Function of Clock Genes : Examination in Cultured Suprachiasmatic Nucleus

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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.