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Stimulatory and inhibitory effects of serotonergic hallucinogens on spinal mono-and polysynaptic reflex pathways in the rat
Neuropharmacology Vol. 31, No. Great Britain
0028-3908/92 $5.00 + 0.00
7, PP. 635-642,1992
Pergamon Press Ltd
Printed in
STIMULATORY AND INHIBITORY EFFECTS OF SEROTONERGIC HALLUCINOGENS ON SPINAL MONOAND POLYSYNAPTIC REFLEX PATHWAYS IN THE RAT J. YAMAZAKI, H. ONO and T. NAGAO Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Accepted 8 December 1991)
Summary-The effects of two 5-HT-related hallucinogens on rat spinal mono- and polysynaptic reflex pathways in the rat were investigated. S-Methoxy-N,N-dimethyltryptamine (5-MeODMT, 1 and lOOpg/kg, iv.), an indolealkylamine agent, produced a dose-dependent decrease in the monosynaptic reflex, whereas I-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, l-100 yg/kg), a phenylalkylamine agent, produced a dose-dependent increase in the monosynaptic reflex. Both agents increased the polysynaptic reflex. The 5-HT, receptor antagonists ketanserin (100 fig/kg) and ritanserin (100 pg/kg) blocked the effects of DOI on the monosynaptic reflex but only partially blocked the 5-MeODMT-induced effect on the monosynaptic reflex. These antagonists inhibited the change in polysynaptic reflex, induced by DOI but not by 5-MeODMT. Neither propranolol (1 mg/kg) nor 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, 1 mg/kg) antagonized the effect of either agent. 5-Methoxy-N,N-dimethyltryptamine and DOI increased the excitability of motoneurons and this effect was inhibited by ketanserin. These results indicate that the two types of hallucinogens possess both common and distinct characteristics, with regard to their action on the spinal reflex: (1) both increase the activity of motoneurons through 5-HT, receptors but (2) only 5-MeODMT has an inhibitory action on the pathway of the monosynaptic reflex. Key words-5-HT, 5-HT, receptor, 5-methoxy-N,N-dimethyltryptamine, phenyl)-2-aminopropane, monosynaptic reflex, hallucinogens.
There is evidence that certain phenylalkylamines and indolealkylamines possess hallucinogenic effects (Glennon, Titeler and McKenney, 1984). 5-MethoxyN,N-dimethyltryptamine (S-MeODMT), one of the indolealkylamine hallucinogens, has high affinity for serotonin, (S-HT,) receptors as well as 5-HT, receptors (McKenna, Repke, Lo and Peroutka, 1990). I-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a prototypic phenylalkylamine hallucinogen, also has high affinity for S-HT, receptors and is considered a useful tool for investigating S-HT,-mediated responses (Glennon, Seggel, Soine, He&k-Davis, Lyon and Titeler, 1988). In addition, there appears to be a correlation between the affinity of these agents for 5-HT, receptors and their hallucinogenic potencies (Glennon et al., 1984). 5-Hydroxytryptamine neurons the somata of which lie in the raphe regions of the pons and medulla, project to the ventral horn of the spinal cord (Brodal, Taber and Walberg, 1960; Dahlstriim and
Fuxe, 1964), where they are thought to regulate motor function. In this laboratory, all the 5-HT agonists tested so far, including hallucinogenic agents such as 5-MeODMT and lysergic acid diethylamide (LSD), had common characteristic effects on spinal reflexes in rats with acute spinal transection, i.e. inhibition of the amplitude of the spinal monosynaptic reflex (MSR) and enhancement of the polysynaptic reflex (PSR) (Kaneko, Ono and Fukuda, 1984, 635
I-(2,5-dimethoxy-4-iodo-
1987; Nagano, Ono, Ozawa and Fukuda, 1987; Nagano, Ono and Fukuda, 1988). On the other hand, it has been reported that 5-HT agonists enhance the excitability of spinal motoneurons in rats (Barasi and Roberts, 1974; Takahashi and Berger, 1990). These findings indicate the existence of multiple 5-HTmediated motor functions in monosynaptic reflex pathways. Recently, Wing, Tapson and Geyer (1990) reported that the behavioral profiles of 5-HT, and 5-HT, agonists could be differentiated, using hallucinogenic agents such as 5-MeODMT and DOI. As in the case of the behavioral study, these types of hallucinogenic agents may exert different electrophysiological actions on spinal monosynaptic reflexes, through multiple subtypes of 5-HT receptors. Therefore, the present experiments were designed to investigate the effects of DO1 in comparison with those of 5MeODMT on monosynaptic reflexes and the excitability of motoneurons, and to determine whether the electrophysiological actions of these agents could be differentiated in the monosynaptic reflex pathway in the rat. METHODS Surgery Male Wistar rats, weighing 250-350 g, were anesthetized with urethane (1 g/kg, i.p.) and a-chloralose
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(25 mg/kg, i.p.) and then artificially ventilated. The vagus nerves were cut bilaterally in the cervical region and the spinal cord was transected at the Cl level. A laminectomy was performed in the lumbosacral region. The ventral and dorsal roots of segment L5 were isolated and a pouch of skin was formed at the site of the dissection, to allow the exposed tissues to be covered with liquid paraffin, which was kept at 36 k 0S”C. The rectal temperature of the animal was maintained at 36°C with a heating pad. During the measurement of excitability of the motoneuron pool, the rats were immobilized with D-tubocurarine chloride (2 mg/kg, i.p.). Recording of segmental spinal reflexes
The dorsal root of segment L5 was placed on a silver-silver chloride wire electrode for stimulation (0.2 Hz, 0.05 msec duration, 10 V supramaximal square-wave pulses; Nihon Kohden, SEN-3301), through an isolation unit. The reflex potentials were recorded from the ipsilateral L5 ventral root, mounted on a silver-silver chloride wire electrode. These were amplified (Nihon Kohden, AVB-10) and
displayed on an oscilloscope (Nihon Kohden, VC10). Eight consecutive responses were averaged every minute (Nihon Kohden, DAT-1100) and the analog output was recorded (Nihon Kohden, RJG-4122). Excitability of motoneurons
The excitability of the somata of motoneurons was measured (Ono, Fukuda and Kudo, 1979). A tungsten microelectrode (tip diameter less than 20 pm) was insulated by cashew, except at its tip. The electrode was inserted vertically, 1.8 mm into a motoneuron pool, from the spot between the entry zones of the dorsal roots of L4 and L5, and 0.8-l.Omm lateral to the midline. The motoneuron pool was stimulated with negative pulses (0.2 Hz, 0.05 msec) through the electrode. The orthodromic action potentials, which reflect the excitability of the motoneuron somata and synaptic response, were recorded from the ventral root of L5, using a bipolar silver-silver chloride wire electrode. The intensity of stimulation was adjusted to the voltage (l-3 V) that caused the amplitude of synaptic response to be maximal and the amplitude of the response of
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Fig. 1. Effectsof S-MeODMT and DOI on mono- and polysynaptic reflexes.(A) Representative averaged monosynaptic and polysynaptic reflexpotentials, elicited by electrical stimulation of the dorsal root. Reflex responses were obtained just before (0 min) intravenous administration of SMeODMT (1 pg/kg) and DOI (10 pg/kg) and 2 and 10min after administration of drug. (B) Effects of 5-MeODMT (circle, 1 pg/kg; square, 100 pg/kg) on the monosynaptic reflex (open symbols) and the polysynaptic reflex (solid symbols). (C,D) Effectsof DOI (triangle, 1 pg/kg; circle, 10 pg/kg; square, 100 pg/kg) on the monosynaptic (C) and the polysynaptic reflex (D). Drugs were injected at 0 min. Ordinates: reflex amplitude (means f SE of 4 experiments) expressed as a percentage of the value just prior to drug administration.
