ARTICLE INFO

Article Type

Original Research

Authors

Talaei   S.A. (*)
Mohammadifar   M. (1)
Azami   A. (2)
Salami   M. (1)






(*) Physiology Research Center, Kashan University of Medical Sciences, Kashan, Iran
(1) Physiology Research Center, Kashan University of Medical Sciences, Kashan, Iran
(2) Anatomical Sciences Research Center, Kashan University of Medical Sciences, Kashan, Iran

Correspondence

Address: Physiology Research Center, Kashan University of Medical Sciences, Qotb e Ravandi Blvd., Kashan, Iran
Phone: +983155621157
Fax: +983155621157
talaei@kaums.ac.ir

Article History

Received:  February  23, 2015
Accepted:  May 29, 2015
ePublished:  September 20, 2015

BRIEF TEXT


… [1] The critical period for brain development in Wistar rats is considered to be about 6 weeks [2]. Mammals rely more on visual system to communicate with their environment, and signals received from that, especially in the critical period of brain development, have important role in the maturation of brain circuits [3]. The role of evolution of these messages in the critical period has been investigated and proven in various studies [4, 5].

Changes in the way of receiving visual messages can affect the brain development [6-8]. … [9-14] The expression of melatonin receptors in the mammalian nervous system changes with the changing age [15]. The amount of expression of these neurohormone receptors is different in different phases of the circadian rhythm [16]. So far, the changes in the expression of hormone receptors have not been studied in relation to age in the critical period of brain development in the hippocampus, and it is not clear whether these changes are affected by the changes in visual experience or not. … [17, 18]

The purpose of this study was to investigate the effect of interaction of age and visual deprivation during critical period of brain development on expression of melatonin receptors in hippocampus of the rats.

This is an experimental study.

36 male Wistar rats kept at animal home at 22 ± 2°C and 55 ± 5% humidity with free access to water and food were studied.

Animals were chosen randomly.

All used chemicals were purchased from Sigma Company (Aldrich; USA). Animals, in terms of lighting conditions of their maintenance, were divided in two main groups: light room (LR) and dark room (DR). LR animals were kept in the natural condition of animal home from their birth to the moment of experiment, i.e. 12 hours of lightness and 12 hours of darkness; and DR rats were placed in complete darkness from their birth to the end of experiment (24 hours). Each main group was divided in to three subgroups (each had 6 rats). Animals in one of these subgroups were enrolled in the study at the age of two weeks (2WDR and 2WLR). The second subgroup was entered the study at the age of four weeks (4WDR and 4 WLR), and the third group was enrolled the study at the age of six weeks (6WDR and 6WLR). To evaluate the expression of genes related to receptors, RT-PCR method was used. Using the kit (Rosche; Germany), cDNA was prepared from samples. PCR for target genes as well as HPRT gene was carried out using thermo-cycler (PeqSTAR 96X, Peqlab Co.; Germany). PCR product was got electrophoresis upon medium of 2% agarose gel, and having been stained with Ethidium Bromide, bands were observed with UV tech device and photographed. The related images were analyzed using IamgeJ 1.48 software and the ratio of expression of each gene to HPRT gene was calculated. In order to study the protein expression of the mentioned receptors, also, Western Blot technique was used. Bradford method was used to measure the amount of extracted protein. Laemmli was added to the samples and the samples were got electrophoresis at 120 volts for 2 hours. To transfer beta actin, MT1, and MT2 proteins to PVDF paper (Polyvinyl Din Fluride, Roche Company; Germany), semi-arid transfer technique was used in 10 volts for 35 minutes. Then, blots were blocked with prepared 5% skim milk in TBST (Tris-Buffered Saline and Tween) for one hour. In the next stage, blots were incubated with primary antibody with dilutions of 1:5000, 1:500, and 1:200 for beta actin (Abcam; USA), MT1 and MT2 (Santa Cruz; USA) at 4°C overnight, respectively. Then blots were washed in TBST and incubated with second antibody (Abcam; USA) with dilution 1:3000 for one hour at room temperature. Blots, again, were washed in TBST (three phases, each 10 minutes) and PBS (one phase for 10 minutes) and having been stained with the solution of chemiluminescence (AceGlow, Peqlab Co.; Germany), they were placed in darkroom in vicinity of photographic film and then the films appeared. The films were scanned and images were analyzed using Image J 1.48 software and the ratio of expression of each protein to Beta actin protein was calculated. Data was analyzed using One-way Multivariate Analysis of Variance and Tukey test. “P<0.05” was considered significant.

