Methamphetamine and dopamine receptor D1 regulate entrainment of murine circadian oscillators

Abstract

We investigated the effect of methamphetamine (MA) injections on the circadian organization of behavior and individual tissues in the mouse. Scheduled, daily injections of MA resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. Daily MA also shifted the peak time of PER2 expression in the liver, pituitary, and salivary glands. It has been suggested that reward pathways, and dopamine signaling in particular, may underlie the effects of MA on the circadian system. To test this hypothesis, we examined the effect of the D1 receptor antagonist SCH23390 (SCH) on circadian rhythms. The MA-induced shift in the phase of pituitary and salivary glands was attenuated by pretreatment with the D1 antagonist SCH23390 (SCH). Interestingly, daily SCH, administered alone, also affected some circadian oscillators. The livers and lungs (but not pituitaries or salivary glands) of mice treated with daily injections of SCH displayed disrupted rhythms of PER2 expression, suggesting that D1 receptor signaling is important for entrainment of these organs. From these results, we conclude that MA has widespread effects within the circadian system, and that these effects are mediated, at least in part, by the dopaminergic system. This study also identifies a role for dopamine signaling in normal entrainment of circadian oscillators.

Figure 1. Circadian locomotor activity.

Wheel running actograms from representative animals in the saline-injected (A), unhandled (B), and MA-injected (C) conditions. Mice in the injected conditions received 10 daily injections at ZT7. The pink bar indicates the time of injection (or time of environmental chamber opening in the case of unhandled mice); day 0 is the first day of injections. The left-hand panels depict daily wheel running beginning 5 days prior to the start of injections. The right-hand panels show enlarged activity data from the area outlined in the left panel (the final 5 days of injections). Methamphetamine injection resulted in an acute increase in locomotor activity with clear anticipatory activity in the minutes prior to the scheduled injection and an increase in activity following injection.

Figure 2. Average activity profiles from mice receiving scheduled daily saline (black), left unhandled (gray), receiving scheduled daily MA (2 mg/kg) injections (MA, red), receiving daily D1 receptor antagonist injections (SCH, blue), and receiving pretreatment with a D1 receptor antagonist followed by MA (SCH+MA, aqua).

Activity data are averaged from 5 min bins across days 4 days prior to the start of injections (day −5 to −2, where day 0 is the first day of injections; Fig. 2A and 2B) or the last 7 days of injections (day 3 to 9; Fig. 2C and 2D). Data are expressed as percent of total daily activity, and zeitgeber time (ZT) is along the x-axis. The time of MA injection (ZT7) is indicated by the pink vertical line. A) and B) illustrate the full 24h activity profiles, plotted as the mean percent of total daily activity. C) and D) are enlarged data from ZT6-11, plotted as the mean ± SEM percent of total daily activity. Note the robust increase in activity level beginning immediately prior to and extending for 3–4h hours following injection in the MA group (D).

Figure 3. Scheduled daily MA injections result in anticipatory and induced activity.

A) Injection anticipatory activity. For each mouse, activity level from ZT6-7 (1h prior to injection) was scored (as percent of average daily activity) on the last 7 days of injection and compared to that animal’s activity level from ZT6-7 on 4 days prior to the start of the injection paradigm (ZT6-7 corresponds to the area to the left of the pink vertical bar in Fig. 2B, D). The difference in activity levels are presented as mean ± SEM. Different letters above bars represent significant differences (p<0.05) between groups (i.e., “a c” differs from “b c” but not from bars designated as “a” or “c” alone, “b” differs from “a c” and “a,” “c” differs from bars labeled “a” alone). B) Injection induced activity. For each mouse, activity level from ZT7-11 (4h post injection) was scored (as percent of average daily activity) on the last 7 days of injection and compared to the activity level from ZT7-11 on 4 days prior to the start of the injection paradigm (ZT7-11 corresponds to the area to the right of the pink vertical bar in Fig. 2B, D). The difference in activity levels are presented as mean ± SEM. Different letters above bars represent significant differences (p<0.05).

Figure 4. MA and SCH injections affect the phase of PER2::LUC expression in circadian oscillators.

Rayleigh plots of the phases of peak PER2::LUC expression in liver, lung, pituitary gland, and salivary gland. Rayleigh plots can be read as a 24h clock, with ZT0 at the top and ZT12 at the bottom of the circle. Arrows represent the average peak phase of PER2::LUC expression (mean vectors for the circular distributions) of each group. The length of the vector represents the strength of the phase clustering while the angle of the vector represents the mean phase. Individual data points are plotted outside the circle. The pink box at ZT7 indicates the timing of MA injection for MA-treated groups. A) Data from groups that were saline-injected, unhandled, injected with MA (MA), or injected with SCH23390 alone (SCH). MA resulted in a significant phase advance in liver and pituitary gland (*, p<0.05 compared to saline, unhandled, and SCH groups). In salivary gland, MA advanced phase relative to unhandled controls (*b, p<0.05) whereas SCH significantly delayed phase compared to all other groups (saline, unhandled, or MA-injected). SCH administration resulted in desynchrony among livers (†, Rayleigh p>0.10). SCH administration also resulted in arrhythmicity in 4/6 lungs. Lungs from SCH-treated animals also failed to demonstrate significant synchrony (†a, Rayleigh p>0.10), although this was probably the result of decreased statistical power due to the low number of remaining rhythmic cultures. B) Data from groups that were pretreated with SCH before MA injection (SCH+MA) or injected with MA alone (same data as A). SCH pretreatment significantly attenuated the MA-induced phase shift in pituitary and salivary gland (*, p<0.05) and disrupted synchrony among livers (†, Rayleigh p>0.10). SCH+MA also resulted in arrhythmicity in 2/5 lungs.

Figure 5. Representative PER2::LUC traces from liver, lung, pituitary, and salivary gland.

Traces depict baseline subtracted, detrended bioluminescence (PER2::LUC) from mice in the saline-injected (black), unhandled (gray), MA-injected (red), SCH-injected (blue), and SCH-pretreated (SCH+MA, aqua) conditions.