The 5-HT Syndrome in Rats as Tool for the Screening of Psychoactive Drugs

Transcription

1 Drug Development Research (1984) Current Trends Review The 5-HT Syndrome in Rats as Tool for the Screening of Psychoactive Drugs R. Ortmann Research Department, Pharmaceuticals Division, Ciba-Geigy Limited, CH 4002-Basel, Switzerland NTRODUCTON METHODS EFFECTS OF NHBTORS OF MAO-A AND 5-HT-UPTAKE DFFERENTATON BETWEEN NHBTORS OF MAO-A AND OF THE 5-HT REUPTAKE SYSTEM P-ADRENOCEPTOR AGONSTS Mechanism of Action 5-HT-ANTAGONSTS 5-HT-AGONSTS Characterization of Behavioral Effects ACKNOWLEDGMENTS REFERENCES ABSTRACT Ortmann, R.: The 5-HT syndrome in rats as tool for the screening of psychoactive drugs. Drug Dev. Res , The use of the behavioral 5-HT syndrome in rats to characterize psychoactive drugs interacting with the central serotonin system is described. The potentiation of Ld-HTPinduced excitation can be used to detect drugs inhibiting central monoamine oxidase (MAO) A or the 5-HT uptake system and to define their mechanism of action. n addition to their potency, the biological half-life of these types of drugs can also be measured. The mechanism of action by agonists like salbutamol or clenbuterol potentiate behavioral effects of Ld-HTP is discussed. The 5-HT syndrome is also a convenient behavioral test system to screen for central 5-HT antagonists and 5-HT agonists. Different procedures to characterize the 5-HT specificity of behavioral effects induced by putative 5-HT agonists are described and their relevance discussed. Received final version May 22, 1984; accepted May 22, Address reprint requests to R. Ortmann, Research Department, Pharmaceuticals Division, Ciba-Geigy Limited, CH4002-Base1, Switzerland Alan R. Liss, nc.

2 594 Ortmann Key words: 5-HT syndrome, 5-HT agonists, 5-HT antagonists, MA0 inhibitors, 5-HT-uptake inhibitors, 6-adrenoceptor agonists, rats NTRODUCTON When rats are injected with compounds increasing dramatically serotonergic transmission in the CNS they show a behavioral syndrome which consists mainly of tremor, hindlimb abduction, lateral head weaving, reciprocal forepaw padding, and Straub tail [Jacobs, The behavioral motor syndrome (5-HT syndrome) can be elicited by injecting, e.g., the serotonin precursor L-5-hydroxytryptophan (Lj-HTP), by 5-HT agonists like 5-methoxy-N,N dimethyltryptamine (5-MeODMT) or quipazine. 5-HT-releasing compounds like p-chloroamphetamine or fenfluramine have also been shown to induce the 5-HT syndrome [Trulson and Jacobs, The syndrome has been shown in a variety of studies to be rather specific for the stimulation of central serotonergic synapses [Jacobs, 1976; Sloviter et al., 1978; Ortmann et al., 19801, possibly located in the brain stem and spinal cord [Jacobs and Klemfuss, For the screening of psychoactive drugs behavioral models like the 5-HT syndrome offer several advantages: They are fast, require no elaborate equipment, provide information on CNS permeability and half-life of drugs in vivo, and can be quantitated to generate ED50 or Cs0 values. This paper reviews the possibilities to use the 5-HT syndrome in rats to identify drugs interacting with the central 5-HT system and to characterize their mechanism of action. METHODS The behavioral syndrome induced by L-5-HTP in rats has been used in our laboratory to study the interaction of drugs with the central 5-HT system of rats [Ortmann et al., 1980, 1981a,b, The method employed for the evaluation of the intensity of behavioral stimulation has been described in detail [Ortmann et al., For the evaluation of potentiating effects we observed and scored the intensity of the following symptoms: tremor (score 0-2), hindlimb abduction (score 0 or l), lateral head weaving (score 0 or l), and forepaw padding (score 0-2). Each rat was scored during three 20 minute intervals after the injection of a threshold dose of L-5-HTP (see Table 1). Cumulative scores for each rat were calculated at the end of the observation period and expressed as total mean score for the group (maximal 18 points) f SD. The percent behavioral stimulation was calculated according to the following equation: mean score - control score % stimulation = x control score Statistical analysis was performed using the Newman-Keuls test according to Zivin and Bartko [ For the quantitation of 5-HT-antagonistic activity only tremor and forepaw padding were scored after the injection of a dose of L-5-HTP producing a maximal behavioral effect [see Table 4; Ortmann et al., The accumulated behavioral score of treated groups was compared with vehicle-treated controls and the effect expressed as percent inhibition. The pharmacological tools used during these studies are summarized in Table 1. For the studies of 5-HT agonists we observed the behavior of the animals during 1 hour after injection of the drug. n addition to the four symptoms mentioned above, we observed the absence or presence of ptosis, backward movements, and salivation. The results were expressed as the percent of the maximal possible intensity (score) of each of the symptoms. n addition we tried to classify qualitatively the behavioral effects observed as similar or different from the typical 5-HT syndrome induced by L-5-HTP.

