Circadian clocks consist of an ingenious autoregulatory feedback loop whereby the cyclically expressed products of the clock gene are able to inhibit their own expression<1. Here we follow the rhythmic expression of the clock gene mPer1 in the brain of a living mouse. This model system enables real-time gene expression to be monitored in the intact brain under physiological conditions.
We have previously produced transgenic mice carrying a luciferase (luc) reporter gene under the control of the promoter of the oscillating clock gene mPer1 (refs 2, 3). Using a two-dimensional photon-counting camera, we were able to detect a day–night variation in luciferase-mediated bioluminescence in the suprachiasmatic nucleus (SCN)3, the mammalian brain's circadian centre4, in fresh brain slices from these mPer1–luc mice.
We inserted a polymer optical fibre (500 μm in diameter; 0.5 numerical aperture) just above the SCN of a mPer1–luc transgenic mouse using a stereotaxic frame; the other end of the optical fibre was connected to a photomultiplier tube operating as part of a photon-counting apparatus (Fig. 1a). As luciferase requires a substrate for light emission, we continuously infused luciferin dissolved in artificial cerebrospinal fluid through a lateral ventricle. The substrate concentration in the cerebrospinal fluid, sampled from the contralateral ventricle, reached a plateau at about 1.3 mM within 3 hours and remained more or less constant.
With this system, we continuously recorded light emission from the SCN of the transgenic mice in vivo under constant darkness. The luminescence showed a clear circadian fluctuation (Fig. 1b), with a 1.5- to 2.5-fold amplitude and peaks and troughs at circadian time (CT) 4–6 and CT15–20, respectively (where CT0 is subjective dawn and CT12 is subjective dusk).
This temporal profile of bioluminescence strikingly resembles the expression profile of native mPer1 messenger RNA. The phase lag between transcription of luciferase mRNA and the production of luminescence was only 0–2 h. The circadian period calculated from the intervals between bioluminescence peaks was about 24 h, which is close to the period of locomotor activity of this transgenic mouse line (24.0 ± 0.05 h; n = 7).
To confirm that this fluctuation in luminescence represents an endogenous oscillation, we recorded the luminescence from two groups of mice housed for at least two weeks under alternating 12-hour light–dark cycles, with lights on at either 7:00 h (LD-entrained mice) or 19:00 h (DL-entrained mice). Both groups were hooked up to the monitoring system at 14:00 h and the luminescence emitted was recorded under constant darkness. As expected, the luminescence recorded from LD-entrained mice oscillated in the opposite phase to that of DL-entrained animals (Fig. 1c).
Capturing the effect from a luciferase reporter transgene with optical-fibre- mediated photon-counting technology offers a way to measure real-time gene expression profiles in the intact brain under physiological conditions. To our knowledge, this is the first time that a ticking clock gene has been monitored in a living mammal. Real-time optical imaging of gene expression, in combination with real-time measurement of electrophysiological, metabolic and behavioural parameters, should advance the study of mammalian brain function.
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Yamaguchi, S., Kobayashi, M., Mitsui, S. et al. View of a mouse clock gene ticking. Nature 409, 684 (2001). https://doi.org/10.1038/35055628
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