A Reduced Extracellular Serotonin Level 18 Increases the 5-HT1A PET Ligand F-MPPF Binding in the Rat Hippocampus Luc Zimmer, PharmD, PhD1,2; Latifa Rbah, MS1; Fabrice Giacomelli, MS3; Didier Le Bars, PharmD, PhD1; and Bernard Renaud, PhD2 1Centre d’Exploration et de Recherche Me´dicales par E´ mission de Positons, Biomedical Cyclotron, Lyon, France; 2Institut National de la Sante´ et de la Recherche Me´dicale, Unite´ 512, Neurochimie et Neuropharmacologie, Lyon 1 University, Lyon, France; and 3Cyclotron Research Center, Lie`ge University, Lie`ge, Belgium 4,2Ј-(Methoxyphenyl)-1-[2Ј-(N-2Љ-pyridinyl)-p-fluorobenz- The serotonin-1A (5-HT1A) receptor has been implicated amido]ethylpiperazine (18F-MPPF) is a radiotracer used in clini- in various affective disorders such as anxiety and depression cal PET studies for the visualization of serotonin-1A (5-HT1A) (1–4). Thus, the functional imaging of these receptors by receptors. In a previous study, we demonstrated that a rapid PET may have important implications for our understanding enhancement of extracellular serotonin concentrations influ- of the role of this receptor in those pathologies and their ences 18F-MPPF–specific binding. Because endogenous sero- tonin is significantly decreased in some pathologies, the aim of therapeutics. this study was to determine whether 18F-MPPF is sensitive to Several radioligands have been developed for the imag- depletion of this neurotransmitter. Methods: Using the ␤-micro- ing and quantification of 5-HT1A receptors using PET and probe, an original ␤ϩ-sensitive intracerebral probe, and micro- have been tested in humans (5). Recently, the selective dialysis, the effect of decreased serotonin on the specific bind- 5-HT1A antagonist, 4,2Ј-(methoxyphenyl)-1-[2Ј-(N-2Љ-pyr- 18 ing of F-MPPF to 5-HT1A receptors was investigated in the idinyl)-p-fluorobenzamido]ethylpiperazine (MPPF), has hippocampus of the anesthetized rat. Extracellular serotonin successfully been labeled with 18F-fluorine, resulting in the was pharmacologically decreased in the hippocampus after a 18F-fluoro analog, 18F-MPPF (6). Animal experiments have single injection of p-ethynylphenylalanine ([p-EPA] 5 mg/kg), a shown a regional distribution of this radioligand that con- new tryptophan hydroxylase inhibitor. Results: Our results curs well with known 5-HT receptor densities (6–10). In showed that the 18F-MPPF–specific binding was significantly 1A 18 enhanced after the decrease of extracellular serotonin. These a recent study, we demonstrated that the F-MPPF–specific results were confirmed by the 18F-MPPF distribution in cerebral binding was decreased after a fenfluramine-induced seroto- tissues (hippocampus-to-cerebellum ratio) and by the decrease nin increase (11). of the extracellular 18F-MPPF collected in hippocampal dialy- Because recent theories of the pathophysiology of depres- sates. Conclusion: This study further supports the view that sion have put much emphasis on deficiency of serotoniner- 18F-MPPF binding potential is increased in the hippocampus if gic function (12,13), it was of great interest to know the the endogenous serotonin is pharmacologically decreased after vulnerability of 18F-MPPF to this neurotransmitter reduction a p-EPA injection. This phenomenon will be an additional factor but little information is currently available concerning this 18 in the interpretation of the results from F-MPPF clinical PET parameter. A recent study of 6 healthy volunteers showed studies. that 18F-MPPF binding was not affected after a dietary Ј Key Words: serotonin receptors; p-ethynylphenylalanine; 4,2 - depletion of tryptophan, supposed to reduce the serotonin (methoxyphenyl)-1-[2Ј-(N-2Љ-pyridinyl)-p-fluorobenzamido]- ethylpiperazine; serotonin; microdialysis; ␤-microprobe synthesis (14). However, the methodologic limit inherent in this clinical study was the lack of effective control of the J Nucl Med 2003; 44:1495–1501 brain serotonin depletion. Therefore, this preclinical study was aimed at determin- ing whether 18F-MPPF–specific binding in the rat hip- pocampus is influenced after a controlled depletion of se- rotonin. For this purpose, we used a new tryptophan hydroxylase inhibitor, the p-ethynylphenylalanine (p-EPA), that we previously characterized (15). Accordingly, we de- Received Jan. 13, 2003; revision accepted Apr. 21, 2003. For correspondence or reprints contact: Luc Zimmer, PharmD, PhD, Centre termined in the rat hippocampus (a) the ability of p-EPA to d’Exploration et de Recherche Me´ dicales par E´ mission de Positons, Biomed- 18 ical Cyclotron, 59 Boulevard Pinel, F-69003 Lyon, France. decrease the extracellular serotonin, (b) the F-MPPF tissue E-mail. [email protected] distribution after p-EPA–induced serotonin depletion, (c) DEPLETION OF SEROTONIN AND 18F-MPPF BINDING • Zimmer et al. 1495 18 the dynamic F-MPPF binding using a new intracerebral medium ϩ 2.2 mmol/L CaCl2)at1.0␮L/min using a microsyringe ␤-sensitive detector (the ␤-microprobe) in control rats in pump. Body temperature was maintained