Immunohistochemical analysis of 1A type melatonin receptors density in the supraoptic nucleus neurons of the hypothalamus of white rats in altered photoperiod
Introduction. Circadian rhythms organization in biological systems depends on the interaction between central units of control after oscillatory processes in the body and brain structures intermediaries in the form of so-called functional chronobiological blocks [Hyldebrandt and et al., 2006]. One of these blocks is formed by the relationship between SON and pineal gland [Reiter et al., 2014]. Through the melatonin receptors the hormone controls the state of the hypothalamic-pituitary system and the activity of the endocrine glands [Ishii et al., 2015].
Objective. To provide the quantitative circadian characteristic of melatonin receptor density in the neurons of the supraoptic nucleus of the rats’ hypothalamus by means of immunohistochemical techniques combined with computer microdensitometry.
Materials and methods. Experiments were conducted on 40 outbred white mature male rats weighing 0,15-0,18 kg. The animals were kept in cages at constant temperature, humidity and free access to water and food. Experimental animals were divided into 2 series each with 2 groups (10 animals each) which were under conditions of standard light regime - 12.00L: 12.00D (light from 08.00AMto 08.00 PM, fluorescent lamps LB-40, the room was lit at the animals level at 200 lux) and hyperilluminated one (day and night light (24.00L: 00T) fluorescent lamps LB-40, room light at animals level was 500 lux) for 7 days. In order to detect differences in circadian melatonin receptors and taking into consideration the cyclical production of melatonin euthanasia of rats was performed with 12-hour interval (02.00AM and 02.00 PM) by decapitation on the 8th day. Chosen dates for the experiment were due to different functional activity of the pineal gland and production of leading chronobiotic - MT in these time periods. Commission on bioethical expertise of Bukovinian State Medical University found that all stages of the experiment were conducted in compliance with the essential requirements of the Helsinki Declaration and the requirements of the Council of Europe on Human Rights and Biomedicine (1977), the provisions of the WHO International Code of Medical Ethics (1983) and the laws of Ukraine (protocol number 22 of November 28, 2007).
For immunohistochemical study fragments of brain cortex with supraoptic nucleus area of the hypothalamus were fixed in 10% solution of neutral buffered formalin for 22 hours. After that a rapid dehydration in ascending concentrations of alcohol was performed, then it was embedded in paraffin at 580C, followed by obtaining histological sections 5 microns thick.
In order to perform immunohistochemical methods we used polyclonal antibodies to melatonin receptor 1A manufactured by Abcam (UK) and streptavidin-biotin visualization system LSAB2 (peroxidase label + diaminobenzidine) manufactured by Chemicon International Inc. (USA). We followed the protocol of standardization methods for all sections at most. Additional nuclei staining was performed with Mayer hematoxylin.
Quantitative research of the staining intensity was conducted as follows. First, using a microscope lens x 40 we received digital copies of optical image which were further analyzed by a licensed copy of the computer program "VideoTesT - Size 5.0" (OOO VideoTest, Russia) that is we conducted the computer microdensitometry. The analysis was performed on the basis of measurements by means of microprobe technique in the field of positive staining in terms of "optical density" (in relative units with a range of 0-1, where "0" corresponds to the absolute optical transparency microprobe, and "1" - absolute optical opacity). The intensity of the specific staining (Score "optical density") was identified with the degree of density of melatonin receptors.
Taking into consideration the need to perform multiple statistical comparisons of averages in statistical samplings and to determine the differences between sets of criteria we used Newman-Keuls test.
Results. The indices of optical density of specific M1A neurocytes of SON staining obtained in the intact group (at 02.00 AM- 0,488 ± 0,0024, at 02.00 P.M. - 0,464 ± 0,0023, p = 0.002) and in animals subjected to light stress (at 02.00 AM-0,295 ± 0, 0019, at 02.00 P.M.- 0,286 ± 0,0018, p = 0,012) had a probable value and were characterized by a clear diurnal periodicity. In the group of animals with pineal gland hypofunction modulation (at 02.00 A.M.- 0,216 ± 0,0017, at 02.00 P.M. - 0,214 ± 0,0021, p> 0,05).
Conclusions. The density of 1A melatonin receptors in rat’s hypothalamic neurons of SON are normally characterized by a accurate circadian rhythm. The highest density of receptors is observed at 02.00 AM, and at 02.00 PM it is significantly lower (p = 0.002).
Immunohistochemical studies revealed that under inhibition of pineal gland activity the circadian rhythm of melatonin receptors density in neurons of supraoptic nuclei of the hypothalamus gets disturbed, which is characterized by an incredible difference of indices in the tested periods of the day.
We are planning further immunohistochemical analysis with a light deprivation of rats for possible disturbances in circadian rhythm of density of 1A melatonin receptors in rat’s hypothalamic neurons of SON.