Elsevier

Neuroscience

Volume 277, 26 September 2014, Pages 595-609
Neuroscience

Expression of mu opioid receptor in dorsal diencephalic conduction system: New insights for the medial habenula

https://doi.org/10.1016/j.neuroscience.2014.07.053Get rights and content

Highlights

  • Mu opioid receptors (MORs) are most dense and understudied in the MHb–IPN pathway.

  • MOR-mcherry knock-in mice reveal receptor expression with cellular resolution.

  • Most substance P neurons of MHb express MOR-mcherry in both cell bodies and terminals.

  • Only a subset of cholinergic neurons in MHb expresses MOR-mcherry.

  • MOR-mcherry is also strongly expressed upstream and downstream the MHb–IPN pathway.

Abstract

The habenular complex, encompassing medial (MHb) and lateral (LHb) divisions, is a highly conserved epithalamic structure involved in the dorsal diencephalic conduction system (DDC). These brain nuclei regulate information flow between the limbic forebrain and the mid- and hindbrain, integrating cognitive with emotional and sensory processes. The MHb is also one of the strongest expression sites for mu opioid receptors (MORs), which mediate analgesic and rewarding properties of opiates. At present however, anatomical distribution and function of these receptors have been poorly studied in MHb pathways. Here we took advantage of a newly generated MOR-mcherry knock-in mouse line to characterize MOR expression sites in the DDC.

MOR-mcherry fluorescent signal is weak in the LHb, but strong expression is visible in the MHb, fasciculus retroflexus (fr) and interpeduncular nucleus (IPN), indicating that MOR is mainly present in the MHb–IPN pathway. MOR-mcherry cell bodies are detected both in basolateral and apical parts of MHb, where the receptor co-localizes with cholinergic and substance P (SP) neurons, respectively, representing two main MHb neuronal populations. MOR-mcherry is expressed in most MHb-SP neurons, and is present in only a subpopulation of MHb-cholinergic neurons. Intense diffuse fluorescence detected in lateral and rostral parts of the IPN further suggests that MOR-mcherry is transported to terminals of these SP and cholinergic neurons. Finally, MOR-mcherry is present in septal regions projecting to the MHb, and in neurons of the central and intermediate IPN.

Together, this study describes MOR expression in several compartments of the MHb–IPN circuitry. The remarkably high MOR density in the MHb–IPN pathway suggests that these receptors are in a unique position to mediate analgesic, autonomic and reward responses.

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

References (91)

  • R.J. Goody et al.

    Quantitative autoradiographic mapping of opioid receptors in the brain of delta-opioid receptor gene knockout mice

    Brain Res

    (2002)
  • S. Ikemoto

    Brain reward circuitry beyond the mesolimbic dopamine system: a neurobiological theory

    Neurosci Biobehav Rev

    (2010)
  • P.W. Kalivas

    Interactions between neuropeptides and dopamine neurons in the ventromedial mesencephalon

    Neurosci Biobehav Rev

    (1985)
  • M.D. Kawaja et al.

    Substance P immunoreactivity in the rat interpeduncular nucleus: synaptic interactions between substance P-positive profiles and choline acetyltransferase- or glutamate decarboxylase-immunoreactive structures

    Neuroscience

    (1991)
  • B.L. Kieffer et al.

    Opioid receptors: from binding sites to visible molecules in vivo

    Neuropharmacology

    (2009)
  • B.L. Kieffer et al.

    Exploring the opioid system by gene knockout

    Prog Neurobiol

    (2002)
  • I. Kitchen et al.

    Quantitative autoradiographic mapping of mu-, delta- and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene

    Brain Res

    (1997)
  • L. Lecourtier et al.

    A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition

    Neurosci Biobehav Rev

    (2007)
  • Y.A. Lee et al.

    Habenula and ADHD: convergence on time

    Neurosci Biobehav Rev

    (2013)
  • P.E. Lutz et al.

    Opioid receptors: distinct roles in mood disorders

    Trends Neurosci

    (2013)
  • R. Maldonado et al.

    RP 67580, a selective antagonist of neurokinin-1 receptors, modifies some of the naloxone-precipitated morphine withdrawal signs in rats

    Neurosci Lett

    (1993)
  • J. Menard et al.