3 5-HT Syndrome in Rats 595 TABLE 1. The Doses of L5-HTP, Clorgyline, and CGP 6085 A Used to Study Potentiation or nhibition of the 5-HT Syndrome* Pretreatment drug (mg/kg) L-5-HTP (mg/kg ip) Type of drug which shows an effect L-5-HTP potentiation nhibitor of MAO-A, 5-HT reuptake Clorgyline, 10 sc 3/5 P-Agonist, inhibitor of 5-HT-uptake and MAO-B CGP 6085 A 10 ip 20 nhibitor of MAO-A L-5-HTP inhibition HT-Antagonists CGP 6085 A 10 ip 50 5-HT-Antagonists *Clorgyline and CGP 6085 A (Waldmeier et al., 1979) were administered 2 hours before the indicated doses of L-5-HTP. The doses of L-5-HTP used for the potentiation tests represent threshold doses producing no or only weak behavioral effects under the described experimental conditions. The L-5-HTP doses used for the inhibition tests produced a maximal 5-HT syndrome. TABLE 2. Effect of Harmaline on the Potentiating Effect of Pargyline on Ld-HTP-nduced Behavioral Excitation* Percentage L-5-HTP Score of maximum Drug mg/kg P (mg/kg) (X f SD) stimulation P Saline f 1.2 Pargy line Saline f 2.2 Pargyline 20 + Harmaline & NS *Pargyline and harmaline were injected ip in the indicated doses 24 hours before the application of L-5-HTP (the behavioral effects were evaluated as described in Methods). EFFECTS OF NHBTORS OF MAO-A AND 5-HT-UPTAKE Our first experiments with a series of inhibitors of monoamineoxidase (MAO) and of 5- HT-reuptake showed that both drug classes potentiated strongly the behavioral effects of L-5- HTP under the described experimental conditions. Further experiments with MAO- and 5-HTuptake inhibitors showed that reliable ED50 values could be produced for the potentiation of behavioral signs induced by 100 mg/kg ip LJ-HTP [Ortmann et al., When these EDSO values were compared [Ortmann et al., with the potency of the drugs to inhibit MAO-A or the 5-HT reuptake system of rat brain in an ex vivo assay, a good correlation was found for both MAO-A inhibitors (N = 12; r = 0.89) and specific inhibitors of the 5-HT uptake (N = 9; r = 0.88), while the inhibitory potency of MAO-B inhibitors determined ex vivo did not correlate at all with the behavioral data [N = 12; r = 0.2; Ortmann et al., This correlation of the behavioral test results with biochemical ex vivo investigations in rat brain suggested that the model could be used to monitor activity of central 5-HT synapses in vivo. Another piece of evidence that the potentiation of the behavioral effects of L-5-HTP is mediated by inhibition of the A but not the B form of MA0 came from an experiment summarized in Table 2. Harmaline, a specific and reversible inhibitor of MAO-A, has been

4 5% Ortmann TABLE 3. Behavioral Characterization of Drugs as nhibitor of MAO-A, MAO-B, or 5-HT-Reuptake* Percentage Pretreatment L-5-HTP of maximum Drug Mg/kg (mg/kg) (mg/kg) stimulation P N m-cpp 10 ip m-cpp 10 ip Clorgyline 10 sc m-cpp 10 ip CGP 6085 A 10 ip 20 9 NS 6 Trazodone 30 ip Clorgyline 10 sc Deprenyl 1 sc NS 6 Deprenyl 1 sc Clorgyline 10 sc CGP A 10 Po CGP A 10 po CGP 6085 A 10 ip CGP A 100 po Clorgyline 10 sc 3 17 NS 6 CGP A 50 ip Clorgyline 10 sc *Clorgyline and CGP 6085 A were given 2 hours before, trazodone and m-cpp 30 minutes before the indicated dose of L-5-HTP to groups of six rats (the experiments were evaluated as described in Methods). shown to increase the specificity of the mixed inhibitor pargyline for MAO-B, when it is injected together with pargyline 24 hours before the test [Fuller and Hemrick, The data in Table 2 show that 24 hours after its application 20 mg/kg ip pargyline potentiates strongly the behavioral effect of 50 mg/kg ip L-5-HTP. This potentiating effect of pargyline is blocked by coadministration of 30 mg/kg harmaline. The reversible MAO-A inhibitor harmaline protects the A form of the