Neuropharmacology 44 (2003) 653–661 www.elsevier.com/locate/neuropharm Regional brain uptake of the muscarinic ligand, [ 18 F]FP-TZTP, is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice E.M. Jagoda a , D.O. Kiesewetter a , K. Shimoji a , L. Ravasi a , M. Yamada b , J. Gomeza b , J. Wess b , W.C. Eckelman a,* a PET Department, Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA b Laboratory of Bioorganic Chemistry (NIDDK), National Institutes of Health, Bethesda, MD 20892, USA Received 11 April 2001; received in revised form 29 November 2002; accepted 7 January 2003 Abstract A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP- TZTP) radiolabeled with the positron emitting radionuclide 18 F ([ 18 F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [ 18 F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [ 18 F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P  0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothala- mus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [ 18 F]FP- TZTP uptakes in the same brain regions were not significantly decreased at P  0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P  0.05. Given the fact that large decreases in [ 18 F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [ 18 F]FP-TZTP preferentially labels M2 receptors in vivo.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Muscarinic receptors; KO mice; Cerebral blood flow; Saturability 1. Introduction Our goal is to develop receptor-specific ligands lab- eled with a positron emitting radionuclide in order to follow biochemical changes as a function of disease by external imaging using positron emission tomography (PET). Postmortem quantitation of muscarinic receptor subtypes in Alzheimer’s patients indicate a selective loss of M2 subtype in cortical regions while the M1 subtype was preserved (Aubert et al., 1992a; Quirion et al., 1989; Rodriguez-Puertas et al., 1997). Therefore, we sought to develop an M2 selective ligand labeled with a positron- emitting radionuclide since that would be most sensitive * Corresponding author. Tel.: +1-301-496-6455; fax: +1-301-402- 3521. E-mail address: eckelman@nih.gov (W.C. Eckelman). 0028-3908/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0028-3908(03)00050-9 to M2 receptor subtype density changes in the living human brain. This, in turn, would allow the study of the progression of Alzheimer’s dementia, early diagnosis, and monitoring of drug therapies using non-invasive external imaging. We have developed a radioligand that is a fluorinated analog of a compound that showed M2 selectivity in bio- logical assays (Sauerberg et al., 1992). This compound, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6- tetrahydro-1-methyl-pyridine (FP-TZTP) has been radi- olabeled with F-18, a positron emitting radionuclide with a half life of 109.7 min (Kiesewetter et al., 1995). Ex vivo autoradiography of rat brain using no carrier added [ 18 F]FP-TZTP confirmed a distribution of radioactivity in gray matter which was characteristic of M2 receptor density (Kiesewetter et al., 1999). This distribution of radioactivity is in good agreement with the distribution as described in immunocytochemical localization studies