Introduction

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The effects of the neurotransmitter serotonin (5-HT) are mediated by at least 14 different receptors: one ligand-gated ion channel (the 5-HT₃ receptor) and 13 G protein-coupled receptors (Boess and Martin, 1994; Hoyer and Martin, 1997). Of these, at least five are coupled to inhibition of adenylyl cyclase (5-HT_1A, 5-HT_1B, 5-HT_1D, 5-HT_1E, 5-HT_1F), three are linked to phosphoinositide hydrolysis (5-HT_2A, 5-HT_2B, 5-HT_2C), and three have been shown to stimulate adenylyl cyclase activity (5-HT₄, 5-HT₆, 5-HT₇). Unlike the classical 5-HT receptors, the 5-HT₆ receptor was not recognized as a pharmacological entity in physiological or radioligand binding experiments before its cloning from rat striatal cDNA (Monsma et al., 1993; Ruat et al., 1993). The highest levels of 5-HT₆ receptor mRNA were detected in the olfactory tubercle, nucleus accumbens, striatum, and hippocampus (Monsma et al., 1993; Ruat et al., 1993; Ward et al., 1995; Gérard et al., 1996). To determine the distribution of 5-HT₆ receptor protein, Gérard et al. (1997) raised polyclonal antibodies against a synthetic peptide corresponding to part of the carboxyl-terminal domain of the 5-HT₆ receptor. They observed high levels of 5-HT₆ receptor-like immunoreactivity in olfactory tubercle, piriform cortex, nucleus accumbens, islands of Calleja, striatum, hippocampus (CA1 and dentate gyrus), and the molecular layer of the cerebellum.

Many nonselective compounds, including several tricyclic antidepressant drugs, antipsychotic agents, and tryptamine and ergoline derivatives interact with the 5-HT₆ receptor, as was shown in binding studies on recombinant rat and human receptors using [³H]LSD, ¹²⁵I-LSD, and [³H]5-HT as radioligands (Monsma et al., 1993; Roth et al., 1994; Boess et al., 1997; reviewed in Sleight et al., 1997).

Putative 5-HT₆-like receptors positively coupled to adenylyl cyclase have been described in the mouse neuroblastoma-derived cell lines N18TG2 and NCB20 (Berry-Kravis and Dawson, 1983; Conner and Mansour, 1990; Unsworth and Molinoff, 1994). In addition, responses with a 5-HT₆ receptor-like profile have been observed in pig caudate membranes (Schoeffter and Waeber, 1994) and in mouse embryonic striatal neurons (Sebben et al., 1994).

Until recently, the physiological role of the 5-HT₆ receptor was not known, because of the lack of selective ligands. However, the functional significance of this receptor has been investigated by using intracerebroventricular injections of 5-HT₆ receptor-specific antisense oligonucleotides. This treatment, which should abolish or reduce the expression of 5-HT₆ receptor protein, produced a behavioral syndrome consisting of yawning, stretching, and chewing (Bourson et al., 1995; Sleight et al., 1996).

Recently, we have described two selective 5-HT₆ receptor antagonists, Ro 04-6790 and Ro 63-0563, and demonstrated the ability of Ro 04-6790 to produce behavioral effects similar to those observed after antisense treatment (Sleight et al., 1998). In the current study, we present the first selective 5-HT₆ receptor radioligand [³H]Ro 63-0563 and demonstrate that it labels recombinant rat and human 5-HT₆ receptors as well as 5-HT₆ receptor binding sites in rat and porcine striatal membranes.