Effect of hallucinogens on spinal reflexes
motoneuron somata to be 80-100% of the amplitude of the synaptic response. The responses were amplified, averaged and recorded, as described above. Materials
The compounds used were as follows: 5-methoxyN,N-dimethyltryptamine (5MeODMT, Sigma); l(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) and 3-tropanyl-3,5dichlorobenzoate (MDL 72222) (Research Biochemical Inc.); ketanserin tartrate and ritanserin (Kyowa Hakko); (+)propranolol hydrochloride (Sumitomo Chemical); urethane (Wake); cr-chloralose and rr-tubocurarine chloride (Tokyo Kasei). Urethane and cr-chloralose were dissolved in distilled water. Test compounds were dissolved in physiological saline, except for ketanserin tartrate, ritanserin and MDL 72222, which were dissolved in distilled water and then injected into a femoral vein. Each antagonist was administered 10min before the injection of an agonist. All doses are expressed in terms of the weight of the forms of the drugs indicated above.
631
the drug. No facilitatory effect of 5-MeODMT on the amplitude of the monosynaptic reflex was observed in the present study. The phenylalkylamine DO1 increased both the mono- and polysynaptic reflexes in a dose-dependent manner (Figs lA, C and D). While 1 pg/kg of DO1 had no effect on either reflex, a dose of lOpg/kg
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Results are expressed as means + SE. Statistical comparisons between control and treated groups were carried out by using two-way analysis of variance (ANOVA) (treatment x times) with repeated measures, followed by post -hoc Bonferroni’s multiple t-test (two-tailed) at each time point, using pooled error mean square (Figs 2 and 3). One-way ANOVA and post-hoc Bonferroni’s multiple t-test (two-tailed) were applied to comparisons between control and treated groups (Fig. 4). Student’s t-test or Welch’s t-test, in the case of significantly different variance, was conducted to compare the control group with the treated group (Fig. SD).
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5-Methoxy-N,N-dimethyltryptamine decreased the monosynaptic reflex and increased the polysynaptic reflex in a dose-dependent manner (Figs 1A and B). A small dose of 5-MeODMT (1 pg/kg, i.v.) produced a maximum decrease of 65% in the monosynaptic reflex and a maximum increase of 45% in the polysynaptic reflex, 2 min after administration. The effects on both reflexes were short-lasting and recovery was observed within 20min. Administration of a large dose of 5-MeODMT (lOOpg/kg) produced a greater inhibition of the monosynaptic reflex (85%) and a greater augmentation of the polysynaptic reflex (110%) (Fig. 1B). The amplitude of the monosynaptic and polysynaptic reflexes gradually recovered but neither reflex returned to baseline levels within 45 min after administration of
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Fig. 2. Effectsof prior administration of ketanserin tartrate (1 mg/kg, i.v.) (B) and ritanserin (I mg/kg) (C) on the monosynaptic (open symbols) and polysynaptic reflexes (solid symbols) changes, induced by 5-MeODMT (1 pg/kg). Ordinates: reflex amplitude (means + SE of 4-g experiments), expressed as a percentage of the value just prior to the injection of S-MeODMT. *P < 0.05, **P < 0.01; significant difference between control (A) and each of the antagonist-treated groups (two-way ANOVA with repeated measures, followed by post-hoc Bonferroni’s multiple ttest).
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produced maximum increases in the mono- and polysynaptic reflexes of 25 and 65%, respectively. These effects remained for over 45 min after administration of DOI. Larger doses of DO1 (lOOpg/kg) induced an even greater (65-75%) and longer-lasting increase in the monosynaptic reflex. The same dose of DO1 produced a similar increase in the polysynaptic reflex to lOpg/kg of DOI.
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4. Effects of prior administration of several 5-HT antagonists on the reflex changes induced by 5-MeODMT (1 pg/kg, iv.) (A) and DOI (lOpg/kg) (B). Each column represents the reflex response amplitude 2 min (A) or 5 min (B) after injection of agonist . Values are means f SE of 3-8 experiments. The reflex amplitude is expressed as a percentage of the value just prior to the injection of each agonist. Open and solid columns represent the effects on the monosynaptic reflex and the polysynaptic reflex, respectively. The following antagonists were administered 10 min before the agonist: ritanserin (100 pg/kg, RITO.1; 1 mg/kg, RITl), ketanserin tartrate (1 mg/kg, KETl), (+)propranolol (1 mg/kg, PROl) and MDL 72222 (1 mg/kg, MDLl). *P < 0.05, **P < 0.01; significant difference between control (CONT) and each of the antagonist-treated groups (one-way ANOVA, followed by post-hoc Bonferroni’s multiple t-test).
