TEMPORAL RELATIONSHIPS BETWEEN THE DIURNAL RHYTHM OF HYPOTHALAMIC CORTICOTROPHIN RELEASING
FACTOR, PITUITARY CORTICOTROPHIN AND PLASMA CORTICOSTERONE IN THE RAT GUY
IXART, ALAIN SZAFARCZYK, JEAN-LUC BELUGOU
AND IVAN ASSENMACHER Laboratoire de Neuroendocrinologie, ERA 85 du CNRS, Université de
F 34060
Montpellier, France
Montpellier II,
(Received 16 February 1976) SUMMARY
Plasma corticosterone (fluorometric assay), pituitary ACTH (bioassay using isolated adrenal cells) and hypothalamic corticotrophin releasing factor (CRF) (bioassay using isolated pituitary cells) were measured singly in groups of six female rats which were killed at 11.00, 15.00, 19.00, 21.00, 23.00, 01.00, 03.00, 05.00, 07.00 and 11.00 h, after 5 weeks of adaptation to a photoperiod of 12 h light:12 h darkness. Locomotor activity was recorded continuously, using actographic cages, and the waking/sleep pattern was recorded by electroencephalography from chronically implanted control rats during the first hours of the light span. The three hormones measured fluctuated with a 24 h rhythmicity, with extreme values ranging between 4\m=.\12\m=+-\1\m=.\42and 31\m=.\78\m=+-\194 (s.e.m.) \g=m\g/100 ml for corticosterone, 4486 \m=+-\269 20and 1270 \m=+-\39\g=m\u. ACTH and 16629 \m=+-\882\g=m\u./mgpituitary for ACTH, and 439 \m=+-\ cells. production/hypothalamus/105 pituitary The onset of the ascending phase of the rhythm started during the first 2 h of light for CRF, 2 h later for ACTH, and again 2 h later for corticosterone. Similarly, the estimated acrophase of the rhythms occurred respectively, 9\m=.\4(CRF), 10\m=.\3 (ACTH) and 14\m=.\4h (corticosterone) after onset of light. These phase relationships point to a central origin of the adrenal rhythm. The diurnal activation of CRF at the very beginning of the light phase was concomitant with an almost immediate reduction of the locomotor activity and onset of sleep. These correlations favour the hypothesis of a common temporal control of both the adrenal and the sleep/waking rhythms.
INTRODUCTION
The occurrence of a marked diurnal rhythm in the plasma concentrations of glucocorticoids is a well-established process that has been verified in most species hitherto studied, namely man, monkey, rat, mouse, rabbit, and among birds: quail, the passerine Zonotrichia albicollis, duck and pigeon (Boissin, Nouguier-Soulé & Assenmacher, 1976). It is also generally accepted that the corticosteroid rhythm is inversely related to the photoperiod in diurnal and nocturnal animals respectively, since the adrenal rhythm oscillates in very close temporal relation to the sleep/wake or locomotor activity rhythm. The corticosteroid level in the circulation usually rises during the resting hours of the sleep/wake cycle, to culminate in maxi¬ mum concentrations by the time of the onset of the daily phase of activity of the animal.
More recently a similar diurnal cycle pattern in secretory activity has been demonstrated for plasma or pituitary content of corticotrophin (ACTH) in man, rats and quail (Boissin et al. 1976), and for hypothalamic corticotrophin releasing factor (CRF) concentrations in rats and pigeons (Sato & George, 1973). These studies did not accurately define the precise relationships at the three levels of the hypothalamic-pituitary-adrenocortical system; either the three cycles were not measured simultaneously in the same experiment, or the method¬
ology
used
was
not relevant.
However, precise knowledge of the sequence of events in the daily activation at the various levels of the adrenocorticotrophic system might throw some light on the mechanism whereby the production of corticosteroids increases while the animal is resting. The present set of experiments
was
designed along these lines.
