Expression of mu opioid receptor in dorsal diencephalic conduction system: New insights for the medial habenula
Introduction
The dorsal diencephalic conduction (DDC) is a highly conserved pathway present in all vertebrates, which interconnects limbic forebrain structures (septum, pallidum, striatum, lateral hypothalamus) to mid- and hindbrain regions including raphe and tegmental nuclei, locus coeruleus, ventral tegmental area (VTA) and the interpeduncular nucleus (IPN). The habenular complex (Hb) is central to DDC (Sutherland, 1982, Bianco and Wilson, 2009). The key role of this highly conserved epithalamic structure in integrating cognitive with emotional and sensory processing has raised increasing interest (Klemm, 2004, Lecourtier and Kelly, 2007, Hikosaka et al., 2008, Ikemoto, 2010, Darcq et al., 2011, Goutagny et al., 2013, Lee and Goto, 2013), and Hb contribution to motivational processes and value-based decision-making has been established (Hikosaka, 2010). The Hb comprises two nuclei, the medial habenula (MHb) and the lateral habenula (LHb), which show distinct anatomy and connectivity within brain networks, and there is some evidence of interconnections between the two nuclei (Sutherland, 1982, Kim and Chang, 2005). The LHb, which has pallidal and hypothalamic afferences and mainly projects to midbrain and hindbrain structures such as the raphe nuclei and VTA (Lecourtier and Kelly, 2007, Jhou et al., 2009, Kaufling et al., 2009, Kim, 2009), was shown involved in aversion and behavioral avoidance (Lammel et al., 2012, Stamatakis and Stuber, 2012, Ilango et al., 2013). The MHb receives septal inputs and projects primarily to the IPN, and has been less studied (Viswanath et al., 2013). This nucleus was proposed to regulate inhibitory controls, cognition-dependent executive functions, place aversion learning (Darcq et al., 2011, Kobayashi et al., 2013) and the nicotine withdrawal syndrome (Dani and De Biasi, 2013, Kobayashi et al., 2013, Dao et al., 2014). A recent report showed that post-natal ablation of MHb cells in transgenic mice induces an abnormal phenotype, characterized by impulsive and compulsive behavior, environmental maladaptation and learning deficits (Kobayashi et al., 2013), underscoring functional implication of this structure in a broad range of behaviors.
MHb expresses remarkably high levels of mu opioid receptors (MORs). This G-protein-coupled receptor belongs to the opioid system, which plays a major role in pain control and autonomic functions, and modulates affective behavior and neuroendocrine physiology (Kieffer and Evans, 2009, Lutz and Kieffer, 2013). The MOR has unambiguously been established as the molecular target for opiate drugs, and mediates all the biological activities of morphine including both therapeutic and adverse effects (Matthes et al., 1996, Charbogne et al., 2014). Further, this receptor mediates reinforcing properties of non-opioid drugs of abuse including alcohol, cannabinoids, and nicotine (Kieffer and Gaveriaux-Ruff, 2002), as well as motivation for food (Papaleo et al., 2007) and social interactions (Moles et al., 2004), indicating a major role in reward processing. The broad MOR distribution throughout the brain supports all these roles (Erbs et al., 2014). Studies of this receptor have traditionally focused on the well described nociceptive and reward circuitries, but there is also some evidence for MOR-mediated morphine analgesia at the level of MHb (Terenzi et al., 1990, Terenzi and Prado, 1990, Darcq et al., 2012). Neuroanatomical distribution and function of MOR in the MHb, however, have been little explored despite strongest expression levels compared to all other brain areas (Kitchen et al., 1997) (reviews in Le Merrer et al., 2009, Lutz and Kieffer, 2013).
MOR distribution throughout the nervous system has been reported at RNA and protein levels (for a review Le Merrer et al., 2009). Low MOR mRNA abundance has rendered in situ hybridization experiments on mouse brain truly challenging, and data have been mainly reported from rat (George et al., 1994, Mansour et al., 1994). Also, protein detection at neuron level has been difficult due to poor availability of specific antibodies in tissue sections. MOR protein levels have otherwise been mapped and quantified using ligand autoradiography (Kitchen et al., 1997, Slowe et al., 1999, Goody et al., 2002), however this approach does not allow cellular resolution and the precise MOR distribution within MHb and associated circuitry is unknown. Our laboratory has recently generated knock-in mice expressing MOR in fusion with the red fluorescent protein mcherry (MOR-mcherry) in place of the native receptor. This mouse has enabled characterizing MOR distribution throughout the entire brain, as well as MOR colocalization with the delta opioid receptor with cellular resolution (Erbs et al., 2014). In the present study, we used this unique tool to investigate MOR distribution in neurons of the two Hb nuclei, and characterized MOR-expressing neuronal populations in the MHb–IPN pathway. Our data demonstrate prominent MOR expression in different subregions of MHb and IPN, and provides a basis for MOR-mediated mechanisms operating at the level of both substance P (SP)-ergic and cholinergic systems, in interaction with glutamatergic transmission in the MHb.
Section snippets
Animals
MOR-mcherry knock-in mice were generated by homologous recombination, as done earlier by our laboratory to generate DOR-eGFP knock-in mice, expressing delta opioid receptor coupled to a green fluorescent protein (Scherrer et al., 2006). In these mice, the mcherry cDNA was introduced into exon 4 of the MOR gene, in frame and 5′ of the stop codon, as described in Erbs et al., 2014. This C-terminal construct was designed to allow correct native-like MOR expression at sub-cellular level (see (Erbs
Results
The MOR-mcherry fluorescent signal is widely present throughout the brain (Fig. 1A, for detailed distribution see Erbs et al., 2014). In the DDC pathway, intense fluorescence was observed at the level of the Hb (Fig. 1C, D), efferent fibers forming the fasciculus retroflexus (fr, Fig. 1A, B), and the IPN (Fig. 1E, F) representing the main projection area of the medial Hb. Notably, the red fluorescent signal was detectable with three different patterns. The signal may appear as intense
Discussion
The Hb is an important relay structure between forebrain septal and pallidal regions, and mid/hindbrain areas that include VTA, IPN, raphe and tegmental nuclei. LHb and MHb nuclei, the two Hb nuclei, regulate monoamine and cholinergic transmission (Lee and Goto, 2011, Lammel et al., 2012, Kobayashi et al., 2013), and are key structures in the modulation of affective and cognitive functions. LHb has been most extensively studied but little is known about MHb. Interest for the medial nucleus,
Conclusion
Altogether, this report demonstrates that MOR is distributed all along the MHb–IPN pathway and associated circuitry. In addition to the well-described expression of this receptor in dopaminergic mesolimbic circuits, associated to LHb, MOR should therefore also be considered a prominent player in MHb-related networks. Fig. 5 summarizes the anatomy of MOR expression sites within MHb and LHb circuitries and their main connections as reported by other reports (Sutherland, 1982, Groenewegen et al.,
Acknowledgments
We thank E. Darcq for critical reading of the manuscript. We thank our funding sources including the Centre National de la Recherche Scientifique – France, the Institut National de la Santé et de la Recherche Médicale (INSERM) – France, the Université de Strasbourg – France. The Agence Nationale pour la Recherche (IMOP), the National Institutes of Health – United States (NIDA DA-05010) and the Stefan and Shirley Hatos Center for Neuropharmacology – United States. O. Gardon was supported by the
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Present address: Department of Neurosciences, UCSD, 9500 Gilman Drive, La Jolla, CA 92093, USA.
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Present address: Institut des Neurosciences Cellulaires et Intégratives UPR3212, 5 rue Blaise Pascal, F-67084 Strasbourg cedex 03, France.