Brain Research, 169 (1979) 531-544 © Elsevier/North-HollandBiomedicalPress
531
EFFECTS OF PARACHLOROPHENYLALANINE AND 5, 6-DIHYDROXYTRYPTAMINE ON THE FREE-RUNNING RHYTHMS OF LOCOMOTOR ACTIVITY AND PLASMA CORTICOSTERONE IN THE RAT EXPOSED TO CONTINUOUS LIGHT
KEN-ICHI HONMA, KENJI WATANABEand TSUTOMU HIROSHIGE Department of Physiology, Hokkaido University School of Medicine, Sapporo 060 (Japan)
(Accepted October 12th, 1978)
SUMMARY Parachlorophenylalanine (PCPA) and 5,6-dihydroxytryptamine (5,6-DHT), depletors of brain serotonin, were administered to the rat and circadian rhythms of locomotor activity and plasma corticosterone were determined simultaneously in individual rats in light-dark cycles (LD) and in 200 lux continuous light (LL). Freerunning periods and acrophases on the 12th day in LL (LL12) were calculated by a least squares spectrum method. In PCPA-treated rats which showed 70 % depletion of brain serotonin, circadian rhythms of locomotor activity in LL and of plasma corticosterone and ACTH in LD disappeared for several days after the drug injection. Circadian rhythms of locomotor activity reappeared after the LL7 day and free-ran with a phase shift. Free-running periods of these rats did not differ significantly from that of control rats. However, the acrophase of PCPA-treated group on the LL11 day was 5 h advanced as compared to that of control. Circadian rhythm of plasma corticosterone in the PCPA-treated rats was detected on the LL12 day but their peak times were distributed around 24:00 h instead of 08:00 h observed in control rats. The 5,6-DHT-treated rats which showed only 40 % depletion of brain serotonin exhibited normal free-running rhythms in both locomotor activity and plasma corticosterone in LL and no difference in the acrophases of these functions on the LL12 day as compared to controls. These results suggest that PCPA affects the circadian clock (or clocks) itself in such a way that it blocks the clock to free-run or at least it effectively shortens the free-running periods of locomotor activity and plasma corticosterone in the rat. INTRODUCTION Although numerous reports have implicated some essential roles of brain serotonergic neurons in the regulation of sleep'wakefulness cycle and of CRF-ACTH
532 secretion a,13,14, there is little evidence for the serotorlergic neurons involved in the circadian oscillation mechanism per se (the circadian clock). Administration of drugs that affect the metabolism of central serotonergic neurons or selective destruction of the central nervous system have been reported to change the circadian rhythms o f sleep-wakefulness16,19 and plasma corticosterone 17,24-~6. But the effects of these drugs were transient and apparently did not correlate with brain serotonin content 27. Therefore the role of brain serotonergic system in the circadian rhythm was questionedl, ~. These previous studies, however, were performed on animals exclusively in light-dark cycles (LD) and the results were evaluated only on the data of group. One could not obtain from the data of group an insight into whether the circadian clock or a transmission pathway was affected by the drug treatment. In this study circadian rhythms of locomotor activity and plasma corticosterone were determined in individual rats in LL as well as in LD and free-running periods and acrophases of these functions were calculated in individual rats which were treated with PCPA and 5,6-DHT, respectively. The purpose of this study was to estimate through these analyses the site of action of the drugs used and to evaluate the role of central serotonergic neurons in the operation of the circadian clock. MATERIALS AND METHODS
Animals and experimental conditions. Male adult rats of Wistar strain, weighing 300-350 g, were used. Animals were bred and reared in a light conditioned room, where environmental conditions were kept constant (temperature 22 4- 1 °C, humidity 60 ~ , light 06:30-18:30 h). Light intensity at the surface of rat cages was 200 lux. Rats were housed in individual cages for 2 weeks prior to the beginning of the experiments. After weaning, all the rats were fed commercial chow and water ad libitum. Measurement of biological rhythms and brain amines. (1) Spontaneous locomotor activity. Spontaneous locomotor activity of the rat was measured by a F A R A D Animex type S. This instrument measures the changes in capacitance of a resonance circuit installed beneath a cage. The amount of activity was expressed by counts, which were recorded when the animal moved. (2) Plasma ACTH and corticosterone. A serial blood sampling was performed in individual rats at 