	Experimental Procedures

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Materials. HeLa cells expressing human 5-HT₆ receptors were obtained from Dr. David Sibley (National Institutes of Health, Bethesda, MD) under licensing agreement. 5-HT was purchased from Fluka (Buchs, Switzerland); ergotamine from Sigma (Buchs, Switzerland); mesulergine, metergoline, methysergide, lisuride, methiothepin, clozapine, amitriptyline, ritanserin, mianserin, and pargyline from Research Biochemicals (Natick, MA). Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin, streptomycin, and geneticin were obtained from Gibco Life Technologies (Basel, Switzerland). Ro 20-1724, Ro 63-0563 (Fig. 1), Ro 04-6790 (Fig. 1), and LSD were synthesized at F. Hoffmann-La Roche AG (Basel, Switzerland). [³H]Ro 63-0563 (specific activity, 29 Ci/mmol) was kindly prepared by Dr. Philipp Huguenin at F. Hoffmann-La Roche (Basel).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structures of selective 5-HT6 receptor antagonists Ro 04-6790 and Ro 63-0563.

Preparation of membranes from cells expressing recombinant 5-HT₆ receptor. Membranes were prepared from HEK 293 cells stably transfected with the rat 5-HT₆ receptor (Boess et al., 1997) or HeLa cells stably expressing a human 5-HT₆ receptor clone (Kohen et al., 1996). HEK 293 cells were grown in Dulbecco's modified Eagle medium + 10% fetal bovine serum containing penicillin (100 IU/ml), streptomycin (100 µg/ml), and 0.5 mg/ml geneticin in a humidified atmosphere (5% CO₂). HeLa cells were grown in exactly the same conditions except that geneticin was omitted from the media. The cells were detached with phosphate-buffered saline containing 1 mM EDTA, washed with phosphate-buffered saline (Gibco Life Technologies) by two centrifugations (10 min, 500 × g) and the resulting pellet was resuspended in ice-cold 50 mM Tris·HCl, pH 7.4, containing 10 mM MgCl₂ and 0.5 mM EDTA by using a Polytron homogenizer (15 sec at maximal speed) at a concentration corresponding to 2 × 10⁶ cells/ml. This homogenate was centrifuged at 100,000 × g for 60 min, the resulting pellet was resuspended in the same buffer to obtain a concentration corresponding to 4 × 10⁷ cells/ml, and aliquots were stored at 80°.

Preparation of rat and porcine striatal membranes. Before dissection, rat brains (male Ibm:RoRo; Biological Research Laboratories, Füllinsdorf, Switzerland) and porcine brains (from a local abattoir) were kept on ice. Striatal tissue (in 3-g aliquots) was homogenized in 26 ml of 0.32 M sucrose with a glass/teflon homogenizer and centrifuged for 10 min at 1000 × g (4°). The pellet was discarded and the supernatant centrifuged for 30 min at 25,000 × g. The pellet was resuspended in 26 ml of 50 mM Tris·HCl, pH 7.4, centrifuged for 15 min at 25,000 × g, resuspended in 50 mM Tris·HCl, incubated for 15 min at 37° and centrifuged for 15 min at 25,000 × g. The pellet was washed two times by resuspension in 50 mM Tris·HCl and centrifugation for 15 min at 25,000 × g and the final pellet was stored frozen at 80°.

[³H]Ro 63-0563 binding assays. Membranes prepared from cells expressing recombinant rat and human 5-HT₆ receptor were resuspended in assay buffer (50 mM Tris·HCl, 10 µM pargyline, 5 mM MgCl₂, 0.5 mM EDTA, and 0.1% ascorbic acid, pH 7.4). Binding assays consisted of 100 µl of membrane suspension (corresponding to 4 × 10⁵ cells per assay tube), 50 µl of [³H]Ro 63-0563 (specific activity, 29 Ci/mmol), and 50 µl of displacing drug or assay buffer (final assay volume, 200 µl). Nonspecific binding was measured in the presence of 10 µM methiothepin. Saturation experiments were performed using eight concentrations of [³H]Ro 63-0563 (final concentrations of 0.31 nM to 40 nM). In competition assays, seven to 14 concentrations of the displacing ligands were tested (3 × 10¹¹ to 10⁴ M) at a final [³H]Ro 63-0563 concentration of 5 nM. At this concentration, specific binding corresponded to 70% of total binding. Incubations were performed at room temperature for 80 min. Association and dissociation experiments were conducted in the presence of 5 nM [³H]Ro 63-0563. Association experiments were carried out by incubating samples with [³H]Ro 63-0563 in the absence or presence of 10 µM methiothepin for 1 min to 120 min. Dissociation was initiated by addition of 10 µM methiothepin after equilibration with [³H]Ro 63-0563 for 120 min and allowed to proceed for 1 to 120 min.

