Research Paper The habenula as a critical node in chronic stress-related anxiety Luis R. Jacinto, Rui Mata, Ashley Novais, Fernanda Marques, Nuno Sousa ⁎ Life and Health Sciences Research Institute, School of Medicine, University of Minho, Braga, Portugal ICVS/3B's – PT Government Associate Laboratory, Braga/Guimarães, Portugal abstract article info Article history: Received 13 August 2016 Received in revised form 2 December 2016 Accepted 5 December 2016 Available online 7 December 2016 The habenula is activated in response to stressful and aversive events, resulting in exploratory inhibition. Al- though possible mechanisms for habenula activation have been proposed, the effects of chronic stress on the habenular structure have never been studied. Herein, we assessed changes in volume, cell density and dendritic structure of habenular cells after chronic stress exposure using stereological and 3D morphological analysis. This study shows for the first time that there is a hemispherical asymmetry in the medial habenula (MHb) of the adult rat, with the right MHbcontaining more neurons than its left counterpart. Additionally, it shows that chronic stress induces a bilateral atrophy of both the MHb and the lateral habenula (LHb). This atrophy was accompanied by a reduction of the number of neurons in the right MHb and the number of glial cells in the bilateral LHb, but not by changes in the dendritic arbors of multipolar neurons. Importantly, these structural changes were correlated with elevated levels of serum corticosterone and increased anxious-like behavior in stressed animals. To further assess the role of the habenula in stress-related anxiety, bilateral lesions of the LHb were performed; interesting- ly, in lesioned animals the chronic stress protocol did not trigger increases in circulating corticosterone or anx- ious-like behavior. This study highlights the role of the habenula in the stress responses and how its sub- regions are structurally impacted by chronic stress with physiological and behavioral consequences. © 2016 Published by Elsevier Inc. Keywords: Habenula Stress Anxiety Stereology Corticosterone 1. Introduction The habenula is a small bilateral brain region that together with the pineal gland forms the epithalamus and is phylogenetically preserved in almost all vertebrates (Andres et al., 1999; Stephenson-Jones et al., 2012). The habenular nuclei, which consist of the medial and lateral sub-regions (MHb and LHb, respectively), have been described as a relay interface between the basal ganglia and the limbic system since forebrain projections from both converge there (Herkenham and Nauta, 1977) and are able to modulate downstream limbic midbrain do- pamine and serotonin circuits (Hikosaka et al., 2008; Hikosaka 2010; Stephenson-Jones et al., 2012; Zhao et al., 2015). This modulation can both promote or inhibit release of dopamine and serotonin in the brain since the habenula is strongly connected with the ventral tegmen- tal area (VTA) – via the rostromedial tegmental nucleus (rMTG) – and the raphe nucleus (RN) – via the interpeduncular nucleus (IPN) (Christoph et al., 1986; Ferraro et al., 1996; Lecourtier et al., 2008; Zhao et al., 2015) –, the major sources of dopaminergic and serotonergic projections in the brain, respectively (Vertes, 1991; Russo and Nestler, 2013). Although the LHb appears to be the only habenula sub-region ca- pable of directly controlling dopamine release, serotonergic modulation is likely more complex as both structures exert influence on the IPN – the MHb directly and the LHb indirectly – and the RN (Hikosaka et al., 2008; Hikosaka, 2010; Stephenson-Jones et al., 2012; Zhao et al., 2015), suggesting that the combined output of both structures is partic- ularly critical for the serotonergic system. Moreover, the MHb also pro- jects to the LHb (Kim and Chang, 2005) and both sub-structures share several efferent inputs (Herkenham and Nauta, 1977) further highlight- ing the complexity of the combined net output of the habenula This crit- ical modulatory role of the monoamines circuits has been linked to several different behaviors and conditions (Hikosaka, 2010; Fakhoury and López, 2014; Proulx et al., 2014), therefore attracting increased at- tention from researchers focusing on mood disorders, depression and stress (Hikosaka, 2010). The particular involvement of the habenula in the stress response was initially attributed to the observation that habenular neurons were activated by a variety of stressors (Wirtshafterm et al., 1994) and aversive/unpleasant stimuli including both the presence of punish- ment and the absence of reward (Matsumoto and Hikosak, 2009). Addi- tionally, stimulation of the habenula produced autonomic responses similar to those observed in emotional stress (Ootsuka and Mohammed, 2015) and LHb activity was correlated with behavioral in- hibition in normal and stressful contexts (Lee and Huang, 1988). This in- hibitory role has been linked to the modulation of dopamine and serotonin release in the brain and has been additionally shown in sever- al lesion (Murphy et al., 1996; Yang et al., 2008; Tian and Uchida, 2015), Experimental Neurology 289 (2017) 46–54 ⁎ Corresponding author at: Life and Health Sciences Research Institute (ICVS), School of Medicine, Universidade do Minho, Campus de Gualtar, 4710-103 Braga, Portugal. E-mail address: njcsousa@med.uminho.pt (N. Sousa). http://dx.doi.org/10.1016/j.expneurol.2016.12.003 0014-4886/© 2016 Published by Elsevier Inc. Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr