5-Hydroxytryptamine 3 (5-HT 3 ) Receptors Mediate Spinal 5-HT Antinociception: An Antisense Approach

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1 /01/ $3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 2 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3227/ JPET 298: , 2001 Printed in U.S.A. 5-Hydroxytryptamine 3 (5-HT 3 ) Receptors Mediate Spinal 5-HT Antinociception: An Antisense Approach DENNIS PAUL, DONGDONG YAO, PEIMIN ZHU, LERNA D. MINOR, and MEREDITH MASON GARCIA The Department of Pharmacology and Experimental Therapeutics and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center (D.P., D.Y., P.Z., L.D.M.); and Department of Otolaryngology, Tulane University Medical College (M.M.G.), New Orleans, Louisiana Received August 16, 2000; accepted April 26, 2001 This paper is available online at ABSTRACT To examine the role of the 5-hydroxytryptamine 1B (5-HT 1B ) and 5-HT 3 receptor subtypes in the analgesia produced by 5-HT (serotonin) agonists, we assessed the effect of antisense oligodeoxynucleotides (AODNs) designed to knock down the number of these receptor subtypes on analgesia produced by intrathecal (i.t.) 5-HT, the 5-HT 1B receptor agonist, 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline maleate (CGS-12066A), and the 5-HT 3 receptor agonist, 2-methyl-5-HT. Groups of mice (n 17 20) were injected i.t. on days 1, 3, and 5 with one of the AODNs, a mismatch oligo, or saline. On day 6, all mice were injected i.t. with 70.5 nmol of 5-HT, 44.4 nmol of CGS-12066A, or 49 nmol of 2-methyl- 5-HT by lumbar puncture. Following testing, spinal cords were rapidly removed and prepared for receptor binding assays. Treatment with AODN for 5-HT 1B receptors produced a 70% Spinal administration of 5-HT and 5-HT agonists produces antinociception (Alhaider et al., 1993). The use of agonist and antagonist drugs purported to be selective for the various 5-HT receptors has provided evidence for spinal 5-HT 1A, 5-HT 1B, and 5-HT 3 receptors in antinociception in models of acute nociceptive pain (Crisp and Smith, 1989; Alhaider and Wilcox, 1993; Alhaider et al., 1993; Xu et al., 1994). 5-HT 1A and 5-HT 1B receptors appear to have reciprocal roles in the mediation of spinal reflexes (el-yassir et al., 1988; Eide et al., 1990; Murphy and Zemlan, 1990). In the spinal cord, 5-HT 3 receptors are located primarily on the axon terminals of the sensory primary neurons that terminate in the substantia gelatinosa of the dorsal horns (Hamon et al., 1989). In contrast, 5-HT 1B receptors are autoreceptors on the neurons of the descending inhibitory serotonergic tract from the nucleus raphe magnus to the spinal dorsal horn (Glaum and Anderson, 1988). This research was supported by National Institutes of Health-National Institute on Drug Abuse Grant DA07379 (to D.P.). These data were presented in part at the 28th Annual Meeting of the Society for Neuroscience, Los Angeles, CA. reduction in ligand binding to this receptor subtype. After treatment with AODN for 5-HT 3 receptors, ligand binding to this receptor subtype was undetectable. In mice tested with i.t. 5-HT, tail-flick analgesia was attenuated only in mice treated with the 5-HT 3 receptor AODN. Mice treated with the AODN designed to knock down 5-HT 1B receptors or with its mismatch oligo were not significantly different from controls. In mice tested with i.t. administration of CGS-12066A, none of the oligo treatments produced a significant attenuation of analgesia. In mice tested with i.t. administration of 2-methyl-5-HT, only 5-HT 3 receptor AODN attenuated analgesia. Thus, 5-HT and 2-methyl-5-HT analgesia are mediated by the 5-HT 3 receptor subtype. However, spinal CGS-12066A analgesia appears not to be mediated by either the 5-HT 1B or the 5-HT 3 receptor subtypes. Several groups have examined the role of 5-HT 3 receptors in tail-flick analgesia. Glaum et al. (1990) and Crisp et al. (1991) used highly selective 5-HT 3 receptor antagonists to block the analgesia produced by i.t. injection of 5-HT or 2-methyl-5-HT, an agonist with a 5- to 10-fold selectivity for 5-HT 3 receptors. Both authors concluded that this receptor subtype is of primary importance for spinal analgesia produced by 5-HT agonists. Although the agonists used have only a 4- to 5-fold selectivity for 5-HT 3 receptors over other 5-HT receptor subtypes, the antagonists used were greater than 60-fold selective. Moreover, the doses used were similar to doses used to attribute other drug effects to mediation by 5-HT 3 receptors. The localization of the 5-HT 3 receptors on axon terminals (Hamon et al., 1989) puts this receptor subtype in a prime location for the modulation of sensory neurons. Thus, the evidence for involvement of 5-HT 3 receptors is strong. Other investigators have examined the role of 5-HT 1B receptors in spinal analgesia (Alhaider and Wilcox, 1993; Alhaider et al., 1993; Hain et al., 1999). These authors also used agonists and antagonists to characterize the role of 5-HT 1B ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); ODN, oligodeoxynucleotide; AODN, antisense ODN; CGS-12066A, 7-trifluoromethyl- 4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline maleate; i.t., intrathecal. 674

