Regulatory Peptides, 28 (1990) 233-239
233
Elsevier REGPEP 00904
Cerebrospinal fluid immunoreactive fl-endorphin concentration is increased by voluntary exercise in the spontaneously hypertensive rat Pavel Hoffmann 1, Lars Terenius 2 and Peter Thor6n 1 1Department of Physiology, University of G6teborg, GSteborg and 2Department of Drug Dependence Research, Karolinska Institute, Stockholm (Sweden) (Received 20 November 1989; revised version received and accepted 19 January 1990)
Key words: Exercise; Rat; Cerebrospinal fluid; fl-Endorphin
Summary The effect of voluntary exercise on cerebrospinal fluid (CSF) levels ofimmunoreactive fl-endorphin has been studied in the spontaneously hypertensive rat (SHR). The exercise consisted of 5-6 weeks of spontaneous running in wheels and the average running distance was 3.5 + 0.4 km/24 h. CSF samples were obtained under anaesthesia from the cisterna magna. Five experimental groups were examined, four groups of runners and one group of sedentary controls. The runners were sampled either (a)shortly (0-3 h) after termination of exercise, or after the wheel had been locked for (b) 24, (c) 48 or (d) 96 h. The runners in group a had significantly higher immunoreactive fl-endorphin levels than the controls. The levels remained increased as compared with controls after 24 and 48 h of enforced abstinence but had returned to control after 96 h. The data indicate that voluntary exercise induces adaptive changes in central fl-endorphin systems.
Introduction Long-lasting exercise is known to lower blood pressure in the post-exercise period, both in hypertensive man [ 1] and in the spontaneously hypertensive rat (SH rat) [2]. Exercise-induced pain threshold elevation has also been described, both in humans and Correspondence: Pavel Hoffmann, Department of Physiology, University of G6teborg, Box 33031, S-40033 G6teborg, Sweden. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
234
in the rat [3,4]. In animal studies, both effects can be abolished by naloxone treatment, thus indicating an involvement of endogenous opioid systems. Similar effects of naloxone on pain threshold are also suggested in man [3]. Running has also been reported to give positive mood changes, almost euphoria, and to reduce anxiety in man [5-7]. These effects have commonly been referred to as 'runner's high' or 'endorphin calm'. Similar effects have also been observed in animal experiments. Thus, SH rats exercised in the present model exhibited less exploratory behaviour and a tendency towards decreased aggression compared to sedentary SH rat controls [8]. Several investigators have also shown an increased level of plasma ~-endorphin or other opioid peptides after physical exercise [9-11 ]. However, fl-endorphin in plasma mainly derives from the pituitary gland [ 12,13]. Since the blood-brain barrier is poorly permeable to circulating peptides, they are not likely to reach CNS centres directly influencing behaviour, pain sensitivity or blood pressure control. Few studies have attempted to study the involvement of CNS endorphins directly during exercise for a long period of time. Blake et al. [ 14] found an increased level of fl-endorphin in specific brain regions in rats after exercise and Christie and Chesher [15] reported increased enkephalin receptor binding after swimming in rats. Further, Sforzo et al. [ 16] found a change in opioid receptor occupation in several brain regions in the post-exercise period, suggestive of decreased receptor occupancy by endogenous opioids. Previous animal studies demonstrating cenu:al nervous system opioid activation have, however, used forced exercise. The aim of this study was to investigate whether CSF fl-endorphin concentration as an index of central activation of fl-endorphin pathways, is influenced by voluntary exercise for a long period of time.
Materials and Methods
Animals 45 male spontaneously hypertensive rats (SH rats), delivered by MOllegaards Breeding Centre, Denmark, were used. At the end of exercise, their average body weight was 330 + 5 g, with no significant differences between groups. All animals were placed in individual cages of the same size (40 × 24 × 15 cm). Throughout the experiment they had free access to food and water. All groups were kept in the same room, where the temperature was kept at 24 °C, the relative humidity was 50-60Y/o, and the light was on in 12 h periods, 7 : 00 a.m.-7 : 00 p.m. The experiments were approved by the Animal Ethics Committee of the University of GSteborg. Physical exercise A wheel ( ~ 22.5 cm), to which the rat had free access was attached to one side of each exercise group cage. To turn, the wheel had to be loaded with the weight of the animal. Wheel revolutions were automatically registered by a microprocessor and printed out every 24 h. Both the running apparatus and the spontaneous running behaviour in the SH rats are described in detail by Shyu et al. [17].
