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Effect of chronic tryptophan depletion on the circadian rhythm of wheel-running activity in rats
Physiology & Behavior, Vol. 55, No. 6, pp. 1005-1013, 1994 Copyright Q 1994 ElsevierScience Ltd Printed in the USA. All rights reserved 0031-9384/94 $6.00 + .OO
Pergamon 0031-9384(93)EOOSV
Effect of Chronic Tryptophan Depletion on the Circadian Rhythm of Wheel-Running Activity in Rats KAZUAKI Department
KAWAI,’
NOR10
YOKOTA
AND
SHIGETO
YAMAWAKI
of Neurology and Psychiatry, Hiroshima University, School of Medicine, l-2-3 Kasumi, Minami-ku, Hiroshima, 734, Japan Received
25 September
1992
KAWAI, K., N. YOKOTA AND S. YAMAWAKI. Effect of chronic tryptophan depletion on the circadian rhythm of wheelrunning activity in rats. PHYSIOL BEHAV 55(6) 1005-1013, 1994.-The effect of chronic treatment with a tryptophan (TRP)free diet on the free-running circadian wheel-running rhythm and the central serotonergic system was investigated in blinded male rats. The long-term TRP-free diet did not change Periods of activity, but disordered their patterns. This seemed to be due to masking, entrainment, enhancement of the morning activity, and obscuring of the activity onset as well as appearance of some periodic activities within the subjective night. A long-term TRP-free diet decreased the concentration of TRP, J-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HJAA) in all brain regions tested: frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons. Density of 5-HT,, receptor binding was significantly decreased in the frontal cortex and hypothalamus, whereas no significant change was observed in the density of 5-HT, receptor binding in all regions. These results suggest that the period of primary circadian pacemaker is not affected, but its oscillation, as well as the coupling strength between the primary and secondary pacemakers, is weakened by the dysfunction of the serotonergic system caused by chronic TRP depletion.
Tryptophan
Serotonin
Free-running rhythm
Wheel running
IT has been suggested that mammals have circadian pacemakers
in the suprachiasmatic nucleus (SCN) in the hypothalamus (16,27). Furthermore, the SCN contains high levels of serotonin (5hydroxytryptamine, 5-I-IT) under the control of 5-HT neurons ascending from the raphe nuclei in the midbrain (2,20,35). Therefore, it is possible that the 5-HT system may affect the generation and entrainment of the circadian rhythm. Several researchers have investigated the relationship between the free-running circadian rhythm and the 5-HT system. It has been speculated that the 5-HT system does not affect the basic circadian pacemaker (40). Some investigators have reported that neither generation nor period of free-running rhythm are altered after lesions of the raphe nuclei (3,32) or the administration of 5-HT neurotoxin (13,21,23,30). In contrast, others have reported that large midbrain lesions disrupt the free-running rhythm (17) and serotonergic drugs cause a change of the free-running period (6,13,38). Recently, it has been reported that the 5-HT content in the SCN correlates negatively with the free-running period (29). Therefore, the role of the 5-HT system has remained uncertain with respect to regulation of the circadian rhythm. Tryptophan (TRP) is an essential amino acid and a precursor of 5-HT. The chronic ingestion of diets lacking TRP decreases the 5-HT level in the brain (8,39). In this study, we have produced a dysfunction of the 5-I-IT system in the brain by the administra-
Blinded rat
tion of a TRP-free diet, and evaluated the effects of chronic TRP depletion on the free-running rhythm of wheel-running activity in rats. METHOD
Free-Running Rhythm of Wheel-Running Activity Male Wistar rats (8 weeks old) were purchased from Charles River Japan Inc. and individually housed in cages (15 X 28.5 x 17 cm) equipped with a running wheel (circumference 1 m). They were fed commercial chow (Oriental Yeast Co., Ltd., Tokyo) and water ad lib. The numbers of revolutions of the wheel were recorded every 30 min by a personal computer (PC-8801 MK2; NEC Co., Ltd., Tokyo) connected to a running wheel. After 2 weeks, each animal was enucleated while under pentobarbital anesthesia. On the 65th or 66th day after enucleation, the animals were divided into two groups. Those in the TRP-free group ingested a TRP-free diet composed of 17.65% casein hydrolysate, 0.30% DL-methionine, 61.35% sucrose, 10.00% cottonseed oil, 5.00% cellulose powder, 3.50% AIN mineral, and 2.20% vitamin mixture (Oriental Yeast Co., Ltd.). Those in the control group received commercial chow. The experiments were carried out twice. Three animals in the control group and four in the TRP-
‘ To whom requests for reprints should be addressed. 1005
free group were first examined during the 153 days from January to August, 1990, and six animals in each group were monitored during the 111 days from December, 1990 to June, 1991. The experimental room was maintained in a 12-h light/12-h dark photoperiod (light on at 0830, light off at 2030; light phase 200 lux, dark phase 5 lux). Room temperature was kept at 23 +2°C during the first experiment and 24 -+ 2°C for the second one. The diurnal variation remained about 4°C in both experiments. Room humidity was kept at SO t 9% in the first and 55 t 10% in the second, but no diurnal variation existed. Because neither the room nor the cages were made absolutely soundproof, we entered the room at random times and touched the cages only for cleaning and feeding. Furthermore, we did not handle the animals at all. However, some other nonenucleated animals were maintained in the same room during both experiments. The periods of the wheel-running rhythm were calculated by a chi-square periodogram (31) for three samples of 50 consecutive days. The first sample was from the 15th to the 64th day after blindness. The second and third samples were from the first to the 50th day and from the 61st to the 110th day after feeding by the TRP-free diet, respectively. The periods of time in the control group were calculated to coincide with the three samples of the TRP-free group. Determination of TRP, 5-HT, and 5-Hydroxyindoleacetic (5-H&4) in the Brain
duced pressure, followed by two washings with 5 ml of ICC-cold SO mMTris-HCl buffer. Radioactivity was determined by a liquid scintillation counter. All experiments were done in duplicate. Specific binding was defined as the total binding minus the nonspecific counts obtained in the presence of 100 FM T-FIT (Wako Pure Chemical Industries, Ltd., Osaka). The 5-l-iT2 receptor binding assay was pcrformcd according to the procedure of Leysen et al. (18) with some modifications. In brief, 1.O nM [“Hlketanserin (27 19.5 GBqimmol; Janssen Biotech, N. V.) was added to tubes containing 300 1.11of membrane preparations and incubated (37”C, 15 min) in a final volume of 500 ~1 of 50 n-N Tris-HCl buffer (pH 7.4). Specific binding was defined as the total binding minus the nonspecific counts obtained in the presence of 1 (LM mianserin (Organon Co., Ltd.. Japan), the same procedure as for the S-HT,* receptor binding assay. The protein concentration was quantified according to Lowry et al. ( 19). Statist&&
Analysis
Statistical analysis was performed by a paired t-test compared within each group. Student’s t-test was used for comparison between the two groups.