2 Knockdown of 5-HT 1B and 5-HT 3 Receptors and Antinociception 675 receptors in spinal analgesia. However, highly selective 5-HT 1B agonists and antagonists are not available. Moreover, the doses of the 5-HT 1B agonists used in early studies were much greater than necessary to elicit other 5-HT 1B - mediated effects: 40 nmol (Alhaider and Wilcox, 1993) versus 6 nmol (Gradin and Persson, 1993), and the doses of 5-HT 1B antagonists were also excessive. Moreover, if 5-HT 1B receptors functioned as autoreceptors, stimulation of this subtype would tend to inhibit the descending modulatory system. Therefore, the role for 5-HT 1B receptors in spinal 5-HT analgesia is more controversial than that of 5-HT 3 receptors. In recent years, approaches that use antisense oligodeoxynucleotides (AODNs) to modify gene expression in vivo have been described (Pasternak and Standifer, 1995). An AODN is a synthetic single-stranded DNA molecule with a sequence complementary to a specific mrna sequence. It is designed to specifically interact and hybridize with a particular sequence of mrna so that the mrna is either degraded by RNase or prevented from being translated into protein, or both. DNA is composed of a sense strand and an antisense strand. Messenger RNA is copied from the antisense strand, and its sequence is identical to the sense strand, except for the thymine bases, which are replaced by a pyrimidine base, uracil. Theoretically then, an antisense strand would be expected to hybridize with the mrna. The specificity of the interaction between the oligonucleotide and its target mrna is based on the Watson-Crick model of base pairing, which allows precise hybridization of nucleic acids based on base stacking and hydrogen bonding. The mrna sequence to which an AODN is complementary must also be free of secondary structure. In vivo administration of AODNs has been used to reduce the number of opioid, -aminobutyric acid B, neuropeptide Y, N-methyl-D-aspartate, dopamine, and 2A receptors in the central nervous system (Wahlestedt et al., 1993; Zhou et al., 1994; Pasternak and Standifer, 1995; Bilsky et al., 1996). The administration of AODNs in vivo also has been used to identify the functional importance of several receptor subtypes (Wahlestedt et al., 1993; Zhou et al., 1994). This approach is of particular merit when investigating the physiological role of novel receptors or receptor subtypes for which a selective antagonist has not been developed. With regard to 5-HT receptor subtypes, 5-HT 3 antagonists are suitable for pharmacological characterization of drug effects, but 5-HT 1B antagonists are not (Alhaider et al., 1993). Accordingly, we re-examined the issue of which receptor subtypes mediate the analgesia produced by spinal 5-HT, the 5-HT 1B agonist CGS-12066A, and the 5-HT 3 agonist 2-methyl-5-HT, using an in vivo antisense approach. Materials and Methods Drugs and Reagents. CGS-12066A was purchased from Research Biochemicals Inc. (Natick, MA). Tris was purchased from Bio-Rad (Richmond, CA). Radioligands were purchased from NEN Life Science Products (Boston, MA). All other drugs and reagents were purchased from Sigma (St. Louis, MO). All doses of drugs were calculated as the salt form. Subjects. All procedures were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and adhered to the International Association for the Study of Pain Guidelines on Animal Experimentation. Male CD-1 mice (Charles River, Boston, MA) were housed 10 to a cage and maintained on a 12-h light/dark cycle with ad libitum access to food and water. Intrathecal injections were made using a 10- l Hamilton syringe fitted to a 30-gauge needle with PE10 tubing (Hylden and Wilcox, 1980). Doses of agonist drugs were selected from preliminary dose-response curves as a dose that produced analgesia in approximately 60 to 70% of mice. Antisense Design, Synthesis and Treatment. AODN sequences spanned the