Standards of evidence in chronobiology: A response

Abstract

A number of recent studies have debated the existence and nature of clocks outside the suprachiasmatic nucleus that may underlie circadian rhythms in conditions of food entrainment or methamphetamine administration. These papers claim that either the canonical clock genes, or the circuitry in the dorsomedial nucleus of the hypothalamus, may not be necessary for these forms of entrainment. In this paper, we review the evidence necessary to make these claims. In particular, we point out that it is necessary to remove classical conditioning stimuli and interval timer (homeostatic) effects to insure that the remaining entrainment is due to a circadian oscillator. None of these studies appears to meet these criteria for demonstrating circadian entrainment under these conditions. Our own studies, which were discussed in detail by a recent Review in these pages by Mistlberger and colleagues, came to an opposite conclusion. However, our studies were designed to meet these criteria, and we believe that these methodological differences explain why we find that canonical clock gene Bmal1 and the integrity of the dorsomedial nucleus are both required to produce true circadian entrainment under conditions of restricted feeding.

Figure 1

A comparison of the data in figure 3B (upper line) and the original (incorrect) supplementary fig. S3B (lower line) in the Fuller et al. [2] paper, on the day in which fig. S3B contained a "gap". The images have been cut directly from the online figures, resized to cover the same time period, and aligned by eye. The red vertical lines marking the feeding time (the offset in the incorrect figure S3B due to not being corrected for the correct time of day) are clear. A piece of a red arrow that marks the food deprivation day is also seen toward the left in the upper register. The "gap" period is the blank area to the left of the red line in the lower register. Note that the lower register (the day in which mean body temperature was 0.06°C lower because of the missing data in the gap period) shows more time periods when the body temperature exceeded the mean (marked by gray or black boxes, depending upon how high the temperature was). Although the differences are subtle, the two plots do not "appear to be identical" as claimed by Mistlberger [1].

Figure 2

Graphs of body temperature for the animals in the corrected suplementary figure S3 in Fuller et al. [2]. The blue line represents the heterozygote animal shown in figures S3A and C, and the red line illustrates the Bmal1 -/- animal with an injection of AAV-Cre into the suprachiasmatic nucleus, shown in figures S3B and D, across the entire five day period in restricted feeding from which the summary graphs in panels C and D were derived. Note that the heterozygote animal (blue) had a normal circadian variation in body temperature, and a robust spike in temperature peaking just around the onset of time of feeding (arrows), as shown in the summary figure S3C. The animal with the injection of AAV-Cre into the suprachiasmatic nucleus had reconstitution of the daily circadian pattern, but no evidence of the anticipatory increase in body temperature prior to feeding, although there was an increase each day after feeding, consistent with the summary figure S3D.

Figure 3

A full set of forebrain autoradiograms on x-ray film from a Bmal1 -/- animal in restricted feeding who received an injection of AAV-Bmal1 into the suprachiasmatic nucleus bilaterally. The box with solid lines identifies a section at the level of the SCN showing hybridization over this nucleus, and only this nucleus. The box with dashed lines represents a section at the level of the DMH, showing lack of hybridization.

Figure 4

A full set of forebrain autoradiograms on x-ray film from a Bmal1 -/- animal in restricted feeding with an injection of AAV-Bmal1 into the dorsomedial hypothalamic nucleus bilaterally. The box with solid lines identifies a section at the level of the DMH, showing selective hybridization over this nucleus and only this nucleus. The box with dashed lines demonstrates a section at the level of the SCN, showing lack of hybridization.

Figure 5

Weights of Bmal1 +/- (blue) and -/- animals (red) across the food restriction experiments. Each animal is plotted as a single line, with the heterozygotes in blue and the homozygotes in red. The Bmal1 -/- animals are smaller, but both groups maintain their body weight well on the restricted feeding protocol that we used.