Effects of S-HT antagonists on the reflex responses to S-MeODMT and DOI
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TlME (min) Fig. 3. Effects of prior administration of ketanserin tartrate (1 mg/kg, i.v.) (B) and ritanserin (lOOpg/kg) (C) on the monosynaptic (open symbols) and polysynaptic reflexes (solid symbols) changes induced by DOI (10 pg/kg). Ordinates: reflex amplitude (means f SE of 4-8 experiments), expressed as a percentage of the value, just prior to injection of DOI. *P
The effects of several 5-HT anatogonists were examined on the reflex changes induced by 5MeODMT (1 pg/kg, i.v.) and DO1 (lOpg/kg), in order to investigate the subtypes of 5-HT receptors involved. The 5-HT, antagonist ketanserin (1 mg/kg), when administered alone, had no effect on either the mono- or polysynaptic reflex. Prior administration of ketanserin (1 mg/kg, iv.; 10 min), partially inhibited the effect of 5-MeODMT on the monosynaptic reflex, but not on the polysynaptic reflex (Figs 2A, B and 4A). The augmentation of both reflexes produced by DO1 was abolished by pretreatment with ketanserin (Figs 3A, B and 4B). Another 5-HT, antagonist, ritanserin (1 mg/kg), which had no effect on the reflexes, when administered alone, inhibited the effect of 5-MeODMT on the monosynaptic reflex (Figs 2C and 4A), although at a dose of 100 pg/kg, ritanserin
639
Effect of hallucinogens on spinal reflexes
had no significant antagonistic action on the response to 5-MeODMT (Fig. 4A). Neither dose of ketanserin produced significant inhibition of the effect of DO1 on the polysynaptic reflex. On the other hand, prior administration of 100 pg/kg of ritanserin abolished the increases in the amplitudes of the mono- and polysynaptic reflexes, induced by DO1 (Figs 3C and 4B). (+)Propranolol (1 mg/kg), used as a S-HT, blocker, had a slight inhibitory effect on both reflexes, when used alone (monosynaptic reflex, - 8.7 f 2.9%, N = 6; polysynaptic reflex, -5.6 f 1.9%, N = 6). This was probably due to its stabilizing action. Pretreatment with propranolol had no effect on the change in either the monosynaptic or polysynaptic reflex, induced by either agonist (Figs 4A and B). Similarly, MDL 72222 (1 mg/kg), a 5-HT, blocker, which alone had no effect, did not significantly alter
the monosynaptic or polysynaptic responses to either agonist (Figs 4A and B). Effects of 5-MeODMT and DOI on motoneuron excitability The excitability of motoneurons was assessed by measuring the orthodromic action potentials from the L5 ventral root evoked by electrical stimulation of the motoneuron pool. As shown in Fig. 5A, the first response (motoneuron somata), with a latency of less than 0.4 msec was considered to be discharge evoked by direct stimulation of motoneurons. The second response (synaptic response), was considered to be a monosynaptically evoked discharge, as judged by its latency (1.3 msec). An occasional third response was considered to be a polysynaptically evoked discharge, also judged
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Fig. 5. Effects of 5-MeODMT and DO1 on the excitability of motoneurons. (A) Representative averaged motoneuron somata (MN) and synaptic response (MS) potentials, elicited by electrical stimulation of the motoneuron pool. Motoneuron somata (MN) and synaptic response (MS) indicate the responses of motoneurons to direct stimulation and to monosynaptic stimulation of the motoneuron pool, respectively. Responses were obtained just before (0 min) intravenous administration of 5-MeODMT (10 ,ug/kg, iv.) and DO1 (100 pg/kg) and 2 and 10 min after administration of drug. (B,C) Effects of 5-MeODMT (circle; 1 pg/kg, square; 10w/kg) (I9 and DOI WJ &kg) (C) on MN (solid symbols) and MS (open symbols). Each drug was injected at 0 min. Ordinates: potential amplitude (means + SE of 4 or 5 experiments), expressed as a percentage of the value just prior to administration of drug. (D) Influence of prior administration of ketanserin tartrate (1 mg/kg). KET: ketanserin was injected 10min before the administration of each agonist. CONT: control experiments. The amplitude of the motoneuron somata (MN) and synaptic response (MS) were calculated as a percentage of the value, just prior to the injection of each agonist. Solid and open columns indicate the response of the amplitudes, of the motoneuron somata and synaptic response, respectively, 2 min after injection of 5-MeODMT or 5 min after injection of DOI (means k SE of 3-6 experiments). lP -z 0.05; significant difference between control and a ketanserin-treated group (Student’s or Welch’s t-test).
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by its time to peak (2.7msec). S-Methoxy-N,Ndimethyltryptamine (1 pg/kg, i.v.) had no effect on the response of the motoneuron somata, but decreased the synaptic response by a maximum 40% 2min after administration (Fig. SB), as was also shown for the monosynaptic reflex (Fig. 1B). A IO-fold larger dose of 5-MeODMT (10 pg/kg), however, augmented the response of the motoneuron somata by 60% but depressed the synaptic response by 75% (Fig. 5B). On the other hand, DO1 (lOOpg/kg) augmented both responses by 70 and 40%, respectively (Fig. SC). The influence of ketanserin on the increase in motoneuron excitability produced by 5-MeODMT and DO1 was examined. Ketanserin (1 mg/kg) administered alone did not affect either response. The amplitudes of the response of the motoneuron somata and synaptic response, 2 min after the injection of ketanserin, were 97.9 + 1.6% (N = 6) and 98.3 f 3.1% (N = 6) of the value just before the injection, respectively. Prior administration of ketanserin abolished the increase in amplitude of the response of the motoneuron somata, produced by 5MeODMT (lOpg/kg) and DO1 (lOOpg/kg) (Fig. 5D). Ketanserin also attenuated the inhibition of the amplitude of the synaptic response, produced by 5-MeODMT and its augmentation by DO1 (Fig. 5D). DISCUSSION
The effect of DOI, a phenylalkylamine hallucinogen, was compared with that of 5-MeODMT, an indolealkylamine hallucinogen, on segmental spinal reflexes, in acutely spinalized rats. Different electrophysiological actions of these two hallucinogenic were found. 5-Methoxy-N,N-dimethylagents tryptamine inhibited the amplitude of the monosynaptic reflex and increased the amplitude of the polysynaptic reflex (Fig. 1B). These results are consistent with previous studies employing 5-MeODMT, LSD, L-5-hydroxytryptophan (L-5-HTP) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (Kaneko et al., 1984, 1987; Nagano et al., 1987, 1988). On the other hand, DO1 did not attenuate, but rather increased the monosynaptic reflex, as well as the polysynaptic reflex, in a dose-dependent manner (Figs IC and D). These effects were similar to those produced by the structurally related hallucinogen DOM (I-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane) (Hasebe and Ono, unpublished observation). It has been proposed that 1-(2,5-dimethoxyphenyl)-2-aminopropane derivatives, such as DOI, act as 5-HT2 receptor agonists in the central nervous system (Glennon, Mckenney, Lyon and Titeler, 1986; Maura, Roccatagliata, Ulivi and Raiteri, 1988; Rasmussen, Glennon and Aghajanian, 1986). In the present study, ketanserin (Leysen, Awouters, Kennis, Laduron, Vandenberk and Janssen, 1981) abolished DOI-induced augmentation of the mono- and polysynaptic reflexes, when administered intravenously, at
et
al.