	Results

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[³H]Ro 63-0563 binding to recombinant rat 5-HT₆ receptor. At a concentration of 5 nM, 70% of total [³H]Ro 63-0563 binding to membranes prepared from HEK 293 cells stably expressing the rat 5-HT₆ receptor was prevented by the presence of 10 µM methiothepin. No specific binding was detected to nontransfected HEK 293 cells. Specific binding reached a maximum within 30 min with an association rate constant of k₁ = 6.01 ± 0.36 × 10⁶ M¹ min¹ (mean ± standard error, three experiments). The binding of [³H]Ro 63-0563 was reversible (dissociation rate constant k₁ = 6.75 ± 0.36 × 10² min¹, mean ± standard error, four experiments) and apparently to a single site (Fig. 2). The dissociation constant (K_d = k₁/k₁) determined from these values was 11.2 nM. In equilibrium binding studies with recombinant rat 5-HT₆ receptor, specific binding of [³H]Ro 63-0563 was saturable in the range of 0.31 to 40 nM, whereas nonspecific binding increased linearly with increasing ligand concentration (Fig. 3). [³H]Ro 63-0563 labeled a single binding site with an equilibrium dissociation constant (K_d) of 6.8 ± 0.9 nM (mean ± standard error, eleven experiments) and a B_max of 2.17 ± 0.09 pmol/mg protein (five experiments).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Association and dissociation kinetics of [3H]Ro 63-0563 binding to rat 5-HT6 receptors stably expressed in HEK 293 cells, performed as described in Experimental Procedures. The data shown (k1 = 5.37 × 106M1 min1, k1 = 7.23 × 102 min1) are representative of three similar experiments, each performed in triplicate.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Equilibrium binding of [3H]Ro 63-0563 to rat 5-HT6 receptor stably expressed in HEK 293 cells, performed as described in Experimental Procedures. , Total binding; , nonspecific binding; , specific binding. Inset, Scatchard transformation of the saturation binding data. The data shown are from one of 11 similar experiments, each performed in triplicate.

[³H]Ro 63-0563 binding to recombinant human 5-HT₆ receptor. In membranes prepared from HeLa cells stably expressing the human 5-HT₆ receptor, [³H]Ro 63-0563 also labeled a single binding site. The binding of [³H]Ro 63-0563 was rapid (association rate constant k₁ = 1.59 ± 0.20 × 10⁷ M¹ min¹, mean ± standard error, three experiments) and reversible (dissociation rate constant k₁ = 4.79 ± 0.38 × 10² min¹, mean ± standard error, three experiments) (results not shown). The affinity constant (K_d = k₁/k₁) determined from these values was 3.0 nM. In equilibrium binding studies with recombinant human 5-HT₆ receptor, [³H]Ro 63-0563 labeled a single binding site with a K_d of 4.96 ± 0.97 nM (mean ± standard error, six experiments) and a B_max of 1.59 ± 0.04 pmol/mg protein (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Equilibrium binding of [3H]Ro 63-0563 to human 5-HT6 receptor stably expressed in HeLa cells, performed as described in Experimental Procedures. , Total binding; , nonspecific binding; , specific binding. Inset, Scatchard transformation of the saturation binding data. The data shown are from one of six similar experiments, each performed in triplicate.

