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Biphasic effects of 8-OH-DPAT on endurance of treadmill performance in the male rat1
European Neuropsychopharmacology 7 (1997) 89–94
Biphasic effects of 8-OH-DPAT on endurance of treadmill performance in the male rat 1 Sven Ahlenius*, Petra Kaur, Peter Salmi ¨ ¨ , Sweden Biochemical and Behavioral Pharmacology, Astra Arcus AB, S-151 85 Sodertalje Received 27 February 1996; accepted 3 October 1996
Abstract Administration of the 5-HT 1A receptor agonist 8-OH-DPAT produced a biphasic pattern of effects on endurance performance of rats walking on top of a treadmill drum (Ø5166 mm, 16 rpm; ¯8 m min 21 ), with enhanced performance at a low dose (0.1 mg kg 21 s.c.) followed by impairment (0.2–0.8 mg kg 21 ). The partial 5-HT 1A receptor agonist (2)-pindolol improved the performance in the low dose range (0.5–2.0 mg kg 21 s.c.), whereas a higher dose (8 mg kg 21 ) was ineffective. The 5-HT 1A receptor antagonist WAY-100,635 produced an impaired performance at a low dose (12.5 mg kg 21 s.c.), with a recovery of performance at higher doses (50–200 mg kg 21 ). It is suggested that the inhibition of central serotonergic neurotransmission produced by stimulation or blockade of 5-HT 1A auto- and post-synaptic receptors, respectively, results in an improved endurance performance on the treadmill, whereas stimulation of postsynaptic 5-HT 1A receptors has the opposite action. In support of this contention, the impaired performance produced by a high dose of 8-OH-DPAT (0.8 mg kg 21 ) was antagonized by pretreatment with (2)-pindolol (2 mg kg 21 s.c.). 1997 Elsevier Science B.V. Keywords: Treadmill locomotion; Physical exercise; Serotonin receptors; 8-OH-DPAT; Brain; (Rat)
1. Introduction In a series of experiments, we have examined the effects of the 5-hydroxytryptamine 1A (5-HT 1A ) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (Arvidsson et al., 1981; Hjorth et al., 1982) on spontaneous (open-field) and trained (treadmill) locomotion in the rat. It was found that the systemic administration of 8-OHDPAT, or local brain injections, in doses producing a characteristic stereotyped forward locomotion, did not cause any signs of motor incoordination or impairment, as assessed in a brief (2.5 min, 8 m min 21 ) treadmill test (Hillegaart et al., 1989a,b). However, the interesting possibility that these animals may, in fact, display an improved treadmill performance was not revealed under these conditions. There is evidence to suggest that physical exercise *Corresponding author. Presented at the VIIIth ECNP Congress, Venice, Italy, Sept 30–Oct 4 1995 (Ahlenius and Salmi, 1995b).
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results in increased brain 5-HT synthesis and turnover (see Chaouloff, 1989) and there appears to be a correlation between increased availability of brain 5-HT and the onset of fatigue in animals and man (Bailey et al., 1992, 1993a,b; Wilson and Maughan, 1992; Wilson and Marsden, 1996). Stimulation of 5-HT 1A autoreceptors in the somato-dendritic region, by means of local application of 8-OH-DPAT into midbrain raphe nuclei, has been shown to result in decreased firing (deMontigny et al., 1984; Sprouse and Aghajanian, 1988), synthesis (Hillegaart et al., 1990; Invernizzi et al., 1991) and release (Hutson et al., 1989; Bonvento et al., 1992) of forebrain 5-HT. These effects, produced by local brain infusions of 8-OH-DPAT, are similar to those seen after its systemic administration (see Dourish et al., 1987). Consequently, inhibition of central serotonergic neurotransmission, produced by 8-OHDPAT, could be expected to alleviate fatigue from physical exercise. The evidence presented above, prompted the present study where the systemic effects of 8-OH-DPAT on physical endurance performance were examined by testing
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rats on a treadmill, at a speed of 8 m min 21 . For comparison, the 5-HT 1A receptor antagonists (2)-pindolol (see Hoyer et al., 1994) and WAY-100,635 (Forster et al., 1995) were also included.
2. Materials and methods
2.1. Animals Adult male Wistar rats (280–320 g) were used (B&K Universal AB, Sollentuna, Sweden). The animals arrived in the laboratory at least 10 days before being used in experiments and were housed, five per cage (Makrolon IV), under controlled conditions of temperature (2160.48C), relative humidity (55–65%) and light–dark cycle (12:12 h, lights off 06.00 h). Food (R36, Ewos, ¨ ¨ Sodertalje, Sweden) and tap water were available ad libitum in the home cage.
Fig. 1. Schematic drawing of the treadmill equipment used in this study (from Ahlenius and Hillegaart, 1986).
2.2. Drugs
2.4. Statistics
(6)-8-Hydroxy-2-(di-n-propylamino)tetralin HBr, mol. wt. 328.29, (Research Biochemicals Incorporated, Natick, MA, USA), (2)-pindolol, mol. wt. 248.33 (RBI), (6) ´ betaxolol HCl, mol. wt. 343.90 (Synthelabo, Paris, France) and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2pyridinyl) cyclohexane carboxamide 3HCl, mol. wt. 549.97 (WAY-100,635, prepared for Astra Arcus). (2)Pindolol was dissolved in a minimal amount of glacial acetic acid and the final volume was made up with isotonic glucose. The other compounds were dissolved in physiological saline, and injections were made by the subcutaneous (s.c.) route, at a constant volume of 2 ml kg 21 . Controls received the appropriate vehicle.
Non-parametric procedures were used throughout. Thus, the median was used as the central value and the variability is shown as the semi-interquartile range. Statistical analysis was performed by means of the Kruskal-Wallis 2-way ANOVA, followed by the Mann-Whitney U-test for individual comparisons, as described in text and figures (see Siegel, 1956).
