Elsevier

Neuropharmacology

Volume 56, Issue 1, January 2009, Pages 247-253
Neuropharmacology

Demonstration of functional α4-containing nicotinic receptors in the medial habenula

https://doi.org/10.1016/j.neuropharm.2008.08.021Get rights and content

Abstract

The medial habenula (MHb) exhibits exceptionally high levels of nicotinic acetylcholine receptors (nAChRs), but it remains unclear whether all expressed nAChR subunit mRNAs are translated to form functional receptors. In particular α4 subunits have not been reported to have any functional role, despite strong α4 mRNA expression in the ventrolateral MHb. We studied a strain of knock-in mice expressing fluorescent α4 nAChRs (α4YFP), as well as a knock-in strain expressing hypersensitive α4 nAChRs (α4L9′A). In α4YFP mice, there was strong fluorescence in the ventrolateral MHb. In hypersensitive α4L9′A mice, injections of a low dose of nicotine (0.1 mg/kg) led to strong c-fos expression in only the ventrolateral region of the MHb, but not in the MHb of wild-type (WT) mice. In MHb slice recordings, ventrolateral neurons from α4L9′A mice, but not from WT mice, responded robustly to nicotine (1 μM). Neurons in the medial aspect of the MHb had >10-fold smaller responses. Thus α4 nAChRs contribute to the selective activation of a subset of MHb neurons. Subunit composition analysis based on gain-of-function knock-in mice provides a useful experimental paradigm.

Introduction

Nicotinic acetylcholine receptors (nAChRs) are promising drug targets in the treatment of several CNS disorders such as Alzheimer's disease, schizophrenia, neuropathic pain and substance abuse (Arneric et al., 2007). Successful treatments will require discovery of agents that select among multiple subtypes of nAChRs (Role, 1992, Gotti et al., 2007). To choose appropriate drugs and to avoid off-target effects, one must understand how each nAChR is distributed among cell types and contributes to neuronal function and behavior. Despite much progress (e.g., Luetje, 2004), these tasks remain challenging because we lack tools to unambiguously identify the subunit make-up of particular nAChRs. Novel subunit-specific toxins (Janes, 2005) and gene-specific knockout models (Cordero-Erausquin et al., 2000, Picciotto et al., 2001) have confirmed roles for certain subunits; however, the expression of multiple subunits within individual cells renders subunit compensation a possibility in knockout studies (e.g., Brussaard et al., 1994).

Definitive identification of nAChR subtypes is especially problematic for neurons in the medial habenula (MHb), which expresses the highest levels of nAChRs in the brain (Marks et al., 1998). MHb expresses mRNAs encoding several different subunits (α3–α7 and β2–β4) (Sheffield et al., 2000). Pharmacological data suggest that heteromeric nAChRs, composed of a minimum of α3 and β4 subunits (α3β4), are a major functional subtype in this nucleus (Mulle and Changeux, 1990, Quick et al., 1999, Perry et al., 2002, Whiteaker et al., 2002). While the function(s) of this small epithalamic nucleus is not well understood (Lecourtier and Kelly, 2007), α3β4-nAChRs are important for sensitization and self-administration of opioids in rats (Glick et al., 2006, Taraschenko et al., 2007). It is not yet known how other nAChR subunits contribute to the heterogeneous MHb nAChR receptor population (Connolly et al., 1995). This paper examines the expression of functional α4 subunits, whose mRNA is also found within the MHb (Wada et al., 1989, Pauly et al., 1996). α4 nAChRs are thought to be necessary and sufficient for the expression of behaviors underlying nicotine addiction including reward, tolerance and sensitization (Tapper et al., 2004, Maskos et al., 2005), but not withdrawal (Salas et al., 2004). Also, α4β2 receptors are associated with monogenic epilepsies (Phillips et al., 1995, Fonck et al., 2003, Fonck et al., 2005).

Here we combined conventional approaches with genetic strategies that use two lines of knock-in mice. One line expresses fluorescently tagged α4 nAChRs (α4YFP, Nashmi et al., 2007); the other expresses hypersensitive α4 nAChRs bearing a leucine to alanine substitution at the M2 9′ position (α4L9′A, Tapper et al., 2004, Fonck et al., 2005). The results show the distribution and function of MHb α4 nAChRs.

Section snippets

Materials and methods

Experiments involving mice were performed following the guidelines established by each institution's Animal Care Committee. Mutant mouse lines were genotyped by PCR analysis of tail DNA.

Distribution of α4 mRNA and expression of α4YFP receptors

The distribution of α4 mRNA and α4 protein was assessed by in situ hybridization in C57BL/6J mice and by fluorescence of tissue from α4YFP mice, respectively. The signal from hybridized α4 mRNA was concentrated in the ventrolateral region of the MHb, whereas no signal was detected in the dorsal–medial aspect of the structure (Fig. 1A and B). A similar distribution pattern of α4 mRNA within the MHb of C57BL/6J mice appears in the data of Wada et al. (1989) and of the Allen Brain Atlas (//www.brain-map.org

Discussion

These studies illustrate how genetic approaches using knock-in mice that express fluorescent or hypersensitive nicotinic receptors can help identify the functional contribution of individual subunit genes to heteromultimeric receptors in specified neurons. Our results indicate that functional nAChRs containing α4 subunits are selectively localized to cells in the ventrolateral region of the MHb. This conclusion is based on a consistent set of four observations: the patterns of α4 mRNA labeling

Acknowledgements

This work was supported by PHS grants NS31669, DA17173, DA17279, California Tobacco-Related Disease Research Project, and the Philip Morris External Research Fund. R.N. was supported by a NARSAD Young Investigator Award.

