J o ~ t r n u lof N~.ui.orh~niisrr).1976. Vol 26. pp. 951-955. Pergamon Press. Printed in Great Britain.
RESPONSE OF RAT BRAIN INDOLES AND MOTOR ACTIVITY TO SHORT LIGHT-DARK CYCLES' GEORGIANA HANSELMA"and A. A. BORBELY' Institute of Pharmacology, University of Zurich, Zurich, Switzerland (Received 17 July 1975. Accepted 20 October 1975)
Abstract-Since the vigilance states of the rat can be largely controlled by one hour light-one hour dark cycles, we investigated the effect of this photoperiod on the rat brain indoles and motor activity. Groups of 9 rats were killed 15-20 min or 45-50 min after the onset of the 1-hour light or 1-hour dark period. Serotonin (5HT) and 5-hydroxyindoleaceticacid were determined fluorometrically in cortex, hypothalamus and brain stem. Tryptophan was determined fluorometrically in serum, cortex and hypothalamus. Cortical tryptophan was highest at the end of the dark period, whereas cortical 5-HT and 5-hydroxyindoleacetic.acidwere highest at the beginning of the light period. Motor activity was high during darkness and low during light. When the biochemical results were compared to the motor activity records of the individual animals, the decrease in motor activity at the onset of light correlated significantly with the cortical 5-hydroxyindoleacetic acid/S-HT ratio in the animals killed at the beginning of the light period. The results indicate a rapid response of the cortical indoles to the onset of light and support the hypothesis that the induction of sleep is related to the brain indole metabolism.
THEVIGILANCE states as well as the motor and consummatory behaviour of the rat can be entrained by 1 hour light-1 hour dark cycles (LD 1 :1) (BORB~LY & HUSTON,1974; E ~ R B B L Y et al., 1975). Slow wave sleep is the sleep state which is most immediately affected by a change in the lighting conditions; it is rapidly increased during the 1-hour light period and rapidly reduced during the 1-hour dark period. The enhancement of sieep by light is most prominent if the light intensity is high, and if its influence occurs during the circadian phase of minimum sleep (BORR ~ L Y . 1975). On the basis of these results we were interested to test the hypothesis proposed by JOUVET (1972) that serotonergic mechanisms play a central role in the induction of sleep. The hypothesis rests largely on the observations that destruction of serotonin (5-HTj containing neurons of the raphe nuclei, or reduction of 5-HT synthesis by p-chlorophenylalanine @CPA), significantly reduce sleep in the cat. The generality of these findings has been challenged by RECHTSCHAFFEN et al. (1973) who reported inconclusive effects of pCPA on sleep in the rat. However, it is doubtful whether the administration of pCPA consti-
* Preliminary reports of this work were presented at the Second International Congress of Sleep Research, 1975, and at the First European Neurosciences Meeting, 1975. To whom all correspondence should be sent. Abbreuiarions used: 5-HIAA, 5-hydroxyindoleaceticacid; 5-HT, 5-hydroxytryptamine; NAT, serotonin-N-acetyltransferase; OPT, o-phthalaldehyde; pCPA, p-chlorophenylalanine; TRY, tryptophan; LD 1 : 1, 1 h light-l h dark; EL, EL, beginning or end of light period; BD, ED, beginning or end of dark period. 95 I
tutes a n optimal approach for studying relations between brain 5-HT and sleep: (1) The use of 6-fluorotryptophan, another tryptophan hydroxylase inhibitor, reduced motor activity in the rat, whereas pCPA produced hyperactivity (JACOBS et al., 1974); (2) The effects of pCPA are not restricted to the central neret d., 1973) nor strictly vous system (RECHTSCHAFFEN to an inhibition of tryptophan hydroxylase ( I L I i L L m ef al., 1970); (3) The long-term effects of this agent may induce compensatory mechanisms, since a gradual recovery of sleep may occur despite continuously low cerebral 5-HT levels (DEMENT, 1969). The LD 1 :1 schedule appeared to be well suited for reliably inducing sleep by a natural variable and thereby testing its effect on brain indoles. O n the basis of the 5-HT hypothesis it could be expected that the induction of sleep by light is accompanied or preceded by changes in brain indole metabolism. We therefore measured the levels of tryptophan (TRY), 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) in four parts of the brain in animals which were killed at the beginning or the end of the light period (BL or EL), or at the beginning or the end of the 'dark (BD or ED). Since we did not want to handle the animals prior to the time of death, we did not inject labelled 5-HT precursors to estimate 5-HT turnover. Motor activity was recorded for each animal as an indicator of the vigilance state. MATERIALS AND METHODS Materials. We obtained 5-HT-creatinine sulphatc from
Hoffmann-LaRoche, Basel; 5-HIAA. HCI, L-cysteine and o-phthalaldehyde (OPT) from Fluka AG, Buchs; L-TRY IIom E. Merck, Darmstadt and Norharman from Sigma,
952
GEORCIANA HANSELMA"and A. A. BORBBLY
