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Pharmacological characterization of release-regulating serotonin autoreceptors in rat cerebellum
317
European Journal of Pharmacology, 126 (1986) 317-321
Elsevier
Short communication P H A R M A C O L O G I C A L CHARACTERIZATION OF R E L E A S E - R E G U L A T I N G S E R O T O N I N A U T O R E C E P T O R S IN RAT C E R E B E L L U M GIAMBATTISTABONANNO, GUIDO MAURA and MAURIZIO RAITERI * lstituto di Farmacologia e Farmacognosia, Unioersitit di Genova, Viale Cembrano 4, 16148 Genooa, Italy
Received 15 May 1986, accepted 27 May 1986
G. BONANNO, G. MAURA and M. RAITERI, Pharmacological characterization of release-regulating serotonin autoreceptors in rat cerebellum, European J. Pharmacol. 126 (1986) 317-321. The release of [3H]5-hydroxytryptamine ([3H]5-HT) evoked by 15 mM KCI in superfused rat cerebellum synaptosomes was inhibited by 5-HT (pEC30 = 8.73). Methiothepin antagonized 5-HT (PA2= 9.28); ketanserin, methysergide, cinanserin and spiperone were ineffective. The receptors involved were activated (pEC30 = 8.90) by the 5-HT1 agonist 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-lH-indole (RU 24969). (-)Propranolol shifted to the right (pA 2 = 8.05) the dose-response curve of 5-HT. The 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was ineffective. In conclusion, autoreceptors are present on 5-HT nerve endings in rat cerebellum and appear to belong to the 5-HTIB subtype. Serotonin autoreceptors Rat cerebellum
Serotonin release
5-HT receptor subtypes
1. Introduction Studies with rat brain synaptosomes (Cerrito and Raiteri, 1979) and slices (G~Sthert and Weinheimer, 1979) showed that serotonin (5-HT) can inhibit its own release through the activation of autoreceptors located on serotonergic axon terminals. Evidence is now accumulating for the existence of different 5-HT receptor types and subtypes in the central nervous system. In particular, the 5-HT 1 binding sites were reported to be heterogeneous and could be subdivided into 5-HTIA , 5-HTIB and 5-HT1c (see, for instance, Hoyer et al., 1985). The possibility that this multiplicity of binding sites reflects the existence of multiple 5-HT receptors has to be investigated in studies in which receptor-mediated functional responses are measured. Although 5-HT autoreceptors have been identified in various areas of the rat brain including * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Superfused synaptosomes
hypothalamus, cerebral cortex and spinal cord they have been characterized pharmacologically in terms of 5-HT receptor subtype only in the cerebral cortex (Middlemiss, 1984; Engel et al., 1986). The cerebellum of the rat contains the lowest levels of 5-HT binding sites (Peroutka and Snyder, 1981). However, fluorescent autoradiographic and immunohistochemical studies have revealed 5-HT fibers and varicosities in the cerebellar cortex of the rat. Contrary to classical views suggesting that raphe neurons are the source of 5-HT to the cerebellum, it has been reported recently (Bishop and Ho, 1985) that in rat cerebellum the majority of the serotonergic afferents do not originate in the raphe nuclei but in the nucleus reticularis gigantocellularis, the nucleus reticularis paragigantocellularis and the nucleus reticularis pontis oralis, three discrete areas of the brainstem reticular formation. The paucity of 5-HT binding sites and the possible different source of innervation with respect to other regions of the CNS prompted us to investigate the existence of 5-HT autoreceptors on serotonergic nerve terminals in rat cerebel-
318 lum and to determine their pharmacological properties as subtypes of the 5-HT receptor.
2. Materials and methods
2.1. Preparation of synaptosomes Crude synaptosomes were prepared as previously described (Raiteri et al., 1983). Briefly, adult male Sprague-Dawley rats (200-250 g) were killed by decapitation; the cerebellum was rapidly removed and homogenized in 40 volumes of 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was centrifuged (5 min, 1000 × g) and synaptosomes were isolated from the supernatant by centrifugation at 12 000 × g for 20 min. The pellet was then resuspended in a physiological salt solution having the following composition (mM): NaC1 125, KC1 3, MgSO4, 1.2, CaC12 1.2, NaHEPO 4 1.0, NaHCO 3 22, glucose 10 (aeration with 95% 02 and 5% CO 2 at 37°C); pH 7.2-7.4.