MATERIALS AND METHODS
A colony of 1000 female Sprague-Dawley (Oncins-France Strain A) rats each weighing 180-190 g, was housed five animals to a cage. Rat chow (Usine d'Alimentation Rationnelle, Paris) and water were available ad libitum. The room was kept at 23 °C, with fluorescent lighting (600 lx; 6000 °K) controlled by an automatic timing device providing a 12 h light: 12 h darkness regimen, with the light span from 19.00 to 07.00 h. The animals were handled gently over 5 weeks, in order to adapt them to the operator and to the guillotine. After 5 weeks of adaptation to the environment, the rats were killed by decapitation at 4 h intervals during the dark phase of the photoperiod, and at 2 h intervals during the light phase, i.e. at 11.00, 15.00, 19.00, 21.00, 23.00, 01.00, 03.00, 05.00, 07.00 and again at U.OOh. At 19.00 and 07.00 h, death occurred 5 min before the switch from darkness to light or light to darkness respectively. At each sampling time, six rats (three per cage, the last two animals of each cage being disregarded for this experiment to avoid interference of stress) were killed. The blood was collected on solid heparin and centrifuged, and indi¬ vidual plasma samples were stored at 30 °C before measurement of corticosterone. Immediately after death, the brain was removed and the medio-basal hypothalamus including the median eminence was cut off, put into 150 /tl 0-1 M-HC1 and immediately frozen on solid C02. An equivalent amount of cortical tissue served as the control. The anterior pituitary was similarly dissected, immersed in 0-5 ml 0-1 M-HC1 and frozen. Plasma corticosterone was determined by an improved fluorometric method after preliminary bidimensional thin-layer chromatography (Szafarczyk, Moretti, Boissin & —
Assenmacher, 1974).
Pituitary ACTH was measured by a micro-bioassay first described by Swallow & Sayers (1969), and modified by Sayers, Swallow & Giordano (1971) and Sayers, Beali, Seeling & Cummings (1973). This assay is based on the corticosterone secretion of isolated adrenal cells that are incubated for 2 h together with the probe samples in a Dubnoff incubator at 37 °C in an atmosphere of 95 % 02:5 % C02 (carbogene), before measuring corticosterone production. USP-Corticotrophin (American Roland Corporation) was used as standard. Hypothalamic CRF was measured by the in-vitro bioassay of Portanova & Sayers (1973), using isolated pituitary cells that were first pre-incubated for 10 min in a Dubnoff incubator at 37 °C in a carbogene atmosphere, and then incubated for another 30 min with the probe sample (hypothalamus, cortex, or synthetic lysine-vasopressin). The supernatant was assayed for its ACTH content, and since no standard CRF was available the CRF potency of the extracts was expressed in terms of µ . ACTH produced by 105 isolated cells during the 40 min incubation test. Enumeration of the isolated cells was performed with a haemocytometer after methylene-blue staining. For the mathematical analysis of the results, the least square method (Halberg, Tong &
used and carried out on an IBM 360/65 Computer. The periodicity the basic parameters of each rhythm ; the circadian amplitude, the mean analysis provided level or mesor, and the phase. The locomotor activity rhythm of the animals was recorded in actographic cages (Szafarczyk, Nouguier-Soulé & Assenmacher, 1974) on three groups of rats living in the same room as those used in the hormonal study. Finally, in order to explore the sleep/wake state of the rats during the first hours of the daily resting phase (light phase) the electroencephalogram patterns were studied from a group of three chronically implanted control rats exposed to the same environmental conditions. The electrodes were located in the cortex, hippocampus, and neck and eye muscles.
Johnson, 1965)
was
RESULTS
Diurnal rhythm ofplasma corticosterone Figure profile of corticosterone rhythm, with the following main characteristics: a marked amplitude, since the corticosterone concentrations were fluctuating between 4-12 ± 1-42 and 31-78 ± 1-94 (s.e.m.) /¿g/100 ml plasma; the onset of the increasing phase of the rhythm occurring between 4 and 6 h after the initiation of the lightspan of the photoperiod; the peak values of the rhythm located at the very end of the light1 illustrates the classical
phase.
Diurnal rhythm of pituitary ACTH content The amplitude of the diurnal fluctuations of the pituitary content of ACTH was remarkably high since the values rose from a minimum of 4-5 ± 0-2 mu. ACTH/mg pituitary to a maximum of 16-6 ±0-3 mu./mg. On the other hand, the daily increase in pituitary ACTH took place between 2 and 4 h after the onset of the light phase while the maximal levels coincided with the end of this
phase.