St. Louis. All reagents used in the biochemical analyses were of fluorometric grade or of the highest grade avail150 able. Environmental conditions and schedule. Thirty-six adult 100 male Sprague-Dawley rats of the SIV-50strain weighing 25C300 g were used. The animals were housed individually $ 50 in transparent plastic bins, had unlimited access to food and water, and were maintained in a separate room with 5 0 g 200 temperature regulated at 22 2°C. They were exposed to 0 an LD 12:12 photoperiod (light from loo0 to 2200) for 5 150 at least 1 week before being exposed to the LD 1 :1 cycles. 3 Twenty-two hours before killing the cages were placed in 100 a special chamber with an LD 1 : I photoperiod. For the BL and EL groups the first hour of this schedule was light, 50 for the BD and ED groups the first hour was dark. This schedule was used so that all animals would be killed as 0 6 7 9 nearly as possible at the same point in their circadian cycle. Time of day, h Animals were killed within the first day of exposure to the LD 1 : 1 cycles since previous results (BORBBLY & Hus- FIG.1. Average motor activity values of the four experTON, 1974; BORBBLY et al., 1975) had shown that animals imental groups plotted for successive 5-min periods during are quickly entrained to the short light cycles and that the last 2 h before death. Motor activity is expressed as by the second day a drift in the circadian activity is already a percentage of the average value between o400 and 0800. apparent. The cages in the LD 1 :1 chamber rested on Letters indicate the time of death: BL, beginning of light; force recorders (BORBELYet al., 1975) whose integrated out- EL, end of light; BD, beginning of dark: ED, end of dark. put was continuously recorded on analogue magnetic tape Shading indicates the dark period. All points are plotted as a measure of motor activity.. Animals were killed in at the beginning of the 5-min period, thus, the points plotgroups of three at 08150820 (BL or BD groups) or at ted at 0700 and 0800 represent the values for 0 7 W 7 0 5 08450850 (EL or ED groups). This hour was at the end and 0 8 W 8 0 5 respectively. For each g o u p n = 9. of the circadian active period during which the entrainment of the sleep cycles by the photoperiod is optimal (BORB~LY et al., 1975). tings for excitation and emission were 352 nm and 473 nm Selection and preservation of tissue. Cervical blood was respectively. collected, centrifuged at 20,000 g for 20 min and the serum Data analyses and statistics. The motor activity records decanted into plastic vials and stored at -20°C until pro- on analogue magnetic tape were converted 10 a digital cessed for total TRY. The brains were rapidly removed form (AD conversion rate approx 30 Hz) and integrated and dissected on an iced platform. Each brain was halved for successive periods of 5 min. For each animal the averalong the midline then sectioned into cortex, brain stem age motor activity between 0400 and 0800 was computed and hypothalamus according to GLOWINSKI & IVERSeN and defmed as 100%. The percentage values at various (1966). The fiontal cortex was separated from the rest of time periods before death were used for further computhe cortex according to QUAY(1968). The pineal glands tation (Fig. 1).The two-tailed Wilcoxon rank-sum test was were removed for separate 5-HT analysis, but since no used to determine significant differences. variation was seen among the four groups thcse results will not be further discussed. The cerebellum was disRESULTS carded. All sections were immediately frozen on solid C02, wrapped in foil and stored at -20°C until analysed for Effect of lightdark cycles on motor activity. For 5-HTand 5-HIAA or for TRY. each of the four groups motor activity was enhanced Biochemical analyses. Brain regions were analysed in during the dark and reduced during the light (Fig. four groups (BL, EL, BD, and ED) of eight. For each brain 1). This finding confirms the results of previous experregion. the four groups were analysed in succession so that iments ( B ~ R B ~& LY HUSTON,1974; BORBBLYet al., not more than 6 days elapsed between the processing of 1975). The motor activity records of the individual the first and the last groups. 5-HT and 5-HIAA were deteranimals showed considerable variability, whereas the mined in one half of the cortex, hypothalamus, and brain stem after butanol-heptane extraction according to the group averages showed only minor variation. Effect of light-dark cycles on brain indoles. The most fluorometric method of CURZON& GREEN (1970). Both halves of the frontal cortex were used for the determination striking results were obtained in the cortex (Fig. 2). of 5-HT and 5-HIAA. Each sample was processed in tripli- Here the TRY levels were highest in the ED group cate: one probe, one internal standard and one tissue and the 5-HT, 5-HIAA, and the ratio 5-HTAA/S-HT blank. External standards were also used for comparison (an estimate of indole turnover) were highest in the of fluorescence from day to day. Fluorescence was read BL group. For each of these four parameters the difon a Farrand spectrofluorometer with excitation waveferences were significant against each of the other length set at 360 nm and emission wavelength at 470 nm three groups. The measurements made in the serum (uncorrected). TRY was determined in the serum, cortex and hypothalamus fluorometrically according to DENCKLA and the other brain regions are shown in Table 1. & DEWEY, (1967). Again all samples were processed in trip- The levels of 5-HT were also highest in the BL group licate and both internal (L-TRY) and external (norharman) in both hypothalamus and brain stem, though in standards were used. Uncorrected spectrofluorometer set- neither of these regions was the difference significant
953
lndole and motor activity response to short photoperiod
