Resetting the rat circadian clock by ultra-short light flashes

We examined the effects that ultra-brief, intense, light flashes have on the rat circadian clock, the suprachiasmatic nucleus of the hypothalamus (SCN). We found that as few as five intense flashes, each 10-micros in duration (1 per s), can produce both phase shifts in free-running activity rhythms and Fos expression in the SCN in rats kept in constant darkness. After pre-exposure to such flashes, phase shifts in response to a continuous light pulse delivered 2 h later were potentiated, but Fos expression in the SCN was decreased as following pre-exposure to continuous light. These results show that flashes induce behavioral and cellular effects indicative of clock resetting similar to those induced by light stimuli of longer duration. Extremely brief but intense, light stimuli may be much more important to clock resetting than had been previously known.     

Resetting of a feeding-entrainable circadian clock in the rat.

Reentrainment of anticipatory activity (AA) after phase shifts of food access was studied in rats with lesions of the suprachiasmatic nuclei. Eight- or 10-h phase delays of feeding time resulted in delaying transients of AA. Twelve-h phase shifts and some 10-h phase advances resulted in rapid reentrainment (2-3 days) without visible transients. Most phase advances of 10 h resulted in delaying transients while 8-h advances induced transients with advancing and delaying components in a number of rats. Split transients were not prevented by advancing mealtime in 1-h steps. Phase shifts of food in multiple steps failed to accelerate delay shifts but retarded advance shifts. After the first 8-h phase delay shift, increased activity reappeared at the preshift phase of AA, simultaneously with anticipation of the phase-shifted meal time. The observation of split transients indicates that two or more circadian oscillators mediate entrainment to mealtime, and the reappearance of AA at a previously established phase suggest the possibility that this system has a memory of phase displacement.     

Resetting of a circadian clock by food pulses.

Rats with lesions of the suprachiasmatic nuclei were exposed to daily feeding until anticipatory activity (AA) developed. Meals were then phase advanced or delayed and presented for 1-4 consecutive days. The phase of the circadian pacemaker was assessed during probes of total food deprivation before or after 8 days of intervening ad lib feeding. One or two food pulses caused phase delays for one cycle but were insufficient to reset the feeding entrainable pacemaker. Complete or partial resetting to 6- or 9-h advances or delays was observed in some rats after three food pulses and in all rats after four food pulses. In some rats, phase shifts of meals appeared to induce both advancing and delaying transients, and two bouts of AA appeared during food-deprivation probes. This suggests that the feeding entrainable pacemaker consists of two or more oscillators which became uncoupled after phase shifts. The persistence of AA at the preshift phase observed after initial phase delays, concomitantly with AA to the phase shifted meals, also suggests the presence of a second oscillator.     

Shaping the light/dark pattern for circadian adaptation to night shift work

This is the second in a series of simulated night shift studies designed to achieve and subsequently maintain a compromise circadian phase position between complete entrainment to the daytime sleep period and no phase shift at all. We predict that this compromise will yield improved night shift alertness and daytime sleep, while still permitting adequate late night sleep and daytime wakefulness on days off. Our goal is to delay the dim light melatonin onset (DLMO) from its baseline phase of ∼ 21:00 to our target of ∼ 3:00. Healthy young subjects (n = 31) underwent three night shifts followed by two days off. Two experimental groups received intermittent bright light pulses during night shifts (total durations of 75 and 120 min per night shift), wore dark sunglasses when outside, slept in dark bedrooms at scheduled times after night shifts and on days off, and received outdoor light exposure upon awakening from sleep. A control group remained in dim room light during night shifts, wore lighter sunglasses, and had unrestricted sleep and outdoor light exposure. After the days off, the DLMO of the experimental groups was ∼ 00:00-1:00, not quite at the target of 3:00, but in a good position to reach the target after subsequent night shifts with bright light. The DLMO of the control group changed little from baseline. Experimental subjects performed better than control subjects during night shifts on a reaction time task. Subsequent studies will reveal whether the target phase is achieved and maintained through more alternations of night shifts and days off.     

Conditioned stimulus control in the rat circadian system depends on clock resetting during conditioning

The authors examined the ability of a conditioned stimulus (CS; mild air disturbance) previously paired with an entraining light pulse to reset the circadian pacemaker in rats. Rats were entrained to a single 30-min light stimulus delivered every 25 hr or 24 hr (T cycle). Each daily light presentation was paired with the CS. After at least 20 days of stable entrainment to each of the T cycles, the rats were allowed to free run and were then presented with the CS at circadian time 15. CS-induced phase shifts in wheel-running activity rhythms were taken as evidence for conditioning. For the most part, conditioning occurred after CS-light pairings on the 25-hr but not 24-hr T cycle. The results suggest that CS control of the circadian clock phase depends on the effect that the entraining light pulse has on the clock during conditioning.     

