Point mutant mice with hypersensitive α4 nicotinic receptors show dopaminergic deficits and increased anxiety

Abstract

Knock-in mice were generated that harbored a leucine-to-serine mutation in the ␣4 nicotinic receptor near the gate in the channel pore. Mice with intact expression of this hypersensitive receptor display dominant neonatal lethality. These mice have a severe deficit of dopaminergic neurons in the substantia nigra, possibly because the hypersensitive receptors are continuously activated by normal extracellular choline concentrations. A strain that retains the neo selection cassette in an intron has reduced expression of the hypersensitive receptor and is viable and fertile. The viable mice display increased anxiety, poor motor learning, excessive ambulation that is eliminated by very low levels of nicotine, and a reduction of nigrostriatal dopaminergic function upon aging. These knock-in mice provide useful insights into the pathophysiology of sustained nicotinic receptor activation and may provide a model for Parkinson's disease.T he mechanism leading from nicotine intake to addiction is not known in detail, but it begins with the activation of neuronal nicotinic acetylcholine receptors (nAChRs). Nicotine elicits dopamine release in several regions of the brain, presumably leading to the reward, motor, and addictive effects (1, 2). The highest-affinity and most abundant nicotine binding in the brain corresponds to the ␣4␤2 nAChR (3). The ␣4 subunit is the principal partner for the ␤2 subunit in brain; ␤2-containing receptors play an important role in nicotine self-administration, in nicotine-stimulated electrophysiological responses in midbrain neurons, and in nicotine-stimulated dopamine release in the ventral striatum (4, 5). The ␣4 subunit and tyrosine hydroxylase are colocalized in dopaminergic neurons (6). Epidemiological studies show that smokers have a lower incidence of Parkinson's disease (7,8), suggesting a protective effect of nicotine via modulation of the dopaminergic system. Both ␣4 and ␤2 knockout mice show only subtle alterations in their physiology or behavior until they reach old age (4, 9, 10). We have used a complementary strategy to understand the roles of ␣4-containing nAChRs. We reasoned that gain of function mutations might generate more noticeable phenotypes, and that these phenotypes would gain relevance from the fact that nicotine and some candidate analgesics (11) are agonists. We have generated lines of knock-in mice by introducing a point mutation into the M2 transmembrane region of the ␣4 subunit to produce a hypersensitive receptor. Materials and MethodsXenopus Oocyte Injections and Electrophysiology. The ␣4 and ␤2 subunits were subcloned into pAMV-PA (12). Capped mRNA transcripts were prepared, and ␣4͞␤2 (2 ng, 1:1) or ␣4L9ЈS͞␤2 (0.2-0.5 ng, 1:1) were microinjected into Xenopus oocytes (12). Twenty-four to seventy-two hours later, two-electrode voltage clamp recordings (12) were made in solutions containing zero Ca 2ϩ .Knock-in Mouse Construction. A 129͞SvJ ␣4 genomic clone containing exon 5 and the L9ЈS mutation was inserted into pKO Scrambler V907 (Lexicon-Genetics, The Woodland...

Genetic variability in nicotinic acetylcholine receptors and nicotine addiction: Converging evidence from human and animal research

“…This SNP was associated with changes in the functional properties of α4 containing receptors: nicotine-induced 86 Rb + efflux was significantly greater in mice that possess the A529 variant [36]. Additionally, mice carrying the A529 variant of this SNP were less sensitive to the effects of nicotine on locomotor activity (measured on the Y-maze), body temperature, and respiration when compared to mice that have the T529 variant [172,173].…”

“…To date, knockout mice have been generated that lack the α3 [191], α4 [108], α5 [183], α6 [19], α7 [123], α9 [177], β2 [129,190], β3 [27], and β4 [190] nAChR subunits, and subsequent research has demonstrated that the deletion of these genes will alter many of the effects of nicotine in mice. Additionally, KI mice with point mutations that produce hypersensitive nAChRs have been generated for the α4 [86,167] and α7 subunits [124]. Although genetically modified mice can help to determine the possible functions of a given gene, there are some limitations to using this approach [20,162].…”

“…In addition to α4 subunit KO mice, two lines of α4 KI mice have been generated that have either a leucine to serine (Leu9'Ser) or a leucine to alanine (Leu9'Ala) point mutation in the pore-forming M2 transmembrane domain of the α4 subunit [86,167]. In Leu9'Ser mice, this point mutation produces α4-containing nAChRs with a ~30-fold increase in sensitivity to acetylcholine and nicotine.…”

“…In Leu9'Ser mice, this point mutation produces α4-containing nAChRs with a ~30-fold increase in sensitivity to acetylcholine and nicotine. The mutation is lethal to homozygous mice but not to heterozygous mice that possess a neomycin resistance cassette [86]. Subsequent research has demonstrated that Leu9'Ser heterozygous mice are more sensitive to the antinociceptive effects of nicotine than WT mice [29].…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Knockin mouse models that contain a gain-of-function mutation in the 9' position of the M2 domain include those for nAChR subunits α4 [130,131], α6 [132], α7 [133] and α9 [134]. Transgenic approaches have also been used to study mutations associated with congenital myasthenic syndromes (in the α1, δ and ε subunit) [135,136] and mutations associated with nocturnal frontal lobe epilepsy (in the α4 subunit) [137][138][139].…”