235
Experimental procedures and groups All exercise groups were allowed to run for 5-6 weeks, while the non-exercising control rats were kept in the same room for the same length of time. Five different groups of SH rats were studied: (a) Runners ( n - - 8 ) . These SH rats were allowed to run for 5-6 weeks and the cerebrospinal fluid (CSF) samples were taken in the morning after the last night of running. (b) Runners, 24 h abstinent (n = 8). These animals were treated in the same way as the animals in group a, eccept that the running wheels were locked for 24 h before CSF sampling. (c) and (d) Runners, 48 h (n -- 8) and 96 h (n - 7) abstinent respectively. These animals were also treated in the same way as the animals in group a but in these groups the running wheels were locked for 48 and 96 h, respectively, before the CSF sampling. (e) Sedentary controls (n = 14). These SH rats were treated in the same way as the runners in group a but they had no access to a running wheel.
Withdrawal of cerebrospinalfluid The rats were rapidly anaesthetised with pentobarbitone (75 mg. k g - 1 body weight, i.p.). A dorsal skin incision was made to expose the calvarium and a dental burr was used to drill a small hole in the midline of the skull just in front of the external occipital crest. Through this hole the cisterna magna was cannulated, using a 23 gauge cannula. The cannula was directed along the inner aspect of the occipital bone, 8.5 mm from the skull surface. A similar method of cannulating the cisterna magna is described in detail by Scheinin [18]. The CSF was slowly withdrawn, using a Hamilton syringe connected to a cannula via a long PE-25 catheter. A mean volume of 140 + 5/~1 was collected. The procedure lasted less than 1 min from the time when the rat was fully anaesthetised until CSF was withdrawn. The CSF was immediately centrifuged and mixed with an equal volume of 0.1 M HC1 and frozen in liquid nitrogen.
[3-Endorphin assay The CSF samples were diluted with a pyridine formic acid buffer (pyridine 0.015 M, formic acid 0.1 M) and run through a 1 ml SP-Sephadex C-25 column equilibrated with the same buffer. The column was washed with pyridine formate buffers (8 ml 0.1 M pyridine, 0.1 M formic acid, then 4 ml 0.35 M pyridine, 0.35 M formic acid) and finally eluted with 4 ml 1.6 M pyridine, 1.6 M formic acid buffer. The eluate was evaporated in vacuo in a Speed-Vac evaporator. The residue was dissolved in 100 #1 methanol: 0.1 M HC1 (1:1) and divided into 25 #1 aliquots; three aliquots were analyzed in triplicate by radioimmunoassay. Samples with a known content of fl-endorphin were run in parallell with the experimental samples. The antiserum 42F was raised against human/~-endorphin and it showed no crossreaction with enkephalins, dynorphins or substance P but it fully cross-reacted with /~-lipotropin. The immunoreactive material has not been characterized chemically in these rats and it is not known whether the chemical nature changes with exercise. Samples were incubated for 24 h, then 5000 cpm ~/sI-/~-endorphin was added and
236
incubation continued for 20 h. Incubation was terminated by treatment with dextrancoated charcoal to separate antibody-bound and free peptide. After centrifugation, the supernatant was counted in a gamma spectrometer. The fl-endorphin RIA procedure is described in detail by O'Brien et al. [ 19]. In the exercise groups, each CSF sample was analysed separately. In the control SHR, the CSF samples from two rats were pooled and analysed together due to the low total fl-endorphin content, which was close to the detection limit of the RIA used (2.0 fmol fl-endorphin). The interassay variation was 10~. All values are corrected for recovery, which was 70-90~o.
Statistical analysis Comparisons between runners (a) and controls were performed using Student's t-test for nonpaired data. To analyse the time-course, one-way ANOVA for independent measures was used. In both tests, a P value of less than 0.05 was considered statistically significant. All values are expressed as mean + S.E.
