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Brain Research 740 Ž1996. 245–252 Research report Effects of tryptophan andror acute running on extracellular 5-HT and 5-HIAA levels in the hippocampus of food-deprived rats Romain Meeusen a a, ) , Katrien Thorre´ b, Francis Chaouloff d , Sophie Sarre b, Kenny De Meirleir a , Guy Ebinger c , Yvette Michotte b Departments of Human Physiology and Sports Medicine, Vrije UniÕersiteit Brussel, Laarbeeklaan 101, B-1090 Brussels, Belgium b Departments of Pharmaceutical Chemistry and Drug Analysis, Vrije UniÕersiteit Brussel, Brussels, Belgium c Department of Neurology, Academic Hospital, Vrije UniÕersiteit Brussel, Brussels, Belgium d Laboratoire Genetique du Stress, INSERM CJF 94-05, UniÕersite´ de Bordeaux, Bordeaux, France ´ ´ Accepted 9 July 1996 Abstract The present microdialysis study has examined whether exercise-elicited increases in brain tryptophan availability Žand in turn 5-HT synthesis. alter 5-HT release in the hippocampus of food-deprived rats. To this end, we compared the respective effects of acute exercise, administration of tryptophan, and the combination of both treatments, upon extracellular 5-HT and 5-hydroxyindoleacetic acid Ž5-HIAA. levels. All rats were trained to run on a treadmill before implantation of the microdialysis probe and 24 h of food deprivation. Acute exercise Ž12 mrmin for 1 h. increased in a time-dependent manner extracellular 5-HT levels Žmaximal increase: 47%., these levels returning to their baseline levels within the first hour of the recovery period. Besides, exercise-induced increases in extracellular 5-HIAA levels did not reach significance. Acute administration of a tryptophan dose Ž50 mgrkg i.p.. that increased extracellular 5-HIAA Žbut not 5-HT. levels in fed rats, increased within 60 min extracellular 5-HT levels Žmaximal increase: 55%. in food-deprived rats. Whereas 5-HT levels returned toward their baseline levels within the 160 min that followed tryptophan administration, extracellular 5-HIAA levels rose throughout the experiment Žmaximal increase: 75%.. Lastly, treatment with tryptophan Ž60 min beforehand. before acute exercise led to marked increases in extracellular 5-HT and 5-HIAA levels Žmaximal increases: 100% and 83%, respectively. throughout the 240 min that followed tryptophan administration. This study indicates that exercise stimulates 5-HT release in the hippocampus of fasted rats, and that a pretreatment with tryptophan Žat a dose increasing extracellular 5-HT levels. amplifies exercise-induced 5-HT release. Keywords: Microdialysis; Hippocampus; Extracellular 5-HT; Extracellular 5-HIAA; Tryptophan administration; Exercise; Food deprivation 1. Introduction Among animal models of psychiatric disorders, starvation-induced hyperactivity has received a great deal of attention because it may be endowed with features observed in anorexia nervosa w2,3,19,38x andror obsessivecompulsive disorder w1x. This model is based on the observation that fed rats given access to running wheels progressively increase their activity; however, if food-restricted, the rats develop high running wheel activity at the expand of feeding Žthereby leading to marked weight losses and possibly death.. A great number of studies has investigated the transmitters putatively responsible for this ‘runners high’, especially the opioidergic w19x, catecholaminergic and serotonergic systems w38x. As far as serotonergic systems are concerned, increases in hypothalamus 5-hydroxy) Corresponding author. Fax: q32 Ž2. 477-4607. tryptamine Ž5-HT. turnover have been found in rats undergoing the starvation-induced hyperactivity paradigm w38x. In keeping with the observation that chronic inhibition of 5-HT synthesis by para-chlorophenylalanine ŽPCPA. w1x and repeated administration of tryptophan Žthe precursor of 5-HT. w2x respectively exacerbates and diminuates the development of starvation-induced hyperactivity, it has been proposed that serotonergic systems may play a key role in this model w1,2,38x. This suggestion is reinforced by the early findings that food-deprivation w11,30x and acute running in trained animals w11,12x both increase in a time-dependent manner brain tryptophan availability Žthrough lipolysis-mediated changes in the blood concentration of the so-called free tryptophan portion.. Moreover, the combination of both treatments Ži.e. acute running following food deprivation. leads to marked increases in brain tryptophan that suggest synergistic influences of these two lipolytic situations w11x. 0006-8993r96r$15.00 Copyright q 1996 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 0 8 7 2 - 4 246 R. Meeusen et al.r Brain Research 740 (1996) 245–252 In keeping with the early observation that the activity of tryptophan hydroxylase, the rate-limiting enzyme in 5-HT biosynthesis, is only partly saturated under basal conditions w7x, it is logical to