doses sufficient to antagonize responses mediated by 5-HT, receptors in the brainstem (Davies, Wilkinson and Roberts, 1988). The more selective 5-HT, antagonist, ritanserin (Leysen, Gommeren, Van Gompel, Wynants, Janssen and Laduron, 1985), also inhibited the DOI-induced increase in the mono- and polysynaptic reflexes (Fig. 3C). In contrast to the antagonistic effects of ketanserin and ritanserin, neither propranolol, a /3-adrenergic blocker that inhibits 5-HT,-mediated behavioral responses (Costain and Green, 1978), nor MDL 72222, a selective 5-HT, antagonist (Fozard, 1984), blocked the effects of DO1 on spinal reflexes (Fig. 4B). These results indicate that DO1 increased both reflexes through the activation of 5-HT, receptors. The ventral root discharge, evoked by stimulation of the ventral horn was measured in order to investigate which part of the monosynaptic reflex pathway was responsible for the facilitatory action of DOI. The drug DO1 enhanced the motoneuron somata response, which is a non-synaptic event (Figs 5A and C). This finding suggests that DO1 potentiated the monosynaptic reflex by increasing the excitability of the motoneuron somata or dendrites. It is noteworthy that 5-MeODMT also enhanced the motoneuron somata response, in spite of the reduced monosynaptic reflex (Figs 1B and 5B). Therefore, an increase in motoneuron excitability appears to be a common feature of these two hallucinogenic agents. The observed increase in the motoneuron somata response is consistent with previous electrophysiological studies, which have shown that 5-HT and 5-HT agonists enhance the excitability of motoneurons in uiuo (White and Neuman, 1980, 1983) and in oitro (Connell and Wallis, 1988, 1989; Wang and Dun, 1990). In addition, studies with facial motoneurons in the rat have shown that 5-HT increased glutamic acid-induced firing and elicited membrane depolarization (McCall and Aghajanian, 1979; VanderMaelen and Aghajanian, 1980). Both 5MeODMT and DOM were also shown to have a depolarizing action on these neurons and to increase excitability (VanderMaelen and Aghajanian, 1982; Rasmussen and Aghajanian, 1990). The ability of ketanserin to inhibit the motoneuron somata responses to DO1 and 5-MeODMT (Fig. 5) indicates the existence of excitatory 5-HT, receptors, presumably on spinal motoneuron somata or dendrites. However, it has been reported that 5-HT increases the excitability of motoneurons, by acting on a 5-HT,-like or 5-HT,* receptor (Roberts, Davies, Girdlestone and Foster, 1988; Takahashi and Berger, 1990). On the other hand, it is reported that in cells of the dorsal root ganglion (DRG) of the rat, 5-HT-induced depolarization, accompanied by increased input resistance, is mediated by 5-HT2 receptors (Todorovic and Anderson, 1990). Although the present results do not exclude the involvement of these receptors in the excitatory effect on the monosynaptic reflex pathway, they
Effect of hallucinogens on spinal reflexes
indicate a role for 5-HT, receptors in the excitation of motoneurons. The monosynaptic reflex pathway is recognized as a monosynaptic pathway from group Ia afferent fibers to motoneurons. Since S-MeODMT did not exert an inhibitory effect on the excitability of the motoneuron somata (Fig. 5B), it may decrease the amplitude of the monosynaptic reflex by inhibiting synaptic transmission from primary afferent terminals to motoneurons. In in vitro spinal cord preparations of the frog, 5-HT has been shown to elicit depolarization of primary afferent fibers and dorsal root ganglion cells (Holtz and Anderson, 1984) and to shorten the Ca*+ spike of dorsal root ganglion cells (Holtz, Shefner and Anderson, 1986), suggesting the possibility of decreased release of transmitter from group Ia afferent fibers. 5-Methoxy-N,N-dimethyltryptamine shows high affinity for both 5-HT, and 5-HT, sites (McKenna et al., 1990). The subtype of receptor mediating the inhibitory action of 5-MeODMT on the monosynaptic reflex does not appear to be identical to the 5-HT, site, because the 5-HT,-selective agonist DO1 failed to inhibit the monosynaptic reflex. Recently, it has been reported that 5-HT, receptor agonists reduce the calcium component of sensory neuron action potentials (Scroggs and Anderson, 1990). In the present study, the 5-MeODMT-induced decrease in the monosynaptic reflex was partially inhibited by the 5-HTr antagonists ketanserin and ritanserin (Fig. 4A). On the other hand, both propranolol and MDL 72222 were ineffective in blocking the effect of 5-MeODMT on the monosynaptic reflex. Therefore, the subtype of 5-HT receptor, mediating the inhibitory action of 5-MeODMT at presynaptic sites, remains to be clarified by using other selective ligands. 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (10 pg/kg) enhanced the polysynaptic reflex to a greater extent than the monosynaptic reflex (Figs 1C and D). 5-Methoxy-N,N-dimethyltryptamine augmented the polysynaptic reflex in spite of its inhibition of the monosynaptic reflex (Fig. 1B). These results suggest that both compounds had a stronger influence on polysynaptic pathways. In conclusion, the hallucinogenic 5-HT agonists, 5-MeODMT and DOI, increased the excitability of motoneurons through 5-HT, receptors. 5-MethoxyN,N-dimethyltryptamine inhibited the monosynaptic reflex, while DO1 increased it. These results suggest that these two hallucinogenic agents differentiate excitatory and inhibitory actions, mediated by various 5-HT receptors in the spinal monosynaptic reflex pathway in the rat. Acknowledgements-The authors are grateful to Kyowa Hakko for providing ketanserin tartrate and ritanserin. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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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 S-HT2 receptors. Life Sci. 2S: 1015-1022. Leysen J. E., Gommeren W., Van Gompel P., Wynants J., Janssen P. F. M. and Laduron P. M. (1985) Recentorbinding properties in vitro and in vivo of t&an&n. Avery potent and long acting serotonin-S, antagonist. Molec. Pharmac. 27: 60&611.
Maura G., Roccatagliata E., Ulivi M. and Raiteri M. (1988) Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT, and 5-HT, receptors. Eur. J. Pharmac. 145: 31-38.
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McCall R. B. and Aghajanian G. K. (1979) Serotonergic facilitation of facial motoneuron excitation. Bruin Res. 169: 11-27. McKenna D. J., Repke D. B., Lo L. and Peroutka S. J. (1990) Differential interactions of indolealkylamines with S-hydroxytryptamine receptor subtypes. Neuropharmacolog> 29: -193-198.
Naeano N.. Ono H. and Fukuda H. (1988) Functional s:gnificance of subtypes of S-HT receptors in the rat spinal reflex pathway. Gen. Pharmac. 19: 789-793. Nagano N., Ono H., Ozawa M. and Fukuda H. (1987) Sensitivity of spinal reflexes to TRH and 5-HT in 5,6dihydroxytryptamine-treated rats. Eur. J. Pharmac. 139: 315-321.