2.3. Treadmill locomotion
The administration of the 5-HT 1A receptor agonist 8OH-DPAT resulted in a biphasic pattern of effects on the endurance of treadmill performance in the rat. Thus, there was an increase in the duration of the treadmill performance at lower doses, with a statistically significant peak at 0.1 mg kg 21 , followed by a decrease in the performance that was statistically significantly below that of controls at the dose of 0.8 mg kg 21 (Fig. 2). WAY-100,635 tended to produce a biphasic pattern of effects, but in a direction opposite to that observed for 8-OH-DPAT, i.e., decreased performance at a low dose (12.5 mg kg 21 ), followed by a normalization of performance at higher doses (50–200 mg kg 21 ). However, the performance of animals given these highest two doses, although not statistically significant from controls, did not differ in statistical terms from the performance in the group of animals given the 12.5 mg dose (Fig. 3). Doses above 200 mg kg 21 produced a clear suppression of the treadmill performance (data not shown).
The animals were trained for two consecutive days to walk on a treadmill (see Fig. 1), at a speed of approximately 8 m min 21 (Ø5166 mm, 16 rpm) (see Ahlenius and Hillegaart, 1986). On days 1 and 2, the animals were trained for 3 min in the morning (09.00–12.00) and in the afternoon (15.00–17.00). Experiments were performed on the day immediately following training and all animals were subjected to a pre-test before being given any treatment. Those animals that failed to walk continuously for 3 min in this pre-test were excluded from the experiment. After being transferred to the observation room, the animals were allowed at least 15 min of habituation and were housed in a ventilated cabinet in-between training or testing sessions, providing the same environmental conditions as in the animal’s quarters. The time the animals remained on the treadmill was recorded up to a maximum of 40 min. Animals were used once only.
3. Results
3.1. Effects of 8 -OH-DPAT and WAY-100,635
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3.2. Effects of (2)-pindolol In the test model used here, the 5-HT 1A receptor antagonist (2)-pindolol produced effects that were similar to those described for 8-OH-DPAT above (Fig. 4). The duration of treadmill performance was enhanced with a maximal effect at 2 mg kg 21 and, within the dose range tested here (0.5–8.0 mg kg 21 ), never went below the performance level of vehicle-treated controls.
3.3. Antagonism by (2)-pindolol, but not betaxolol, of 8 -OH-DPAT-induced impairment of treadmill performance
Fig. 2. Effects of 8-OH-DPAT on endurance performance of rats on a treadmill. 8-OH-DPAT was administered s.c. 10 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 6–17 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, *P,0.05, ***P,0.01.
Fig. 3. Effects of WAY-100,635 on endurance performance of rats on a treadmill. WAY-100,635 was administered s.c. 10 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 8–14 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, **P,0.025.
Also in this experiment, 8-OH-DPAT (at a dose of 0.8 mg kg 21 ) produced a marked and statistically significant decrease in the duration of the treadmill performance, in comparison with vehicle-treated controls. (2)-Pindolol (2 mg kg 21 ) by itself produced an increased duration of the treadmill performance that was not statistically significant, although it was comparable numerically to results presented in Fig. 4. When combined with 8-OH-DPAT, (2)pindolol antagonized the 8-OH-DPAT-induced deficit in the treadmill performance. In fact, the animals treated with both (2)-pindolol and 8-OH-DPAT uniformly performed at maximal efficacy (Fig. 5). In contrast to effects produced
Fig. 4. Effects of (2)-pindolol on endurance performance of rats on a treadmill. (2)-Pindolol was administered s.c. 30 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 5–7 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, *P,0.05, **P,0.025.
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by (2)-pindolol, the administration of betaxolol (8 mg kg 21 ) did not antagonize the 8-OH-DPAT-induced deficit in treadmill performance, nor did betaxolol by itself affect the behavior (Fig. 6).
4. Discussion
Fig. 5. Antagonism by (2)-pindolol of the 8-OH-DPAT-induced decrease in endurance performance of rats on a treadmill. (2)-Pindolol (2 mg kg 21 s.c.) was administered 30 min, and 8-OH-DPAT (0.8 mg kg 21 s.c.) was administered 20 min, before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 5–12 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, ***P,0.01.
Fig. 6. Failure to antagonize the 8-OH-DPAT-induced decrease in endurance performance of rats on a treadmill by pretreatment with betaxolol. Betaxolol (8 mg kg 21 s.c.) was administered 30 min, and 8-OH-DPAT (0.8 mg kg 21 s.c.) was administered 20 min, before treadmill observations started. The figure shows medians6the semiinterquartile range, based on results from 5–8 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, ***P,0.01.
These results demonstrate that the systemic administration of the 5-HT 1A receptor agonist 8-OH-DPAT produces a biphasic pattern of effects on treadmill endurance performance in the rat. Thus, lower doses (about 0.1 mg kg 21 ) enhance, whereas higher doses (above 0.4 mg kg 21 ) reduce the time the animals stay on the treadmill, in comparison with the performance of saline-treated controls. It is suggested that this enhanced and impaired endurance performance produced by 8-OH-DPAT is due to the inhibition and stimulation of brain serotonergic neurotransmission, respectively. The improvement in endurance performance produced by low doses of 8-OH-DPAT is thus probably explained by inhibition of firing and by decreased synthesis and release of 5-HT in serotonergic neurons (see Section 1), resulting in less stimulation at postsynaptic 5-HT receptors, whereas the impaired performance at higher doses most likely is due to stimulation of postsynaptic 5-HT 1A receptors. In support of direct stimulation of postsynaptic 5-HT 1A receptors by 8-OH-DPAT, it has been shown that 8-OH-DPAT-induced serotonin behavioral syndrome, as well as locomotor hyperactivity in reserpinetreated animals, is sensitive to (2)-pindolol pretreatment (Tricklebank et al., 1984; Ahlenius and Salmi, 1995a). In further support of the interpretation that a low dose of 8-OH-DPAT produces improved treadmill performance through stimulation of inhibitory serotonergic autoreceptors, administration of the 5-HT 1A receptor antagonist WAY-100,635 resulted in an impaired performance. At higher doses, there was a tendency for a gradual improvement in performance, presumably reflecting blockade of postsynaptic 5-HT 1A receptors. The fact that these latter effects did not result in a performance that was improved above control levels may be related to a lack of receptor selectivity for WAY-100,635 with increasing doses. Thus, for example, dopamine receptor blocking properties appear as a prominent feature at doses at, or above, 200 mg kg 21 (Ahlenius et al., in preparation). On the other hand, it has been shown that administration of the 5-HT 2 receptor agonist, quipazine, to rats also results in an impaired performance, whereas an improved performance was seen with the 5-HT 2 receptor antagonist, LY-53,857 (Bailey et al., 1993b). Since the 5-HT 2 receptor is localized primarily on the postsynaptic neuron, the effects of these agents, in all probability, are related to stimulation and blockade of postsynaptic receptors, respectively (see Hoyer et al., 1994). It should be noted, however, that in reserpine-treated rats, the 8-OH-DPAT-
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induced hyperactivity could not be antagonized by pretreatment with the 5-HT 2A / C receptor antagonist, ritanserin (Ahlenius and Salmi, 1995a), but there is other evidence that some aspects of behavioral effects of 8-OH-DPAT, in normal animals, are partially sensitive to ritanserin (Hillegaart et al., 1996). Further studies are needed, however, to clarify the possible involvement of 5-HT 2 receptors in the effects reported here. The 5-HT 1A receptor antagonist, (2)-pindolol (see Hoyer et al., 1994), by itself, enhanced the treadmill performance in this study. This effect is probably due to the weak intrinsic activity of this agent at 5-HT 1A autoreceptors (Hjorth and Carlsson, 1985). At the same time, the impairment of treadmill performance caused by a high 21 dose of 8-OH-DPAT (0.8 mg kg ) was fully antagonized by pretreatment with (2)-pindolol. Somewhat unexpectedly, it appears that the combined (2)-pindolol–8-OH-DPAT treatment resulted in maximal performance, with less variability than that seen after administration of a low dose of either 8-OH-DPAT or (2)-pindolol by themselves. A possible explanation for this observation could be that the postsynaptic 5-HT 1 receptor blockade by (2)-pindolol is backed up by inhibition of serotonergic firing and release at the somato-dendritic level, where both 8-OH-DPAT and (2)-pindolol act as agonists. The fact that (2)-pindolol fully antagonized postsynaptic effects produced by a high dose of 8-OH-DPAT is in good agreement with available evidence (e.g. Tricklebank et al., 1984; Ahlenius and Salmi, 1995a). (2)-Pindolol is a b-blocker and is clinically used as an antihypertensive agent (see Hoffman and Lefkowitz, 1990). That such properties of (2)-pindolol did not directly influence the result obtained here is shown by the fact that a high dose (8 mg kg 21 ) of the selective bblocker, betaxolol (see Hoffman and Lefkowitz, 1990), did not, by itself, affect the treadmill performance, nor did it antagonize the effects produced by 8-OH-DPAT. It has been shown that 5-HT 1A receptor agonists affect glucose utilization (Chaouloff and Jeanrenaud, 1987; ¨ Uvnas-Moberg et al., 1996), and such effects could possibly interact with well-known effects of exercise on plasma levels of glucose and free fatty acids. The doses of 8-OH-DPAT needed to produce such an effect, however, appear to be much higher than the doses of 8-OH-DPAT (0.1 mg kg 21 s.c.) associated with increased endurance in the present study. Furthermore, in a recent study using 5-HT 2 receptor agonists and antagonists, the resulting effects on endurance performance on a treadmill appear to be unrelated to neuroendocrine effects (Bailey et al., 1993a). The findings of the latter study, together with results from a study on exercising volunteers who were taking the serotonin selective neuronal reuptake inhibitor, paroxetine (Wilson and Maughan, 1992), provide evidence to suggest that the observed, or perceived, fatigue is, in fact, central in origin. In some laboratory models, 8-OH-DPAT behaves as a
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partial dopamine D 2 receptor agonist (e.g. Ahlenius et al., 1989). Its intrinsic activity, however, is too low to stimulate motor activity in reserpine-treated rats (Ahlenius and Salmi, 1995a). Thus, although it is unclear if such properties of 8-OH-DPAT may play a role in the present context, it should be noted that the effects of 8-OH-DPAT, and other serotonergic agents, on treadmill performance, could be, at least partially, mediated indirectly via brain dopaminergic mechanisms (see Chaouloff, 1989).
5. Conclusion Stimulation of serotonergic 5-HT 1A cell body autoreceptors by means of the agonist, 8-OH-DPAT, or the partial agonist, (2)-pindolol, enhances endurance performance of rats on a treadmill, whereas an impairment was seen with the 5-HT 1A receptor antagonist WAY-100,635. With increasing doses, 8-OH-DPAT will also stimulate postsynaptic 5-HT 1A receptors, resulting in an impaired performance of the rat on the treadmill, with this effect being antagonized by pretreatment with (2)-pindolol. These results suggest that inhibition of brain serotonergic neurotransmission, obtained via activation of inhibitory autoreceptors, results in improved physical endurance. The results also provide an important example of a biphasic behavioral effect of a 5-HT 1A receptor agonist in the rat, possibly related to preferential stimulation of pre- or post-synaptic serotonergic receptors.
Acknowledgments ´ (6)-Betaxolol was generously supplied by Synthelabo, Paris, France. For expert and unfailing help with experimental animals, we would like to thank Mr Thomas Andersson and his staff at Astra Arcus AB. Figures were prepared using SigmaPlot (Jandel GmbH, Erkrath, Germany) and final prints were prepared by Lindfors and ¨ ¨ Andersson Photosetting, Sodertalje, Sweden.