observe that food deprivation, acute exercise, or the combination of both increase 5-HT synthesis Žand metabolism. w11x. However, the extent to which precursor-induced changes in synthesis have functional consequences on 5-HT release has been the matter of debate w26,31x, including in exercise models w8,36x. On the one hand, treatments that decrease brain tryptophan availability andror 5-HT synthesis Žsuch as PCPA. decrease evoked release of 5-HT w22,34,40x. On the other hand, in vivo experiments using push-pull, voltammetry, and more recently microdialysis techniques have found that tryptophan administration increases w6,44,49x or does not affect w16,34,42x extracellular 5-HT levels. The use of superfused brain slices has also led to contradictory results w18,40x, whereas behavioural experiments have suggested that tryptophan, at the opposite of 5-hydroxytryptophan, does not promote the so-called 5-HT syndrome w25x. At the opposite to what can be observed under basal conditions, there is a general agreement that tryptophan administration increases release of 5-HT under depolarizing conditions Žw18,42x; but see w40x. or when 5-HT metabolism into 5-HIAA is prevented w25,18,34x. Taken together, these results have suggested that under basal conditions Ži.e. without any electrical stimulation of serotonergic neurones: see below., increases in tryptophan lead to increases in 5-HT synthesis that are immediately buffered by accelerated metabolism into 5-hydroxyindoleacetic acid Ž5HIAA. andror intraneuronal storage of 5-HT w31x. In addition to the aforementioned situations, there are Žless specific. events during which administration of tryptophan also leads to increases in 5-HT release. Hence, stressful situations such as immobilisation w29x, streptozotocin-diabetes w5x, or food deprivation w41x allow the recognition of increases in extracellular 5-HT levels upon exogenous tryptophan administration. In keeping with this last observation, it can thus be suggested that exercise-induced increases in brain tryptophan availability Žand in turn 5-HT synthesis. lead to a marked release of 5-HT in food-deprived rats. If true, this could suggest that starvation-induced hyperactivity leads to profound changes in 5-HT release, an event which would underlie the reciprocal relationship that has been observed, through pharmacological approaches, between central serotonergic activity and vulnerability to starvation-induced hyperactivity. Taking into account this uncertainty, we have measured, by means of microdialysis, the influence of one acute running session upon extracellular 5-HT and 5-HIAA levels in the hippocampus of food-deprived rats. To get an insight into the metabolic effects of running Žrather than novelty stress-related influences., rats were trained to run on the treadmill before acute exercise, and only those which were not reluctant to run during the training sessions were kept for the final experiments. Moreover, be- cause previous studies have suggested that tryptophan administration may diminish through feedback mechanisms cell firing of serotonergic neurones w45x, we have also compared the effects of tryptophan and tryptophan plus exercise upon extracellular 5-HT and 5-HIAA levels. 2. Materials and methods 2.1. Animals Male albino Wistar rats Ž200–300 g. were used housed two per cage. When treadmill adaptation started Žsee further., the animals were adapted to individualised housing. The animals were kept on a 12:12 h lightrdark cycle Žlights on at 06.00 h. and were fed a standard diet with free access to food and water. All experiments started in the morning between 09.00 and 09.30 h. The protocols were carried out according to the national rules on animal experiments and were approved by the Ethics Committee of the Faculty of Medicine and Pharmacy of the Vrije Universiteit Brussel. 2.2. Exercise training Animals used for the exercise experiments Žsee below. were placed on the treadmill four to five times in order to adapt them to the experimental situation. Each time they exercised for 15 to 30 min in order to familiarize them with running. Animals reluctant to run during this testing period, were not used in the experiment. 