Journal of Neuroscience Methods 194 (2010) 21–27 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing Iciar P. López a,1 , Pascal Salin b,1 , Philippe Kachidian b , Pedro Barroso-Chinea a , Alberto J. Rico a , Virginia Gómez-Bautista a , Lorena Conte-Perales a , Patrice Coulon c , Lydia Kerkerian-Le Goff b , José L. Lanciego a,∗ a Area de Neurociencias, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra and Centro de Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Pamplona, Spain b Developmental Biology Institute of Marseille-Luminy, UMR 6216 CNRS-Université de la Méditerranée, Marseille, France c Laboratoire de la Plasticité et Physio-Pathologie de la Motricité, UMR 6196 CNRS-Université de la Méditerranée, Marseille, France article info Article history: Received 30 September 2009 Received in revised form 12 January 2010 Accepted 13 January 2010 Keywords: Trans-synaptic tracing Dendritic spines PHA-L BDA Confocal microscope abstract Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of con- nected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibod- ies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris- leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further char- acterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The inherent complexity of brain circuits often requires imple- menting demanding multiple tracing paradigms (for a review, see Lanciego and Wouterlood, 2006; Lanciego et al., 2000). In such studies and regardless of the experimental design, it is ultimately necessary to unequivocally determine whether or not a given axon terminal contacts a post-synaptic element. Anterograde tracers define individual fibers, varicosities and axon terminals well, such as PHA-L (Gerfen and Sawchenko, 1984) and BDA (Veenman et al., 1992) and therefore, pre-synaptic elements can be adequately iden- tified. However, the currently available retrograde tracers are not so effective. Indeed, unless demanding procedures like intracellu- ∗ Corresponding author at: Basal Ganglia Neuromorphology Laboratory, Neuro- sciences Division, Center for Applied Medical Research, University of Navarra, Spain. Tel.: +34 948 194 700x2002; fax: +34 948 194 715. E-mail address: jlanciego@unav.es (J.L. Lanciego). 1 These authors participated equally in this work. lar injections are undertaken (Buhl and Lubke, 1989), the kind of labeling one might expect from retrograde tracing of projection neurons is at best, a granular-like accumulation of the retrograde marker within the cell soma, and sometimes, the thickest main dendrites. In other words, retrograde tracers fail to define the post- synaptic element, even when using the most sensitive tracers such as Fluoro-Gold (Schmued and Fallon, 1986). When retrograde trac- ers are combined with anterograde tracers it is possible to visualize areas that overlap between anterogradely labeled terminal fields and retrogradely labeled neurons, even though defining presump- tive contacts is frequently hampered by the failure of accurately elucidate the post-synaptic element. However, this can be over- come by employing intracellular filling of neurons with Lucifer Yellow combined with either BDA or PHA-L tracing and electron microscopy (Wouterlood et al., 1990, 1992; Jorritsma-Byham et al., 1994). In summary, the detailed analysis of anatomical interactions in neuronal circuits requires the use of retrograde tracers to ensure labeled neurons are visualized in their entirety in a Golgi-like fashion, which must also be compatible with other existing tools 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.01.015