enzyme from the irreversible inactivation by pargyline and therefore leaves the A form fully active 24 hours after application. The deamination of the MAO-A substrate 5-HT is therefore not impaired in these animals and hence no behavioral potentiation of L-5-HTP is observed. DFFERENTATON BETWEEN NHBTORS OF MAO-A AND OF THE 5-HT REUPTAKE SYSTEM Since oxidation of 5-HT by the A form of MA0 and its reuptake into nerve endings are the two major pathways for inactivation of released 5-HT, we tried to differentiate behaviorally between these inactivating systems. We found that only 5-HT-uptake inhibitors potentiate the effect of 3 mg/kg ip L-5-HTP in MAO-A-inhibited rats (clorgyline 10 mg/kg sc), while MAO- A inhibitors were inactive under these experimental conditions [Ortmann et al., t was reasoned that after blocking MAO-A (the major degrading enzyme of 5-HT [Yang and Neff, 1974]), 5-HT reuptake would represent the main course to inactivate neuronally released 5- HT. As an example, Table 3 shows that in addition to its 5-HT agonistic activity m- chlorophenylpiperazine (m-cpp) possesses also 5-HT-uptake inhibiting activity, as shown by Garattini et al. [ in rat platelets. m-cpp potentiated L-5-HTP only in clorgyline-pretreated rats, while it showed no potentiating activity in animals pretreated with the specific 5-HTreuptake inhibitor CGP 6085 A [Waldmeier et al., 19791, The same stimulating effect was found when clorgyline-treated rats received trazodone or the MAO-B inhibitor deprenyl and a threshold dose of L-5-HTP. Deprenyl had no such effect in rats which only received 100 mg/ kg ip L-5-HTP (Table 3). t was assumed that MAO-B contributes to the oxidative degradation of 5-HT only when the A form of the enzyme of MA0 is inhibited and high synaptic concentrations of 5-HT are obtained [Ortmann et al.,

5 5-HT Syndrome in Rats 597 When drugs were tested in rats injected with the specific 5-HT uptake inhibitor CGP 6085 A and a dose of 20 mg/kg LJ-HTP, the opposite situation was observed (Table 3): Only MAO-A inhibitors potentiated under these conditions the behavioral effect of LJ-HTP by blocking the only major pathway of degradation of 5-HT left after inactivation of the 5-HT reuptake system. By testing drugs under both experimental conditions (i.e., pretreatment with clorgyline or CGP 6085 A) the potencies of drugs to inhibit MAO-A or 5-HT uptake can be thus independently determined. This is shown for the MAO-A inhibitor CGP A (Table 3). This drug combines a potent and reversible inhibition of MAO-A with an inhibition of the 5-HT-uptake system at higher doses [Waldmeier et al., 1983, Waldmeier and Baumann, Table 3 shows that without any pretreatment 10 mg/kg CGP A strongly potentiates the behavioral effect of 100 mg/kg ip L-5-HTP. n the presence of CGP 6085 A the same dose of CGP A still potentiates L-5-HTP s behavioral activity. This result confirms that at the dose of 10 mgikg PO the compound has strong central MAO-A inhibitory but no 5-HT-uptake inhibitory activity, because only MAO-A inhibitors were found to be active under these experimental conditions. n the presence of clorgyline a potentiation of L-5-HTP effects is seen only after ip administration of 50 mg/kg CGP A. Oral administration of up to 100 mg/kg of the compound induced only weak effects (Table 3). These behavioral results found with high doses of CGP A in clorgyline-pretreated animals represent the inhibition of 5- HT uptake measured for CGP A in rat brain [EDSO = 30 mg/kg PO, Waldmeier et al., 1983, Waldmeier and Baumann, Clorgyline showed the same ED50 for the potentiation of L-5-HTP-induced excitation when given 2 or 48 hours before the injection of L-5-HTP. This corresponds to the long-lasting inhibition of MAO-A activity found in biochemical