Diurnal rhythm
of hypothalamic CRF content An initial experiment was designed to assess the specificity of the bioassay used. In this experiment the CRF potency of hypothalamic extracts was compared with that of: (1) equivalent (20 mg) extracts from cortical regions of the brain; and (2) addition of 100 mu. synthetic lysine-vasopressin. Figure 2 shows that, while the two latter treatments induced a significant but moderate stimulating effect on ACTH release by the isolated pituitary cells, the hypothalamic extract displayed a CRF potency that was almost twice that of the others. We concluded from this experiment that we were measuring a specific CRF activity in the hypothalamus, which occurred at a high concentration in that part of the brain. Regarding the variations of hypothalamic CRF activity over the 24 h period, it can be seen from Fig. 1 that the activity followed a definite diurnal profile, with values fluctuating between 439 ±20 and 1270 ±39/tu. ACTH/hypothalamus/105 pituitary cells. On the other hand a significant (50 %) increase in hypothalamic CRF concentration occurred within the first 2 h of the light phase. After a 4 h plateau, the CRF potency increased further to reach a maximum 2 h before the end of the light period. Phase relationships between the CRF, ACTH and corticosterone rhythms It can be seen from the preceding results that the onset of the daily increase in any hormonal parameter occurred in the sequence: CRF, ACTH and corticosterone, at approximately 2 h intervals. Another way to calculate the sequence of events is based on a mathematical calculation
(least-square-method) of the acrophases (calculated position of the peaks) in relation to the diurnal rhythms (Table 1). The periodicity analysis demonstrated acrophases located respectively 9-4 (CRF), the beginning of the light period.
that the three rhythms studied had their 10-3 (ACTH) and 14-4 h (corticosterone) after
Fig. I. Diurnal rhythm in hypothalamic corticotrophin releasing factor (CRF) ( ), pituitary ACTH (O) and plasma corticosterone (A) in rats exposed to 12 h light and 12 h darkness. Values are means
±s.e.m. of 6 rats.
Diurnal rhythm in locomotor activity The shape of the diurnal variations of locomotor activity conforms to the classical pattern in rats. During the light span of the photoperiod the animals displayed very reduced activity and the small amount of waves of moderate electrical activity recorded from the actographic cages was possibly indicative of short periods of awakening. In contrast to the quiescent light phase of the cycle, the dark phase corresponded to high locomotor activity, which began about 1 h before darkness. There were several (usually three to four) waves of peak activity throughout the dark phase which ended abruptly when the light was switched on.
500
400
È
-
2 300 a o
200
F
100
FACTOR, PITUITARY CORTICOTROPHIN AND PLASMA CORTICOSTERONE IN THE RAT GUY
IXART, ALAIN SZAFARCZYK, JEAN-LUC BELUGOU
AND IVAN ASSENMACHER Laboratoire de Neuroendocrinologie, ERA 85 du CNRS, Université de
F 34060
Montpellier, France
Montpellier II,
(Received 16 February 1976) SUMMARY
Plasma corticosterone (fluorometric assay), pituitary ACTH (bioassay using isolated adrenal cells) and hypothalamic corticotrophin releasing factor (CRF) (bioassay using isolated pituitary cells) were measured singly in groups of six female rats which were killed at 11.00, 15.00, 19.00, 21.00, 23.00, 01.00, 03.00, 05.00, 07.00 and 11.00 h, after 5 weeks of adaptation to a photoperiod of 12 h light:12 h darkness. Locomotor activity was recorded continuously, using actographic cages, and the waking/sleep pattern was recorded by electroencephalography from chronically implanted control rats during the first hours of the light span. The three hormones measured fluctuated with a 24 h rhythmicity, with extreme values ranging between 4\m=.\12\m=+-\1\m=.\42and 31\m=.\78\m=+-\194 (s.e.m.) \g=m\g/100 ml for corticosterone, 4486 \m=+-\269 20and 1270 \m=+-\39\g=m\u. ACTH and 16629 \m=+-\882\g=m\u./mgpituitary for ACTH, and 439 \m=+-\ cells. production/hypothalamus/105 pituitary The onset of the ascending phase of the rhythm started during the first 2 h of light for CRF, 2 h later for ACTH, and again 2 h later for corticosterone. Similarly, the estimated acrophase of the rhythms occurred respectively, 9\m=.\4(CRF), 10\m=.\3 (ACTH) and 14\m=.\4h (corticosterone) after onset of light. These phase relationships point to a central origin of the adrenal rhythm. The diurnal activation of CRF at the very beginning of the light phase was concomitant with an almost immediate reduction of the locomotor activity and onset of sleep. These correlations favour the hypothesis of a common temporal control of both the adrenal and the sleep/waking rhythms.