References Ahlenius, S. and Hillegaart, V. (1986) Involvement of extrapyramidal motor mechanisms in the suppression of locomotor activity by antipsychotic drugs: A comparison between the effects produced by pre- and post-synaptic inhibition of dopaminergic neurotransmission. Pharmacol. Biochem. Behav. 24, 1409–1415. ¨ Ahlenius, S., Hillegaart, V. and Wijkstrom, A. (1989) Evidence for selective inhibition of limbic forebrain dopamine synthesis by 8-OHDPAT in the rat. Naunyn-Schmiedeberg’s Arch. Pharmacol. 339, 551–556. Ahlenius, S. and Salmi, P. (1995a) Antagonism of reserpine-induced suppression of spontaneous motor activity by stimulation of 5-HT 1A receptors in rats. Pharmacol. Toxicol. 76, 149–156. Ahlenius, S. and Salmi, P. (1995b) Enhanced duration of treadmill
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locomotion induced by 8-OH-DPAT in rats. Eur. Neuropsychopharmacol. 5, 403. Arvidsson, L.-E., Hacksell, U., Nilsson, J.L.G., Hjorth, S., Carlsson, A., ¨ H. (1981) 8-Hydroxy-2-(diLindberg, P., Sanchez, D. and Wikstrom, n-propylamino)tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist. J. Med. Chem. 24, 921–923. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1992) Effect of increased brain serotonergic activity on endurance performance in the rat. Acta Physiol. Scand. 145, 75–76. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1993a) Neuroendocrine and substrate responses to altered brain 5-HT activity during prolonged exercise to fatigue. J. Appl. Physiol. 74, 3006–3012. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1993b) Serotonergic agonists and antagonists affect endurance performance in the rat. Int. J. Sports Med. 14, 330–333. Bonvento, G., Scatton, B., Claustre, Y. and Rouquier, L. (1992) Effect of local injection of 8-OH-DPAT into the dorsal or median raphe nuclei on extracellular levels of serotonin in serotonergic projection areas in the rat brain. Neurosci. Lett. 137, 101–104. Chaouloff, F. (1989) Physical exercise and brain monoamines: A review. Acta Physiol. Scand. 137, 1–13. Chaouloff, F. and Jeanrenaud, B. (1987) 5-HT 1A and a 2 -adrenergic receptors mediate the hyperglycemic and hypoinsulinemic effects of 8-hydroxy-2-(di-n-propylamino)tetralin in the conscious rat. J. Pharmacol. Exp. Ther. 243, 1159–1166. Dourish, C.T., Ahlenius, S. and Hutson, P.H. (Eds.) (1987) Brain 5-HT 1A Receptors: Behavioral and Neurochemical Pharmacology. Ellis Horwood, Chichester. Forster, E.A., Cliffe, I.A., Bill, D.J., Dover, G.M., Jones D., Reilly, Y. and Fletcher, A. (1995) A pharmacological profile of the selective silent 5-HT 1A receptor antagonist, WAY-100635. Eur. J. Pharmacol. 281, 81–88. Hillegaart, V., Ahlenius, S. and Larsson, K. (1989a) Effects of local application of 5-HT into the median and dorsal raphe nuclei on male rat sexual and motor behavior. Behav. Brain Res. 33, 279–286. Hillegaart, V., Estival, A. and Ahlenius, S. (1996) Evidence for specific involvement of 5-HT 1A and 5-HT 2A / C receptors in the expression of patterns of spontaneous motor activity in the rat. Eur. J. Pharmacol. 295, 155–161. Hillegaart, V., Hjorth, S. and Ahlenius, S. (1990) Effects of 5-HT and 8-OH-DPAT on forebrain monoamine synthesis after local application into the median and dorsal raphe nuclei of the rat. J. Neural Transm. Gen. Sect. 81, 131–145. Hillegaart, V., Wadenberg, M.-L. and Ahlenius, S. (1989b) Effects of 8-OH-DPAT on motor activity in the rat. Pharmacol. Biochem. Behav. 32, 797–800. Hjorth, S. and Carlsson, A. (1985) (2)Pindolol stereospecifically inhibits rat brain serotonin (5-HT) synthesis. Neuropharmacology 24, 1143– 1146.
¨ Hjorth, S., Carlsson, A., Lindberg, P., Sanchez, D., Wikstrom, H., Arvidsson, L.-E., Hacksell, U. and Nilsson J.L.G. (1982) 8-Hydroxy2-(di-n-propylamino)-tetralin, 8-OH-DPAT, a potent and selective simplified ergot congener with central 5-HT-receptor stimulating activity. J. Neural Transm. 55, 169–188. Hoffman, B.B. and Lefkowitz, R.J. (1990) Adrenergic receptor antagonists. In: Goodman, A.G., T.W. Rall, A.S. Nies and P. Taylor (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics. Pergamon Press, New York, pp. 221–243. Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R. and Humphrey, P.A. (1994) VII. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol. Rev. 46, 157–203. Hutson, P.H., Sarna, G.S., O’Connell, M.T. and Curzon, G. (1989) Hippocampal 5-HT synthesis and release in vivo is decreased by infusion of 8-OH-DPAT into the nucleus raphe dorsalis. Neurosci. Lett. 100, 276–280. Invernizzi, R., Carli, M., Di Clemente, A. and Samanin, R. (1991) Administration of 8-hydroxy-2-(di-n-propylamino)tetralin in raphe nuclei dorsalis and medianus reduces serotonin synthesis in the rat brain: Differences in potency and regional sensitivity. J. Neurochem. 56, 243–247. deMontigny, C., Blier, P. and Chaput, Y. (1984) Electrophysiologically identified serotonin receptors in the rat CNS. Neuropharmacology 23, 1511–1520. Siegel, S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Sprouse, J.S. and Aghajanian, G.K. (1988) Responses of hippocampal pyramidal cells to putative serotonin 5-HT 1A and 5-HT 1B agonists: A comparative study with dorsal raphe neurons. Neuropharmacology 27, 707–715. Tricklebank, M.D., Forler, C. and Fozard, J.R. (1984) The involvement of subtypes of the 5-HT 1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat. Eur. J. Pharmacol. 106, 271–282. ¨ Uvnas-Moberg, K., Ahlenius, S., Alster, P. and Hillegaart, V. (1996) Effects of 5-HT and dopamine agonists, selective for different receptor subtypes, on glucose, insulin and glucagon plasma levels in the rat. Neuroendocrinology 63, 269–274. Wilson, W.M. and Marsden, C.A. (1996) In vivo measurement of extracellular serotonin in the ventral hippocampus during treadmill running. Behav. Pharmacol. 7, 101–104. Wilson, W.M. and Maughan, R.J. (1992) Evidence for a possible role of 5-hydroxytryptamine in the genesis of fatigue in man: Administration of paroxetine, a 5-HT re-uptake inhibitor, reduces the capacity to perform prolonged exercise. Exp. Physiol. 77, 921–924.
