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Functional significance of subtypes of 5-HT receptors in the rat spinal reflex pathway
Gen. Pharmac. Vol. 19, No. 6, pp. 789-793, 1988 Printed in Great Britain
0306-3623/88 $3.00+0.00 Pergamon Press plc
FUNCTIONAL SIGNIFICANCE OF SUBTYPES OF 5-HT RECEPTORS IN THE RAT SPINAL REFLEX PATHWAY N o s u o NAGANO, HIDEKI ONO and HIDEOMI FUKUDA Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-I, Bunkyo-ku, Tokyo 113, Japan [Tel: (03) 812-2111] (Received I 1 January 1988) Abstraet--l. The functional significance of subtypes of 5-hydroxytryptamine (5-HT) receptors was studied in the rat spinal reflex pathway. 2. Ketanserin had no effect on the mono- (MSR) or polysynaptic reflex (PSR) in spinal rats, but decreased the PSR in intact rats. 3. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) decreased the MSR and increased the PSR in spinal rats. 4. Ketanserin antagonized the effects of 5-MeODMT without antagonizing the effects of 8-OH-DPAT. 5. Cinanserin had similar effects to those of ketanserin. 6. These results suggest that both 5-HT~A and 5-HT2 receptors mediate MSR inhibition and PSR augmentation in the spinal reflexes of spinal rats, and that the 5-HT2 receptor has a supraspinal tonic excitatory influence on the PSR in intact rats.
5,6-dihydroxytryptamine (5,6-DHT)- or 6-hydroxydopamine (6-OHDA)- treated rats. The effects of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) and cinanserin, a classical 5-HT agonist and antagonist respectively, were also studied.
INTRODUCTION
It is widely acknowledged that subtypes of 5-hydroxytryptamine (5-HT) receptors exist in the rat brain. On the basis of radioligand binding studies, Peroutka and Snyder (1979) classified 5-HT binding sites into 5-HT~, labelled by [aH]5-HT with high affinity and 5-HT2, labelled by [3H]spiperone with high affinity. Further study has also suggested the existence of two classes of 5-HTI site named 5-HT~A and 5-HTIB (Pedigo et al., 1981). In addition, there have been some attempts to relate several forms of behaviour, occurring following administration of 5-HT precursors or agonists, with subtypes of 5-HT receptors. It has been reported that reciprocal forepaw treading (Tricklebank et al., 1985a) and temperature change (Goodwin and Green, 1985) in rats are correlated with activation of the 5-HTIA receptor and that "wetdog" shaking behaviour in rats (Yap and Taylor, 1983; Lucki et al., 1984) and head twitching behaviour in mice (Green et al., 1983; Goodwin and Green, 1985) are correlated with the 5-HT2 receptor. Furthermore, it has been suggested that myoclonus in the guinea pig is 5-HT 1receptor-mediated (Luscombe et at., 1984). The spinal reflex, which is composed of the monosynaptic reflex (MSR) and the polysynaptic reflex (PSR) is a clear and simple tool for evaluating the activity of neurotransmitters or neuromodulators in the spinal cord. We have demonstrated that 5-HT agonists decreased the MSR and increased the PSR in spinal rats (Nagano et al., 1987). In the present study, the functional significance of subtypes of the 5-HT receptor was studied in the rat spinal reflex using 8-hydroxy-2-(di-n-propylamino)tetralin(8-OHDPAT), a selective 5-HT~A agonist, and ketanserin, a 5-HT2 antagonist. In addition, we examined whether the effect of ketanserin is mediated by its inhibitory action at the 5-HT2 receptor or ~1-adrenoceptor, using
MATERIALS AND METHODS
MSR and PSR recording The spinal reflex was recorded in intact (non-spinalized) and spinal rats. Male Wistar rats, weighing 310--380g, were anesthetized with urethane (1 g/kg, i.p.) and ~-chloralose (25 mg/kg, i.p.) and then artificially ventilated. In spinal rats, the vagus nerves were cut bilaterally in the cervical region, and the spinal cord was transected at the CI level. Laminectomy was performed in the lumbo-sacral region. Ventral and dorsal roots below L4 were cut bilaterally, and the dorsal and ventral roots of L5 were isolated. A skin pouch was then formed at the site of the dissection and the exposed tissues were covered with liquid paraffin to prevent drying. The temperatures of the paraffin pool and rectum were kept at 36 + 0.5~ The dorsal and ventral roots of L5 were placed on bipolar silver-silver chloride wire electrodes for stimulation (0.2 Hz, 0.05 reset, 10 V; Nihon Kohden SEN-7103) and recording, respectively. The reflex potentials were amplified (Nihon Kohden AVB-10), displayed on an oscilloscope (Nihon Kohden VC-10) and averaged 8 times by an averager (Nihon Kohden DAT-II00), the analog output of which was recorded (Nihon Kohden WT-625G). The reflex amplitude (the height of the MSR and the PSR) was measured. Intracisternal injection of 5,6-DHT and 6-OHDA In some experiments, 5,6-DHT or 6-OHDA treatment was performed. Male Wistar rats, weighing 280-320 g, were anesthetized with ether. 5,6-DHT creatinine sulphate (75 #g as free base) was dissolved in 0.9% saline containing 0.2 mg/ ml ascorbic acid and injected intracisternally in a volume of 20 #1, using a Hamilton syringe. Rats were pretreated with desipramine-HCl (25 mg/kg, i.p.) 60 min before administration of 5,6-DHT to prevent uptake of the neurotoxin into noradrenergic neurons. 6-OHDA hydrobromide (100/zg)
789
790
NOBUO NAGANOet al.
