Systems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Cell division in many mammalian tissues is associated with specific times of day, but just how the circadian clock controls this timing has not been clear. Here, we show in the regenerating liver (of mice) that the circadian clock controls the expression of cell cycle-related genes that in turn modulate the expression of active Cyclin B1-Cdc2 kinase, a key regulator of mitosis. Among these genes, expression of wee1 was directly regulated by the molecular components of the circadian clockwork. In contrast, the circadian clockwork oscillated independently of the cell cycle in single cells. Thus, the intracellular circadian clockwork can control the cell-division cycle directly and unidirectionally in proliferating cells.

Individual cellular clocks in the suprachiasmatic nucleus (SCN), the circadian center, are integrated into a stable and robust pacemaker with a period length of about 24 hours. We used real-time analysis of gene expression to show synchronized rhythms of clock gene transcription across hundreds of neurons within the mammalian SCN in organotypic slice culture. Differentially phased neuronal clocks are topographically arranged across the SCN. A protein synthesis inhibitor set all cell clocks to the same initial phase and, after withdrawal, intrinsic interactions among cell clocks reestablished the stable program of gene expression across the assemblage. Na+-dependent action potentials contributed to establishing cellular synchrony and maintaining spontaneous oscillation across the SCN.