Responsiveness of the aging circadian clock to light

The present study assessed whether advances in sleep times and circadian phase in older adults might be due to decreased responsiveness of the aging circadian clock to light. Sixteen young (29.3 ± 5.6 years) and 14 older adults (67.1 ± 7.4 years) were exposed to 4 h of control dim (10 lux) or bright light (3500 lux) during the night. Phase shifts of the melatonin rhythm were assessed from the nights before and after the light exposure. Bright light delayed the melatonin midpoint in both young and older adults (p < 0.001). Phase delays for the older subjects were not significantly different from those of the young subjects for either the bright or dim light conditions. The magnitude of phase delays was correlated with both sleep offset and phase angle in the older, but not the younger subjects. The present results indicate that at light intensities commonly used in research as well as clinical practice older adults are able to phase delay to the same extent as younger subjects.     

Reduced light sensitivity of the circadian clock in a hypopigmented mouse mutant

Pink-eyed dilution (p/p) is a recessive mutation in mice which results in reduced pigmentation of the retinal pigment epithelium, as well as alterations in visual pathways and function. We investigated whether this mutation also affects light information reaching the circadian clock. Entrainment to a 12 h light 12 h dark cycle and the free-running period in constant darkness were not affected by this mutation. Phase shifts in response to 1 h light pulses consisting of bright white light at either circadian time 16 or 24 also did not differ between mutant and wild-type C57BL/6J mice. However, when 5 min, 502 nm light pulses of 1.2 × 10^-1 W/cm² or 4 × 10^-2 W/cm² were given at circadian time 16, the mutant mice responded with significantly smaller phase shifts than the wild-type mice. When animals were transferred to constant light, the free-running period of wild-type mice was longer than that of mutant mice, a finding which is consistent with a sensitivity difference between mutant and wild-type mice. Horseradish peroxidase tracing of retinal innervation of the hypothalamic suprachiasmatic nuclei (SCN) — the location of a circadian pacemaker — revealed a reduced innervation of the SCN in mutant mice compared with wild-type mice. The total volume of the SCN, as determined by neutral red stain, was also reduced in mutant mice, although not to as great an extent as the retinal innervation. Taken together, these results indicate that while basic characteristics of circadian clock function are not altered by the pink-eyed dilution mutation, the sensitivity of the clock to light is reduced. Whether the reduced retinal innervation results in the decreased sensitivity of the clock to light, or whether the decreased sensitivity is a result of other unknown changes in the light input to the clock remains to be determined.     

Rapid reentrainment of the circadian clock itself, but not the measurable activity rhythms to a new light-dark cycle in the rat.

Experiments were performed to determine if the circadian clock reentrains more quickly to an 8-hour phase shift in light-dark (LD) cycles than does the overt rhythm of activity. To investigate the reentrainment of the clock itself to an 8-hour advance or delay in the LD cycle, the rats were released into constant darkness only two or three days after a shift in LD cycle, and the amount of the phase shift of the clock itself was estimated from where free-running rhythm started by backward extrapolation. If the circadian clock could rapidly reset itself to the new LD cycle, it was predicted that the free-running rhythm of activity would start from near the dark period of the new LD cycle rather than the preceding one. When rats were released into constant darkness three days after the LD cycle was advanced by 8 hours, the activity of the free-running rhythm started near time of dark period of the new LD cycle in all rats (n = 16). When rats (n = 24) were released into constant darkness two days after the LD cycle was advanced by 8 hours, 12 rats started the activity near time of dark period of the new LD cycle, while 9 rats started the activity near time of dark period of the preceding LD cycle. The remaining 3 rats showed the activity of the free-running rhythm near intermediate phase (transient phase). On the other hand, when the rats were not released into constant darkness after LD cycle was advanced by 8 hours, it took 6.4 days for activity rhythm to reentrain to the advanced LD cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leptin phase-advances the rat suprachiasmatic circadian clock in vitro

The suprachiasmatic nucleus (SCN) controls circadian rhythms in mammals. The SCN may also participate in regulating body metabolism and energy. Similar to other hypothalamic nuclei, the SCN have been reported to contain glucose-sensitive neurons and receptors for the adipose tissue hormone, leptin. Here we investigated leptin effects on the SCN clock. Our results demonstrate that the SCN circadian clock, when isolated in vitro, can be phase advanced by leptin in a dose-dependent fashion that does not require non-SCN hypothalamic tissue. Phase advances are induced at all circadian times except late subjective night. These data suggest that peripheral signals of energy and metabolism directly modulate the circadian pacemaker in mammals.     

