splenic mass and volume, and a parallel increase in fat accumulation, and testicular growth in birds under migratory state. The gene expression assay revealed an upregulation of MEL1A and MEL1B mRNA levels in both the tissues in MIG. Additionally, we found a nocturnal increase of splenic IL1B expression, and IL1B, IL6, and TLR4 expression in the blood. The mRNA expression of melatonin receptors and proinflammatory cytokine showed a positive correlation. These results suggest that melatonin relays the photoperiodic signal to peripheral immune organs, which shows LHS-dependent changes in mRNA expression of immune genes.

Novel Therapeutics for Treating Sleep Disorders: New Perspectives on Maydis stigma

Sleep is a restorative period that plays a crucial role in the physiological functioning of the body, including that of the immune system, memory processing, and cognition. Sleep disturbances can be caused by various physical, mental, and social problems. Recently, there has been growing interest in sleep. Maydis stigma (MS, corn silk) is a female maize flower that is traditionally used as a medicinal plant to treat many diseases, including hypertension, edema, and diabetes. It is also used as a functional food in tea and other supplements. β-Sitosterol (BS) is a phytosterol and a natural micronutrient in higher plants, and it has a similar structure to cholesterol. It is a major component of MS and has anti-inflammatory, antidepressive, and sedative effects. However, the potential effects of MS on sleep regulation remain unclear. Here, we investigated the effects of MS on sleep in mice. The effects of MS on sleep induction were determined using pentobarbital-induced sleep and caffeine-induced sleep disruption mouse models. MS extracts decreased sleep latency and increased sleep duration in both the pentobarbital-induced sleep induction and caffeine-induced sleep disruption models compared to the positive control, valerian root extract. The butanol fraction of MS extracts decreased sleep latency time and increased sleep duration. In addition, β-sitosterol enhances sleep latency and sleep duration. Both MS extract and β-sitosterol increased alpha activity in the EEG analysis. We measured the mRNA expression of melatonin receptors 1 and 2 (MT1/2) using qRT-PCR. The mRNA expression of melatonin receptors 1 and 2 was increased by MS extract and β-sitosterol treatment in rat primary cultured neurons and the brain. In addition, MS extract increased the expression of clock genes including per1/2, cry1/2, and Bmal1 in the brain. MS extract and β-sitosterol increased the phosphorylation of ERK1/2 and αCaMKII. Our results demonstrate for the first time that MS has a sleep-promoting effect via melatonin receptor expression, which may provide new scientific evidence for its use as a potential therapeutic agent for the treatment and prevention of sleep disturbance.

Retinal and extra-retinal photoreceptor responses and reproductive performance of Japanese quail (Coturnix coturnix japonica) following exposure to different photoperiodic regime

Japanese quail is a truly photoperiodic avian species. In general long days are gonado-stimulatory and short days are gonado-inhibitory for this poultry bird. To investigate the correlation of retinal and extra-retinal photoreceptors with different photoperiodic conditions quail were divided into 2 groups and kept under long day (16L: 8D) and short day (8L: 16D) condition separately to develop photosensitivity and scotosensitivity respectively. Transfer of long day quail to intermediate day-length (13.5L: 10.5D) developed photorefractoriness (relative) and prolonged exposure to short photoperiodic conditions led the birds to develop scotorefractoriness. Increased expression of mRNA and immunosignaling of photoreceptors rhodopsin, transducin in eye and hypothalamus while decreased mRNA expression of melatonin receptors (Mel1b, Mel1c) were noted in the eyes of photosensitive (PS) and scotorefractory (SR) quail compared to photorefractory (PR) and scotosensitive (SS) birds respectively. Decreased expression of hypothalamic GnIH and melatonin receptors mRNA was observed in PS and SR birds compared to PR and SS birds respectively. Modulation of retinal and extra retinal photoreceptors leads to increased spermatogenesis as well as mRNA expression of steroidogenic genes and androgen receptor in the testis of sexually active PS and SR quail. These results led us to conclude that gonadal stimulation in PS as well as SR quail is outcome of activated retinal and extra retinal photoreceptors which lowered melatonin receptors and GnIH expression. Contrarily testicular inhibition in PR and SS is the outcome of decreased photoperception. It is suggested that decreased photoperception in SS quail increases after prolong exposure of the short day (in SR) leading to increased activity of HPG axis.