Results
CSF /3-endorphin level was increased by voluntary wheel-running exercise Immunoreactive fl-endorphin concentration was significantly increased by voluntary long-lasting exercise in wheels. Thus, the concentration of CSF fl-endorphin in the runners (group a) was 30.8 + 5.4 fmol/ml, as compared to 16.8 + 2.4 fmol/ml in the sedentary control group (e), (P < 0.05, Fig. 1.). The mean running distance in the runners' group was 3.3 + 0.5 km/24 h during the last week. There was no correlation between the mean running activity during the last week in each animal and the CSF 40-
E o E
o
30-
20-
,/////A u. 0
10-
Controls
Runners
Fig. !. C S F i m m u n o r e a c t i v e fl-endorphin levels, measured by R I A , in r u n n e r s a n d s e d e n t a r y controls.
Voluntary exercise for 5-6 weeks significantlyincreased CSF fl-endorphin concentration in the SHR. Asterisk indicates a significantdifferencefrom the control group * P ~ 0.05).
237
50-
_~ E
4030"
? ,,
20" 10-
0-
6
2~
4's
12
9~
Time (hour)
Fig. 2. Time-course of the CSF immunoreactive p-endorphin elevation during enforced abstinence aider voluntary exercise for 5-6 weeks. Note that the level is still increased 24 and 48 h after termination of exercise. (F = 2.74, * P < 0.05). The intermittent line and shaded area indicate the fl-endorphin concentration (mean + S.E.) in the sedentary control group.
fl-endorphin level. Similarly, there was no correlation between running distance in the last night and the endorphin level.
fl-Endorphin levels were still increased 24 and 48 h of abstinence from wheel-running The CSF fl-endorphin concentration was still increased in SH rats which had their running wheel locked for 24 or 48 h before the CSF sampling. The fl-endorphin concentration in the 24-(b) and 48-h(c) abstinent groups was 3 8 . 1 _ 8.1 and 2 8 . 0 _ 3.0fmol/ml, respectively, as compared to 16.8 __ 2.4 in the controls(e), (P _< 0.05, Fig. 2). In the 96-h abstinent group (d), the fl-endorphin concentration was 20.3 + 3.1 fmol/ml, thus having returned to a level similar to that of the controls. The mean running distance in groups b, c and d was similar to that in group a, being 3.9 + 0.9, 3.6 + 0.7 and 3.4 _ 0.6 km/24 h respectively, in the last week.
Discussion The present results indicate that voluntary long-duration running exercise in SH rats has an effect on CSF immunoreactive fl-endorphin levels. The levels were significantly increased in runners compared to sedentary controls and the elevation was still present 24 and 48 h after termination of exercise. However, 96 h post-exercise, the concentration of CSF fl-endorphin was back to the level of non-exercising control rats. The SH rat was chosen in this study since this rat strain shows a completely spontaneous running behavior in wheels, which is in contrast to studies using forced exercise. Although altered opiate receptor binding in brains from hypertensive compared to normotensive rats has been demonstrated [20], preliminary results indicate that a similar increase in CSF fl-endorphin following voluntary exercise is seen in normotensive rats (Daneryd et al., unpublished observations). Voluntary running in the present exercise model has previously been shown to give a post-exercise drop in blood pressure [2] and an increase in pain threshold [4]. Both
238
these effects could be antagonised by naloxone, thus indicating an involvement of endogenous opioid systems. Exercising SH rats have also been found to exhibit less exploratory behaviour in an open-field test and to be less aggressive than sedentary control animals [8]. Similar behavioural post-exercise effects are commonly referred to as 'endorphin calm'. The results in this study show more directly an influence of exercise on the brain fl-endorphin system. However, if the increased CSF concentration of immunorective fl-endorphin reflects increase of opioid peptide release, this does