activity. No significant correlations could be found between the percentage of motor activity of the individual animals on the one hand, and the concentrations of TRY, 5-HT or 5-HIAA on the other. The only significant correlation was seen in the BL group between the percentage decrease in motor activity for the last two 5-min periods before death and the ratio 5-HIAA/5-HT in the cortex. ( P < 0.05; rank correlation test). DISCUSSION
It has been shown in previous studies that the concentration of 5-HT in various brain regions is higher during the 12-h light phase than during the 12-h dark period (QUAY,1968; HERYet al., 1972). However, in Ih FIG.2. Average cortical indole levels for each of the four those studies the influence of light per se cannot be experimental groups defined in Fig. 1. The cortex [minus differentiated from the effect of an endogenous circathe frontal area (QUAY,1968)] was sectioned according dian rhythm on the brain indoles. In the pineal gland, & IVERSEN (1966). One half was analysed for example, the latter effect has been shown to persist to GLOWINSKI & DEWEY, 1967) and the even during continuous darkness or in blinded rats for tryptophan (TRY) (DENCKLA & GREEN, 1970). other half for 5-HT and 5-HIAA (CURZON (QUAY,1964; SNYDER et al., 1965). In the present exShading indicates the dark period. The value shown for periments the influence of a circadian variable was 5-HIAA at BD represents the average of 7 animals; For all other bars n = 8. Brackets indicate SEM. Asterisk minimized by killing the animals during the same indicates significant difference compared to each of the hour of the day. Under these standardized conditions other groups; TRY, P < 0.02: 5-HT and 5-HIAA, P < 0.01 a rapid rise of 5-HT levels in the various brain regions, particularly in the cortex, followed the onset (Wilcoxon rank-sum test). of light. Compared to the end of the dark period, cortical 5-HT increased by more than 60% and with respect to all other groups. The levels of TRY 5-HIAA by 1850/,, whereas TRY decreased by more in the serum and the hypothalamus were highest in than 35% (Fig. 2). These findings indicate that 5-HT the EL group rather than in the ED group as in the turnover is massively increased during the 15 min of cortex. Again these differences were significant only exposure to light. The fact that the light-induced changes of indole when compared to the lowest levels. 5-HTAA and the ratio S-HTAA/S-HT showed much greater variability levels were most prominent and consistent in the corfrom region to region so that it is not possible to tex is in accordance with previous reports. Thus H ~ R Y provide a summarizing statement for these results. et al., (1972) found that the high rate of conversion Correlation between indole concentration and motor of C3H]TRY to C3H]5-HT during the light period was TABLE I . AVERAGEVALUES OF INDOLE LEVELS AND THE AVERAGE OF THE
INDIVIDUAL RATIOS FOR SERUM AND THREE BRAIN REGIONS ~
~~
TRY Serum
RL
EL Hypothalamus
Brainstem
Frontal Cortex
BD ED BL EL ED ED BL EL RD
118 k 20 174 f 22*d 152 f 19 115 k 9 75 f 6 85 k 8*c,d 56k6. 60 f 5
Not measured
3.1 2 0.3 2.8 f 0.2 2.2 f 0.2*a 3.0 0 2 4.3 f 0.2 4.3 f 0.2 4.0 +_ 0 2
t
ED
BL EL BD ED
5-HT
Not measured
3.4 f 0.3 3.2 0.3 3-6 f 0.4 2.9 f 0.3
5-HIAA
4.5 f 0.5 3.7 f 0.5 4.4 k 0.5 3.4 f 0.2 3.6 0 3 5.0 i: 0 8 (7) 3.8 +_ 0.2
t
*
3.7 f 0.3 3.4 0.2 3.9 & 0.7 3.3 f 0.3
5-HJAAp-HT ~~~
5-HIA.4/5-HT
1.4 f 0 1 1.3 i 0 2 2.1 & 0.3 1.1 i 04*b.c 0 9 f 0.5 1.2 f 0.2 (7) 1.0 +_ 0 1
t 1.1 f 0.1 1.1 f 0.1
1.0 & 0 1 1.2 f 0.1
All values are expressed as nmol/ml serum or nmol/g tissue i SEM. Group designation is the same as in Fig. 1. Except where indicated by the number in parentheses, n = 8. * Significant at least at the level P < 0.05; a-against all other groups; b-against BL; c--against BD; d-against ED. f Samples lost in processing.
954
GEORCIANA HANSELMANN and A. A. BORBELY
most noticeable in the cortex, and BLONDAUX et al. In all four experimental groups the average motor (1975), when measuring the changes in injected activity was high during darkness and low during C3H]TRY metabolism in cats with isthmic lesions, light, a result which confirms our previous findings also found the greatest increase in conversion rates (BORBBLY & HUSTON,1974). However, when the in the cortex. Since cortical indole levels are lower records of the individual animals were compared, no than in the other brain regions tested, light-induced correlation was seen between the biochemical and bechanges may be more readily apparent in the cortex. havioural data. It is not very surprising that the Moreover, in the hypothalamus, a substantial amount motor activity, as determined for successive 5-min of 5-methoxytryptamine, which has the same fluores- periods by our recording system, shows no significant cence characteristics as 5-HT when reacted with OPT correlation with regional indole levels at the time of (GREEN et al., 1973), may also serve to mask the fluc- death. Even if a relationship exists between indole tuations of 5-HTin this region. levels in specific parts of the brain and certain aspects The rate of 5-HT synthesis in the brain depends of behaviour, it is difficult to record the appropriate on both the availability of the substrate TRY and behaviour during the pertinent period of time, and the activity of the hydroxylating enzyme (tryptophan then select the relevant parts of the brain for the 5-mono-oxygenase, tryptophan 5-hydroxylase, EC determination of indoles. On the basis of these nega1.14.16.4). Some workers have suggested that brain tive findings we can, therefore, by no means exclude TRY concentration is the rate-limiting factor in the the existence of a correlation between indole levels formation of 5-HT (e.g. FERNSIROM & WURTMAN, and behaviour. 