investigations [Maitre et al., and indicates that this behavioral test can also be used to determine the half-life of the biological activity of inhibitors of MAO-A. The effect of MA0 inhibitors in other psychopharmacological test systems was found to have a much shorter half-life than the inhibition of MA0 [Maitre et al., The fact that the threshold dose of LJ-HTP in combination with clorgyline is much lower (3 mg/kg ip) than with the 5-HT-uptake inhibitor CGP 6085 A (20 mg/kg ip) deserves a comment. t suggests that synaptic concentrations of 5-HT may be higher in the presence of a MAO-A inhibitor than of a compound inhibiting 5-HT reuptake. This could then explain why a desensitization of 5-HT receptors was observed in some binding and behavioral experiments only after chronic administration of MA0 inhibitors, but not after 5-HT-uptake inhibitors [e.g., Savage et al., 1980; Lucki and Fraser, Chronic elevation of synaptic 5-HT levels which are high enough to induce a down regulation of 5-HT receptors may only be achieved under certain experimental conditions by MAO-A-, but not by 5-HT-reuptake inhibitors. Given without L-5-HTP, neither MAO-A- nor L-5-HTP-uptake inhibitors alone induce any behavioral signs of the 5-HT syndrome. This suggests that synaptic 5-HT levels reached after inhibition of MAO-A or of the 5-HT reuptake system are not sufficient to induce the 5- HT syndrome. The combination of a very potent MAO-A inhibitor like clorgyline with a strong inhibitor of the 5-HT uptake system (CGP 6085 A) was however shown to generate the 5-HT syndrome without the additional injection of Ld-HTP [Ortmann et al., nterestingly, there are clinical reports that the combination of these two drug classes can produce a 5- HT syndrome in man [nsel et al., P-ADRENOCEPTOR AGONSTS Another drug class which was found to potentiate L-5-HTP-induced excitation in rats consists of P-adrenoceptor agonists like salbutamol, clenbuterol, or fenoterol [Ortmann et al., 1981al. ndications for the interaction of these drugs with the 5-HT system have also come from biochemical studies, which showed that 5-HT turnover in rat brain is increased after acute or chronic peripheral injection of salbutamol maldmeier, 1981; Erdoe et al., 1982;

6 598 Ortmann Sugrue, or terbutaline [Hallberg et al., The increased 5-HT turnover produced by clenbuterol was shown by Nimgaonkar et al. [1983] to be caused by an increase of tryptophan concentration in the brain, due to increased levels of free tryptophan in plasma. The potentiation of L-5-HTP-induced excitation by P-adrenoceptor agonists was mainly seen in MAO-A-inhibited rats. The degree of behavioral stimulation by this class of drugs tended to vary in different experiments and was not as reproducible as the potentiating effect of inhibitors of MAO-A or of the 5-HT-uptake system [Ortmann et al., 1981al. Mechanism of Action The potentiating effect of salbutamol was also seen after central application, pointing to a central action of salbutamol. Lesion studies using icv injection of 6-OHDA, showed that intact central noradrenergic pathways were not necessary for the potentiation of L-5-HTPinduced excitation, while the destruction of 5-HT containing neurons by icv injection of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) impaired the effect of salbutamol. Studies with different adrenoceptor antagonists did not yield unequivocal evidence for the mediation of salbutamol s potentiating effect by While peripheral injections of 10 mg/ kg ip (-)-propranolol completely blocked the potentiating effect of salbutamol, practolol was not effective after ip injections up to 100 mg/kg ip. Both (-)-propran0101 and practolol did, however, not block the behavioral potentiation of salbutamol, when they were