INTRODUCTION
The occurrence of a marked diurnal rhythm in the plasma concentrations of glucocorticoids is a well-established process that has been verified in most species hitherto studied, namely man, monkey, rat, mouse, rabbit, and among birds: quail, the passerine Zonotrichia albicollis, duck and pigeon (Boissin, Nouguier-Soulé & Assenmacher, 1976). It is also generally accepted that the corticosteroid rhythm is inversely related to the photoperiod in diurnal and nocturnal animals respectively, since the adrenal rhythm oscillates in very close temporal relation to the sleep/wake or locomotor activity rhythm. The corticosteroid level in the circulation usually rises during the resting hours of the sleep/wake cycle, to culminate in maxi¬ mum concentrations by the time of the onset of the daily phase of activity of the animal.
More recently a similar diurnal cycle pattern in secretory activity has been demonstrated for plasma or pituitary content of corticotrophin (ACTH) in man, rats and quail (Boissin et al. 1976), and for hypothalamic corticotrophin releasing factor (CRF) concentrations in rats and pigeons (Sato & George, 1973). These studies did not accurately define the precise relationships at the three levels of the hypothalamic-pituitary-adrenocortical system; either the three cycles were not measured simultaneously in the same experiment, or the method¬
ology
used
was
not relevant.
However, precise knowledge of the sequence of events in the daily activation at the various levels of the adrenocorticotrophic system might throw some light on the mechanism whereby the production of corticosteroids increases while the animal is resting. The present set of experiments
was
designed along these lines.
MATERIALS AND METHODS
A colony of 1000 female Sprague-Dawley (Oncins-France Strain A) rats each weighing 180-190 g, was housed five animals to a cage. Rat chow (Usine d'Alimentation Rationnelle, Paris) and water were available ad libitum. The room was kept at 23 °C, with fluorescent lighting (600 lx; 6000 °K) controlled by an automatic timing device providing a 12 h light: 12 h darkness regimen, with the light span from 19.00 to 07.00 h. The animals were handled gently over 5 weeks, in order to adapt them to the operator and to the guillotine. After 5 weeks of adaptation to the environment, the rats were killed by decapitation at 4 h intervals during the dark phase of the photoperiod, and at 2 h intervals during the light phase, i.e. at 11.00, 15.00, 19.00, 21.00, 23.00, 01.00, 03.00, 05.00, 07.00 and again at U.OOh. At 19.00 and 07.00 h, death occurred 5 min before the switch from darkness to light or light to darkness respectively. At each sampling time, six rats (three per cage, the last two animals of each cage being disregarded for this experiment to avoid interference of stress) were killed. The blood was collected on solid heparin and centrifuged, and indi¬ vidual plasma samples were stored at 30 °C before measurement of corticosterone. Immediately after death, the brain was removed and the medio-basal hypothalamus including the median eminence was cut off, put into 150 /tl 0-1 M-HC1 and immediately frozen on solid C02. An equivalent amount of cortical tissue served as the control. The anterior pituitary was similarly dissected, immersed in 0-5 ml 0-1 M-HC1 and frozen. Plasma corticosterone was determined by an improved fluorometric method after preliminary bidimensional thin-layer chromatography (Szafarczyk, Moretti, Boissin & —
Assenmacher, 1974).
Pituitary ACTH was measured by a micro-bioassay first described by Swallow & Sayers (1969), and modified by Sayers, Swallow & Giordano (1971) and Sayers, Beali, Seeling & Cummings (1973). This assay is based on the corticosterone secretion of isolated adrenal cells that are incubated for 2 h together with the probe samples in a Dubnoff incubator at 37 °C in an atmosphere of 95 % 02:5 % C02 (carbogene), before measuring corticosterone production. USP-Corticotrophin (American Roland Corporation) was used as standard. Hypothalamic CRF was measured by the in-vitro bioassay of Portanova & Sayers (1973), using isolated pituitary cells that were first pre-incubated for 10 min in a Dubnoff incubator at 37 °C in a carbogene atmosphere, and then incubated for another 30 min with the probe sample (hypothalamus, cortex, or synthetic lysine-vasopressin). The supernatant was assayed for its ACTH content, and since no standard CRF was available the CRF potency of the extracts was expressed in terms of µ . ACTH produced by 105 isolated cells during the 40 min incubation test. Enumeration of the isolated cells was performed with a haemocytometer after methylene-blue staining. For the mathematical analysis of the results, the least square method (Halberg, Tong &
used and carried out on an IBM 360/65 Computer. The periodicity the basic parameters of each rhythm ; the circadian amplitude, the mean analysis provided level or mesor, and the phase. The locomotor activity rhythm of the animals was recorded in actographic cages (Szafarczyk, Nouguier-Soulé & Assenmacher, 1974) on three groups of rats living in the same room as those used in the hormonal study. Finally, in order to explore the sleep/wake state of the rats during the first hours of the daily resting phase (light phase) the electroencephalogram patterns were studied from a group of three chronically implanted control rats exposed to the same environmental conditions. The electrodes were located in the cortex, hippocampus, and neck and eye muscles.