1971; TACLIAMONTE et al., 1973), whereas others have During the LD 1 :1 schedule, the onset of light inproposed that only TRY that has been newly taken creased cortical 5-HT and 5-HIAA, reduced motor up into the brain is available for 5-HT synthesis activity, and induced slow wave sleep (BORBELYet (HERYet al., 1972; HAMONet al., 1974). In our study a]., 1975). Towards the end of the light period, when neither cortical nor hypothalamic TRY levels changed the animal was usually inactive and exhibited sleep, in parallel with the 5-HT levels, indicating that the the cortical level of the two indoles did not differ substrate concentration does not constitute the only from the values during darkness. These findings suprate-limiting factor of 5-HT synthesis. Due to the brev- port the hypothesis that the induction of sleep, but ity of the LD cycles employed, we cannot rule out not the maintenance of sleep, depends on an increase the possibility that a process initiated during the dark of 5-HT metabolism (JOUVET, 1972; PETITJEAN et a[., period, resulting in high cortical TRY levels at ED, 1975). The fact that the ratio 5-HIAA/S-HT was corcontributes substantially to the high levels of 5-HT related with the reduction of motor activity after the and 5-HIAA during BL. However, light itself could onset of light lends further support to that assumpaffect cerebral 5-HT levels by altering the activity of tion. Catecholamines, which have not been detertryptophan 5-hydroxylase, or the catabolizing enzymes mined in the present study, may play a major role monoamine oxidase (amine: oxygen oxidoreductase in the induction of wakefulness and motor activity (deaminating) (flavin-containing),EC 1.4.3.4) or sero- by darkness and may in turn influence the serotonertonin N-acetyltransferase (EC 2.3.1.5: NAT). The gic system (STEINet al., 1974). effects of short light periods on the latter enzyme in On the other hand, light may influence brain inthe pineal gland during a critical period in the dark doles and behaviour by largely independent mechare well documented (DEGUCHI & AXELROD, 1972; anisms. Preliminary results indicate that LD-induced KLEIN& WELLER,1972), though it is not known changes of motor activity during the LD 1:1 schedule whether NAT is relevant for the metabolism of extra- are not abolished after administration of p-chloropineal 5-HT (ELLISON et al., 1972). Nevertheless, the phenylalanine (300 mg/kg, intraperitoneally ;BORBELY, light-induced effects on the activity of a rate-limiting unpublished observation). In view of these findings, enzyme in the pineal gland (AXELROD, 1974) may and of the conflicting evidence described in the introserve as a model of the mechanisms affecting extra- duction, the original 5-HT hypothesis of sleep may pineal indole metabolism. have to be modified. An increase of 5-HT metabolism We measured serum total TRY primarily as an ad- may well be a sufficient, but not a necessary condition ditional control, since this parameter is more indica- for the induction of sleep. tive of eating behaviour than of the amount of TRY available to the brain (PEREZ-CRUET et al., 1972; KNOTT& CURZON1972; LIPSEIT et al., 1973). Previous work from our laboratory (BORBBLY & HUSTON, 1974) showed that feeding behaviour was not Acknowledgements-The authors would like to thank Prof. DT.W. L~CHTENSTE~GER for making his laboratory availentrained as readily by the short LD cycles as motor able to us, F. HEFTIfor his many helpful suggestions conactivity. Toward the end of the active period of the cerning the biochemical determinations and E. PICHLER, circadian cycle the feeding behaviour is high enough M. HINKKANEN and M. HELMEYfor technical assistance so that it may continue into the light period. The and help in preparing the manuscript. This work was aided high serum TRY at EL may thus merely represent by support from the Swiss National Science Foundation, Grant Nos. 3.2120.73 and 3.5350.71. a delay of nutrient absorption from the gut.
Indole and motor activity response to short photoperiod REFERENCES J. (1974) Science, N.Y 184, 1341-1348. AXELROD F. & PUJOLJ.-F. (1975) BLONDAUX C., BUDAM., PETITJEAN Brain Res. 88, 425437. BORBBLY A. A. (1975) Neurosci. Lett. 1, 67-71. BORRBLY A. A. & HUSTONJ. P. (1974) Physiol. Behau. 13, 795-802. BORBBLY A. A,, HUSTON J. P. & WASERP. G. (1975) Brain RPS. 95, 87-101. CURZON G. & GREEN A. R. (1970) Br. J . Phartmc. Chemother. 39, 653-655. DECUCHI T. & AXELROD J. (1972) Proc. natn. Acad. Sci. U.S.A. 69, 2547-2550. DEMENT W. C. (1969) in Sleep, Physiology and Pathology, a Symposium (KALESA., ed.) pp. 245-265. Lippencott, Philadelphia. DENCKLA W. D. & DEWEY H. K. (1967) J. Lab. clin. Med. 69, 16&169. ELLISONN., WELLERJ. L. & KI.EIND. C. (1972) J . Neurochem. 19, 133S1341. FERNSTROM J. D. & WURTMAN R. J. (1971) Science, N.Y 173, 149-152. GLOWINSKI J. & IVERSEN L. L. (1966) J . Neurochern. 13, 655-659. GREEN A. R., KOSLOWS. H. & COSTAE. (1973) Brain Res. 51, 371-374. HAMONM., Borrrtcor~s., MOROT-GAUDRY Y., HBRY F. & GLOWINSKI J. (1974) in Serotonin, New Vistas (COSTA E., GESSAG. L. & SaNDLEn M., eds.) Adv. hiochein. PSVchopharmac. 11, 153-162. Raven Press. New York.