2 or 4 h intervals to obtain a circadian rhythm of plasma A C T H and corticosterone. When a sampling at 2 h intervals was done, 1 ml of 0.5 ~o of bupivacaine hydrochloride (Marcain, Yoshitomi, Tokyo) was injected subcutaneously at the base of the tail at 06:00 h on the experimental day as a local anesthetic. The tail end was cut by razor and a few drops of blood were collected into a capillary tube. When plasma A C T H was simultaneously determined, blood was collected into a plastic microtube of Beckman (400 /~1) containing 5 /~1 of 6 ~ Na2-EDTA. The blood sampling was finished within 2 rain. Blood was centrifuged immediately and plasma was stored in --80 °C until the assay of plasma A C T H and corticosterone. Plasma A C T H was assayed by A C T H radioimmunoassay kit (CIS France). Plasma corticosterone was measured by the method of Glick et al. 4 and in part by a competitive protein binding assay 20, using the plasma of rhesus monkey as a source of transcortin. The two methods gave a negligible difference
533 in plasma corticosterone levels determined. (3) Brain norepinephrine and serotonin. Animals were sacrificed between 12.00 and 13.00 h by decapitation. Brain tissue was quickly removed and sectioned on a dry-ice plate following the method of Glowinski and IversenS. Samples were stored in --20 °C until used. Brain norepinephrine and serotonin were determined by the method of Maickel et al. is. Experimentalprocedures. Two series of experiments were performed. Experiment 1.30 mg/100 g body weight ofp-chlorophenylalanine methylester.HC1 (Sigma) was injected intraperitoneally to rats and 3 days after the first injection a second injection of the same dose was done. 5 rats were used for the determination of plasma ACTH and corticosterone 3 days after the first injection (LD3) and 12 days (LD12) in LD. A serial blood sampling was performed at 4 h intervals starting from 08:00 to 04:00 h on the following day. Plasma was divided into each assay tube (100 #1 for ACTH and 10 /~1 for corticosterone). 19 rats were exposed to 200 lux continuous light. PCPA was injected to 10 rats at 08:00 h on the first day of exposure to constant light (LL1) and the LL4 day. On the LL12 day, serial blood sampling was done in both PCPA-treated and non-treated rats, starting from 08:00 to 04:00 h on the following day at 4 h intervals. Locomotor activity of 6 rats in both groups was monitored continuously from the onset of the experiment. 40 rats were used for the determination of brain monoamine contents after PCPA injection. To know a time course of the drug effect, rats were decapitated 3, 5, 9 and 12 days after PCPA treatment in LD. Decapitation was performed at 12:00 h. Non-treated 10 rats were sacrificed in LD as a control. Experiment 2. 5,6-Dihydroxytryptamine creatinine sulfate (Sigma)was administered intraventricularly to the rat. 6 rats were anesthetized by pentobarbital and 75 #g of 5,6-DHT dissolved in 10/~1 of 0.9 ~o saline solution containing 0.5 ~o ascorbic acid was injected into the lateral ventricle of the brain by means of a stereotactic apparatus. The other 6 rats were injected with the solvent as a control on the same surgical procedures. 2 and 6 days after the operation (PO2 and PO6), serial blood sampling was performed at 2 h intervals starting from 08:00 to 06:00 h on the following day in LD. Then the rats were exposed to 200 lux continuous light. On the LL12 day, blood samples were obtained by a serial sampling on the same schedule as performed in LD. A few days after, rats were sacrificed by decapitation and brain tissue was removed for the determination of brain monoamine contents. Locomotor activity of the rats was monitored throughout the experiment. Statistical analysis. A least squares spectrum method was used for the calculation of free-running circadian parameters (mesor, amplitude and period) of locomotor activity8. To estimate an acrophase of the circadian rhythm, the first step of cosinor analysis was modified 9. The measured variable (Yi) is a function of the time of observation (ti), according to the following formula, Yi = Co + C cos (2 ~r x ti/T q- 99). Here, Co indicates a mesor, C an amplitude, 99 a phase-angle to an appropriate reference and T a period. Co, C and 99 are calculated by the least squares spectrum method. 99 is given as follows,
534
[n l q~ = tan -1
-\
Yl sin 2~ xi/n i=O
)
I; Yi cos 2z~ xi/n /i=O
To test a difference between two mean values, Student's t-test was used. RESULTS