References (36)

  • E.B. Sheffield et al.

    Nicotinic acetylcholine receptor subunit mRNA expression and channel function in medial habenula neurons

    Neuropharmacology

    (2000)
  • N.J. Woolf et al.

    Cholinergic systems in the rat brain: II. Projections to the interpeduncular nucleus

    Brain Research Bulletin

    (1985)
  • A.B. Brussaard et al.

    Developmental regulation of multiple nicotinic AChR channel subtypes in embryonic chick habenula neurons: contributions of both the α2 and α4 subunit genes

    Pflugers Archives

    (1994)
  • J.G. Connolly et al.

    Heterogeneity of neuronal nicotinic acetylcholine receptors in thin slices of rat medial habenula

    Journal of Physiology

    (1995)
  • A. Contestabile et al.

    Afferent connections of the interpeduncular nucleus and the topographic organization of the habenulo-interpeduncular pathway: an HRP study in the rat

    Journal of Comparative Neurology

    (1981)
  • C. Fonck et al.

    Increased sensitivity to agonist-induced seizures, Straub tail, and hippocampal theta rhythm in knock-in mice carrying hypersensitive α4 nicotinic receptors

    Journal of Neuroscience

    (2003)
  • C. Fonck et al.

    Novel seizure phenotype and sleep disruptions in knock-in mice with hypersensitive α4 nicotinic receptors

    Journal of Neuroscience

    (2005)
  • C.W. Luetje

    Getting past the asterisk: the subunit composition of presynaptic nicotinic receptors that modulate striatal dopamine release

    Molecular Pharmacology

    (2004)
  • Cited by (21)

    • Habenular synapses and nicotine

      2019, Neuroscience of Nicotine: Mechanisms and Treatment
    • The α4β2 nicotinic acetylcholine receptor modulates autism-like behavioral and motor abnormalities in pentylenetetrazol-kindled mice

      2016, European Journal of Pharmacology
      Citation Excerpt :

      This structure is critically placed between the limbic and cortical networks, allowing it to propagate epileptic activity and facilitate epileptogenesis (Pollock et al., 2014). The medial habenula displays high expression levels of neuronal nicotinic acetylcholine receptors, and shows increased c-fos expression following PTZ-induced kindling in mice (Bastlund et al., 2005; Fonck et al., 2009). Neuronal nicotinic acetylcholine receptor systems in both animals and humans have major roles in normal cognitive function and in cognitive impairment, including learning, memory and ADHD.

    • Diversity of native nicotinic receptor subtypes in mammalian brain

      2015, Neuropharmacology
      Citation Excerpt :

      The drawback of the immunoprecipitation studies is that their spatial resolution is only regional but the recent generation of Kin mouse strains expressing α3, α4, α6, β2, β3, or β4 nAChR subunits fused to green fluorescent protein (GFP) has made it possible to determine the precise localisation of the receptors -containing these subunits (Fowler and Kenny, 2012). Using a combined electrophysiological/genetic approach and two lines of Kin mice (one expressing fluorescently tagged α4* nAChRs (α4YFP, Nashmi et al., 2007), and the other hypersensitive α4* nAChRs bearing a leucine to alanine substitution at the M2 9′ position (α4L9′A, Tapper et al., 2004; Fonck et al., 2005), Fonck et al. (2009) demonstrated that nAChRs containing α4 subunits are selectively localised to cells in the ventrolateral region of the MHb, thus confirming previous ISH results (Le Novere et al., 1996). Shih et al. (2014) extended this study and used many more GFP-tagged mice to demonstrate high α3 and β4 nAChR subunit levels throughout the ventral MHb, which is consistent with the findings of previous immunoprecipitation and Western blotting (Yeh et al., 2001; Gahring et al., 2004,), radioligand binding (Salas et al., 2004), ISH (Wada et al., 1989; Le Novère et al., 1996; Winzer-Serhan and Leslie, 1997), and transgenic studies (Frahm et al., 2011).

    • Haloperidol modulates midbrain-prefrontal functional connectivity in the rat brain

      2013, European Neuropsychopharmacology
      Citation Excerpt :

      In our covariation analysis we used the whole habenular complex, this is by itself a comparatively small region in the rat brain, the further division of which would produce ROIs that would likely be undersized for a fair judgement of results, given the acquisition resolution of the fMRI data and residual inter-animal variations remaining after spatial co-registration. The lateral habenula (LHb) is an important relay center between the limbic forebrain and the midbrain (ventral tegmental area, SN, raphe nuclei) and is involved in negative reward processing (Hikosaka, 2010; Haber and Knutson, 2010) and depression (Sartorius and Henn, 2007; Sartorius et al., 2010), whereas the function of the medial habenula (MHb) is presumed to be more associated with regulation of the sleep-wake cycle, learning and memory (Fonck et al., 2009). Haloperidol induced an increased correlation of habenula with several structures, most notably the bed nucleus of stria terminalis (BNST), CPu, posterodorsal hippocampus and the secondary motor cortex.

    • Recent advances in gene manipulation and nicotinic acetylcholine receptor biology

      2011, Biochemical Pharmacology
      Citation Excerpt :

      In addition, mouse lines expressing multiple different tagged subunits should provide information on assembly partners. Since the first report of targeted homologous recombination in mouse embryonic stem cells by Thomas and Capecchi in 1987 [47], the homologous recombination technique has been extensively used to engineer knock-out mice [48]. More recently, this method has also been used to generate knock-in mice in which gene sequences are altered rather than deleted.

    View all citing articles on Scopus
    1

    Present address: AstraZeneca Pharmaceuticals LP, Wilmington, DE 19850, USA.

    View full text