Insights into the neurobiology of the nicotinic cholinergic system and nicotine addiction from mice expressing nicotinic receptors harboring gain-of-function mutations

Abstract

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated, cation-selective ion channels expressed throughout the brain. Although these channels have been investigated for several decades, it is still challenging 1) to identify the important nAChR subunits in cholinergic transmission and nicotine dependence and 2) to develop nAChR subtype-specific ligands. To overcome these challenges, we and others have studied mice expressing mutant, gain-of-function nAChR subunits. In this review, we discuss this research approach and the results it has yielded to date. Gain-of-function mutations, including those in nAChR subunits, provide an approach that is complementary to loss-of-function studies such as gene knockouts; the former allows one to answer questions of sufficiency and the latter addresses questions of necessity. Mutant mice expressing gain-of-function nAChR subunits are commonly produced using traditional gene targeting in embryonic stem cells, but novel approaches such as bacterial artificial chromosome transgenesis have yielded important insights as well. α7 nAChRs were the first nAChRs to be targeted with a gain-of-function mutation, followed by a pair of α4 nAChR gain-of-function mutant mice. These α4 nAChR gain-of-function mice (α4 L9'S mice, followed by α4 L9'A mice) provided an important system to probe α4 nAChR function in vivo, particularly in the dopamine reward system. α6 nAChR gain-of-function mice provided the first robust system allowing specific manipulation of this receptor subtype. Other targeted mutations in various nAChR subunits have also been produced and have yielded important insights into nicotinic cholinergic biology. As nAChR research advances and more details associated with nAChR expression and function emerge, we expect that existing and new mouse lines expressing gain-of-function nAChR subunits will continue to provide new insights.

Fig. 1.

nAChR M2 domain. nAChRs are composed of an N-terminal ligand binding domain, three adjacent transmembrane segments (M1–M3), a large cytoplasmic intracellular loop, and a fourth transmembrane segment (M4). Mouse M2 transmembrane protein sequences were aligned for the following nAChR subunits between the 2′ and 19′ positions: α3, α4, α5, α6, α7, β2, β3, and β4. The 9′ Leu residue is highly conserved across nAChRs and is often mutated to Ser or Thr when creating nAChR gain-of-function mutant mice.

Fig. 2.

Effect of a transmembrane (TM) 2 mutation on nAChR sensitivity. A, the particular nAChR subtype of interest will often, broadly speaking, have a sensitivity to ligand similar to that of other nAChR subtypes. In the absence of ligands that specifically activate the nAChR subtype of interest, an alternative approach to isolating the biological action of the receptor is to increase the sensitivity of the receptor by 10- to 100-fold. B, in the presence of a sufficient fraction of nAChR subunits of interest harboring a TM2 sensitizing mutation, receptor sensitivity is dramatically increased and the action of the receptor can be pharmacologically isolated relative to other nAChRs.

Fig. 3.

DA transmission regulated by the nicotinic cholinergic system: insights from hypersensitive nAChR mice. A, DA neurons express both α4*(non-α6), α6*(non-α4), and α4α6* nAChRs on their soma and dendrites, and a fraction of DA neurons also express α7 nAChRs. α7 nAChRs are also known to be expressed on glutamatergic terminals that synapse onto DA neurons. Midbrain GABAergic neurons express α4*(non-α6) nAChRs. Cholinergic input to the ventral midbrain is capable of activating all of these nAChRs. In striatum, DA terminals also express α4*(non-α6), α6*(non-α4), and α4α6* nAChRs, which respond to local ACh to modulate DA release. Striatal α4*(non-α6) nAChRs are known to participate in cholinergic regulation of GABA release. In striatum, α7 nAChRs are expressed in GABAergic and cholinergic cells, but are largely excluded from DA terminals. B, in α4 L9′A mice, selective activation of α4* nAChRs largely recapitulates the action of nicotine, producing both activation of DA neurons via the action of somatodendritic α4* nAChRs and attenuation of DAergic activity through stimulation of inhibitory GABAergic interneurons. Selective α4* activation with nicotine produces nicotine reward as well as behavioral sensitization and tolerance. In α6 L9′S mice, selective activation of α6* nAChRs can specifically activate DA neurons and cause elevated DA release and locomotor hyperactivity.