Ono H., Fukuda H. and Kudo Y. (1979) Mechanisms of depressant action of baclofen on the spinal reflex in the rat. Neuropharmacology 18: 647653. Rasmussen K. and Aghajanian G. K. (1990) Serotonin excitation of facial motoneurons: receptor subtype characterization. Synapse 5: 324-332. Rasmussen K., Glennon R. A. and Aghajanian G. K. (1986) Phenethylamine hallucinogens in the locus coeruleus: potency of action correlates with rank order of 5-HT, binding affinity. Eur. J. Pharmac. 132: 7942.
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Scroggs R. S. and Anderson E. G. (1990) 5-HT, receptor agonists reduce the Ca*+ component of sensory neuron action potentials. Eur. J. Pharmac. 178: 229-232. Takahashi T. and Berger A. J. (1990) Direct excitation of rat spinal motoneurones by serotonin. J. Physiol. 423: 63-76. Todorovic S. M. and Anderson E. G. (1990) Pharmacological characterization of 5-hydroxytryptamine, and S-hydroxvtryptaminel receptors in rat dorsal root ganglion cells.~J.w>harmac. exp.- Ther. 254: 109-l 15. - VanderMaelen C. P. and Aghaianian G. K. (1980) Intracellular studies showing modulation of ‘facial motoneurone excitability by serotonin. Nature 287: 346-347. VanderMaelen C. P. and Aghajanian G. K. (1982) Intracellular studies on the effects of systemic administration of serotonin agonists on rat facial motoneurons. Eur. J. Pharmac. 78: 233-236.
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STIMULATORY AND INHIBITORY EFFECTS OF SEROTONERGIC HALLUCINOGENS ON SPINAL MONOAND POLYSYNAPTIC REFLEX PATHWAYS IN THE RAT J. YAMAZAKI, H. ONO and T. NAGAO Department of Toxicology and Pharmacology, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Accepted 8 December 1991)
Summary-The effects of two 5-HT-related hallucinogens on rat spinal mono- and polysynaptic reflex pathways in the rat were investigated. S-Methoxy-N,N-dimethyltryptamine (5-MeODMT, 1 and lOOpg/kg, iv.), an indolealkylamine agent, produced a dose-dependent decrease in the monosynaptic reflex, whereas I-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, l-100 yg/kg), a phenylalkylamine agent, produced a dose-dependent increase in the monosynaptic reflex. Both agents increased the polysynaptic reflex. The 5-HT, receptor antagonists ketanserin (100 fig/kg) and ritanserin (100 pg/kg) blocked the effects of DOI on the monosynaptic reflex but only partially blocked the 5-MeODMT-induced effect on the monosynaptic reflex. These antagonists inhibited the change in polysynaptic reflex, induced by DOI but not by 5-MeODMT. Neither propranolol (1 mg/kg) nor 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, 1 mg/kg) antagonized the effect of either agent. 5-Methoxy-N,N-dimethyltryptamine and DOI increased the excitability of motoneurons and this effect was inhibited by ketanserin. These results indicate that the two types of hallucinogens possess both common and distinct characteristics, with regard to their action on the spinal reflex: (1) both increase the activity of motoneurons through 5-HT, receptors but (2) only 5-MeODMT has an inhibitory action on the pathway of the monosynaptic reflex. Key words-5-HT, 5-HT, receptor, 5-methoxy-N,N-dimethyltryptamine, phenyl)-2-aminopropane, monosynaptic reflex, hallucinogens.
There is evidence that certain phenylalkylamines and indolealkylamines possess hallucinogenic effects (Glennon, Titeler and McKenney, 1984). 5-MethoxyN,N-dimethyltryptamine (S-MeODMT), one of the indolealkylamine hallucinogens, has high affinity for serotonin, (S-HT,) receptors as well as 5-HT, receptors (McKenna, Repke, Lo and Peroutka, 1990). I-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a prototypic phenylalkylamine hallucinogen, also has high affinity for S-HT, receptors and is considered a useful tool for investigating S-HT,-mediated responses (Glennon, Seggel, Soine, He&k-Davis, Lyon and Titeler, 1988). In addition, there appears to be a correlation between the affinity of these agents for 5-HT, receptors and their hallucinogenic potencies (Glennon et al., 1984). 5-Hydroxytryptamine neurons the somata of which lie in the raphe regions of the pons and medulla, project to the ventral horn of the spinal cord (Brodal, Taber and Walberg, 1960; Dahlstriim and
Fuxe, 1964), where they are thought to regulate motor function. In this laboratory, all the 5-HT agonists tested so far, including hallucinogenic agents such as 5-MeODMT and lysergic acid diethylamide (LSD), had common characteristic effects on spinal reflexes in rats with acute spinal transection, i.e. inhibition of the amplitude of the spinal monosynaptic reflex (MSR) and enhancement of the polysynaptic reflex (PSR) (Kaneko, Ono and Fukuda, 1984, 635
I-(2,5-dimethoxy-4-iodo-
1987; Nagano, Ono, Ozawa and Fukuda, 1987; Nagano, Ono and Fukuda, 1988). On the other hand, it has been reported that 5-HT agonists enhance the excitability of spinal motoneurons in rats (Barasi and Roberts, 1974; Takahashi and Berger, 1990). These findings indicate the existence of multiple 5-HTmediated motor functions in monosynaptic reflex pathways. Recently, Wing, Tapson and Geyer (1990) reported that the behavioral profiles of 5-HT, and 5-HT, agonists could be differentiated, using hallucinogenic agents such as 5-MeODMT and DOI. As in the case of the behavioral study, these types of hallucinogenic agents may exert different electrophysiological actions on spinal monosynaptic reflexes, through multiple subtypes of 5-HT receptors. Therefore, the present experiments were designed to investigate the effects of DO1 in comparison with those of 5MeODMT on monosynaptic reflexes and the excitability of motoneurons, and to determine whether the electrophysiological actions of these agents could be differentiated in the monosynaptic reflex pathway in the rat. METHODS Surgery Male Wistar rats, weighing 250-350 g, were anesthetized with urethane (1 g/kg, i.p.) and a-chloralose
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(25 mg/kg, i.p.) and then artificially ventilated. The vagus nerves were cut bilaterally in the cervical region and the spinal cord was transected at the Cl level. A laminectomy was performed in the lumbosacral region. The ventral and dorsal roots of segment L5 were isolated and a pouch of skin was formed at the site of the dissection, to allow the exposed tissues to be covered with liquid paraffin, which was kept at 36 k 0S”C. The rectal temperature of the animal was maintained at 36°C with a heating pad. During the measurement of excitability of the motoneuron pool, the rats were immobilized with D-tubocurarine chloride (2 mg/kg, i.p.). Recording of segmental spinal reflexes
The dorsal root of segment L5 was placed on a silver-silver chloride wire electrode for stimulation (0.2 Hz, 0.05 msec duration, 10 V supramaximal square-wave pulses; Nihon Kohden, SEN-3301), through an isolation unit. The reflex potentials were recorded from the ipsilateral L5 ventral root, mounted on a silver-silver chloride wire electrode. These were amplified (Nihon Kohden, AVB-10) and