injected icv up to 100 pg per rat [unpublished observation]. Practolol crosses the blood-brain barrier only poorly [Scales and Cosgrave, and possesses higher affinity for the 6,-adrenergic than for the &-receptor [Minneman et al., Taken together, these results could possibly indicate that peripheral &-receptors are mediating the behavioral effect of salbutamol, since 1) the peripheral P -specific antagonist practolol did not antagonize salbutamol s behavioral effect while (-)-propranolol did block it and 2) both (-)-propran0101 and practolol were inactive when injected directly into the brain. n addition, it was shown that clenbuterol has only low affinity for the 5-HT2 receptor labeled by H-spiperone in rat cortex membranes [Green et al., 1983bl. This receptor type supposedly mediates the behavioral 5-HT syndrome induced by serotonergic agonists [Peroutka et al., 1981; Ortmann et al., 1982; Green et al., 1983al. Therefore clenbuterol or other 0- adrenoceptor agonists probably do not exert their behavioral activity by direct stimulation of postsynaptic 5-HTz receptors. This assumption seems to be supported by the finding that 5,7- DHT lesions decrease the potentiating effect of salbutamol on L-5-HTP-induced excitation in rats. The behavioral effect of L-5-HTP and the 5-HT agonist 5-MeODMT has been shown to be increased in 5,7-DHT-lesioned animals, supposedly due to induction of 5-HT receptor supersensitivity [Ortmann et al., 1981b. The exact mechanism whereby P-adrenoceptor agonists exert their behavioral stimulation of the central serotonergic system of the rat remains to be elucidated. Up to now it is not entirely clear what kind of receptor type is involved in the mediation of the effect and if this proposed receptor is centrally or peripherally located. The recent finding that /3-adrenoceptor agonists increase the level of central L-tryptophan and central 5-HT turnover [Nimgaonkar et al., can possibly also explain most of the behavioral findings generated with various 0- adrenoceptor agonists [Ortmann et al., 1981; Nimgaonkar et al., HT-ANTAGONSTS There are numerous examples for the determination of central anti-5-ht activity using the inhibition of different behavioral signs in mice [e.g., Corne et a]., and rats induced by serotonergic agonists, like L-5-HTP [Ortmann et al., 19821, tryptamine [Leysen et al., 19781, mescaline [Niemegeers et al., 19831, or tryptophan in MAO-inhibited rats (Deakin and Green, Besides known 5-HT antagonists like cinanserine, methysergid, or mianserin, neuroleptics [Maj et al., 1978bl and some tricyclic antidepressants [e.g., Maj et al., 1979;

7 5-HT Syndrome in Rats 599 HEAD TREMOR LATERAL HEADMOVEMENTS FOREPAW PADDNG % N H B T 0 N D 3 10 CNANSERN MG/KG P Fig. 1. nhibition by cinanserin of behavioral signs induced by L-5-HTP in rats. The indicated doses of cinanserin were injected ip 30 minutes before 250 mg/kg ip L-5-HTP into groups of six rats. The intensity of head tremor, lateral head movements, and forepaw padding was compared with a control group which received physiological saline solution instead of cinanserin. The behavioral score of the control group was set as 100%. Delini-Stula et al., were shown to antagonize certain behavioral symptoms induced by serotonergic agonists. Some of these studies tried to correlate the inhibitory activity found in behavioral tests with results of other test procedures to determine central 5-HT-antagonistic activity of drugs [Leysen et al., 1978; Peroutka et al., 1981; Ortmann et al., 1982; Niemegeers et al., Leysen et al. [1978] reported that the potencies of drugs to prevent tryptamineinduced seizures in rats and to displace 3H-spiperone from binding sites in frontal cortex in vitro correlated highly. We showed that the potency of