initiation codon for each receptor. All unmodified (phosphodiester) ODNs were synthesized by the LSU Health Sciences Center Core Labs using an Expedite nucleic acid synthesis system (Applied Biosystems, Foster City, CA) and purified using reverse-phase liquid chromatography. The oligos were diluted in sterile water, and concentrations were confirmed by spectrophotometry. Base sequences (5 to 3 ) for ODNs were as follows: 5-HT 1B antisense: CTGCTCCTCCATAGCTCT; 5-HT 1B mismatch: CTGTC- CCTCCATAGTCCT; 5-HT 3 antisense: CGGGATGCAGAGCCGCAT; 5-HT 3 mismatch: CGGGAGTCAAGGCCGCAT. ODNs were injected i.t. at a dose of 20 g/2 l on days 1, 3, and 5. Mice were tested for tail-flick analgesia on day 6. Tail-Flick Analgesia. Analgesia was assessed quantally using the tail-flick assay (D Amour and Smith, 1941). The latency to withdraw the tail from a focused light stimulus was measured electronically using a photocell. Baseline latencies ( sec) were determined for all animals before 5-HT agonist treatments as the mean of two trials. 5-HT agonists were injected i.t. 10 min before testing. Mice having a tail-flick latency that was at least double their predrug baseline were considered to be analgesic. A 12-s cutoff was used to minimize tissue damage. 5-HT 1B Receptor Binding. To prepare tissue for the 5-HT 1B receptor binding assay, mice used in the tail-flick experiments were killed by cervical dislocation; then, spinal cords were rapidly removed and placed in 50 mm treated Tris-HCl buffer (50 mm Tris- HCl, 1 mm EDTA, 100 mm NaCl) at a ratio of 1:50 (w/v). Five spinal cords were pooled and homogenized with a Tekmar Tissumizer (Tekmar, Cincinnati, OH) for 20 s at setting 50. The homogenate was incubated at room temperature with 0.1 mm phenylmethylsulfonyl fluoride (protease inhibitor) for 15 min and then centrifuged at 10,000g for 40 min at 4 C. The pellets were resuspended with 0.32 M sucrose (1:6, w/v) and stored at 75 C until use. 5-HT 1B receptors were detected using 100 pm [ 125 I]iodocyanopindolol in 10 mm Tris buffer with 30 M isoproterenol, 150 mm NaCl, and 10 M pargyline, ph 7.7, at 23 C (Hoyer et al., 1985). Assay volume was 250 l. All determinations were in triplicate with three replications. Incubations were at 37 C for 90 min. Nonspecific binding was determined with 100 M 5-HT. Assays were terminated by rapid filtration across GF-B filters (Brandel Inc., Gaithersburg, MD) that were treated with 2% polyethylenimine and washed twice with 5 ml of 10 mm Tris buffer. Radioactivity remaining on the filters was determined using a Beckman gamma counter (Beckman Instruments, Palo Alto, CA). For each binding assay, a 0.5-ml aliquot of the tissue homogenate was stored at 20 C for subsequent protein determination according to the method of Lowry et al. (1951). Mean dry weight of tissue averaged 0.29 mg/tube. In preliminary saturation studies, we found that [ 125 I]iodocyanopindolol bound to a single site with a K D value of 0.18 nm, comparable to 0.23 nm found by Hoyer et al. (1985). The B max value in spinal cord was pmol/mg of protein. Specific binding was 48 to 52% of total binding at 100 pm. Levels of binding were comparable for data from the saturation curves and the single-concentration binding from the treated animals, indicating that our tissue preparation was sufficient to remove any residual drug. 5-HT 3 Binding Assay. For the 5-HT 3 receptor binding assay, spinal cords of mice used in the behavioral experiments, and an additional 10 mice per treatment group, were rapidly removed, weighed, and placed in ice-cold 50 mm Tris/Krebs buffer (50 mm Tris, 118 mm NaCl, 4.75 mm KCl, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4, 2.5 mm CaCl 2, 25 mm NaHCO 3, and 11 mm glucose, ph 7.6) con-

3 676 Paul et al. taining 0.2 M EDTA and 4 M NaCl. Nine to 10 spinal cords were pooled and homogenized for each tissue preparation. Phenylmethylsulfonyl fluoride (100 mm) was added, and the homogenate was allowed to sit at room temperature for 15 min. It was then centrifuged twice at 48,000g for 20 min. The pellet was resuspended at a ratio of 1:50 in the Tris/Krebs buffer. 