was dissolved in 0.9% saline containing 0.1 mg/ml ascorbic acid and similarly injected.
ketanserin-T
1 mg/kg,
i.v.
MSR
Drugs
The drugs used were 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT-HBr,Research Bioehemicals Inc.), 5-methoxy-N,N-dimethyltryptamine (5-MeODMT, Sigma), ketanserin tartrate (ketanserin-T, Kyowa Hakko), cinanserin-HC1 (Squibb), desipramine-HC1 (CIBA-Geigy), 5,6-dihydroxytryptamine creatinine sulphate (5,6-DHT, _ ~ _ [0.5 mV Sigma), 6-hydroxydopamine hydrobromide (6-OHDA, Sigma), urethane (Wako) and ~-chloralose (Tokyo Kasei). i The test drugs were dissolved in physiological saline, apart 2 ms from ketanserin which was dissolved in distilled water, and injected into the femoral vein. Fig. 1. Effects of ketanserin-T (1 mg/kg, i.v.) on the MSR and the PSR in intact rats. Representative 8-times-averaged responses (MSR and PSR) elicited by electrical stimulation of the L5 dorsal root are illustrated. The left spinal reflexes RESULTS are those just before administration of the drug while the The MSR and the PSR elicited by electrical reflexes on the right are those 2 min after administration of the drug. stimulation of the L5 dorsal root were recorded from the ipsilateral L5 ventral root. The latencies to the peaks of the MSR and the PSR were about 2 and Cinanserin-HC1 (5 mg/kg, i.v.) had no effect on the 3 reset, respectively. Representative 8-times-averaged MSR but decreased the PSR to approx. 80% in intact responses (MSR and PSR) are illustrated (Fig. 1). rats (Fig. 2D). The above-mentioned experiments were Ketanserin-T (1 mg/kg, i.v.) had no effect on the 'performed using intact rats, while those described MSR, but decreased the PSR to approx. 50% in below involved spinal rats to evaluate the activities of intact rats (Figs 1 and 2A). A high dose of ketanserin- 5-HT agonists and antagonists in the spinal cord. T (5mg/kg, i.v.) slightly decreased the MSR and 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) decreased the markedly decreased the PSR in intact rats (Fig. 2B). MSR to 50% and increased the PSR to 150-160% in The PSR inhibition of ketanserin showed no recovery spinal rats (Fig. 3A). The peak effects of 8-OHwithin 120 rain after administration of the drug. The DPAT on both the MSR and the PSR occurred at inhibitory effect of ketanserin-T (1 mg/kg, i.v.) on 5 min after drug administration. The effects of 8-OHthe PSR was attenuated by 5,6-DHT or 6-OHDA DPAT on the reflexes showed no recovery within 45 min after administration of the drug. administered 2 weeks previously (Fig. 2C).
A
ketanserin-T 1 mg/kg, i.v.
C ketanserin-T 1 mg/"kg, i'.v .
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Fig. 2. Effects of ketanserin-T (1 and 5 mg/kg, i.v.) (A and B) and cinanserin-HCl (5 mg/kg, i.v.) (D) on the MSR (O) and the PSR (Q) in intact rats, and the effects of ketanserin-T (1 mg/kg, i.v.) on the PSR in 5,6-DHT-treated rats (O) and 6-OHDA-treated (0) rats (C). Each point represents the reflex amplitude (mean +_SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to drug injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P < 0.05 (two-tailed Student's t-test) as compared with the effect of ketanserin-T (1 mg/kg, i.v.) on the PSR in intact rats.
5-HT receptors and spinal reflexes
5-MeODMT had similar effects to those of 8-OHDPTAT. 5-MeODMT (1 #g/kg, i.v.) decreased the MSR to 60-70% and increased the PSR to 130150% in spinal rats (Fig. 4A). The peak effects of 5-MeODMT on both the MSR and the PSR were transient, but the effects continued for about 30 min. Ketanserin alone had no effect on either the MSR or the PSR in spinal rats (Figs 3B and 4B). Preadministration of ketanserin-T (1 mg/kg, i.v., 10 min before) did not influence the effects of 8-OH-DPATHBr (0.1 mg/kg, i.v.) on either the MSR or the PSR (Fig. 3B). In contrast, the effects of 5-MeODMT on both the MSR and the PSR were significantly antagonized by preadministration of ketanserin-T (1 mg/kg, i.v., 10 min before) (Fig. 4B).