Acid
After termination of the behavioral experiment, all rats were killed by decapitation. Their brains were removed immediately, placed on ice to be dissected by hand with a blade, and divided into the frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons according to the method of Glowinski and Iversen (11). A part of each region was frozen at -80°C for radioligand receptor assay. Each tissue sample was homogenized (Polytron, set at 8, 10 s) in 20 vol. (w/v) of 0.1 N perchloric acid solution. After centrifugation at 20,000 X g, 2°C for 15 min, the resulting supernatants were filtrated on millipore filters (pore size 0.33 pm) and separated into TRP, S-HT, and S-HIA. by high performance liquid chromatography with electrochemical detection (Shimazu Co., Ltd., Kyoto). The mobile phase was 50 mM phosphate buffer (pH 3.4) containing 12% methanol and 85 mg/ 1 octanesulfonic acid. The flow rate was 0.8 ml/min through a column 7 pm in diameter (Hiber LiChrosorb RP-18; Kantokagaku Co., Ltd., Tokyo). Assays of 5-HT,, and 5-HT, Receptor Binding Sites in the Brain Frozen samples of each region were homogenized (Polytron, set at 8, 10 s) in 15 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4, and centrifuged (49,000 x g,4°C 15 min). The pellets were suspended in 15 ml of 50 mM Tris-HCl buffer and incubated at 37°C for 20 min to exclude endogenous S-HT. After recentrifugation (49,000 x g,4°C 15 min), the resulting pellets were SUSpended again in 100 vol. (w/v) of the same buffer and used for the receptor binding assay. The 5-HTiA receptor binding assay was performed according to the procedure of Peroutka (24) with some modifications. In brief, 1.0 r&f [‘H]&hydroxy-2-(di-n-propylamine)-tetralin ([3H]-8-OH-DPAT; 6.77 TBq/mmol; Amersham) was added to tubes containing 300 ~1 of the membranous preparations, and then incubated (37°C 1.5 min) in a final volume of 500 ~1 of 50 mM Tris-HCl buffer (pH 7.4) containing 20 /JM pargyline, 4 mM CaC12, and 0.1% ascorbic acid. The incubation was stopped by rapid filtration on Whatman GF/B glass fiber filters under re-
RESULTS
Wheel-Running
Activity
In the first experiment, three animals in the control group and four in the TRP-free group were examined. Figures 1 and 2 show their double-plotted records. All animals housed in the cages were entrained to the light/dark cycle of the experimental room and had a stable wheel-running rhythm at first. After bilateral optical enucleation, they took about 2 weeks before their rhythms began to free-run. Their periods of free-running rhythms were longer than 24 h, with the exception of the TRP-free rat 4 (see Table 1). In the control group (Fig. l), the free-running periods of control rats 1 and 2 were stable during the observation. In the control rat 3, the period began to lengthen after 2 months following blindness and then shortened again. The onset of activity corresponded approximately to the onset of light phase after 5 months following blindness. In the TRP-free group (Fig. 2), three animals (TRP-free rats 1, 2, and 3), for which free-running periods were longer than 24 h, took 1 month of feeding on the TRP-free diet before their patterns of activity became disordered. After 2 months of the TRP-free diet, they demonstrated increases in activities that corresponded to the onset of the light phase, as well as a distinct, non-24-h free-running rhythm. The free-running rhythms of the TRP-free rats 1 and 2 were obscured after 4 months of the TRPfree diet, because the total daily counts of wheel-running activity were extremely reduced. In the TRP-free rat 4, for which freerunning period was shorter than 24 h, the period began to lengthen after feeding on the TRP-free diet. After 2 months of beginning the TRP-free diet, its period reached about 24 h, and its onset of activity corresponded approximately to the onset of the light phase. Six animals in each group were examined in the second experiment. Figures 3 and 4 show their double-plotted records. After bilateral optical enucleation, most of the animals had freerunning rhythms similar to those in the first experiment. HOWever, during the 60-80 days after blindness, the onsets of activity of the control rats 6 and 9 fitted the same phases as those before blindness. In the control group (Fig. 3), the free-running periods for one-half of the animals (control rats 4, 6, and 8) clearly
CIRCADIAN
RHYTHM IN TRYPTOPHAN
1007
DEPLETION Control-3
Control-2
Control-l
D
6
I 10 20 30
40
40 50 60 70 60 90 100 110 120 130 140 150 160 170 160 190 200 210 220
FIG. 1. Continuous recordings of wheel-running activity in the control group in the first experiment. The chart is double-plotted. Arrow B indicates the day on which rats were blinded. The D indicates the dav after blindness. The horizontal bar indicates the dark period of the day.
lengthened after 60-80 days following blindness. The activity patterns of all animals were stable during the observation. In the TRP-free group (Fig. 4), the periods for one-half of the animals (TRP-free rats $7, and 10) also lengthened immediately after feeding on the TRP-free diet, whereas the activity patterns of all these animals were disordered. Similar to the result of the first experiment, the TRP-free rat 6 showed an increase in activity near the beginning of the light phase. Most of the animals had activity patterns that were different from the results of the first experiment. Four animals (TRP-free rats 5, 7, 9, and 10) had the enhancement of morning activity, that is to say, the appearance of most wheel-running activity during the latter half of the subjective night, but a separate, smaller activity onset was present. In the TRP-free rat 9, the onset of activity became obscure. The TRP-free rat 8 expressed bouts of activity with a period of 25.40 h, which was additional periodic activity within only the subjective night. After 2 months of the TRP-free diet, four animals (TRP-free rats 5, 8, 9, and 10) expressed similar periodic activities within the subjective night, with periods ranging from 25.15 to 25.40 h. The free-running rhythms of all the animals were obscured after 3 months of feeding, because the total amount of daily counts of the wheel-running activity was extremely reduced. In both experiments, the chi-square periodogram analysis in the TRP-free group indicated that all of these peaks appeared to be multiple and lower in amplitude after feeding on a TRP-free diet than before (data not shown).
Table 1 shows the primary free-running periods corresponded to the highest peak in each periodogram calculated for the three samples. As described in the Method section, the first sample was from the 15th to the 64th day after blindness prior to a TRP-free diet. The second and third samples were from the first to the 50th day and from the 61st to the 110th day after feeding on a TRP-free diet, respectively. The three samples in the control group corresponded with those of the TRP-free group. In both groups, the mean time periods of the second and third samples significantly lengthened compared with that of the first sample [control group: t(8) = 3.36 and 5.52, p < 0.01; TRP-free group: r(9) = 2.32 and 2.94, p < 0.051. However, the periods showed no difference between the control and TRP-free groups for any sample,t(l7) = 0.310.62, p > 0.05. Figure 5 shows the mean daily counts of the wheel-running activity for the same three samples. In the control group, the counts of the second sample were significantly reduced compared with that of the first, t(8) = 4.68, p < 0.01. In both groups, the counts of the third sample were significantly reduced compared with those of the others [control group: t(8) = 4.81 and 4.37,~ < 0.01; TRP-free group: t(9) = 4.21 and 4.25, p < 0.01, respectively]. On the other hand, the counts of the second and third samples in the TRP-free group significantly increased compared with those of the control group [second sample: r(l1) = 2.74, p < 0.05; third sample: t(l1) = 3.27,~ < 0.011.
1008
KAWAI. TRP free-l
TRP free-2
YOKOTA rw
TRP free-3
ANI)
LAMAWAKI
free-4
0
+a
1
’
*I3
+B
10
10
M
m
30
30
40
4
50
50 00
00
1
T
T
1
1
10 M
20
1 10
10
i
20
30
30
40
40 50
50
00
00
70
70
10
IO m
(&
150
:.-
:-
_-
;;-.
_
FIG. 2. Continuous recordings of wheel-running activity in the TRP-free group in the first experiment. The chart is double-plotted. Arrow B indicates the day on which rats were blinded. Arrow T indicates the time Period of the feeding on a TRP-free diet. The D indicates the day after blindness and the day after beginning the TRP-free diet. The horizontal bar indicates the dark period of the day.
Contents of TRF’, 5-HT, and 5-H&4
in the Brain
The contents of TRP, WIT, and 5HL4A were determined for four regions (frontal cortex, hippocampus, midbrain, and pons) in animals fed a TRP-free diet for 111 consecutive days, and for five regions (frontal cortex, hippocampus, thalamus, hypothalamus, and pons) for 153 consecutive days. As shown in Table 2, their contents significantly decreased at all regions in the TRPfree group after both feedings [ill days: 49-10) = 4.69-14.19, p < 0.01; 153 days: t(5) = 8.16-29.56,~ < 0.011 and they all gradually decreased to a similar degree in each region. The mean TRP contents for 111 days became 32.3% and those for 153 days were 22.5% of control values. The mean 5-HT contents were 44.9% and 27.3% and the mean 5-HIAA contents were 25.8% and 18.1%, respectively. Density of 5-HT, and 5-HT2 Receptor Binding in the Brain The 5-HTIA and 5-HTz receptor binding sites were determined for six regions (frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons) in animals fed a TRP-free diet for 111 consecutive days, and for three regions (frontal cortex, hippocampus, and pons) for 153 consecutive days. As shown in Table 3, the density of 5-HTIA receptor binding significantly decreased in the frontal cortex [ill days: ~‘(10)= 3.27, p < 0.01; 153 days: t(5) = 5.75,~ < 0.011 and hypothalamus, l(l0) = 2.79, p < 0.05. On the other hand, the density of 5-HTz receptor bind-
ing did not significantly change in any regions for 111 days, t(9-
10) = 0.23-1.56, p > 0.05, and for 153 days, 42-5) 2.84, p > 0.05.
= 0.03-
DISCUSSION
Long-term feeding with a TRP-free diet similarly decreased the contents of TRP, 5-HT, and 5-HIAA in all the evaluated regions of the brain. These results suggest that chronic TRP depletion decreases central 5-HT synthesis. It has been already reported that chronic consumption of a corn diet, which contains low levels of TRP, reduces the contents of TRP and 5-HT to 4060% and 70-80%, respectively, of control values in young adult rat brains (8,39). Our results suggest that 5-HT synthesis may be more disordered than those of these previous reports, because the contents of TRP and 5-H-T are reduced by 20-30% and 30-44% of controls, respectively. The decrease of 5-HT contents may differ in relation to the degree and interval of TRP intake restriction, as well as the age of the animals. The changes of receptor binding sites related to 5-HT differed in tested classes and regions, although the contents of 5-HT were reduced to a similar degree in all regions. The density of ~-HTLA, receptor binding decreased only in the frontal cortex and hypothalamus, whereas the density of 5-HTz receptor binding did not change. To our knowledge, no other investigators have reported the effect of TRP depletion on the receptors related to 5-HT. On
CIRCADIAN
RHYTHM IN TRYPTOPHAN
1009
DEPLETION
FIG. 3. Continuous recordings of wheel-running activity in the control group in the second experiment. See Fig. 1 legend.