5-HT 3 receptors were detected using 3 nm [ 3 H]zacopride (Barnes et al., 1988; Glaum and Anderson, 1988). Assay volume was 1 ml. All determinations were in triplicate with three replications. Incubations were carried out at 37 C for 60 min. Nonspecific binding was determined using 30 M tropisetron. Assays were terminated by rapid filtration over GF-B filters treated with 2% polyethylenimine. Filters were transferred to 7-ml polypropylene vials and 5 ml of Econoscint (ICN, Costa Mesa, CA) scintillation fluor added. Radioactivity was determined using a Beckman 2600 liquid scintillation spectrometer. For each binding assay, a 0.5-ml aliquot of the tissue homogenate was stored at 20 C for protein determination. Tissue dry weight averaged 1.15 mg/tube. In preliminary saturation studies, we found that [ 3 H]zacopride bound to a single site with a K D value of 1.4 nm, comparable to 0.75 nm found by Barnes et al. (1988). B max in spinal cord was pmol/mg of protein. Specific binding was 62 to 68% of total binding at 3 nm [ 3 H]zacopride. As with the 5-HT 1B receptor binding assay, levels of binding were comparable for data from the saturation curves and the single-concentration binding from the treated animals, indicating that our tissue preparation was sufficient to remove any residual drug. All receptor binding data were normalized for tissue concentration and were analyzed by experimenters blinded to the treatment condition. Saturation curves were analyzed using nonlinear regression. Single-concentration studies of AODN-treated spinal cords were analyzed using a one-way analysis of variance. In vivo studies were analyzed using the Fisher s exact test, but the experimenters were not blind to the treatment. Results Pretreatment of mice with the AODN for 5-HT 1B receptors produces a 70% attenuation (p 0.001) of [ 125 I]iodocyanopindolol binding for this subtype (Fig. 1). The AODN for 5-HT 3 receptors produced a slight reduction in 5-HT 1B binding (p 0.05). This result was similar to that produced by the two mismatch treatments. Pretreatment of mice with the AODN for 5-HT 3 receptors reduced [ 3 H]zacopride binding to undetectable levels (p ; Fig. 1). Similar to the results with [ 125 I]iodocyanopindolol binding, the AODN for 5-HT 1B receptors produced a slight reduction in 5-HT 3 binding (p 0.05). This result was similar to that produced by the two mismatch treatments. For each of the in vivo experiments, results represent pooling of data from two separate experiments that produced similar results. Spinal administration of 5-HT (70.5 nmol, i.t.) produced analgesia in 64% of mice pretreated with saline (Fig. 2). At higher doses, 5-HT produced spontaneous tail-flicks. Pretreatment with the AODN for 5-HT 3 receptors significantly attenuated analgesia produced by i.t. 5-HT. Pretreatment with the AODN for 5-HT 1B receptors did not affect spinal 5-HT analgesia significantly. Likewise, pretreatment with the mismatch oligos for these two AODNs had no significant effect on analgesia. Spinal administration of CGS-12066A (44.4 nmol, i.t.) produced analgesia in 50% of saline-pretreated control mice (Fig. 3). At doses higher than this, spontaneous tail-flicks interfered with the testing procedure. Surprisingly, none of the oligo treatments had a significant effect on CGS-12066Aproduced analgesia. Spinal administration of 2-methyl-5-HT (49 nmol, i.t.) produced analgesia in 67% of mice pretreated with saline (Fig. 4). Doses higher than this also produced spontaneous tailflicks. Like animals tested with 5-HT, pretreatment with the AODN for 5-HT 3 receptors significantly attenuated analgesia produced by i.t. 2-methyl-5-HT, whereas pretreatment with the AODN for 5-HT 1B receptors had no significant effect. Fig. 1. Antisense knockdown of spinal 5-HT 1B and 5-HT 3 receptors. Groups of mice were treated on days 1, 3, and 5 with saline, AODN for 5-HT 1B receptors, mismatch ODN for 5-HT 1B receptors, AODN for 5-HT 3 receptors, or mismatch ODN for 5-HT 3 receptors. On day 6, spinal cords were rapidly removed and processed for 5-HT 1B receptor binding with 100 pm 125 I-cyanopindolol or 5-HT 3 receptor binding with 3 nm [ 3 H]zacopride. Specific binding in control spinal cords was 12,330 dpm/mg tissue for 5-HT 1B binding and 260 dpm/mg tissue for 5-HT 3 binding. Data represent mean percent control specific binding S.E.M. Only the AODN for 5-HT 1B receptors significantly reduced 125 I-cyanopindolol binding. Likewise, only the AODN for 5-HT 3 receptors significantly reduced [ 3 H]zacopride binding. Fig. 2. Effect of antisense treatment on intrathecal 5-HT-induced analgesia. Groups of mice (n 18) were treated on days 1, 3, and 5 with saline, AODN for 5-HT 1B receptors, mismatch ODN for 5-HT 1B receptors, AODN for 5-HT 3 receptors, or mismatch ODN for 5-HT 3 receptors. On day 6, all mice were injected with 5-HT (70.5 nmol, i.t.). Ten minutes later, all mice were tested for tail-flick analgesia. Only the AODN for 5-HT 3 receptors produced a significant attenuation of 5-HT analgesia.