not necessarily indicate an increased receptor activation. For instance, Puttfarcken et al. [21] and Morris and Herz [22] could show opioid receptor desensitisation and a reduction in receptor number after chronic agonist treatment. The changes in fl-endorphin levels are not likely to be isolated. Preliminary data indicate persistent effects on serotonin levels and turnover in running SH rats, which effects were maintained for at least 48 h of enforced abstinence (Hoffmann et al. unpublished observations). Other opioid peptide systems may also be involved. The pattern observed here is, however, different from that observed during detoxification of methadone-maintained human heroin addicts. During methadone maintenance, their CSF levels of fl-endorphin as well as receptor-assayed proenkephalin- and prodynorphin-derived opioid peptides were not significantly different from those in healthy volunteers. In withdrawal, all measured opioid peptides in CSF dropped to a minimum level at about 30 h, which was followed by a rebound increase to essentially normal levels at 50-80 h [ 19]. Common to the present study and the study by O'Brien and co-workers [ 19] is the protracted nature of the effects and the slowly normalising levels ofCSF opioid peptides. It is interesting that maximum withdrawal to opiates is observed at about 36 h of abstinence. When exercise is abruptly stopped, for over 24 h, the SH rats exhibit significantly increased aggression, possibly corresponding to a withdrawal reaction. This post-exercise aggression reached a maximum 3-6 days after termination of exercise [8]. In conclusion, this study indicates that voluntary exercise in SH rats gives rise to elevated concentrations ofCSF fi-endorphin. The changes in CSF endorphin level may, together with changes in monoamine metabolism, be the biochemical correlate of previously observed effects on behaviour, pain threshold and blood pressure in running animals and man.
Acknowledgements This study was supported by the Swedish Medical Research Council (No 3766 and 4764), by Idrottens Forskningsr~td (No. 52/89) and by the National Institute on Drug Abuse, Rockville, MD. The skilful technical assistance of Mrs. Ehsabeth PoUak and Mrs. Ingrid Eriksson is also gratefully acknowledged.
239
References 1 Wilcox, R. G., Bennet, T., Brown, A. M. and MacDonald, I. A., Is exercise good for high blood pressure ?, Br. Med. J., 285 (1982) 767-769. 2 Shyu, B.C. and Thor~n, P., Circulatory events following spontaneous muscle exercise in normotensive and hypertensive rats, Acta Physiol. Scand., 128 (1986) 515-524. 3 Olausson, B., Eriksson, E., Ellmarker, L., Rydenhag, B., Shyu, B.C. and Andersson, S.A., Effects of naloxone on dental pain threshold after muscle exercise and low frequency TNS: a comparative study in man, Acta Physiol. Scand., 126 (1986) 299-305. 4 Shyu, B.C., Andersson, S.A. and Thor6n, P., Endorphin mediated increase in pain threshold induced by long - lasting exercise in rats, Life Sci., 10 (1982) 833-840. 5 Farrel, P. A., Gustafson, A. B., Morgan, W. P. and Pert, C. B., Enkephaiins, catecholamines, and psychological mood alternations: effects of prolonged exercise, Med. Sci. Sports Exerc., 19 (1987) 347-353. 6 Wagemaker, H. and Goldstein, L., The runner's high. J. Sports Med., 20 (1980) 227-229. 7 Janal, M. N., Colt, E. W. D., Clark, W. C. and Glusman, M., Pain sensitivity, mood and plasma endocrine levels in man following long-distance running: Effects of naloxone, Pain, 19 (1984) 13-25. 8 Hoffmann, P., Thor6n, P. and Ely, D., Effect of voluntary exercise on open-field behavior and on aggression in the spontaneously hypertensive rat (SHR), Behav. Neural Biol., 47 (1987) 346-355. 9 Colt, E. W. D., Wardlaw, S.H. and Frantz, A. G., The effect of running on plasma/~-endorphin, Life Sci., 28 (1981) 1637-1640. 