2.3. Surgery and intrahippocampal dialysis Animals were anaesthetised with a mixture of ketamine and diazepam Ž50 mgrkg: 5 mgrkg i.p.. and placed on a stereotaxic frame. The skull was exposed and a guide cannula ŽCMA Microdialysis, Stockholm, Sweden. was implanted through a bore hole in the left ventral hippocampus Ž x: y4.6, y: y5.6, z: q4.6. according to the coordinates described by Paxinos and Watson w37x. The animals were allowed to recover from surgery for two days, then the microdialysis probe with a membrane length of 3 mm ŽCMA Microdialysis. was inserted and the animals were placed on the treadmill. The microdialysis probes were connected to a microinfusion pump ŽBAS, USA. and perfused with a modified Ringer’s solution Ž147.5 mmolrl Naq, 4 mmolrl Kq, 2.2 mmolrl Ca2q, 153.7 mmolrl Cly. at a constant flow rate of 1 m lrmin. From that moment the rats were food deprived, and stayed on the treadmill until the end of the experiment. The sampling of the dialysates began 24 h after probe implantation. The microdialysis samples were collected every 20 min. The day of the experiment samples were collected for at least 2 h Žsix samples. to verify stable basal conditions before one of the following experimental procedures was started. R. Meeusen et al.r Brain Research 740 (1996) 245–252 2.4. Experimental procedures For the exercise experiments, after at least 2 h of baseline collections, the animals ran for 60 min at a moderate speed Ž12 mrmin. on a motor driven horizontal treadmill ŽOmnitech electronics, Columbus, Ohio, USA.. An adjustment was made to the treadmill in order to attach the counter-balance arm of the microdialysis system. Sampling continued during recovery from exercise Ž120 min., total collection time being at least 300 min. In another series of experiments, after at least 2 h of baseline collections, Žresting. animals received either saline or L-tryptophan Ž50 mgrkg.. Sampling continued during 140 min Ž7 collection samples.. Lastly, one experiment was aimed at measuring the effects of tryptophan in exercising animals. To this end, again after at least 2 h of baseline collections, rats were pretreated Ži.p.. either with saline or L-tryptophan Ž50 mgrkg.. Sixty min after this injection, the animals started running during 60 min at a moderate speed Ž12 mrmin.. Sampling continued for 2 h following exercise, total collection time being at least 360 min. 2.5. Analytical procedures The microbore liquid chromatography with dual electrochemical detection assay that was used for the determination of 5-HT and 5-HIAA has previously been described 247 by Sarre et al. w39x. In summary, this system consists of a LC-pump connected to a Sepstik w splitter kit ŽB.A.S., USA., to obtain a microflow over a microbore column Ž100 = 1 mm i.d., C 18 , 3 m m.. This column is coupled directly to a low dispersion injection valve with a 10 m l loop. The connection with the electrochemical cell is made by fused silica Ž50 m m i.d... The electrochemical cell of the detector ŽLC 4B, BAS, USA. consists of two working electrodes positioned in parallel in such a way that separate potentials Ž625 and 500 mV. can be set versus the AgrAgCl reference electrode. The cell volume is reduced, using a 16 m m gasket. A dual channel integration computer program is used to integrate the chromatograms ŽIntegration Pack w Kontron, Milan, Italy.. The mobile phase consisted of 95% acetate-citrate buffer Ž0.1 mM sodium acetate, 20 mM citric acid, 0.1 mM octanesulfonic acid, 0.1 mM Na 2 EDTA and 1 mM dibutylamine, pH 4.2. and 5% MeOH. The flow rate was set at 0.7 mlrmin, yielding 58 m lrmin over the microbore column. The limit of detection of the assay was 0.6 fmol on column. 2.6. Statistics 5-HT could be measured in the dialysates without the use of a reuptake inhibitor added to the perfusion fluid. The extracellular concentrations of 5-HT and 5-HIAA in the dialysates, not corrected for in vivo recovery, are expressed as percentages of the baseline value Ž8.5 " 1.5 Fig. 1. Respective effects of exercise Ž12 mrmin. on extracellular 5-HT Župper panel. and 5-HIAA Žlower panel. levels in the hippocampus of the food-deprived rat. Values are the mean " S.E.M. of five animals. Before the onset of exercise at least six samples Ž120 min. were collected Ždata of three collections are shown.. ) P - 0.05 ŽANOVA for repeated measures with Fischer PLSD. for the differences with the last pre-exercise sample. 248 R. Meeusen et al.r Brain Research 740 (1996) 245–252 fmolr20 ml and 9 " 2.9 pmolr20 ml for 5-HT and 5HIAA in 24 animals.. The latter was the average concentration of the dialysate 5-HT or 5-HIAA levels that were obtained during the first 120 min of collection. Data, given as means " S.E.M., were analysed either through repeated measures ANOVA followed, if significant, by Fischer PLSD-test Žexercise., or through two-way ANOVA for repeated measures Žtreatment= time. Žtryptophan, tryptophan and exercise. followed, if significant, by Fischer PLSD-tests. 3. Results 3.1. Effects of acute exercise on extracellular 5-HT and 5-HIAA leÕels in the hippocampus of 24-h food-depriÕed rats As shown in Fig. 1, a 60-min exercise session at 12 mrmin increased in a time-dependent manner extracellular 5-HT levels, an effect that could still be observed during the first 20-min recovery sample. Besides, exercise-induced elevation in 5-HIAA levels, which yielded a maximal increase of 26% at the end of exercise, did not reach statistical significance. 