Bulyk R. Ye., Burachyk A. I., BulykТ. S., Kryvchanska M. I., Vlasova K. V.

MORPHOLOGY

Scentific article

The mechanisms of circadian pacemaker activity of the neuronal systems of the suprachiasmatic nucleus (SCN) of the hypothalamus are currently deeply researched. Meanwhile, information concerning the effects of photoperiod modifications (in particular, constant illumination or darkness) on the activities of specific structures involved in the formation of circadian rhythms remains relatively limited. We studied the circadian variation of the cytometric characteristics of the SCN neurons of the hypothalamus of rats, as well as the effect of experimental changes in the light-dark cycle and the introduction of exogenous melatonin on these parameters. Three groups of animals (12 rats each) were kept for seven days under normal photoperiod conditions (12.00L:12.00D, control group), constant illumination and prolonged darkness. The fourth group of animals under experimental conditions, like the rats of group 2, was injected intraperitoneally with melatonin (Sigma, USA, purification rate 99.5%) at a dose of 1.0 mg/kg, 1.0 ml of solvent (0.9% ethanol solution in NSS). To study the morphometric characteristics of the hypothalamus neurons, multiple histological sections were prepared according to a standard procedure on a rotary microtome. Morphometric analysis of the hypothalamus neurons and quantitative analysis of the content of RNA in them were performed on a VIDAS-386 computer system for digital image analysis in the visible region. Analyzing the morphometric parameters of neurons of the hypothalamus, the daily dynamics of the indices was found. Thus, compared to the daytime period (2 p.m.), till 2 a.m., a significant increase (by 7.8±1.5%) of the body area of the SCN neurons was observed, due to the growth of the cell nucleus area. In turn, the increase in the area of the nucleus of the neuron was due to the probable increase in the area of its nucleolus, which was 5.60±0.237 μm 2 . At the same time, at night, the observed nuclear-cytoplasmic ratio (N:C Ratios) in pacemaker neurons were 1.7±0.05% and significantly higher than in the daytime. While also, the specific volume of the neuron core increased by 18.2±2.16%, and the cytoplasm, on the contrary, decreased by 14.2±1.98%. These changes were combined with an increase in the concentration of RNA in the nuclei by 7.3±1.5%, as well as with an increase in the concentration of RNA in the nucleoli of neurons by 8.5±1.7% and the area occupied by them by 26.5±5.2% in comparison with the daily period. With the purpose of deeper analysis of place and role of the pineal gland, the leading neuroendocrine transducer of circadian periodism, in the functioning of the hypothalamus SCN, we performed morphometric studies of these nuclei under conditions of constant darkness and prolonged illumination (simulation of epiphyseal hyper- and hypofunction of the animals, respectively). In contrast to light deprivation, a prolonged light regime causes a more pronounced desynchronosis of the morphofunctional activity of SCN neurons, reduces the concentration of RNA in their structures, which is likely to be caused by a disruption of the synthesis of corresponding c-Fos proteins involved in the realization of the temporal organization of biological systems. In order to correct the disruptions caused by prolonged stay of rats at constant illumination, we used exogenous melatonin as one of the drugs. With the introduction of in dole on the background of light stress, a tendency to normalization of the parameters of the area of SCN neurons appeared about 2 p.m. In particular, during the day step of the study, the area of the neuron was 37.06±0.49 μm 2, of the nucleus, 23.59±0.374 μm 2 , of the nucleolus, 4.10±0.065 μm 2 . The indicated change in the size of the SCN neuron at 2 p. m. was due to an increase in the area of the nucleus and nucleolus. N:C Ratios were within 1.75±0.028 units, the specific volume of the nucleus was 63.7±1.02%, and the cytoplasm was 36.4±0.66%. As to the concentration of RNA in the structures of the pacemaker cells of the SCN, it was 0.273±0.0029 AU in the daytime step in the nucleus, 0.378±0.0031 AU in the nucleolus, and 0.146 ± 0.0015 AU in the cytoplasm. As to the comparison group, the N:C Ratio was proved higher in this diurnal period. At the same time, the concentration of RNA in the components of the SCN neurons also increased synchronously. However, according to morphometric measurements, the use of the hormone did not normalize the circadian rhythm of activity of pacemaker cells of the investigated nuclei. This gives grounds to state that the light regime is the dominant factor in the development of circadian rhythms.

