The methamphetamine-sensitive circadian oscillator does not employ canonical clock genes

Abstract

The "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus controls most behavioral, physiological, and molecular circadian rhythms in mammals. However, there are other, still unidentified, circadian oscillators that are able to carry out some SCN functions. Here we show that one of these, the methamphetamine-sensitive circadian oscillator (MASCO), which generates behavioral rhythms in the absence of the SCN, is based on an entirely different molecular mechanism. We tested mice lacking, or with mutations of, genes that form the canonical circadian machinery. In all cases, animals that were arrhythmic as a consequence of genetic defect expressed circadian locomotor rhythms when treated with methamphetamine. These results strongly support the hypothesis that the mechanism generating MASCO does not involve the molecular feedback loops that underlie canonical circadian rhythmicity. The properties of MASCO may provide insight into the evolution of circadian mechanisms. Importantly, MASCO may play a role in addiction to psychostimulants.

Fig. 1.

Clock Δ19 mice on methamphetamine (MA). Sample actograms (A and C) and periodograms (B and D) from a ClockΔ19 heterozygous (A and B) and a ClockΔ19 homozygous mouse (C and D). Actograms are double plotted. The arrow indicates the period of drug administration. τ in h is listed along the bottom of each actogram. Baseline τ was calculated in DD before drug administration. The methamphetamine τs were calculated using the last 2 weeks of drug treatment and are represented in the periodograms (B and D). Methamphetamine use significantly lengthened τ in Clock-mutant mice (F (1, 9) = 5.863, P < 0.05). The effects of methamphetamine administration did not differ by genotype (heterozygote or homozygous Clock mutation).

Fig. 2.

Tau-mutant mice on methamphetamine (MA). Sample actograms (A and C) and periodograms (B and D) from a Tau heterozygous mutant (A and B) and a Tau homozygous mutant (C and D). Actograms are double plotted. The arrow indicates the period of drug administration (0.005% methamphetamine). τ in h is listed along the bottom of each actogram. Baseline τ was calculated in DD before drug administration. The methamphetamine τs were calculated using the last 2 weeks of drug treatment and are represented in the periodograms (B and D). Methamphetamine use significantly lengthened τ in Tau-mutant mice (F (1, 19) = 18.104, P < 0.05). The effects of methamphetamine administration did not differ by genotype (heterozygote or homozygous Tau mutation). Note that the heterozygote animal (A) was on methamphetamine for many days before a substantial lengthening of the free-running period was achieved.

Fig. 3.

SCNX Tau-mutant mice on methamphetamine (MA). (A) Sample actogram from an SCN-lesioned Tau homozygous mutant mouse. The data from (A) are replotted in (B); the x-axis has been changed to 37.17 h to aid in visualizing rhythmicity. Actograms are double plotted. The arrow indicates the period of drug administration (0.005% methamphetamine). Baseline τ was calculated in DD before drug administration. The methamphetamine τ was calculated using the last 2 weeks of drug treatment and is represented in the periodogram (C). A significant peak is observed at 37.17 h.

Fig. 4.

Npas2^−/− mice on methamphetamine (MA). Sample actograms (A and C) and periodograms (B and D) from an intact Npas2^−/− mouse (A and B) and an SCNX Npas2^−/− mouse (C and D). Actograms are double plotted. The arrow indicates the period of drug administration (0.005% methamphetamine). τ in h is listed along the bottom of each actogram. Baseline τ was calculated in DD before drug administration. Methamphetamine τs were calculated using the last 2 weeks of drug treatment and are represented in the periodograms (B and D). A significant peak is observed at 24.33 h for the intact mouse (B). Methamphetamine use significantly lengthened τ in intact Npas2^−/− mice (t = −4.384, P < 0.05). The SCNX animal had no significant free-running period in DD before drug administration; however, note the weak 24-h rhythm following methamphetamine removal. During methamphetamine treatment a significant peak is observed at 27.67 h with a smaller peak at 34.24 h (D).

Fig. 5.

Per1^−/−/Per2^−/− mice on methamphetamine (MA). Sample actogram (A) and periodogram (B) from a Per1^−/−/Per2^−/− mouse. The actogram is double plotted. The arrow indicates the period of drug administration (0.005% methamphetamine). τ in h is listed along the bottom of the actogram. Methamphetamine τ was calculated using the last 2 weeks of drug treatment and is represented in the periodogram (B). A significant peak is observed at 28.33 h.

Fig. 6.

Bmal1^−/− mouse on methamphetamine (MA). Double-plotted actogram from a Bmal1-knockout mouse depicting infrared activity data before methamphetamine treatment (A) and in the initial period of 0.005% methamphetamine water administration (B). The first day of the actogram in (B) is the first day of 0.005% methamphetamine availability; before that point the animal was consuming 0.0035% methamphetamine water. The τ in h is listed along the side of the actogram. The abrupt change in period that occurred toward the middle of the record was spontaneous. (C) A single-plotted actogram of the initial period of 0.005% methamphetamine treatment (when the observed τ was 23.83 h). The x-axis has been changed to 23.83 h to aid in visualizing rhythmicity. (D) A single-plotted actogram of the latter portion of (B) with the x-axis changed to 36.67 h to aid in visualizing rhythmicity. (E) Periodogram analysis from this mouse after ≈2.5 weeks of 0.005% methamphetamine treatment. This periodogram covers the last 10 days of the record shown in (B); a significant peak is observed at 36.67 h.

Fig. 7.

Cry1^−/−/Cry2^−/− mouse on methamphetamine (MA). Double-plotted actograms from an intact Cry1^−/−/Cry2^−/− mouse before methamphetamine treatment (A) and during the last 2 weeks of drug treatment (B). This animal had no significant free-running period in DD before drug administration but takes on a long free-running period in the presence of 0.005% methamphetamine, as shown in the periodogram (C). A significant peak is observed at 29.17 h.

Fig. 8.

SCNX Cry1^−/−/Cry2^−/− mouse on methamphetamine (MA). Actograms from an SCNX Cry1^−/−/Cry2^−/− mouse before methamphetamine treatment (A) and during the last 2 weeks of drug treatment (B). This animal had no significant free-running period in DD before drug administration but takes on a long free-running period in the presence of 0.005% methamphetamine. The data from (B) are replotted in (C) with the x-axis changed to 26.66 h to aid in visualizing the 2 periods present. (D) Periodogram analysis of these data shows a significant peak at 30.33 h and a smaller peak at 26.66 h.