3.2. Effects of tryptophan on extracellular 5-HT and 5HIAA leÕels in the hippocampus of 24-h food-depriÕed rats As shown in Fig. 2, a 50 mgrkg dose of tryptophan increased extracellular 5-HT levels Žmaximal increase: 55%., this effect being significant in the second, third and fourth dialysate samples that followed tryptophan administration. On the other hand, the latter treatment increased 5-HIAA levels throughout the 160 min of experiment Žmaximal increase: 75% at the end of the experiment., this change being already observed in the second dialysate sample that followed tryptophan administration ŽFig. 2.. By comparison, administration of a 50 mgrkg dose of tryptophan to fed rats did not affect extracellular 5-HT levels, although it increased extracellular 5-HIAA levels Žmaximal increase: 90%. in a time-dependent manner similar to that observed in food-deprived rats Ždata not shown.. 3.3. Effects of tryptophan and exercise on extracellular 5-HT and 5-HIAA leÕels in the hippocampus of 24-h food-depriÕed rats Administration of tryptophan Ž50 mgrkg. increased extracellular 5-HT and 5-HIAA levels, compared with saline administration, including during the exercise session Fig. 2. Respective effects of saline and tryptophan Ž50 mgrkg. on extracellular 5-HT Župper panel. and 5-HIAA Žlower panel. levels in the hippocampus of the food-deprived rat. Values are the mean " S.E.M. of four animals. Before injection Žarrow. at least six samples Ž120 min. were collected Ždata of three collections are shown.. Two-way ANOVA Žtreatment= time.: ) significant different from baseline, a significant difference between saline and tryptophan Ž P - 0.05.. R. Meeusen et al.r Brain Research 740 (1996) 245–252 249 Fig. 3. Respective effects of combined tryptophan Ž50 mgrkg. and exercise Ž12 mrmin. on extracellular 5-HT Župper panel. and 5-HIAA Žlower panel. levels in the hippocampus of the food-deprived rat. Before injection Žarrow. at least six samples Ž120 min. were collected Ždata of three collections are shown.. Values are the mean " S.E.M. of 5–6 animals. Two-way ANOVA Žtreatment= time.: ) significant different from baseline, a significant difference between saline and tryptophan Ž P - 0.05.. and the recovery period ŽFig. 3.. Actually, 5-HT levels reached a plateau Žmaximal increase: 100%. at the end of exercise whereas 5-HIAA levels reached a plateau Žmaximal increase: 80%. during exercise recovery ŽFig. 3.. 4. Discussion On the basis of exercise-induced increases in tryptophan and 5-HIAA levels in whole brain, brain regions, or cerebrospinal fluid Žfor reviews: w8,36x., it has been suggested that such a paradigm increases brain 5-HT synthesis and metabolism. Actually, experiments aimed at determining the mechanisms underlying exercise-induced increases in central tryptophan levels have shown that lipolysis plays a key role w11,12x. Thus, because free fatty acids displace tryptophan from its binding to albumin w35x, lipolysis-induced increases in circulating free fatty acids increase the free Žas opposed to the albumin-bound. portion of tryptophan w11,12x. A similar picture may be observed in fooddeprived rats w11,30x. Exercise having little effects on those circulating amino acids competing with tryptophan for entry into the brain w4,12x, lipolysis-induced increases in circulating free tryptophan lead to parallel increases in brain tryptophan and in turn 5-HT synthesisrmetabolism w11,12x. In keeping with the observation that Ži. the aforementioned changes take place in trained rats w11,12x, and Žii. human volunteers performing exercise display also increases in circulating free fatty acids and in free tryptophan w4,14,21x, this suggests that it is exercise per se, rather than the psychological aspects of this stress-like procedure, that underlies the aforementioned changes in central serotonergic systems. However, whether exerciseinduced increases in 5-HT synthesis are associated with effective changes in 5-HT release has been the matter of debate w8,9x. As indicated in Section 1, tryptophan-induced increases in 5-HT synthesis in undisturbed animals may lead to increased metabolism into 5-HIAA without functional consequences on transmission w16,18,34,42x. Whether this lack of direct relationship is due to the opposite circadian fluctuations between synthesis and release of 5-HT w26x and the circadian-dependent relationship between 5-HT release and arousal w50x is worth mentioning. On the other hand, it has been suggested that synthesis of 5-HT, which increases 5-HT release during stressful events, serves only 250 R. Meeusen et al.r Brain Research 740 (1996) 245–252 to replenish serotonergic neurones following depolarisation-induced release of the amine w9x. The latter hypothesis would thus suggests that synthesis of 5-HT would only serve to avoid depletion of the amine in serotonergic neurones. Past studies have suggested, through indirect indices, that exercise may increase 5-HT release. Thus, a nonselective 5-HT2A receptor antagonist blocked exercise-induced prolactin release w15x, whereas