Johnson, 1965)
was
RESULTS
Diurnal rhythm ofplasma corticosterone Figure profile of corticosterone rhythm, with the following main characteristics: a marked amplitude, since the corticosterone concentrations were fluctuating between 4-12 ± 1-42 and 31-78 ± 1-94 (s.e.m.) /¿g/100 ml plasma; the onset of the increasing phase of the rhythm occurring between 4 and 6 h after the initiation of the lightspan of the photoperiod; the peak values of the rhythm located at the very end of the light1 illustrates the classical
phase.
Diurnal rhythm of pituitary ACTH content The amplitude of the diurnal fluctuations of the pituitary content of ACTH was remarkably high since the values rose from a minimum of 4-5 ± 0-2 mu. ACTH/mg pituitary to a maximum of 16-6 ±0-3 mu./mg. On the other hand, the daily increase in pituitary ACTH took place between 2 and 4 h after the onset of the light phase while the maximal levels coincided with the end of this
phase.
Diurnal rhythm
of hypothalamic CRF content An initial experiment was designed to assess the specificity of the bioassay used. In this experiment the CRF potency of hypothalamic extracts was compared with that of: (1) equivalent (20 mg) extracts from cortical regions of the brain; and (2) addition of 100 mu. synthetic lysine-vasopressin. Figure 2 shows that, while the two latter treatments induced a significant but moderate stimulating effect on ACTH release by the isolated pituitary cells, the hypothalamic extract displayed a CRF potency that was almost twice that of the others. We concluded from this experiment that we were measuring a specific CRF activity in the hypothalamus, which occurred at a high concentration in that part of the brain. Regarding the variations of hypothalamic CRF activity over the 24 h period, it can be seen from Fig. 1 that the activity followed a definite diurnal profile, with values fluctuating between 439 ±20 and 1270 ±39/tu. ACTH/hypothalamus/105 pituitary cells. On the other hand a significant (50 %) increase in hypothalamic CRF concentration occurred within the first 2 h of the light phase. After a 4 h plateau, the CRF potency increased further to reach a maximum 2 h before the end of the light period. Phase relationships between the CRF, ACTH and corticosterone rhythms It can be seen from the preceding results that the onset of the daily increase in any hormonal parameter occurred in the sequence: CRF, ACTH and corticosterone, at approximately 2 h intervals. Another way to calculate the sequence of events is based on a mathematical calculation
(least-square-method) of the acrophases (calculated position of the peaks) in relation to the diurnal rhythms (Table 1). The periodicity analysis demonstrated acrophases located respectively 9-4 (CRF), the beginning of the light period.
that the three rhythms studied had their 10-3 (ACTH) and 14-4 h (corticosterone) after
Fig. I. Diurnal rhythm in hypothalamic corticotrophin releasing factor (CRF) ( ), pituitary ACTH (O) and plasma corticosterone (A) in rats exposed to 12 h light and 12 h darkness. Values are means
±s.e.m. of 6 rats.
Diurnal rhythm in locomotor activity The shape of the diurnal variations of locomotor activity conforms to the classical pattern in rats. During the light span of the photoperiod the animals displayed very reduced activity and the small amount of waves of moderate electrical activity recorded from the actographic cages was possibly indicative of short periods of awakening. In contrast to the quiescent light phase of the cycle, the dark phase corresponded to high locomotor activity, which began about 1 h before darkness. There were several (usually three to four) waves of peak activity throughout the dark phase which ended abruptly when the light was switched on.
500
400
È
-
2 300 a o
200
F
100