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Fig. 4. Effects of 5-MeODMT (1/~g/kg, i.v.) on the MSR ( 9 and the PSR (O) (A), and the influences of preadministration (10 min before) of ketanserin-T (I mg/kg, i.v.) (B) or cinanserin-HC1 (5 mg/kg, i.v.) (C) on the effects of 5-MeODMT (I #g/kg, i.v.) on the MSR (O) and the PSR (O) in spinal rats. Each point represents the reflex amplitude (mean + SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to 5-MeODMT injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P < 0.05, **P < 0.01 (twotailed Student's t-test) as compared with the effects of 5-MeODMT (1 pg/kg, i.v.) alone on the MSR and PSR in spinal rats.
8-OH-DPAT-HBr 0.i mg/kg, i.v.
C cinanserin-HCl
-i0
i.v. 40
50
Fig. 3. Effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the MSR (O) and the PSR (O) (A), and the influences of preadministration (10 min before) of ketanserin-T (1 mg/kg, i.v.) (B) or cinanserin-HC1 (5 mg/kg, i.v.) (C) on the effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the MSR ( 9 and the PSR (Q) in spinal rats. Each point represents the reflex amplitude (mean __+SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to 8-OH-DPAT injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P <0.05, **P < 0 . 0 ! (two-tailed Student's t-test) as compared with the effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) alone on the MSR and the PSR in spinal rats.
Cinanserin alone also had no effect on either the MSR or the PSR in spinal rats (Figs 3C and 4C). Preadministration of cinanserin-HCl (5 mg/kg, i.v., 10min before) attenuated the excitatory effect of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the PSR but not its inhibitory effect on the MSR (Fig. 3C). The effects of 5-MeODMT (1 #g/kg, i.v.) on both the MSR and the PSR were significantly antagonized by the preadministration of cinanserin-HC1 (5 mg/kg, i.v., 10min before) (Fig. 4C). DISCUSSION
Ketanserin-T (1 mg/kg, i.v.), a 5-HT 2 antagonist, decreased the PSR in intact rats (Figs 1 and 2A)
792
NoBuo NAGANOet aL
and had no effect on the spinal reflexes in spinal rats (Figs 3B and 4B). These results suggest that ketanserin blocks supraspinal tonic excitatory effects on the PSR. Furthermore, supraspinal tonic effects on the PSR are considered to be at least 5-HT 2 receptormediated. However, it has been reported that ketanserin, which shows markedly greater affinity for the 5-HT 2 than for the 5-HT~ receptor, also has appreciable cq-adrenoceptor affinity (Leysen et al., 1981). Therefore, we examined whether the inhibitory effect of ketanserin on the PSR was mediated at the 5-HT 2 receptor or the aq-adrenoceptor. The PSR inhibition caused by ketanserin was attenuated by treatment with either 5,6-DHT or 6-OHDA (Fig. 2C). It may be considered that ketanserin has no inhibitory action at the 5-HT2 receptor in 5,6-DHT-treated rats, since 5,6-DHT treatment causes depletion of 5-HT following destruction of descending serotonergic neurons (Nobin et al., 1973). Therefore, the PSR inhibition caused by ketanserin in 5,6-DHT~ rats is considered to be due to its blockade of descending Ctl-adrenoceptor-mediated activity. On the other hand, the PSR inhibition caused by ketanserin in 6-OHDA-treated rats is considered to be due to its blockade of descending 5-HT2 receptor-mediated activity, since 6-OHDA causes depletion of noradrenaline in the spinal cord (Kostrzewa and Jacobowitz, 1974). Thus, these results suggest that the inhibitory effect of ketanserin on the PSR is mediated by its antagonistic action at both the 5-HT2 and ~-adrenergic receptors. A high dose of ketanserin-T (5 mg/kg, i.v.) slightly decreased the MSR (Fig. 2B). It has been reported that ~t~-blockers, such as phenoxybenzamine and chlorpromazine, decrease the MSR in the rat spinal reflex (Hino et al., 1984). Therefore, the effect of ketanserin-T (5 mg/kg, i.v.) on the MSR may be considered to be mediated by its antagonistic action at the cq-adrenoceptor. Cinanserin, which is a 5-HT2 antagonist and has less cq-adrenoceptor affinity than ketanserin (Leysen et al., 1981), had no effect on the MSR and decreased the PSR in intact rats (Fig. 2D). This result supports the hypothesis that supraspinal tonic excitatory effects on the PSR are at least 5-HT2 receptor-mediated. 