a series of neuroleptics and 5-HTantagonists to inhibit tremor and forepaw padding induced by 250 mg/kg ip Ld-HTP correlated well (r = 0.8; N = 22) with the potencies of these drugs to displace 3H-spiperone from its binding site in frontal cortex in vivo [Ortmann et al., n the same antagonists like (-)-propranolol or oxprenolol were found to be inactive to antagonize behavioral signs of excitation induced by LJ-HTP up to high doses (30 mg/kg ip). These results were interpreted as support for the contention that the behavioral signs induced by these compounds were mediated by central 5-HT2 receptors [Peroutka and Snyder, 1979; Peroutka et al., The finding that 5-HT antagonists with specificity for the 5-HT2 receptor like ketanserine or pirenperone are potent inhibitors of behavioral 5-HT-mediated effects [Niemegeers et al., 1983; Green et al., 1983a1 support this hypothesis. The lack of specific 5-HT1 antagonists makes it, however, difficult to exclude a possible role of 5-HT1-receptors for the mediation of the 5-HT syndrome. During our studies we found that the behaviorat signs induced by 250 mglkg ip L-5- HTP (tremor, lateral head movements, forepaw padding, hindlimb abduction) were affected to a different degree by many of the drugs tested. Figure 1 shows the effect of cinanserin on three behavioral symptoms observed. While tremor and forepaw padding were the most susceptible to inhibition by both 5-HT antagonists and neuroleptics, lateral head movements were difficult to block even with high doses of the 5-HT antagonists. For the quantitative analysis of 5-HT antagonistic activity of drugs we therefore analyzed only the inhibition of tremor and forepaw

8 600 Ortmann padding induced by L-5-HTP. Dose-response curves generated for various antagonists were usually rather flat and maximal effects often did not exceed 70-80% [Ortmann et al., The observation that some of the 5-HT-antagonist may possess mixed agonistic/antagonistic properties could possibly explain some of these results [Colpaert et al., Tricyclic antidepressant drugs did not antagonize in a reproducible, dose-dependent manner tremor or forepaw padding induced by 250 mg/kg ip L-5-HTP. The 5-HT-antagonistic activity of these drugs could be possibly masked by a concomitant inhibition of the 5-HTreuptake system. This activity could then counteract the 5-HT-antagonism by increasing the concentration of neuronal 5-HT. We therefore also measured the L-5-HTP antagonistic activity of antidepressants in rats pretreated with a maximal dose of the specific 5-HT uptake inhibitor CGP 6085 A; 50 mg/kg ip L-5-HTP produced maximal signs of excitation in rats treated with 10 mg/kg ip CGP 6085 A (Table 1). Using this experimental procedure, we could demonstrate the central 5-HT antagonistic activity exerted by trazodone [Baran et al., and other compounds with strong antiserotonergic activity like mianserin [Maj et al., 1978a; Table 41. m-cpp did not antagonize the behavioral effects of L-5-HTP, indicating that the 5-HTantagonistic property of trazodone does not reside in the m-cpp moiety. Tricyclic antidepressants did not however show a consistent and reproducible inhibition of these symptoms even under these conditions. t therefore appears that the 5-HT-uptake inhibitory activity of the tricyclic antidepressants is not responsible for the lack of 5-HT-antagonistic activity seen under our experimental conditions. The finding that amitryptiline and nortryptiline are potent inhibitors of head twitches induced by DL-5-HTP in mice [Fuxe et al., suggests that this test system could be more suited to defined 5-HT-antagonistic activity of tricyclic antidepressant drugs than L-5-HTP-induced induced tremor or forepaw padding in rats. The reasons for these differences in