Segregated pathways to the vomeronasal amygdala: differential projections from the anterior and posterior divisions of the accessory olfactory bulb Alicia Mohedano-Moriano, 1, * Palma Pro-Sistiaga, 1, * Isabel U ´ beda-Ban ˜o ´n, 1 Carlos Crespo, 2 Ricardo Insausti 1 and Alino Martinez-Marcos 1 1 Laboratorio de Neuroanatomı ´a Humana, Departamento de Ciencias Me ´ dicas, Facultad de Medicina, Centro Regional de Investigaciones Biome ´ dicas, Universidad de Castilla-La Mancha, 02006 Albacete, Spain 2 Departamento de Biologı ´a Celular, Facultad de Ciencias Biolo ´ gicas, Universidad de Valencia, 46100 Burjassot, Spain Keywords: G-protein-coupled receptor, pheromone, rat, vomeronasal receptor Abstract Apically and basally located receptor neurons in the vomeronasal sensory epithelium express G i2a - and G oa -proteins, V1R and V2R vomeronasal receptors, project to the anterior and posterior accessory olfactory bulb and respond to different stimuli, respectively. The extent to which secondary projections from the two portions of the accessory olfactory bulb are convergent in the vomeronasal amygdala is controversial. This issue is addressed by using anterograde and retrograde tract-tracing methods in rats including electron microscopy. Injections of dextran-amines, Fluoro Gold, cholera toxin-B subunit and Fast Blue were delivered to the anterior and posterior accessory olfactory bulb, bed nucleus of the stria terminalis, dorsal anterior amygdala and bed nucleus of the accessory olfactory tract ⁄ anteroventral medial amygdaloid nucleus. We have demonstrated that, apart from common vomeronasal-recipient areas, only the anterior accessory olfactory bulb projects to the bed nucleus of the stria terminalis, medial division, posteromedial part, and only the posterior accessory olfactory bulb projects to the dorsal anterior amygdala and deep cell layers of the bed nucleus of the accessory olfactory tract and the anteroventral medial amygdaloid nucleus. These results provide evidence that, excluding areas of convergence, the V1R and V2R vomeronasal pathways project to specific areas of the amygdala. These two vomeronasal subsystems are therefore anatomically and functionally separated in the telencephalon. Introduction Parallel to the olfactory system, most mammals possess a vomeronasal system that includes a vomeronasal organ, an accessory olfactory bulb (AOB) and some vomeronasal-recipient amygdaloid structures (Halp- ern & Martinez-Marcos, 2003). The olfactory system is able to sense a large number of odorants (Malnic et al., 1999), including some volatile substances that arise from pheromones (Lin et al., 2004); whereas the vomeronasal system detects mainly (Luo et al., 2003), but not exclusively, pheromones (Restrepo et al., 2004; Baxi et al., 2006). In many mammals, the vomeronasal system is anatomically and functionally dichotomous (Halpern et al., 1998a,b). Two classes of vomeronasal receptor neurons, apically and basally placed in the sensory epithelium, express G i2a - and G oa -proteins (Berghard & Buck, 1996; Jia & Halpern, 1996), V1R and V2R vomeronasal receptors (Dulac & Axel, 1995; Herrada & Dulac, 1997; Matsunami & Buck, 1997; Ryba & Tirindelli, 1997), respond to different stimuli (Del Punta et al., 2002a) and project to specific glomeruli in the anterior and posterior AOB (Belluscio et al., 1999; Rodriguez et al., 1999), respectively. The two portions of the AOB are also functionally different (Sugai et al., 1997). The multiple apical dendrites of mitral- tufted cells tend to restrict their distribution to glomeruli innervated by axons of sensory neurons expressing the same vomeronasal receptor within one of the divisions of the AOB (Jia & Halpern, 1997; Del Punta et al., 2002b; Wagner et al., 2006). How this segregated information is transferred to the vomeronasal amygdala is a matter of controversy. In opossums, the posterior AOB receives differential centrifugal inputs from the posteromedial cortical amygdaloid nucleus (PMCo; Martinez-Marcos & Halpern, 1999a). Also in opossums, differential projections from the posterior AOB to deep cell layers of the medial amygdaloid complex (Me) have been reported (Martinez-Marcos & Halpern, 1999b). Anterograde (von Campenhausen & Mori, 2000) and retrograde (Salazar & Brennan, 2001) tracing studies, however, have not revealed divergent projections from the two portions of the AOB in mice. In the present report we show, for the first time, vomeronasal- recipient structures in the telencephalon of rats receiving specific inputs from the anterior and posterior divisions of the AOB. Accordingly, apart from a common vomeronasal pathway, two separate pathways can be traced from V1R and V2R receptors to given areas in the vomeronasal amygdala. Materials and methods Thirty-six adult male and female Sprague–Dawley rats from the University Hospital of Albacete were used in the present study. Animals were maintained under veterinary supervision in a 12 h light : dark Correspondence: Dr A. Martinez-Marcos, as above. E-mail: Alino.Martinez@uclm.es *A.M.-M. and P.P.-S. have equally contributed to this work. Received 24 July 2006, revised 6 February 2007, accepted 8 February 2007 European Journal of Neuroscience, Vol. 25, pp. 2065–2080, 2007 doi:10.1111/j.1460-9568.2007.05472.x ª The Authors (2007). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd European Journal of Neuroscience