Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety - PubMed (original) (raw)

. 2001 Feb 27;98(5):2786-91.

doi: 10.1073/pnas.041582598. Epub 2001 Feb 20.

J Schwarz, P Deshpande, S Schwarz, M W Nowak, C Fonck, R Nashmi, P Kofuji, H Dang, W Shi, M Fidan, B S Khakh, Z Chen, B J Bowers, J Boulter, J M Wehner, H A Lester

Affiliations

Point mutant mice with hypersensitive alpha 4 nicotinic receptors show dopaminergic deficits and increased anxiety

C Labarca et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Knock-in mice were generated that harbored a leucine-to-serine mutation in the alpha4 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.

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Figures

Figure 1

Figure 1

Physiological design, recombinant construction, and genomic characterization of the α4 knockin mouse strains. Agonist concentration-response relations of WT and mutated (α4L9′S) rat α4β2 receptors expressed in oocytes (five oocytes for each curve). (A) Acetylcholine. (B) Nicotine. (C) Choline. The choline responses of the WT receptor were not studied systematically, because there is no response at choline concentrations up to 1 mM, and higher concentrations of choline block the channel. (Insert) Time course of the response to 30 μM choline, showing partial desensitization. (D) Targeting construct containing exon 5 with the Leu9′Ser mutation, the neomycin resistance gene (neo) flanked by loxP sites, the diphtheria toxin A chain gene (DT), and the pKO V907 vector (pKO). (E) Deletion of the neo cassette by transfecting the neo-intact ES cells with a cytomegalovirus-Cre plasmid generates neo-deleted ES cell lines. (F) Southern blot analysis of genomic DNA from five embryonic stem-cell clones following digestion with _Bam_HI and _Eco_RI restriction endonucleases and hybridization with a flanking genomic fragment as a probe, indicated in E. Lines analyzed in lanes 1, 2, 4, and 5 contain DNA from two WT genes. Lane 3 shows a line with one WT gene (9.7 kb, Bam_HI–_Bam_HI as indicated in_E) and one mutant gene (7.7 kb,_Bam_HI–_Eco_RI in E). (G) Sequence analysis of DNA extracted from WT, heterozygous (het), and homozygous (hom) neo-intact mice. The WT sequence at nucleotide position 142, corresponding to the codon at position 9′ in the M2 region, is CTT, encoding leucine; the mutant sequence is TCT, encoding serine.

Figure 2

Figure 2

Detection and probable pathophysiological basis of dopaminergic neuron deficits in mutant mice. (A) Substantia nigra of WT (Left) and homozygous neo-intact ED 18 embryos, double stained for the α4 subunit (green) and for TH (red). (B) Tyrosine hydroxylase staining of substantia nigra of WT (Left) and heterozygous neo-deleted (Right) ED 18 embryos. (C) Cell counts of TH-positive neurons in substantia nigra of ED 16 to ED 18 embryos from WT, neo-intact, and neo-deleted mice. The heterozygote (het) cell counts do not differ significantly from WT, but both the homozygous (homo) neo-intact (P < 0.01, t test) and the neo-deleted cell counts (P < 0.05,t test) differ significantly from WT. (D) Whole-cell voltage-clamp recording of responses to two consecutive puffs of choline (100 μM, 20 ms) in neuron-like cells differentiated from ED 16 midbrain neuronal progenitor cells. Upper trace, cell from a WT embryo; lower trace, cell derived from a heterozygous neo-intact ED 16 embryo. (E) Mean ± SEM of responses in neuron-like cells derived from heterozygous animals (n = 5 cells) but little or no response in cells from WT animals (n = 7 cells; significant difference, P < 0.05, t test).

Figure 3

Figure 3

Spontaneous and drug-modulated locomotion of WT and heterozygous (het) neo-intact mutants. (A) No treatment. Heterozygotes showed significantly higher locomotion than WT mice at the beginning of the experiment (P < 0.001). (B) Nicotine, 0.02 mg/kg, was injected 30 min after the start of behavioral monitoring. The plot shows data averaged over the time periods, 10 min before (BL, baseline) and 5–15 min after injection. Heterozygous mice showed a significant reduction of locomotor activity after nicotine injection (P < 0.05). There was no significant difference in noninjected animals (right-hand bars), nicotine-injected control animals, or saline-injected WT or heterozygous animals (data not shown). (C) Heterozygotes were impaired compared with WTs on the accelerating rotarod (P < 0.012) when tested for three sequential days (n = 20–22 of each genotype). (D and E) The effect of amphetamine on locomotion of WT and heterozygous (het) mice at two ages. Before drug administration, animals were allowed to habituate for 30 min. Ten male WT and 10 male heterozygous mice showing at least a 3-fold increased activity over baseline in response to amphetamine at 3 months of age were selected for longitudinal follow-up studies. (D) Comparison in amphetamine responses for heterozygotes at 3 months vs. 11 months of age. (E) The average activity is plotted for the period between 5 and 45 min after injection. The response at 11 months declines significantly compared with the response at 3 months in heterozygous mice [F(1,9) = 12.72, P < 0.01] but not in WT mice.

Figure 4

Figure 4

Increased anxiety in α4 heterozygotes (het) compared with WT mice and in the elevated plus maze (A) and mirrored chamber (B) (n = 22 mice of each genotype) (1, 2). (A) Heterozygotes were significantly more anxious, as measured by percentage of entrances into the open arms (P < 0.01), percentage of time in the open arms (P < 0.002), percentage of time in the closed arms (P < 0.02), and entrances to the end of the open arms (P < 0.005). (B) Heterozygotes were significantly more anxious in the mirrored chamber, as measured by latency to enter the mirrored chamber (P < 0.026), percentage of time in the mirrored chamber (P < 0.03), entrances into the mirrored chamber (P < 0.02), but not by the number of entries into the mirrored passage.

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