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