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HEny F.,KOUEK E. & GLOWINSKI J. (1972) Bruin Res. 43, 445-465. JACOBS B. L., EUBANKS E. E. & WISE W. D. (1974) Neuro/ ' / J O ; . / ~ I O C . O / O ( /13, ? . 575-583. Joi.v.t:i M. (1972) Ergebn. Physiol. 64, 166307. KLFIND. C. & WELLEKJ. L. (1972) Science, N.Y 177, 532-533. KNOTTP. J. & CURZONG. (1972) Nature, Lond. 239, 452453. B. K.: WURTMAN R. J. & MUNRO LrPPSETT D., MADRAS H. N. (1973) Life Sci. 12, 57-64. W. R. & MAIKEL MILLERF. P.; COXR. H., JR., SNODCRASS R . P. (1970) Biochem. Pharmac. 19, 435--442. PEREZ-CRUET J., TACLIAMONTE A,, TAGLIAMONTE P. & GESSAG. L. (1972) Li&e Sci. 11, 31-39. PETITJEAN F., S A K AK., ~ BLOVDAUX C. & JOUVET M. (1975) Brain Res. 88, 439453. QUAYW. B. (1964) Progr. Brain Res. 8, 61-63. QUAYW. B. (1968) Arn. J . Physio[. 215, 1448-1453. A., LOVELLR. A.. FREEDMAN D. X.. RECHTSCHAFFEN WHITEHEAD w. E. & ALDRICH, M . (1973) i n So-otonin and Belzaz;ior (BARCHAS J. & USDIN E., eds.) pp. 401418. Academic Press, New York. SNYDERS. H., ZWEICM., AXELRODJ. & FISCHER J. E. (1965) Proc. natn. Acad. Sci. U.S.A. 53, 301-305. J.-F. (1974) Brain Rrs. 72, STEIN D., JOUVETM. & PUJOL 366365. TACLIAMONTE A., BIGGIOG., VARCIUL. & GESSAG. L. (1973) J . Neurochem. 20, 909-912.
RESPONSE OF RAT BRAIN INDOLES AND MOTOR ACTIVITY TO SHORT LIGHT-DARK CYCLES' GEORGIANA HANSELMA"and A. A. BORBELY' Institute of Pharmacology, University of Zurich, Zurich, Switzerland (Received 17 July 1975. Accepted 20 October 1975)
Abstract-Since the vigilance states of the rat can be largely controlled by one hour light-one hour dark cycles, we investigated the effect of this photoperiod on the rat brain indoles and motor activity. Groups of 9 rats were killed 15-20 min or 45-50 min after the onset of the 1-hour light or 1-hour dark period. Serotonin (5HT) and 5-hydroxyindoleaceticacid were determined fluorometrically in cortex, hypothalamus and brain stem. Tryptophan was determined fluorometrically in serum, cortex and hypothalamus. Cortical tryptophan was highest at the end of the dark period, whereas cortical 5-HT and 5-hydroxyindoleacetic.acidwere highest at the beginning of the light period. Motor activity was high during darkness and low during light. When the biochemical results were compared to the motor activity records of the individual animals, the decrease in motor activity at the onset of light correlated significantly with the cortical 5-hydroxyindoleacetic acid/S-HT ratio in the animals killed at the beginning of the light period. The results indicate a rapid response of the cortical indoles to the onset of light and support the hypothesis that the induction of sleep is related to the brain indole metabolism.
THEVIGILANCE states as well as the motor and consummatory behaviour of the rat can be entrained by 1 hour light-1 hour dark cycles (LD 1 :1) (BORB~LY & HUSTON,1974; E ~ R B B L Y et al., 1975). Slow wave sleep is the sleep state which is most immediately affected by a change in the lighting conditions; it is rapidly increased during the 1-hour light period and rapidly reduced during the 1-hour dark period. The enhancement of sieep by light is most prominent if the light intensity is high, and if its influence occurs during the circadian phase of minimum sleep (BORR ~ L Y . 1975). On the basis of these results we were interested to test the hypothesis proposed by JOUVET (1972) that serotonergic mechanisms play a central role in the induction of sleep. The hypothesis rests largely on the observations that destruction of serotonin (5-HTj containing neurons of the raphe nuclei, or reduction of 5-HT synthesis by p-chlorophenylalanine @CPA), significantly reduce sleep in the cat. The generality of these findings has been challenged by RECHTSCHAFFEN et al. (1973) who reported inconclusive effects of pCPA on sleep in the rat. However, it is doubtful whether the administration of pCPA consti-
* Preliminary reports of this work were presented at the Second International Congress of Sleep Research, 1975, and at the First European Neurosciences Meeting, 1975. To whom all correspondence should be sent. Abbreuiarions used: 5-HIAA, 5-hydroxyindoleaceticacid; 5-HT, 5-hydroxytryptamine; NAT, serotonin-N-acetyltransferase; OPT, o-phthalaldehyde; pCPA, p-chlorophenylalanine; TRY, tryptophan; LD 1 : 1, 1 h light-l h dark; EL, EL, beginning or end of light period; BD, ED, beginning or end of dark period. 95 I
tutes a n optimal approach for studying relations between brain 5-HT and sleep: (1) The use of 6-fluorotryptophan, another tryptophan hydroxylase inhibitor, reduced motor activity in the rat, whereas pCPA produced hyperactivity (JACOBS et al., 1974); (2) The effects of pCPA are not restricted to the central neret d., 1973) nor strictly vous system (RECHTSCHAFFEN to an inhibition of tryptophan hydroxylase ( I L I i L L m ef al., 1970); (3) The long-term effects of this agent may induce compensatory mechanisms, since a gradual recovery of sleep may occur despite continuously low cerebral 5-HT levels (DEMENT, 1969). The LD 1 :1 schedule appeared to be well suited for reliably inducing sleep by a natural variable and thereby testing its effect on brain indoles. O n the basis of the 5-HT hypothesis it could be expected that the induction of sleep by light is accompanied or preceded by changes in brain indole metabolism. We therefore measured the levels of tryptophan (TRY), 5-HT, and 5-hydroxyindoleacetic acid (5-HIAA) in four parts of the brain in animals which were killed at the beginning or the end of the light period (BL or EL), or at the beginning or the end of the 'dark (BD or ED). Since we did not want to handle the animals prior to the time of death, we did not inject labelled 5-HT precursors to estimate 5-HT turnover. Motor activity was recorded for each animal as an indicator of the vigilance state. MATERIALS AND METHODS Materials. We obtained 5-HT-creatinine sulphatc from