the hypophagic effect of the 5-HT uptake blockerr5-HT releaser dextrofenfluramine proved more consistent in exercising animals w10x. Since the completion of these studies, more direct analyses have been conducted with microdialysis w23,24,51x. In one study, it was found that extracellular 5-HT levels in the ventral horn of the spinal cord did not change during exercise, but began to drop on cessation of the stimulus w24x. In keeping with the findings that Ži. treadmill locomotion stimulates Žin a speed-dependent manner. the discharge rate of serotonergic caudal raphe neurons w28,46x, and Žii. locomotion may be associated with a rapid reuptake of 5-HT w47x, it has been suggested that exercise increases both release and reuptake of 5-HT w24x. Interestingly, one study reported walking-induced increases in the extracellular 5-HT levels in the frontal cortex of Žuntrained. rats w32x whereas another study reported running-induced increases in extracellular 5-HIAA levels in the latter region, but not in the raphe dorsalis Ži.e. a serotonergic cell body-enriched region. w13x. Lastly, two recent studies Žpublished while this manuscript was in preparation. related to extracellular 5-HT levels in the ventral funiculus of the spinal cord w23x and the hippocampus w51x have shown that exercise increases extracellular 5-HT levels. The present study, which was conducted in food-deprived rats, confirms those findings in fed rats. Hence, all these studies Žincluding the present one. reveal that extracellular 5-HT levels rise in a time-dependent manner during exercise, but return progressively toward baseline levels on immediate cessation of the exercise. In keeping with these results, it is noteworthy that behavioural activity Žincluding locomotion. has been reported to correlate positively with hippocampal extracellular 5-HT levels w33x. Besides, the maximal increase in 5-HT levels differed markedly among studies, a difference which is likely to be due to the training andror exercise protocols and to the region analysed therein. It has been claimed that extracellular 5-HT levels rise in poor runners, but not in good runners w51x. The lack of clear interindividual differences in the present study renders however this hypothesis unlikely. On the other hand, it has been reported that exercise either increases extracellular 5-HIAA levels, with a pattern parallel to that observed with 5-HT levels w23x, or not w51x. The present study reports intermediate results, i.e. extracellular 5-HIAA levels followed a pattern similar to those observed with 5-HT, but nowhere were the changes in 5-HIAA levels significantly different from the pre-exercise period. On the basis of the parallelism between extracellular 5-HT and 5-HIAA levels, it has been claimed that extracellular 5-HIAA levels reflect 5-HT release, at least during exercise w23x. As underlined recently w48x, extracellular 5-HIAA levels reflect the combination of synthesis, intraneuronal metabolism, metabolism following release of the parent amine, and probenecid-sensitive efflux, thereby indicating the difficulty to acknowledge the functional significance of extraneuronal 5-HIAA levels. On the other hand, one crucial question is the extent to which exercise-induced increases in synthesis may participate in the aforementioned increases in extracellular 5-HT levels. Because parallel experiments aimed at measuring tryptophan, 5-HT and 5-HIAA in hippocampal tissues were not performed, it is impossible to provide a clearcut answer. It is our belief that future experiments should investigate whether 5-HT synthesis participates Žpartly or totally. in the exercise-induced changes in extracellular levels of 5-HT, or if at the opposite 5-HT synthesis serves only to replenish depolarisation-induced release of 5-HT Žsee above.. As indicated in Section 1, the hypothesis that central serotonergic systems may play a key role in the starvation-induced hyperactivity model w1,2,38x has prompted us to analyse the effects of running in food-deprived rats. Although it is clear that the combination of food deprivation and exercise Žas used herein. is a paradigm that does not strictly parallel hyperactivity-induced fasting, we felt that at least in a first stage, assessing the question of the functional effects of food deprivation and exercise on extracellular 5-HT levels would help in our comprehension of the mechanisms underlying food restriction-induced hyperactivity. However, because activity on a running wheel, at the opposite of treadmill running, does not affect central tryptophan levels but decreases hippocampal 5-HT and 5-HIAA levels w17,27x, it could be argued that the pattern of extracellular 5-HT levels may vary between treadmill exercise and spontaneous wheel running. Besides the observation that spontaneous wheel running may actually increase 5-HT release from the hippocampus w27x, it is important to state