the other hand, some investigators have reported that the administration of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (DHT), causes the density of 5-HT’1Areceptor binding to decrease only in the raphe nuclei and striatum, but not in other brain regions (12,36,37). The treatment of this drug is also reported to have no effect on the density of U-IT, receptor binding in all brain regions (9,25). Our results suggest that chronic TRP depletion and the administration of the 5-I-IT neurotoxin may affect the 5-I-lTrA receptor system with the distinct mechanism, yielding different results in brain regions, although neither may affect the 5-I-IT, receptor system. However, it will be necessary to conduct further studies on this point. Roth the primary free-running periods and the wheel-running activity were altered after blindness in each group (Table 1 and Fig. 5). Recently, it is reported that the activity of wheel running produces a feedback on a circadian pacemaker. The increase in activity causes a phase shift in the hamster (26,34). The activity correlates negatively with free-running periods in the rat (29). Our results may support that activity affects the free-running period in the rat, but does not induce phase shift. However, this
TRP fr”-5
TRP
fma-s
conclusion requires circumspection, because one-half of the animals have clearly and suddenly lengthened periods at a point of about 60-80 days after blindness for the second experiment (Figs. 3 and 4). This phenomenon is suspected to be due to unknown environmental factors in the experimental room; however, the cause is unclear. Therefore, it is uncertain in this study whether or not activity produces feedback to a pacemaker. The primary free-running periods were not different between the control and TRP-free group during any sample interval, although the wheel-running activities in the TRP-free group increased compared with those in the control group. These results may suggest that the free-running period is not affected by chronic TRP depletion, when the effects on pacemaker are considered as a whole, including the increase in activity due to a TRP-free diet. It has been reported that the continuous treatment with TRP lengthens the free-running period of spontaneous optic nerve impulses in the isolated Aplysia eye (7). No investigators, however, have reported on the effect of TRP depletion on the free-running period. Several investigators have tried to determine the relationship of the 5-HT system and the free-running periods
TRP h-7
FIG. 4. Continuous recordings of wheel-running activity in the TRP-free group in the second experiment. See Fig. 2 legend.
1011)
KAWAI.
1
TABLE PERlOD
OF
WHEEL-RUNNING
RHYTHM
BY
CHI-SQUARE
PERIODOGRAM Rat No.
Control group 1 2 3 4 5 6 7 8 9 Mean + SE
Before
I -SO
Days
hl-IIODays
24.35 24.30 24.00 23.75 24.30 24.00 24.15 24.15 23.95 24.11 ? 0.07
24.60 24.45 24.10 24.00 24.45 24.05 24.20 24.20 23.90 24.22 t- 0.08*
24.60 24.45 24.05 24.00 24.50 24.15 24.30 24.15 24.15 24.26 -t 0.07*
24.30 24.45 24.35 23.50 24.05 24.30 23.65 23.95 24.20 23.75 24.05 2 0.10
24.50 24.50 24.35 23.50 24.15 24.30 24.15 23.90 24.50 23.90 24.18 t O.lOt
24.45 24.45 24.55 24.00 24.00 24.60 24.35 24.00 25.15 23.95 24.35 5 0.12.t
TRP-free group
1 2 3 4 5 6 7 8 9 10 Mean t SE
The values represent a period (hours) of the highest peak in each sample interval. Before indicates the first sample from the 15th to the 64th day after blindness before feeding on a TRP-free diet. l-50 days indicates the second sample from the first to the 50th day after feeding on a TRP-free diet. 61-110 days indicates the third sample from the 61st to the 110th day after feeding on a TRP-free diet. The three samples in the control group correspond with those of the TRP-free group. *t Indicate the results compared with those of Before by a paired Itest: *p < 0.01, tp < 0.05.
of wheel running in rodents. Some of these reports suggest that the central 5-HT system may affect these free-running periods because a negative correlation exists between 5-HT contents in the SCN and free-running periods (29), and because the periods are shortened or lengthened by the administration of the S-HT synthesis inhibitor, parachlorophenylalanine (13), or the monoamineoxidase inhibitor, clorgyline (6,38), respectively. However, others have reported that the free-running periods are not affected by the administration of the 5-HT neurotoxin, 5,6- or 5,7-DHT (13,21,23,30). Contradictory results have also been reported that lesions of raphe nuclei interfere with the expression of the free-running rhythm (17) or affect neither generation nor period of the free-running rhythm (3,32). Therefore, the role of the 5-HT system has remained uncertain with respect to the circadian system. Our results suggest that the period of primary circadian pacemaker may not be affected by the dysfunction of 5-HT system due to the lack of TRP, a precursor of 5-HT. The activity patterns were disordered after a TRP-free diet, although the periods were not affected. The peaks of chi-square periodograms became multiple and low. In the first experiment, most of the animals had increases in activities with about a 24h period, which were different from free-running rhythms. Their activities seem to be due to the expression of masking effects, because the onsets of their activities coincide with the onset of
YOKOTA
AND
\r /\MAWAki
the light phase. The phenomenon in the TRP-free rat -I stems 10 be due to the expression of entrainment effects, because the onset of its activity coincides with that of other animals (Fig. 2). The control rat 3 is also suspected to have been entrained after i months following blindness, because it then has an activity pattern similar to the TRP-free rat 4 (Fig. 1). All the animals in the TRP-free group expressed these masking or entrainment effects, whereas only one rat did in the control group. These results suggest that chronic TRP depletion may make animals susceptible to subtle environmental factors, because the masking and entrainment effects are expressed by the periodic change of environmental factors. It is speculated that chronic TRP deplction may make the oscillation of the primary circadian pacemaker weakened. The environmental factors producing the masking or cntrainment effects have been reported to be the temperature cycles (lo), the light cycles (l), and the periodic social interaction (22). In this study, they appear to be related with the lighting being on in the experimental room, because the onsets of activity are all near the beginning of the light phase. However, the light cannot have affected the animals directly because they had been enucleated. The two following factors may be suspected. The first one is the changes of noise, because neither the room nor the cages are made perfectly soundproof, and because some other nonenucleated animals, as maintained in the same room. may convey the photic cues to the enucleated animals via a noise due to a change in activity level during the light and dark phases. The second is the temperature cycle, as the diurnal variation remained in the room temperature. In the second experiment, only the TRP-free rat 6 expressed the masking effect. Most of the animals expressed the enhancement of morning activity, the obscuring of the activity onset, and the secondary periodic bout activities only within subjective nights, with periods ranging from 25.15 to 25.40 h (Fig. 4). These changes of activity pattern within subjective night were expressed only in the TRP-free group, not in the control group. These phenomena have been previously reported in untreated blinded hamsters (5) and clorgyline-treated hamsters (6). In these reports, a multioscillator system is hypothesized that the primary pacemaker, which divides into the subjective night and day. mod-
4wa
1
b 0 BdOW
I-50
daya
51-110 day*
FIG. 5. Change of mean daily counts of wheel-running activity at the three samples of 50 consecutive days. Before indicates the first sample from the 15th to the 64th day after blindness and before feeding on the TRP-free diet. l-50 days indicates the second sample from the first to the 50th day after feeding on the TRP-free diet. 61-110 days indicates the third sample from the 61st to the 110th day after feeding on the TRPfree diet. Open circle indicates means of the TRP-free group. Closed circle indicates means of the control group. The vertical bars represent SE. Significant difference between the Go groups by Student’s r-test: *p < 0.05, **p < 0.01. Significant difference by a paired f-test: “I, < 0.01 vs. Before, “p < 0.01 vs. Before and l-50 days.
CIRCADIAN RHYTHM IN TRYPTOPHAN
1011
DEPLETION TABLE 2
CONTENTS OF TRP, 5-HT, AND 5-HIAA IN VARIOUS REGIONS OF RAT BRAIN BY HI’LC-ECD
111 Days Frontal cortex Control
9.47 2 0.29
TRP-free
2.99 k 0.77”
diet
Hippocampus Control TRP-free
9.05 5 0.33 diet
Thalamus Control TRP-free
3.02 k 0.62* n.d.
diet
Hypothalamus Control
5-HL4A
5-HT
TRP 111 Days
153 Days
16.48 ? 0.26 3.56 2 0.36: 17.76 + 0.59 3.80 2 0.39*
111 Days
153 Days
153 Days
2.44 t 0.20
2.42 2 0.03
1.34 + 0.05
1.15 + 0.05
1.03 2 0.17*
0.60 + 0.09*
0.27 2 0.06*
0.13 2 0.05*
1.99 + 0.11
1.52 k 0.10
1.61 5 0.05
1.34 2 0.04
1.01 2 0.18*
0.48 ? 0.07*
0.44 5 0.11*
0.24 2 0.04*
n.d.
2.12 ? 0.16
n.d.
1.40 k 0.02
n.d.
15.66 2 0.60
3.69 2 0.27*
n.d.
0.59 2 0.11*
n.d.
0.32 k 0.06;
TRP-free
diet
n.d. n.d.
16.52 + 0.34 3.97 ? 0.33*
n.d. n.d.
