Finally our results suggest that R may
Finally, our results suggest that σ2R may, at least partially, mediate the hunger-suppressive action of amphetamine by interacting with orexigenic receptors in CRF2R-OX1R heteromer contexts. Despite extensive evidence supporting the formation of GPCR oligomers in heterologous systems, the lack of appropriate methodology makes controversial their existence in their native environment. A specific pharmacological property for the heteroreceptor complex is critical for identify such signaling complexes in native tissue. In the present study, we observed that Orexin A caused an increase of extracellular dopamine and glutamate levels in the VTA that was counteracted by a previous VTA infusion of a CRF2R selective antagonist. These microdialysis experiments demonstrate the negative cross-antagonism previously described in vitro and confirm that this dimeric entity is functionally relevant in vivo. To our knowledge, this is the first report showing in vivo GPCR heteromer identification by cross-antagonism. Thus, the present study conveys a novel approach to add to those that allow identification of GPCR heteromers in natural sources (Franco et al., 2016).
Materials and methods
Acknowledgements Supported by CiberNed’s intramural program (Ref. No. PI2016/02) and by grants from Spanish Ministerio de Economía, Industria y Competitividad (BFU2015-64405-R, and SAF2017-84117-R; they may include FEDER funds).
Introduction Corticotropin-releasing factor (CRF) is a hypothalamic neurohormone, but also an extrahypothalamic neurotransmitter, that regulates the neuroendocrine, autonomic and behavioral stress reponses (Bale et al., 2002, Bale and Vale, 2004, Vale et al., 1981). The actions of CRF are mediated by two distinct G protein-coupled receptors, CRF receptor type 1 (CRF1) and CRF receptor type 2 (CRF2) (Chang et al., 1993, Lovenberg et al., 1995). CRF1 is expressed abundantly in the central nervous system (CNS), including the cerebral cortex, lrrk2 and striatum (Van Pett et al., 2000). CRF2 is expressed predominantly in the periphery, and limited centrally to subcortical regions, such as the hypothalamus, hippocampus and amygdala (Van Pett et al., 2000). Originally, it was suggested that CRF1 and CRF2 mediate antagonistic effects in the CNS, since stimulation of CRF1 provoked activation of the HPA axis, anxiety and depression, and increase of locomotor activity (at least in a familial environment), whereas stimulation of CRF2 evoked anxiolytic and antidepressant effects, and decrease of locomotor activity (Bale et al., 2002, Bale and Vale, 2004, Vale et al., 1981). Recently, it was demonstrated that the role of CRF receptors in the stress responses is not a matter of simple dualism, but it depends upon the brain regions and neuron populations being activated (Henckens et al., 2016, Janssen and Kozicz, 2013). Nicotine is the main psychoactive component of tobacco that causes addiction. Besides the regulation of the stress responses, CRF has been implicated in nicotine addiction based on several lines of evidence (Bruijnzeel and Gold, 2005, Sarnyai et al., 2001). First, acute administration of nicotine, like any other stressor, evokes a dose-dependent activation of the hypothalamic–pituitary–adrenal (HPA) axis that is initiated by hypothalamic CRF (Bruijnzeel and Gold, 2005, Sarnyai et al., 2001). Second, nicotine withdrawal syndrome resembles the behavioral stress response that is mediated by extrahypothalamic CRF (Bruijnzeel and Gold, 2005, Sarnyai et al., 2001). Third, exposure to stressors is one of the leading causes of nicotine relapse (Bruijnzeel and Gold, 2005, Sarnyai et al., 2001). Finally, both CRF receptors participate to the acute, chronic and withdrawal actions of nicotine (Bruijnzeel et al., 2009, Bruijnzeel, 2012, George et al., 2007, Kamdi et al., 2009, Marcinkiewcz et al., 2009). The actions of nicotine are mediated by nicotinic acetylcholine receptors (nAchRs) that are considered ligand-gated ion channels composed of pentameric combinations of α and β subunits, since normally they respond to acetycholine and allow natrium or calcium ions to enter the cells (Benowitz, 2010). Based on their primary sites of expression, nAchRs are classified into two subtypes: muscle-type nicotinic receptors found in neuromuscular junctions and neuronal-type nicotinic receptors found on neuronal bodies and nerve terminals (Benowitz, 2010). The most abundant neuronal nAchRs are α4β2, α3β4 and α7 located both pre- and postsynaptically where they can influence the release of other neurotransmitters, such as dopamine, glutamate and GABA (Benowitz, 2010).