Effect Of Monochromatic Light On Circadian Clock System Of Pineal Gland And Hypothalamus In The Chicks

In avian,melatonin synthesized and secreted rhythmically with a low level in daytime and a high level in nighttime,which was regulated by the biological clock in circadian system.The pineal gland and the hypothalamus belong to the master clocks of avian biological clock system,and they both contain independent pacemakers with interaction to each other.The oscillation of biological pacemaker depends on a negative feedback regulated loop containing a series of positive clock genes(cClock,cBmall and cBmal2)and negative clock genes(cCryl,cCry2,cPer2 and cPer3).The previous study reported that GL improved the level of plasma melatonin in P14 chick.However,it is unknown whether the effect of monochromatic light on the plasma melatonin was regulated through the circadian clock system.Therefore,in this study AA broilers at PO were used to reared in WL(400-760 nm),RL(660 nm);GL(560 nm)and BL(480 nm)with LD 12:12,respectively.At P3,pineal and sham-operation was taken.Then,to be reared at P14,chicks were killed at 6 timepoints every 4 hours in 24 h.Their pineal gland and the hypothalamus were used to study the effect of different monochromatic light on the master clock in circadian clock system.Further,the mechanism of the effect of monochromatic light on MEL secretion in pineal gland and the influence of MEL on the expression of clock genes in hypothalamus were discussed.The results showed as follows:1 The circadian pattern of clock genes in chick pineal gland and hypothalamusThe positive clock genes and negative clock gene oscillated with a remarkable circadian rhythmicity in pineal gland and hypothalamus.Except cPers,the pattern of other clock genes all exhibited an increasing in the daytime and decreasing in the nighttime.The similar pattern between two pacemakers was the acrophases of cCrys as well as cPers,respectively.The different pattern seemed that the amplitude of cClock in pineal gland was twice as that in hypothalamus,while these of cBmals and cCryl in pineal gland were half of these in hypothalamus.The acrophases of cClock,cBmals and cPer3 in pineal gland occurred later than those of hypothalamus.2 The effect of monochromatic light on circadian system in pineal glandThe pattern of clock genes:When chick were reared in the different monochromatic light,the pattern of their cdock genes showed strong rhythmical,except that the rhythmicity of cCry2 tended to be damped(R2 = 0.243,P = 0.033).Compared with WL,GL enhance the mRNA level expression of positive clock genes and reduced these of negative clock gene,while RL showed an opposite role to GL.Meanwhile,the mesor and amplitude of positive clock genes were high and these of negative ones were low in GL.The acrophases of positive clock genes delayed in BL by(0.2 h-0.8 h),and these of negative clock genes were earlier by(0.1 h-1.5 h).CLOCK and BMAL1 also expressed with a high level in GL,MEL secretion:GL increased the mRNA level of cAanat in pineal gland,and improved the level of plasma MEL by 1.48%-30.93%(P = 0.000-0.655,ANOVA);RL reduced the secretion of MEL by 5.6%-23.4%(P = 0.000-0.006,ANOVA).In different monochromatic light conditions,the oscillation of the mRNA level of cAanat in pineal gland and the level of plasma MEL showed obvious circadian rhythmicity.The mesor and amplitude in GL was higher than other light condition,but lowest in RL.The pattern of opsins:The pattern of cOpnp in WL and GL was high in day and low in nigh,but with an opposite pattern in BL and RL.The rhythmicity of cOpnp was disrupted in RL(R2=0.2,P=0.425).cOpn4-I showed a low expression in day and high in night.GL had a best oscillation rhythmicity and amplitude than these of other light conditions.The pattern of cOpn4-2 showed a high level in day and low in night with rhythmicity.BL has a lowest oscillation rhythmicity and amplitude than these of other light conditions.cCreb oscillated with strong rhythmicity.3 The effect of monochromatic light on clock genes in hypothalamusIn the different monochromatic light,the pattern of clock genes in hypothalamus showed clear rhythmicity.GL improved the expression of cClock,cBmall and cCryl with a high mesor and amplitude,and decreased those of cBmal2 and cCry2.RL showed an opposite role to GL.Meawhile,the acrophase of clock genes in RL was more variable than that of GL and BL.Resemble to mRNA level,CLOCK and BMAL1 showed a high expression in GL.The positive neuron of CLOCK in chick hypothalamus was located in SCN(vSCN and mSCN)and non-SCN region such as AM,PVN,PerVN and MB.4 The effect of monochromatic light on clock genes in hypothalamus mediated by MELThe effect of PINX on plasma MEL level:In different monochromatic light conditions,chick plasma MEL level was lower in PINX than in sham-operation.The expression of plasma MEL showed a clear rhythmicity.GL also showed a high level than other light conditions 7.0%-55.52%(P = 0.000-0.039,ANOVA).The effect of PINX on clock genes expression:With a decreasing in plasma MEL level,the amplitude of cClock and cPer2 was lower than that of Sham.cBmall showed a slight variation in mesor and amplitude compared with Sham,and the acrophase in RL and BL was later than that in Sham by 2 h or early 2 h.The rhythmicity of cBmal2 and cCry2 was loss in RL.PINX decreased the expression of cCryl,but increased that in RL.RL increased cPer3 expression in RL by PINX,CLOCK and BMAL1 showed a similar pattern with their mRNA level.CLOCK was found in vSCN,PerVN and PVN.The effect of PINX on MEL receptors expression:cMel1a,cMel1b and cMel1c exhibited a strong oscillation rhythmicity.The expression of the three receptors was reduced in PINX with a low amplitude than intact and SHAM group 35.43%-37.98%(P = 0.000,AVONA),12.55%-34.55%(P =0.000-0.032,AVONA)and 4.39%-23.71%(P = 0.000-0.999,AVONA).cMellb and cMellc oscillated without rhythmicity by PINX.The type of MEL receptor to mediate the effect of MEL:Add 250 pg/mL MEI to hypothalamus cell in vitro,cClock has a significantly high level than the group without MEL by 45.63%-45.81%(P=0.003-0.606,ANOVA).When adding Mella/Mellb non-selective antagonist Luzindole,cClock was lower than MEL group 30.6%-31.4%(P = 0.004-0.01,ANOVA).When adding Mel lb selective antagonist 4P-PDOT,the expression to cClock was lower than add MEL 23.6%(P = 0.009,ANOVA).wnen aaaing Mienc selective antagonist Prazosin,the expression to cClock was higner man non-MEL group 44.37%-47.6%(P = 0.002-0.009,ANOVA).Conclusion:Seven clock genes showed strong clear oscillation rhythmicity in pineal gland and hypothalamus of P14 chick.GL improved positive clock genes expression and reduced that of negative clock genes through cOpnp and cOpn4-1 to enhance cAanat mRAN level in pineal gland leading to a high level of plasma MEL level.Also,CLOCK was found in vSCN,PerVN and PVN both in intact and PINX.The effect of monochromatic light on clock genes in hypothalamus was mediated by MEL.MEL increased expression of clock genes in hypothalamus through the receptor Mel la and Mellb.