Pharmacological profile of [³H]Ro 63-0563 binding to rat and human 5-HT₆ receptors. Table 1 shows the pK_i values and Hill slopes measured in competition assays with various 5-HT₆ receptor ligands in the presence of 5 nM [³H]Ro 63-0563. The pharmacological profile of [³H]Ro 63-0563 binding to the recombinant rat 5-HT₆ receptor (methiothepin > LSD > clozapine > lisuride > ergotamine > 5-HT > amitriptyline ~ metergoline ~ mianserin ~ ritanserin > methysergide > mesulergine) was similar to that determined with [³H]LSD using the same cells (Table 1). The Hill slopes of both antagonists (e.g., methiothepin, clozapine, mianserin, ritanserin) and agonists (e.g., 5-HT, LSD, lisuride), as well as those of unlabeled Ro 63-0563 and the structurally related 5-HT₆ receptor-selective antagonist Ro 04-6790, were close to one and competition curves with 14 ligand concentrations did not suggest the presence of multiple binding sites (Fig. 5). The affinities of the ligands tested at the human 5-HT₆ receptor were very similar to those determined for the recombinant rat 5-HT₆ receptor (correlation coefficient r = 0.97, 14 experiments) (Table 1; Fig. 6).

View this table: [in this window] [in a new window]   TABLE 1 Pharmacological profile of [3H]Ro 63-0563 binding pKi (* or pKd) values (mean ± standard error, > three experiments) and Hill slopes measured with rat 5-HT6 receptor expressed in HEK 293 cells and human 5-HT6 receptor expressed in HeLa cells and porcine striatal membranes. Data from [3H]LSD and [3H]5-HT binding studies with recombinant rat 5-HT6 receptor (Boess et al., 1997 and aSleight et al., 1998) are presented for comparison.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Pharmacological profile of [3H]Ro 63-0563 binding to rat 5-HT6 receptors stably expressed in HEK 293 cells. Competition experiments with LSD (), clozapine (), Ro 04-6790 (), 5-HT (), and ritanserin () were performed as described in Experimental Procedures. The data shown are from one of three or four similar experiments, each performed in triplicate.

[³H]Ro 63-0563 binding to 5-HT₆ receptor-like binding sites in rat striatum. In equilibrium binding studies with rat striatal membranes, [³H]Ro 63-0563 labeled a single binding site with a K_d of 11.7 ± 1.1 nM (mean ± standard error, four experiments) and a B_max of 175 ± 26 fmol/mg protein (Fig. 7). Competition assays were carried out in the presence of 2 nM [³H]Ro 63-0563. At this concentration, specific binding corresponded to 20% of total binding. In an initial characterization with a limited set of ligands (tested at seven different concentrations in the range of 10¹⁰ to 10⁴ M), we measured the following pK_i values (three or four experiments, mean ± standard error) for Ro 04-6790 (7.10 ± 0.36), 5-HT (7.31 ± 0.43), amitriptyline (6.90 ± 0.53), clozapine (7.50 ± 0.14), and methiothepin (7.98 ± 0.05). Comparison with the pK_i values measured at the recombinant rat receptor (Table 1) showed a statistically significant correlation (r = 0.9, six experiments, p < 0.01), indicating that this [³H]Ro 63-0563 binding site had a pharmacological profile similar to the 5-HT₆ receptor. Because of the relatively large quantity of tissue needed per assay, a more detailed pharmacological characterization was carried out using porcine striatal tissue.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Pharmacological profile of [3H]Ro 63-0563 binding to human 5-HT6 receptors stably expressed in HeLa cells. Competition experiments with LSD (), clozapine (), Ro 04-6790 (), 5-HT (), and ritanserin () were performed as described in Experimental Procedures. The data shown are from one of three or four similar experiments, each performed in triplicate.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Equilibrium binding of [3H]Ro 63-0563 to 5-HT6 receptor-like binding sites in rat striatal membranes, performed as described in Experimental Procedures. Inset, Scatchard transformation of the saturation binding data. The data shown are from one of three similar experiments, each performed in triplicate.