Biphasic effects of 8-OH-DPAT on endurance of treadmill performance in the male rat 1 Sven Ahlenius*, Petra Kaur, Peter Salmi ¨ ¨ , Sweden Biochemical and Behavioral Pharmacology, Astra Arcus AB, S-151 85 Sodertalje Received 27 February 1996; accepted 3 October 1996
Abstract Administration of the 5-HT 1A receptor agonist 8-OH-DPAT produced a biphasic pattern of effects on endurance performance of rats walking on top of a treadmill drum (Ø5166 mm, 16 rpm; ¯8 m min 21 ), with enhanced performance at a low dose (0.1 mg kg 21 s.c.) followed by impairment (0.2–0.8 mg kg 21 ). The partial 5-HT 1A receptor agonist (2)-pindolol improved the performance in the low dose range (0.5–2.0 mg kg 21 s.c.), whereas a higher dose (8 mg kg 21 ) was ineffective. The 5-HT 1A receptor antagonist WAY-100,635 produced an impaired performance at a low dose (12.5 mg kg 21 s.c.), with a recovery of performance at higher doses (50–200 mg kg 21 ). It is suggested that the inhibition of central serotonergic neurotransmission produced by stimulation or blockade of 5-HT 1A auto- and post-synaptic receptors, respectively, results in an improved endurance performance on the treadmill, whereas stimulation of postsynaptic 5-HT 1A receptors has the opposite action. In support of this contention, the impaired performance produced by a high dose of 8-OH-DPAT (0.8 mg kg 21 ) was antagonized by pretreatment with (2)-pindolol (2 mg kg 21 s.c.). 1997 Elsevier Science B.V. Keywords: Treadmill locomotion; Physical exercise; Serotonin receptors; 8-OH-DPAT; Brain; (Rat)
1. Introduction In a series of experiments, we have examined the effects of the 5-hydroxytryptamine 1A (5-HT 1A ) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (Arvidsson et al., 1981; Hjorth et al., 1982) on spontaneous (open-field) and trained (treadmill) locomotion in the rat. It was found that the systemic administration of 8-OHDPAT, or local brain injections, in doses producing a characteristic stereotyped forward locomotion, did not cause any signs of motor incoordination or impairment, as assessed in a brief (2.5 min, 8 m min 21 ) treadmill test (Hillegaart et al., 1989a,b). However, the interesting possibility that these animals may, in fact, display an improved treadmill performance was not revealed under these conditions. There is evidence to suggest that physical exercise *Corresponding author. Presented at the VIIIth ECNP Congress, Venice, Italy, Sept 30–Oct 4 1995 (Ahlenius and Salmi, 1995b).
1
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results in increased brain 5-HT synthesis and turnover (see Chaouloff, 1989) and there appears to be a correlation between increased availability of brain 5-HT and the onset of fatigue in animals and man (Bailey et al., 1992, 1993a,b; Wilson and Maughan, 1992; Wilson and Marsden, 1996). Stimulation of 5-HT 1A autoreceptors in the somato-dendritic region, by means of local application of 8-OH-DPAT into midbrain raphe nuclei, has been shown to result in decreased firing (deMontigny et al., 1984; Sprouse and Aghajanian, 1988), synthesis (Hillegaart et al., 1990; Invernizzi et al., 1991) and release (Hutson et al., 1989; Bonvento et al., 1992) of forebrain 5-HT. These effects, produced by local brain infusions of 8-OH-DPAT, are similar to those seen after its systemic administration (see Dourish et al., 1987). Consequently, inhibition of central serotonergic neurotransmission, produced by 8-OHDPAT, could be expected to alleviate fatigue from physical exercise. The evidence presented above, prompted the present study where the systemic effects of 8-OH-DPAT on physical endurance performance were examined by testing
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rats on a treadmill, at a speed of 8 m min 21 . For comparison, the 5-HT 1A receptor antagonists (2)-pindolol (see Hoyer et al., 1994) and WAY-100,635 (Forster et al., 1995) were also included.
2. Materials and methods
2.1. Animals Adult male Wistar rats (280–320 g) were used (B&K Universal AB, Sollentuna, Sweden). The animals arrived in the laboratory at least 10 days before being used in experiments and were housed, five per cage (Makrolon IV), under controlled conditions of temperature (2160.48C), relative humidity (55–65%) and light–dark cycle (12:12 h, lights off 06.00 h). Food (R36, Ewos, ¨ ¨ Sodertalje, Sweden) and tap water were available ad libitum in the home cage.
Fig. 1. Schematic drawing of the treadmill equipment used in this study (from Ahlenius and Hillegaart, 1986).
2.2. Drugs
2.4. Statistics
(6)-8-Hydroxy-2-(di-n-propylamino)tetralin HBr, mol. wt. 328.29, (Research Biochemicals Incorporated, Natick, MA, USA), (2)-pindolol, mol. wt. 248.33 (RBI), (6) ´ betaxolol HCl, mol. wt. 343.90 (Synthelabo, Paris, France) and N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2pyridinyl) cyclohexane carboxamide 3HCl, mol. wt. 549.97 (WAY-100,635, prepared for Astra Arcus). (2)Pindolol was dissolved in a minimal amount of glacial acetic acid and the final volume was made up with isotonic glucose. The other compounds were dissolved in physiological saline, and injections were made by the subcutaneous (s.c.) route, at a constant volume of 2 ml kg 21 . Controls received the appropriate vehicle.
Non-parametric procedures were used throughout. Thus, the median was used as the central value and the variability is shown as the semi-interquartile range. Statistical analysis was performed by means of the Kruskal-Wallis 2-way ANOVA, followed by the Mann-Whitney U-test for individual comparisons, as described in text and figures (see Siegel, 1956).
2.3. Treadmill locomotion
The administration of the 5-HT 1A receptor agonist 8OH-DPAT resulted in a biphasic pattern of effects on the endurance of treadmill performance in the rat. Thus, there was an increase in the duration of the treadmill performance at lower doses, with a statistically significant peak at 0.1 mg kg 21 , followed by a decrease in the performance that was statistically significantly below that of controls at the dose of 0.8 mg kg 21 (Fig. 2). WAY-100,635 tended to produce a biphasic pattern of effects, but in a direction opposite to that observed for 8-OH-DPAT, i.e., decreased performance at a low dose (12.5 mg kg 21 ), followed by a normalization of performance at higher doses (50–200 mg kg 21 ). However, the performance of animals given these highest two doses, although not statistically significant from controls, did not differ in statistical terms from the performance in the group of animals given the 12.5 mg dose (Fig. 3). Doses above 200 mg kg 21 produced a clear suppression of the treadmill performance (data not shown).
