Function of Clock Genes : Examination in Cultured Suprachiasmatic Nucleus

在多电极培养板上培养新生大鼠或小鼠的视交叉上核(SCN)，观察单个视交叉上神经元的时钟基因功能。1.BMAL1基因在单个SCN神经元昼夜放电节律中的作用将反义寡核苷酸应用于表现出较强昼夜放电节律的SCN神经元，并在单个SCN神经元水平上检测其对节律参数如相位、周期和幅度的影响。BMAL1基因的10个μM反义寡核苷酸以相位依赖的方式改变单个SCN神经元的放电节律。结果提示，BMAL1基因在SCN神经元水平上在环境系统中起着关键作用。2.单个SCN神经元水平的突变表型从新生时钟突变小鼠的切片上切取，连续监测5天以上的自发放电。无论基因型如何，SCN切片中的大多数SCN神经元都表现出明显的昼夜节律。纯合子、杂合子和野生型小鼠的平均放电节律昼夜节律周期分别为27.2、24.8和23.5h。这些周期与各基因型小鼠跑轮活动的昼夜节律周期相似。各组放电节律的波幅无明显差异。结果清楚地表明，时钟突变延长了单个SCN神经元的周期，但不影响节律的产生和幅度。

Clock gene functions were examined in single suprachiasmatic neurons by culturing suprachiasmatic nucleus (SCN) of new born rats or mice on an multi electrode dish. Spontaneous firing of single neurons was monitored continuously for up to several weeks and circadian rhythms in firing rate were analyzed.1.BMAL1 gene function in the circadian firing rhythms of individual SCN neuronsAntisense oligonucleotide was applied on SCN neurons showing robust circadian firing rhythms, and effects on rhythm parameters, such as phase, period, and amplitude, were examined at the level of single SCN neurons. Ten μM antisense oligonucleotide of BMAL1 gene phase-dependently phase shifted the firing rhythm of individual SCN neurons. The result suggests the critical role of BMAL1 gene in the circadiant system at the level of the SCN neuron.2.Phenotype of mutation at the level of single SCN neuronsA slice including the SCN was dissected from newborn Clock mutant mice and spontaneous firing was monitored continuously for over 5 days. Irrespective of genotype, most SCN neurons in an SCN slice showed significant circadian firing rhythms. Mean circadian periods of firing rhythms in homozygous, heterozygous, and wild type mice were 27.2, 24.8 and 23.5 h, respectively. The periods were similar to the circadian period of wheel-running activity of each genotype mice. The amplitude of firing rhythms were not different among them. Present result clearly showed that Clock mutaion lengthend the period of individual SCN neurons, but did not affects the rhythm generation or its amplitude.