2.2. Experiments of release Synaptosomes were incubated 15 rain at 37°C in an atmosphere of 95% 02 and 5% C O 2 with [3H]5-HT (final concentration 0.06 /~M). After incubation, aliquots of the synaptosomal suspensions were distributed into parallel superfusion chambers thermostated at 37°C (Raiteri et al., 1983) and superfusion was started, at a rate of 0.6 ml/min, with standard medium aerated with 5% CO 2 in 02. After 20 min of equilibration, collection of superfusate samples was started (t = 0) according to the following scheme: one single fraction from t = 0 to t = 17 then one fraction for 2 min from t = 17 to t = 19 (basal release) and one 2 min fraction from t - - 2 0 to t = 22, corresponding to the maximal release observed during high K + exposure (15 mM KC1, substituting an equimolar concentration of NaC1, was introduced at t = 18). When 5-HT was used as an agonist, citalopram (a 5-HT uptake inhibitor) was present from the beginning of superfusion. The antagonists were added 8 min before depolarization and the agonists were added concomitantly with high K +. The amount of radioactivity released into each
fraction was expressed as a percentage of the total synaptosomal tritium at the start of the fraction considered. The K+-evoked release was calculated by subtracting the basal release from the post-depolarization fraction having the highest tritium content. Under the experimental conditions described the radioactivity released by high K + consisted for the major part of unmetabolized [3H]5-HT, as shown previously by chromatographic analysis (Raiteri et al., 1983). Therefore, in the remainder of the text, we refer to K+-evoked tritium release over baseline as K+-evoked [3H]5-HT release. The pA 2 values of the antagonists were calculated according to Furchgott (1972).
2.3. Drugs and chemicals [1,2-3H(N)]5-HT creatinine sulfate (spec. act. 30.1 Ci/mmol) was obtained from New England Nuclear (Boston, MA). 8-OH-DPAT was purchased from RBI (Wayland, MA), 5-HT creatinine sulfate from Calbiochem (Los Angeles, CA). The following compounds were gifts: 8-OHDPAT (Dr. D. Middlemiss, Merrell, Strasbourg, France), RU 24969 (Roussel Uclaf, Romainville, France), citalopram (Lundbeck, Copenhagen, Denmark), methiothepin (Hoffman-La Roche, Basel, Switzerland), methysergide (Sandoz, Basel, Switzerland), ketanserin and spiperone (Janssen, Beerse, Belgium), (-)propranolol (ICI, MacClasfield,. Great Britain), cinanserin (Squibb, Princeton, N J).
3. Results
3.1. Effects of 5-HT and of 5-HT antagonists on the K +-evoked [3H]5-HT release After labeling with [3H]5-HT, cerebellar synaptosomes were depolarized in superfusion with 15 mM KC1. The K+-induced release of [3H]5-HT was completely C a 2 +-dependent (not shown). When prelabeled synaptosomes were exposed to exogenous 5-HT, the K+-evoked release of [3H]5-HT was reduced in a concentration-dependent way (fig. 1). The pEC30 value (negative loga-
319 6O
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.-T--
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I 0001 0.01 0.03 0.1 . . . . i 0 .0 0 1
i 0.01
i 0.1
Serotonin concentration (pM) Fig. 1. Concentration-dependent inhibition by 5-HT of the K+-evoked release of [3H]5-HT: effects of methiothepin and of (-)propranolol. Crude synaptosomes were incubated 15 min at 37°C with [3H]5-HT and superfused as described under Methods. Depolarization was carried out with 15 mM KC1. Exogenous 5-HT was added concomitantly with 15 mM KC1. The antagonists were present starting 8 rain before depolarization. Fractions of superfusate were collected and analyzed as described in detail in the Methods section. Means +S.E.M, of 6-8 experiments run in triplicate are presented. (©) 5-HT; (12) 5-HT+0.006/tM methiothepin; (zx)5-HT+0.1 /~M ( - )propranolol.
r i t h m of the c o n c e n t r a t i o n causing 30% i n h i b i t i o n o f the K ÷ - e v o k e d release) a m o u n t e d to 8.73. T h e c o n c e n t r a t i o n - r e s p o n s e curve of 5 - H T was shifted to the right b y m e t h i o t h e p i n in a p a r a l l e l way. T h e a p p a r e n t p A 2 value of m e t h i o t h e p i n was calcul a t e d from the curves shown in the figure at the level of the EC30 values of 5 - H T (which represent r o u g h l y the h a l f - m a x i m u m effect) a n d a m o u n t e d to 9.28. K e t a n s e r i n , methysergide, cinancerin or spipero n e c o u l d n o t c o u n t e r a c t 5 - H T even at concentrations two o r d e r s of m a g n i t u d e higher t h a n that of the agonist ( d a t a n o t shown).