suprachiasmatic nuclei, hypothalamus, morphofunctional condition, light deprivation,permanent lighting, melatonin

1. Arushanyan EB, Shchetinin EV. Melatonin kak universal’nyy modulyator lyubykh patologicheskikh protsessov. Patologicheskaya fiziologiya i eksperimental’naya terapiya. 2016;60(1):79-88. [in Russian].
2. Zamorskiy II, Sopova IYu, Khavinson VKh. Vliyanie melatonina i epitalamina na soderzhanie produktov belkovoy i lipidnoy peroksidatsii v kore bol’shikh polushariy i gippokampe mozga krys v usloviyakh ostroy gipoksii. Byulleten’ eksperimental’noy biologii i meditsiny. 2012;154(7):59-61. [in Russian]. 
3. Karachentsev YuI, Kravchun NA, redaktory. Pineal’naya zheleza i gipotalamo-gipofizarno-tireoidnaya sistema: vozrastnye i khronobiologicheskie aspekty. Khar’kov: S. A. M.; 2013. 264 s. [in Russian].
4. Tymofii OV, Bulyk RIe, Lomakina YuV. Efekty melatoninu na ekspresiiu hena c-Fos u neironakh medial’noho dribnoklitynnoho sub’iadra paraventrykuliarnoho yadra lipotalamusa schuriv pry zminenomu fotoperiodi. Svit medytsyny ta biolohii. 2015;2(Ch 2):187-91. [in Ukrainian].
5. Khavinson VKh, Lin’kova NS, Kvetnoy IM, Kvetnaya TV, Polyakova VO, Korf Kh. Molekulyarno-kletochnye mekhanizmy peptidnoy regulyatsii sinteza melatonina v kul’ture pinealotsitov. Byulleten’ eksperimental’noy biologii i meditsiny. 2012;153(2):223-6. [in Russian]. 
6. Bedont JL, Blackshaw S. Constructing the suprachiasmatic nucleus: a watchmaker’s perspective on the central clockworks. Front Syst Neurosci. 2015 May 8;9:74.
7. Bedont JL, Newman EA, Blackshaw S. Patterning, specification, and differentiation in the developing hypothalamus. Wiley Interdiscip Rev Dev Biol. 2015 Sep-Oct;4(5):445-68.
8. Fernandez F, Lu D, Ha P, Costacurta P, Chavez R, Heller HC, et al. Circadian rhythm. Dysrhythmia in the suprachiasmatic nucleus inhibits memory processing. Science. 2014 Nov 14;346(6211):854-7. 
9. Kiessling S, Sollars PJ, Pickard GE. Light stimulates the mouse adrenal through a retinohypothalamic pathway independent of an effect on the clock in the suprachiasmatic nucleus. PLoS One [Internet]. 2014 Mar 21 [cited 2018 Jan 11];9(3):e92959. 
10. Venegas C, García JA, Escames G, Ortiz F, López A, Doerrier C, et al. Extrapineal melatonin: analysis of its subcellular distribution and daily fluctuations. J Pineal Res. 2012 Mar;52(2):217-27.
11. Wang JL, Lim AS, Chiang WY, Hsieh WH, Lo MT, Schneider JA, et al. Suprachiasmatic neuron numbers and rest-activity circadian rhythms in older humans. Ann Neurol. 2015 Aug;78(2):317-22.

«Bulletin of problems biology and medicine» Issue 1 Part 1 (142), 2018 year, 264-269 pages, index UDK 591.185.6

10.29254/2077-4214-2018-1-1-142-264-269