10 Gambert, S.R., Garthwaite, T. L., Pontzer, C. H., Cook, E. E., Tristani, F. E., Duthie, E.H., Martinson, D.R., Hagen, T.C. and McCarty, D.J., Running elevates plasma/~-endorphin immunoreactivity and ACTH in untrained human subjects, Proc. Soc. Exp. Biol. Med., 168 (1981) 1-4. 11 Goldfarb, A.H., Hatfield, B.D., Sforzo, G.A. and Flyrm, M.G., Serum/~-endorphin levels during a graded exercise test to exhaustion, Med. Sci. Sports Exerc., 19 (1987) 78-82. 12 Guillemin, R., Vargo, T., Rossier, J., Minick, S., Ling, N., Rivier, C., Vale, W. and Bloom, F.,/~-Endorphin and adrenocorticotropin are secreted concomittantly by the pituitary gland, Science, 197 (1977) 1367-1369. 13 Mains, R.E., Eipper, B.A. and Ling, N., Common precursor to corticotropins and endorphins, Proc. Natl. Acad. Sci. USA, 74 (1977) 3014-3018. 14 Blake, M.J., Stein, E.A. and Vomachka, A.J., Effects of exercise training on brain opioid peptides and serum LH in female rats, Peptides 5 (1984) 953-958. i 5 Christie, M.J. and Chesher, G.B., [3H]Leu-enkephalin binding following chronic swim-stress in mice, Neurosci. Lett., 36 (1983) 323-328. 16 Sforzo, G.A., Seeger, T,F., Pert, C.B., Pert, A. and Dotson, C.O., In vivo opioid receptor occupation in the rat brain following exercise, Med. Sci. Sports Exerc., 18 (1986) 380-384. 17 Shyu, B.C., Andersson, S.A. and Thoren, P., Spontaneous running in wheels: A computer assisted method for measuring physiological parameters during exercise in rodents, Acta Physiol. Scand., 121 (1984) 103-109. 18 Scheinin, H., Monitoring of drug-induced changes in monoamine metabolites in rat cerebrospinal fluid. Thesis (1986) Department of Pharmacology, University of Turku, Turku, Finland. 19 0'Brien, C.P., Terenius, L.Y. Nyberg, F., McLellan, A.T. and Eriksson, I., Endogenous opioids in cerebrospinal fluid of opioid-dependent humans, Biol. Psychiatry 24 (1988) 649-662. 20 Zamir, N., Segal, M. and Simantov, R., Opiate receptor binding in the brain of the hypertensive rat, Brain Res., 213 (1981) 217-222. 21 Puttfarcken, P.S., Werling, L.L. and Cox, B.M., Effects of chronic morphine exposure on opioid inhibition of adenylyl cyclase in 7315c cell membranes: A useful model for the study of tolerance of # opioid receptors, Mol. Pharmacol., 33 (1988) 520-527. 22 Morris, B.J. and Herz, A., Control of opiate receptor number in vivo: Simultaneous k-receptor downregulation and #-receptor up-regulation following chronic agonist/antagonist treatment, Neuroscience, 29 (1989) 433-442.
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Elsevier REGPEP 00904
Cerebrospinal fluid immunoreactive fl-endorphin concentration is increased by voluntary exercise in the spontaneously hypertensive rat Pavel Hoffmann 1, Lars Terenius 2 and Peter Thor6n 1 1Department of Physiology, University of G6teborg, GSteborg and 2Department of Drug Dependence Research, Karolinska Institute, Stockholm (Sweden) (Received 20 November 1989; revised version received and accepted 19 January 1990)
Key words: Exercise; Rat; Cerebrospinal fluid; fl-Endorphin
Summary The effect of voluntary exercise on cerebrospinal fluid (CSF) levels ofimmunoreactive fl-endorphin has been studied in the spontaneously hypertensive rat (SHR). The exercise consisted of 5-6 weeks of spontaneous running in wheels and the average running distance was 3.5 + 0.4 km/24 h. CSF samples were obtained under anaesthesia from the cisterna magna. Five experimental groups were examined, four groups of runners and one group of sedentary controls. The runners were sampled either (a)shortly (0-3 h) after termination of exercise, or after the wheel had been locked for (b) 24, (c) 48 or (d) 96 h. The runners in group a had significantly higher immunoreactive fl-endorphin levels than the controls. The levels remained increased as compared with controls after 24 and 48 h of enforced abstinence but had returned to control after 96 h. The data indicate that voluntary exercise induces adaptive changes in central fl-endorphin systems.