4 Knockdown of 5-HT 1B and 5-HT 3 Receptors and Antinociception 677 Fig. 3. Effect of antisense treatment on intrathecal CGS-12066A-induced analgesia. Groups of mice (n 18) were treated as described in Fig. 3. On day 6, all mice were injected with CGS-12066A (44.4 nmol, i.t.). Ten minutes later, all mice were tested for tail-flick analgesia. None of the treatments significantly affected CGS-12066A-induced analgesia. Fig. 4. Effect of antisense treatment on intrathecal 2-methyl-5-HT-induced analgesia. Groups of mice (n 18) were treated as described in Fig. 3. On day 6, all mice were injected with 2-methyl-5-HT (49 nmol, i.t.). Ten minutes later, all mice were tested for tail-flick analgesia. Only the AODN for 5-HT 3 receptors produced a significant attenuation of 2-methyl-5-HT analgesia. Pretreatment with the mismatch oligos for the two AODNs had no significant effect on analgesia. Discussion A valuable axiom in pharmacology is that confidence in your data should be only as strong as the selectivity of your drugs. In light of pharmacological characterization using putative selective agonists and antagonists, several investigators have implicated both 5-HT 1B and 5-HT 3 receptors in the mediation of spinal 5-HT analgesia (Eide et al., 1990; Glaum et al., 1990; Alhaider and Wilcox, 1993; Alhaider et al., 1993; Hain et al., 1999). Although the selectivity of 5-HT 3 receptor antagonists used in these studies appears adequate, the selectivity of the 5-HT 1B agonists and antagonists was insufficient for definitive attribution of effects to this receptor subtype (Alhaider et al., 1993). In preliminary testing, we found that although i.t. CGS-12066A was analgesic, another 5-HT 1B receptor agonist, CP93129, was not (data not shown). Likewise, Crisp et al. (1991) found no elevation of tail-flick latency by intrathecally administered trifluoromethylphenyl piperazine. Moreover, doses of 5-HT 1B receptor agonists used in both our studies (44.4 nmol) and those of Alhaider et al. (1993) (45 nmol) appeared to be much higher than those used to characterize other spinal 5-HT 1B receptor-mediated effects (6 nmol; Gradin and Persson, 1993). The AODN approach is of particular merit when investigating the physiological role of novel receptors or receptor subtypes for which a selective antagonist has not been developed. For example, the AODN strategy has been used to block effects of 5-HT 1A and 5-HT 6 receptor stimulation (Bourson et al., 1995; Sleight et al., 1996). Because of the lack of selective compounds, functional studies of 5-HT 6 receptors have used the AODN approach almost exclusively (Bourson et al., 1995; Sleight et al., 1996). Following this strategy, we have demonstrated the usefulness of a 5-HT 3 receptor AODN to block the analgesia produced by intrathecal administration of 5-HT or the 5-HT 3 receptor agonist, 2-methyl-5-HT. In contrast, AODN treatment that reduced 5-HT 1B receptors by 70% did not block analgesia produced by spinal administration of 5-HT, 2-methyl-5-HT, or the 5-HT 1B receptor agonist, CGS-12066A. These results are evidence that analgesia produced by 5-HT and 2-methyl-5-HT is primarily attributable to an action at the 5-HT 3 receptor subtype, but the analgesia produced by CGS-12066A is not attributable to either the 5-HT 1B or the 5-HT 3 receptor subtype. This latter finding is consistent with the results of Pickard et al. (1996), who demonstrated that the effects of CGS-12066A on light-induced phase shifts of the circadian activity rhythm and induction of c-fos expression in the suprachiasmatic nucleus were not antagonized by the 5-HT 1 antagonist, methiothepin. An alternative mechanism of the analgesic effects of CGS A is suggested by the results of Durham and Russo (1999). These authors found that administration of CGS A inhibits the release of calcitonin gene-related peptide from sensory primary afferent neurons. Release of this neuropeptide has been correlated with the mediation of nociception (Van Rossum et al., 1997). The findings that the AODN for 5-HT 1B receptors did not significantly attenuate 5-HT 3 receptor binding and conversely, the AODN for 5-HT 3 receptors did not significantly attenuate 5-HT 1B receptor binding, attest to the selectivity of these two binding assays. 