Paraquat induced impaired reproductive function and modulation of retinal and extra-retinal photoreceptors in Japanese quail (Coturnix coturnix japonica)

Paraquat (PQ) being a potent herbicide, causes toxic effect on growth, development and reproduction of plant as well as in animals. In this study we have mainly focused on the toxic effect of PQ on photoperception via different photoreceptors present in retina, pineal and hypothalamus and thereby its effect on hypothalamic – pituitary – gonadal (HPG) axis. PQ was administered i.p.10 mg/kg body weight daily for 1 week in poultry birds Japanese quail (Coturnix coturnix japonica). Our findings clearly indicated decrease in immunoreactivity of retinal and extra retinal photoreceptors (Iodopsin, rhodopsin and transducin) following PQ treatment in comparison to control group. Increased immunoreactivity of GnIH was observed in testis and epididymis of PQ treated group. Decreased mRNA expression of photoreceptors (rhodopsin and melanopsin), steroidogenic genes, androgen receptor, GnRH-I were found in PQ treated group while increased mRNA expression of melatonin receptors (Mel 1a R, Mel 1b R, Mel 1c R) and GnIH were found in PQ treated group. Thus, from the present results it may be concluded that PQ treatment alters the photoperception via altering the expression of photoreceptors and also modulates the HPG axis thereby alters the reproductive functions in Japanese quails.

P.211 - Paravertebral muscles in adolescent idiopathic scoliosis: mRNA expression of melatonin receptors 1A/1B, calmodulin, and estrogen receptor-2

P.211 - Paravertebral muscles in adolescent idiopathic scoliosis: mRNA expression of melatonin receptors 1A/1B, calmodulin, and estrogen receptor-2

Effects of Lycium barbarum. polysaccharide on type 2 diabetes mellitus rats by regulating biological rhythms.

Type 2 diabetes mellitus (T2DM) is associated with circadian disruption. Our previous experimental results have showed that dietary Lycium barbarum. polysaccharide (LBP-4a) exhibited hypoglycemic and improving insulin resistance (IR) activities. This study was to explore the mechanisms of LBP-4a for improving hyperglycemia and IR by regulating biological rhythms in T2DM rats. The rats of T2DM were prepared by the high-sucrose-fat diets and injection of streptozotocin (STZ). The levels of insulin, leptin and melatonin were measured by enzyme linked immunosorbent assay (ELISA). The effect of LBP-4a on mRNA expression of melatonin receptors (MT2) in epididymal adipose tissue was evaluated by RT-PCR. The expression of CLOCK and BMAL1 in pancreatic islet cells was detected by Western blotting. Our data indicated that the 24-hr rhythm of blood glucose appeared to have consistent with normal rats after gavaged administration of LBP-4a for each day of the 4 weeks, and the effects of hypoglycemia and improving hyperinsulinemia in T2DM rats treated at high dose were much better than that at low dose. The mechanisms were related to increasing MT2 level in epididymal adipose tissue and affecting circadian clocks gene expression of CLOCK and BMAL1 in pancreatic islet cells. LBP-4a administration could treat T2DM rats. These observations provided the background for the further development of LBP-4a as a potential dietary therapeutic agent in the treatment of T2DM.

Kisspeptin regulates the somatic growth-related factors of the cinnamon clownfish Amphiprion melanopus

This study aimed to test the effects of kisspeptin (Kiss) on somatic growth in the cinnamon clownfish Amphiprion melanopus. We investigated the effects of Kiss treatment on the growth by measuring the mRNA expressions of the growth hormone (GH), insulin-like growth hormone factor (IGF-I), somatolactin (SL), and melatonin receptor (MT). The expression levels of GH and SL of the pituitary gland and IGF-I of the liver increased after Kiss treatment (in vivo and in vitro). In addition, the MT mRNA expression increased in the pituitary gland and brain after Kiss treatment (in vivo and in vitro). These results support the hypothesis that Kiss directly regulates the somatic growth-related factors, such as GH, SL, and MT, and IGF-I in the cinnamon clownfish. Further, injection of Kiss resulted in significantly higher levels of plasma melatonin than that in the control. We, therefore, conclude that Kiss plays a role in modulating growth and artificially induced rapid growth in cinnamon clownfish.