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) decreased the MSR and increased the PSR in spinal rats (Fig. 3A). A radioligand binding study has shown that 8-OHDPAT has negligible affinity for the 5-HT2 receptor but very high affinity for the 5-HT~A receptor (Middlemiss and Fozard, 1983). Therefore, this result strongly suggests that the 5-HT~A receptor in the spinal cord is involved in MSR inhibition and PSR augmentation. This hypothesis is supported by the result that the effects of 8-OH-DPAT-HBr (0.1 mg/ kg, i.v.) on both the MSR and the PSR were not antagonized by preadministration of ketanserin-T (1 mg/kg, i.v.), a 5-HT2 antagonist (Fig. 3B). The PSR augmentation by 8-OH-DPAT-HBr (0.1 mg/ kg, i.v.) was attenuated by preadministration of cinanserin-HCl (5 mg/kg, i.v.) (Fig. 3C). This result may be due to the fact that cinanserin has affinity not only for the 5-HT2 but also for the 5-HT~ receptor (Leysen et al., 1981). 5-MeODMT (1 lt/kg, i.v.), a non-selective 5-HT agonist, had similar effects to those of 8-OH-DPAT in spinal rats (Fig. 4A). The effects of 5-MeODMT
(1 ttg/kg, i.v.) on the MSR and the PSR were significantly antagonized by the preadministration of ketanserin-T (1 mg/kg, i.v.) (Fig. 4B) or cinanserinHCI (5 mg/kg, i.v.) (Fig. 4C). Although 5-MeODMT has high affinity for the 5-HT l receptor rather than for the 5-HT 2 receptor (Tricklebank et al., 1985a), these results suggest that the effects of 5-MeODMT are mediated by the 5-HT2 receptor. Therefore, it is suggested that the 5-HT2 receptor in the spinal cord is also involved in MSR inhibition and the PSR augmentation in spinal rats. In the radioligand binding studies, it has been reported that there are low levels of 5-HT2-binding sites in the rat spinal cord (Monroe and Smith, 1983; Pazos et al., 1985). On the contrary, Iverfeldt et al., (1986) have suggested that there are functional 5-HT2 receptors in the rat spinal cord, since the release of substance P from slices of the ventral spinal cord was enhanced by 5-HT and the enhancement was completely blocked by 5-HT 2 antagonists including ketanserin. Moreover, in behavioural studies, similar results, that 5-HT2 antagonists antagonize the response induced by 5-MeODMT but not those induced by 8-OH-DPAT, have been reported (Tricklebank et al., 1985b; Goodwin and Green, 1985). The present study suggests that the spinal reflex is a precise and quantitative pharmacological model for evaluating the activity of 5-HT in the spinal cord. More selective 5-HT drugs (especially 5-HT~ antagonists and 5-HT2 agonists) will be required in order to elucidate the physiological and functional significance of 5-HT receptor subtypes in the central nervous system. Acknowledgements--This work was supported in part by
Research Grants from the Ministry of Education, Science and Culture of Japan and Takeda Science Foundation. We thank Kyowa Hakko, Squibb and CIBA-Geigy for providing ketanserin-T, cinanserin-HCl and desipramine-HCl, respectively. REFERENCES
Goodwin G. M. and Green A. R. (1985) A behavioural and biochemical study in mice and rats of putative selective agonists and antagonists for 5-HT 1and 5-HT2 receptors. Br. J. Pharmac. 84, 743-753. Green A. R., O'Shaughnessy K., Hammond M., Schfichter M. and Grahame-Smith D. G. (1983) Inhibition of 5hydroxytryptamine-mediated behaviour by the putative 5-HT2 antagonist pirenperone. Neuropharmacology 22, 573-578. Hino M., Ono H. and Fukuda H. (1984) Supraspinal tonic influence on spinal reflexes and involvement in the effect of chlorpromazine. Genl Pharmac. 15, 155-158. Iverfeldt K., Peterson L., Brodin E., Ogren S. and Bartfai T. (1986) Serotonin type-2 receptor mediated regulation of substance P release in the ventral spinal cord and the effects of chronic antidepressant treatment. NaunynSchmiedeberg's Arch. Pharmac. 333, 1-6. Kostrzewa R. M. and Jacobowitz D. M. (1974) Pharmacological actions of 6-hydroxydopamine. Pharmac. Rev. 26, 199-288.
