Reentrainment Of Locomotor Activity And Expression Of mPer1 mRna To Successive 8Hr Advanced Ld Cycle

As described above, we found that the drinking-water administration of mela-tonin ameliorated the delay of re-entrainment to an 8-hr advanced LD cycle in both SAMP8 and SAMR1. Thus, melatonin may accelerate the re-entrainment to LD shifts in mice, as observed in melatonin-containing tube-implanted rats (8). However, we do not know whether the melatonin-induced acceleration means the acceleration of an overt rhythm such as locomotion or that of clock gene expression. Expression of mPerl mRNA in the SCN is under the control of the clock genes and this expression is good marker for the circadian rhythm (13). Therefore, we expected to examine the reduction of the mRNA expression of mPerl in the SCN at ZT4 (the peak time of the expression of mPer1 mRNA in normal LD cycle, 13) in successive 8-hr advanced mice. We expected to observe the normalization of the expression of mPerl mRNA at ZT4 in the melatonin-treated animals, because melatonin accelerated the re-entrainment.

The experimental schedule of re-entrainment is shown by a horizontal bar in Figure 3A. The LD cycle in the housing room was advanced by 8 hr. At this schedule, the phase advance was conducted 3 times with 3-day intervals from Day 2; therefore, LD cycle returned to the previous initial LD cycle after 10 days. Melatonin (13.3 (g/ml, 0.2% ethanol) or vehicle (0.2% ethanol) was administered in the animals' drinking water. In this experiment, we used young ddY mice (6-8 weeks old). The mice had free access to food and the drinking water. The measurement of locomotion was the same as described earlier. LD-shifted mice exhibited significantly low locomotor activity during the last 3 days (Figure 3A and 3B, p < 0.05 vs. non-shifted animals, Dunnett's test). In contrast, the melatonin-treated LD-shifted mice showed a recovery of the reduction of locomotion.

Figure 2. Effect of melatonin application on Ca2+ influx into the SCN cells. A shows the time course of changes in the ratio (340 nm/380 nm) and B shows the summarized data. The numbers of SCN slices are shown in parentheses; the columns and bars means and S.E.M., respectively. Melatonin (0.1 and 1 mM) significantly increased the Ca2+ influx (Dunnett's test, *p < 0.05).

Figure 2. Effect of melatonin application on Ca2+ influx into the SCN cells. A shows the time course of changes in the ratio (340 nm/380 nm) and B shows the summarized data. The numbers of SCN slices are shown in parentheses; the columns and bars means and S.E.M., respectively. Melatonin (0.1 and 1 mM) significantly increased the Ca2+ influx (Dunnett's test, *p < 0.05).

Reentrainment

Figure 3. Re-entrainment of locomotor activity rhythm to three-times 8-hr advanced light-dark cycles and the effect of melatonin drinking water on re-entrainment in ddY mice. The second and third vertical bars show the schedule of the light-dark cycle. Melatonin ameliorated the deficit of re-entrainment in shifted animals (third actogram in A and B). In panel B, the numbers of mice are shown in parentheses; the columns and bars means and S.E.M., respectively. **p < 0.01 (Student's t-test).

Normal Shifted Shifted

+ melatonin

Figure 3. Re-entrainment of locomotor activity rhythm to three-times 8-hr advanced light-dark cycles and the effect of melatonin drinking water on re-entrainment in ddY mice. The second and third vertical bars show the schedule of the light-dark cycle. Melatonin ameliorated the deficit of re-entrainment in shifted animals (third actogram in A and B). In panel B, the numbers of mice are shown in parentheses; the columns and bars means and S.E.M., respectively. **p < 0.01 (Student's t-test).

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