062 — (ROC0074) mRNA expression of melatonin receptors in rat hippocampus during the chronic phase of pilocarpine-induced temporal lobe epilepsy

062 — (ROC0074) mRNA expression of melatonin receptors in rat hippocampus during the chronic phase of pilocarpine-induced temporal lobe epilepsy

Melatonin receptor (MTNR1A and MTNR2B) expression during the breeding season in the yak (Bos grunniens)

Melatonin plays key roles in a wide range of mammalian body functions, which are mediated by the melatonin-specific cell surface receptor (MTNR1A and MTNR1B). To better understand the role of MTNR in the yak (Bos grunniens), we determined the melatonin receptor mRNA expression level. The analysis showed that the MTNR mRNA expression level was higher in the pineal gland tissue than in the hypothalamus, pituitary gland, and ovary (P < 0.01) during the breeding season. Immunofluorescence analyses showed that yak MTNR was located in the pinealocyte, synaptic ribbon, and synaptic spherules of the pineal gland and that melatonin interacts via nerve fibres. In the hypothalamus, MTNR was located in the magnocellular neurons and parvicellular neurons. MTNR was localized in acidophilic cells and basophilic cells in the pituitary gland. In the ovary, MTNR was present in the ovarian follicle, corpus luteum, and Leydig cells.  

Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish

The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin–melatonin receptor system may play a role in predicting the moon phase through changes in night brightness.

The presence of melatonin receptors and inhibitory effect of melatonin on hydrogen peroxide‐induced endothelial nitric oxide synthase expression in bovine cerebral blood vessels

Melatonin plays a key role in a variety of important physiological functions including influencing cerebral blood vessels. Therefore, in the present study, we have identified the existence of melatonin receptors and test the effect of melatonin on hydrogen peroxide-induced endothelial nitric oxide synthase (eNOS) expression in bovine cerebral arteries. The results indicate that mt1A melatonin receptor mRNA is expressed in bovine cerebral arteries. The relative levels of mt1A melatonin receptor mRNA expression in anterior, posterior, middle and vertebral cerebral arteries were compared. The data show the highest and lowest levels of mRNA expressions in the middle and vertebral cerebral arteries, respectively. The maximal number (B(max)) of different types of melatonin receptors in various regions of cerebral arteries were identified and further characterized by using the selective 2-[(125)I] iodomelatonin binding assay. Saturation studies revealed that the binding represented a single site of high affinity binding for the melatonin receptor with the highest and lowest binding capacities in the middle and vertebral arteries, respectively. In order to elaborate the functional significance of melatonin in cerebral blood vessels, hydrogen peroxide- induced induction in eNOS protein level and phosphorylation of calcium/calmodulain-dependent protein kinase II (phospho-CaMKII) were demonstrated in the bovine isolated cerebral arteries with these effect being abolished by melatonin. This is the first evidence showing expression of mt1A melatonin receptor in the bovine cerebral arteries. However, further studies are necessary to delineate the role of melatonin and its receptors in regulating physiology of the cerebral vessels.

Asymmetric Expression of Melatonin Receptor mRNA in Bilateral Paravertebral Muscles in Adolescent Idiopathic Scoliosis

Comparison of melatonin receptor mRNA expression in bilateral paravertebral muscles in adolescent idiopathic scoliosis (AIS). OBJECTIVES.: To investigate the change of melatonin receptor mRNA expression in bilateral paravertebral muscles in AIS, congenital scoliosis (CS), and control in order to analyze its association to the pathogenesis of AIS. Muscle imbalance and asymmetry of stretch receptors in the paravertebral muscles of patients with AIS were supposed to have a large role to play in the development and production of the deformity. Melatonin is a focus of studies of the mechanism underlying the development of scoliosis, and there is no research on the expression of melatonin receptors in the paravertebral muscles of patients with AIS. Twenty cases with average age of 15.1 +/- 2.2 years and average Cobb angle of 56.2 degrees +/- 16.1 degrees, including 10 cases with Cobb angle >50 degrees and 10 cases with Cobb angle < or =50 degrees, were included in AIS group. The apical vertebrae were from T6 to T11. Twelve cases with an average age of 11.6 +/- 3.2 years and average Cobb angle of 59.2 degrees +/- 33.3 degrees were included in CS group. The apical vertebrae were from T7 to T12. Ten cases without scoliosis were in the control group. The mRNA expression of melatonin receptor subtype MT1 and MT2 was detected by the RT-PCR method. The MT2 mRNA expression on the concave side of the paravertebral muscle was higher than that on the convex side in AIS and CS groups (P < 0.05), but the MT1 mRNA expression showed no significant difference (P > 0.05). In the AIS group, the ratio of MT2 mRNA expression on the concave side compared with the convex side in cases with Cobb angle >50 degrees and cases with Cobb angle < or =50 degrees showed no significant difference (P > 0.05). The MT1 and MT2 mRNA expression showed no significant difference in control group (P > 0.05). The melatonin receptor expression in bilateral paravertebral muscles in AIS is asymmetric, which may be a secondary change. The bilateral asymmetry in force exerted on the scoliotic spine may be the cause.

