Journal of Molecular Neuroscience 105 Volume 30, 2006 *Author to whom all correspondence and reprint requests should be addressed. E-mail: bmolles@pasteur.fr Targeted In Vivo Expression of Nicotinic Acetylcholine Receptors in Mouse Brain Using Lentiviral Expression Vectors B. E. Molles,* ,1 U. Maskos, 1 S. Pons, 1 M. Besson, 1,2 B. P. Guiard, 2 J.-P. Guilloux, 2 A. Evrard, 1 A. Cormier, 1 M. Mameli-Engvall, 1 I. Cloëz-Tayarani, 1 H. Nakatani, 1 N. Dufour, 4 A.-P. Bemelmans, 4 J. Mallet, 4 P. Cazala, 3 A. M. Gardier, 2 V. David, 3 P. Faure, 1 S. Granon, 1 and J.-P. Changeux 1 1 Unité Récepteurs et Cognition, Institut Pasteur, 75724 Paris Cedex 15, France; 2 Laboratoire de Neuropharmacologie EA3544, Faculté de Pharmacie, University Paris-Sud, 92296 Châtenay- Malabry Cedex, France; 3 Laboratoire de Neurosciences Cognitives, University de Bordeaux-I, 33405 Talence Cedex, France; and 4 Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégéneratifs (LGN), Hôpital de la Pitié-Salpêtrière, 75013 Paris, France Introduction Nicotinic acetylcholine receptors (nAChRs) in the brain exhibit diverse functional properties and ubiq- uitous distribution. Yet, except for providing a recep- tor for the exogenously applied nicotine of tobacco products, their role in the normal functioning of the brain has remained elusive. We have used a lenti- viral expression vector to re-express the 2 subunit specifically in the ventral tegmental area (VTA) of 2 –/– mice. The viral vector efficiently expresses 2- subunit protein leading to new nAChR-binding sites. VTAneurons transduced by the lentiviral vector are responsive to intravenous nicotine when analyzed using in vivo electrophysiology. Nicotine-induced dopamine release from the nucleus accumbens (NuAcc) was also restored in re-expressing 2 –/– mice. Intra-VTA injection of nicotine was found to be reinforcing in both wild-type and 2-subunit re- expressing 2 –/– mice, but not in 2 –/– mice. Fur- thermore, in the absence of applied nicotine, the spontaneous slow exploratory behavior of the mice was restored, whereas fast navigation did not change. This latter behavioral analysis suggests a role for 2* nAChR, specifically expressed in the VTA, in mam- malian cognitive function. Journal of Molecular Neuroscience Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN0895-8696/06/30:105–106/$30.00 JMN (Online)ISSN 1559-1166 DOI 10.1385/JMN/30:1-2:105 ORIGINAL ARTICLE Results and Discussion Lentiviral-based expression vectors (Naldini et al., 1996) used in the present work were based on the previously described pTRIP U3 (Sirven et al., 2001), with the phosphoglycerate kinase promoter used to drive expression of the transgene. Our lenti- viral expression vector contains a bicistronic cassette (Maskos et al., 2005) simultaneously expressing the mouse 2 subunit and the enhanced green fluores- cent protein (eGFP) using an intervening internal ribosome entry site sequence (Clontech). Lentivec- tors were stereotaxically injected into the VTA of both wild-type and 2 –/– mouse brains. One month after injection of lentivector, GFP fluorescence is observed in both dopaminergic and nondopamin- ergic VTA neurons (Maskos et al., 2005). [ 125 I]Epiba- tidine autoradiography on brain slices (Zoli et al., 1998) reveals the presence of re-expressed high-affin- ity nAChR at the injection site in the VTA, as well as in the mesolimbic projection, the axon terminals in the NuAcc, and in some, but not all of, the other areas receiving projections from the VTA. 2 –/– mice injected with 2+eGFP-expressing lentivector (VEC mice) were then compared with wild-type (WT) or 2 –/– (KO) mice that had been