Elliott G. Marchant

Entrainment and phase shifting of circadian rhythms in mice by forced treadmill running

Daily schedules of spontaneous, drug-, or novelty-induced running can entrain circadian rhythms in rodents. Forced running, by contrast, has been reported to have weak or no effects, although a thorough comparative study in a single species is lacking. To fill this gap, drinking or activity rhythms were monitored in C57 mice subjected to daily, 3-h bouts of forced treadmill running or to 3-h daily access to home cage running wheels. Entrainment to treadmill running was observed in 17/27 mice, and to restricted wheel access in 11/20 mice. Entrainment was affected by availability of a home cage wheel (e.g., 14/16 mice with no wheel entrained to treadmill running). Phase angle of entrainment was related to prior circadian period (tau), and tau following entrainment exhibited aftereffects. No mice entrained to a 3-h daily schedule of water access, suggesting that entrainment to scheduled running was not related to water or associated food intake. Phase shifts in response to single 3-h bouts of treadmill running or wheel access were small and not reliably induced. The entrainment paradigm is thus recommended for further study of behavioral effects on the mouse circadian system; forced running, in particular, offers several methodological advantages. The results do not support prior suggestions that forced and voluntary activity differ in value as nonphotic zeitgebers.

Anticipation and entrainment to feeding time in intact and SCN-ablated C57BL/6j mice.

To characterize properties and mechanisms of non-photic entrainment of circadian rhythms, the effects of scheduled feeding were assessed in intact and suprachiasmatic nuclei (SCN) ablated C57BL/6j mice. During ad libitum food access, mice with no or partial SCN damage exhibited free-running activity and drinking rhythms, whereas mice with complete ablations were arrhythmic. When food was restricted to 4 h/day for 5-9 weeks, intact and partial SCN-ablated mice exhibited anticipatory activity to mealtime, concurrent with free-running rhythms. In some cases, free-running rhythms became entrained to feeding time; this was more prevalent in intact than partial ablated mice and was related to free-running period. Free-running phase or period were modified in other cases, revealing a phase-response profile consistent with other non-photic zeitgebers. Five of 12 mice with complete or near complete SCN ablations showed anticipatory activity. Mice that failed to anticipate were less active generally and sustained larger lesions. Sites of damage unique to non-anticipators were not evident. The results indicate that the SCN is not necessary for anticipatory rhythms in mice, but that cell populations distributed across several hypothalamic areas may be important for at least some behavioral markers of this circadian function.

Discrimination of circadian phase in intact and suprachiasmatic nuclei-ablated rats

This study examined whether the circadian system of rats can serve as a consulted clock for discriminating time of day. Food restricted rats housed in activity wheels were trained to lever press for food in a two-lever T-maze in which the left arm was correct in a morning feeding session, and the right arm in an afternoon session (7 h interval). All six rats learned the task (discrimination ratios > chance on 85-95% of sessions) and exhibited anticipatory wheel running prior to most sessions. Performance was not disrupted by inverting the LD cycle or by omitting 1-3 sessions, indicating that learning was not dependent on light-dark cues, alternation strategies, or physiological states associated with intermeal interval. Five of six additional rats with ablations of the suprachiasmatic nucleus light-entrainable pacemaker acquired the discrimination, indicating that time-of-day cues can be provided by another circadian pacemaker (likely food-entrainable). The results provide the first clear evidence that the circadian system in a mammal can function as a consulted clock that provides discriminative time cues for cognitive processes subserving behavioral plasticity.

Enhanced food-anticipatory circadian rhythms in the genetically obese Zucker rat.

This study examines the effects of the leptin receptor mutation in obese Zucker rats on entrainment of food-anticipatory rhythms to daily feeding schedules. Leptin is secreted by adipocytes in proportion to fat content, exhibits a daily rhythm in plasma that is synchronized to feeding time, and inhibits activity of arcuate neuropeptide Y neurons that stimulate feeding behavior and regulate metabolism. Activity within this neuropeptide Y system is enhanced by food deprivation and attenuated by overfeeding and diet-induced obesity. Diet-induced obesity, in turn, attenuates food-anticipatory rhythms. If the effects of obesity on food-entrained rhythms are mediated by leptin inhibition of neuropeptide Y neurons, then these rhythms may be enhanced in leptin-insensitive Zucker obese rats. Alternatively, if daily rhythms of leptin mediate the generation or entrainment of these rhythms, Zucker rats may fail to anticipate daily feedings. Zucker obese and lean rats received food for 3 h/day during the midlight period. Both groups exhibited significant food-anticipatory activity that persisted during three cycles of food deprivation, but this rhythm was significantly more robust in obese rats, when expressed as anticipation and persistence ratios, and as peak values. Anticipatory rhythms did not persist in either group when food was provided ad lib. These results indicate that central actions of leptin may mediate the inhibitory effects of obesity on the expression of food-anticipatory rhythms in rats, but do not mediate the inhibitory effects of ad lib food access, and do not serve as necessary internal entrainment cues or clock components for the food-entrainable circadian system.

Morphine phase-shifts circadian rhythms in mice : role of behavioural activation

The effect of morphine on circadial wheel-running rhythms of C57BL/6j mice was examined. Mice received morphine (25 mg kg−1, i.p.) or saline at eight different circadian phases in constant dark. Morphine injections in the middle of the inactive period induced significant advance phase shifts, whereas injections at other times induced small delay shifts or no responses. This phase–response relationship was not altered by optic enucleation. Morphine also induced hyperactivity. Restriction of activity prevented phase shifts. The results indicate that morphine shifts circadian rhythms by its effects on behaviour, rather than by a direct action on the circadian pacemaker. Morphine may represent a useful tool for further study of behaviourally induced phase-resetting in this species.