the susceptibility for inhibition found for different behavioral signs in mice and rats are not clear. By taking the discussed precautions, the 5-HT syndrome induced by L-5-HTP in rats can be a suitable procedure to determine central 5-HT antagonistic activity of drugs. This antagonistic activity can also be determined with drugs possessing MAO-A- or 5-HT-reuptake inhibitory properties. The dose-response curves produced in our laboratory for different antagonists were generally flatter than those found in the LS-HTP potentiation test. This makes a quantitative analysis for 5-HT antagonists more difficult than for drugs potentiating L-5-HTP behavioral signs. 5-HT-AGONSTS The 5-HT syndrome induced in rats by different serotonergic compounds has become an important and simple tool for the screening of drugs which stimulate central serotonin pathways. Beside 5-HT, L-5-HTP, and substituted tryptamines, phenylpiperazines like m-chlorophenylpiperazine [m-cpp; Maj and Lewandowska, 19801, m-trifluoromethylphenylpiperazine, quipazine [Fuller et al., 1981; Green et al., 19761, MK 212 [Clineschmidt, 19791, 8-hydroxy-2-(di-n-propy1amino)tetralin [Arvidsson et al., 19811, a piperidinyl indole derivative [RU 24969; Euvrard and Boissier, 19801, and oxaflozane [Hache et al., have been described to induce behavioral signs of excitation which are partially comparable to the typical 5-HT motor syndrome induced by L-5-HTP or 5-MeODMT. Characterization of Behavioral Effects Table 5 describes the behavioral signs induced by some of these compounds and compares them with the behavioral effects of LJ-HTP. The syndrome induced by quipazine and most other agonists can be described as L-5-HTP-like consisting mainly of tremor, hindlimb abduction, forepaw padding, and only little backward motion. m-cpp and m- trifluoromethylphenylpiperazine, however, induce mainly a hunched position, backward motion, and ptosis, with only few L-5-HTP-like symptoms, like forepaw padding or tremor.

9 5-HT Syndrome in Rats 601 TABLE 4.5-HT-Antagonistic Effects of Drugs in Rats Pretreated With CGP 6085 A L-5-HTP Percentage of Dw3 mgk (mgk) inhibition P Trazodone m-cpp LO 50 0 NS Mianserin *Ten milligrams per kilogram CGP 6085 A was given 2 hours, the antagonists 0.5 hours before L-5-HTP. The behavioral score of control groups, which did not receive the antagonists was taken as 100% and the % inhibition by the antagonists calculated accordingly (statistical analysis was done as described in Methods). TABLE 5. Qualitative Characterization of Behavioral Symptoms nduced by 5- HT Agonists in Rats* HTP-like L-5-HTP 5-MeODMT p-c1- Amphetamine Fenfluramine Quipazine Dose range (mgikg ip) Prominent behavioral signs Duration of symptoms (min) Forepaw padding (piano play- ca 90 ing), lateral head movements, ca 15 tremor, hindlimb abduction, ca 60 backward motion, wet dog ca 60 shakes, salivation, hyper- ca 60 activity Not HTP-like m-cpp m-trifluoromethylphenylpiperazine Crouched position, extension of hindlimbs to the front, backward movements (intensified by tactile stimuli) ca 60 ca 60 Trazodone 3-30 Crouched position, head down, slow movements, backward movements induced by tactile stimuli MK Crouched position, extension of hindlimbs to the front, salivation, ptosis, backward movements induced by tactile stimuli; at 30 ip: tremor, dyspnoe *The absence or presence of various behavioral signs and their duration were observed as described in Methods. A quantitative analysis was not attempted. This qualitative analysis suggests that an atypical 5-HT syndrome induced by compounds like trazodone or m-cpp is mediated by a different neurotransmitter system or by different 5-HT receptors than those mediating the typical 5-HT syndrome induced by, e.g., L-5-HTP or quipazine.

