Bobu Corina, Sandu Cristina, Laurent Virginie, Felder-Schmittbuhl Marie-Paule, Hicks David
Department of Neurobiology of Rhythms, Institute for Cellular and Integrative Neurosciences, Strasbourg, France.
Mol Vis. 2013 May 21;19:1060-73. Print 2013.
Prolonged periods of constant lighting are known to perturb circadian clock function at the molecular, physiological, and behavioral levels. However, the effects of ambient lighting regimes on clock gene expression and clock outputs in retinal photoreceptors--rods, cones and intrinsically photosensitive retinal ganglion cells--are only poorly understood.
Cone-rich diurnal rodents (Muridae: Arvicanthis ansorgei) were maintained under and entrained to a 12 h:12 h light-dark cycle (LD; light: 300 lux). Three groups were then examined: control (continued maintenance on LD); animals exposed to a 36 h dark period before sampling over an additional 24 h period of darkness (DD); and animals exposed to a 36 h light period before sampling over an additional 24 h period of light (300 lux, LL). Animals were killed every 3 or 4 h over 24 h, their retinas dissected, and RNA extracted. Oligonucleotide primers were designed for the Arvicanthis clock genes Per1, Per2, Cry1, Cry2, and Bmal1, and for transcripts specific for rods (rhodopsin), cones (short- and mid-wavelength sensitive cone opsin, cone arrestin, arylalkylamine N-acetyltransferase) and intrinsically photosensitive retinal ganglion cells (melanopsin). Gene expression was analyzed by real-time PCR.
In LD, expression of all genes except cone arrestin was rhythmic and coordinated, with acrophases of most genes at or shortly following the time of lights on (defined as zeitgeber time 0). Arylalkylamine N-acetyltransferase showed maximal expression at zeitgeber time 20. In DD conditions the respective profiles showed similar phase profiles, but were mostly attenuated in amplitude, or in the case of melanopsin, did not retain rhythmic expression. In LL, however, the expression profiles of all clock genes and most putative output genes were greatly altered, with either abolition of daily variation (mid-wavelength cone opsin) or peak expression shifted by 4-10 h.
These data are the first to provide detailed measures of retinal clock gene and putative clock output gene expression in a diurnal mammal, and show the highly disruptive effects of inappropriate (nocturnal) lighting on circadian and photoreceptor gene regulation.
已知长时间持续光照会在分子、生理和行为水平上扰乱昼夜节律钟功能。然而，环境光照模式对视网膜光感受器（视杆细胞、视锥细胞和内在光敏性视网膜神经节细胞）中生物钟基因表达和生物钟输出的影响却知之甚少。
将富含视锥细胞的昼行性啮齿动物（鼠科：安氏中仓鼠）饲养在12小时光照 - 12小时黑暗周期（LD；光照强度约为300勒克斯）下并使其同步化。然后检查三组动物：对照组（继续维持在LD条件下）；在额外24小时黑暗期采样前暴露于36小时黑暗期的动物（DD）；在额外24小时光照期（约300勒克斯，LL）采样前暴露于36小时光照期的动物。在24小时内每3或4小时处死动物，解剖其视网膜并提取RNA。为安氏中仓鼠的生物钟基因Per1、Per2、Cry1、Cry2和Bmal1以及视杆细胞（视紫红质）、视锥细胞（短波长和中波长敏感视锥蛋白、视锥细胞抑制蛋白、芳基烷基胺N - 乙酰转移酶）和内在光敏性视网膜神经节细胞（黑视蛋白）的特异性转录本设计寡核苷酸引物。通过实时PCR分析基因表达。
在LD条件下，除视锥细胞抑制蛋白外的所有基因表达均具有节律性且相互协调，大多数基因的峰值相位出现在光照开启时或之后不久（定义为授时因子时间0）。芳基烷基胺N - 乙酰转移酶在授时因子时间20时表达最高。在DD条件下，各自的表达谱显示出相似的相位特征，但幅度大多减弱，或者对于黑视蛋白而言，不再保持节律性表达。然而，在LL条件下，所有生物钟基因和大多数假定输出基因的表达谱都发生了很大变化，要么每日变化消失（中波长视锥蛋白），要么峰值表达提前4 - 10小时。
这些数据首次提供了昼行性哺乳动物视网膜生物钟基因和假定生物钟输出基因表达的详细测量结果，并显示了不适当（夜间）光照对昼夜节律和光感受器基因调控的高度破坏作用