Effect o f PCPA on the free-running circadian rhythms o f locomotor activity and plasma corticosterone A time course of hypothalamic amine contents after the administration of PCPA was shown in Table I. PCPA reduced brain serotonin content to 30 9/00of control value for at least 5 days. Serotonin content recovered gradually afterward and reached a normal level 12 days after the first injection of the drug. Fig. 1 illustrates the effect of PCPA on the circadian rhythms of plasma A C T H and corticosterone in LD. Three days after the injection, circadian rhythms of both functions were abolished and flattened at the mean level of the group. In individuals, some fluctuation of plasma A C T H and corticosterone was observed but marked circadian rhythm was not detected in all rats. 12 days after the first injection, entrained circadian rhythms of plasma A C T H and corticosterone appeared in both individual rats and mean levels of 5 rats. Fig. 2 illustrates free-running rhythms of locomotor activity of PCPA-treated and non-treated rats in 200 lux continuous light. PCPA was administered on the LLI day and the LL4 day. A circadian rhythm of locomotor activity of a PCPA-treated rat (no. 1) was damped from the time of drug administration and did not manifest itself for about 6 days in LL. From the LL7 day, circadian rhythm of locomotor activity was restored and ran with a period slightly longer than 24 h (phase-shift). In control rat (no. 1), a marked free-running circadian rhythm was observed from the onset of continuous light and showed a phase-shift. TABLEI
Time course of hypothalamic amine contents after the administration of PCPA PCPA was injected intraperitoneally in doses of 30 mg/100 g body weight, twice. Amine contents (mean values ± S.E. of the mean) were given in terms of/~g/g wet tissue weight. Numbers in parentheses indicate number of rats sacrificed.
Days after the first injection
Serotonin
Norepinephrine
Control PCPA-treated rats 3 days 5 days 9 days 12 days
1.54 ~c 0.04 (10)
1.72 ± 0.08 (10)
0.48 4- 0.02 (10)* 0.45 :t= 0.03 (8)* 0.88 =l_0.09 (8)* 1.43 ± 0.09 (11)
1.51 :k 0.21 (10) -1.64 :~- 0.12 (8) --
* P < 0.001 vs control.
535 PCPA
N=5
LD 3
No.2
N0.1 2OO
,40
40
200 40
200
20
100 20
100
"0 uI
ixi -d o. o
3
el 20
100
i
-I-
%
%,1
i
LD12 40
200
40
1200 40,
2oo
20
100 20;
=100
-D
m
,g ~
E
100
2O
nl n
'-r. i
12oo
2400
120O
24OO
I~,~ ~,~
12oo
240o
Fig. 1. Effects of PCPA on the circadian rhythms of plasma ACTH and corticosterone in LD in both grouped and individual rats. Two typical individuals are shown. Blood samplings were performed 3 days (LD3) and 12 days (LD 12) after the drug injection. Open circles indicate values of plasma corticosterone and closed circles those of plasma ACTH. Vertical lines represent S.E. of the mean. Black horizontal bars in the bottom of each column indicate a dark period in a day. Human ACTH 1-39 was used as a standard in the assay.
Fig. 3 shows circadian rhythms of locomotor activity and plasma corticosterone determined simultaneously in individual rats on the LL12 day. Circadian rhythm of plasma corticosterone was observed in both rats but the peak of the rhythm was detected at 24:00-04:00 h in the PCPA-treated rat while the peak was seen at 12:00 h in the control rat. Fluctuations of the mean levels of plasma corticosterone in individual rats are demonstrated in Fig. 4. In control groups, the peak of the rhythm was observed at 08:00 h, indicating that about 16 h of phase-shift took place in LL (the peak of the rhythm existed at 16:00 h in LD). In contrast, the peak of plasma corticosterone rhythm in PCPA-treated group was seen at 24:00 h. This means only a 8 h phase-shift occurred in PCPA-treated group during the same period in LL. The distribution of the peak time of plasma corticosterone in individual rats on the LL12 day was shown in Table II. The mode of the distribution in control rats was 08:00 h and that in PCPAtreated was 24:00 h. Table I I I summarizes free-running circadian parameters (period, amplitude and mesor) of locomotor activity and its acrophase on the LL11 day. The peak time was also given of plasma corticosterone observed on the LL12 day in 12 individual rats of control and PCPA-treated group. In these rats locomotor activity and plasma corticosterone were simultaneously determined, since serial blood sampling per se was shown to not essentially affect the circadian features of the locomotor activity rhythm lo. Free-running period of the locomotor activity of PCPA-treated group was
536 25.1 i 0.1 h, which was not significantly different from that of control group. However, an acrophase of locomotor activity on the LL11 day was 08:45 ~ 38 min in PCPA-treated group and 13:44 ± 45 min in control, the difference being about 5 h and highly significant (P < 0.001).