3.2. Effects of R U 24969 and of 8-OH-DPA T at the cerebellar autoreceptors F i g u r e 2 illustrates the effects of these two 5 - H T r e c e p t o r agonists o n the K + - e v o k e d release of [3H]5-HT. A c o n c e n t r a t i o n - d e p e n d e n t inhibi-
0.1 03
---
RU 24969 Methiothepin
1
8 - OH - D PAT
Drug concentration(pM) Fig. 2. Effect of RU 24969 and of 8-OH-DPAT on the K +evoked release of [3H]5-HT. Experimental details as in the legend to fig. 1. The data presented are means + S.E.M. of 6-8 experiments in triplicate.
tion of release was o b s e r v e d with R U 24969 (pEC30 = 8.90). In contrast, 8 - O H - D P A T was ineffective at 1 # M , a c o n c e n t r a t i o n 1000 times higher than that p r o d u c i n g , in the case of 5 - H T or R U 24969, a b o u t 25-30% i n h i b i t i o n of [3H]5-HT release.
3.3. Effect of (-)propranolol on the inhibition of [3H]5-HT release by 5-HT W h e n ( - ) p r o p r a n o l o l was used as a 5 - H T 1 a n t a g o n i s t the c o n c e n t r a t i o n - r e s p o n s e curve of 5 - H T was shifted to the right in a parallel w a y (fig. 1). T h e a p p a r e n t p A 2 values for ( - ) p r o p r a n o l o l a m o u n t e d to 8.05. C o n c e n t r a t i o n s of ( - ) p r o p r a n o l o l higher t h a n 0.5 /xM caused a decrease of the K + - e v o k e d [3H]5-HT release (not shown).
4. D i s c u s s i o n
R e c e p t o r s in the central nervous system have b e e n studied f u n c t i o n a l l y b y a p p l y i n g various exp e r i m e n t a l a p p r o a c h e s in which a r e c e p t o r - m e d i a ted response is m e a s u r e d . T r a n s m i t t e r release can b e e x p l o i t e d as a f u n c t i o n a l test in the identification a n d c h a r a c t e r i z a t i o n of C N S receptors. In
320
particular, the method used in the present work has allowed the identification and characterization of 5-HT autoreceptors in a region, the cerebellum, which is very poor in 5-HT binding sites. Serotonergic afferents originating largely from the medullary and pontine reticular formation reach the cerebellar cortex (Bishop and Ho, 1985). It was shown by autoradiography that some of these projections terminate in the granule cell layer, where they appear to synapse on granule cell dendrites. The finding that 5-HT receptors are present on isolated glutamatergic nerve terminals (Maura et al., in press) may suggest that 5-HT fibers also reach the molecular layer, where glutamic acid appears to be released from the parallel fibers. The results of the present investigation show that release-regulating, methiothepin-sensitive 5HT autoreceptors exist in rat cerebellum and are located on serotonergic nerve endings. Ketanserin, a sPecific ligand for the 5-HT2 sites could not antagonize 5-HT. Methysergide, cinanserin and spiperone which have been considered by many investigators as preferential 5-HT2 antagonists, although with somewhat less selectivity, behaved like ketanserin. Thus, the autoreceptors in rat cerebellum do not belong to the 5-HT2 type. Being activated by nanomolar concentrations of 5-HT, they could be considered as 5-HT1 receptors. The existence in the CNS of three subtypes of the 5-HT1 site, named 5-HTIA, 5-HT1B and 5-HTlc has been proposed on the basis of binding studies (Hoyer et al., 1985). Recently, 8-OH-DPAT was reported to selectively displace [3H]5-HT from the 5-HTIA site in the rat frontal cortex (Middlemiss and Fozard, 1983). This compound was, however, ineffective at the 5-HT autoreceptors regulating 5-HT release from cerebellar synaptosomes, similarly to what had been found with cortical slices (Middlemiss, 1984). On the other hand, (-)propranolol antagonized 5-HT at the autoreceptors in slices of cerebral cortex (Middlemiss, 1984) and in synaptosomes from cerebellum (fig. 1). This compound had been found to stereoselectively displace [3H]5-HT from the 5-HT~ sites showing equal and high affinity for the 5 - H T I A and the 5-HT m subsites, but low affinity for the 5-HTlc (Hoyer et al., 1985).