Hoffmann-LaRoche, Basel; 5-HIAA. HCI, L-cysteine and o-phthalaldehyde (OPT) from Fluka AG, Buchs; L-TRY IIom E. Merck, Darmstadt and Norharman from Sigma,
952
GEORCIANA HANSELMA"and A. A. BORBBLY
St. Louis. All reagents used in the biochemical analyses were of fluorometric grade or of the highest grade avail150 able. Environmental conditions and schedule. Thirty-six adult 100 male Sprague-Dawley rats of the SIV-50strain weighing 25C300 g were used. The animals were housed individually $ 50 in transparent plastic bins, had unlimited access to food and water, and were maintained in a separate room with 5 0 g 200 temperature regulated at 22 2°C. They were exposed to 0 an LD 12:12 photoperiod (light from loo0 to 2200) for 5 150 at least 1 week before being exposed to the LD 1 :1 cycles. 3 Twenty-two hours before killing the cages were placed in 100 a special chamber with an LD 1 : I photoperiod. For the BL and EL groups the first hour of this schedule was light, 50 for the BD and ED groups the first hour was dark. This schedule was used so that all animals would be killed as 0 6 7 9 nearly as possible at the same point in their circadian cycle. Time of day, h Animals were killed within the first day of exposure to the LD 1 : 1 cycles since previous results (BORBBLY & Hus- FIG.1. Average motor activity values of the four experTON, 1974; BORBBLY et al., 1975) had shown that animals imental groups plotted for successive 5-min periods during are quickly entrained to the short light cycles and that the last 2 h before death. Motor activity is expressed as by the second day a drift in the circadian activity is already a percentage of the average value between o400 and 0800. apparent. The cages in the LD 1 :1 chamber rested on Letters indicate the time of death: BL, beginning of light; force recorders (BORBELYet al., 1975) whose integrated out- EL, end of light; BD, beginning of dark: ED, end of dark. put was continuously recorded on analogue magnetic tape Shading indicates the dark period. All points are plotted as a measure of motor activity.. Animals were killed in at the beginning of the 5-min period, thus, the points plotgroups of three at 08150820 (BL or BD groups) or at ted at 0700 and 0800 represent the values for 0 7 W 7 0 5 08450850 (EL or ED groups). This hour was at the end and 0 8 W 8 0 5 respectively. For each g o u p n = 9. of the circadian active period during which the entrainment of the sleep cycles by the photoperiod is optimal (BORB~LY et al., 1975). tings for excitation and emission were 352 nm and 473 nm Selection and preservation of tissue. Cervical blood was respectively. collected, centrifuged at 20,000 g for 20 min and the serum Data analyses and statistics. The motor activity records decanted into plastic vials and stored at -20°C until pro- on analogue magnetic tape were converted 10 a digital cessed for total TRY. The brains were rapidly removed form (AD conversion rate approx 30 Hz) and integrated and dissected on an iced platform. Each brain was halved for successive periods of 5 min. For each animal the averalong the midline then sectioned into cortex, brain stem age motor activity between 0400 and 0800 was computed and hypothalamus according to GLOWINSKI & IVERSeN and defmed as 100%. The percentage values at various (1966). The fiontal cortex was separated from the rest of time periods before death were used for further computhe cortex according to QUAY(1968). The pineal glands tation (Fig. 1).The two-tailed Wilcoxon rank-sum test was were removed for separate 5-HT analysis, but since no used to determine significant differences. variation was seen among the four groups thcse results will not be further discussed. The cerebellum was disRESULTS carded. All sections were immediately frozen on solid C02, wrapped in foil and stored at -20°C until analysed for Effect of lightdark cycles on motor activity. For 5-HTand 5-HIAA or for TRY. each of the four groups motor activity was enhanced Biochemical analyses. Brain regions were analysed in during the dark and reduced during the light (Fig. four groups (BL, EL, BD, and ED) of eight. For each brain 1). This finding confirms the results of previous experregion. the four groups were analysed in succession so that iments ( B ~ R B ~& LY HUSTON,1974; BORBBLYet al., not more than 6 days elapsed between the processing of 1975). The motor activity records of the individual the first and the last groups. 