Locomotor Activity (counts/15rain.) PCPA
treated
Rat
Control
:::::::::::::::::::::::::::::::
Rat
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LD
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~h I
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_ J_
t
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l ........
ml
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,.,.I
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Fig. 2. Free-running rhythms of locomotor activity in 200 lux LL. PCPA was injected on the LLI day and the LL4 day in PCPA-treated rat (no. 1). Abscissa indicates a time of day, starting from 16: 30 h to 16: 30 h on the following day. Shadowed portion indicates a dark period in LD. Locomotor activity was expressed by histogram, recorded by counts per 15 rain.
537
i,o 1000~ CtttCt LL12
Contro[ No.1
~ 20 t
!,° t 2000
i%~L 1000~
,iI
¢ I"'~ t ' ~ ' I ' LL 1 2
Fig. 3. Simultaneously determined circadian rhythms of locomotor activity and plasma corticosteron¢ on the LL12 day in a PCPA-treated rat (no. 1) and in a control (no. 1). Sampling times, indicated by arrows, started from 0 8 : 0 0 h at 4 h intervals.
Control LL12
PCPA LL 12
N=9
N=10
20
m 20 U nl
10
E e~
%
i
1200
i
24OO
I
120o
I
24OO
Fig. 4. Circadian rhythms of plasma corticosterone on the LLI2 day in both PCPA-treated and nontreated groups. Abscissa indicates a time of day. Vertical lines indicate S. E. of the mean. P values against the same points of control group were expressed in some points of PCPA-treated group.
538 TABLE I1
Distribution of the acrophase (peak time) of the plasma corticosterone level determined on the 12th day after exposure to continuous light (LLI2 day) Acrophase (h)
Control (9)
PCPA-treated (10)
08:00 12:00 16:00 20:00 24:00 04:00
6 1 0 0 0 2
0 2 0 2 5-6* 1-0"
* One rat showed the acrophase at both 24:00 and 04:00.
TABLE llI
Free-running circadian parameters (period, amplitude and mesor) of locomotor activity and its acrophase on the LLI1 day The peak times of plasma corticosterone were observed on the LL 12 day in individual rats in which both functions were determined simultaneously.
Free-running parameters Period PCPA-treated rats No. 1 25.0 2 25.1 3 25.6 4 24.6 5 25.2 6 25.3 25.1 i 0 . 1 Controlrats No.l 25.7 2 25.2 3 25.0 4 25.4 5 25.2 6 25.4 25.3 ± 0.1
Amplitude
Acrophase on L L l l
Mesor
Peak time of corticosterone on LL12
399 137 124 87 147 180 179 ± 46
416 418 356 440 331 334 383 ~_ 19
10:38 06:57 09:40 06:58 09:53 08:26 08:45 ± 00:38*
24:00 or 04:00 20:00 20: 00 12:00 24:00 24:00
237 215 178 86 240 228 197 ± 24
323 341 373 271 394 333 339
16:07 12:14 11:43 15:28 12:27 14:22 13:44 ± 00:45
12:00 08:00 08 : 00 08 : 00 08:00 08:00
17
* P < 0.001 vs corresponding control values.
Effect o f 5 , 6 - D H T on the free-running circadian rhythms o f locomotor activity and plasma corticosterone Th e effects o f i n t r a v e n t r i c u l a r a d m i n i s t r a t i o n o f 5 , 6 - D H T on the e n t r a i n e d circadian r h y t h m s o f l o c o m o t o r activity an d p l a s m a c o r t i c o s t e r o n e in a rat was d e m o n s t r a t e d in Fig. 5. T h e circadian r h y t h m s o f b o t h functions were disturbed f o r a b o u t 2 days after the drug administration. But 6 days after the injection (PO6), circadian r h y t h m s were restored. H y p o t h a l a m i c serotonin c o n t e n t was reduced to 60 ~o o f c o n t r o l value in the 5 , 6 - D H T - t r e a t e d rats.