The animals were trained for two consecutive days to walk on a treadmill (see Fig. 1), at a speed of approximately 8 m min 21 (Ø5166 mm, 16 rpm) (see Ahlenius and Hillegaart, 1986). On days 1 and 2, the animals were trained for 3 min in the morning (09.00–12.00) and in the afternoon (15.00–17.00). Experiments were performed on the day immediately following training and all animals were subjected to a pre-test before being given any treatment. Those animals that failed to walk continuously for 3 min in this pre-test were excluded from the experiment. After being transferred to the observation room, the animals were allowed at least 15 min of habituation and were housed in a ventilated cabinet in-between training or testing sessions, providing the same environmental conditions as in the animal’s quarters. The time the animals remained on the treadmill was recorded up to a maximum of 40 min. Animals were used once only.
3. Results
3.1. Effects of 8 -OH-DPAT and WAY-100,635
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3.2. Effects of (2)-pindolol In the test model used here, the 5-HT 1A receptor antagonist (2)-pindolol produced effects that were similar to those described for 8-OH-DPAT above (Fig. 4). The duration of treadmill performance was enhanced with a maximal effect at 2 mg kg 21 and, within the dose range tested here (0.5–8.0 mg kg 21 ), never went below the performance level of vehicle-treated controls.
3.3. Antagonism by (2)-pindolol, but not betaxolol, of 8 -OH-DPAT-induced impairment of treadmill performance
Fig. 2. Effects of 8-OH-DPAT on endurance performance of rats on a treadmill. 8-OH-DPAT was administered s.c. 10 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 6–17 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, *P,0.05, ***P,0.01.
Fig. 3. Effects of WAY-100,635 on endurance performance of rats on a treadmill. WAY-100,635 was administered s.c. 10 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 8–14 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, **P,0.025.
Also in this experiment, 8-OH-DPAT (at a dose of 0.8 mg kg 21 ) produced a marked and statistically significant decrease in the duration of the treadmill performance, in comparison with vehicle-treated controls. (2)-Pindolol (2 mg kg 21 ) by itself produced an increased duration of the treadmill performance that was not statistically significant, although it was comparable numerically to results presented in Fig. 4. When combined with 8-OH-DPAT, (2)pindolol antagonized the 8-OH-DPAT-induced deficit in the treadmill performance. In fact, the animals treated with both (2)-pindolol and 8-OH-DPAT uniformly performed at maximal efficacy (Fig. 5). In contrast to effects produced
Fig. 4. Effects of (2)-pindolol on endurance performance of rats on a treadmill. (2)-Pindolol was administered s.c. 30 min before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 5–7 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, *P,0.05, **P,0.025.
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by (2)-pindolol, the administration of betaxolol (8 mg kg 21 ) did not antagonize the 8-OH-DPAT-induced deficit in treadmill performance, nor did betaxolol by itself affect the behavior (Fig. 6).
4. Discussion
Fig. 5. Antagonism by (2)-pindolol of the 8-OH-DPAT-induced decrease in endurance performance of rats on a treadmill. (2)-Pindolol (2 mg kg 21 s.c.) was administered 30 min, and 8-OH-DPAT (0.8 mg kg 21 s.c.) was administered 20 min, before treadmill observations started. The figure shows medians6the semi-interquartile range, based on results from 5–12 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, ***P,0.01.
Fig. 6. Failure to antagonize the 8-OH-DPAT-induced decrease in endurance performance of rats on a treadmill by pretreatment with betaxolol. Betaxolol (8 mg kg 21 s.c.) was administered 30 min, and 8-OH-DPAT (0.8 mg kg 21 s.c.) was administered 20 min, before treadmill observations started. The figure shows medians6the semiinterquartile range, based on results from 5–8 animals per group. Statistical differences from saline-treated controls are indicated in the figure. ns P.0.05, ***P,0.01.
These results demonstrate that the systemic administration of the 5-HT 1A receptor agonist 8-OH-DPAT produces a biphasic pattern of effects on treadmill endurance performance in the rat. Thus, lower doses (about 0.1 mg kg 21 ) enhance, whereas higher doses (above 0.4 mg kg 21 ) reduce the time the animals stay on the treadmill, in comparison with the performance of saline-treated controls. It is suggested that this enhanced and impaired endurance performance produced by 8-OH-DPAT is due to the inhibition and stimulation of brain serotonergic neurotransmission, respectively. The improvement in endurance performance produced by low doses of 8-OH-DPAT is thus probably explained by inhibition of firing and by decreased synthesis and release of 5-HT in serotonergic neurons (see Section 1), resulting in less stimulation at postsynaptic 5-HT receptors, whereas the impaired performance at higher doses most likely is due to stimulation of postsynaptic 5-HT 1A receptors. In support of direct stimulation of postsynaptic 5-HT 1A receptors by 8-OH-DPAT, it has been shown that 8-OH-DPAT-induced serotonin behavioral syndrome, as well as locomotor hyperactivity in reserpinetreated animals, is sensitive to (2)-pindolol pretreatment (Tricklebank et al., 1984; Ahlenius and Salmi, 1995a). In further support of the interpretation that a low dose of 8-OH-DPAT produces improved treadmill performance through stimulation of inhibitory serotonergic autoreceptors, administration of the 5-HT 1A receptor antagonist WAY-100,635 resulted in an impaired performance. At higher doses, there was a tendency for a gradual improvement in performance, presumably reflecting blockade of postsynaptic 5-HT 1A