Circadian rhythms are integral to eukaryotic life, structuring temporal patterns of physiology and behavior. These cycles are not a passive response to the external environment, but are driven by an internal biological clock with an endogenous period approximating 24 hours. This biological clock and its constituent outputs are represented by regular cycles in gene expression that continue in the absence of any environmental stimuli. The suprachiasmatic nuclei of the hypothalamus (SCN) are the site of a central circadian clock in the mammal. The aim of our current study is to identify and characterise genes that are components of the central oscillator or that are elements of the input or output (clock controlled genes) pathways of the mammalian circadian clock. Time-of-day specific cDNA libraries have been constructed from mRNA extracted from the SCN (0.8 mm3 punched tissue sections) and the cerebella of 2-month old male C57BL/6 mice maintained on a 12:12 light:dark cycle. Animals were sacrificed in three different groups: 0.2–0.7 h after lights on (zeitgeber time [ZT] 0.2–0.7, morning), ZT5.5–6 (midday) and ZT11–11.5 (evening, 0.5–1 h before lights off) (ZT0=lights on). These timepoints and light–dark cycle (entrainment) conditions were chosen since (1) known clock genes and clock-regulated genes peak in expression at various time points after the dark/light transitions, and (2) the amplitudes of rhythmicity are greatest under entrainment conditions. By collecting tissue under entrainment conditions we have been able to use light-induced immediate early genes such as c-fos as internal controls to validate the method. For each collection, brain tissue from 90–120 animals was extracted and frozen rapidly (approximately 1.5 min between sacrifice and freezing of each tissue punch), yielding amounts of poly A+ RNA sufficient to construct representative libraries containing even rare transcripts.

Data will be presented describing differential gene expression in the mammalian brain based on location (e.g. SCN versus cerebellum) and time-of-day (e.g. midday versus evening). We anticipate that microarray screening will identify novel clock genes, whose expression changes as a function of time of day, and thus in turn will reveal components of the central oscillator, or components of the input or output pathways to the clock.