4.12 2 0.14 1.22 2 0.14*
n.d. n.d.
2.23 2 0.08 0.44 2 0.06’
Midbrain Control TRP-free
diet
7.34 + 0.24 2.18 ? 0.45*
n.d. n.d.
2.81 t 0.10 1.10 ? 0.16*
n.d. n.d.
2.17 ? 0.11 0.62 5 0.15’
n.d. n.d.
Pons Control TRP-free
diet
7.46 2 0.26 2.42 5 0.51*
14.07 2 0.13 3.11 * 0.30*
3.58 ? 0.25 1.60 2 0.30*
2.58 2 0.10 0.59 * 0.10*
2.81 2 0.26 0.83 2 0.19*
1.84 2 0.04 0.35 2 0.06*
The values represent mean + SE of concentration (nmoI/g tissue). 111 days: control n = 5-6, free diet n = 4. n.d.: not determined. * Indicates results compared with each control by Student’s f-test: p < 0.01.
TRP-free
diet n = 6. 153 days: control n = 3, TRP-
treated rats (14). It is speculated that the coupling of pacemakers in infantile rats is unstable and the secondary oscillations in methamphetamine-treated rats are enhanced, respectively. Our results
ulates the secondary bout oscillators by providing a window during which their expression is feasible. A similar phenomenon has also been reported in infantile rats (15) and methamphetamine-
TABLE 3 DENSITY OF 5-HT,, AND 5-HT, RECEPTOR BINDING IN VARIOUS REGIONS OF RAT BRAIN 5-HT,A 111 Days
WIT, 153 Days
111 Days
153 Days
215.70 % 18.08 164.16 ? 1.81
Frontal cortex Control TRP-free diet Hippocampus
20.97 ? 0.98 15.83 5 1.23*
29.93 + 1.82 15.88 + 1.62*
181.80 + 4.31 196.85 + 9.68
Control TRP-free Thalamus
54.60 5 1.95 50.13 2 3.16
64.83 2 3.12 60.75 2 1.25
32.94 ? 2.68 28.46 2 2.29
18.82 t 19.93 2
diet
Control TRP-free diet Hypothalamus
5.05 t 0.52 4.83 + 0.27
n.d. n.d.
30.48 2 2.27 31.61 2 3.64
n.d. n.d.
Control TRP-free Midbrain
diet
14.17 * 1.19 10.47 2 0.60.t
n.d. n.d.
32.42 5 2.44 29.69 2 1.12
n.d. n.d.
Control TRP-free Pons
diet
9.47 5 0.48 8.76 5 0.47
n.d. n.d.
20.74 ? 2.77 20.50 2 3.50
n.d. nd.
diet
11.39 t 0.66 9.82 ? 0.63
4.46 % 0.92 4.79 2 0.45
30.20 5 2.30 31.60 -c 3.83
6.01 + 6.03 2
Control TRP-free
1.30 4.74
0.51 0.57
The values represent mean 2 SE of specific binding (fmoI/mg protein) at 1.0 r&f of [3H]DPAT (5-HT,J and [3H]ketanserin (5-I-IT,). 111 days: control n = 6, TRP-free diet n = 5-6. 153 days: control n = 3, TRPfree diet n = 4. nd.: not determined. * Indicate results compared with each control by Student’s I-test: *p < 0.01, tp < 0.05.
1011
KAWAl.
suggest that the coupling between the primary pacemaker and the secondary bout oscillator may be strong and stable in adult rats (from 8 to 33 or 39 weeks old). However, under a dysfunction of the 5-HT system due to the lack of TRP, the secondary bout oscillations may be relatively enhanced due to the decrease of coupling strength. Different results were obtained in the first and second experiments. The difference of room temperature and humidity in each experiment may have affected the oscillation and the coupling on the pacemakers. If chronic TRP depletion makes the oscillation of the primary circadian pacemakers and/or the coupling weakened, these changes may be related to the downregulation of 5-HT,, receptor in hypothalamus containing the SCN, following decrease in 5HT content. The chronic treatment of clorgyline, which caused the similar phenomena, is reported to cause 5-HT level to return towards control level after initiate increase (4) and to make a downregulation of 5-HT, receptor (28). Recently, the treatments
YOKOTA
AND YAMAWAKI
of 5-HT,+, agonists are reported to induced a phase advance oi free-running rhythm in hamster (33). Further studies will be r-cquired to clarify this point. In conclusion,
our results
suggest
that the dysfunction
of the
5-HT system due to the lack of TRP may make the oscillation of primary circadian pacemaker and the coupling strength between the primary and secondary pacemakers weakened, without causing any change in the period of primary pacemaker. These changes may be due to the downregulation of 5-HT,,, receptor system.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Kiyohisa Takahashi, Head of the Division of Mental Disorder Research, National Institute of Neuroscience. NCNP, Japan, for his invaluable advice. They also thank Dr. Akihiko Seo, Department of Public Health, Hiroshima University School of Medicine, Japan, for valuable help in the experiment,
REFERENCES 1. Aschoff, J.; von Goetz. C. Maskine of circadian activitv rhvthms in hamsters by darkness. J. Comp. Ph;siol. [A] 162:559:562: 1988. 2 Azmitia, E. C.; Segal, M. An autoradiographic analysis of the differential ascending projections of the dorsal and median raphe nuclei in the rat. J. Comp. Neurol. 179641-668; 1978. 3 Block, M.; Zucker, I. Circadian rhythms of rat locomotor activity after lesions of the midbrain raphe nuclei. J. Comp. Physiol. 109:235-247; 1976. 4 Campbell, I. C.; Robinson, D. S.; Lovenberg, W.; Murphy, D. L. The effects of chronic regimens of clorgyline and pargyline on monoamine metabolism in the rat brain. J. Neurochem. 32:49-55; 1979. 5. Davis, F. C.; Menaker, M. Hamsters through time’s window: Temporal structure of hamster locomoter rhythmicity. Am. J. Physiol. 239:R149-R155; 1980. 6. Duncan, W. C.; Tamarkin, L.; Sokolove, P. G.; Wehr, T. A. Chronic clorgyline treatment of the Syrian hamster: An analysis of effects on the circadian pacemaker. J. Biol. Rhythms 3:305-322; 1988. 7. Eskin, A. Differential effects of amino acids on the period of the circadian rhythm from the Aplysiu eye. J. Neurobiol. 13:231-239; 1982. 8. Fernstron, J. D.; Wurtman, R. J. Effect of chronic corn consumption on serotonin content of rat brain, Nature New Biol. 234:62-64; 1971. 9. Fischette, C. T.; Neck, B.; Renner, K. Effects of 5,7-dihydroxytryptamine on serotonini and serotonin, receptors throughout the rat central nervous system using quantitative autoradiography. Brain Res. 421:263-279; 1987. 10. Francis, A. J. P.; Coleman, G. J. The effect of ambient temperature cycles upon circadian running and drinking activity in male and female laboratory rats. Physiol. Behav. 43:471-477; 1988. in 11. Glowinski, J.; Iversen, L. L. Regional studies of catecholamines the rat brain-I: The disposition of [3H]norepinephrine, [‘Hldopamine and [3H]dopa in various regions of the brain. J. Neurochem. 13:655669; 1966. 12 Hall, M. D.; El Mestikawy, S.; Emerit, M. B.; Pichat, L.; Hamon, M.; Gozlan, H.; [3H]8-hydroxy-2-(di-n-propylamino) tetralin binding to pre- and postsynaptic 5-hydroxytryptamine sites in various regions of the rat brain. J. Neurochem. 44:1685-1696; 1985. 13 Honma, K.; Watanabe, K.; Hiroshige, T. 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. Brain Res. 169:531-544; 1979. 14. Honma, K.; Honma, S.; Hiroshige, T. Disorganization of the rat activity rhythm by chronic treatment with methamphetamine. Physiol. Behav. 38:687-695; 1986. 15. Honma, S.; Honma, K. Interaction between the circadian and ultradian rhythms of spontaneous locomotor activity in rats during the
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early developmental period. In: Schulz, H.; Lavie, P., eds. Ultradian rhythms in physiology and behavior. Berlin: Springer-Verlag; 1985:95-109. Inouye, S. T.; Kawamura, H. Characteristics of a circadian pacemaker in the suprachiasmatic nucleus. J. Comp. Physiol. 146:153160; 1982. Levine, J. D.; Rosenwasser, A. M.; Yanovski, J. A.; Adler, N. T. Circadian activity rhythms in rats with midbrain raphe lesions. Brain Res. 384:240-249; 1986. Leysen, J. E.; Niemegeers, C. J. E.; Van Nueten, J. M.; Laduron. P. M. [3H]ketanserin (R 41468): A selective ‘H-ligand for serotoninz receptor binding sites. Mol. Pharmacol. 21:301-314; 1982. Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265275; 1951. Moore, R. Y.; Halaris, A. E.; Jones, B. E. Serotonin neurons of midbrain raphe: Ascending projections. J. Comp. Neurol. 180:417438; 1978. Morin, L. P.; Blanchard, J. Depletion of brain serotonin by 5,7-DHT modifies hamster circadian rhythm response to light, Brain Res. 566:173-185; 1991. Mrosovsky, N. Phase response curves for social entrainment. J. Comp. Physiol. [A] 162:35-46; 1988. Ohi, K.; Hayashi, S.; Takahashi, K. Development of circadian rhythms in rats with lesions of serotonergic system. Physiol. Behav. 44:393-403; 1988. Peroutka, S. J. Pharmacological differentiation and characterization of 5-I-l-r,A, 5-HTin, and 5-HTic binding sites in rat frontal cortex. J. Neurochem. 47:529-539; 1986. Quik, M.; Azmitia, E. Selective destruction of the serotonergic fibers of the fomix-fimbria and cingulum bundle increases 5-HT, but not 5-HT, receptors in rat midbrain. Eur. J. Pharmacol. 90:377-384; 1983. Reebs, S. G.; Mrosovsky, N. Effects of induced wheel running on the circadian activity rhythms of Syrian hamster: Entrainment and phase response curve. J. Biol. Rhythms 4:39-48; 1989. Rusak, B.; Zuker, I. Neural regulation of circadian rhythms. Physiol. Rev. 59:449-526; 1979. Savage, D. D.; Mendels, J.; Frazer, A. Monoamine oxidase inhibitors and serotonin uptake inhibitors: Differential effects on [3H]serotonin binding sites in rat brain. J. Pharmacol. Exp. Ther. 212:259-263; 1980. Shioiri, T.; Takahashi, K.; Yamada, Y.; Takahashi, S. Motor activity correlates negatively with free-running period, while positively with serotonin contents in SCN in free-running rats. Physiol. Behav. 49:779-786; 1991. Smale, L.; Michels, K. M.; Moore, R. Y.; Morin, L. P. Destruction of the hamster serotonergic system by 5,7-DHT: Effects on circadian
