Most organisms living on earth have an internal clock and thus circadian rhythm represents a basic feature of life. In mammals, the central oscillator resides in the small paired oval-shaped suprachiasmatic nucleus (SCN)—densely packed neurons in the hypothalamus—which work to control circadian functioning. Circadian oscillation is initially driven in the cellular core oscillator by a transcription/translation-based core feedback loop of a set of clock genes. In this paper, we visualized this clock gene expression by using a highly sensitive CCD camera, and showed the synchronized rhythms of clock gene transcription in hundreds of SCN neurons with distinctive topographic orientation.

The successful methodology is derived from the following three key points:

1. genetically engineered animals:
   • For detecting the circadian oscillation, we used mPer1 because it is a center molecule of core clock oscillation and its mRNA expression in the SCN is really robust. We first confirmed that the 5’ upstream region of mPer1 promoter has a promoter activity, and generates the transgenic mice carrying this mPer1 promoter fused to the firefly luciferase gene. By inserting an optical fiber into the brain just on the SCN of these transgenic mice, we succeeded in detecting the luciferase luminescence rhythm in freely moving mice.
2. detection camera:
   • We adopted the very sensitive cooled cryogenic CCD camera with its original incubator, which was designed by Dr. Masaki Kobayashi, a specialist in biomedical optics. The weakness of the enzyme luciferase for use as biological material is the low intensity of its luminescence. We conquered this weakness by increasing the detection sensitivity with a low background.
3. slice culture techniques:
   • We had already developed the slice culture system for SCN about 10 years ago. By combining these methods, we have been able to visualize weak luminescent cell rhythms with the highest quality.

The feature of the circadian system is the prevalence of the oscillation at the levels of genes reflects at cell, tissue, and system levels. How are individual cellular clocks driven by the oscillatory molecular loop encoded by clock genes integrated into a stable and robust pacemaker with a period length of ~24 hours? This was investigated in the SCN, the mammalian master clock. We used real-time analysis of gene expression to show synchronized rhythms of clock gene transcription across hundreds of neurons within the SCN in the organotypic slice culture. Differentially phased neuronal clocks are topographically arranged across the SCN. A protein synthesis inhibitor cycloheximide set all cell clocks to the same initial phase, and following withdrawal, intrinsic interactions among cell clocks re-establish the stable program of gene expression across the assemblage. Tetrodotoxin-induced desynchronicity and the suppression of the cell-clock rhythm, demonstrates that the neuronal network properties dependent on sodium dependent action potentials play a dominant role in both establishing cellular synchrony and maintaining spontaneous oscillation across the SCN. The genotype-specific circadian period which arises from the coupling of multiple SCN cellular oscillators contrasts with many other rhythmic tissues (e.g., the heart), in which the fastest cells set the rate. Thousands of clock oscillating cells in the SCN generate standard internal time, and spread out the time signal throughout the entire body. Finally, circadian changes resulted in accompanying changes in behavior and hormone secretion.

I first encountered SCN in 1983 at Kyoto in Dr. Yasuhiko Ibata’s lab at the Department of Anatomy and Neurobiology of the Kyoto Prefectural University of Medicine. At that time, many brain-gut peptides were discovered, and we were screening their expression in the brain by using immunocytochemistry. I found that an antiserum against VIP could detect a cluster of neurons in the SCN with the distinct topography inside this nucleus. Then, by our original quantitative histochemical methods, we found that VIP immunoreactivity showed the diurnal change in the SCN, although, at that time, the change of gene expression in physiological conditions was not commonly known. We succeeded in developing a slice culture of SCN to detect peptide release in the early 1990s through a collaboration with Dr. Shin-Ichi Inouye, a Professor of Biology and Director of the Research Institute for Time Studies at Yamaguchi University in Ube City. In 1997, collaborating with Dr. Hajime Tei of the University of Tokyo, we found mPer1, a mammalian first homologue of Drosophila period gene. Although I technically use morphology, physiology, neurochemistry, cell biology, and molecular biology, I am always primarily interested in SCN, the master of circadian rhythms.