Phase shifts in the circadian rhythm in plasma concentrations of melatonin in rams induced by a 1-hour light pulse

The effect of a 1-hr light pulse, given at night, on the timing of the circadian rhythm in the plasma concentration of melatonin was examined in Soay rams to investigate the mechanisms involved in determining the duration of the nocturnal peak in melatonin secretion. Animals (n = 8) were housed under short days (LD 8:16) or long days (LD 16:8) and received a light pulse at various times of night. They were released into constant dim red light (DD) on day 1. Blood samples were collected hourly for 30 hr from 1000 hr on day 3, and the plasma concentration of melatonin was determined by radioimmunoassay to assess the timing of the melatonin peak. Control animals (n = 8) were maintained under the same conditions but received no light pulse. Under short days, a light pulse given early in the night caused a phase delay in the melatonin peak, and a light pulse given in the late night caused a phase advance. The mean duration of the melatonin peak was slightly reduced following a light pulse in the early or late night, and slightly increased following a pulse given near the middle of the night. Under long days, both light-pulse treatments given at night caused a phase delay in the melatonin peak, but there was no significant change in duration of the melatonin peak. The duration of the melatonin peak at day 3 under DD in the control animals was similar for all treatments, regardless of the previous entraining photoperiod (mean duration: 12.6-14.8 hr) and was similar to that under short days (14.6 hr), but was significantly longer than that under long days (8.2 hr). Information on the phase response curve in the Soay ram and on the period of the circadian oscillator governing the melatonin rhythm (c 23.0 hr under DD) predicts a close phase relationship between the end of the light phase and the onset of the melatonin peak as observed under normal 24-hr LD cycles. The current results also indicate that light acts to entrain the circadian rhythm influencing the onset and offset of melatonin secretion, and thus dictates the duration of the melatonin peak.     

Immunoneutralization of corticotropin-releasing hormone prevents the diurnal surge of ACTH

To determine whether CRH is required for the evening rise in plasma ACTH, rats were injected at 0800 hr with CRH antiserum (anti-CRH) or normal rabbit serum (NRS). Blood samples were taken through venous catheters at 0800 hr before treatment and at 1300, 1700, and 2100 hr. Plasma was assayed for immunoreactive ACTH and corticosterone. There was no significant difference in pretreatment values between the two groups. Immunoneutralization of CRH abolished the rise in plasma ACTH seen at 1700 hr in the NRS group but had little effect on earlier levels. The diurnal elevation in plasma corticosterone continued after anti-CRH treatment, but peak levels occurred earlier. Plasma ACTH and corticosterone were significantly correlated at the time of the diurnal surge, but not at 0800 hr or 1300 hr in the NRS controls or at any time point in the anti-CRH group. These results suggest that CRH is required for the diurnal surge of plasma ACTH. They also confirm previous observations by others that the adrenal cortex does not require active CRH or a diurnal surge of ACTH in order to exhibit a significant diurnal increase in secretion of corticosterone, and that factors other than CRH may be relatively more active than CRH in regulation of ACTH secretion during the time of circadian inactivity.     

5-methoxypsoralen: Effect on the noradrenaline circadian rhythm of sleep-deprived subjects

5-methoxypsoralen (5-MOP), a serotonin analogue, stimulates the secretion of melatonin, which plays an important role in circadian rhythm regulation. Melatonin production is essentially controlled by noradrenaline (NA). To investigate the effect of 5-MOP on the 24 hr NA rhythm, hourly plasma NA concentrations measured over 24 hr in 7 healthy young subjects who took 40 mg 5-MOP orally at 21:00 hr the evening before were compared with values obtained in drug-free subjects. All subjects were sleep-deprived and under conditions in which sympathetic nervous system activation was repeated every hour over 24 hr. In both series of patients, a significant difference was observed between mean morning values and mean night values, reflecting persistence of a circadian rhythm. In subjects given 5-MOP, a significant difference was also observed between mean afternoon values and mean night values. However, MANOVA analysis failed to find any difference between the two series of subjects. Acute administration of 5-MOP thus had no significant effect on the NA circadian rhythm under our study conditions.     

Cocaine-induced stimulation of the rat hypothalamic-pituitary-adrenal axis is progressively attenuated following hourly-interval regimens of the drug.