5-HT and 5-HIAA were deteranimals showed considerable variability, whereas the mined in one half of the cortex, hypothalamus, and brain stem after butanol-heptane extraction according to the group averages showed only minor variation. Effect of light-dark cycles on brain indoles. The most fluorometric method of CURZON& GREEN (1970). Both halves of the frontal cortex were used for the determination striking results were obtained in the cortex (Fig. 2). of 5-HT and 5-HIAA. Each sample was processed in tripli- Here the TRY levels were highest in the ED group cate: one probe, one internal standard and one tissue and the 5-HT, 5-HIAA, and the ratio 5-HTAA/S-HT blank. External standards were also used for comparison (an estimate of indole turnover) were highest in the of fluorescence from day to day. Fluorescence was read BL group. For each of these four parameters the difon a Farrand spectrofluorometer with excitation waveferences were significant against each of the other length set at 360 nm and emission wavelength at 470 nm three groups. The measurements made in the serum (uncorrected). TRY was determined in the serum, cortex and hypothalamus fluorometrically according to DENCKLA and the other brain regions are shown in Table 1. & DEWEY, (1967). Again all samples were processed in trip- The levels of 5-HT were also highest in the BL group licate and both internal (L-TRY) and external (norharman) in both hypothalamus and brain stem, though in standards were used. Uncorrected spectrofluorometer set- neither of these regions was the difference significant
953
lndole and motor activity response to short photoperiod
activity. No significant correlations could be found between the percentage of motor activity of the individual animals on the one hand, and the concentrations of TRY, 5-HT or 5-HIAA on the other. The only significant correlation was seen in the BL group between the percentage decrease in motor activity for the last two 5-min periods before death and the ratio 5-HIAA/5-HT in the cortex. ( P < 0.05; rank correlation test). DISCUSSION
It has been shown in previous studies that the concentration of 5-HT in various brain regions is higher during the 12-h light phase than during the 12-h dark period (QUAY,1968; HERYet al., 1972). However, in Ih FIG.2. Average cortical indole levels for each of the four those studies the influence of light per se cannot be experimental groups defined in Fig. 1. The cortex [minus differentiated from the effect of an endogenous circathe frontal area (QUAY,1968)] was sectioned according dian rhythm on the brain indoles. In the pineal gland, & IVERSEN (1966). One half was analysed for example, the latter effect has been shown to persist to GLOWINSKI & DEWEY, 1967) and the even during continuous darkness or in blinded rats for tryptophan (TRY) (DENCKLA & GREEN, 1970). other half for 5-HT and 5-HIAA (CURZON (QUAY,1964; SNYDER et al., 1965). In the present exShading indicates the dark period. The value shown for periments the influence of a circadian variable was 5-HIAA at BD represents the average of 7 animals; For all other bars n = 8. Brackets indicate SEM. Asterisk minimized by killing the animals during the same indicates significant difference compared to each of the hour of the day. Under these standardized conditions other groups; TRY, P < 0.02: 5-HT and 5-HIAA, P < 0.01 a rapid rise of 5-HT levels in the various brain regions, particularly in the cortex, followed the onset (Wilcoxon rank-sum test). of light. Compared to the end of the dark period, cortical 5-HT increased by more than 60% and with respect to all other groups. The levels of TRY 5-HIAA by 1850/,, whereas TRY decreased by more in the serum and the hypothalamus were highest in than 35% (Fig. 2). These findings indicate that 5-HT the EL group rather than in the ED group as in the turnover is massively increased during the 15 min of cortex. Again these differences were significant only exposure to light. The fact that the light-induced changes of indole when compared to the lowest levels. 5-HTAA and the ratio S-HTAA/S-HT showed much greater variability levels were most prominent and consistent in the corfrom region to region so that it is not possible to tex is in accordance with previous reports. Thus H ~ R Y provide a summarizing statement for these results. et al., (1972) found that the high rate of conversion Correlation between indole concentration and motor of C3H]TRY to C3H]5-HT during the light period was TABLE I . AVERAGEVALUES OF INDOLE LEVELS AND THE AVERAGE OF THE
INDIVIDUAL RATIOS FOR SERUM AND THREE BRAIN REGIONS ~
~~
TRY Serum
RL
EL Hypothalamus
Brainstem
Frontal Cortex
BD ED BL EL ED ED BL EL RD
118 k 20 174 f 22*d 152 f 19 115 k 9 75 f 6 85 k 8*c,d 56k6. 60 f 5
Not measured
3.1 2 0.3 2.8 f 0.2 2.2 f 0.2*a 3.0 0 2 4.3 f 0.2 4.3 f 0.2 4.0 +_ 0 2
t
ED
BL EL BD ED
5-HT
Not measured
3.4 f 0.3 3.2 0.3 3-6 f 0.4 2.9 f 0.3
5-HIAA
4.5 f 0.5 3.7 f 0.5 4.4 k 0.5 3.4 f 0.2 3.6 0 3 5.0 i: 0 8 (7) 3.8 +_ 0.2
t
*
3.7 f 0.3 3.4 0.2 3.9 & 0.7 3.3 f 0.3
5-HJAAp-HT ~~~
5-HIA.4/5-HT
1.4 f 0 1 1.3 i 0 2 2.1 & 0.3 1.1 i 04*b.c 0 9 f 0.5 1.2 f 0.2 (7) 1.0 +_ 0 1
t 1.1 f 0.1 1.1 f 0.1
1.0 & 0 1 1.2 f 0.1
All values are expressed as nmol/ml serum or nmol/g tissue i SEM. Group designation is the same as in Fig. 1. Except where indicated by the number in parentheses, n = 8. * Significant at least at the level P < 0.05; a-against all other groups; b-against BL; c--against BD; d-against ED. f Samples lost in processing.