Cholecystokinin concentration in specific brain areas of rats fed during the light or dark phase of the circadian cycle

Measurement of peptide concentration in specific areas can be used as an initial investigative method for identifying brain sites in which the peptides may be acting. In this study cholecystokinin (CCK) concentration in specific hypothalamic and hindbrain areas of male Sprague-Dawley rats was measured in order to determine whether changes occurred as a result of feeding activity during different portions of the circadian cycle. Three groups of 40 rats each were studied: Group 1 were fasted 16 hr during the dark phase then sacrificed immediately or after a 20 min light phase meal. Group 2 were fasted 16 hr during the light phase then sacrificed immediately after lights out or after a 20 min dark-onset meal. Group 3 were fed ad lib and sacrificed immediately after light out or after a 20 min dark-onset meal. CCK was extracted from dissected areas and concentration was measured by RIA. There was no difference in CCK concentration of any of the 9 brain areas in rats fasted during the dark phase and fed during the light phase. In rats fasted during the light phase CCK concentration of the paraventricular nucleus (PVN) was greater in those that subsequently ate a meal at dark-onset than in those that did not eat (p less than 0.05). In ad lib fed rats CCK concentration was less in the anterior hypothalamus (AH) and greater in the supraoptic nucleus (SON) in rats that ate a dark-onset meal than in rats that did not (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat circadian rhythms entrain to a descending saw-tooth light intensity cycle.

We reexamined whether rat circadian rhythms entrained to the light intensity cycle of a descending saw-tooth (ST-d) form, in which illuminance decreased rectilinearly from 300 lx to 0 lx in 24 h, and abruptly returned to 300 lx (lights-on time). Ambulation, drinking and subcutaneous body temperature were simultaneously monitored in 5 intact, 5 pinealectomized and 5 orchiectomized rats. Additionally, sleep was monitored in the intact rats. In all the rats, entrainment was confirmed during 65 days' exposure to the ST-d cycle. The waveforms of the entrained rhythms were much modified compared with those during LD 12:12. The estimated activity periods of the entrained rhythms straddled the lights-on time of the ST-d cycle. In all the groups of rats, administration of a single ST-d cycle in constant dim red light produced only delay shifts irrespective of its circadian phase, and there was no significant circadian variation in the magnitude of phase shifts. The results indicate that rat circadian rhythms entrain to the ST-d cycle with an unexpected phase position, which cannot be explained by the phase-response curve.     

Brief constant light accelerates serotonergic re-entrainment to large shifts of the daily light/dark cycle

Brief (∼2 day) constant light exposure (LL_b) in hamsters dramatically enhances circadian phase-resetting induced by the 5-HT receptor agonist, (±)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) and other nonphotic stimuli. The present study was undertaken to determine if LL_b can also amplify phase-resetting responses to endogenous 5-HT and accelerate re-entrainment to large-magnitude advance and delay shifts of the light/dark (LD) cycle. First, central serotonergic activity was increased by i.p. injection of l-tryptophan±the 5-HT reuptake inhibitor fluoxetine. Hamsters under LD or exposed to LL_b received vehicle or drugs during the early morning, and phase-shifts of the locomotor activity rhythm were measured after release to constant darkness. Neither drug phase-shifted animals not exposed to LL_b (P>0.5 vs. vehicle); however in animals receiving LL_b,l-tryptophan with and without fluoxetine produced large phase-advance shifts (means=2.5±0.4 h and 2.6±0.2 h, respectively; both P<0.035 vs. vehicle). Next, the effects of LL_b combined with 8-OH-DPAT or l-tryptophan+fluoxetine on serotonergic re-entrainment to 10 h phase-advance and phase-delay shifts of the LD cycle were assessed. In groups not exposed to LL_b, vehicle controls re-entrained slowly to the advance and delay shifts (means=16±1 and 24±4 days, respectively), but those treated with 8-OH-DPAT re-entrained faster (means=11±2 and 9±2 days, respectively; both P<0.05 vs. vehicle). In groups exposed to LL_b, vehicle controls re-entrained slowly to the advance and delay shifts (means=15±2 and 25±3 days, respectively); however those receiving 8-OH-DPAT rapidly re-entrained to the delay and advance shifts, with the majority (75%) requiring only 1–2 days (means=2±1 and 4±2 days, respectively; both P<0.05 vs. vehicle). Animals exposed to LL_b and treated with l-tryptophan+fluoxetine also exhibited accelerated re-entrainment to a 10 h advance shift (mean=5±2 days; P<0.05 vs. vehicle). Thus through enhancing serotonergic phase-resetting, LL_b facilitates rapid re-entrainment to large shifts of the LD cycle which offers a potential approach for treating circadian-related desynchronies.     