displayed on an oscilloscope (Nihon Kohden, VC10). Eight consecutive responses were averaged every minute (Nihon Kohden, DAT-1100) and the analog output was recorded (Nihon Kohden, RJG-4122). Excitability of motoneurons
The excitability of the somata of motoneurons was measured (Ono, Fukuda and Kudo, 1979). A tungsten microelectrode (tip diameter less than 20 pm) was insulated by cashew, except at its tip. The electrode was inserted vertically, 1.8 mm into a motoneuron pool, from the spot between the entry zones of the dorsal roots of L4 and L5, and 0.8-l.Omm lateral to the midline. The motoneuron pool was stimulated with negative pulses (0.2 Hz, 0.05 msec) through the electrode. The orthodromic action potentials, which reflect the excitability of the motoneuron somata and synaptic response, were recorded from the ventral root of L5, using a bipolar silver-silver chloride wire electrode. The intensity of stimulation was adjusted to the voltage (l-3 V) that caused the amplitude of synaptic response to be maximal and the amplitude of the response of
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Fig. 1. Effectsof S-MeODMT and DOI on mono- and polysynaptic reflexes.(A) Representative averaged monosynaptic and polysynaptic reflexpotentials, elicited by electrical stimulation of the dorsal root. Reflex responses were obtained just before (0 min) intravenous administration of SMeODMT (1 pg/kg) and DOI (10 pg/kg) and 2 and 10min after administration of drug. (B) Effects of 5-MeODMT (circle, 1 pg/kg; square, 100 pg/kg) on the monosynaptic reflex (open symbols) and the polysynaptic reflex (solid symbols). (C,D) Effectsof DOI (triangle, 1 pg/kg; circle, 10 pg/kg; square, 100 pg/kg) on the monosynaptic (C) and the polysynaptic reflex (D). Drugs were injected at 0 min. Ordinates: reflex amplitude (means f SE of 4 experiments) expressed as a percentage of the value just prior to drug administration.
Effect of hallucinogens on spinal reflexes
motoneuron somata to be 80-100% of the amplitude of the synaptic response. The responses were amplified, averaged and recorded, as described above. Materials
The compounds used were as follows: 5-methoxyN,N-dimethyltryptamine (5MeODMT, Sigma); l(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) and 3-tropanyl-3,5dichlorobenzoate (MDL 72222) (Research Biochemical Inc.); ketanserin tartrate and ritanserin (Kyowa Hakko); (+)propranolol hydrochloride (Sumitomo Chemical); urethane (Wake); cr-chloralose and rr-tubocurarine chloride (Tokyo Kasei). Urethane and cr-chloralose were dissolved in distilled water. Test compounds were dissolved in physiological saline, except for ketanserin tartrate, ritanserin and MDL 72222, which were dissolved in distilled water and then injected into a femoral vein. Each antagonist was administered 10min before the injection of an agonist. All doses are expressed in terms of the weight of the forms of the drugs indicated above.
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the drug. No facilitatory effect of 5-MeODMT on the amplitude of the monosynaptic reflex was observed in the present study. The phenylalkylamine DO1 increased both the mono- and polysynaptic reflexes in a dose-dependent manner (Figs lA, C and D). While 1 pg/kg of DO1 had no effect on either reflex, a dose of lOpg/kg
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Results are expressed as means + SE. Statistical comparisons between control and treated groups were carried out by using two-way analysis of variance (ANOVA) (treatment x times) with repeated measures, followed by post -hoc Bonferroni’s multiple t-test (two-tailed) at each time point, using pooled error mean square (Figs 2 and 3). One-way ANOVA and post-hoc Bonferroni’s multiple t-test (two-tailed) were applied to comparisons between control and treated groups (Fig. 4). Student’s t-test or Welch’s t-test, in the case of significantly different variance, was conducted to compare the control group with the treated group (Fig. SD).
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5-Methoxy-N,N-dimethyltryptamine decreased the monosynaptic reflex and increased the polysynaptic reflex in a dose-dependent manner (Figs 1A and B). A small dose of 5-MeODMT (1 pg/kg, i.v.) produced a maximum decrease of 65% in the monosynaptic reflex and a maximum increase of 45% in the polysynaptic reflex, 2 min after administration. The effects on both reflexes were short-lasting and recovery was observed within 20min. Administration of a large dose of 5-MeODMT (lOOpg/kg) produced a greater inhibition of the monosynaptic reflex (85%) and a greater augmentation of the polysynaptic reflex (110%) (Fig. 1B). The amplitude of the monosynaptic and polysynaptic reflexes gradually recovered but neither reflex returned to baseline levels within 45 min after administration of
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Fig. 2. Effectsof prior administration of ketanserin tartrate (1 mg/kg, i.v.) (B) and ritanserin (I mg/kg) (C) on the monosynaptic (open symbols) and polysynaptic reflexes (solid symbols) changes, induced by 5-MeODMT (1 pg/kg). Ordinates: reflex amplitude (means + SE of 4-g experiments), expressed as a percentage of the value just prior to the injection of S-MeODMT. *P < 0.05, **P < 0.01; significant difference between control (A) and each of the antagonist-treated groups (two-way ANOVA with repeated measures, followed by post-hoc Bonferroni’s multiple ttest).
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produced maximum increases in the mono- and polysynaptic reflexes of 25 and 65%, respectively. These effects remained for over 45 min after administration of DOI. Larger doses of DO1 (lOOpg/kg) induced an even greater (65-75%) and longer-lasting increase in the monosynaptic reflex. The same dose of DO1 produced a similar increase in the polysynaptic reflex to lOpg/kg of DOI.
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4. Effects of prior administration of several 5-HT antagonists on the reflex changes induced by 5-MeODMT (1 pg/kg, iv.) (A) and DOI (lOpg/kg) (B). Each column represents the reflex response amplitude 2 min (A) or 5 min (B) after injection of agonist . Values are means f SE of 3-8 experiments. The reflex amplitude is expressed as a percentage of the value just prior to the injection of each agonist. Open and solid columns represent the effects on the monosynaptic reflex and the polysynaptic reflex, respectively. The following antagonists were administered 10 min before the agonist: ritanserin (100 pg/kg, RITO.1; 1 mg/kg, RITl), ketanserin tartrate (1 mg/kg, KETl), (+)propranolol (1 mg/kg, PROl) and MDL 72222 (1 mg/kg, MDLl). *P < 0.05, **P < 0.01; significant difference between control (CONT) and each of the antagonist-treated groups (one-way ANOVA, followed by post-hoc Bonferroni’s multiple t-test).