Leysen J. E., Awouters F., Kennis L., Laduron P. M., Vandenberk J. and Janssen P. A. J. (1981) Receptor binding profile of R 41 468, a novel antagonist at 5-HT2 receptors. Life Sci. 28, 1015-1022. Lucki I., Nobler M. S. and Frazer A. (1984) Differential
5-HT receptors and spinal reflexes actions of serotonin antagonists on two behavioral models of serotonin receptor activation in the rat. J. Pharmac. exp. Ther. 228, 133-139. Luscombe G., Jenner P. and Marsden C. D. (1984) Correlation of [3H]5-hydroxytryptamine (5HT) binding to brain stem preparations and the production and prevention of myoelonus in guinea pig by 5HT agonists and antagonists. Fur. J. Pharmac. 104, 235-244. Middlemiss D. N. and Fozard J. R. (1983) 8-Hydroxy2-(di-n-propylamino)tetralin discriminates between subtypes of the 5-HT 1 recognition site. Eur. J. Pharmac. 90, 151-153. Monroe P. J. and Smith D. J. (1983) Characterization of multiple [3H]5-hydroxytryptamine binding sites in rat spinal cord tissue. J. Neurochem. 41, 349-355. Nagano N., Ono H., Ozawa M. and Fukuda H. (1987) Sensitivity of spinal reflexes to TRH and 5-HT in 5,6-dihydroxytryptamine-treated rats. Eur. J. Pharmac. 139, 315-321. Nobin A., Baumgarten H. G., Bjbrklund A,, Lachenmayer L. and Stenevi U. (1973) Axonal degeneration and regeneration of the bulbo-spinal indolamine neurons after 5,6-dihydroxytryptamine treatment. Brain Res. 56, 1-24. Pazos A., Cortts R. and Palacios J. M. (1985) Quantitative
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autoradiographie mapping of serotonin receptors in the rat brain. Brain Res. 346, 231-249, Pedigo N, W., Yamamura H. I. and Nelson D. L (1981) Discrimination of multiple [3H]5-hydroxytryptamine binding sites by the neuroleptic spiperone in rat brain. J. Neurochem. 36, 220-226. Peroutka S. J. and Snyder S. H. (1979) Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. Molec. Pharmac, 16, 687--699. Tricklebank M. D., Forler C., Middlemiss D, N. and Fozard J. R. (1985a) Subtypes of the 5-HT receptor mediating the behavioural responses to 5-methoxy-N,N-dimethyltryptamine in the rat. Eur. J. Pharmac. 117, 15-24. Tricklebank M. D., Forler C. and Fozard J. R. (1985b) The involvement of subtypes of the 5-HT~ receptor and of catecholaminergie systems in the behavioural response to 8-hydroxy-2-(di-n-propylamino)tetralin in the rat, Eur. J. Pharmac. 106, 271-282. Yap C. Y. and Taylor D. A. (1983) Involvement of 5-HT2 receptors in the wet-dog shake behaviour induced by 5-hydroxytryptophan in the rat. Neuropharmacology 22, 801-804.
0306-3623/88 $3.00+0.00 Pergamon Press plc
FUNCTIONAL SIGNIFICANCE OF SUBTYPES OF 5-HT RECEPTORS IN THE RAT SPINAL REFLEX PATHWAY N o s u o NAGANO, HIDEKI ONO and HIDEOMI FUKUDA Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-I, Bunkyo-ku, Tokyo 113, Japan [Tel: (03) 812-2111] (Received I 1 January 1988) Abstraet--l. The functional significance of subtypes of 5-hydroxytryptamine (5-HT) receptors was studied in the rat spinal reflex pathway. 2. Ketanserin had no effect on the mono- (MSR) or polysynaptic reflex (PSR) in spinal rats, but decreased the PSR in intact rats. 3. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) decreased the MSR and increased the PSR in spinal rats. 4. Ketanserin antagonized the effects of 5-MeODMT without antagonizing the effects of 8-OH-DPAT. 5. Cinanserin had similar effects to those of ketanserin. 6. These results suggest that both 5-HT~A and 5-HT2 receptors mediate MSR inhibition and PSR augmentation in the spinal reflexes of spinal rats, and that the 5-HT2 receptor has a supraspinal tonic excitatory influence on the PSR in intact rats.
5,6-dihydroxytryptamine (5,6-DHT)- or 6-hydroxydopamine (6-OHDA)- treated rats. The effects of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) and cinanserin, a classical 5-HT agonist and antagonist respectively, were also studied.