The role of multiple (iv) injections of cocaine on the rat hypothalamic-pituitary-adrenal (HPA) axis was examined using four different temporal regimens of drug exposure. In intact rats, cocaine (5 mg/kg) consistently stimulated the secretion of adrenocorticotropin hormone (ACTH) and corticosterone over a 6 hr interval regimen. In all experimental groups, administration of the vehicle alone failed to measurably alter the secretion of the aforementioned hormones. When rats where exposed to the drug over a 4 hr interval regimen, a modest attenuation of ACTH, but not corticosterone, secretion was observed following the third and last cocaine injection. To test whether the attenuation of ACTH secretion to cocaine administration was caused by corticosterone-mediated negative feedback, the response of intact and adrenalectomized (ADX) rats over 2 hr and 1 hr interval regimens was compared. In intact rats, both drug interval regimens resulted in a significant attenuation of ACTH secretion following, the second and third injections of the drug. ADX rats, on the other hand, exhibited significant increases in ACTH levels following either interval regimens, though we observed a modest blunting of pituitary responsiveness to the 1 hr regimen. From these results we conclude that in intact rats the activity of the HPA axis is significantly attenuated in response to multiple, acute cocaine injections, and that this decreased response may be at least in part caused by a negative corticoid feedback mechanism.     

ACTH-provoked cortisol peaks during sleep and their effect on the endogenous secretory activity

The effects of low physiological doses of ACTH on nocturnal plasma cortisol patterns were investigated in six male subjects. Concomitant sleep EEG recordings were analysed in relation to the cortisol level. 250 ng ACTH1-24 (Synacthène), injected through an indwelling catheter at a period of low adreno-cortical activity (2400 h), induced a cortisol peak followed by a four to five-hour period without any cortisol secretion. The same dose of ACTH injected at 0430 h, when cortisol secretory activity was high, did not entirely abolish endogenous secretion, which was diminished for a shorter time (2.5 hr). The ACTH-provoked cortisol peaks of comparable size to endogenous secretory peaks, can suppress cortisol secretion for several hours. This suppressive capacity depends on timing in relation to high or low secretory activity periods. However, spontaneous cortisol peaks have no appreciable effect on further secretory episodes. This difference in suppressive capacities suggests that the 24 hr cortisol rhythm is regulated independently of such feedback mechanisms.     

Pulsatile ACTH secretion: variation with time of day and relationship to cortisol

It has long been known that ACTH is secreted in an episodic fashion demonstrating circadian and ultradian rhythms. High intensity venous sampling has recently revealed that in addition to these larger ultradian fluctuations in hormone levels, plasma ACTH in rats demonstrates high frequency, low amplitude oscillations which have been called "micropulses." These micropulses were not detected in previous studies due to sampling intervals of greater than 5 minutes. To investigate the presence of these ACTH micropulses in a primate species, blood samples were drawn from six chair-restrained rhesus monkeys at one-minute intervals for up to 70 minutes and plasma was assayed for immunoreactive ACTH. To assess the variation in ACTH micropulse parameters with time of day and the relationship to cortisol secretion, four of the monkeys were sampled for three 70-minute periods beginning at 0530, 1100, and 1730 hours, and plasma was assayed for immunoreactive ACTH and cortisol. Analysis of the data revealed that ACTH and cortisol are secreted in micropulses in rhesus monkeys with marked individual variation in the pattern of secretion and a concurrence of approximately 75% of ACTH and cortisol micropulses. Difference in pulse amplitude but not frequency appeared to contribute to the circadian variation in mean ACTH levels and a sampling interval of two minutes appeared to be adequate for accurately identifying micropulses of ACTH.     

Diurnal rhythm of drinking activity in the house musk shrew]

A diurnal rhythm of drinking activity in 7 male and 6 female house musk shrews (Jic: SUN) aged about one year was observed over a period of 10 days under a schedule of 12 hr light and 12 hr darkness (light on at 07:00). In general, the pattern of drinking activity was similar among both sexes, with around 24-hr diurnal rhythm. A few typical drinking patterns of these animals were represented as follows: 1) Drinking interval was very close in the dark phase, while it was a little too sparse in the light phase (n = 4). 2) Its interval remains stationary through a whole day (n = 5). 3) Drinking was performed between the latter half of light and the first half of dark phases (n = 4).     

Circadian rhythm of serotonin binding in rat brain--II. Influence of sleep deprivation and imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P less than 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed through the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak value of 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

Effect of ACTH on circadian periodicity of nuclear volume and mitotic division in the zona fasciculata externa of the adrenal cortex of mice.

In cells of the zona fasciculata externa of the adrenal cortex of mice, the maximal value of nuclear volume is observed in evening and night time, while the mitotic peak occurs in the early part of the day. Ten day subcutaneous injection of 1.5 units of ACTH twice in 24 hr produced nuclear hypertrophy and stimulation of mitotic activity of cells of the zona fasciculata externa. The circadian periodicity of nuclear volume in mice injected with ACTH is disturbed, while the circadian rhythm of mitotic activity is retained.     