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and response to triazolam. Brain Res. 515:9-19; 1990. Sokolove, P. G.; Bushell, W. N. The chi square periodogram: Its utility for analysis of circadian rhythms. J. Theor. Biol. 72:131-160; 1978. Szafarczyk, A.; Ixart, G.; Alonso, G.; Malaval, F.; Nouguier-Soule, J.; Assenmacher, I. Effects of raphe lesions on circadian ACTH, corticosterone and motor activity rhythms in free-running blinded rats. Neurosci. Lett. 23:87-92; 1981. Tominaga, K.; Shibata, S.; Ueki, S.; Watanabe, S. Effects of 5-I-K,* receptor agonists on the circadian rhythm of wheel-running activity in hamsters. Eur. J. Pharmacol. 214:79-84; 1992. Turek, F. W. Effects of stimulated physical activity on the circadian pacemaker of vertebrates. J. Biol. Rhythms 4:135-147; 1989. Van de Kar, L. D.; Lorens, S. A. Differential serotonergic innervation of individual hypothalamic nuclei and other forebrain regions by the dorsal and median midbrain raphe nuclei. Brain Res. 162:4554; 1979.
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36. VergC, D.; Daval, G.; Marcinkiewicz, M.; et al. Quantitative autoradiography of multiple 5-HT1 receptor subtypes in the brain of control or 5,7-dihydroxytryptamine-treated rats. J. Neurosci. 6:34743482; 1986. 37. Weissmann-Nanopoulos, D.; Mach, E.; Magre, J.; Demassey, Y.; Pujol, J. F. Evidence for the localization of 5HTlA binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain. Neurochem. Int. 7:1061-1072; 1985. 38. Wirz-Justice, A.; Campbell, I. C. Antidepressant drugs can slow or dissociate circadian rhythm. Experientia 38:1301-1309; 1982. 39. Zambotti, F.; Carruba, M.; Vicentini, L.; Mantegazza, P. Selective effect of a maize diet in reducing serum and brain tryptophan contents and blood and brain serotonin levels. Life Sci. 17:1663-1670; 1975. 40. Zuker, I.; Rusak, B.; King, R. G., Jr. Neural bases for circadian rhythms in rodent behavior. In: Riesen, A. H.; Thompson, R. F., eds. Advances in psychobiology. New York: Wily; 1976:35-74.
Pergamon 0031-9384(93)EOOSV
Effect of Chronic Tryptophan Depletion on the Circadian Rhythm of Wheel-Running Activity in Rats KAZUAKI Department
KAWAI,’
NOR10
YOKOTA
AND
SHIGETO
YAMAWAKI
of Neurology and Psychiatry, Hiroshima University, School of Medicine, l-2-3 Kasumi, Minami-ku, Hiroshima, 734, Japan Received
25 September
1992
KAWAI, K., N. YOKOTA AND S. YAMAWAKI. Effect of chronic tryptophan depletion on the circadian rhythm of wheelrunning activity in rats. PHYSIOL BEHAV 55(6) 1005-1013, 1994.-The effect of chronic treatment with a tryptophan (TRP)free diet on the free-running circadian wheel-running rhythm and the central serotonergic system was investigated in blinded male rats. The long-term TRP-free diet did not change Periods of activity, but disordered their patterns. This seemed to be due to masking, entrainment, enhancement of the morning activity, and obscuring of the activity onset as well as appearance of some periodic activities within the subjective night. A long-term TRP-free diet decreased the concentration of TRP, J-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HJAA) in all brain regions tested: frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons. Density of 5-HT,, receptor binding was significantly decreased in the frontal cortex and hypothalamus, whereas no significant change was observed in the density of 5-HT, receptor binding in all regions. These results suggest that the period of primary circadian pacemaker is not affected, but its oscillation, as well as the coupling strength between the primary and secondary pacemakers, is weakened by the dysfunction of the serotonergic system caused by chronic TRP depletion.
Tryptophan
Serotonin
Free-running rhythm
Wheel running
IT has been suggested that mammals have circadian pacemakers
in the suprachiasmatic nucleus (SCN) in the hypothalamus (16,27). Furthermore, the SCN contains high levels of serotonin (5hydroxytryptamine, 5-I-IT) under the control of 5-HT neurons ascending from the raphe nuclei in the midbrain (2,20,35). Therefore, it is possible that the 5-HT system may affect the generation and entrainment of the circadian rhythm. Several researchers have investigated the relationship between the free-running circadian rhythm and the 5-HT system. It has been speculated that the 5-HT system does not affect the basic circadian pacemaker (40). Some investigators have reported that neither generation nor period of free-running rhythm are altered after lesions of the raphe nuclei (3,32) or the administration of 5-HT neurotoxin (13,21,23,30). In contrast, others have reported that large midbrain lesions disrupt the free-running rhythm (17) and serotonergic drugs cause a change of the free-running period (6,13,38). Recently, it has been reported that the 5-HT content in the SCN correlates negatively with the free-running period (29). Therefore, the role of the 5-HT system has remained uncertain with respect to regulation of the circadian rhythm. Tryptophan (TRP) is an essential amino acid and a precursor of 5-HT. The chronic ingestion of diets lacking TRP decreases the 5-HT level in the brain (8,39). In this study, we have produced a dysfunction of the 5-I-IT system in the brain by the administra-
Blinded rat
tion of a TRP-free diet, and evaluated the effects of chronic TRP depletion on the free-running rhythm of wheel-running activity in rats. METHOD
Free-Running Rhythm of Wheel-Running Activity Male Wistar rats (8 weeks old) were purchased from Charles River Japan Inc. and individually housed in cages (15 X 28.5 x 17 cm) equipped with a running wheel (circumference 1 m). They were fed commercial chow (Oriental Yeast Co., Ltd., Tokyo) and water ad lib. The numbers of revolutions of the wheel were recorded every 30 min by a personal computer (PC-8801 MK2; NEC Co., Ltd., Tokyo) connected to a running wheel. After 2 weeks, each animal was enucleated while under pentobarbital anesthesia. On the 65th or 66th day after enucleation, the animals were divided into two groups. Those in the TRP-free group ingested a TRP-free diet composed of 17.65% casein hydrolysate, 0.30% DL-methionine, 61.35% sucrose, 10.00% cottonseed oil, 5.00% cellulose powder, 3.50% AIN mineral, and 2.20% vitamin mixture (Oriental Yeast Co., Ltd.). Those in the control group received commercial chow. The experiments were carried out twice. Three animals in the control group and four in the TRP-
‘ To whom requests for reprints should be addressed. 1005
free group were first examined during the 153 days from January to August, 1990, and six animals in each group were monitored during the 111 days from December, 1990 to June, 1991. The experimental room was maintained in a 12-h light/12-h dark photoperiod (light on at 0830, light off at 2030; light phase 200 lux, dark phase 5 lux). Room temperature was kept at 23 +2°C during the first experiment and 24 -+ 2°C for the second one. The diurnal variation remained about 4°C in both experiments. Room humidity was kept at SO t 9% in the first and 55 t 10% in the second, but no diurnal variation existed. Because neither the room nor the cages were made absolutely soundproof, we entered the room at random times and touched the cages only for cleaning and feeding. Furthermore, we did not handle the animals at all. However, some other nonenucleated animals were maintained in the same room during both experiments. The periods of the wheel-running rhythm were calculated by a chi-square periodogram (31) for three samples of 50 consecutive days. The first sample was from the 15th to the 64th day after blindness. The second and third samples were from the first to the 50th day and from the 61st to the 110th day after feeding by the TRP-free diet, respectively. The periods of time in the control group were calculated to coincide with the three samples of the TRP-free group. Determination of TRP, 5-HT, and 5-Hydroxyindoleacetic (5-H&4) in the Brain
duced pressure, followed by two washings with 5 ml of ICC-cold SO mMTris-HCl buffer. Radioactivity was determined by a liquid scintillation counter. All experiments were done in duplicate. Specific binding was defined as the total binding minus the nonspecific counts obtained in the presence of 100 FM T-FIT (Wako Pure Chemical Industries, Ltd., Osaka). The 5-l-iT2 receptor binding assay was pcrformcd according to the procedure of Leysen et al. (18) with some modifications. In brief, 1.O nM [“Hlketanserin (27 19.5 GBqimmol; Janssen Biotech, N. V.) was added to tubes containing 300 1.11of membrane preparations and incubated (37”C, 15 min) in a final volume of 500 ~1 of 50 n-N Tris-HCl buffer (pH 7.4). Specific binding was defined as the total binding minus the nonspecific counts obtained in the presence of 1 (LM mianserin (Organon Co., Ltd.. Japan), the same procedure as for the S-HT,* receptor binding assay. The protein concentration was quantified according to Lowry et al. ( 19). Statist&&
Analysis
Statistical analysis was performed by a paired t-test compared within each group. Student’s t-test was used for comparison between the two groups.