Introduction Long-lasting exercise is known to lower blood pressure in the post-exercise period, both in hypertensive man [ 1] and in the spontaneously hypertensive rat (SH rat) [2]. Exercise-induced pain threshold elevation has also been described, both in humans and Correspondence: Pavel Hoffmann, Department of Physiology, University of G6teborg, Box 33031, S-40033 G6teborg, Sweden. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
234
in the rat [3,4]. In animal studies, both effects can be abolished by naloxone treatment, thus indicating an involvement of endogenous opioid systems. Similar effects of naloxone on pain threshold are also suggested in man [3]. Running has also been reported to give positive mood changes, almost euphoria, and to reduce anxiety in man [5-7]. These effects have commonly been referred to as 'runner's high' or 'endorphin calm'. Similar effects have also been observed in animal experiments. Thus, SH rats exercised in the present model exhibited less exploratory behaviour and a tendency towards decreased aggression compared to sedentary SH rat controls [8]. Several investigators have also shown an increased level of plasma ~-endorphin or other opioid peptides after physical exercise [9-11 ]. However, fl-endorphin in plasma mainly derives from the pituitary gland [ 12,13]. Since the blood-brain barrier is poorly permeable to circulating peptides, they are not likely to reach CNS centres directly influencing behaviour, pain sensitivity or blood pressure control. Few studies have attempted to study the involvement of CNS endorphins directly during exercise for a long period of time. Blake et al. [ 14] found an increased level of fl-endorphin in specific brain regions in rats after exercise and Christie and Chesher [15] reported increased enkephalin receptor binding after swimming in rats. Further, Sforzo et al. [ 16] found a change in opioid receptor occupation in several brain regions in the post-exercise period, suggestive of decreased receptor occupancy by endogenous opioids. Previous animal studies demonstrating cenu:al nervous system opioid activation have, however, used forced exercise. The aim of this study was to investigate whether CSF fl-endorphin concentration as an index of central activation of fl-endorphin pathways, is influenced by voluntary exercise for a long period of time.
Materials and Methods
Animals 45 male spontaneously hypertensive rats (SH rats), delivered by MOllegaards Breeding Centre, Denmark, were used. At the end of exercise, their average body weight was 330 + 5 g, with no significant differences between groups. All animals were placed in individual cages of the same size (40 × 24 × 15 cm). Throughout the experiment they had free access to food and water. All groups were kept in the same room, where the temperature was kept at 24 °C, the relative humidity was 50-60Y/o, and the light was on in 12 h periods, 7 : 00 a.m.-7 : 00 p.m. The experiments were approved by the Animal Ethics Committee of the University of GSteborg. Physical exercise A wheel ( ~ 22.5 cm), to which the rat had free access was attached to one side of each exercise group cage. To turn, the wheel had to be loaded with the weight of the animal. Wheel revolutions were automatically registered by a microprocessor and printed out every 24 h. Both the running apparatus and the spontaneous running behaviour in the SH rats are described in detail by Shyu et al. [17].
235
Experimental procedures and groups All exercise groups were allowed to run for 5-6 weeks, while the non-exercising control rats were kept in the same room for the same length of time. Five different groups of SH rats were studied: (a) Runners ( n - - 8 ) . These SH rats were allowed to run for 5-6 weeks and the cerebrospinal fluid (CSF) samples were taken in the morning after the last night of running. (b) Runners, 24 h abstinent (n = 8). These animals were treated in the same way as the animals in group a, eccept that the running wheels were locked for 24 h before CSF sampling. (c) and (d) Runners, 48 h (n -- 8) and 96 h (n - 7) abstinent respectively. These animals were also treated in the same way as the animals in group a but in these groups the running wheels were locked for 48 and 96 h, respectively, before the CSF sampling. (e) Sedentary controls (n = 14). These SH rats were treated in the same way as the runners in group a but they had no access to a running wheel.
Withdrawal of cerebrospinalfluid The rats were rapidly anaesthetised with pentobarbitone (75 mg. k g - 1 body weight, i.p.). A dorsal skin incision was made to expose the calvarium and a dental burr was used to drill a small hole in the midline of the skull just in front of the external occipital crest. Through this hole the cisterna magna was cannulated, using a 23 gauge cannula. The cannula was directed along the inner aspect of the occipital bone, 8.5 mm from the skull surface. A similar method of cannulating the cisterna magna is described in detail by Scheinin [18]. The CSF was slowly withdrawn, using a Hamilton syringe connected to a cannula via a long PE-25 catheter. A mean volume of 140 + 5/~1 was collected. The procedure lasted less than 1 min from the time when the rat was fully anaesthetised until CSF was withdrawn. The CSF was immediately centrifuged and mixed with an equal volume of 0.1 M HC1 and frozen in liquid nitrogen.
