Nicotinic-agonist stimulated ⁸⁶Rb⁺ efflux and [³H]epibatidine binding of mice differing in β2 genotype

Abstract

Nicotinic acetylcholine receptor function and binding was measured in 12 brain regions from mice differing in β2 subunit expression. Function was measured by on-line detection of ⁸⁶Rb⁺ efflux stimulated under conditions that measure two pharmacologically distinct nicotinic responses: (1) stimulation with 10 μM nicotine, a response that is relatively sensitive to inhibition by the antagonist, dihydro-β-erythroidine (DHβE); and (2) stimulation with 10 μM epibatidine in the presence of 2 μM DHβE, a response that is relatively resistant to inhibition by DHβE. Deletion of the β2 subunit profoundly reduced both DHβE-sensitive and -resistant ⁸⁶Rb⁺ efflux in each brain region and essentially eliminated activity in regions such as cerebral cortex and thalamus. However, residual activity was observed in regions such as olfactory bulbs and inferior colliculus. [³H]Epibatidine binding was measured under conditions that allow estimation of both high- and low-affinity sites. High-affinity sites sensitive to inhibition by the nicotinic agonist, cytisine, were virtually eliminated in every region by the β2 null mutation. In contrast, only a subset of the high-affinity sites insensitive to inhibition by cytisine were eliminated in β2 null mutants, suggesting receptor heterogeniety. Similarly, low affinity [³H]epibatidine binding was heterogeneous in that a fraction of the sites required the β2 subunit. Many remaining sites were sensitive to inhibition by α-bungarotoxin indicating that a subset of the low affinity [³H]epibatidine binding are of the α7* subtype. Distinct regional variation was observed among the 12 brain regions. These studies confirm important roles for β2-containing receptors in mediating pharmacologically distinct functions and as components of several identifiable binding sites.

Introduction

Nicotinic cholinergic receptors (nAChR) comprise a large and diverse family of ligand gated ion channels that are found at the neuromuscular junction, in the autonomic nervous system, and in the central nervous system (McGehee and Role, 1995, Lindstrom, 1999). nAChR diversity arises from the existence of many different receptor subunits that, when expressed in heterologous systems, such as Xenopus oocytes, as combinations of a single α and a single β exhibit physiological and pharmacological properties that depend upon both the α and β subunits (Luetje and Patrick, 1991, Cachelin and Jaggi, 1991, Gross et al., 1991, Couturier et al., 1990, Séguéla et al., 1993, Chavez-Noriega et al., 1997, Vibat et al., 1995, Fenster et al., 1997). nAChR diversity is futher expanded by the fact that the receptors are likely to be pentameric (Anand et al., 1991, Cooper et al., 1991) and that more than two different types of subunit may be present in each nAChR (Conroy et al., 1992, Ramirez-Latorre et al., 1996, Wang et al., 1996, Groot-Kormelink et al., 1998). Indeed, physiological and pharmacological properties are altered by the expression of a third type of subunit. Thus the potential for nAChR diversity in the CNS is very large, although restricted expression of some subunits limits the diversity.

Functional nAChR have been identified in the CNS using electrophysiological and biochemical approaches, both of which demonstrate nAChR diversity. For example, using whole cell patch clamp methods, pharmacological and physiological differences have been observed in hippocampal cells in culture (Alkondon and Albuquerque, 1993, Alkondon and Albuquerque, 1995) and have been confirmed in hippocampal cells in situ (Alkondon et al., 1997). Several unique pharmacological subtypes have also been identified in the medial habenula/interpedunctular nucleus pathway (Mulle et al., 1991, McGehee et al., 1995). A major functional role of nAChR appears to be the modulation of neurotransmitter release that has been demonstrated using electrophysiological (McGehee et al., 1995, Gray et al., 1996, Lena and Changeux, 1997, Alkondon et al., 1997), biochemical (reviewed in Kaiser and Wonnacott, 1999), and in vivo microdialysis (Imperato et al., 1986, Nisell et al., 1994, Benwell et al., 1995, Picciotto et al., 1998) methods. Differing pharmacological properties exhibited by the various measurements are highly suggestive of nAChR diversity.

Inasmuch as nAChR are ligand-gated ion channels, receptor function has also been measured using agonist-stimulated ion flux. Our laboratory has used nAChR-mediated ⁸⁶Rb⁺ efflux from mouse brain synaptosomes as a general method to measure receptor function. This method has recently been used to identify two pharmacologically distinct responses in mouse brain (Marks et al., 1999) and should be generally applicable for other nAChRs, as well.

One powerful approach to investigate nAChR diversity and the role of distinct nAChR subunits in receptor function is to study null mutant mice. Null mutants have been described for three major CNS nAChR subunits: β2 (Picciotto et al., 1995), α4 (Marubio et al., 1999), and α7 (Orr-Urtreger et al., 1997), as well as for α3 (Xu et al., 1999a) and β4 (Xu et al., 1999b). The β2, β4, α4 and α7 null mutants are viable, but the α3 null mutants do not survive to adulthood and suffer severe functional deficits in autonomic function. Initial studies using the null mutants have yielded valuable information. Fast desensitizing nAChR-mediated currents in hippocampus, postulated to be mediated by α7 nAChR (Alkondon and Albuquerque, 1993), are eliminated in α7 null mutant mice, as is [¹²⁵I]α-bungarotoxin binding (Orr-Urtreger et al., 1997). Both the β2 (Picciotto et al., 1995, Zoli et al., 1998) and the α4 (Marubio et al., 1999) null mutants lose the vast majority of their high affinity [³H]nicotinic binding sites, which had been previously postulated to be primarily α4β2 nAChR (Whiting and Lindstrom, 1988, Flores et al., 1992). Both mutations also eliminate functional nicotinic responses in several thalamic nuclei, which normally display robust activity (Picciotto et al., 1995, Marubio et al., 1999). However, a subset of high affinity [³H]epibatidine binding sites persist in distinct brain nuclei of α4 and β2 null mutant mice, further demonstrating that [³H]epibatidine binds to several nAChR subtypes (Zoli et al., 1998). Elimination of α4 or β2 subunits also alters specific physiological and behavioral responses including avoidance learning (Picciotto et al., 1995), nicotine self-administration, nicotine-stimulated dopamine release in vivo (Picciotto et al., 1998), and nicotine-induced antinociception (Marubio et al., 1999). The β2 null mutation also greatly reduces both nAChR-mediated GABA release (Lu et al., 1998) and nAChR-mediated ⁸⁶Rb⁺ efflux (Marks et al., 1999) in whole mouse brain synaptosomes. Therefore, investigations using nAChR null mutant mice have been very informative both in confirming postulates about the composition of functional nAChR and in revealing nAChR responses that do not require the subunit of interest.

The results reported here make use of β2 null mutant mice to investigate two pharmacologically distinct components of ⁸⁶Rb⁺ efflux in 12 brain regions. The studies also extend observations about the subtypes of [³H]epibatidine binding sites expressed in these brain areas. The results indicate that the β2 subunit is required for the function and binding of many, but not all, nAChR and that the distribution of those nAChR subtypes that require the β2 subunit varies markedly among the brain regions.