Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice - PubMed (original) (raw)

Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice

S E Hamilton et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

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Figures

Figure 1

Figure 1

Targeting the m1 gene by homologous recombination. Restriction endonuclease map of the wild-type m1 mAChR gene (Top), the targeting vector (Bottom), and the mutant allele (Middle) resulting from homologous recombination at the m1 locus. (Inset) Example of genomic Southern blot used to determine mouse genotypes. The 1.8-kb _Kpn_I–_Bam_HI fragment, which includes the entire coding region of m1, was used as a probe. _Bam_HI digests of mouse tail DNA yield a 3.1-kb band in the wild type (+/+) and a 5.1-kb band in the m1 mutant (−/−). Both bands are present in the heterozygote (+/−). tk, Herpes simplex virus thymidine kinase gene cassette; neo, gene cassette for neomycin resistance; pGEM, intervening plasmid sequences.

Figure 2

Figure 2

Light microscopic localization of m1–m4 in the hippocampus of m1 knockout (−/−, Left) and wild-type mice (+/+, Right). Note the absence of m1 immunoreactivity in the hippocampus and overlying cortex from the mutant mice when compared with the dark staining evident in the wild-type mice. Minimal background staining in the m1-deficient mice was similar to controls in which the primary antibody was omitted for mutant and wild-type brain sections. CA1 and CA3, regions of Ammon’s horn; DG, dentate gyrus. (Bar = 500 μm.)

Figure 3

Figure 3

Concentration of total mAChRs and specific m1–m4 subtypes in mouse forebrain and cerebellum. Total mAChR numbers were determined by [3H]QNB binding assays and protein concentrations were measured by the Lowry assay on tissue homogenates from the forebrain (A) and cerebellum (B). [3H]QNB-labeled receptors were solubilized and immunoprecipitated with subtype-specific antisera from mouse forebrain (C) and cerebellum (D). In all cases values are the mean ± SD, n = 4.

Figure 4

Figure 4

Modulation of M current by a muscarinic agonist is missing in the −/− mouse. (A) Time course of action of Oxo and angiotensin II. Oxo (10 μM) had a large and partially reversible inhibitory effect on the M current in a SCG cell from a +/+ mouse but had no effect on M current in a SCG neuron from a −/− mouse. The inhibitory action of angiotensin II (500 nM) was observed in both cells. (B) Current traces from the cells in A 4 s before (thin traces) and after 1 min of Oxo application (thick traces). The holding current and the relaxing tail current are Oxo-sensitive only in the cell from the +/+ mouse. (C) Oxo strongly inhibited M current in all cells from +/+ mice (n = 7) but had no effect in cells from −/− mice (n = 9).

Figure 5

Figure 5

Absence of pilocarpine-induced seizures in m1 mutant mice. Mice were injected with 200 mg/kg (A and C) or 300 mg/kg (B and D) pilocarpine (i.p.) and observed for 45 min. The percentage of mice experiencing at least one tonic-clonic seizure is shown in A and B. Seizure severities and behavior ratings assigned for the entire 45-min period are presented for these mice in C and D. Values are expressed as the mean ± SD for each genotype.

Figure 6

Figure 6

Assessment of kainic acid-induced seizures in wild-type and m1 mutant mice. Mice were injected with kainic acid (s.c.) at 25 mg/kg (A) (n = 9) or 35 mg/kg (B) (n = 9) and observed for 45 min. Mice were assigned ratings for their behavior during each 5-min period. Values represent the mean ± SEM for each genotype.

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References

    1. Messer W S, Bohnett M, Stibbe J. Neurosci Lett. 1990;116:184–189. - PubMed
    1. Messer W S, Stibbe J R, Bohnett M. Brain Res. 1991;564:66–72. - PubMed
    1. Velaquez-Moctezuma J, Gillin J C, Shiromani P J. Brain Res. 1989;503:128–131. - PubMed
    1. Bartolini A, Ghelardini C, Fantetti L, Malcangio M, Malmberg-Aiello P, Giotti A. Br J Pharmacol. 1992;105:77–82. - PMC - PubMed
    1. Turski L, Ikonomidou C, Turski W A, Bortolotto A A, Cavalheiro E A. Synapse. 1989;3:154–171. - PubMed

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