Differences in cortisol, aldosterone and testosterone responses to ACTH 1-17 administered at two different times of day

The effects of 100 micrograms, i.m. of the analog ACTH 1-17 administered at 0800 and 1800 on the secretion of cortisol, aldosterone and testosterone have been studied in normal subjects: 8 male and 8 female. The group as a whole and the males had significantly greater absolute and percent increments in plasma cortisol after administration at 1800. In the females, there was only a greater percent increment in cortisol after the evening administration. The heptadecapeptide always significantly stimulated serum aldosterone, with no difference between the two times of administration. In the females, ACTH 1-17 significantly stimulated testosterone, with a more protracted secretion after the evening administration. In the males, there was always a significant testosterone decrease after the administration of the drug, with no difference between morning and evening. In conclusion, 100 micrograms i.m. of the analog ACTH 1-17 stimulates cortisol secretion more when given during the circadian nadir of plasma cortisol, but only in men. ACTH 1-17 increases testosterone in women and decreases it in men, whereas it seems to increase aldosterone secretion in both sexes.     

Circadian and circahemidian rhythms in plasma prolactin of the ram: seasonal changes

The existence of a circadian rhythm in plasma prolactin of the ram is controversial. Differences among authors can be related to both data sampling (e.g. interindividual changes, time of day, time of year, sampling interval among others) and statistical analyses. To test this hypothesis six adult "Préalpes du Sud" rams were studied individually during 72 hr in January (8 hr of light-16 hr of darkness), April (13L-11D), June (16L-8D) and September (13L-11D). Blood was sampled (vacutainer) from a jugular vein every hour, centrifuged and plasma samples stored at -20 degrees C until prolactin determinations (radioimmunoassay) were made. Individual time series were analysed according to three complementary methods: display of raw data (chronogram), best fitting cosine functions with different period tau (iterative cosinors) and power spectra. Seasonal changes in the 24 hr mean (peak time in June) were confirmed. A circahemidian rhythm (tau = 12 hr) and a circadian rhythm (tau = 24 hr) were validated, respectively in January and April while time series documented in June and September exhibited no rhythmic organization. It seems, therefore, that animals adjusted their rhythmic patterns of prolactin secretion to the increasing (January, April) rather than decreasing (June, September) photofraction (duration of the light span/24 hr).     

Daily profiles of aldosterone levels during normal, high and low sodium intake in patients with essential hypertension.

The episodic secretion of aldosterone depends on the dietary sodium intake, alterations in posture and follows ACTH circadian rhythm. Aldosterone daily profiles have been studied in 23 supine essential hypertensive patients on normal sodium intake. Secretory pulses at a frequency of two to five pulses per 12 hr have occurred, independent of PRA levels. Among 13 patients with normal PRA two lost pulsatility when sodium was loaded (10 g/24 hr) and the same happened with two others on sodium restricted diet (2 g/24 hr). These results suggest a profound effect of dietary sodium intake on the pulsatile pattern of aldosterone secretion, particularly in normal PRA essential hypertension.     

Responses of plasma adrenocorticotropin and cortisol to intravenous injection of synthetic ovine corticotropin releasing factor in the morning and early evening in normal human subjects

This study was designed to compare the responsiveness of adrenocorticotropin (ACTH) and cortisol secretion to corticotropin-releasing factor (CRF) in the morning and early evening in normal human subjects. Synthetic ovine CRF (1.0 micrograms/kg) or normal saline, was administered as an i.v. bolus injection to six normal males at 900 h and 1700 h. Blood samples were obtained before and 15, 30, 60, 90 and 120 min after CRF or saline injection. Significant increases in plasma ACTH and cortisol levels were observed in all subjects at the both time of testing after CRF injection. The net increments in the areas under the concentration curve (areas in the CRF experiment minus those in the saline control experiment) were not statistically different for both ACTH (mean +/- SEM: 41.0 +/- 10.6 pg/ml h in the morning: 51.1 +/- 8.9 pg/ml h in the evening) and cortisol (mean +/- SEM: 28.5 +/- 5.0 micrograms/dl h in the morning; 36.2 +/- 4.0 micrograms/dl h in the evening). Also no significant difference was observed in net increment, peak level and the ratio of peak level to the basal level of ACTH and cortisol after CRF injection. There were no appreciable changes in plasma concentrations of growth hormone, thyroid-stimulating hormone or prolactin, although slight but statistically significant rises in plasma levels of luteinizing hormone and follicle-stimulating hormone were observed. These results suggest that there is no significant difference in responsiveness of the pituitary-adrenal axis to CRF in the morning (900 h) and early evening (1700 h), and thus the time of day will not necessarily have to be considered when CRF is used between these times in a clinical test to evaluate pituitary ACTH reserve.     