[³H]Ro 63-0563 binding to 5-HT₆ receptor-like binding sites in porcine striatum. Specific binding of [³H]Ro 63-0563 to porcine striatal membranes reached a maximum within 30 min with an association rate constant of k₁ = 6.75 ± 1.74 × 10⁷ M¹ min¹ (mean ± standard error, three experiments). The binding of [³H]Ro 63-0563 was reversible (dissociation rate constant k₁ = 1.28 ± 0.33 × 10¹ min¹, mean ± standard error, three experiments). The affinity constant (K_d = k₁/k₁) determined from these values was 2 nM. In equilibrium binding studies with porcine striatal membranes, [³H]Ro 63-0563 labeled a single binding site with a K_d of 8.01 ± 0.63 nM (mean ± standard error, 16 experiments) and a B_max of 130 ± 25 fmol/mg protein (eight experiments) (Fig. 8). The affinities of a range of 5-HT₆ receptor ligands at this [³H]Ro 63-0563 binding site were similar to those found for the recombinant rat and human 5-HT₆ receptor labeled with [³H]Ro 63-0563. There was a statistically significant (p < 0.01) correlation of the pK_i values of the 14 ligands tested at the binding site in porcine striatal membranes with the values measured for both rat (correlation coefficient r = 0.76) and human recombinant 5-HT₆ receptor (r = 0.78). High concentrations of all ligands tested displaced [³H]Ro 63-0563 binding to the same level as the nonspecific binding defined in the presence of 10 µM methiothepin. In addition, the Hill slopes of the ligands tested were not significantly different from one (i.e., the mean Hill slope value and unity were within 2 standard deviation units from each other), which suggests that [³H]Ro 63-0563 labels a homogenous population of binding sites (Table 1, Fig. 9).

View larger version (24K): [in this window] [in a new window]   Fig. 8.   Equilibrium binding of [3H]Ro 63-0563 to 5-HT6 receptor-like binding sites in porcine striatal membranes, performed as described in Experimental Procedures. Inset, Scatchard transformation of the saturation binding data. The data shown are from one of eight similar experiments, each performed in triplicate.

View larger version (28K): [in this window] [in a new window]   Fig. 9.   Pharmacological profile of [3H]Ro 63-0563 binding to 5-HT6 receptor-like binding sites in porcine striatal membranes. Competition experiments with LSD (), clozapine (), Ro 04-6790 (), 5-HT (), and ritanserin () were performed as described in Experimental Procedures. The data shown are from one of three to six similar experiments, each performed in triplicate.

	Discussion

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Ro 63-0563 is a high affinity 5-HT₆ receptor antagonist with more than 100-fold selectivity for the 5-HT₆ receptor compared with 69 other receptors and binding sites (Sleight et al., 1998). [³H]Ro 63-0563 binds with nanomolar affinity to a single binding site in membranes prepared from HEK 293 cells stably expressing recombinant rat 5-HT₆ receptors (K_d = 7 nM) and HeLa cells stably expressing recombinant human 5-HT₆ receptors (K_d = 5 nM). The number of binding sites labeled by [³H]Ro 63-0563 in HEK 293 cells stably expressing rat 5-HT₆ receptor (2.17 pmol/mg protein) is similar to that measured previously with the (nonselective) agonist radioligands [³H]5-HT (2.54 pmol/mg protein) and [³H]LSD (2.22 pmol/mg protein) in the same cell line (Boess et al., 1997). This suggests that these radioligands do not distinguish between different agonist high and low affinity states of the recombinant rat 5-HT₆ receptor expressed in HEK 293 cells.

