Reconsideration of 5-Hydroxytryptamine (5-HT)7 Receptor Distribution Using [H]5-Carboxamidotryptamine and [H]8-Hydroxy-2-(di-n-propylamino)tetraline: Analysis in Brain of 5-HT1A Knockout and 5-HT1A/1B Double-Knockout Mice

The characterization and anatomical distribution of 5-hydroxytryptamine (5-HT)7 receptor binding sites in brain tissue has been hampered by the lack of a specific radioligand. In the present autoradiographic study, we took advantage of 5-HT1A knockout and 5-HT1A/1B double-knockout mice to revisit the pharmacological characterization and anatomical localization of 5-HT7 binding sites in mouse brain using [H]5-carboxamidotryptamine (5-CT) and [H]8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT). The distribution pattern of [H]5-CT binding sites (2 nM) in the brain of mice lacking the 5-HT1A/1B receptor was scarce and confined to the septum, globus pallidus, thalamus, hypothalamus, amygdala, cortex, and substantia nigra. The low densities of [H]5CT binding sites detected in septum, thalamus, hypothalamus, amygdala, and cortex were displaced by 10 M of the selective 5-HT7 receptor antagonist (R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl)pyrrolidine-1-sulfonyl) phenol (SB-269970). The SB-269970insensitive [H]5-CT binding sites detected in globus pallidus and substantia nigra of 5-HT1A/1B knockout mice were displaced by N-[3-(2-dimethylamino)ethoxy-4-methoxy-phenyl]-2 -methyl-4 (5-methyl-1,2,4-oxadiazol-3-yl)-(1,1 -biphenyl)-4-carboxamide hydrochloride (SB-216641) (1 M), demonstrating the 5-HT1D nature of these binding sites. In contrast to the low densities of [H]5-CT binding sites, high-to-moderate densities of [H]8OH-DPAT binding sites (10 nM) were found throughout the brain of 5-HT1A and 5-HT1A/1B knockout mice (olfactory system, septum, thalamus, hypothalamus, amygdala, CA3 field of the hippocampus, cortical mantle, and central gray). These [H]8-OHDPAT binding sites were displaced by 10 M SB-269970, risperidone, and methiothepin but not by pindolol, N-tert-butyl-3[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide (WAY100135), or citalopram. We conclude that despite its high affinity for the 5-HT7 receptor in tissue homogenates, [ H]5-CT is not a good tracer for measuring 5-HT7 receptor binding sites autoradiographically. Also, the lower affinity ligand [H]8-OHDPAT is a much better tracer for autoradiographic studies at the 5-HT7 receptor binding sites. The 5-HT7 receptor is the most recently identified member of the family of G protein-coupled 5-HT receptors (for review, see Vanhoenacker et al., 2000). The 5-HT7 receptor has been cloned from rat (Lovenberg et al., 1993; Ruat et al., 1993; Shen et al., 1993), mouse (Plassat et al., 1993), human (Bard et al., 1993), and guinea pig (Tsou et al., 1994). The receptor is positively coupled to adenylate cyclase, preferentially via Gs. It exhibits a high degree of interspecies homology (approximately 95%) but a low sequence homology with other 5-HT receptors ( 40%). In rat and human, three different splice variants have been described to date, which are thought to have similar pharmacology and function (Heidmann et al., 1997; Krobert et al., 2001). The pharmacological profile of the 5-HT7 receptor is consistent across all tested species and is characterized by high affinity for the 5-HT1 agonists 5-CT, 5-HT, and 8-OH-DPAT; and the 5-HT2 antagonists ritanserin, metergoline, mesulergine, and risperidone. Possible physiological functions for this receptor have been suggested, including relaxation in several vascular preparations (Bard et al., 1993; Shen et al., 1993) and circadian rhythm control via the suprachiasmatic nucleus of the hypothalamus (Lovenberg et al., 1993; Gannon, 2001). Because 8-OH-DPAT has been shown to exhibit a relatively high affinity for the 5-HT7 receptor, multiple functions formerly ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; 5-CT, 5-carboxamidotryptamine; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetraline; SB-269970, (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl) phenol; WAY-100135, N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin1-yl]-2-phenylpropanamide; GR125743, N-[4-methoxy-3-(4-methyl piperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide; SB-216641, N-[3-(2dimethylamino)ethoxy-4-methoxy-phenyl]-2 -methyl-4 -(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1 -biphenyl)-4-carboxamide hydrochloride. 