Transient circadian internal desynchronization after light-dark phase shift in monkeys.

In four conscious chair-acclimatized squirrel monkeys (Saimiri sciureus) studied with lights on (600 lx) from 0800 to 2000 h daily (LD 12:12), prominent 24-h rhythms in feeding, drinking, activity, body temperature, and urinary potassium, sodium, and water excretion were seen. When the monkeys were subjected to 36 h of darkness followed by 36 h of light each variable demonstrated a circadian rhythm which was not passively dependent on the light-dark cycle. After the 24-h light-dark cycle was abruptly phase-delayed by 8 h, all the rhythms resynchronized with the new light-dark cycle phase, demonstrating that light-dark cycles are an effective zeitgeber. However, the resynchronization of the rhythms of feeding, drinking, activity, and body temperature was 90% complete within approximately 2 days while the 90% resynchronization of the urinary rhythms took approximately 5 days. These results suggest that the circadian timing system in S. sciureus may consist of several spontaneously oscillating units which can become transiently uncoupled during pertubations of environmental time cues.     

Influence of age on behavioural response in the light/dark paradigm.

We have compared the performance of male Swiss mice at different ages (correlated with different body weight; 12-34 g) in the light/dark test of anxiety. Mice received saline only. The best age at which control values were optimum was that of 4 weeks old. Mice at this age spent 58% of the total test duration in the dark compartment. The oldest mice (i.e., 8 weeks old) exhibited an increase in total activity characterised by increase in movements in each compartment, together with an increase in the number of transitions. An age-related effect was found suggesting caution when interpreting the results of mice in the light/dark paradigm, the best period being that of 4 weeks.     

Impact of a circadian clock on the timing of human sleep

This paper redescribes some recordings of human sleep and waking made in several laboratories during the past decade under conditions of temporal isolation. Since 1972 it has been noticed that sleep duration depends mainly on the timing of prior sleep onset relative to a rhythm of 24- to 25-h duration. The present paper emphasizes four additional points: 1) that the dependence sometimes includes a remarkable discontinuity, 2) that such dependence is characteristic of a rhythmically modulated threshold process; 3) that the rhythm's period gradually changes in some experiments; and 4) that no comparable regularity has been detected in the timing of sleep onset. This last impugns the reliability of models that treat sleep onset and wake onset as complementary but comparable processes.     

Adaptation to a visuomotor shift depends on the starting posture

Previous studies have shown that human subjects can adapt to a new visuomotor relationship that depends on the trajectory of the arm. However, these studies have not distinguished between hand- and joint-based learning models. We have examined whether different endpoint kinematics are necessary to obtain a differential visuomotor shift. The joint trajectory was varied by changing the initial posture, while maintaining a similar finger trajectory. After learning, maximum after-effects were found when movement began with the posture used during exposure to the visuomotor shift and decreased with the difference between initial and trained posture. This was shown to be independent of the final posture attained. Our results show that adaptation to a visual remapping cannot be due to the recoding of a desired final posture and depends on the arm trajectory in joint space.     

Control of rat hypothalamic pro-opiomelanocortin neurons by a circadian clock that is entrained by the daily light-off signal.

Previous studies have clearly demonstrated that the immediate-early gene, c-fos can regulate, through its protein product Fos, the expression of the pro-opiomelanocortin gene. In the present study, immunohistochemistry for Fos and beta-endorphin was used to assess the basal activity of hypothalamic pro-opiomelanocortin-producing neurons throughout a 12 h light/12 h dark cycle. Here, we showed that Fos is undetectable in most beta-endorphin neurons from late morning until 30 min after light offset in the evening, whereas Fos is spontaneously expressed in these neurons after 1 h following dark onset. The number of beta-endorphin neurons expressing Fos increases continuously during the first half of the dark phase, is maximal at the middle of this phase and decreases through late night and early morning, reaching a nadir 2-3 h after light onset. Acute shifts of lighting parameters allowed us to demonstrate that the light-off signal per se is neither sufficient nor necessary for Fos expression in beta-endorphin neurons. However, when recurrent, this signal is able to entrain Fos expression after a period of adaptation to the new light/dark schedule. Moreover, an expression of Fos in beta-endorphin neurons persists during subjective night in rat exposed to constant light or constant dark for two to three days. Thus, the occurrence of the daily rhythmic increase in the expression of Fos protein in hypothalamic pro-opiomelanocortin neurons exclusively at (subjective) night suggests that these neurons are, most likely, controlled by a (circadian) nocturnal oscillator. Our data also reveal an interesting property of this oscillator: its entrainment by the daily light-to-dark transition signal.