[3-Endorphin assay The CSF samples were diluted with a pyridine formic acid buffer (pyridine 0.015 M, formic acid 0.1 M) and run through a 1 ml SP-Sephadex C-25 column equilibrated with the same buffer. The column was washed with pyridine formate buffers (8 ml 0.1 M pyridine, 0.1 M formic acid, then 4 ml 0.35 M pyridine, 0.35 M formic acid) and finally eluted with 4 ml 1.6 M pyridine, 1.6 M formic acid buffer. The eluate was evaporated in vacuo in a Speed-Vac evaporator. The residue was dissolved in 100 #1 methanol: 0.1 M HC1 (1:1) and divided into 25 #1 aliquots; three aliquots were analyzed in triplicate by radioimmunoassay. Samples with a known content of fl-endorphin were run in parallell with the experimental samples. The antiserum 42F was raised against human/~-endorphin and it showed no crossreaction with enkephalins, dynorphins or substance P but it fully cross-reacted with /~-lipotropin. The immunoreactive material has not been characterized chemically in these rats and it is not known whether the chemical nature changes with exercise. Samples were incubated for 24 h, then 5000 cpm ~/sI-/~-endorphin was added and
236
incubation continued for 20 h. Incubation was terminated by treatment with dextrancoated charcoal to separate antibody-bound and free peptide. After centrifugation, the supernatant was counted in a gamma spectrometer. The fl-endorphin RIA procedure is described in detail by O'Brien et al. [ 19]. In the exercise groups, each CSF sample was analysed separately. In the control SHR, the CSF samples from two rats were pooled and analysed together due to the low total fl-endorphin content, which was close to the detection limit of the RIA used (2.0 fmol fl-endorphin). The interassay variation was 10~. All values are corrected for recovery, which was 70-90~o.
Statistical analysis Comparisons between runners (a) and controls were performed using Student's t-test for nonpaired data. To analyse the time-course, one-way ANOVA for independent measures was used. In both tests, a P value of less than 0.05 was considered statistically significant. All values are expressed as mean + S.E.
Results
CSF /3-endorphin level was increased by voluntary wheel-running exercise Immunoreactive fl-endorphin concentration was significantly increased by voluntary long-lasting exercise in wheels. Thus, the concentration of CSF fl-endorphin in the runners (group a) was 30.8 + 5.4 fmol/ml, as compared to 16.8 + 2.4 fmol/ml in the sedentary control group (e), (P < 0.05, Fig. 1.). The mean running distance in the runners' group was 3.3 + 0.5 km/24 h during the last week. There was no correlation between the mean running activity during the last week in each animal and the CSF 40-
E o E
o
30-
20-
,/////A u. 0
10-
Controls
Runners
Fig. !. C S F i m m u n o r e a c t i v e fl-endorphin levels, measured by R I A , in r u n n e r s a n d s e d e n t a r y controls.
Voluntary exercise for 5-6 weeks significantlyincreased CSF fl-endorphin concentration in the SHR. Asterisk indicates a significantdifferencefrom the control group * P ~ 0.05).
237
50-
_~ E
4030"
? ,,
20" 10-
0-
6
2~
4's
12
9~
Time (hour)
Fig. 2. Time-course of the CSF immunoreactive p-endorphin elevation during enforced abstinence aider voluntary exercise for 5-6 weeks. Note that the level is still increased 24 and 48 h after termination of exercise. (F = 2.74, * P < 0.05). The intermittent line and shaded area indicate the fl-endorphin concentration (mean + S.E.) in the sedentary control group.
fl-endorphin level. Similarly, there was no correlation between running distance in the last night and the endorphin level.