receptors. The fact that these latter effects did not result in a performance that was improved above control levels may be related to a lack of receptor selectivity for WAY-100,635 with increasing doses. Thus, for example, dopamine receptor blocking properties appear as a prominent feature at doses at, or above, 200 mg kg 21 (Ahlenius et al., in preparation). On the other hand, it has been shown that administration of the 5-HT 2 receptor agonist, quipazine, to rats also results in an impaired performance, whereas an improved performance was seen with the 5-HT 2 receptor antagonist, LY-53,857 (Bailey et al., 1993b). Since the 5-HT 2 receptor is localized primarily on the postsynaptic neuron, the effects of these agents, in all probability, are related to stimulation and blockade of postsynaptic receptors, respectively (see Hoyer et al., 1994). It should be noted, however, that in reserpine-treated rats, the 8-OH-DPAT-
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induced hyperactivity could not be antagonized by pretreatment with the 5-HT 2A / C receptor antagonist, ritanserin (Ahlenius and Salmi, 1995a), but there is other evidence that some aspects of behavioral effects of 8-OH-DPAT, in normal animals, are partially sensitive to ritanserin (Hillegaart et al., 1996). Further studies are needed, however, to clarify the possible involvement of 5-HT 2 receptors in the effects reported here. The 5-HT 1A receptor antagonist, (2)-pindolol (see Hoyer et al., 1994), by itself, enhanced the treadmill performance in this study. This effect is probably due to the weak intrinsic activity of this agent at 5-HT 1A autoreceptors (Hjorth and Carlsson, 1985). At the same time, the impairment of treadmill performance caused by a high 21 dose of 8-OH-DPAT (0.8 mg kg ) was fully antagonized by pretreatment with (2)-pindolol. Somewhat unexpectedly, it appears that the combined (2)-pindolol–8-OH-DPAT treatment resulted in maximal performance, with less variability than that seen after administration of a low dose of either 8-OH-DPAT or (2)-pindolol by themselves. A possible explanation for this observation could be that the postsynaptic 5-HT 1 receptor blockade by (2)-pindolol is backed up by inhibition of serotonergic firing and release at the somato-dendritic level, where both 8-OH-DPAT and (2)-pindolol act as agonists. The fact that (2)-pindolol fully antagonized postsynaptic effects produced by a high dose of 8-OH-DPAT is in good agreement with available evidence (e.g. Tricklebank et al., 1984; Ahlenius and Salmi, 1995a). (2)-Pindolol is a b-blocker and is clinically used as an antihypertensive agent (see Hoffman and Lefkowitz, 1990). That such properties of (2)-pindolol did not directly influence the result obtained here is shown by the fact that a high dose (8 mg kg 21 ) of the selective bblocker, betaxolol (see Hoffman and Lefkowitz, 1990), did not, by itself, affect the treadmill performance, nor did it antagonize the effects produced by 8-OH-DPAT. It has been shown that 5-HT 1A receptor agonists affect glucose utilization (Chaouloff and Jeanrenaud, 1987; ¨ Uvnas-Moberg et al., 1996), and such effects could possibly interact with well-known effects of exercise on plasma levels of glucose and free fatty acids. The doses of 8-OH-DPAT needed to produce such an effect, however, appear to be much higher than the doses of 8-OH-DPAT (0.1 mg kg 21 s.c.) associated with increased endurance in the present study. Furthermore, in a recent study using 5-HT 2 receptor agonists and antagonists, the resulting effects on endurance performance on a treadmill appear to be unrelated to neuroendocrine effects (Bailey et al., 1993a). The findings of the latter study, together with results from a study on exercising volunteers who were taking the serotonin selective neuronal reuptake inhibitor, paroxetine (Wilson and Maughan, 1992), provide evidence to suggest that the observed, or perceived, fatigue is, in fact, central in origin. In some laboratory models, 8-OH-DPAT behaves as a
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partial dopamine D 2 receptor agonist (e.g. Ahlenius et al., 1989). Its intrinsic activity, however, is too low to stimulate motor activity in reserpine-treated rats (Ahlenius and Salmi, 1995a). Thus, although it is unclear if such properties of 8-OH-DPAT may play a role in the present context, it should be noted that the effects of 8-OH-DPAT, and other serotonergic agents, on treadmill performance, could be, at least partially, mediated indirectly via brain dopaminergic mechanisms (see Chaouloff, 1989).
5. Conclusion Stimulation of serotonergic 5-HT 1A cell body autoreceptors by means of the agonist, 8-OH-DPAT, or the partial agonist, (2)-pindolol, enhances endurance performance of rats on a treadmill, whereas an impairment was seen with the 5-HT 1A receptor antagonist WAY-100,635. With increasing doses, 8-OH-DPAT will also stimulate postsynaptic 5-HT 1A receptors, resulting in an impaired performance of the rat on the treadmill, with this effect being antagonized by pretreatment with (2)-pindolol. These results suggest that inhibition of brain serotonergic neurotransmission, obtained via activation of inhibitory autoreceptors, results in improved physical endurance. The results also provide an important example of a biphasic behavioral effect of a 5-HT 1A receptor agonist in the rat, possibly related to preferential stimulation of pre- or post-synaptic serotonergic receptors.
Acknowledgments ´ (6)-Betaxolol was generously supplied by Synthelabo, Paris, France. For expert and unfailing help with experimental animals, we would like to thank Mr Thomas Andersson and his staff at Astra Arcus AB. Figures were prepared using SigmaPlot (Jandel GmbH, Erkrath, Germany) and final prints were prepared by Lindfors and ¨ ¨ Andersson Photosetting, Sodertalje, Sweden.