Taken together, the results allow the conclusion that the release-regulating 5-HT autoreceptor in rat cerebellum is pharmacologically similar to that in the cerebral cortex and, therefore, in analogy to what has been proposed for the latter (Middlemiss, 1984; Engel et al., 1986), could be classified as a 5-HTm receptor subtype. Interestingly, the 5-HT receptors sited on glutamatergic terminals in rat cerebellum and which are postsynaptic with respect to the 5-HT autoreceptors also belong to the 5-HT1 type but not to the 5-HTla subtype (Maura et al., in press).
Acknowledgements This work was supported by grants from the Italian National Research Council and from the Italian Ministry of Education. The authors with to thank Miss Graziella Pellegrini for technical assistance and Mrs. Maura Agate for preparing the typescript.
References Bishop, G.A. and R.H. Ho, 1985, The distribution and origin of serotonin immunoreactivity in the rat cerebellum, Brain Res. 331, 195. Cerrito, F. and M. Raiteri, 1979, Serotonin release is modulated by presynaptic autoreceptors, European J. Pharmacol. 57, 427. Engel, G., M. Grthert, D. Hoyer, E. Schlicker and K. Hillenbrand, 1986, Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1a binding sites, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 1. Furchgott, R.F., 1972, The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory, in: Handbook of Experimental Pharmacology. Catecholamines, Vol. XXXIII, eds. H. Blaschko and E. Muscholl (Springer, Berlin, Heidelberg, New York) p. 283. G~thert, M. and G. Weinheimer, 1979, Extraceilular 5-hydroxytryptamine inhibits 5-hydroxytryptamine release from rat brain cortex slices, Naunyn-Schmiedeb. Arch. Pharmacol. 310, 93. Hoyer, D., G. Engel and H.O. Kaikman, 1985, Characterization of the 5-HT1B recognition site in rat brain: binding studies with (-)[125H]iodocyanopindolol, European J. Pharmacol. 118, 1. Maura, G., A. Ricchetti and M. Raiteri, Serotonin inhibits the depolarization-evoked release of endogenous glutamate from rat cerebellar nerve endings, Neurosci. Lett. (in press).
321 Middlemiss, D.N., 1984, 8-Hydroxy-2-(di-n-propylamino)tetralin is devoid of activity at the 5-hydroxytryptamine autoreceptor in rat brain. Implications for the proposed link between the autoreceptor and the [3H]5-HT recognition site, Naunyn-Schmiedeb. Arch. Pharmacol. 327, 18. Middlemiss, D.N. and J.R. Fozard, 1983, 8-Hydroxy-2-(di-npropylamino)-tetralin discriminates between subtypes of the 5-HT 1 recognition site, European J. Pharmacol. 90, 151.
Peroutka, S.J. and S.H. Snyder, 1981, Two distinct serotonin receptors: regional variations in receptor binding in mammalian brain, Brain Res. 208, 339. Raiteri, M., G. Maura and P. Versace, 1983, Functional evidence for two stereochemically different alpha-2 adrenoceptors regulating central norepinephrine and serotonin release, J. Pharmacol. Exp. Ther. 224, 679.