539
5,6-DHT LD I=O2
~ 2o
PO 6
P
531
EFFECTS OF PARACHLOROPHENYLALANINE AND 5, 6-DIHYDROXYTRYPTAMINE ON THE FREE-RUNNING RHYTHMS OF LOCOMOTOR ACTIVITY AND PLASMA CORTICOSTERONE IN THE RAT EXPOSED TO CONTINUOUS LIGHT
KEN-ICHI HONMA, KENJI WATANABEand TSUTOMU HIROSHIGE Department of Physiology, Hokkaido University School of Medicine, Sapporo 060 (Japan)
(Accepted October 12th, 1978)
SUMMARY Parachlorophenylalanine (PCPA) and 5,6-dihydroxytryptamine (5,6-DHT), depletors of brain serotonin, were administered to the rat and circadian rhythms of locomotor activity and plasma corticosterone were determined simultaneously in individual rats in light-dark cycles (LD) and in 200 lux continuous light (LL). Freerunning periods and acrophases on the 12th day in LL (LL12) were calculated by a least squares spectrum method. In PCPA-treated rats which showed 70 % depletion of brain serotonin, circadian rhythms of locomotor activity in LL and of plasma corticosterone and ACTH in LD disappeared for several days after the drug injection. Circadian rhythms of locomotor activity reappeared after the LL7 day and free-ran with a phase shift. Free-running periods of these rats did not differ significantly from that of control rats. However, the acrophase of PCPA-treated group on the LL11 day was 5 h advanced as compared to that of control. Circadian rhythm of plasma corticosterone in the PCPA-treated rats was detected on the LL12 day but their peak times were distributed around 24:00 h instead of 08:00 h observed in control rats. The 5,6-DHT-treated rats which showed only 40 % depletion of brain serotonin exhibited normal free-running rhythms in both locomotor activity and plasma corticosterone in LL and no difference in the acrophases of these functions on the LL12 day as compared to controls. These results suggest that PCPA affects the circadian clock (or clocks) itself in such a way that it blocks the clock to free-run or at least it effectively shortens the free-running periods of locomotor activity and plasma corticosterone in the rat. INTRODUCTION Although numerous reports have implicated some essential roles of brain serotonergic neurons in the regulation of sleep'wakefulness cycle and of CRF-ACTH
532 secretion a,13,14, there is little evidence for the serotorlergic neurons involved in the circadian oscillation mechanism per se (the circadian clock). Administration of drugs that affect the metabolism of central serotonergic neurons or selective destruction of the central nervous system have been reported to change the circadian rhythms o f sleep-wakefulness16,19 and plasma corticosterone 17,24-~6. But the effects of these drugs were transient and apparently did not correlate with brain serotonin content 27. Therefore the role of brain serotonergic system in the circadian rhythm was questionedl, ~. These previous studies, however, were performed on animals exclusively in light-dark cycles (LD) and the results were evaluated only on the data of group. One could not obtain from the data of group an insight into whether the circadian clock or a transmission pathway was affected by the drug treatment. In this study circadian rhythms of locomotor activity and plasma corticosterone were determined in individual rats in LL as well as in LD and free-running periods and acrophases of these functions were calculated in individual rats which were treated with PCPA and 5,6-DHT, respectively. The purpose of this study was to estimate through these analyses the site of action of the drugs used and to evaluate the role of central serotonergic neurons in the operation of the circadian clock. MATERIALS AND METHODS
Animals and experimental conditions. Male adult rats of Wistar strain, weighing 300-350 g, were used. Animals were bred and reared in a light conditioned room, where environmental conditions were kept constant (temperature 22 4- 1 °C, humidity 60 ~ , light 06:30-18:30 h). Light intensity at the surface of rat cages was 200 lux. Rats were housed in individual cages for 2 weeks prior to the beginning of the experiments. After weaning, all the rats were fed commercial chow and water ad libitum. Measurement of biological rhythms and brain amines. (1) Spontaneous locomotor activity. Spontaneous locomotor activity of the rat was measured by a F A R A D Animex type S. This instrument measures the changes in capacitance of a resonance circuit installed beneath a cage. The amount of activity was expressed by counts, which were recorded when the animal moved. (2) Plasma ACTH and corticosterone. A serial blood sampling was performed in individual rats at 2 or 4 h intervals to obtain a circadian rhythm of plasma A C T H and corticosterone. When a sampling at 2 h intervals was done, 1 ml of 0.5 ~o of bupivacaine hydrochloride (Marcain, Yoshitomi, Tokyo) was injected subcutaneously at the base of the tail at 06:00 h on the experimental day as a local anesthetic. The tail end was cut by razor and a few drops of blood were collected into a capillary tube. When plasma A C T H was simultaneously determined, blood was collected into a plastic microtube of Beckman (400 /~1) containing 5 /~1 of 6 ~ Na2-EDTA. The blood sampling was finished within 2 rain. Blood was centrifuged immediately and plasma was stored in --80 °C until the assay of plasma A C T H and corticosterone. Plasma A C T H was assayed by A C T H radioimmunoassay kit (CIS France). Plasma corticosterone was measured by the method of Glick et al. 4 and in part by a competitive protein binding assay 20, using the plasma of rhesus monkey as a source of transcortin. The two methods gave a negligible difference