at 37°C Ϯ 1°C through- comparison with p-EPA–treated rats, and (d) the extracel- out the test period using a thermostatically controlled heating lular free 18F-MPPF using microdialysis during this phar- blanket (CMA/Microdialysis). After a 2-h equilibrium period, 10- macologic challenge. Finally, we discussed several mecha- min dialysate samples were collected and were defined as basal level before drug (p-EPA, 5 mg/kg intraperitoneally) or saline nisms underlying the sensitivity of 18F-MPPF to serotonin injection (control rats). The serotonin content in dialysates was depletion. assayed using HPLC with an electrochemical detector (Antec Leyden). We used a C18 reverse-phase column (Uptisphere ODB, MATERIALS AND METHODS 3 ␮m, 100 ϫ 2 mm; Interchim); the mobile phase, delivered at a rate of 0.3 mL/min, was composed of 75 mmol/L NaH2PO4, 0.1 Drugs mmol/L ethylenediaminetetraacetic acid, 0.3 mmol/L octanesulfo- Free p-EPA was synthesized as described (16), and the chemical nic acid, and 18% methanol (pH 4.3). In these conditions, the Ͼ purity of the final compound was 99%. Briefly, p-EPA was retention time for serotonin was 5.0 min, and its detection limit produced by the Heck reaction of trimethylsilylacetylene with was 0.5 pg per sample (10 ␮L). N-tert-butyloxy-carbonyl-4-iodo-L-phenylalanine methyl ester fol- lowed by removal of the protecting groups. The p-EPA was Determination of 18F-MPPF Binding dissolved in saline before animal injections. The ␤-microprobe apparatus, previously named “SIC” (19), is 18F-MPPF was synthesized with a radiochemical yield of 25% the prototype of a ␤-sensitive microprobe stereotactically im- (decay corrected) in an automated synthesizer (17), using the planted in the rat brain, designed and manufactured by the Institut chemical pathway previously described (8). Chemical and radio- of Physique Nucle´aire (Orsay, France). The sensitive end of the chemical purity were Ͼ98% as determined by high-performance probe consists of a 1-mm-length and 1-mm-diameter plastic scin- liquid chromatography (HPLC). Specific activity from the injected tillating fiber, allowing definition of the detection volume sur- radiotracer ranged from 74 ϫ 103 MBq/␮mol to 148 ϫ 103 rounding the probe. The detection tip is coupled to a single-photon MBq/␮mol (2–4 Ci/␮mol). counting photomultiplier (R7400P; Hamamatsu). An interface module ensures the readout of the photomultiplier signal through Animal Procedures an amplifier integrator and the radiotracer kinetics are visualized in A total of 44 male Sprague–Dawley rats (Elevage De´pre´) real time. weighing 300–400 g were used in this study. All experimental After rat anesthesia and catheterization of the tail vein, 1 ␤-mi- procedures were in compliance with European Economic Com- croprobe was implanted in the hippocampus, and the second was mission guidelines and directives (86/09/EEC). During all exper- implanted in the cerebellum. The coordinates of implantation were iments, the rats were anesthetized by a single intraperitoneal in- as follows: A/P Ϫ5.0, L/M 5.0, and V/D Ϫ8.0 (hippocampus); A/P jection of urethane (Sigma-Aldrich) at a dose of 1.7 g/kg body Ϫ12.0, L/M 3.0, and V/D Ϫ4.0 (cerebellum), from the bregma weight and remained anesthetized throughout all procedures. A point and the dura, respectively. Body temperature was maintained catheter was inserted in the tail vein, allowing the injection of the at 37°C Ϯ 1°C throughout the test period using a thermostatically radioactive tracer (18F-MPPF). After all experiments, the rats were controlled heating blanket (CMA/Microdialysis). sacrificed by a KCl intravenous injection. ␤-Microprobe acquisition was performed 2 h after implantation of the probes according to our previous studies (11,19). This time 18 F-MPPF Tissue Distribution period corresponds with the neurotransmission stabilization period Nine anesthetized rats were injected intraperitoneally with p- (20). For each acquisition, 37 MBq 18F-MPPF (in a volume of 0.4 EPA (5 mg/kg) and 9 were injected with saline (control rats). Four mL saline) were injected via the tail vein over a 45-s period. This hours after p-EPA (or saline) injection, each rat received a 37-MBq activity corresponded to a stable content of 250–500 pmol. The 18 F-MPPF intravenous injection. The rats were killed by decapi- time course of radioactivity was studied for 90 min using 10-s time 18 ϭ tation at 30, 45, and 60 min after F-MPPF injection (n 3 per integration acquisition. time for p-EPA–treated and control rats). The brains were carefully In a first group of rats, each anesthetized rat received a p-EPA removed and immediately dissected on an ice-cooled glass plate. injection (5 mg/kg intraperitoneally in 0.3 mL of saline) followed The hippocampus and the cerebellum were dissected free-hand. at4hbya37-MBq 18F-MPPF injection. The 18F-MPPF binding The dissected tissues were blotted and rinsed with water to re- was measured with the ␤-microprobe in the hippocampus and the moved adhering blood and placed in preweighed counting vials.