Diurnal rhythm of plasma immunoreactive corticotropin-releasing factor in normal subjects

Plasma corticotropin-releasing factor (CRF), corticotropin (ACTH) and cortisol levels were simultaneously determined by radioimmunoassays at 0600 h, 1200 h, 1800 h and 2200 h in six normal subjects, in order to examine whether the diurnal rhythm in plasma CRF exists and how it correlates to the diurnal rhythm in plasma ACTH and cortisol concentration. The highest CRF level was observed at 0600 h (7.0 +/- 1.2 pg/ml) and significantly lower levels (p less than 0.01) at 1800 h (1.7 +/- 0.2 pg/ml) and 2200 h (1.9 +/- 0.4 pg/ml). A clear diurnal rhythm was demonstrated in plasma ACTH and cortisol levels, with the highest values at 0600 h (44.6 +/- 8.1 pg/ml and 15.9 +/- 2.0 micrograms/dl, respectively) and the lowest at 2200 h (12.3 +/- 2.8 pg/ml and 4.6 +/- 1.0 micrograms/ml, respectively). These results suggest that the diurnal rhythm in ACTH and cortisol is under the regulation, at least in part, of the diurnal rhythm in CRF secretion.     

Opiate receptor blockade and diurnal pituitary and adrenal hormone levels

Peripheral pituitary hormone levels exhibit circadian variations though the mechanism of these changes is unknown. In order to investigate the possible role of endogenous opiates in such changes we have studied the influence of opiate receptor blockade with naloxone (6.8 mg) on pituitary hormones in the morning and again in the evening in six normal male volunteers. Basal ACTH, cortisol, aldosterone and prolactin were higher in the morning than in the evening. Following naloxone at 0700h both ACTH and cortisol rose indicating a tonic inhibition of ACTH by endogenous opiates at that time. At 2230h cortisol rose following naloxone but ACTH did not, suggesting that endogenous opiates do not play an important role in the diurnal rhythm of this hormone and consistent with the suggestion that endogenous opiates can effect cortisol levels independently of their action on ACTH. Neither aldosterone nor prolactin were influenced by naloxone. In contrast TSH was unaffected by naloxone in the morning but fell in the evening (mean + SE decrement over 120 min -0.6 +/- 0.3 mU/l as compared with the control +0.6 +/- 0.4 mU/l; p less than 0.01). Thus, endogenous opiates probably tonically stimulates TSH levels in the evening when TSH may increase and possibly play a role in the circadian rhythm of TSH.     

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.     

Adrenocortical responsiveness to the synthetic ACTH 1-17 analogue given at different circadian stages

The study concerns the adrenocortical glucocorticoid responsiveness to the ACTH 1-17 analogue given at different times of the day, namely near to the zenith and the nadir of the circadian curve of plasma cortisol. Two schedules of administration of the heptadecapeptide have been performed: a pulse i.v. injection of 4 microgram; b. i.m. injection of 100 microgram. Both the doses were given to the same subject in the morning and in the evening of different days. The chrono-sensitivity of the adrenal cortex to ACTH 1.17 analogue is well evident after pulse stimulation by a micro-dose of the heptadecapeptide; in fact the plasma cortisol increase from basal values is significantly higher in evening than in the morning (p less than 0.001). The cortico-stimulatory effect of the higher dose of ACTH 1.17 analogue lasts about 12 h. and then plasma and urinary glucocorticoids take the usual circadian pattern again.     

The relative Zeitgeber strength of lights-on and lights-off is changed in old mice