125 I-Cyanopindolol could not be binding to a subpopulation of 5-HT 3 receptors, and [ 3 H]zacopride could not be binding to a subpopulation of 5-HT 1B receptors. Because 30% of 5-HT 1B receptors remained following AODN treatment, it could be argued that if there are a considerable number of spare receptors in a spinal 5-HT 1B analgesia system, a significant reduction in analgesia would not be expected. We feel that this interpretation is unlikely to be true because CGS-12066A is considered to be a low-efficacy agonist (Neale et al., 1987). Consequently, this stimulation of all or nearly all of the spinal 5-HT 1B receptors would be required for maximum analgesia. A 70% reduction in receptors would be expected to produce a significant reduction in this drug s analgesic effect. Using the most selective agonists and antagonists avail-

5 678 Paul et al. able, several laboratories have concluded that both 5-HT 1B and 5-HT 3 receptors are involved in serotonin-produced analgesia (Glaum et al., 1990; Crisp et al., 1991; Alhaider and Wilcox, 1993; Alhaider et al., 1993; Hain et al., 1999). Our results using an antisense strategy are inconsistent with this interpretation. We propose that 5-HT 3 receptors are important, but 5-HT 1B receptors are of relatively little importance, at least in this model of analgesia. These results do not preclude involvement of other 5-HT receptor subtypes, although only these two subtypes have been implicated in previous studies. In contrast, Hain et al. (1999), using a pharmacological approach, have proposed that the involvement of 5-HT 1B receptors is strain-dependent. DBA/2 mice, which have relatively high levels of this receptor, were sensitive to i.p. administration of CGS In contrast, C57BL/6 mice, which are deficient in functional 5-HT 1B receptors, were less sensitive. Unfortunately, these authors did not demonstrate antagonism of the CGS12066 analgesia with either an antagonist or antisense. In addition, Crisp et al. (1991) reported that the 5-HT 1B receptor agonist trifluoromethylphenyl piperazine was analgesic in a pindolol-reversible manner in the hot-plate test, but not the tail-flick test, in rats. Therefore, it would be of interest to assess the relative importance of these two receptor subtypes in other species and in other analgesic assays not involving a spinal reflex. For example, 5-HT 1B receptors may be important in the modulation of antinociception in the rat hot-plate test. Nevertheless, a role for 5-HT 1B receptors in the modulation of nociception does not fit with neuroanatomical and physiological data regarding this subtype. If 5-HT 1B receptors are autoreceptors on the neurons of the descending inhibitory serotonergic tract from the nucleus raphe magnus to the spinal dorsal horn (Glaum and Anderson, 1988), then stimulation of these receptors would be expected to attenuate the activity of the descending inhibitory system. The expected action would be hyperalgesia or allodynia, rather than analgesia. In summary, the analgesia produced by spinal injection of 5-HT in CD-1 mice appears to be mediated primarily by 5-HT3 receptors, as does the analgesia produced by 2-methyl- 5-HT. Because neither AODN treatment significantly attenuated the analgesia produced by CGS-12066A, this effect is not mediated by 5-HT 1B receptors or 5-HT 3 receptors. However, it is not clear whether this analgesic effect is mediated by the release of calcitonin gene-related peptide, stimulation of another 5-HT receptor subtype, or some other receptor that may be stimulated by CGS-12066A. 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