Lateral Habenula and the Rostromedial Tegmental Nucleus Innervate Neurochemically Distinct Subdivisions of the Dorsal Raphe Nucleus in the Rat Chemutai Sego, 1,2 Luciano Gonc ¸alves, 1,3 Leandro Lima, 1 Isadora C. Furigo, 1 Jose Donato Jr, 1 and Martin Metzger 1 * 1 Department of Physiology & Biophysics, Institute of Biomedical Sciences, University of S~ ao Paulo, 05508-900 S~ ao Paulo, Brazil 2 Department of Anatomy, Institute of Biomedical Sciences, University of S~ ao Paulo, 05508-900 S~ ao Paulo, Brazil 3 Department of Human Anatomy, Federal University of the Tri^ angulo Mineiro, 38025-180 Uberaba, Brazil ABSTRACT The lateral habenula (LHb) is an epithalamic structure differentiated in a medial (LHbM) and a lateral division (LHbL). Together with the rostromedial tegmental nucleus (RMTg), the LHb has been implicated in the processing of aversive stimuli and inhibitory control of monoamine nuclei. The inhibitory LHb influence on mid- brain dopamine neurons has been shown to be mainly mediated by the RMTg, a mostly GABAergic nucleus that receives a dominant input from the LHbL. Interestingly, the RMTg also projects to the dorsal raphe nucleus (DR), which also receives direct LHb projections. To compare the organization and transmitter phenotype of LHb pro- jections to the DR, direct and indirect via the RMTg, we first placed injections of the anterograde tracer Phaseo- lus vulgaris leucoagglutinin into the LHb or the RMTg. We then confirmed our findings by retrograde tracing and investigated a possible GABAergic phenotype of DR- projecting RMTg neurons by combining retrograde trac- ing with in situ hybridization for GAD67. We found only moderate direct LHb projections to the DR, which mainly emerged from the LHbM and were predominantly directed to the serotonin-rich caudal DR. In contrast, RMTg projections to the DR were more robust, emerged from RMTg neurons enriched in GAD67 mRNA, and were focally directed to a distinctive DR subdivision immuno- histochemically characterized as poor in serotonin and enriched in presumptive glutamatergic neurons. Thus, besides its well-acknowledged role as a GABAergic con- trol center for the ventral tegmental area (VTA)–nigra complex, our findings indicate that the RMTg is also a major GABAergic relay between the LHb and the DR. J. Comp. Neurol. 522:1454–1484, 2014. V C 2013 Wiley Periodicals, Inc. INDEXING TERMS: Raphe nuclei; serotonin, glutamate, anxiety; reward; stress The lateral habenula (LHb) and the newly discovered rostromedial tegmental nucleus (RMTg; Jhou et al., 2009a,b), also known as the tail of the ventral tegmen- tal area (tVTA; Kaufling et al., 2009), are now consid- ered key components of a brain circuit implicated in the inhibitory control of monoamine nuclei (Hikosaka, 2010; Lavezzi and Zahm, 2011; Bourdy and Barrot, 2012). The LHb is an evolutionarily conserved epithala- mic structure (Stephenson-Jones, 2012) composed of 10 distinct subnuclei (Andres et al., 1999; Geisler et al., 2003). Nowadays, it is well established that LHb neurons encode disappointment and expectation of negative conditions (Ullsperger and von Cramon, 2003; Shepard et al., 2006) and are primarily excited by reward omission and aversive stimuli and outcomes (Matsumoto and Hikosaka, 2007, 2009). Consistent with a role in encoding disappointments, the LHb has been implicated in a broad array of functions and path- ologic conditions (for reviews, see Lecourtier and Kelly, 2007; Hikosaka et al., 2008; Hikosaka, 2010; Shelton et al., 2012). Notably, several studies point to a promi- nent role of the LHb in mechanisms of stress and pain, as well as in major depressive disorder (Caldecott- Grant sponsor: FAPESP; Grant numbers: 2012/02388-3; 2011/ 03292-7; 2010/18086-0; Grant sponsor: CNPq; Grant numbers: 14199/2012-7; 143177/2008-7. *CORRESPONDENCE TO: Martin Metzger, PhD, Dept. of Physiology & Biophysics, Institute of Biomedical Sciences I, University of S~ ao Paulo, USP, Av. Prof. Lineu Prestes 1524, 05508-900 S~ ao Paulo, SP, Brazil. E-mail: metzger@icb.usp.br Received May 7, 2013; Revised December 23, 2013; Accepted December 23, 2013. DOI 10.1002/cne.23533 Published online December 28, 2013 in Wiley Online Library (wileyonlinelibrary.com) V C 2013 Wiley Periodicals, Inc. 1454 The Journal of Comparative Neurology | Research in Systems Neuroscience 522:1454–1484 (2014) RESEARCH ARTICLE