that both the speed and the duration of the running wheel sessions in fed rats are far less important than the ones measured in the starvation-induced hyperactivity paradigm. However, a clearcut answer will only arise from studies using microdialysis in wheel running rats, i.e., a somewhat complex protocol to set up. As stated in Section 1, numerous studies w16,18,34,42x but not all w6,40,44,49x have found that tryptophan administration yields increases in 5-HT release only under stimulated conditions. The present study confirms this hypothesis because tryptophan administration did not increase extracellular 5-HT levels in fed rats although a similar treatment increased extracellular 5-HT levels in food-deprived rats, thus confirming data from Ref. w41x. Interestingly, extracellular 5-HIAA levels rose to similar extent independently from the metabolic state of the animal, thereby suggesting that under certain conditions Že.g. tryptophan administration to fed rats., extracellular 5-HIAA R. Meeusen et al.r Brain Research 740 (1996) 245–252 levels are not indices of 5-HT release. It has been claimed that the differential effect of tryptophan upon extracellular 5-HT levels Žaccording to the metabolic state of the animals. may lie into a better entry of tryptophan into the brain w41x. However this is unlikely because Ži. the intrinsic effectiveness of tryptophan uptake was found to be decreased in 24-h food deprived rats, compared with fed rats w11x, and Žii. increasing the dose of tryptophan up to 100 mgrkg did not prove more effective than a 50 mgrkg dose upon extracellular 5-HT levels w42x. Actually, another explanation would lie into the suggestion that food deprivation actually increases the firing rate of serotonergic neurones, thereby allowing de novo synthesis of 5-HT to Žpartly or totally. increase 5-HT release. Hence, this effect of tryptophan would thus resemble that observed in tryptophan-injected rats undergoing other stressors such as immobilisation w29x or streptozotocin-diabetes w5x. On the other hand, the possibility that tryptophan is effective in food-deprived rats Žcompared to fed rats. because the former rats are more reactive than the latter ones to the paradigm used Ži.e. handling, injection. cannot be discarded. Past works have shown that tryptophan administration may, under certain conditions, decrease serotonergic nerve firing w45x, an effect thought to be mediated by 5-HT release, and in turn, stimulation of Ž5-HT1A . autoreceptors endowed with self-inhibitory functions upon serotonergic nerve firing w43x. In keeping with this possibility, we investigated whether a pretreatment with tryptophan Žat a dose that increases 5-HT release. would diminish exercise-induced increases in extracellular 5-HT levels. Actually, tryptophan and exercise-induced increases in extracellular 5-HT levels were merely additive throughout the experiment. This could suggest two complementary mechanisms, Ži. tryptophan increasing synthesis Žand release. in neurons initially depolarised by food deprivation, and in turn Žii. exercise amplifying serotonergic nerve depolarisation. For some unknown reason, saline-pretreated runner rats displayed increased extracellular 5HIAA levels whereas these levels did not change significantly in our initial experiments in saline-treated rats or in exercised rats. Whether this difference is due to a synergistic interaction between the stress of the injection procedure and exercise is a possibility which merits consideration. Taken together, the results from the present study show that exercise in food-deprived rats is associated with increases in extracellular 5-HT levels in the hippocampus, thus suggesting sustained 5-HT release. However, because rats were exercised during the inactive phase of their circadian cycle Ži.e. when serotonergic firing rate is the lowest w28x., it remains to be discovered wether the amplitude of these increases would be more affected during the circadian cycle. Actually, this experiment would also help to solve the question regarding the origin Žexercise andror arousal. of the aforementioned increases in 5-HT release. This study also shows that tryptophan administration to 251 food-deprived rats, but not to fed rats, increases hippocampal 5-HT release, thereby confirming past results, whilst tryptophan administration prior to running amplifies, rather than lowers, exercise-induced increases in extracellular 5-HT levels. These results suggest that starvation-induced hyperactivity may be associated with increased hippocampal release of 5-HT. In keeping with this possibility, the observation that administration of valine, an amino acid competing with tryptophan for entry into the brain, decreases evoked, but not basal release of 5-HT in the hippocampus w22x and exercise-induced increases in 5-HT synthesis w11,20x is worth mentioning. If release of 5-HT is involved in the exacerbation of the starvation-induced hyperactivity syndrome, these results suggest that the use of valine Žor some other neutral amino acid. could prove of most interest. References w1x Altemus, M., Glowa, J.R., Galliven, E., Leong, Y.M. and Murphy, D.L., Effects of serotonergic agents on food-restriction-induced hyperactivity, Pharmacol. Biochem. BehaÕ., 53 Ž1996. 