Circadian rhythm from the eye of Aplysia: temperature compensation of the effects of protein synthesis inhibitors

Abstract

1. The circadian rhythm of compound action potentials (CAP) frequency recorded from the isolated eye of Aplysia in culture medium and darkness was subjected to step and pulse treatments with anisomycin, a protein synthesis inhibitor. 2. The step application of anisomycin and its continued presence in the culture medium lengthened the period of the rhythm in a dose-dependent manner. At 10(−8) M the period was increased from the normal 26.5 h to about 28 h and at 10(−7) M the period was lengthened to 31 h or longer. At 10(−6) M the rhythm was suppressed but the CAP activity continued without the cyclic variations in CAP frequency. 3. Six-hour pulses of anisomycin at 10(−6) M caused phase-dependent phase-shifts of the rhythm. Maximum phase delays of 15 h were obtained at CT (circadian time) 2 and maximum phase advances of 4 h were obtained at CT 6. The phase response curves at 12, 15 and 17 degrees C were essentially identical. 4. Anisomycin appears to act rather selectively on the circadian clock mechanism. It does not alter the CAP amplitude and duration and it does not alter the bursting pacemaker mechanism of the optic nerve CAP or central neurones. 5. The results support the hypothesis that the synthesis of a protein or polypeptide on eucaryotic ribosomes is an essential part of the circadian clock timing mechanism. The sensitivity of the clock to anisomycin is the same at three different temperatures (12, 15 and 17 degrees C) within the physiological range of temperatures for Aplysia, as expected for a clock whose period length is temperature compensated (Q10 1.02) over that same range. 6. At the critical phases of CT 1-4, anisomycin pulses often caused unusual perturbations of the rhythm. These effects are consistent with the hypothesis that the circadian rhythm is a multioscillator system.