References Ahlenius, S. and Hillegaart, V. (1986) Involvement of extrapyramidal motor mechanisms in the suppression of locomotor activity by antipsychotic drugs: A comparison between the effects produced by pre- and post-synaptic inhibition of dopaminergic neurotransmission. Pharmacol. Biochem. Behav. 24, 1409–1415. ¨ Ahlenius, S., Hillegaart, V. and Wijkstrom, A. (1989) Evidence for selective inhibition of limbic forebrain dopamine synthesis by 8-OHDPAT in the rat. Naunyn-Schmiedeberg’s Arch. Pharmacol. 339, 551–556. Ahlenius, S. and Salmi, P. (1995a) Antagonism of reserpine-induced suppression of spontaneous motor activity by stimulation of 5-HT 1A receptors in rats. Pharmacol. Toxicol. 76, 149–156. Ahlenius, S. and Salmi, P. (1995b) Enhanced duration of treadmill
94
S. Ahlenius et al. / European Neuropsychopharmacology 7 (1997) 89 – 94
locomotion induced by 8-OH-DPAT in rats. Eur. Neuropsychopharmacol. 5, 403. Arvidsson, L.-E., Hacksell, U., Nilsson, J.L.G., Hjorth, S., Carlsson, A., ¨ H. (1981) 8-Hydroxy-2-(diLindberg, P., Sanchez, D. and Wikstrom, n-propylamino)tetralin, a new centrally acting 5-hydroxytryptamine receptor agonist. J. Med. Chem. 24, 921–923. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1992) Effect of increased brain serotonergic activity on endurance performance in the rat. Acta Physiol. Scand. 145, 75–76. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1993a) Neuroendocrine and substrate responses to altered brain 5-HT activity during prolonged exercise to fatigue. J. Appl. Physiol. 74, 3006–3012. Bailey, S.P., Davis, J.M. and Ahlborn, E.N. (1993b) Serotonergic agonists and antagonists affect endurance performance in the rat. Int. J. Sports Med. 14, 330–333. Bonvento, G., Scatton, B., Claustre, Y. and Rouquier, L. (1992) Effect of local injection of 8-OH-DPAT into the dorsal or median raphe nuclei on extracellular levels of serotonin in serotonergic projection areas in the rat brain. Neurosci. Lett. 137, 101–104. Chaouloff, F. (1989) Physical exercise and brain monoamines: A review. Acta Physiol. Scand. 137, 1–13. Chaouloff, F. and Jeanrenaud, B. (1987) 5-HT 1A and a 2 -adrenergic receptors mediate the hyperglycemic and hypoinsulinemic effects of 8-hydroxy-2-(di-n-propylamino)tetralin in the conscious rat. J. Pharmacol. Exp. Ther. 243, 1159–1166. Dourish, C.T., Ahlenius, S. and Hutson, P.H. (Eds.) (1987) Brain 5-HT 1A Receptors: Behavioral and Neurochemical Pharmacology. Ellis Horwood, Chichester. Forster, E.A., Cliffe, I.A., Bill, D.J., Dover, G.M., Jones D., Reilly, Y. and Fletcher, A. (1995) A pharmacological profile of the selective silent 5-HT 1A receptor antagonist, WAY-100635. Eur. J. Pharmacol. 281, 81–88. Hillegaart, V., Ahlenius, S. and Larsson, K. (1989a) Effects of local application of 5-HT into the median and dorsal raphe nuclei on male rat sexual and motor behavior. Behav. Brain Res. 33, 279–286. Hillegaart, V., Estival, A. and Ahlenius, S. (1996) Evidence for specific involvement of 5-HT 1A and 5-HT 2A / C receptors in the expression of patterns of spontaneous motor activity in the rat. Eur. J. Pharmacol. 295, 155–161. Hillegaart, V., Hjorth, S. and Ahlenius, S. (1990) Effects of 5-HT and 8-OH-DPAT on forebrain monoamine synthesis after local application into the median and dorsal raphe nuclei of the rat. J. Neural Transm. Gen. Sect. 81, 131–145. Hillegaart, V., Wadenberg, M.-L. and Ahlenius, S. (1989b) Effects of 8-OH-DPAT on motor activity in the rat. Pharmacol. Biochem. Behav. 32, 797–800. Hjorth, S. and Carlsson, A. (1985) (2)Pindolol stereospecifically inhibits rat brain serotonin (5-HT) synthesis. Neuropharmacology 24, 1143– 1146.
¨ Hjorth, S., Carlsson, A., Lindberg, P., Sanchez, D., Wikstrom, H., Arvidsson, L.-E., Hacksell, U. and Nilsson J.L.G. (1982) 8-Hydroxy2-(di-n-propylamino)-tetralin, 8-OH-DPAT, a potent and selective simplified ergot congener with central 5-HT-receptor stimulating activity. J. Neural Transm. 55, 169–188. Hoffman, B.B. and Lefkowitz, R.J. (1990) Adrenergic receptor antagonists. In: Goodman, A.G., T.W. Rall, A.S. Nies and P. Taylor (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics. Pergamon Press, New York, pp. 221–243. Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R. and Humphrey, P.A. (1994) VII. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol. Rev. 46, 157–203. Hutson, P.H., Sarna, G.S., O’Connell, M.T. and Curzon, G. (1989) Hippocampal 5-HT synthesis and release in vivo is decreased by infusion of 8-OH-DPAT into the nucleus raphe dorsalis. Neurosci. Lett. 100, 276–280. Invernizzi, R., Carli, M., Di Clemente, A. and Samanin, R. (1991) Administration of 8-hydroxy-2-(di-n-propylamino)tetralin in raphe nuclei dorsalis and medianus reduces serotonin synthesis in the rat brain: Differences in potency and regional sensitivity. J. Neurochem. 56, 243–247. deMontigny, C., Blier, P. and Chaput, Y. (1984) Electrophysiologically identified serotonin receptors in the rat CNS. Neuropharmacology 23, 1511–1520. Siegel, S. (1956) Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Sprouse, J.S. and Aghajanian, G.K. (1988) Responses of hippocampal pyramidal cells to putative serotonin 5-HT 1A and 5-HT 1B agonists: A comparative study with dorsal raphe neurons. Neuropharmacology 27, 707–715. Tricklebank, M.D., Forler, C. and Fozard, J.R. (1984) The involvement of subtypes of the 5-HT 1 receptor and of catecholaminergic systems in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat. Eur. J. Pharmacol. 106, 271–282. ¨ Uvnas-Moberg, K., Ahlenius, S., Alster, P. and Hillegaart, V. (1996) Effects of 5-HT and dopamine agonists, selective for different receptor subtypes, on glucose, insulin and glucagon plasma levels in the rat. Neuroendocrinology 63, 269–274. Wilson, W.M. and Marsden, C.A. (1996) In vivo measurement of extracellular serotonin in the ventral hippocampus during treadmill running. Behav. Pharmacol. 7, 101–104. Wilson, W.M. and Maughan, R.J. (1992) Evidence for a possible role of 5-hydroxytryptamine in the genesis of fatigue in man: Administration of paroxetine, a 5-HT re-uptake inhibitor, reduces the capacity to perform prolonged exercise. Exp. Physiol. 77, 921–924.