European Journal of Pharmacology, 126 (1986) 317-321
Elsevier
Short communication P H A R M A C O L O G I C A L CHARACTERIZATION OF R E L E A S E - R E G U L A T I N G S E R O T O N I N A U T O R E C E P T O R S IN RAT C E R E B E L L U M GIAMBATTISTABONANNO, GUIDO MAURA and MAURIZIO RAITERI * lstituto di Farmacologia e Farmacognosia, Unioersitit di Genova, Viale Cembrano 4, 16148 Genooa, Italy
Received 15 May 1986, accepted 27 May 1986
G. BONANNO, G. MAURA and M. RAITERI, Pharmacological characterization of release-regulating serotonin autoreceptors in rat cerebellum, European J. Pharmacol. 126 (1986) 317-321. The release of [3H]5-hydroxytryptamine ([3H]5-HT) evoked by 15 mM KCI in superfused rat cerebellum synaptosomes was inhibited by 5-HT (pEC30 = 8.73). Methiothepin antagonized 5-HT (PA2= 9.28); ketanserin, methysergide, cinanserin and spiperone were ineffective. The receptors involved were activated (pEC30 = 8.90) by the 5-HT1 agonist 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-lH-indole (RU 24969). (-)Propranolol shifted to the right (pA 2 = 8.05) the dose-response curve of 5-HT. The 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was ineffective. In conclusion, autoreceptors are present on 5-HT nerve endings in rat cerebellum and appear to belong to the 5-HTIB subtype. Serotonin autoreceptors Rat cerebellum
Serotonin release
5-HT receptor subtypes
1. Introduction Studies with rat brain synaptosomes (Cerrito and Raiteri, 1979) and slices (G~Sthert and Weinheimer, 1979) showed that serotonin (5-HT) can inhibit its own release through the activation of autoreceptors located on serotonergic axon terminals. Evidence is now accumulating for the existence of different 5-HT receptor types and subtypes in the central nervous system. In particular, the 5-HT 1 binding sites were reported to be heterogeneous and could be subdivided into 5-HTIA , 5-HTIB and 5-HT1c (see, for instance, Hoyer et al., 1985). The possibility that this multiplicity of binding sites reflects the existence of multiple 5-HT receptors has to be investigated in studies in which receptor-mediated functional responses are measured. Although 5-HT autoreceptors have been identified in various areas of the rat brain including * To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
Superfused synaptosomes
hypothalamus, cerebral cortex and spinal cord they have been characterized pharmacologically in terms of 5-HT receptor subtype only in the cerebral cortex (Middlemiss, 1984; Engel et al., 1986). The cerebellum of the rat contains the lowest levels of 5-HT binding sites (Peroutka and Snyder, 1981). However, fluorescent autoradiographic and immunohistochemical studies have revealed 5-HT fibers and varicosities in the cerebellar cortex of the rat. Contrary to classical views suggesting that raphe neurons are the source of 5-HT to the cerebellum, it has been reported recently (Bishop and Ho, 1985) that in rat cerebellum the majority of the serotonergic afferents do not originate in the raphe nuclei but in the nucleus reticularis gigantocellularis, the nucleus reticularis paragigantocellularis and the nucleus reticularis pontis oralis, three discrete areas of the brainstem reticular formation. The paucity of 5-HT binding sites and the possible different source of innervation with respect to other regions of the CNS prompted us to investigate the existence of 5-HT autoreceptors on serotonergic nerve terminals in rat cerebel-
318 lum and to determine their pharmacological properties as subtypes of the 5-HT receptor.
2. Materials and methods
2.1. Preparation of synaptosomes Crude synaptosomes were prepared as previously described (Raiteri et al., 1983). Briefly, adult male Sprague-Dawley rats (200-250 g) were killed by decapitation; the cerebellum was rapidly removed and homogenized in 40 volumes of 0.32 M sucrose buffered at pH 7.4 with phosphate. The homogenate was centrifuged (5 min, 1000 × g) and synaptosomes were isolated from the supernatant by centrifugation at 12 000 × g for 20 min. The pellet was then resuspended in a physiological salt solution having the following composition (mM): NaC1 125, KC1 3, MgSO4, 1.2, CaC12 1.2, NaHEPO 4 1.0, NaHCO 3 22, glucose 10 (aeration with 95% 02 and 5% CO 2 at 37°C); pH 7.2-7.4.
2.2. Experiments of release Synaptosomes were incubated 15 rain at 37°C in an atmosphere of 95% 02 and 5% C O 2 with [3H]5-HT (final concentration 0.06 /~M). After incubation, aliquots of the synaptosomal suspensions were distributed into parallel superfusion chambers thermostated at 37°C (Raiteri et al., 1983) and superfusion was started, at a rate of 0.6 ml/min, with standard medium aerated with 5% CO 2 in 02. After 20 min of equilibration, collection of superfusate samples was started (t = 0) according to the following scheme: one single fraction from t = 0 to t = 17 then one fraction for 2 min from t = 17 to t = 19 (basal release) and one 2 min fraction from t - - 2 0 to t = 22, corresponding to the maximal release observed during high K + exposure (15 mM KC1, substituting an equimolar concentration of NaC1, was introduced at t = 18). When 5-HT was used as an agonist, citalopram (a 5-HT uptake inhibitor) was present from the beginning of superfusion. The antagonists were added 8 min before depolarization and the agonists were added concomitantly with high K +. The amount of radioactivity released into each
fraction was expressed as a percentage of the total synaptosomal tritium at the start of the fraction considered. The K+-evoked release was calculated by subtracting the basal release from the post-depolarization fraction having the highest tritium content. Under the experimental conditions described the radioactivity released by high K + consisted for the major part of unmetabolized [3H]5-HT, as shown previously by chromatographic analysis (Raiteri et al., 1983). Therefore, in the remainder of the text, we refer to K+-evoked tritium release over baseline as K+-evoked [3H]5-HT release. The pA 2 values of the antagonists were calculated according to Furchgott (1972).