Acid
After termination of the behavioral experiment, all rats were killed by decapitation. Their brains were removed immediately, placed on ice to be dissected by hand with a blade, and divided into the frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons according to the method of Glowinski and Iversen (11). A part of each region was frozen at -80°C for radioligand receptor assay. Each tissue sample was homogenized (Polytron, set at 8, 10 s) in 20 vol. (w/v) of 0.1 N perchloric acid solution. After centrifugation at 20,000 X g, 2°C for 15 min, the resulting supernatants were filtrated on millipore filters (pore size 0.33 pm) and separated into TRP, S-HT, and S-HIA. by high performance liquid chromatography with electrochemical detection (Shimazu Co., Ltd., Kyoto). The mobile phase was 50 mM phosphate buffer (pH 3.4) containing 12% methanol and 85 mg/ 1 octanesulfonic acid. The flow rate was 0.8 ml/min through a column 7 pm in diameter (Hiber LiChrosorb RP-18; Kantokagaku Co., Ltd., Tokyo). Assays of 5-HT,, and 5-HT, Receptor Binding Sites in the Brain Frozen samples of each region were homogenized (Polytron, set at 8, 10 s) in 15 ml of ice-cold 50 mM Tris-HCl buffer, pH 7.4, and centrifuged (49,000 x g,4°C 15 min). The pellets were suspended in 15 ml of 50 mM Tris-HCl buffer and incubated at 37°C for 20 min to exclude endogenous S-HT. After recentrifugation (49,000 x g,4°C 15 min), the resulting pellets were SUSpended again in 100 vol. (w/v) of the same buffer and used for the receptor binding assay. The 5-HTiA receptor binding assay was performed according to the procedure of Peroutka (24) with some modifications. In brief, 1.0 r&f [‘H]&hydroxy-2-(di-n-propylamine)-tetralin ([3H]-8-OH-DPAT; 6.77 TBq/mmol; Amersham) was added to tubes containing 300 ~1 of the membranous preparations, and then incubated (37°C 1.5 min) in a final volume of 500 ~1 of 50 mM Tris-HCl buffer (pH 7.4) containing 20 /JM pargyline, 4 mM CaC12, and 0.1% ascorbic acid. The incubation was stopped by rapid filtration on Whatman GF/B glass fiber filters under re-
RESULTS
Wheel-Running
Activity
In the first experiment, three animals in the control group and four in the TRP-free group were examined. Figures 1 and 2 show their double-plotted records. All animals housed in the cages were entrained to the light/dark cycle of the experimental room and had a stable wheel-running rhythm at first. After bilateral optical enucleation, they took about 2 weeks before their rhythms began to free-run. Their periods of free-running rhythms were longer than 24 h, with the exception of the TRP-free rat 4 (see Table 1). In the control group (Fig. l), the free-running periods of control rats 1 and 2 were stable during the observation. In the control rat 3, the period began to lengthen after 2 months following blindness and then shortened again. The onset of activity corresponded approximately to the onset of light phase after 5 months following blindness. In the TRP-free group (Fig. 2), three animals (TRP-free rats 1, 2, and 3), for which free-running periods were longer than 24 h, took 1 month of feeding on the TRP-free diet before their patterns of activity became disordered. After 2 months of the TRP-free diet, they demonstrated increases in activities that corresponded to the onset of the light phase, as well as a distinct, non-24-h free-running rhythm. The free-running rhythms of the TRP-free rats 1 and 2 were obscured after 4 months of the TRPfree diet, because the total daily counts of wheel-running activity were extremely reduced. In the TRP-free rat 4, for which freerunning period was shorter than 24 h, the period began to lengthen after feeding on the TRP-free diet. After 2 months of beginning the TRP-free diet, its period reached about 24 h, and its onset of activity corresponded approximately to the onset of the light phase. Six animals in each group were examined in the second experiment. Figures 3 and 4 show their double-plotted records. After bilateral optical enucleation, most of the animals had freerunning rhythms similar to those in the first experiment. HOWever, during the 60-80 days after blindness, the onsets of activity of the control rats 6 and 9 fitted the same phases as those before blindness. In the control group (Fig. 3), the free-running periods for one-half of the animals (control rats 4, 6, and 8) clearly
CIRCADIAN
RHYTHM IN TRYPTOPHAN
1007
DEPLETION Control-3
Control-2
Control-l
D
6
I 10 20 30
40
40 50 60 70 60 90 100 110 120 130 140 150 160 170 160 190 200 210 220
FIG. 1. Continuous recordings of wheel-running activity in the control group in the first experiment. The chart is double-plotted. Arrow B indicates the day on which rats were blinded. The D indicates the dav after blindness. The horizontal bar indicates the dark period of the day.
lengthened after 60-80 days following blindness. The activity patterns of all animals were stable during the observation. In the TRP-free group (Fig. 4), the periods for one-half of the animals (TRP-free rats $7, and 10) also lengthened immediately after feeding on the TRP-free diet, whereas the activity patterns of all these animals were disordered. Similar to the result of the first experiment, the TRP-free rat 6 showed an increase in activity near the beginning of the light phase. Most of the animals had activity patterns that were different from the results of the first experiment. Four animals (TRP-free rats 5, 7, 9, and 10) had the enhancement of morning activity, that is to say, the appearance of most wheel-running activity during the latter half of the subjective night, but a separate, smaller activity onset was present. In the TRP-free rat 9, the onset of activity became obscure. The TRP-free rat 8 expressed bouts of activity with a period of 25.40 h, which was additional periodic activity within only the subjective night. After 2 months of the TRP-free diet, four animals (TRP-free rats 5, 8, 9, and 10) expressed similar periodic activities within the subjective night, with periods ranging from 25.15 to 25.40 h. The free-running rhythms of all the animals were obscured after 3 months of feeding, because the total amount of daily counts of the wheel-running activity was extremely reduced. In both experiments, the chi-square periodogram analysis in the TRP-free group indicated that all of these peaks appeared to be multiple and lower in amplitude after feeding on a TRP-free diet than before (data not shown).
Table 1 shows the primary free-running periods corresponded to the highest peak in each periodogram calculated for the three samples. As described in the Method section, the first sample was from the 15th to the 64th day after blindness prior to a TRP-free diet. The second and third samples were from the first to the 50th day and from the 61st to the 110th day after feeding on a TRP-free diet, respectively. The three samples in the control group corresponded with those of the TRP-free group. In both groups, the mean time periods of the second and third samples significantly lengthened compared with that of the first sample [control group: t(8) = 3.36 and 5.52, p < 0.01; TRP-free group: r(9) = 2.32 and 2.94, p < 0.051. However, the periods showed no difference between the control and TRP-free groups for any sample,t(l7) = 0.310.62, p > 0.05. Figure 5 shows the mean daily counts of the wheel-running activity for the same three samples. In the control group, the counts of the second sample were significantly reduced compared with that of the first, t(8) = 4.68, p < 0.01. In both groups, the counts of the third sample were significantly reduced compared with those of the others [control group: t(8) = 4.81 and 4.37,~ < 0.01; TRP-free group: t(9) = 4.21 and 4.25, p < 0.01, respectively]. On the other hand, the counts of the second and third samples in the TRP-free group significantly increased compared with those of the control group [second sample: r(l1) = 2.74, p < 0.05; third sample: t(l1) = 3.27,~ < 0.011.