The pharmacological profile of the rat 5-HT₆ receptor labeled with [³H]Ro 63-0563 (methiothepin > LSD > clozapine ~ Ro 63-0563 > lisuride > ergotamine ~ Ro 04-6790 > 5-HT > amitriptyline ~ metergoline ~ mianserin ~ ritanserin > methysergide > mesulergine) was very similar to that of the human 5-HT₆ receptor (methiothepin > Ro 63-0563 ~ LSD > lisuride > ergotamine > Ro 04-6790 ~ clozapine > 5-HT ~ metergoline > ritanserin > mianserin ~ amitriptyline > methysergide > mesulergine) (correlation coefficient r = 0.97, 14 experiments). As expected, there was also a significant correlation between the pK_i values for the rat recombinant 5-HT₆ receptor labeled with [³H]Ro 63-0563 measured in the present study and the values obtained with the same cell line using either [³H]LSD (r = 0.87, 14 experiments) or [³H]5-HT (r = 0.92, 12 experiments) as the radioligand (Boess et al., 1997).

Because Northern blots (Monsma et al., 1993), in situ hybridization (Ward et al., 1995), and reverse-transcription polymerase chain reaction (Gérard et al., 1996) had demonstrated especially high levels of 5-HT₆ receptor mRNA in the rat striatum and high levels of 5-HT₆ receptor-like immunoreactivity had also been observed in this brain region (Gérard et al., 1997), we looked for [³H]Ro 63-0563 binding sites in rat striatal membranes. [³H]Ro 63-0563 labeled a single binding site with a K_d of 11.7 nM and a B_max of 175 fmol/mg protein. A comparison of the pK_i and pK_d values of the ligands tested in rat striatal membranes (including [³H]Ro 63-0563) with the values determined for the recombinant rat 5-HT₆ receptor showed a significant correlation (r = 0.9, six experiments, p < 0.01) suggesting that this [³H]Ro 63-0563 binding site actually corresponds to the native rat 5-HT₆ receptor. Because of the relatively large quantity of tissue needed per assay, a more detailed pharmacological characterization was carried out using porcine striatal membranes, a tissue in which 5-HT-mediated stimulation of adenylyl cyclase activity with a 5-HT₆ receptor-like profile has been reported (Schoeffter and Waeber, 1994). The amount of specific binding observed in porcine striatal membranes (20-30%) was generally higher than that measured with rat striatal membranes (10-20%), which facilitated a more detailed characterization of the [³H]Ro 63-0563 binding in the porcine tissue. We did attempt to improve the level of specific binding by changing pH, the buffer used for the binding assay (HEPES instead of Tris), and the concentrations of the various salts in the incubation buffer. None of these changes, however, significantly improved specific binding (results not shown). In equilibrium binding studies [³H]Ro 63-0563 labeled a single binding site in porcine striatal membranes with a K_d of 8 nM and a B_max of 130 fmol/mg protein. The pK_i values of the 5-HT₆ receptor ligands tested at the [³H]Ro 63-0563 binding site in porcine striatal membranes showed a statistically significant correlation with the values measured for both rat (correlation coefficient r = 0.76, 14 experiments, p < 0.01) and human recombinant 5-HT₆ receptor (r = 0.78, 14 experiments, p < 0.01). Although the overall pharmacological profile was quite similar to that of the rat and human receptor, methiothepin, clozapine, and amitriptyline had about 10-fold lower affinity for the binding site in porcine brain. This is in agreement with the low pK_b values of methiothepin and clozapine for the inhibition of 5-HT mediated stimulation of adenylyl cyclase in porcine striatal membranes reported by Schoeffter and Waeber (1994). High concentrations of all ligands tested displaced [³H]Ro 63-0563 binding to the same level as the nonspecific binding defined in the presence of 10 µM methiothepin. The Hill slopes of the ligands tested, including the selective 5-HT₆ receptor ligand Ro 04-6790, were not significantly different from unity, suggesting that [³H]Ro 63-0563 labels a homogenous population of binding sites. Furthermore, we attempted to displace [³H]Ro 63-0563 with 5-HT in both the presence and absence of guanylyl-imidodiphosphate (200 µM). Guanylyl imidodiphosphate had no effect on either the amount of specific binding, the affinity of 5-HT, or the Hill slope of the displacement (results not shown).