533 in plasma corticosterone levels determined. (3) Brain norepinephrine and serotonin. Animals were sacrificed between 12.00 and 13.00 h by decapitation. Brain tissue was quickly removed and sectioned on a dry-ice plate following the method of Glowinski and IversenS. Samples were stored in --20 °C until used. Brain norepinephrine and serotonin were determined by the method of Maickel et al. is. Experimentalprocedures. Two series of experiments were performed. Experiment 1.30 mg/100 g body weight ofp-chlorophenylalanine methylester.HC1 (Sigma) was injected intraperitoneally to rats and 3 days after the first injection a second injection of the same dose was done. 5 rats were used for the determination of plasma ACTH and corticosterone 3 days after the first injection (LD3) and 12 days (LD12) in LD. A serial blood sampling was performed at 4 h intervals starting from 08:00 to 04:00 h on the following day. Plasma was divided into each assay tube (100 #1 for ACTH and 10 /~1 for corticosterone). 19 rats were exposed to 200 lux continuous light. PCPA was injected to 10 rats at 08:00 h on the first day of exposure to constant light (LL1) and the LL4 day. On the LL12 day, serial blood sampling was done in both PCPA-treated and non-treated rats, starting from 08:00 to 04:00 h on the following day at 4 h intervals. Locomotor activity of 6 rats in both groups was monitored continuously from the onset of the experiment. 40 rats were used for the determination of brain monoamine contents after PCPA injection. To know a time course of the drug effect, rats were decapitated 3, 5, 9 and 12 days after PCPA treatment in LD. Decapitation was performed at 12:00 h. Non-treated 10 rats were sacrificed in LD as a control. Experiment 2. 5,6-Dihydroxytryptamine creatinine sulfate (Sigma)was administered intraventricularly to the rat. 6 rats were anesthetized by pentobarbital and 75 #g of 5,6-DHT dissolved in 10/~1 of 0.9 ~o saline solution containing 0.5 ~o ascorbic acid was injected into the lateral ventricle of the brain by means of a stereotactic apparatus. The other 6 rats were injected with the solvent as a control on the same surgical procedures. 2 and 6 days after the operation (PO2 and PO6), serial blood sampling was performed at 2 h intervals starting from 08:00 to 06:00 h on the following day in LD. Then the rats were exposed to 200 lux continuous light. On the LL12 day, blood samples were obtained by a serial sampling on the same schedule as performed in LD. A few days after, rats were sacrificed by decapitation and brain tissue was removed for the determination of brain monoamine contents. Locomotor activity of the rats was monitored throughout the experiment. Statistical analysis. A least squares spectrum method was used for the calculation of free-running circadian parameters (mesor, amplitude and period) of locomotor activity8. To estimate an acrophase of the circadian rhythm, the first step of cosinor analysis was modified 9. The measured variable (Yi) is a function of the time of observation (ti), according to the following formula, Yi = Co + C cos (2 ~r x ti/T q- 99). Here, Co indicates a mesor, C an amplitude, 99 a phase-angle to an appropriate reference and T a period. Co, C and 99 are calculated by the least squares spectrum method. 99 is given as follows,
534
[n l q~ = tan -1
-\
Yl sin 2~ xi/n i=O
)
I; Yi cos 2z~ xi/n /i=O
To test a difference between two mean values, Student's t-test was used. RESULTS
Effect o f PCPA on the free-running circadian rhythms o f locomotor activity and plasma corticosterone A time course of hypothalamic amine contents after the administration of PCPA was shown in Table I. PCPA reduced brain serotonin content to 30 9/00of control value for at least 5 days. Serotonin content recovered gradually afterward and reached a normal level 12 days after the first injection of the drug. Fig. 1 illustrates the effect of PCPA on the circadian rhythms of plasma A C T H and corticosterone in LD. Three days after the injection, circadian rhythms of both functions were abolished and flattened at the mean level of the group. In individuals, some fluctuation of plasma A C T H and corticosterone was observed but marked circadian rhythm was not detected in all rats. 12 days after the first injection, entrained circadian rhythms of plasma A C T H and corticosterone appeared in both individual rats and mean levels of 5 rats. Fig. 2 illustrates free-running rhythms of locomotor activity of PCPA-treated and non-treated rats in 200 lux continuous light. PCPA was administered on the LLI day and the LL4 day. A circadian rhythm of locomotor activity of a PCPA-treated rat (no. 1) was damped from the time of drug administration and did not manifest itself for about 6 days in LL. From the LL7 day, circadian rhythm of locomotor activity was restored and ran with a period slightly longer than 24 h (phase-shift). In control rat (no. 1), a marked free-running circadian rhythm was observed from the onset of continuous light and showed a phase-shift. TABLEI
Time course of hypothalamic amine contents after the administration of PCPA PCPA was injected intraperitoneally in doses of 30 mg/100 g body weight, twice. Amine contents (mean values ± S.E. of the mean) were given in terms of/~g/g wet tissue weight. Numbers in parentheses indicate number of rats sacrificed.