2.3. Drugs and chemicals [1,2-3H(N)]5-HT creatinine sulfate (spec. act. 30.1 Ci/mmol) was obtained from New England Nuclear (Boston, MA). 8-OH-DPAT was purchased from RBI (Wayland, MA), 5-HT creatinine sulfate from Calbiochem (Los Angeles, CA). The following compounds were gifts: 8-OHDPAT (Dr. D. Middlemiss, Merrell, Strasbourg, France), RU 24969 (Roussel Uclaf, Romainville, France), citalopram (Lundbeck, Copenhagen, Denmark), methiothepin (Hoffman-La Roche, Basel, Switzerland), methysergide (Sandoz, Basel, Switzerland), ketanserin and spiperone (Janssen, Beerse, Belgium), (-)propranolol (ICI, MacClasfield,. Great Britain), cinanserin (Squibb, Princeton, N J).
3. Results
3.1. Effects of 5-HT and of 5-HT antagonists on the K +-evoked [3H]5-HT release After labeling with [3H]5-HT, cerebellar synaptosomes were depolarized in superfusion with 15 mM KC1. The K+-induced release of [3H]5-HT was completely C a 2 +-dependent (not shown). When prelabeled synaptosomes were exposed to exogenous 5-HT, the K+-evoked release of [3H]5-HT was reduced in a concentration-dependent way (fig. 1). The pEC30 value (negative loga-
319 6O
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_L
A c o
50
~ sc
c
"I
40
~ 40 N 30
-T-
.-T--
2O
k-,-r
20
/7
I 0001 0.01 0.03 0.1 . . . . i 0 .0 0 1
i 0.01
i 0.1
Serotonin concentration (pM) Fig. 1. Concentration-dependent inhibition by 5-HT of the K+-evoked release of [3H]5-HT: effects of methiothepin and of (-)propranolol. Crude synaptosomes were incubated 15 min at 37°C with [3H]5-HT and superfused as described under Methods. Depolarization was carried out with 15 mM KC1. Exogenous 5-HT was added concomitantly with 15 mM KC1. The antagonists were present starting 8 rain before depolarization. Fractions of superfusate were collected and analyzed as described in detail in the Methods section. Means +S.E.M, of 6-8 experiments run in triplicate are presented. (©) 5-HT; (12) 5-HT+0.006/tM methiothepin; (zx)5-HT+0.1 /~M ( - )propranolol.
r i t h m of the c o n c e n t r a t i o n causing 30% i n h i b i t i o n o f the K ÷ - e v o k e d release) a m o u n t e d to 8.73. T h e c o n c e n t r a t i o n - r e s p o n s e curve of 5 - H T was shifted to the right b y m e t h i o t h e p i n in a p a r a l l e l way. T h e a p p a r e n t p A 2 value of m e t h i o t h e p i n was calcul a t e d from the curves shown in the figure at the level of the EC30 values of 5 - H T (which represent r o u g h l y the h a l f - m a x i m u m effect) a n d a m o u n t e d to 9.28. K e t a n s e r i n , methysergide, cinancerin or spipero n e c o u l d n o t c o u n t e r a c t 5 - H T even at concentrations two o r d e r s of m a g n i t u d e higher t h a n that of the agonist ( d a t a n o t shown).
3.2. Effects of R U 24969 and of 8-OH-DPA T at the cerebellar autoreceptors F i g u r e 2 illustrates the effects of these two 5 - H T r e c e p t o r agonists o n the K + - e v o k e d release of [3H]5-HT. A c o n c e n t r a t i o n - d e p e n d e n t inhibi-
0.1 03
---
RU 24969 Methiothepin
1
8 - OH - D PAT
Drug concentration(pM) Fig. 2. Effect of RU 24969 and of 8-OH-DPAT on the K +evoked release of [3H]5-HT. Experimental details as in the legend to fig. 1. The data presented are means + S.E.M. of 6-8 experiments in triplicate.
tion of release was o b s e r v e d with R U 24969 (pEC30 = 8.90). In contrast, 8 - O H - D P A T was ineffective at 1 # M , a c o n c e n t r a t i o n 1000 times higher than that p r o d u c i n g , in the case of 5 - H T or R U 24969, a b o u t 25-30% i n h i b i t i o n of [3H]5-HT release.