We have previously demonstrated that the apparent affinity of certain compounds, particularly tryptamine derivatives, was dependent upon whether [³H]5-HT or [³H]LSD was used as the radioligand (Boess et al., 1997). Because the purpose of the present study was to determine whether 5-HT₆ receptors are expressed in the rat and porcine striatum, we specifically chose ligands that had consistent affinities with respect to both [³H]5-HT and [³H]LSD binding. It would be interesting, however, to determine the affinity of a number of tryptamine derivatives for the 5-HT₆ receptor labeled with [³H]Ro 63-0563 and compare them with the affinities obtained with [³H]5-HT and [³H]LSD binding.

These results demonstrate for the first time the presence of 5-HT₆ receptor binding sites in membranes prepared from rat and porcine brain. Previously, a [³H]clozapine binding site with a pharmacological profile resembling that of the 5-HT₆ receptor had been identified in rat brain membranes (Glatt et al., 1995). However, 5-HT displayed a very low affinity (K_i = 200,000 nM) for this [³H]clozapine binding site in rat brain compared with an affinity of 150 nM at recombinant rat 5-HT₆ receptors (Glatt et al., 1995), which suggests that these binding sites may not correspond to the 5-HT₆ receptor, but to a distinct receptor with similar pharmacological properties. In contrast, 5-HT has a high affinity for both the recombinant rat and human 5-HT₆ receptor labeled with [³H]Ro 63-0563 (K_i = 34 and 44 nM, respectively), as well as for the [³H]Ro 63-0563 binding sites in rat and porcine striatal membranes (19 and 40 nM, respectively).

Our data show that 5-HT₆ receptors are expressed in the rat and porcine striatum. In agreement with our results, 5-HT-mediated stimulation of adenylyl cyclase activity with a 5-HT₆ receptor-like pharmacological profile has been observed in porcine striatal membrane preparations (Schoeffter and Waeber, 1994) and mouse striatal neurons in primary culture (Sebben et al., 1994). Sebben et al. (1994) studied the affinity of a limited number of compounds for a 5-HT-sensitive [¹²⁵I]LSD binding site in membranes prepared from mouse striatal neurons after 11 days of primary culture and concluded that they interacted with more than one population of binding sites. Our results suggest that at least some of the [¹²⁵I]LSD binding sites did in fact correspond to 5-HT₆ receptors. The number of receptors measured in the membrane preparations of mouse striatal neurons with [¹²⁵I]LSD (B_max = 16 fmol/mg) (Sebben et al., 1994) was approximately 10-fold lower than the value we observed in membranes prepared from freshly dissected rat and porcine striatum (175 and 130 fmol/mg protein, respectively). This may indicate that the expression of 5-HT₆ receptors actually diminishes during primary culture or that species differences exist in the levels of 5-HT₆ receptor expression.

In summary, [³H]Ro 63-0563 is a 5-HT₆ receptor selective radioligand that binds with high affinity to recombinant rat and human 5-HT₆ receptors. The pharmacological profile of the [³H]Ro 63-0563 binding site is similar to that of the 5-HT₆ receptor labeled with either [³H]LSD or [³H]5-HT. We have identified [³H]Ro 63-0563 binding sites with a 5-HT₆ receptor-like pharmacological profile in the rat and porcine striatum, confirming the expression of 5-HT₆ receptors in this brain region in agreement with the distribution of 5-HT₆ receptor mRNA and immunoreactivity. [³H]Ro 63-0563 will therefore be a useful tool to determine the expression level of 5-HT₆ receptors in different brain regions. In addition, our data support the results of studies using antisense oligonucleotides (Bourson et al., 1995; Sleight et al., 1996) and the 5-HT₆ receptor selective antagonist Ro 04-6790 (Sleight et al., 1998) that first suggested the presence of functional 5-HT₆ receptors in the rat brain. Indeed, given the previously reported data from our group (Bourson et al., 1995; Sleight et al., 1996, 1998) and the data reported here, we feel justified in abandoning lower case letters for 5-HT₆ receptor nomenclature.