PHASE OF THE ELECTRICAL ACTIVITY RHYTHM IN THE SCN IN VITRO NOT INFLUENCED BY PREPARATION TIME

Abstract

The mammalian circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, drives daily rhythms in behavioral, physiological, and endocrine functions. The SCN has a genetic basis for rhythm generation and remains rhythmic when it is isolated and kept in constant conditions. This allows for an in vitro analysis of circadian attributes, which is a powerful approach in the study of SCN cellular mechanisms. For studying the phase of the SCN rhythm in vitro, it is important to assess whether preparation of the tissue itself introduces phase shifts. In the present study, we investigated whether preparation of hypothalamic brain slices affects the phase and waveform of the rhythm in electrical impulse frequency of the mouse SCN. Mice were kept under a 12:12 h light-dark cycle, and slices were prepared at six timepoints distributed over the 24 h cycle. We used the peak time and the time of the half-maximum levels in electrical activity as markers for circadian phase. The peak time in electrical activity was observed during the mid-subjective day, irrespective of the time of preparation, at a mean ZT of 5.18 ± 0.20 h (n = 39). After preparation in red light at the end of the subjective night, the circadian phase appeared slightly advanced. When slices were prepared in the dark, using infrared illumination, the ANOVA showed no significant differences in peak times and time of half-maximum values between preparation times. The results affirm the value of the slice preparation for studying the phase of the SCN in vitro. We conclude that the phase and waveform of the electrical activity in the SCN in vitro is unaffected by the time of slice preparation but may be influenced by short light presentation when preparation is performed during the subjective night. (Author correspondence: [email protected])

Key Words:

• Suprachiasmatic nucleus
• Electrophysiology
• Entrainment
• Phase shift
• in vitro

ACKNOWLEDGMENTS

We thank Jan Janse and Sander van Berloo for excellent technical support and assistance. This research was supported by Netherlands Organization for Scientific Research (NWO), program grant nr 805.47.212 ‘From Molecule to Cell’, and by “Entrainment of the circadian clock” – EUCLOCK program of the EU.