10 602 Ortmann 'x H A X N 1 E N S T Y 80 - CONTROLS 5,TDHT LESON FOREPAU PADDNG MG/KG P w Fig. 2. Comparison of the behavioral effects induced by quipazine in 5,7-DHT-lesioned and shamoperated rats. The intensity of tremor of the head, forepaw padding, and extension of hindlimbs were scored in sham-operated and 5,7-DHT-lesioned groups of six rats during three 20-minute intervals after application. The maximal possible behavioral score for each symptom was set as 100%. 5,7-DHT lesions and sham operations were done 4 weeks before the experiment as described elsewhere [Ortmann et al., 1981 bl. n addition to the qualitative behavioral classification of different 5-HT-agonists, we used the 5-HT-antagonist cinanserine to answer the question if the behavioral symptoms are mediated by the 5-HT-system or by other neurotransmitter pathways. We found that tremor and forepaw padding induced of quipazine, p-c1-amphetamine, Ru were clearly antagonized by 30 mg/kg ip cinanserine, while m-cpp- and m-trifluoromethylphenylpiperazineinduced behavioral effects were not changed significantly by this 5-HT antagonist (data not shown). This result indicates that the behavioral signs induced by m-cpp are mediated either by a 5-HT receptor, which is not blocked by cinanserine, or that another neurotransmitter system is mediating the effect. The finding that m-cpp and m-trifluoromethylphenylpiperazine act as antagonists at 5-HT receptors mediating the contraction of rat jugular vein [Cohen and Fuller, indicates that these compounds can indeed differ from other 5-HT-agonists in their affinity for different 5-HT receptors in the CNS or the periphery. To further characterize the behavioral syndrome induced by various putative 5-HT agonists, their behavioral effects were observed in rats injected intracerebrally with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT). This neurotoxin depletes rather specifically central 5-HT and induces a rapid developing supersensitivity toward the behavioral effects of L-5-HTP and 5-MeODMT [Ortmann et al., 1981bl. t was reasoned that the behavioral activity of drugs stimulating postsynaptic receptors would be increased by 5,7-DHT lesions due to the development of supersensitivity, while the effects of 5-HT-releasing agents would be diminished due to the decreased levels of central 5-HT. Figure 2 shows that the behavioral activity of quipazine was indeed higher in 5,7-DHT animals. The behavioral activity of m-cpp was found to be unchanged and the effect of p-chloroamphetamine, a 5-HT-releasing agent, was clearly diminished (results not shown). A similar antagonism of p-chloroamphetamine's behavioral effects were reported for p-chloro-phenylalanine (PCPA), which depletes central 5-HT

11 5-HT Syndrome in Rats TREMOR FOREPAW PADDNG 8 S C 0 R 6 E MNUTES AFTER APPLCATON 1 innn / / n Fig. 3. Effect of clorgyline pretreatment on behavioral effects induced by quipazine in rats. The intensity of head tremor and forepaw padding were determined in groups of six rats which received water (closed bars) or 10 rng/kg sc clorgyline (open bars) 2 hours before 10 mg/kg ip quipazine. The behavioral observation was done in 10-minute intervals until the rats appeared normal. levels to a greater extent than 5,7-DHT [Trulson and Jacobs, The interpretation of these experiments becomes obviously complex, if a drug like, e.g., quipazine, combines a 5-HTreleasing activity with a stimulation of postsynaptic receptors. The result with m-cpp seemed however to confirm the finding that its behavioral effects in rats are not mediated by the release of 5-HT or by the same 5-HT receptor type, mediating the behavioral activity of LJ-HTP or quipazine. A further characterization of the mechanism of action of 5-HT agonists is possible by testing these compounds in clorgyline-pretreated animals. f release of 5-HT is responsible for the behavioral syndrome, the symptoms should be more intense or prolonged in MAO-inhibited rats because the degradation of released 5-HT is impaired. Figure 3 shows that the 5-HT syndrome induced by 10 mg/kg ip quipazine is significantly increased and prolonged in MAO- A-inhibited rats. Similar results were obtained with MK 212, while the atypical syndrome induced by m-cpp and trazodone was unchanged in clorgyline-pretreated rats. Pretreatment of the rats with the specific 5-HT-reuptake inhibitor CGP 6085 A did not change the behavioral effect of any of the 5-HT agonists. These results suggest that part of the behavioral activity of quipazine is exerted by release of 5-HT, possibly due to the blockade of presynaptic 5-HT autoreceptors [Baumann and Waldmeier, ; Baumann, 1982; Martin and Sanders-Bush, 1982; Goethert, An alternative explanation for these results could be that the tested 5- HT-agonists are degraded by MAO-A. An inhibition of this enzyme by clorgyline could therefore prolong or potentiate their behavioral effects. The data emphasize the importance of a careful behavioral characterization of 5-HT agonists in order to determine if their effects are mediated through the 5-HT system. Dopamine-releasing agents can produce a symptomatology which is relatively similar to the 5-HT syndrome. Together with 5-HT receptor binding studies, the behavioral 5-HT syndrome seems to be a convenient screening procedure for the detection of drugs stimulating the central 5-HT system.

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