1008
KAWAI. TRP free-l
TRP free-2
YOKOTA rw
TRP free-3
ANI)
LAMAWAKI
free-4
0
+a
1
’
*I3
+B
10
10
M
m
30
30
40
4
50
50 00
00
1
T
T
1
1
10 M
20
1 10
10
i
20
30
30
40
40 50
50
00
00
70
70
10
IO m
(&
150
:.-
:-
_-
;;-.
_
FIG. 2. Continuous recordings of wheel-running activity in the TRP-free group in the first experiment. The chart is double-plotted. Arrow B indicates the day on which rats were blinded. Arrow T indicates the time Period of the feeding on a TRP-free diet. The D indicates the day after blindness and the day after beginning the TRP-free diet. The horizontal bar indicates the dark period of the day.
Contents of TRF’, 5-HT, and 5-H&4
in the Brain
The contents of TRP, WIT, and 5HL4A were determined for four regions (frontal cortex, hippocampus, midbrain, and pons) in animals fed a TRP-free diet for 111 consecutive days, and for five regions (frontal cortex, hippocampus, thalamus, hypothalamus, and pons) for 153 consecutive days. As shown in Table 2, their contents significantly decreased at all regions in the TRPfree group after both feedings [ill days: 49-10) = 4.69-14.19, p < 0.01; 153 days: t(5) = 8.16-29.56,~ < 0.011 and they all gradually decreased to a similar degree in each region. The mean TRP contents for 111 days became 32.3% and those for 153 days were 22.5% of control values. The mean 5-HT contents were 44.9% and 27.3% and the mean 5-HIAA contents were 25.8% and 18.1%, respectively. Density of 5-HT, and 5-HT2 Receptor Binding in the Brain The 5-HTIA and 5-HTz receptor binding sites were determined for six regions (frontal cortex, hippocampus, thalamus, hypothalamus, midbrain, and pons) in animals fed a TRP-free diet for 111 consecutive days, and for three regions (frontal cortex, hippocampus, and pons) for 153 consecutive days. As shown in Table 3, the density of 5-HTIA receptor binding significantly decreased in the frontal cortex [ill days: ~‘(10)= 3.27, p < 0.01; 153 days: t(5) = 5.75,~ < 0.011 and hypothalamus, l(l0) = 2.79, p < 0.05. On the other hand, the density of 5-HTz receptor bind-
ing did not significantly change in any regions for 111 days, t(9-
10) = 0.23-1.56, p > 0.05, and for 153 days, 42-5) 2.84, p > 0.05.
= 0.03-
DISCUSSION
Long-term feeding with a TRP-free diet similarly decreased the contents of TRP, 5-HT, and 5-HIAA in all the evaluated regions of the brain. These results suggest that chronic TRP depletion decreases central 5-HT synthesis. It has been already reported that chronic consumption of a corn diet, which contains low levels of TRP, reduces the contents of TRP and 5-HT to 4060% and 70-80%, respectively, of control values in young adult rat brains (8,39). Our results suggest that 5-HT synthesis may be more disordered than those of these previous reports, because the contents of TRP and 5-H-T are reduced by 20-30% and 30-44% of controls, respectively. The decrease of 5-HT contents may differ in relation to the degree and interval of TRP intake restriction, as well as the age of the animals. The changes of receptor binding sites related to 5-HT differed in tested classes and regions, although the contents of 5-HT were reduced to a similar degree in all regions. The density of ~-HTLA, receptor binding decreased only in the frontal cortex and hypothalamus, whereas the density of 5-HTz receptor binding did not change. To our knowledge, no other investigators have reported the effect of TRP depletion on the receptors related to 5-HT. On
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RHYTHM IN TRYPTOPHAN
1009
DEPLETION
FIG. 3. Continuous recordings of wheel-running activity in the control group in the second experiment. See Fig. 1 legend.
the other hand, some investigators have reported that the administration of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (DHT), causes the density of 5-HT’1Areceptor binding to decrease only in the raphe nuclei and striatum, but not in other brain regions (12,36,37). The treatment of this drug is also reported to have no effect on the density of U-IT, receptor binding in all brain regions (9,25). Our results suggest that chronic TRP depletion and the administration of the 5-I-IT neurotoxin may affect the 5-I-lTrA receptor system with the distinct mechanism, yielding different results in brain regions, although neither may affect the 5-I-IT, receptor system. However, it will be necessary to conduct further studies on this point. Roth the primary free-running periods and the wheel-running activity were altered after blindness in each group (Table 1 and Fig. 5). Recently, it is reported that the activity of wheel running produces a feedback on a circadian pacemaker. The increase in activity causes a phase shift in the hamster (26,34). The activity correlates negatively with free-running periods in the rat (29). Our results may support that activity affects the free-running period in the rat, but does not induce phase shift. However, this
TRP fr”-5
TRP
fma-s
conclusion requires circumspection, because one-half of the animals have clearly and suddenly lengthened periods at a point of about 60-80 days after blindness for the second experiment (Figs. 3 and 4). This phenomenon is suspected to be due to unknown environmental factors in the experimental room; however, the cause is unclear. Therefore, it is uncertain in this study whether or not activity produces feedback to a pacemaker. The primary free-running periods were not different between the control and TRP-free group during any sample interval, although the wheel-running activities in the TRP-free group increased compared with those in the control group. These results may suggest that the free-running period is not affected by chronic TRP depletion, when the effects on pacemaker are considered as a whole, including the increase in activity due to a TRP-free diet. It has been reported that the continuous treatment with TRP lengthens the free-running period of spontaneous optic nerve impulses in the isolated Aplysia eye (7). No investigators, however, have reported on the effect of TRP depletion on the free-running period. Several investigators have tried to determine the relationship of the 5-HT system and the free-running periods
TRP h-7
FIG. 4. Continuous recordings of wheel-running activity in the TRP-free group in the second experiment. See Fig. 2 legend.
1011)
KAWAI.
1
TABLE PERlOD
OF
WHEEL-RUNNING
RHYTHM
BY
CHI-SQUARE
PERIODOGRAM Rat No.
Control group 1 2 3 4 5 6 7 8 9 Mean + SE
Before
I -SO
Days
hl-IIODays
24.35 24.30 24.00 23.75 24.30 24.00 24.15 24.15 23.95 24.11 ? 0.07
24.60 24.45 24.10 24.00 24.45 24.05 24.20 24.20 23.90 24.22 t- 0.08*
24.60 24.45 24.05 24.00 24.50 24.15 24.30 24.15 24.15 24.26 -t 0.07*
24.30 24.45 24.35 23.50 24.05 24.30 23.65 23.95 24.20 23.75 24.05 2 0.10
24.50 24.50 24.35 23.50 24.15 24.30 24.15 23.90 24.50 23.90 24.18 t O.lOt
24.45 24.45 24.55 24.00 24.00 24.60 24.35 24.00 25.15 23.95 24.35 5 0.12.t
TRP-free group
1 2 3 4 5 6 7 8 9 10 Mean t SE
The values represent a period (hours) of the highest peak in each sample interval. Before indicates the first sample from the 15th to the 64th day after blindness before feeding on a TRP-free diet. l-50 days indicates the second sample from the first to the 50th day after feeding on a TRP-free diet. 61-110 days indicates the third sample from the 61st to the 110th day after feeding on a TRP-free diet. The three samples in the control group correspond with those of the TRP-free group. *t Indicate the results compared with those of Before by a paired Itest: *p < 0.01, tp < 0.05.