123–131. w2x Aravich, P.F., Doerries, L.E. and Rieg, T.S., Exercise-induced weight loss in the rat and anorexia nervosa, Appetite, 23 Ž1994. 196. w3x Aravich, P.F., Rieg, T.S., Ahmed, I. and Lauterio, T.J., Fluoxetine induces vasopressin and oxytocin abnormalities in food-restricted rats given voluntary exercise: relationship to anorexia nervosa, Brain Res., 612 Ž1993. 180–189. w4x Blomstrand, E., Celsing, F. and Newsholme, E.A., Changes in plasma concentrations of aromatic and branched-chain amino acids during sustained exercise in man and their possible role in fatigue, Acta Physiol. Scand., 133 Ž1988. 115–121. w5x Broderick, P.A. and Jacoby, J.H., Diabetes-related changes in Ltryptophan induced release of striatal biogenic amines, Diabetes, 37 Ž1988. 956–960. w6x Carboni, E., Cadoni, C., Tanda, G.L. and Di Chiara, G., Calcium-dependent, tetrodotoxin-sensitive stimulation of cortical serotonin release after a tryptophan load, J. Neurochem., 53 Ž1989. 976–978. w7x Carlsson, A. and Lindqvist, M., The effect of L-tryptophan and psychotropic drugs on the formation of 5-hydroxytryptophan in the mouse brain in vivo, J. Neural Transm., 34 Ž1972. 23–43. w8x Chaouloff, F., Physical exercise and brain monoamines: a review, Acta Physiol. Scand., 137 Ž1989. 1–13. w9x Chaouloff, F., Physiopharmacological interactions between stress hormones and central serotonergic systems, Brain Res. ReÕ., 18 Ž1993. 1–32. w10x Chaouloff, F., Danguir, J. and Elghozi, J.L., Dextrofenfluramine, but not 8-OH-DPAT, affects the decrease in food consumed by rats submitted to physical exercise, Pharmacol. Biochem. BehaÕ., 32 Ž1989. 573–576. w11x Chaouloff, F., Elghozi, J.L., Guezennec, Y. and Laude, D., Effects of conditioned running on plasma, liver and brain tryptophan and on brain 5-hydroxytryptamine metabolism of the rat, Br. J. Pharmacol., 86 Ž1985. 33–41. w12x Chaouloff, F., Kennett, G.A., Serrurier, B., Merino, D. and Curzon, G., Amino acid analysis demonstrates that increased plasma free tryptophan causes the increase of brain tryptophan during exercise in the rat, J. Neurochem., 46 Ž1986. 1647–1650. w13x Clement, H.W., Schafer, F., Ruwe, C., Gemsa, D. and Wesemann, ¨ W., Stress-induced changes of extracellular 5-hydroxyindoleacetic acid concentrations followed in the nucleus raphe dorsalis and the frontal cortex of the rat, Brain Res., 614 Ž1993. 117–124. 252 R. Meeusen et al.r Brain Research 740 (1996) 245–252 w14x Davis, J.M., Bailey, S.P., Woods, J.A., Galiano, F.J., Hamilton, M.T. and Bartoli, W.P., Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during prolonged cycling, Eur. J. Appl. Physiol., 65 Ž1992. 513–519. w15x De Meirleir, K., L’Hermite-Baleriaux, M., L’Hermite, M., Rost, R. and Hollmann, W., Evidence for serotoninergic control of exerciseinduced prolactin release, Horm. Metab. Res., 17 Ž1985. 380–381. w16x De Simoni, M.G., Sokola, A., Fodritto, F., Dal Toso, G. and Algeri, S., Functional meaning of tryptophan-induced increase of 5-HT metabolism as clarified by in vivo voltammetry, Brain Res., 411 Ž1987. 89–94. w17x Elam, M., Svensson, T.H. and Thoren, P., Brain monoamine metabolism is altered in rats following spontaneous, long-distance running, Acta Physiol. Scand., 130 Ž1987. 313–316. w18x Elks, M.L., Youngblood, W.W. and Kizer, J.S., Serotonin synthesis and release in brain slices: independence of tryptophan, Brain Res., 172 Ž1979. 471–486. w19x Epling, W.F. and Pierce, W.D., Activity-based anorexia: a biobehavioral perspective, Int. J. Eating Disord., 7 Ž1988. 475–485. w20x Fernstrom, J. and Wurtman, R., Brain serotonin content: physiological regulation by plasma neutral amino acids, Science, 178 Ž1972. 414–416. w21x Fischer, H.G., Hollmann, W. and De Meirleir, K., Exercise changes in plasma tryptophan fractions and relationship with prolactin, Int. J. Sports Med., 12 Ž1991. 487–489. w22x Gartside, S.E., Cowen, P.J. and Sharp, T., Evidence that the large neutral amino acid L-valine decreases electrically-evoked release of 5-HT in rat hippocampus in vivo, Psychopharmacology, 109 Ž1992. 251–253. w23x Gerin, C., Becquet, D. and Privat, A., Direct evidence for the link between monoaminergic pathways and motor activity. I. A study with microdialysis probes implanted in the ventral funiculus of the spinal cord, Brain Res., 704 Ž1995. 191–201. w24x Gerin, C., Legrand, A. and Privat, A., Study of 5-HT release with a chronically implanted microdialysis probe in the ventral horn of the spinal cord of unrestrained rats during exercise on a treadmill, J. Neurosci. Methods, 52 Ž1994. 