Days after the first injection
Serotonin
Norepinephrine
Control PCPA-treated rats 3 days 5 days 9 days 12 days
1.54 ~c 0.04 (10)
1.72 ± 0.08 (10)
0.48 4- 0.02 (10)* 0.45 :t= 0.03 (8)* 0.88 =l_0.09 (8)* 1.43 ± 0.09 (11)
1.51 :k 0.21 (10) -1.64 :~- 0.12 (8) --
* P < 0.001 vs control.
535 PCPA
N=5
LD 3
No.2
N0.1 2OO
,40
40
200 40
200
20
100 20
100
"0 uI
ixi -d o. o
3
el 20
100
i
-I-
%
%,1
i
LD12 40
200
40
1200 40,
2oo
20
100 20;
=100
-D
m
,g ~
E
100
2O
nl n
'-r. i
12oo
2400
120O
24OO
I~,~ ~,~
12oo
240o
Fig. 1. Effects of PCPA on the circadian rhythms of plasma ACTH and corticosterone in LD in both grouped and individual rats. Two typical individuals are shown. Blood samplings were performed 3 days (LD3) and 12 days (LD 12) after the drug injection. Open circles indicate values of plasma corticosterone and closed circles those of plasma ACTH. Vertical lines represent S.E. of the mean. Black horizontal bars in the bottom of each column indicate a dark period in a day. Human ACTH 1-39 was used as a standard in the assay.
Fig. 3 shows circadian rhythms of locomotor activity and plasma corticosterone determined simultaneously in individual rats on the LL12 day. Circadian rhythm of plasma corticosterone was observed in both rats but the peak of the rhythm was detected at 24:00-04:00 h in the PCPA-treated rat while the peak was seen at 12:00 h in the control rat. Fluctuations of the mean levels of plasma corticosterone in individual rats are demonstrated in Fig. 4. In control groups, the peak of the rhythm was observed at 08:00 h, indicating that about 16 h of phase-shift took place in LL (the peak of the rhythm existed at 16:00 h in LD). In contrast, the peak of plasma corticosterone rhythm in PCPA-treated group was seen at 24:00 h. This means only a 8 h phase-shift occurred in PCPA-treated group during the same period in LL. The distribution of the peak time of plasma corticosterone in individual rats on the LL12 day was shown in Table II. The mode of the distribution in control rats was 08:00 h and that in PCPAtreated was 24:00 h. Table I I I summarizes free-running circadian parameters (period, amplitude and mesor) of locomotor activity and its acrophase on the LL11 day. The peak time was also given of plasma corticosterone observed on the LL12 day in 12 individual rats of control and PCPA-treated group. In these rats locomotor activity and plasma corticosterone were simultaneously determined, since serial blood sampling per se was shown to not essentially affect the circadian features of the locomotor activity rhythm lo. Free-running period of the locomotor activity of PCPA-treated group was
536 25.1 i 0.1 h, which was not significantly different from that of control group. However, an acrophase of locomotor activity on the LL11 day was 08:45 ~ 38 min in PCPA-treated group and 13:44 ± 45 min in control, the difference being about 5 h and highly significant (P < 0.001).
Locomotor Activity (counts/15rain.) PCPA
treated
Rat
Control
:::::::::::::::::::::::::::::::
Rat
:.:.:.:.:.:.-.-.:.-.:.:.:.:...: .~.~:; ..... ..,......,
LD
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,i
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~
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I*
" JIl _~ _j,II.