Sleep deprivation can attenuate light-induced phase shifts of circadian rhythms in hamsters

To determine whether sleep deprivation (SD) affects the response of circadian rhythms to light, hamsters were forced to walk on a slowly rotating treadmill for 6 or 24 h, ending early in the night, with or without a light pulse during the last 30 min. SD alone did not produce a significant phase shift. Light pulses (300 and 50 lx) alone induced significant delay shifts (55 and 35 min, respectively). Twenty-four hours SD significantly attenuated the delay to brighter light and 6 h SD significantly attenuated the delay to moderate light. Sleep loss or attendant low-intensity continuous activity appear to modulate the response of the hamster circadian system to light.

Serotonin antagonists do not attenuate activity-induced phase shifts of circadian rhythms in the Syrian hamster

A variety of observations from several rodent species suggest that a serotonin (5-HT) input to the suprachiasmatic nucleus (SCN) circadian pacemaker may play a role in resetting or entrainment of circadian rhythms by non-photic stimuli such as scheduled wheel running. If 5-HT activity within the SCN is necessary for activity-induced phase shifting, then it should be possible to block or attenuate these phase shifts by reducing 5-HT release or by blocking post-synaptic 5-HT receptors. Animals received one of four serotonergic drugs and were then locked in a novel wheel for 3 h during the mid-rest phase, when novelty-induced activity produces maximal phase advance shifts. Drugs tested at several doses were metergoline (5-HT1/2 antagonist; i.p.), (+)-WAY100135 (5-HT1A postsynaptic antagonist, which may also reduce 5-HT release by an agonist effect at 5-HT1A raphe autoreceptors; i.p.), NAN-190 (5-HT1A postsynaptic antagonist, which also reduces 5-HT release via an agonist effect at 5-HT1A raphe autoreceptors; i.p.) and ritanserin (5-HT2/7 antagonist; i.p. and i.c.v.). Mean and maximal phase shifts to running in novel wheels were not significantly affected by any drug at any dose. These results do not support a hypothesis that 5-HT release or activity at 5HT1, 2 and 7 receptors in the SCN is necessary for the production of activity-induced phase shifts in hamsters.

Serotonin and feedback effects of behavioral activity on circadian rhythms in mice

Wheel running activity can shorten the period (tau) of circadian rhythms in rats and mice. The role of serotonin (5HT), in this effect of behavior on circadian pacemaker function, was assessed by measuring tau during wheel-open and wheel-locked conditions in mice sustaining neurotoxic 5HT lesions directed at the suprachiasmatic nucleus (SCN). Intact mice exhibited a significant lengthening of tau (approximately 10 min) within 3 weeks when running wheels were locked. Mice with immunocytochemically confirmed 5HT depletion showed significantly longer tau than intact mice during wheel access, and did not show a significant change in tau up to 6 weeks after wheels were locked. In these mice, variability of tau across wheel access conditions was similar in magnitude to tau variability in intact mice at two time points without wheel access (+/- 3 min). 5HT-depleted mice also exhibited significantly longer activity periods (alpha), and a significantly delayed peak of activity within alpha. Previous studies show that a delayed peak of activity within alpha is associated with longer tau. Group differences in tau, and apparent failure of wheel-locking to lengthen tau in mice with 5HT lesions, may thus be due to loss of a serotonergic behavioral input pathway to the SCN, or to a lesion-induced change in the waveform of the activity rhythm.

Phase-shifting effects of pituitary adenylate cyclase activating polypeptide on hamster wheel-running rhythms

Pituitary adenylate cyclase activating polypeptide (PACAP38) is a putative neurochemical of the main retinal input to the mammalian circadian pacemaker housed in the suprachiasmatic nucleus (SCN). We assessed the phase-resetting effects of microinjection of PACAP38 into the SCN region on hamster wheel-running rhythms. When administered during the middle of the subjective day, PACAP38 evoked large but transient phase advances ( approximately 60 min), that were followed by small, steady-state phase delays. During the early subjective night, PACAP38 elicited small to moderate phase delays without any detectable concentration-dependence. Late in the subjective night, PACAP38 had no significant effects. Saline microinjection had no effect at any phase tested. These findings show that PACAP38 has small to moderate effects on the phase of the hamster SCN circadian pacemaker, including significant phase delays early in the subjective night.

Nonphotic Phase-Shifting and the Motivation to Run: Cold Exposure Reexamined

Circadian rhythms in rodents can be phase shifted by appropriately timed activity. This may be dependent on motivational context; running induced by a novel wheel is effective, whereas running induced by cold has been inferred to be ineffective. This issue was reexamined using a different cold exposure procedure. On the first day of constant dark, 6 h before usual dark onset, Syrian hamsters were exposed to cold (±4°C) in their home cages, or were confined to novel wheels for 3 h. Activity rhythms were significantly phase advanced by 92 ± 10 min following cold exposure and 86 ± 17 min following novel wheel running, compared to 13 ± 18 min in a control condition. Most hamsters exhibited eating, drinking, and modest levels of wheel running (1367 ± 292 counts/6 h) during and for 3 h after cold exposure. Phase shifts following cold were not affected by food and water deprivation but were significantly attenuated by locking the wheel for 6 h beginning at cold onset (24 ± 12 min). These data indicate that cold-induced running, even at modest levels, is an effective nonphotic Zeitgeber and do not provide support for a hypothesis that motivational contexts determine the phase-shifting value of physical activity.