3.3. Effect of (-)propranolol on the inhibition of [3H]5-HT release by 5-HT W h e n ( - ) p r o p r a n o l o l was used as a 5 - H T 1 a n t a g o n i s t the c o n c e n t r a t i o n - r e s p o n s e curve of 5 - H T was shifted to the right in a parallel w a y (fig. 1). T h e a p p a r e n t p A 2 values for ( - ) p r o p r a n o l o l a m o u n t e d to 8.05. C o n c e n t r a t i o n s of ( - ) p r o p r a n o l o l higher t h a n 0.5 /xM caused a decrease of the K + - e v o k e d [3H]5-HT release (not shown).
4. D i s c u s s i o n
R e c e p t o r s in the central nervous system have b e e n studied f u n c t i o n a l l y b y a p p l y i n g various exp e r i m e n t a l a p p r o a c h e s in which a r e c e p t o r - m e d i a ted response is m e a s u r e d . T r a n s m i t t e r release can b e e x p l o i t e d as a f u n c t i o n a l test in the identification a n d c h a r a c t e r i z a t i o n of C N S receptors. In
320
particular, the method used in the present work has allowed the identification and characterization of 5-HT autoreceptors in a region, the cerebellum, which is very poor in 5-HT binding sites. Serotonergic afferents originating largely from the medullary and pontine reticular formation reach the cerebellar cortex (Bishop and Ho, 1985). It was shown by autoradiography that some of these projections terminate in the granule cell layer, where they appear to synapse on granule cell dendrites. The finding that 5-HT receptors are present on isolated glutamatergic nerve terminals (Maura et al., in press) may suggest that 5-HT fibers also reach the molecular layer, where glutamic acid appears to be released from the parallel fibers. The results of the present investigation show that release-regulating, methiothepin-sensitive 5HT autoreceptors exist in rat cerebellum and are located on serotonergic nerve endings. Ketanserin, a sPecific ligand for the 5-HT2 sites could not antagonize 5-HT. Methysergide, cinanserin and spiperone which have been considered by many investigators as preferential 5-HT2 antagonists, although with somewhat less selectivity, behaved like ketanserin. Thus, the autoreceptors in rat cerebellum do not belong to the 5-HT2 type. Being activated by nanomolar concentrations of 5-HT, they could be considered as 5-HT1 receptors. The existence in the CNS of three subtypes of the 5-HT1 site, named 5-HTIA, 5-HT1B and 5-HTlc has been proposed on the basis of binding studies (Hoyer et al., 1985). Recently, 8-OH-DPAT was reported to selectively displace [3H]5-HT from the 5-HTIA site in the rat frontal cortex (Middlemiss and Fozard, 1983). This compound was, however, ineffective at the 5-HT autoreceptors regulating 5-HT release from cerebellar synaptosomes, similarly to what had been found with cortical slices (Middlemiss, 1984). On the other hand, (-)propranolol antagonized 5-HT at the autoreceptors in slices of cerebral cortex (Middlemiss, 1984) and in synaptosomes from cerebellum (fig. 1). This compound had been found to stereoselectively displace [3H]5-HT from the 5-HT~ sites showing equal and high affinity for the 5 - H T I A and the 5-HT m subsites, but low affinity for the 5-HTlc (Hoyer et al., 1985).
Taken together, the results allow the conclusion that the release-regulating 5-HT autoreceptor in rat cerebellum is pharmacologically similar to that in the cerebral cortex and, therefore, in analogy to what has been proposed for the latter (Middlemiss, 1984; Engel et al., 1986), could be classified as a 5-HTm receptor subtype. Interestingly, the 5-HT receptors sited on glutamatergic terminals in rat cerebellum and which are postsynaptic with respect to the 5-HT autoreceptors also belong to the 5-HT1 type but not to the 5-HTla subtype (Maura et al., in press).
Acknowledgements This work was supported by grants from the Italian National Research Council and from the Italian Ministry of Education. The authors with to thank Miss Graziella Pellegrini for technical assistance and Mrs. Maura Agate for preparing the typescript.
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