INTRODUCTION
It is widely acknowledged that subtypes of 5-hydroxytryptamine (5-HT) receptors exist in the rat brain. On the basis of radioligand binding studies, Peroutka and Snyder (1979) classified 5-HT binding sites into 5-HT~, labelled by [aH]5-HT with high affinity and 5-HT2, labelled by [3H]spiperone with high affinity. Further study has also suggested the existence of two classes of 5-HTI site named 5-HT~A and 5-HTIB (Pedigo et al., 1981). In addition, there have been some attempts to relate several forms of behaviour, occurring following administration of 5-HT precursors or agonists, with subtypes of 5-HT receptors. It has been reported that reciprocal forepaw treading (Tricklebank et al., 1985a) and temperature change (Goodwin and Green, 1985) in rats are correlated with activation of the 5-HTIA receptor and that "wetdog" shaking behaviour in rats (Yap and Taylor, 1983; Lucki et al., 1984) and head twitching behaviour in mice (Green et al., 1983; Goodwin and Green, 1985) are correlated with the 5-HT2 receptor. Furthermore, it has been suggested that myoclonus in the guinea pig is 5-HT 1receptor-mediated (Luscombe et at., 1984). The spinal reflex, which is composed of the monosynaptic reflex (MSR) and the polysynaptic reflex (PSR) is a clear and simple tool for evaluating the activity of neurotransmitters or neuromodulators in the spinal cord. We have demonstrated that 5-HT agonists decreased the MSR and increased the PSR in spinal rats (Nagano et al., 1987). In the present study, the functional significance of subtypes of the 5-HT receptor was studied in the rat spinal reflex using 8-hydroxy-2-(di-n-propylamino)tetralin(8-OHDPAT), a selective 5-HT~A agonist, and ketanserin, a 5-HT2 antagonist. In addition, we examined whether the effect of ketanserin is mediated by its inhibitory action at the 5-HT2 receptor or ~1-adrenoceptor, using
MATERIALS AND METHODS
MSR and PSR recording The spinal reflex was recorded in intact (non-spinalized) and spinal rats. Male Wistar rats, weighing 310--380g, were anesthetized with urethane (1 g/kg, i.p.) and ~-chloralose (25 mg/kg, i.p.) and then artificially ventilated. In spinal rats, the vagus nerves were cut bilaterally in the cervical region, and the spinal cord was transected at the CI level. Laminectomy was performed in the lumbo-sacral region. Ventral and dorsal roots below L4 were cut bilaterally, and the dorsal and ventral roots of L5 were isolated. A skin pouch was then formed at the site of the dissection and the exposed tissues were covered with liquid paraffin to prevent drying. The temperatures of the paraffin pool and rectum were kept at 36 + 0.5~ The dorsal and ventral roots of L5 were placed on bipolar silver-silver chloride wire electrodes for stimulation (0.2 Hz, 0.05 reset, 10 V; Nihon Kohden SEN-7103) and recording, respectively. The reflex potentials were amplified (Nihon Kohden AVB-10), displayed on an oscilloscope (Nihon Kohden VC-10) and averaged 8 times by an averager (Nihon Kohden DAT-II00), the analog output of which was recorded (Nihon Kohden WT-625G). The reflex amplitude (the height of the MSR and the PSR) was measured. Intracisternal injection of 5,6-DHT and 6-OHDA In some experiments, 5,6-DHT or 6-OHDA treatment was performed. Male Wistar rats, weighing 280-320 g, were anesthetized with ether. 5,6-DHT creatinine sulphate (75 #g as free base) was dissolved in 0.9% saline containing 0.2 mg/ ml ascorbic acid and injected intracisternally in a volume of 20 #1, using a Hamilton syringe. Rats were pretreated with desipramine-HCl (25 mg/kg, i.p.) 60 min before administration of 5,6-DHT to prevent uptake of the neurotoxin into noradrenergic neurons. 6-OHDA hydrobromide (100/zg)
789
790
NOBUO NAGANOet al.
was dissolved in 0.9% saline containing 0.1 mg/ml ascorbic acid and similarly injected.
ketanserin-T
1 mg/kg,
i.v.
MSR
Drugs
The drugs used were 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT-HBr,Research Bioehemicals Inc.), 5-methoxy-N,N-dimethyltryptamine (5-MeODMT, Sigma), ketanserin tartrate (ketanserin-T, Kyowa Hakko), cinanserin-HC1 (Squibb), desipramine-HC1 (CIBA-Geigy), 5,6-dihydroxytryptamine creatinine sulphate (5,6-DHT, _ ~ _ [0.5 mV Sigma), 6-hydroxydopamine hydrobromide (6-OHDA, Sigma), urethane (Wako) and ~-chloralose (Tokyo Kasei). i The test drugs were dissolved in physiological saline, apart 2 ms from ketanserin which was dissolved in distilled water, and injected into the femoral vein. Fig. 1. Effects of ketanserin-T (1 mg/kg, i.v.) on the MSR and the PSR in intact rats. Representative 8-times-averaged responses (MSR and PSR) elicited by electrical stimulation of the L5 dorsal root are illustrated. The left spinal reflexes RESULTS are those just before administration of the drug while the The MSR and the PSR elicited by electrical reflexes on the right are those 2 min after administration of the drug. stimulation of the L5 dorsal root were recorded from the ipsilateral L5 ventral root. The latencies to the peaks of the MSR and the PSR were about 2 and Cinanserin-HC1 (5 mg/kg, i.v.) had no effect on the 3 reset, respectively. Representative 8-times-averaged MSR but decreased the PSR to approx. 80% in intact responses (MSR and PSR) are illustrated (Fig. 1). rats (Fig. 2D). The above-mentioned experiments were Ketanserin-T (1 mg/kg, i.v.) had no effect on the 'performed using intact rats, while those described MSR, but decreased the PSR to approx. 50% in below involved spinal rats to evaluate the activities of intact rats (Figs 1 and 2A). A high dose of ketanserin- 5-HT agonists and antagonists in the spinal cord. T (5mg/kg, i.v.) slightly decreased the MSR and 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) decreased the markedly decreased the PSR in intact rats (Fig. 2B). MSR to 50% and increased the PSR to 150-160% in The PSR inhibition of ketanserin showed no recovery spinal rats (Fig. 3A). The peak effects of 8-OHwithin 120 rain after administration of the drug. The DPAT on both the MSR and the PSR occurred at inhibitory effect of ketanserin-T (1 mg/kg, i.v.) on 5 min after drug administration. The effects of 8-OHthe PSR was attenuated by 5,6-DHT or 6-OHDA DPAT on the reflexes showed no recovery within 45 min after administration of the drug. administered 2 weeks previously (Fig. 2C).
