Elsevier

Neuropharmacology

Volume 38, Issue 6, 15 June 1999, Pages 769-783
Neuropharmacology

α3β4 subunit-containing nicotinic receptors dominate function in rat medial habenula neurons

https://doi.org/10.1016/S0028-3908(99)00024-6Get rights and content

Abstract

Regional-specific differences in nicotinic acetylcholine receptors (nAChRs) were examined using the whole-cell patch clamp technique in rat medial habenula (MHb) slices. The majority of cells in the ventral two thirds of the MHb responded robustly to local pressure application of nAChR agonists. Mean agonist potency profiles in the middle and ventral thirds of the MHb were similar: cytisine was the most potent agonist and DMPP the weakest, consistent with a significant contribution of the β4 subunit to functional nAChRs in all areas of the MHb. In acutely isolated MHb neurons, the α3β4-selective toxin α-CTx-AuIB (1 μM) reversibly blocked≈75% of the nicotine-induced currents, as expected for cells solely expressing α3β4 nAChRs. However, the α3β2-selective toxin, α-CTx-MII (100 nM), blocked a variable fraction (0–90%) of the MHb nicotinic response implying that β2 subunits may contribute to some functional receptors. We suggest that the effects of α-CTx-MII may arise from interaction with α3β2β4 subunit-containing nAChRs. This idea is supported by the findings (1) that α-CTx-MII antagonizes receptors comprised of α3, β2 and β4 subunits in Xenopus oocytes, and (2) that a mutant α-CTx-MII toxin[H12A], which blocks α3β2β4 receptors but not α3β2 or α3β4 nAChRs, also reduces nicotinic currents in some MHb neurons. Overall these data imply that most functional nAChRs on MHb cells contain at least α3 and β4 subunits, and that a variable subpopulation additionally contains the β2 subunit.

Introduction

The involvement of neuronal nicotinic acetylcholine receptors (nAChRs) in a variety of cognitive functions and central nervous system (CNS) diseases predicts a large diversity of receptor subtypes (Sargent, 1993, Colquhoun and Patrick, 1997a, Lena and Changeux, 1997). This diversity is possible because of the many ways in which multiple nAChR subunits can be arranged into a pentameric structure (Role, 1992). However, in vivo it is likely that only a fraction of these receptor arrangements exist. In mammalian brain, radiolabeled binding studies have revealed four major classes of nAChRs with regionally distinct distributions: a high affinity [3H]nicotine binding site containing α4 and β2 subunits (Flores et al., 1992), a low affinity nicotinic receptor, which is labeled by α-bungarotoxin (α-BTX) and contains α7 subunits (Clarke et al., 1985, Seguela et al., 1993, Orr-Urtreger et al., 1997), and two additional receptors labeled by [3H]epibatidine with differential sensitivity to cytisine and probably containing α3 and β4 subunits (Perry and Kellar, 1995, Marks et al., 1998, Zoli et al., 1998). For the most part, these binding sites appear analogous to functional nAChR CNS subtypes, typified by those on cultured hippocampal neurons (Albuquerque et al., 1995). Additional functional receptor diversity may be generated by the inclusion of more subunits within these general subtypes (Ramirez-Latorre et al., 1996, Colquhoun and Patrick, 1997b).

The exact subunit make-up of functional nAChRs in the mammalian CNS and their relationship to the regional distribution of subunit mRNAs is unknown. Cells within the mammalian medial habenula (MHb) nucleus express a rich complement of subunit mRNAs: α3, α4, β2 (Wada et al., 1989), β3 (Deneris et al., 1989), β4 (Duvoisin et al., 1989, Dineley-Miller and Patrick, 1992), α7 (Seguela et al., 1993), α5 and α6 (Le Novere et al., 1996). On this basis alone a large variety of nAChRs should exist in this region. Although pharmacological studies have suggested that nicotine-induced responses in rat MHb (McCormick and Prince, 1987) occur via a distinct pharmacological receptor class (Mulle and Changeux, 1990), more than one single channel conductance has been observed, implying the existence of multiple nAChRs (Connolly et al., 1995).

Although absolute determination of the subunit composition of native CNS nAChRs by comparison with known receptors expressed in Xenopus oocytes is not possible (Covernton et al., 1994, Sivilotti et al., 1997), comparative studies can provide evidence in support of subunit composition (Luetje and Patrick, 1991, Colquhoun and Patrick, 1997a). For nAChRs on MHb neurons, the lack of sensitivity to α-BTX and the high potency of cytisine (Mulle and Changeux, 1990) are consistent with the absence of α7 subunits and the presence of β4 subunits, respectively (Couturier et al., 1990, Luetje and Patrick, 1991, Seguela et al., 1993, Wong et al., 1995, Orr-Urtreger et al., 1997). In the present study we have addressed the subunit composition and diversity of nAChRs in the MHb. Our data show that despite a tremendous potential for diversity of receptor subtypes, all functional nAChRs on all MHb neurons likely contain both α3 and β4 subunits.

Section snippets

Isolated medial habenula cells

Neurons were isolated from the habenula region of 10–25 day-old rats using methods described previously (Mulle et al., 1991, Lester and Dani, 1995). A single rat was anesthetized under halothane and decapitated. Following removal of the brain, both habenula nuclei (with as little surrounding tissue as possible) were microdissected in ice-cold saline and then placed immediately into a PIPES-buffered solution containing papain (60–90 U; Worthington Biochemical Corporation, Freehold, NJ), ≈10 mg

Regional agonist pharmacology of functional nAChRs in MHb

Because the sensitivity of nAChRs to different nicotinic agonists is determined in part by the subunit composition, a preliminary estimate of the presence of certain subunits can be obtained by constructing agonist potency profiles (Luetje and Patrick, 1991). The majority of cells (72/74) in the ventral two thirds of the MHb responded with inward currents to brief applications (2 s) of various nicotinic agonists at concentrations of 10 μM (Fig. 3A, B). The deactivation phases after removal of

Discussion

Despite the abundance of functional nAChRs on the soma-dendritic regions of MHb neurons, no clear physiological role for these receptors has yet been discovered (Edwards et al., 1992). However, because of the diversity and non-uniform distribution of mRNAs encoding for multiple nAChR subunits (Deneris et al., 1989, Duvoisin et al., 1989, Wada et al., 1989, Seguela et al., 1993, Le Novere et al., 1996), this area of the brain is a useful model for addressing the molecular nature of the

Acknowledgements

This work was supported by United States Public Health Service Grants NS-31669 and DA-11940 (R.A.J.L.) and the W.M. Keck Foundation #931360. We thank Elise B. Sheffield for contributing to the data on desensitization of α3β4α5 receptors.

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