The expression of MT 1 and MT 2 melatonin receptor mRNA in several rat tissues

The mechanisms that mediate the various effects of melatonin in mammalian tissues are not always known. Therefore, the aim of this study was to investigate whether MT 1 and MT 2 melatonin receptors are expressed in certain tissues of the rat. The expression of MT 1 and MT 2 melatonin receptor mRNA was determined using a real-time quantitative RT-PCR method. In addition, we examined whether mRNA for either subtype of receptor shows any difference in the expression between midnight and noon, similar to the changes in melatonin concentrations in plasma and tissue samples. MT 1 and MT 2 melatonin receptor mRNAs were found in the rat hypothalamus, retina and small intestine. We also showed a low expression of MT 2 mRNA in the rat liver and heart SA node. In the heart apex and the Harderian gland, no appearance of either of the receptor mRNAs was detectable. A significant difference in the expression of MT 1 mRNA between day and night was found in the hypothalamus. In conclusion, our findings suggest that at least some effects of melatonin are mediated through membrane MT 1 and MT 2 receptors in the hypothalamus, the retina and the small intestine. Down-regulation of receptors might be one reason for the difference in the hypothalamic MT 1 melatonin receptor mRNA expression between midnight and noon. In the liver and the heart SA node, the physiological significance of possible MT 2 receptors remains unclear. According to our negative midnight and noon results in the Harderian gland and heart apex melatonin may exert its effect on these tissues by a non-receptor mechanism.

Implication for melatonin and its receptor in the spinal deformities of hereditary Lordoscoliotic Rabbits.

The association between melatonin system and the spontaneous development of the spinal deformities in the Hereditary Lordoscoliotic Rabbit, the natural animal model for idiopathic scoliosis, was studied. To examine the implication for melatonin and its receptor in the spinal deformities of the natural animal model, the Hereditary Lordoscoliotic Rabbit. We previously reported radiologic and histologic studies investigating the etiology of spinal deformities in a breed of Japanese White Rabbit, the Hereditary Lordoscoliotic Rabbit. These animals develop thoracic lordoscoliosis during growth and as such can be used as a model for human idiopathic scoliosis. Although previous studies in chickens have established that pinealectomy produces scoliosis, the cause of the condition is yet to be fully elucidated. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were measured by radioimmunoassay and compared with those of Japanese White Rabbits (controls). The expression of melatonin receptor in the rabbit was detected by homology cloning to access the number of the melatonin receptor mRNA in the rabbit spinal cord by quantitative reverse-transcribed polymerase chain reaction. Serum melatonin levels in Hereditary Lordoscoliotic Rabbits were significant higher than those of controls in each period until 20 weeks. We detected the expression of melatonin receptor mRNA in rabbit spinal cord. However, no significant quantitative differences were found in the level of expression of melatonin mRNA in the spinal cord between Hereditary Lordoscoliotic Rabbits and controls. In relation to the present study, we suggest that causes of spinal deformities in the Hereditary Lordoscoliotic Rabbit may be the result of the contribution of melatonin receptors as well as that of altered serum melatonin levels in the Hereditary Lordoscoliotic Rabbit. Further studies will be required to investigate the expression of melatonin receptor in other tissues of the Hereditary Lordoscoliotic Rabbit as well as to delineate the role of melatonin in the pathogenesis of idiopathic scoliosis.

MT1 melatonin receptor mRNA expression exhibits a circadian variation in the rat suprachiasmatic nuclei

The aim of the present study was to investigate the daily regulation of both MT1 and MT2 melatonin receptor subtype mRNA expression in the rat SCN in order to clarify their role in the daily variation of SCN melatonin receptors. Existing MT1 and MT2 partial clones were extended by PCR to 982 and 522 bp, respectively. However, while the MT1 clone allowed us to set up a highly sensitive in situ hybridization (ISH) method, we could not detect MT2 expression within the SCN. Therefore, our results suggest that only MT1 mRNA can be correlated with 2-iodo-melatonin binding sites in the rat SCN. Investigation of MT1 mRNA expression throughout the 24 h light/dark cycle or in constant darkness clearly showed that in the two conditions, mRNA expression showed a robust rhythm with two peaks, one after the day/night and one after the night/day transitions in LD, and at the beginning of the subjective night and day in DD, respectively. Furthermore, these variations were not linked to the daily changes in melatonin receptor density. Thus, the transcriptional regulation of MT1 receptors does not appear to play a role in the daily regulation of melatonin binding sites availability.