    Lateral and medial septal lesions reduce anxiety in the plus-maze and probe-burying tests

    Physiol Behav

    (1996)
  • N. Michaud et al.

    Cardiovascular and behavioural effects induced by naloxone-precipitated morphine withdrawal in rat: characterization with tachykinin antagonists

    Neuropeptides

    (2003)
  • H. Mizoguchi et al.

    Involvement of multiple micro-opioid receptor subtypes on the presynaptic or postsynaptic inhibition of spinal pain transmission

    Peptides

    (2014)
  • L.P. Morin et al.

    The ascending serotonergic system in the hamster: comparison with projections of the dorsal and median raphe nuclei

    Neuroscience

    (1999)
  • V. Panchal et al.

    Attenuation of morphine withdrawal signs by intracerebral administration of 18-methoxycoronaridine

    Eur J Pharmacol

    (2005)
  • C. Qin et al.

    Neurochemical phenotypes of the afferent and efferent projections of the mouse medial habenula

    Neuroscience

    (2009)
  • J. Ren et al.

    Habenula “cholinergic” neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes

    Neuron

    (2011)
  • T.L. Ripley et al.

    Lack of self-administration and behavioural sensitisation to morphine, but not cocaine, in mice lacking NK1 receptors

    Neuropharmacology

    (2002)
  • S.J. Slowe et al.

    Quantitative autoradiography of mu-, delta- and kappa1 opioid receptors in kappa-opioid receptor knockout mice

    Brain Res

    (1999)
  • R.J. Sutherland

    The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex

    Neurosci Biobehav Rev

    (1982)
  • M.G. Terenzi et al.

    Antinociception induced by stimulation of the habenular complex of the rat

    Brain Res

    (1990)
  • M.G. Terenzi et al.

    Antinociception elicited by electrical or chemical stimulation of the rat habenular complex and its sensitivity to systemic antagonists

    Brain Res

    (1990)
  • T. Yamaguchi et al.

    Distinct roles of segregated transmission of the septo-habenular pathway in anxiety and fear

    Neuron

    (2013)
  • L.M. Yang et al.

    Substance P receptor antagonist in lateral habenula improves rat depression-like behavior

    Brain Res Bull

    (2014)
  • Q. Zhou et al.

    Alteration in the brain content of substance P (1–7) during withdrawal in morphine-dependent rats

    Neuropharmacology

    (1998)
  • H. Aizawa et al.

    Molecular characterization of the subnuclei in rat habenula

    J Comp Neurol

    (2012)
  • H. Beaudry et al.

    Activation of spinal mu- and delta-opioid receptors potently inhibits substance P release induced by peripheral noxious stimuli

    J Neurosci

    (2011)
  • I.H. Bianco et al.

    The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain

    Philos Trans R Soc Lond B Biol Sci

    (2009)
  • A. Bilkei-Gorzo et al.

    Increased morphine analgesia and reduced side effects in mice lacking the tac1 gene

    Br J Pharmacol

    (2010)
  • J.P. Changeux

    Nicotine addiction and nicotinic receptors: lessons from genetically modified mice

    Nat Rev Neurosci

    (2010)
  • W. Chen et al.

    mu-Opioid receptor inhibition of substance P release from primary afferents disappears in neuropathic pain but not inflammatory pain

    Neuroscience

    (2014)
  • A.C. Cuello et al.

    Detection of substance P in the central nervous system by a monoclonal antibody

    Proc Natl Acad Sci U S A

    (1979)
  • J.A. Dani et al.

    Mesolimbic dopamine and habenulo-interpeduncular pathways in nicotine withdrawal

    Cold Spring Harb Perspect Med

    (2013)
  • D.Q. Dao et al.

    Nicotine enhances excitability of medial habenular neurons via facilitation of neurokinin signaling

    J Neurosci

    (2014)
  • Cited by (0)

    Co-first authors.

    Present address: Department of Neurosciences, UCSD, 9500 Gilman Drive, La Jolla, CA 92093, USA.

    §

    Present address: Institut des Neurosciences Cellulaires et Intégratives UPR3212, 5 rue Blaise Pascal, F-67084 Strasbourg cedex 03, France.

    View full text