A
ketanserin-T 1 mg/kg, i.v.
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Fig. 2. Effects of ketanserin-T (1 and 5 mg/kg, i.v.) (A and B) and cinanserin-HCl (5 mg/kg, i.v.) (D) on the MSR (O) and the PSR (Q) in intact rats, and the effects of ketanserin-T (1 mg/kg, i.v.) on the PSR in 5,6-DHT-treated rats (O) and 6-OHDA-treated (0) rats (C). Each point represents the reflex amplitude (mean +_SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to drug injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P < 0.05 (two-tailed Student's t-test) as compared with the effect of ketanserin-T (1 mg/kg, i.v.) on the PSR in intact rats.
5-HT receptors and spinal reflexes
5-MeODMT had similar effects to those of 8-OHDPTAT. 5-MeODMT (1 #g/kg, i.v.) decreased the MSR to 60-70% and increased the PSR to 130150% in spinal rats (Fig. 4A). The peak effects of 5-MeODMT on both the MSR and the PSR were transient, but the effects continued for about 30 min. Ketanserin alone had no effect on either the MSR or the PSR in spinal rats (Figs 3B and 4B). Preadministration of ketanserin-T (1 mg/kg, i.v., 10 min before) did not influence the effects of 8-OH-DPATHBr (0.1 mg/kg, i.v.) on either the MSR or the PSR (Fig. 3B). In contrast, the effects of 5-MeODMT on both the MSR and the PSR were significantly antagonized by preadministration of ketanserin-T (1 mg/kg, i.v., 10 min before) (Fig. 4B).
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Fig. 4. Effects of 5-MeODMT (1/~g/kg, i.v.) on the MSR ( 9 and the PSR (O) (A), and the influences of preadministration (10 min before) of ketanserin-T (I mg/kg, i.v.) (B) or cinanserin-HC1 (5 mg/kg, i.v.) (C) on the effects of 5-MeODMT (I #g/kg, i.v.) on the MSR (O) and the PSR (O) in spinal rats. Each point represents the reflex amplitude (mean + SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to 5-MeODMT injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P < 0.05, **P < 0.01 (twotailed Student's t-test) as compared with the effects of 5-MeODMT (1 pg/kg, i.v.) alone on the MSR and PSR in spinal rats.
8-OH-DPAT-HBr 0.i mg/kg, i.v.
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Fig. 3. Effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the MSR (O) and the PSR (O) (A), and the influences of preadministration (10 min before) of ketanserin-T (1 mg/kg, i.v.) (B) or cinanserin-HC1 (5 mg/kg, i.v.) (C) on the effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the MSR ( 9 and the PSR (Q) in spinal rats. Each point represents the reflex amplitude (mean __+SEM of 4 or 5 experiments) calculated as a percentage of the value just prior to 8-OH-DPAT injection. Where SEM bars are not shown, they lie within the dimensions of the symbols. *P <0.05, **P < 0 . 0 ! (two-tailed Student's t-test) as compared with the effects of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) alone on the MSR and the PSR in spinal rats.