Effects of S-HT antagonists on the reflex responses to S-MeODMT and DOI
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TlME (min) Fig. 3. Effects of prior administration of ketanserin tartrate (1 mg/kg, i.v.) (B) and ritanserin (lOOpg/kg) (C) on the monosynaptic (open symbols) and polysynaptic reflexes (solid symbols) changes induced by DOI (10 pg/kg). Ordinates: reflex amplitude (means f SE of 4-8 experiments), expressed as a percentage of the value, just prior to injection of DOI. *P
The effects of several 5-HT anatogonists were examined on the reflex changes induced by 5MeODMT (1 pg/kg, i.v.) and DO1 (lOpg/kg), in order to investigate the subtypes of 5-HT receptors involved. The 5-HT, antagonist ketanserin (1 mg/kg), when administered alone, had no effect on either the mono- or polysynaptic reflex. Prior administration of ketanserin (1 mg/kg, iv.; 10 min), partially inhibited the effect of 5-MeODMT on the monosynaptic reflex, but not on the polysynaptic reflex (Figs 2A, B and 4A). The augmentation of both reflexes produced by DO1 was abolished by pretreatment with ketanserin (Figs 3A, B and 4B). Another 5-HT, antagonist, ritanserin (1 mg/kg), which had no effect on the reflexes, when administered alone, inhibited the effect of 5-MeODMT on the monosynaptic reflex (Figs 2C and 4A), although at a dose of 100 pg/kg, ritanserin
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had no significant antagonistic action on the response to 5-MeODMT (Fig. 4A). Neither dose of ketanserin produced significant inhibition of the effect of DO1 on the polysynaptic reflex. On the other hand, prior administration of 100 pg/kg of ritanserin abolished the increases in the amplitudes of the mono- and polysynaptic reflexes, induced by DO1 (Figs 3C and 4B). (+)Propranolol (1 mg/kg), used as a S-HT, blocker, had a slight inhibitory effect on both reflexes, when used alone (monosynaptic reflex, - 8.7 f 2.9%, N = 6; polysynaptic reflex, -5.6 f 1.9%, N = 6). This was probably due to its stabilizing action. Pretreatment with propranolol had no effect on the change in either the monosynaptic or polysynaptic reflex, induced by either agonist (Figs 4A and B). Similarly, MDL 72222 (1 mg/kg), a 5-HT, blocker, which alone had no effect, did not significantly alter
the monosynaptic or polysynaptic responses to either agonist (Figs 4A and B). Effects of 5-MeODMT and DOI on motoneuron excitability The excitability of motoneurons was assessed by measuring the orthodromic action potentials from the L5 ventral root evoked by electrical stimulation of the motoneuron pool. As shown in Fig. 5A, the first response (motoneuron somata), with a latency of less than 0.4 msec was considered to be discharge evoked by direct stimulation of motoneurons. The second response (synaptic response), was considered to be a monosynaptically evoked discharge, as judged by its latency (1.3 msec). An occasional third response was considered to be a polysynaptically evoked discharge, also judged
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Fig. 5. Effects of 5-MeODMT and DO1 on the excitability of motoneurons. (A) Representative averaged motoneuron somata (MN) and synaptic response (MS) potentials, elicited by electrical stimulation of the motoneuron pool. Motoneuron somata (MN) and synaptic response (MS) indicate the responses of motoneurons to direct stimulation and to monosynaptic stimulation of the motoneuron pool, respectively. Responses were obtained just before (0 min) intravenous administration of 5-MeODMT (10 ,ug/kg, iv.) and DO1 (100 pg/kg) and 2 and 10 min after administration of drug. (B,C) Effects of 5-MeODMT (circle; 1 pg/kg, square; 10w/kg) (I9 and DOI WJ &kg) (C) on MN (solid symbols) and MS (open symbols). Each drug was injected at 0 min. Ordinates: potential amplitude (means + SE of 4 or 5 experiments), expressed as a percentage of the value just prior to administration of drug. (D) Influence of prior administration of ketanserin tartrate (1 mg/kg). KET: ketanserin was injected 10min before the administration of each agonist. CONT: control experiments. The amplitude of the motoneuron somata (MN) and synaptic response (MS) were calculated as a percentage of the value, just prior to the injection of each agonist. Solid and open columns indicate the response of the amplitudes, of the motoneuron somata and synaptic response, respectively, 2 min after injection of 5-MeODMT or 5 min after injection of DOI (means k SE of 3-6 experiments). lP -z 0.05; significant difference between control and a ketanserin-treated group (Student’s or Welch’s t-test).
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by its time to peak (2.7msec). S-Methoxy-N,Ndimethyltryptamine (1 pg/kg, i.v.) had no effect on the response of the motoneuron somata, but decreased the synaptic response by a maximum 40% 2min after administration (Fig. SB), as was also shown for the monosynaptic reflex (Fig. 1B). A IO-fold larger dose of 5-MeODMT (10 pg/kg), however, augmented the response of the motoneuron somata by 60% but depressed the synaptic response by 75% (Fig. 5B). On the other hand, DO1 (lOOpg/kg) augmented both responses by 70 and 40%, respectively (Fig. SC). The influence of ketanserin on the increase in motoneuron excitability produced by 5-MeODMT and DO1 was examined. Ketanserin (1 mg/kg) administered alone did not affect either response. The amplitudes of the response of the motoneuron somata and synaptic response, 2 min after the injection of ketanserin, were 97.9 + 1.6% (N = 6) and 98.3 f 3.1% (N = 6) of the value just before the injection, respectively. Prior administration of ketanserin abolished the increase in amplitude of the response of the motoneuron somata, produced by 5MeODMT (lOpg/kg) and DO1 (lOOpg/kg) (Fig. 5D). Ketanserin also attenuated the inhibition of the amplitude of the synaptic response, produced by 5-MeODMT and its augmentation by DO1 (Fig. 5D). DISCUSSION
The effect of DOI, a phenylalkylamine hallucinogen, was compared with that of 5-MeODMT, an indolealkylamine hallucinogen, on segmental spinal reflexes, in acutely spinalized rats. Different electrophysiological actions of these two hallucinogenic were found. 5-Methoxy-N,N-dimethylagents tryptamine inhibited the amplitude of the monosynaptic reflex and increased the amplitude of the polysynaptic reflex (Fig. 1B). These results are consistent with previous studies employing 5-MeODMT, LSD, L-5-hydroxytryptophan (L-5-HTP) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (Kaneko et al., 1984, 1987; Nagano et al., 1987, 1988). On the other hand, DO1 did not attenuate, but rather increased the monosynaptic reflex, as well as the polysynaptic reflex, in a dose-dependent manner (Figs IC and D). These effects were similar to those produced by the structurally related hallucinogen DOM (I-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane) (Hasebe and Ono, unpublished observation). It has been proposed that 1-(2,5-dimethoxyphenyl)-2-aminopropane derivatives, such as DOI, act as 5-HT2 receptor agonists in the central nervous system (Glennon, Mckenney, Lyon and Titeler, 1986; Maura, Roccatagliata, Ulivi and Raiteri, 1988; Rasmussen, Glennon and Aghajanian, 1986). In the present study, ketanserin (Leysen, Awouters, Kennis, Laduron, Vandenberk and Janssen, 1981) abolished DOI-induced augmentation of the mono- and polysynaptic reflexes, when administered intravenously, at
et
al.