Melatonin Regulates the mRNA Expression of the mt1 Melatonin Receptor in the Rat Pars tuberalis

The pars tuberalis (PT) of the pituitary is a major neuroendocrine target site for melatonin as it contains a large number of high-affinity melatonin receptors. We have previously shown that melatonin autoregulates the density of its own receptors in the PT. However, whether melatonin regulation includes mRNA expression in vivo is unclear. In the present study we have used quantitative in situ hybridization to (1) follow the daily profile of mt₁ mRNA expression in the rat PT and (2) investigate whether mt₁ mRNA expression could be regulated in vivo by melatonin. We found clear diurnal variations of mt₁ mRNA expression that persist in constant darkness. We also showed, on pinealectomized animals, that the rhythmic pineal melatonin secretion is necessary for the expression of these daily variations. In a second step, we studied the effect of an acute suppression of endogenous melatonin synthesis on mt₁ melatonin receptors by applying a 1-hour light pulse during the night. We found that light induced a dramatic increase in mt₁ mRNA which was totally prevented by a melatonin injection showing that the acute effect of melatonin on the receptor mRNA is strongly inhibitory. A light pulse applied to animals with a chronic absence of melatonin was ineffective showing that light only affects melatonin receptors via the light-induced plasma melatonin suppression. Altogether our results show that melatonin regulates mt₁ melatonin receptor mRNA expression. However, this regulation seems to be complex: acute changes in plasma melatonin concentration regulate negatively the gene transcription, even if the daily endogenous nocturnal melatonin peak seems a prerequisite for variations in its receptor expression.

Melatonin receptor subtype expression in human cerebellum.

We report the distinct cellular distribution in the human cerebellar cortex of the mammalian mt1 (Mel1a) and MT2 (Mel1b) (1) melatonin receptor subtypes. Specific binding of the non-selective radioligand 2-[125I]iodomelatonin to the outer molecular layer was significantly higher than to the granule cell layer. Melatonin receptor subtype expression was assessed by in situ hybridization using selective and specific digoxigenin-labeled antisense oligonucleotide probes. This is the first demonstration of MT2 melatonin receptor mRNA expression in human cerebellar Bergmann glia and astrocytes. On the other hand, the mt1 melatonin receptor mRNA was expressed in both basket-stellate cells and granule cells. We conclude that mt1 and MT2 melatonin receptors are heterogeneously expressed in human cerebellar cortex.

Responsiveness to melatonin and its receptor expression in the aging circadian clock of mice.

This study determined the effect of age on the efficacy of melatonin treatment to phase shift circadian activity rhythms and on melatonin receptor expression in the suprachiasmatic nucleus (SCN) and paraventricular nucleus of the thalamus (PVNT) of C3H/HeN mice. The circadian rhythm of 2-[125I]iodomelatonin binding, assessed at three times of the day [circadian times (CT) 2, 10, and 18], showed a modest age-related decrease in the SCN but not the PVNT of old C3H/HeN mice (24 mo). There was a tendency for age to reduce Mel1a melatonin receptor mRNA expression in the suprachiasmatic nucleus during the day, but not during the night. The magnitude of phase shifts of circadian activity rhythms (advances or delays) induced by administration of melatonin at CT 10 or CT 2 was identical in young and old C3H/HeN mice. Together, these results suggest that the decrease in melatonin receptor expression in the SCN had little effect on melatonin-induced phase shifts of circadian activity rhythms. We conclude that the responsiveness of the circadian timing system to melatonin administration does not decrease with age.

Effect of a light pulse on melatonin receptor density and mRNA expression in Siberian hamster suprachiasmatic nuclei

Melatonin receptors located in the suprachiasmatic nuclei (SCN) have been cloned in mammals hypothalamus. We previously reported a SCN melatonin receptor photic regulation in the rat. The present study's aim was to investigate whether a photic regulation of both melatonin receptor density and mRNA expression takes place in the SCN of a highly photoperiodic species, the Siberian hamster. To achieve this goal, we first set up an in situ hybridization protocol without RNase treatment to allow a highly sensitive and reproducible quantitative analysis of melatonin receptor mRNA expression. Results showed that a 1 h light pulse delivered during night-time did not affect SCN melatonin binding capacities while it induced a strong increase of the melatonin receptor mRNA expression in the SCN of pinealectomized animals.