Cinanserin alone also had no effect on either the MSR or the PSR in spinal rats (Figs 3C and 4C). Preadministration of cinanserin-HCl (5 mg/kg, i.v., 10min before) attenuated the excitatory effect of 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) on the PSR but not its inhibitory effect on the MSR (Fig. 3C). The effects of 5-MeODMT (1 #g/kg, i.v.) on both the MSR and the PSR were significantly antagonized by the preadministration of cinanserin-HC1 (5 mg/kg, i.v., 10min before) (Fig. 4C). DISCUSSION
Ketanserin-T (1 mg/kg, i.v.), a 5-HT 2 antagonist, decreased the PSR in intact rats (Figs 1 and 2A)
792
NoBuo NAGANOet aL
and had no effect on the spinal reflexes in spinal rats (Figs 3B and 4B). These results suggest that ketanserin blocks supraspinal tonic excitatory effects on the PSR. Furthermore, supraspinal tonic effects on the PSR are considered to be at least 5-HT 2 receptormediated. However, it has been reported that ketanserin, which shows markedly greater affinity for the 5-HT 2 than for the 5-HT~ receptor, also has appreciable cq-adrenoceptor affinity (Leysen et al., 1981). Therefore, we examined whether the inhibitory effect of ketanserin on the PSR was mediated at the 5-HT 2 receptor or the aq-adrenoceptor. The PSR inhibition caused by ketanserin was attenuated by treatment with either 5,6-DHT or 6-OHDA (Fig. 2C). It may be considered that ketanserin has no inhibitory action at the 5-HT2 receptor in 5,6-DHT-treated rats, since 5,6-DHT treatment causes depletion of 5-HT following destruction of descending serotonergic neurons (Nobin et al., 1973). Therefore, the PSR inhibition caused by ketanserin in 5,6-DHT~ rats is considered to be due to its blockade of descending Ctl-adrenoceptor-mediated activity. On the other hand, the PSR inhibition caused by ketanserin in 6-OHDA-treated rats is considered to be due to its blockade of descending 5-HT2 receptor-mediated activity, since 6-OHDA causes depletion of noradrenaline in the spinal cord (Kostrzewa and Jacobowitz, 1974). Thus, these results suggest that the inhibitory effect of ketanserin on the PSR is mediated by its antagonistic action at both the 5-HT2 and ~-adrenergic receptors. A high dose of ketanserin-T (5 mg/kg, i.v.) slightly decreased the MSR (Fig. 2B). It has been reported that ~t~-blockers, such as phenoxybenzamine and chlorpromazine, decrease the MSR in the rat spinal reflex (Hino et al., 1984). Therefore, the effect of ketanserin-T (5 mg/kg, i.v.) on the MSR may be considered to be mediated by its antagonistic action at the cq-adrenoceptor. Cinanserin, which is a 5-HT2 antagonist and has less cq-adrenoceptor affinity than ketanserin (Leysen et al., 1981), had no effect on the MSR and decreased the PSR in intact rats (Fig. 2D). This result supports the hypothesis that supraspinal tonic excitatory effects on the PSR are at least 5-HT2 receptor-mediated. 8-OH-DPAT-HBr (0.1 mg/kg, i.v.) decreased the MSR and increased the PSR in spinal rats (Fig. 3A). A radioligand binding study has shown that 8-OHDPAT has negligible affinity for the 5-HT2 receptor but very high affinity for the 5-HT~A receptor (Middlemiss and Fozard, 1983). Therefore, this result strongly suggests that the 5-HT~A receptor in the spinal cord is involved in MSR inhibition and PSR augmentation. This hypothesis is supported by the result that the effects of 8-OH-DPAT-HBr (0.1 mg/ kg, i.v.) on both the MSR and the PSR were not antagonized by preadministration of ketanserin-T (1 mg/kg, i.v.), a 5-HT2 antagonist (Fig. 3B). The PSR augmentation by 8-OH-DPAT-HBr (0.1 mg/ kg, i.v.) was attenuated by preadministration of cinanserin-HCl (5 mg/kg, i.v.) (Fig. 3C). This result may be due to the fact that cinanserin has affinity not only for the 5-HT2 but also for the 5-HT~ receptor (Leysen et al., 1981). 5-MeODMT (1 lt/kg, i.v.), a non-selective 5-HT agonist, had similar effects to those of 8-OH-DPAT in spinal rats (Fig. 4A). The effects of 5-MeODMT
(1 ttg/kg, i.v.) on the MSR and the PSR were significantly antagonized by the preadministration of ketanserin-T (1 mg/kg, i.v.) (Fig. 4B) or cinanserinHCI (5 mg/kg, i.v.) (Fig. 4C). Although 5-MeODMT has high affinity for the 5-HT l receptor rather than for the 5-HT 2 receptor (Tricklebank et al., 1985a), these results suggest that the effects of 5-MeODMT are mediated by the 5-HT2 receptor. Therefore, it is suggested that the 5-HT2 receptor in the spinal cord is also involved in MSR inhibition and the PSR augmentation in spinal rats. In the radioligand binding studies, it has been reported that there are low levels of 5-HT2-binding sites in the rat spinal cord (Monroe and Smith, 1983; Pazos et al., 1985). On the contrary, Iverfeldt et al., (1986) have suggested that there are functional 5-HT2 receptors in the rat spinal cord, since the release of substance P from slices of the ventral spinal cord was enhanced by 5-HT and the enhancement was completely blocked by 5-HT 2 antagonists including ketanserin. Moreover, in behavioural studies, similar results, that 5-HT2 antagonists antagonize the response induced by 5-MeODMT but not those induced by 8-OH-DPAT, have been reported (Tricklebank et al., 1985b; Goodwin and Green, 1985). The present study suggests that the spinal reflex is a precise and quantitative pharmacological model for evaluating the activity of 5-HT in the spinal cord. More selective 5-HT drugs (especially 5-HT~ antagonists and 5-HT2 agonists) will be required in order to elucidate the physiological and functional significance of 5-HT receptor subtypes in the central nervous system. Acknowledgements--This work was supported in part by
Research Grants from the Ministry of Education, Science and Culture of Japan and Takeda Science Foundation. We thank Kyowa Hakko, Squibb and CIBA-Geigy for providing ketanserin-T, cinanserin-HCl and desipramine-HCl, respectively. REFERENCES
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