doses sufficient to antagonize responses mediated by 5-HT, receptors in the brainstem (Davies, Wilkinson and Roberts, 1988). The more selective 5-HT, antagonist, ritanserin (Leysen, Gommeren, Van Gompel, Wynants, Janssen and Laduron, 1985), also inhibited the DOI-induced increase in the mono- and polysynaptic reflexes (Fig. 3C). In contrast to the antagonistic effects of ketanserin and ritanserin, neither propranolol, a /3-adrenergic blocker that inhibits 5-HT,-mediated behavioral responses (Costain and Green, 1978), nor MDL 72222, a selective 5-HT, antagonist (Fozard, 1984), blocked the effects of DO1 on spinal reflexes (Fig. 4B). These results indicate that DO1 increased both reflexes through the activation of 5-HT, receptors. The ventral root discharge, evoked by stimulation of the ventral horn was measured in order to investigate which part of the monosynaptic reflex pathway was responsible for the facilitatory action of DOI. The drug DO1 enhanced the motoneuron somata response, which is a non-synaptic event (Figs 5A and C). This finding suggests that DO1 potentiated the monosynaptic reflex by increasing the excitability of the motoneuron somata or dendrites. It is noteworthy that 5-MeODMT also enhanced the motoneuron somata response, in spite of the reduced monosynaptic reflex (Figs 1B and 5B). Therefore, an increase in motoneuron excitability appears to be a common feature of these two hallucinogenic agents. The observed increase in the motoneuron somata response is consistent with previous electrophysiological studies, which have shown that 5-HT and 5-HT agonists enhance the excitability of motoneurons in uiuo (White and Neuman, 1980, 1983) and in oitro (Connell and Wallis, 1988, 1989; Wang and Dun, 1990). In addition, studies with facial motoneurons in the rat have shown that 5-HT increased glutamic acid-induced firing and elicited membrane depolarization (McCall and Aghajanian, 1979; VanderMaelen and Aghajanian, 1980). Both 5MeODMT and DOM were also shown to have a depolarizing action on these neurons and to increase excitability (VanderMaelen and Aghajanian, 1982; Rasmussen and Aghajanian, 1990). The ability of ketanserin to inhibit the motoneuron somata responses to DO1 and 5-MeODMT (Fig. 5) indicates the existence of excitatory 5-HT, receptors, presumably on spinal motoneuron somata or dendrites. However, it has been reported that 5-HT increases the excitability of motoneurons, by acting on a 5-HT,-like or 5-HT,* receptor (Roberts, Davies, Girdlestone and Foster, 1988; Takahashi and Berger, 1990). On the other hand, it is reported that in cells of the dorsal root ganglion (DRG) of the rat, 5-HT-induced depolarization, accompanied by increased input resistance, is mediated by 5-HT2 receptors (Todorovic and Anderson, 1990). Although the present results do not exclude the involvement of these receptors in the excitatory effect on the monosynaptic reflex pathway, they
Effect of hallucinogens on spinal reflexes
indicate a role for 5-HT, receptors in the excitation of motoneurons. The monosynaptic reflex pathway is recognized as a monosynaptic pathway from group Ia afferent fibers to motoneurons. Since S-MeODMT did not exert an inhibitory effect on the excitability of the motoneuron somata (Fig. 5B), it may decrease the amplitude of the monosynaptic reflex by inhibiting synaptic transmission from primary afferent terminals to motoneurons. In in vitro spinal cord preparations of the frog, 5-HT has been shown to elicit depolarization of primary afferent fibers and dorsal root ganglion cells (Holtz and Anderson, 1984) and to shorten the Ca*+ spike of dorsal root ganglion cells (Holtz, Shefner and Anderson, 1986), suggesting the possibility of decreased release of transmitter from group Ia afferent fibers. 5-Methoxy-N,N-dimethyltryptamine shows high affinity for both 5-HT, and 5-HT, sites (McKenna et al., 1990). The subtype of receptor mediating the inhibitory action of 5-MeODMT on the monosynaptic reflex does not appear to be identical to the 5-HT, site, because the 5-HT,-selective agonist DO1 failed to inhibit the monosynaptic reflex. Recently, it has been reported that 5-HT, receptor agonists reduce the calcium component of sensory neuron action potentials (Scroggs and Anderson, 1990). In the present study, the 5-MeODMT-induced decrease in the monosynaptic reflex was partially inhibited by the 5-HTr antagonists ketanserin and ritanserin (Fig. 4A). On the other hand, both propranolol and MDL 72222 were ineffective in blocking the effect of 5-MeODMT on the monosynaptic reflex. Therefore, the subtype of 5-HT receptor, mediating the inhibitory action of 5-MeODMT at presynaptic sites, remains to be clarified by using other selective ligands. 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (10 pg/kg) enhanced the polysynaptic reflex to a greater extent than the monosynaptic reflex (Figs 1C and D). 5-Methoxy-N,N-dimethyltryptamine augmented the polysynaptic reflex in spite of its inhibition of the monosynaptic reflex (Fig. 1B). These results suggest that both compounds had a stronger influence on polysynaptic pathways. In conclusion, the hallucinogenic 5-HT agonists, 5-MeODMT and DOI, increased the excitability of motoneurons through 5-HT, receptors. 5-MethoxyN,N-dimethyltryptamine inhibited the monosynaptic reflex, while DO1 increased it. These results suggest that these two hallucinogenic agents differentiate excitatory and inhibitory actions, mediated by various 5-HT receptors in the spinal monosynaptic reflex pathway in the rat. Acknowledgements-The authors are grateful to Kyowa Hakko for providing ketanserin tartrate and ritanserin. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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