The difference between the mean values of expression of MT1 receptor gene in the hippocampus of Wistar rats in different groups was significant. MT1 receptor mRNA expression was reached from 1.45 ± 0.07 in the hippocampus of rats in group 2WLR to 1.81 ± 0.04 in the hippocampus of rats in group 6WLR. Statistical analysis showed that the difference between groups 2WLR and 4WLR as well as the difference between groups 4WLR and 6WLR was not significant for relative expression of MT1 receptor gene (Diagram 1A). Keeping animals in absolute darkness from birth reversed the trend of expression of mentioned receptor gene, so that relative mRNA expression of this receptor reached from 1.10 ± 0.03 in group 2WDR to 0.84 ± 0.07 in the group 6WDR. Also, difference between groups 2WDR and 4WDR as well as difference between groups 4WDR and 6WDR was not significant for relative expression of MT1receptor gene. Inter-group comparison of MT1 receptor gene expression showed that the expression of this gene in animals kept in darkness was lower than those of animals kept in light. Along with aging, relative expression of MT2 gene was increased in the hippocampus of animals kept in the lightness and the expression of gene in the groups kept in darkness was reduced (Diagram 1B). In addition, statistical analysis of data showed a significant difference between the mean of relative expression of mentioned subunit gene in different groups of experiment. The mentioned receptor mRNA expression in the hippocampus of Wistar rats was reached from 1.22 ± 0.04 in the hippocampus of rats in group 2WLR to 1.65 ± 0.12 in group 6WLR. Tukey Post-hoc test showed that the difference between groups 2WLR and 4WLR as well as the difference between groups 4WLR and 6WLR was not significant for relative expression of gene in this receptor. Also, the relative mRNA expression of this receptor was reached from 0.93 ± 0.05 in group 2WDR to 0.71 ± 0.04 in group 6WDR. Statistical analysis showed that the difference between groups 2WDR and 4WDR as well as the difference between groups 4WDR and 6 WDR was not significant for relative expression of MT2 receptor gene. Inter-groups comparison of MT2 receptor gene expression indicated that the expression of this gene in animals kept in darkness was lower compared to those at the same age kept in lightness. The difference between the relative expressions of MT1 protein in the hippocampus was significant in different groups. With aging, relative expression of this protein increased about 36% in LR group (Diagram 1C). In other words, the difference between groups 2WLR and 6WLR was significant. However, the difference between groups 2WLR and 4WLR, as well as between 4WLR and 6WLR was not significant. Keeping animals in absolute darkness caused reversal MT2 protein expression in the hippocampus of Wistar rats and its expression was reached from 0.84 ± 0.03 in group 2WDR to 0.56 ± 0.04 in group 6WDR. Inter-group comparison of MT1 receptor protein expression indicated that its expression was lower in animals kept in darkness compared to those with the same age kept in lightness. MT2 receptor protein relative expression in Wistar rats in groups kept in lightness had time-dependent increase and Turkey Post-test results from its expression in the groups kept in darkness revealed significant difference between groups 2WLR and 6WLR. However, the difference between groups 2WLR and 4WLR as well as 4WLR and 6WLR was not significant. Light deprivation of animals led to the 50% reduction in expression of protein in this receptor from 0.73 ± 0.05 in group 2WDR to 0.35 ± 0.03 in group 6WDR (Diagram 1D).Statistical analysis showed a significant difference between the mean of relative expression of the mentioned protein in different groups of experiment. Data analysis showed that the difference between 2WDR and 4WDR groups, as well as between 4WDR and 6WDR, was not significant.

The rate of gene and protein expression of both MT1 and MT2 melatonin receptors in Wistar rats kept in normal condition of animal home was increased about 36% in a time-dependent trend from the age of 2 weeks to the end of 6 weeks. Keeping Wistar rats in absolute darkness caused a reversal trend in the gene and protein expression of both receptors in the hippocampus from the age of 2 to 6 weeks. RT-PCR technique has demonstrated that both melatonin receptors are existed in different areas of the hippocampus in Wistar rats [19]. Although the expression of melatonin receptors in the supra-chiasmatic nucleus (SCN) of the brain has growing trend during fatal period, their expression significantly reduces after birth, while it does not change significantly up to one month and it increases up to one month later, until the brain becomes mature, and then its expression remains constant [20]. MT1 receptor expression in the SCN of hamster decreases from birth up to 60 days [21]. Although the expression of both melatonin receptors in the eyes of the rats has decreasing trend from the age 14 to 60 days, but reduction in expression of MT2 is more obvious [22].

The effect of change in circadian rhythms and change in light reception rhythm on melatonin receptors expression should be studied in visual cortex.

Non-declared

Along with aging in the critical period of brain development, the expression of both melatonin receptors in hippocampus of the rats grows and visual deprivation reverses this trend.

Research Deputy of Kashan University of Medical Sciences is appreciated.

The authors have no conflict of interest.

Principles of working with laboratory animals were observed in accordance with Tehran Treaty as well as instructions approved by Ethics Committee of Research Department of Kashan University of Medical Sciences. The mentioned research project was approved by Ethics Committee of Research Department of Kashan University of Medical Sciences.

This study has been sponsored by Research Department of Kashan University of Medical Sciences.

TABLES and CHARTS

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