of wheel running in rodents. Some of these reports suggest that the central 5-HT system may affect these free-running periods because a negative correlation exists between 5-HT contents in the SCN and free-running periods (29), and because the periods are shortened or lengthened by the administration of the S-HT synthesis inhibitor, parachlorophenylalanine (13), or the monoamineoxidase inhibitor, clorgyline (6,38), respectively. However, others have reported that the free-running periods are not affected by the administration of the 5-HT neurotoxin, 5,6- or 5,7-DHT (13,21,23,30). Contradictory results have also been reported that lesions of raphe nuclei interfere with the expression of the free-running rhythm (17) or affect neither generation nor period of the free-running rhythm (3,32). Therefore, the role of the 5-HT system has remained uncertain with respect to the circadian system. Our results suggest that the period of primary circadian pacemaker may not be affected by the dysfunction of 5-HT system due to the lack of TRP, a precursor of 5-HT. The activity patterns were disordered after a TRP-free diet, although the periods were not affected. The peaks of chi-square periodograms became multiple and low. In the first experiment, most of the animals had increases in activities with about a 24h period, which were different from free-running rhythms. Their activities seem to be due to the expression of masking effects, because the onsets of their activities coincide with the onset of
YOKOTA
AND
\r /\MAWAki
the light phase. The phenomenon in the TRP-free rat -I stems 10 be due to the expression of entrainment effects, because the onset of its activity coincides with that of other animals (Fig. 2). The control rat 3 is also suspected to have been entrained after i months following blindness, because it then has an activity pattern similar to the TRP-free rat 4 (Fig. 1). All the animals in the TRP-free group expressed these masking or entrainment effects, whereas only one rat did in the control group. These results suggest that chronic TRP depletion may make animals susceptible to subtle environmental factors, because the masking and entrainment effects are expressed by the periodic change of environmental factors. It is speculated that chronic TRP deplction may make the oscillation of the primary circadian pacemaker weakened. The environmental factors producing the masking or cntrainment effects have been reported to be the temperature cycles (lo), the light cycles (l), and the periodic social interaction (22). In this study, they appear to be related with the lighting being on in the experimental room, because the onsets of activity are all near the beginning of the light phase. However, the light cannot have affected the animals directly because they had been enucleated. The two following factors may be suspected. The first one is the changes of noise, because neither the room nor the cages are made perfectly soundproof, and because some other nonenucleated animals, as maintained in the same room. may convey the photic cues to the enucleated animals via a noise due to a change in activity level during the light and dark phases. The second is the temperature cycle, as the diurnal variation remained in the room temperature. In the second experiment, only the TRP-free rat 6 expressed the masking effect. Most of the animals expressed the enhancement of morning activity, the obscuring of the activity onset, and the secondary periodic bout activities only within subjective nights, with periods ranging from 25.15 to 25.40 h (Fig. 4). These changes of activity pattern within subjective night were expressed only in the TRP-free group, not in the control group. These phenomena have been previously reported in untreated blinded hamsters (5) and clorgyline-treated hamsters (6). In these reports, a multioscillator system is hypothesized that the primary pacemaker, which divides into the subjective night and day. mod-
4wa
1
b 0 BdOW
I-50
daya
51-110 day*
FIG. 5. Change of mean daily counts of wheel-running activity at the three samples of 50 consecutive days. Before indicates the first sample from the 15th to the 64th day after blindness and before feeding on the TRP-free diet. l-50 days indicates the second sample from the first to the 50th day after feeding on the TRP-free diet. 61-110 days indicates the third sample from the 61st to the 110th day after feeding on the TRPfree diet. Open circle indicates means of the TRP-free group. Closed circle indicates means of the control group. The vertical bars represent SE. Significant difference between the Go groups by Student’s r-test: *p < 0.05, **p < 0.01. Significant difference by a paired f-test: “I, < 0.01 vs. Before, “p < 0.01 vs. Before and l-50 days.
CIRCADIAN RHYTHM IN TRYPTOPHAN
1011
DEPLETION TABLE 2
CONTENTS OF TRP, 5-HT, AND 5-HIAA IN VARIOUS REGIONS OF RAT BRAIN BY HI’LC-ECD
111 Days Frontal cortex Control
9.47 2 0.29
TRP-free
2.99 k 0.77”
diet
Hippocampus Control TRP-free
9.05 5 0.33 diet
Thalamus Control TRP-free
3.02 k 0.62* n.d.
diet
Hypothalamus Control
5-HL4A
5-HT
TRP 111 Days
153 Days
16.48 ? 0.26 3.56 2 0.36: 17.76 + 0.59 3.80 2 0.39*
111 Days
153 Days
153 Days
2.44 t 0.20
2.42 2 0.03
1.34 + 0.05
1.15 + 0.05
1.03 2 0.17*
0.60 + 0.09*
0.27 2 0.06*
0.13 2 0.05*
1.99 + 0.11
1.52 k 0.10
1.61 5 0.05
1.34 2 0.04
1.01 2 0.18*
0.48 ? 0.07*
0.44 5 0.11*
0.24 2 0.04*
n.d.
2.12 ? 0.16
n.d.
1.40 k 0.02
n.d.
15.66 2 0.60
3.69 2 0.27*
n.d.
0.59 2 0.11*
n.d.
0.32 k 0.06;
TRP-free
diet
n.d. n.d.
16.52 + 0.34 3.97 ? 0.33*
n.d. n.d.
4.12 2 0.14 1.22 2 0.14*
n.d. n.d.
2.23 2 0.08 0.44 2 0.06’
Midbrain Control TRP-free
diet
7.34 + 0.24 2.18 ? 0.45*
n.d. n.d.
2.81 t 0.10 1.10 ? 0.16*
n.d. n.d.
2.17 ? 0.11 0.62 5 0.15’
n.d. n.d.
Pons Control TRP-free
diet
7.46 2 0.26 2.42 5 0.51*
14.07 2 0.13 3.11 * 0.30*
3.58 ? 0.25 1.60 2 0.30*
2.58 2 0.10 0.59 * 0.10*
2.81 2 0.26 0.83 2 0.19*
1.84 2 0.04 0.35 2 0.06*
The values represent mean + SE of concentration (nmoI/g tissue). 111 days: control n = 5-6, free diet n = 4. n.d.: not determined. * Indicates results compared with each control by Student’s f-test: p < 0.01.
TRP-free
diet n = 6. 153 days: control n = 3, TRP-
treated rats (14). It is speculated that the coupling of pacemakers in infantile rats is unstable and the secondary oscillations in methamphetamine-treated rats are enhanced, respectively. Our results
ulates the secondary bout oscillators by providing a window during which their expression is feasible. A similar phenomenon has also been reported in infantile rats (15) and methamphetamine-
TABLE 3 DENSITY OF 5-HT,, AND 5-HT, RECEPTOR BINDING IN VARIOUS REGIONS OF RAT BRAIN 5-HT,A 111 Days
WIT, 153 Days
111 Days
153 Days
215.70 % 18.08 164.16 ? 1.81
Frontal cortex Control TRP-free diet Hippocampus
20.97 ? 0.98 15.83 5 1.23*
29.93 + 1.82 15.88 + 1.62*
181.80 + 4.31 196.85 + 9.68
Control TRP-free Thalamus
54.60 5 1.95 50.13 2 3.16
64.83 2 3.12 60.75 2 1.25
32.94 ? 2.68 28.46 2 2.29
18.82 t 19.93 2
diet
Control TRP-free diet Hypothalamus
5.05 t 0.52 4.83 + 0.27
n.d. n.d.
30.48 2 2.27 31.61 2 3.64
n.d. n.d.
Control TRP-free Midbrain
diet
14.17 * 1.19 10.47 2 0.60.t
n.d. n.d.
32.42 5 2.44 29.69 2 1.12
n.d. n.d.
Control TRP-free Pons
diet
9.47 5 0.48 8.76 5 0.47
n.d. n.d.
20.74 ? 2.77 20.50 2 3.50
n.d. nd.
diet
11.39 t 0.66 9.82 ? 0.63
4.46 % 0.92 4.79 2 0.45
30.20 5 2.30 31.60 -c 3.83
6.01 + 6.03 2
Control TRP-free
1.30 4.74
0.51 0.57
The values represent mean 2 SE of specific binding (fmoI/mg protein) at 1.0 r&f of [3H]DPAT (5-HT,J and [3H]ketanserin (5-I-IT,). 111 days: control n = 6, TRP-free diet n = 5-6. 153 days: control n = 3, TRPfree diet n = 4. nd.: not determined. * Indicate results compared with each control by Student’s I-test: *p < 0.01, tp < 0.05.
1011
KAWAl.
suggest that the coupling between the primary pacemaker and the secondary bout oscillator may be strong and stable in adult rats (from 8 to 33 or 39 weeks old). However, under a dysfunction of the 5-HT system due to the lack of TRP, the secondary bout oscillations may be relatively enhanced due to the decrease of coupling strength. Different results were obtained in the first and second experiments. The difference of room temperature and humidity in each experiment may have affected the oscillation and the coupling on the pacemakers. If chronic TRP depletion makes the oscillation of the primary circadian pacemakers and/or the coupling weakened, these changes may be related to the downregulation of 5-HT,, receptor in hypothalamus containing the SCN, following decrease in 5HT content. The chronic treatment of clorgyline, which caused the similar phenomena, is reported to cause 5-HT level to return towards control level after initiate increase (4) and to make a downregulation of 5-HT, receptor (28). Recently, the treatments
YOKOTA
AND YAMAWAKI
of 5-HT,+, agonists are reported to induced a phase advance oi free-running rhythm in hamster (33). Further studies will be r-cquired to clarify this point. In conclusion,
our results
suggest
that the dysfunction
of the
5-HT system due to the lack of TRP may make the oscillation of primary circadian pacemaker and the coupling strength between the primary and secondary pacemakers weakened, without causing any change in the period of primary pacemaker. These changes may be due to the downregulation of 5-HT,,, receptor system.
ACKNOWLEDGEMENTS
The authors are grateful to Dr. Kiyohisa Takahashi, Head of the Division of Mental Disorder Research, National Institute of Neuroscience. NCNP, Japan, for his invaluable advice. They also thank Dr. Akihiko Seo, Department of Public Health, Hiroshima University School of Medicine, Japan, for valuable help in the experiment,
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