954
GEORCIANA HANSELMANN and A. A. BORBELY
most noticeable in the cortex, and BLONDAUX et al. In all four experimental groups the average motor (1975), when measuring the changes in injected activity was high during darkness and low during C3H]TRY metabolism in cats with isthmic lesions, light, a result which confirms our previous findings also found the greatest increase in conversion rates (BORBBLY & HUSTON,1974). However, when the in the cortex. Since cortical indole levels are lower records of the individual animals were compared, no than in the other brain regions tested, light-induced correlation was seen between the biochemical and bechanges may be more readily apparent in the cortex. havioural data. It is not very surprising that the Moreover, in the hypothalamus, a substantial amount motor activity, as determined for successive 5-min of 5-methoxytryptamine, which has the same fluores- periods by our recording system, shows no significant cence characteristics as 5-HT when reacted with OPT correlation with regional indole levels at the time of (GREEN et al., 1973), may also serve to mask the fluc- death. Even if a relationship exists between indole tuations of 5-HTin this region. levels in specific parts of the brain and certain aspects The rate of 5-HT synthesis in the brain depends of behaviour, it is difficult to record the appropriate on both the availability of the substrate TRY and behaviour during the pertinent period of time, and the activity of the hydroxylating enzyme (tryptophan then select the relevant parts of the brain for the 5-mono-oxygenase, tryptophan 5-hydroxylase, EC determination of indoles. On the basis of these nega1.14.16.4). Some workers have suggested that brain tive findings we can, therefore, by no means exclude TRY concentration is the rate-limiting factor in the the existence of a correlation between indole levels formation of 5-HT (e.g. FERNSIROM & WURTMAN, and behaviour. 1971; TACLIAMONTE et al., 1973), whereas others have During the LD 1 :1 schedule, the onset of light inproposed that only TRY that has been newly taken creased cortical 5-HT and 5-HIAA, reduced motor up into the brain is available for 5-HT synthesis activity, and induced slow wave sleep (BORBELYet (HERYet al., 1972; HAMONet al., 1974). In our study a]., 1975). Towards the end of the light period, when neither cortical nor hypothalamic TRY levels changed the animal was usually inactive and exhibited sleep, in parallel with the 5-HT levels, indicating that the the cortical level of the two indoles did not differ substrate concentration does not constitute the only from the values during darkness. These findings suprate-limiting factor of 5-HT synthesis. Due to the brev- port the hypothesis that the induction of sleep, but ity of the LD cycles employed, we cannot rule out not the maintenance of sleep, depends on an increase the possibility that a process initiated during the dark of 5-HT metabolism (JOUVET, 1972; PETITJEAN et a[., period, resulting in high cortical TRY levels at ED, 1975). The fact that the ratio 5-HIAA/S-HT was corcontributes substantially to the high levels of 5-HT related with the reduction of motor activity after the and 5-HIAA during BL. However, light itself could onset of light lends further support to that assumpaffect cerebral 5-HT levels by altering the activity of tion. Catecholamines, which have not been detertryptophan 5-hydroxylase, or the catabolizing enzymes mined in the present study, may play a major role monoamine oxidase (amine: oxygen oxidoreductase in the induction of wakefulness and motor activity (deaminating) (flavin-containing),EC 1.4.3.4) or sero- by darkness and may in turn influence the serotonertonin N-acetyltransferase (EC 2.3.1.5: NAT). The gic system (STEINet al., 1974). effects of short light periods on the latter enzyme in On the other hand, light may influence brain inthe pineal gland during a critical period in the dark doles and behaviour by largely independent mechare well documented (DEGUCHI & AXELROD, 1972; anisms. Preliminary results indicate that LD-induced KLEIN& WELLER,1972), though it is not known changes of motor activity during the LD 1:1 schedule whether NAT is relevant for the metabolism of extra- are not abolished after administration of p-chloropineal 5-HT (ELLISON et al., 1972). Nevertheless, the phenylalanine (300 mg/kg, intraperitoneally ;BORBELY, light-induced effects on the activity of a rate-limiting unpublished observation). In view of these findings, enzyme in the pineal gland (AXELROD, 1974) may and of the conflicting evidence described in the introserve as a model of the mechanisms affecting extra- duction, the original 5-HT hypothesis of sleep may pineal indole metabolism. have to be modified. An increase of 5-HT metabolism We measured serum total TRY primarily as an ad- may well be a sufficient, but not a necessary condition ditional control, since this parameter is more indica- for the induction of sleep. tive of eating behaviour than of the amount of TRY available to the brain (PEREZ-CRUET et al., 1972; KNOTT& CURZON1972; LIPSEIT et al., 1973). Previous work from our laboratory (BORBBLY & HUSTON, 1974) showed that feeding behaviour was not Acknowledgements-The authors would like to thank Prof. DT.W. L~CHTENSTE~GER for making his laboratory availentrained as readily by the short LD cycles as motor able to us, F. HEFTIfor his many helpful suggestions conactivity. Toward the end of the active period of the cerning the biochemical determinations and E. PICHLER, circadian cycle the feeding behaviour is high enough M. HINKKANEN and M. HELMEYfor technical assistance so that it may continue into the light period. The and help in preparing the manuscript. This work was aided high serum TRY at EL may thus merely represent by support from the Swiss National Science Foundation, Grant Nos. 3.2120.73 and 3.5350.71. a delay of nutrient absorption from the gut.
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