::::::: :+::.
: PcPA ~ L L.i_... l ~ sill lJl I:ll~k
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I
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~h I
I L~I
J l " ~
_ J_
t
~PCPA
l ........
ml
~ .... IutJL 4,J..l,--
_~JLt
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It I ~ t ' J
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,~...Jtl.
+~l,"at t I I I ~t I " - a ~ ' ~ ~
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i
_._~. ~ , J L I l l d[ j i " J ~ l b
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,.,.I
t ..i J. 'Ii+"J~: I~, "I"'+ :'+'Y
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No.1
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Fig. 2. Free-running rhythms of locomotor activity in 200 lux LL. PCPA was injected on the LLI day and the LL4 day in PCPA-treated rat (no. 1). Abscissa indicates a time of day, starting from 16: 30 h to 16: 30 h on the following day. Shadowed portion indicates a dark period in LD. Locomotor activity was expressed by histogram, recorded by counts per 15 rain.
537
i,o 1000~ CtttCt LL12
Contro[ No.1
~ 20 t
!,° t 2000
i%~L 1000~
,iI
¢ I"'~ t ' ~ ' I ' LL 1 2
Fig. 3. Simultaneously determined circadian rhythms of locomotor activity and plasma corticosteron¢ on the LL12 day in a PCPA-treated rat (no. 1) and in a control (no. 1). Sampling times, indicated by arrows, started from 0 8 : 0 0 h at 4 h intervals.
Control LL12
PCPA LL 12
N=9
N=10
20
m 20 U nl
10
E e~
%
i
1200
i
24OO
I
120o
I
24OO
Fig. 4. Circadian rhythms of plasma corticosterone on the LLI2 day in both PCPA-treated and nontreated groups. Abscissa indicates a time of day. Vertical lines indicate S. E. of the mean. P values against the same points of control group were expressed in some points of PCPA-treated group.
538 TABLE I1
Distribution of the acrophase (peak time) of the plasma corticosterone level determined on the 12th day after exposure to continuous light (LLI2 day) Acrophase (h)
Control (9)
PCPA-treated (10)
08:00 12:00 16:00 20:00 24:00 04:00
6 1 0 0 0 2
0 2 0 2 5-6* 1-0"
* One rat showed the acrophase at both 24:00 and 04:00.
TABLE llI
Free-running circadian parameters (period, amplitude and mesor) of locomotor activity and its acrophase on the LLI1 day The peak times of plasma corticosterone were observed on the LL 12 day in individual rats in which both functions were determined simultaneously.
Free-running parameters Period PCPA-treated rats No. 1 25.0 2 25.1 3 25.6 4 24.6 5 25.2 6 25.3 25.1 i 0 . 1 Controlrats No.l 25.7 2 25.2 3 25.0 4 25.4 5 25.2 6 25.4 25.3 ± 0.1
Amplitude
Acrophase on L L l l
Mesor
Peak time of corticosterone on LL12
399 137 124 87 147 180 179 ± 46
416 418 356 440 331 334 383 ~_ 19
10:38 06:57 09:40 06:58 09:53 08:26 08:45 ± 00:38*
24:00 or 04:00 20:00 20: 00 12:00 24:00 24:00
237 215 178 86 240 228 197 ± 24
323 341 373 271 394 333 339
16:07 12:14 11:43 15:28 12:27 14:22 13:44 ± 00:45
12:00 08:00 08 : 00 08 : 00 08:00 08:00
17
* P < 0.001 vs corresponding control values.
Effect o f 5 , 6 - D H T on the free-running circadian rhythms o f locomotor activity and plasma corticosterone Th e effects o f i n t r a v e n t r i c u l a r a d m i n i s t r a t i o n o f 5 , 6 - D H T on the e n t r a i n e d circadian r h y t h m s o f l o c o m o t o r activity an d p l a s m a c o r t i c o s t e r o n e in a rat was d e m o n s t r a t e d in Fig. 5. T h e circadian r h y t h m s o f b o t h functions were disturbed f o r a b o u t 2 days after the drug administration. But 6 days after the injection (PO6), circadian r h y t h m s were restored. H y p o t h a l a m i c serotonin c o n t e n t was reduced to 60 ~o o f c o n t r o l value in the 5 , 6 - D H T - t r e a t e d rats.
539
5,6-DHT LD I=O2
~ 2o
PO 6
P