fl-Endorphin levels were still increased 24 and 48 h of abstinence from wheel-running The CSF fl-endorphin concentration was still increased in SH rats which had their running wheel locked for 24 or 48 h before the CSF sampling. The fl-endorphin concentration in the 24-(b) and 48-h(c) abstinent groups was 3 8 . 1 _ 8.1 and 2 8 . 0 _ 3.0fmol/ml, respectively, as compared to 16.8 __ 2.4 in the controls(e), (P _< 0.05, Fig. 2). In the 96-h abstinent group (d), the fl-endorphin concentration was 20.3 + 3.1 fmol/ml, thus having returned to a level similar to that of the controls. The mean running distance in groups b, c and d was similar to that in group a, being 3.9 + 0.9, 3.6 + 0.7 and 3.4 _ 0.6 km/24 h respectively, in the last week.
Discussion The present results indicate that voluntary long-duration running exercise in SH rats has an effect on CSF immunoreactive fl-endorphin levels. The levels were significantly increased in runners compared to sedentary controls and the elevation was still present 24 and 48 h after termination of exercise. However, 96 h post-exercise, the concentration of CSF fl-endorphin was back to the level of non-exercising control rats. The SH rat was chosen in this study since this rat strain shows a completely spontaneous running behavior in wheels, which is in contrast to studies using forced exercise. Although altered opiate receptor binding in brains from hypertensive compared to normotensive rats has been demonstrated [20], preliminary results indicate that a similar increase in CSF fl-endorphin following voluntary exercise is seen in normotensive rats (Daneryd et al., unpublished observations). Voluntary running in the present exercise model has previously been shown to give a post-exercise drop in blood pressure [2] and an increase in pain threshold [4]. Both
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these effects could be antagonised by naloxone, thus indicating an involvement of endogenous opioid systems. Exercising SH rats have also been found to exhibit less exploratory behaviour in an open-field test and to be less aggressive than sedentary control animals [8]. Similar behavioural post-exercise effects are commonly referred to as 'endorphin calm'. The results in this study show more directly an influence of exercise on the brain fl-endorphin system. However, if the increased CSF concentration of immunorective fl-endorphin reflects increase of opioid peptide release, this does not necessarily indicate an increased receptor activation. For instance, Puttfarcken et al. [21] and Morris and Herz [22] could show opioid receptor desensitisation and a reduction in receptor number after chronic agonist treatment. The changes in fl-endorphin levels are not likely to be isolated. Preliminary data indicate persistent effects on serotonin levels and turnover in running SH rats, which effects were maintained for at least 48 h of enforced abstinence (Hoffmann et al. unpublished observations). Other opioid peptide systems may also be involved. The pattern observed here is, however, different from that observed during detoxification of methadone-maintained human heroin addicts. During methadone maintenance, their CSF levels of fl-endorphin as well as receptor-assayed proenkephalin- and prodynorphin-derived opioid peptides were not significantly different from those in healthy volunteers. In withdrawal, all measured opioid peptides in CSF dropped to a minimum level at about 30 h, which was followed by a rebound increase to essentially normal levels at 50-80 h [ 19]. Common to the present study and the study by O'Brien and co-workers [ 19] is the protracted nature of the effects and the slowly normalising levels ofCSF opioid peptides. It is interesting that maximum withdrawal to opiates is observed at about 36 h of abstinence. When exercise is abruptly stopped, for over 24 h, the SH rats exhibit significantly increased aggression, possibly corresponding to a withdrawal reaction. This post-exercise aggression reached a maximum 3-6 days after termination of exercise [8]. In conclusion, this study indicates that voluntary exercise in SH rats gives rise to elevated concentrations ofCSF fi-endorphin. The changes in CSF endorphin level may, together with changes in monoamine metabolism, be the biochemical correlate of previously observed effects on behaviour, pain threshold and blood pressure in running animals and man.
Acknowledgements This study was supported by the Swedish Medical Research Council (No 3766 and 4764), by Idrottens Forskningsr~td (No. 52/89) and by the National Institute on Drug Abuse, Rockville, MD. The skilful technical assistance of Mrs. Ehsabeth PoUak and Mrs. Ingrid Eriksson is also gratefully acknowledged.
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