0022-3565/02/3021-240–248$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 32987/988066 JPET 302:240–248, 2002 Printed in U.S.A. 240 at A PE T Jornals on M ay 3, 2017 jpet.asjournals.org D ow nladed from attributed to a 5-HT1A-like receptor (including adenylate cyclase stimulation in gastrointestinal, cardiovascular, and hypothalamic preparations) may be likely attributable to the 5-HT7 subtype. Atypical antipsychotics, such as clozapine and risperidone, and some antidepressants display high affinity for this receptor. The significance of 5-HT7 receptor blockade with regard to antipsychotic or antidepressant drug action remains to be elucidated (Eglen et al., 1997; Vanhoenacker et al., 2000). The specific distribution of 5-HT7 receptor mRNA in rat and guinea pig brain is well described (To et al., 1995; Gustafson et al., 1996; Mengod et al., 1996; Kinsey et al., 2001; Neumaier et al., 2001). However, the characterization and study of the anatomical distribution of 5-HT7 receptor binding sites in brain tissue have been hampered by the lack of a specific radioligand. Several attempts have been made using available radioligands in rat brain homogenates (Stowe and Barnes, 1998; Hemedah et al., 1999) or tissue sections (To et al., 1995; Waeber and Moskowitz, 1995; Gustafson et al., 1996; Mengod et al., 1996). The autoradiographic studies used a relatively nonselective radioligand (i.e., [H]5-CT) in the presence of drugs to mask other binding sites. Very low densities of 5-HT7 binding sites have been reported in these studies, whereas a relatively high abundance of 5-HT7 receptor mRNA or 5-HT7 immunoreactivity has been reported in rat brain (Gustafson et al., 1996; Mengod et al., 1996; Kinsey et al., 2001; Neumaier et al., 2001). The authors have concluded that the masking compounds added in these autoradiographic studies might have sufficient affinity for the 5-HT7 receptor to occupy part of the 5-HT7 binding sites at the concentration used, thus occluding full visualization of this receptor. An attempt to label 5-HT7 receptors in hamster brain has also been made using [H]8OH-DPAT in the presence of pindolol for 5-HT1A receptor occlusion (Duncan et al., 1999). However, no pharmacological characterization was presented in the latter study. Furthermore, the concentration of [H]8-OH-DPAT (1.8 nM) used by Duncan and colleagues seems to be very low with respect to its moderate affinity for 5-HT7 receptor binding sites in recombinant cell lines or in rat brain tissue homogenates (Ki of 30–65 nM; Lovenberg et al., 1993; Eglen et al., 1997; Krobert et al., 2001). In the present autoradiographic study, we took advantage of 5-HT1A knockout and 5-HT1A/1B knockout mice to revisit the anatomical localization and pharmacological characterization of 5-HT7 binding sites in mouse brain using [ H]5-CT and [H]8-OH-DPAT. We tested the hypothesis whether the 5-HT1A and 5-HT1B/1D masking compounds added in the previous [H]5-CT autoradiographic studies (Waeber and Moskowitz, 1995; Gustafson et al., 1996; Mengod et al., 1996) truly occluded the 5-HT7 receptor binding sites at the concentration used. In mice lacking the 5-HT1A receptor, we performed concentration binding studies to determine the suitable concentration of [H]8-OH-DPAT for labeling of 5-HT7 receptor binding sites. The 5-HT7 binding sites were characterized by measuring the potency of several compounds, including a selective 5-HT7 receptor antagonist (SB269970; Hagan et al., 2000) to inhibit 5-HT7 binding sites. This study provides a detailed anatomical localization of 5-HT7 receptor binding sites throughout the brain of 5-HT1A and 5-HT1A/1B knockout mice and addresses the controversial issue of the atypical feature of [H]5-CT binding sites observed in 5-HT1A receptor-containing regions (Waeber and Moskowitz, 1995; Castro et al., 1997). Experimental Procedures Generation of 5-HT1A Knockout and 5-HT1A/1B DoubleKnockout Mice 5-HT1A receptor knockout mice had been generated as described previously (Ramboz et al., 1998). 5-HT1A/1B double-knockout mice were obtained by first crossing homozygous 5-HT1A knockout mice with previously generated homozygous 5-HT1B receptor knockout mice (Saudou et al., 1994). This first cross led to offspring that were heterozygous for both the 5-HT1A and 5-HT1B mutant alleles. Subsequent breeding of the heterozygote mice led to the generation of double-knockout mice. Southern blot analysis of tail DNA was used to determine which mice were homozygous mutant for both receptors. All the experiments described in this study have been carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of