129–141. w25x Grahame-Smith, D.G., Studies in vivo on the relationship between brain tryptophan, brain 5-HT synthesis and hyperactivity in rats treated with a monoamine oxidase inhibitor and L-tryptophan, J. Neurochem., 18 Ž1971. 1053–1066. w26x Hery, ´ F. and Ternaux, J.P., Regulation of release processes in central serotoninergic neurons, J. Physiol. (Paris), 77 Ž1981. 287–301. w27x Hoffmann, P., Elam, M., Thoren, P. and Hjorth, S., Effects of long-lasting voluntary running on the cerebral levels of dopamine, serotonin and their metabolites in the spontaneously hypertensive rat, Life Sci., 54 Ž1994. 855–861. w28x Jacobs, B.L. and Fornal, C.A., 5-HT and motor control: a hypothesis, Trends Neurosci., 16 Ž1993. 346–352. w29x Kennett, G.A. and Joseph, M.H., The functional importance of increased brain tryptophan in the serotonergic response to restraint stress, Neuropharmacology, 20 Ž1981. 39–43. w30x Knott, P. and Curzon, G., Free tryptophan in plasma and brain tryptophan metabolism, Nature, 239 Ž1972. 452–453. w31x Kuhn, D.M., Wolf, W.A. and Youdim, M.B.H., Serotonin neurochemistry revisited: a new look at some old axioms, Neurochem. Int., 8 Ž1986. 141–154. w32x Kurosawa, M., Okada, K., Sato, A. and Uchida, S., Extracellular release of acetylcholine, noradrenaline and serotonin increases in the cerebral cortex during walking in conscious rats, Neurosci. Lett., 161 Ž1993. 73–76. w33x Linthorst, A.C.E., Flachskamm, C., Muller-Preuss, P., Holsboer, F. ¨ w34x w35x w36x w37x w38x w39x w40x w41x w42x w43x w44x w45x w46x w47x w48x w49x w50x w51x and Reul, J.M.H.M., Effect of bacterial endotoxin and interleukin-1a on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. J. Neurosci., 15 Ž1995. 2920–2934. Marsden, C.A., Conti, J., Strope, E., Curzon, G. and Adams, R.N., Monitoring 5-hydroxytryptamine release in the brain of the freely moving unanaesthetized rat using in vivo voltammetry, Brain Res., 171 Ž1979. 85–99. McMenamy, R.H., Binding of indole analogues to human serum albumin. Effects of fatty acids, J. Biol. Chem., 24 Ž1965. 4235–4243. Meeusen, R. and De Meirleir, K., Exercise and brain neurotransmission, Sports Med., 20 Ž1995. 160–188. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Pirke, K.M., Broocks, A., Wilckens, T., Marquard, R. and Schweiger, U., Starvation-induced hyperactivity in the rat: the role of endocrine and neurotransmitter changes, Neurosci. BiobehaÕ. ReÕ., 17 Ž1993. 287–294. Sarre, S., Michotte, Y., Marvin, C. and Ebinger, G., Microbore liquid chromatography with dual electrochemical detection for the determination of serotonin and 5-hydroxyindoleacetic acid in rat brain dialysates, J. Chromatogr., 582 Ž1992. 29–34. Schaechter, J.D. and Wurtman, R.J., Serotonin release varies with brain tryptophan levels, Brain Res., 532 Ž1990. 203–210. Schwartz, D.H., Hernandez, L. and Hoebel, B.G., Tryptophan increases extracellular serotonin in the lateral hypothalamus of fooddeprived rats, Brain Res. Bull., 25 Ž1990. 803–807. Sharp, T., Bramwell, S.R. and Grahame-Smith, D.G., Effect of acute administration of L-tryptophan on the release of 5-HT in rat hippocampus in relation to serotoninergic neuronal activity: an in vivo microdialysis study, Life Sci., 50 Ž1992. 1215–1223. Sprouse, J.S. and Aghajanian, G.K., Žy.-Propranolol blocks the inhibition of serotonergic dorsal raphe firing by 5-HT1A selective agonists, Eur. J. Pharmacol., 128 Ž1986. 295–298. Ternaux, J.P., Boireau, A., Bourgoin, S., Hamon, M., Hery, F. and Glowinski, J., In vivo release of 5-HT in the lateral ventricle of the rat: effects of 5-hydroxytryptophan and tryptophan, Brain Res., 101 Ž1976. 533–548. Trulson, M.E. and Jacobs, B.L., Dose–response relationships between systemically administered L-tryptophan or L-5-hydroxytryptophan and raphe unit activity in the rat, Neuropharmacology, 15 Ž1976. 339–344. Veasey, S.C., Fornal, C.A., Metzler, C.W. and Jacobs, B.L., Response of serotonergic caudal raphe neurons in relation to specific motor acivities in freely moving cats, J. Neurosci., 15 Ž1995. 5346–5359. Wallis, D.I., 5-HT receptors involved in initiation or modulation of motor patterns: opportunities for drug development, Trends Pharmacol. Sci., 15 Ž1994. 288–292. Westerink, B.H.C., Brain microdialysis and its application for the study of animal behaviour, BehaÕ. Brain Res., 70 Ž1995. 103–124. Westerink, B.H.C. and De Vries, J., Effect of precursor loading on the synthesis rate and release of dopamine and serotonin in the striatum: a microdialysis study in conscious rats, J. Neurochem., 56 Ž1991. 228–233. Wilkinson, L., Auerbach, S. and Jacobs, B.L., Extracellular serotonin levels change with behavioural state but not pyrogen-induced hyperthermia, J. Neurosci., 11 Ž1991. 2732–2741. Wilson, W.M. and Marsden, C.A., In vivo measurement of extracellular serotonin in the ventral hippocampus during treadmill running, BehaÕ. Pharmacol., 7 Ž1996. 101–104.