UNCHANGED DENSITY OF 5-HT 1A AUTORECEPTORS ON THE PLASMA MEMBRANE OF NUCLEUS RAPHE DORSALIS NEURONS IN RATS CHRONICALLY TREATED WITH FLUOXETINE M. RIAD, a L. RBAH, b,c M. VERDURAND, b,c N. AZNAVOUR, c L. ZIMMER b,c AND L. DESCARRIES a * a Departments of Pathology and Cell Biology and of Physiology, and Groupe de recherche sur le système nerveux central, Faculty of Med- icine, Université de Montréal, CP 6128, Succursale Centre-ville, Mon- treal, QC, Canada H3C 3J7 b Laboratory of Neuropharmacology FRE CNRS 3006, Faculty of Phar- macy, Université Lyon 1, Lyon, France c PET Department, CERMEP, 59 Bd Pinel, Lyon, France Abstract—5-HT 1A autoreceptors regulate the firing of 5-HT neu- rons and their release of 5-HT. In previous immuno-electron microscopic studies, we have demonstrated an internalization of 5-HT 1A autoreceptors in the nucleus raphe dorsalis (NRD) of rats, after the acute administration of a single dose of the specific agonist 8-hydroxy-2-(di-n-propylamine)tetralin (8-OH- DPAT) or of the selective 5-HT reuptake inhibitor, fluoxetine. Twenty-four hours after either treatment, the receptors were back in normal density on the plasma membrane of NRD neurons. Here, we examined the subcellular localization of these receptors and the in vivo binding of the 5-HT 1A radio- ligand 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluorobenz- amido]ethylpiperazine labeled with [ 18 F]fluorine ([ 18 F]MPPF) after chronic fluoxetine treatment (10 mg/kg daily for 3 weeks, by minipump). Unexpectedly, after such a treatment, there were no more differences between treated and control rats in either the density of plasma membrane labeling of NRD den- drites, or in the in vivo binding of [ 18 F]MPPF, as measured with -microprobes. This was in keeping with earlier reports of an unchanged density of 5-HT 1A receptor binding sites after chronic fluoxetine treatment, but quite unexpected from the strong electrophysiological and biochemical evidence for a desensitization of 5-HT 1A autoreceptors under such conditions. Indeed, when the fluoxetine-treated rats were challenged with a single dose of 8-OH-DPAT, there was no internalization of the 5-HT 1A autoreceptors, at variance with the controls. Interestingly, several laboratories have reported an uncoupling of 5-HT 1A autoreceptors from their G protein in the NRD of rats chronically treated with fluox- etine. Therefore, the best explanation for our results is that, after repeated internalization and retargeting, functional 5-HT 1A autoreceptors are replaced by receptors uncoupled from their G protein on the plasma membrane of NRD 5-HT neurons. Thus, the regulatory function of these autorecep- tors may depend on a dynamic balance among their produc- tion, activation, internalization and recycling to the plasma membrane in inactivated (desensitized) form. © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: fluoxetine, 5-HT 1A autoreceptors, desensitization, internalization, immunogold, [ 18 F]MPPF. There is considerable pre-clinical and clinical evidence implicating the brain 5-HT systems in the etiopathology of mood disorders (Mann, 1999; Naughton et al., 2000) and demonstrating that an increase in 5-HT neurotransmission underlies the efficacy of a variety of antidepressant treat- ments (reviewed in Blier et al., 1994). Selective 5-HT re- uptake inhibitors (SSRIs), which block the neuronal plasma membrane 5-HT transporter and hence increase extracellular 5-HT in the nucleus raphe dorsalis (NRD) and its territories of projection, have thus become the most frequently prescribed class of drugs in the treatment of major depression (Fuller, 1995). Upon acute administra- tion, however, SSRIs inhibit the activity of 5-HT neurons through the activation of somatodendritic 5-HT 1A autore- ceptors that normally regulate the firing of 5-HT neurons and their release of 5-HT (Gardier et al., 1996). It is only following chronic SSRI administration that 5-HT neurons gradually recover their normal firing rate and 5-HT release as a result of 5-HT 1A autoreceptor desensitization (Cza- chura and Rasmussen, 2000; Albert and Lemonde, 2004). The 2– 4 weeks’ delay in obtaining a clinically effective therapeutic response with SSRIs is believed to be due in large part to adaptive changes taking place in 5-HT system and notably to the time required for achieving this desen- sitization of somatodendritic 5-HT 1A autoreceptors (Artigas et al., 1996). The mechanism by which desensitization of 5-HT 1A autoreceptors (but not heteroreceptors) occurs is not en- tirely clear. Some of the above and other in vivo animal studies have shown that there is no change in the density of 5-HT 1A receptor (5-HT 1A R) binding sites in the NRD following chronic fluoxetine treatment (Le Poul et al., 1995; Li et al., 1996; Hervás and Artigas, 1998; Raap et al., 1999). Using immuno-electron microscopy with specific 5-HT 1A R antibodies, combined to -microprobe measure- ment of the in vivo binding of the 5-HT 1A R radioligand [ 18 F]MPFF (Aznavour and Zimmer, 2007), we have previ- ously demonstrated that, within 1 h after acute treatment with the specific agonist 8-hydroxy-2-(di-n-propylamine) tetralin (8-OH-DPAT) (0.5 mg kg i.v. or i.p.) or with the SSRI fluoxetine (10 mg/kg i.p.), there is a significant decrease in the density of 5-HT 1A R on the plasma membrane of NRD dendrites (internalization), paralleled by a decrease of the in vivo binding of [ 18 F]fluorine-labeled 4,2-(methoxyphenyl)- 1-[2-(N-2-pyridinyl)-p-fluorobenzamido]ethyllpiperazine *Corresponding author. Tel: +1-514-343-7070; fax: +1-514-343-5755. E-mail address: laurent.descarries@umontreal.ca (L. Descarries). Abbreviations: NRD, nucleus raphe dorsalis; PB, phosphate buffer; PBS, phosphate-buffered saline; SSRI, selective 5-HT reuptake inhibitor; 5-HT 1A R, 5-HT 1A receptor; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamine) tetralin; [ 18 F]MPPF, 4,2-(methoxyphenyl)-1-[2-(N-2-pyridinyl)-p-fluoro- benzamido]ethylpiperazine labeled with [ 18 F]fluorine. Neuroscience 151 (2008) 692–700 0306-4522/08$32.00+0.00 © 2007 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.024 692