Effects of Preparation Time on Phase of Cultured Tissues Reveal Complexity of Circadian Organization

Abstract

The phases of central (SCN) and peripheral circadian oscillators are held in specific relationships under LD cycles, but in the absence of external rhythmic input may damp or drift out of phase with each other. Rats exposed to prolonged constant light become behaviorally arrhythmic, perhaps as a consequence of dissociation of phases among SCN cells. We asked whether individual central and peripheral circadian oscillators were rhythmic in LL treated arrhythmic rats and, if rhythmic, what were the phase relationships among them. We prepared SCN, pineal gland, pituitary and cornea cultures from transgenic Per1-luc rats whose body temperature and locomotor activity were arrhythmic, and from several groups of rhythmic rats held in LD, DD, and short-term LL. We measured mPer1 gene expression by recording light output with sensitive photomultipliers. Most of the cultures from all groups displayed circadian rhythms. This could reflect persistent rhythmicity in vivo prior to culture, or, alternatively, rhythmicity that may have been initiated by the culture procedure. To test this we cultured tissues at two different times 12 h apart and asked whether phase of the rhythm was related to culture time. The pineal, pituitary and SCN cultures showed partial or complete dependence of phase on culture time, while peak phases of the cornea cultures were independent of culture time in rhythmic rats, and were randomly distributed regardless of culture time in arrhythmic animals. These results suggest that in behaviorally arrhythmic rats oscillators in the pineal, pituitary and SCN had been arrhythmic or severely damped in vivo while the cornea oscillator was free-running. The peak phases of the SCN cultures were particularly sensitive to some aspect of the culture procedure since rhythmicity of SCN cultures from robustly rhythmic LDentrained rats was strongly influenced when the procedure was carried out at any time except the second half of the day. Keywords circadian organization; Period1; constant light; suprachiasmatic nucleus; pineal; pituitary; cornea; culture While the molecular mechanisms that generate circadian oscillations within cells have been extensively studied (Reppert and Weaver 2002;Lowrey and Takahashi, 2004), understanding of the mechanisms that connect the molecular framework to physiological function and behavior is still limited (Buijs et al., 2003). We have approached these questions using a transgenic rat model carrying a luciferase gene under the control of the mouse Period1 (Per1) promoter. SCN cultures from these animals express circadian oscillations of Per1-luc expression indefinitely, while oscillations in cultured peripheral tissues damp after several cycles (Yamazaki et al., 2000). The phases of central and peripheral circadian oscillators are held in specific relationships under light-dark (LD) cycles (Yamazaki et al., 2000;Abe et al., 1 To whom correspondence should be addressed: Michael Menaker Department of Biology, University of Virginia, Charlottesville, VA 22903; Tel: 434-982-5767; F...

Resetting of central and peripheral circadian oscillators in aged rats

“…Although one could speculate that an increased rate of shifting for arcuate nucleus and pineal gland correlates with decreased circadian amplitude in vivo [33], indirect measurements of rhythm robustness for pineal gland did not reveal any age-related differences in amplitude [37]. It must be noted that culture preparation can affect the circadian phase of some cultured tissues [38]. However the low phase variance among the cultures within each group, and the fact that the mean peak phases were consistent with expected values for transients during resynchronization together suggest that our measurements reflect in vivo phases of these tissues.…”

Retinal pathways influence temporal niche

“…The running-wheel activity of Rpe65 Ϫ/Ϫ ;Opn4 Ϫ/Ϫ ; mPer2 luc mice, control Rpe65 ϩ/Ϫ ;Opn4 ϩ/Ϫ ;mPer2 luc mice, and mPer2 luc mice in LD12:12 was recorded for 3-4 weeks. Animals were anesthetized with CO 2 and decapitated approximately 1 h before lights-off, a time of day chosen for previous experiments of this type to minimize disruption to tissues caused by a light-to-dark transition in the middle of the day (44). For pineal dissection, the pineal on the skull cap was removed and placed in cold Hanks' balanced salt solution (HBSS).…”

Neural correlates of individual differences in circadian behaviour

“…Briefly, mice were sacrificed at ZT7 -10, which does not reset the SCN [25,26]. Brains were sectioned in the coronal plane and three consecutive slices (150 mm) were retained from the rostral, middle and caudal portions of the SCN (figure 1c).…”