The daily activity pattern of old mice is characterized by a decreased amplitude, a phase advance, and less stable relationship between lights-off and the onset of the main activity maximum. When analyzing the possible causes of these changes, it must be remembered that the activity rhythm of laboratory mice is bimodal, with a main peak in the first half of the dark time and a secondary one shortly after lights-on. Thus it seems to be controlled by at least two circadian oscillators--an "evening oscillator" coupled more strongly to lights-off and a "morning oscillator" coupled to lights-on--though both oscillators are also coupled to each other. The objective of the present paper was to investigate the putative changes in the strength of these couplings in HaZ:ICR mice of different ages (adult animals of 20 weeks, n = 12; old mice of 72 and 91 weeks of age, n = 6 each) and kept in a 24 h LD-cycle with a gradually reduced light:dark ratio. In adult mice, lengthening the dark time caused the onset of the main maximum of activity to be delayed in relation to the time of lights-off, while the morning maximum of activity was advanced in relation to lights-on. On average, the sizes of the advance and the delay were equal. As a consequence, the activity pattern did not shift in relation to the middle of the dark time. Lengthening the dark time resulted in a bigger (on average, 1.5 h) difference between the evening and morning activity onsets. Under short photoperiods (< or = 2 h of light) the activity rhythm started to free run, and the difference between evening and morning activity onsets decreased again. The changes obtained in senile mice were similar. However, the limits of entrainment were reached with longer photoperiods compared to adult animals. Also, the phase delay of the activity onset in the evening was much less, nearly zero. As a consequence, the activity pattern as whole phase-advanced in relation to the middle of the dark time. A model was proposed in which lights-off triggers advances of the "evening oscillator," lights-on delays the "morning oscillator," and the two oscillators are coupled with each other. Though it was probably the case, decreased coupling strengths could not be shown with the present experimental approach. However, it was clearly evident that, with increasing age, the advancing effect of lights-off exceeded the delaying effect of lights-on.     

Circadian Rhythm of Serotonin Binding in Rat Brain—II. Influence of Sleep Deprivation and Imipramine

Sleep deprivation (SD) modified the circadian rhythm of specific high affinity serotonin (5-HT) binding to rat brain membranes. In control rats a 24-hr rhythm was evident with a trough at 1000-1200 and a nadir at 0000. During the last 26 hr of a 49 hr SD period, trough and peak values were delayed by 4-6 hr. The 24-hr mean binding was significantly (P < 0.001) different from that of controls. If sleep deprivation was followed by recovery sleep (RS), the normal rhythm of 5-HT binding was obtained already within 1 hr after SD. The effects of SD and RS were ascertained by plasma ACTH and corticosterone assay. No significant change in the hormone rhythms were observed though the mean plasma level of ACTH and corticosterone were enhanced to about 180 and 150%, respectively. Chronic treatment with the antidepressant imipramine resulted in a decrease of the 24-hr mean 5-HT binding by about 50% and a 2-hr delay of peak and trough values. Imipramine treatment decreased the peak valueof 5-HT concentration at 1000 to about 65% and appears to abolish the rhythm of 5-HT concentration.     

Rapid photoperiodic responses in Japanese quail: is daylength measurement based upon a circadian system?

Experimental photoperiods, presented either once only or repeatedly, were used to assess the oscillatory and hourglass properties of the photoperiodic clock in Japanese quail. Gonadectomized quail on 8-hr daylengths respond to a single skeleton photoperiod consisting of two 8-hr light pulses separated by 2 hr of darkness (i.e., LDLD 8:2:8:6) with a marked increase in secretion rate of luteinizing hormone (LH). This response suggests that the second light pulse interacts with a "photoinducible phase" (phi i) lying some 10-16 hr from "dawn" (start of the first light pulse). If, however, groups of quail maintained on 8-hr daylengths are transferred to continuous darkness (DD), and the position of the phi i is sought by a single 8-hr light pulse applied at various times on the first or third day of DD, then an increase in circulating LH is, at best, barely detectable. It would appear that a strongly responsive phi i does not recur rhythmically in DD. Instead, the light pulse apparently acts primarily as a "dawn" signal that triggers a single cycle of photoinducibility, since a second 8-hr light pulse, placed to begin 2 hr after the end of the first, induces a large increase in plasma LH. Similar results are obtained if any single 8-hr light pulse presented to animals held in darkness is preceded, 10 hr earlier, by a short "dawn" light signal. Such dawn signals can be effective when very short; a pulse of only 30 sec can cause a subsequent phi i. The dawn pulse is effective at any circadian phase and leads to a single cycle in photoinducibility. In contrast, a much longer light pulse (perhaps not less than 4 hr) is needed to interact with phi i if significant gonadotropin secretion is to be stimulated. In confirmation of the findings described above, we found that Nanda-Hammer lighting schedules have remarkably little effect in stimulating gonadotropin secretion in gonadectomized quail. There is, for example, a very marked difference between the effectiveness of "resonating" schedules such as LD 6:6, which stimulates a high LH secretion rate since each "inductive" light pulse is preceded by an appropriate "dawn" signal, and a theoretically effective schedule such as LD 6:30, which induces a very small response by comparison. Such schedules (even theoretically noninductive ones) can, however, be made very highly inductive if alternate light pulses are preceded by an appropriately positioned 15-min light pulse to act as "dawn."