Limits...
A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection.

Taranda J, Maison SF, Ballestero JA, Katz E, Savino J, Vetter DE, Boulter J, Liberman MC, Fuchs PA, Elgoyhen AB - PLoS Biol. (2009)

Bottom Line: The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells.One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear.The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

ABSTRACT
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.

Show MeSH

Related in: MedlinePlus

Genetic Engineering and Genotyping of Chrna9L9'T Mutant Mice(A) Gene structures and restriction maps for the targeting construct, wild-type, and recombinant alleles (before and after removal of the neomycin resistance cassette) are shown. Black boxes represent the exons of the Chrna9 gene. The introduction of the mutation is shown by an asterisk (*).(B) An autoradiograph of a genomic Southern blot using DNA prepared from wild-type (left lane) and successfully targeted ES cells (right lane) and hybridized to a 32P-labled DNA probe prepared from the 962-bp SacII-KpnI fragment shown. Wild-type (WT) ES cell DNA yield a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yield a 7,300-bp fragment.(C) Routine genotyping of neo-deleted Chrna9L9′T mutant mice performed by PCR from isolated tail biopsy tissue genomic DNA samples and amplimers flanking the lox P footprint (white box). A representative gel indicating the results for wild-type, heterozygous, and homozygous knockin mice is shown. The PCR fragment length for the wild-type Chrna9 allele is 203 bp, and 269 bp for the mutant allele. MW, molecular weight.(D) A PCR fragment using amplimers flanking the L9′T mutation (Figure 1A) was obtained from genomic DNA isolated from tail biopsies and sequenced to check the integrity of the mutation in each breeding pair. A representative electropherogram indicating the results for wild-type and homozygous knockin mice is shown. The wild-type sequence at nucleotide position 210, corresponding to the codon at position 9′ in the second transmembrane domain, is TTG, encoding leucine; the mutant sequence is ACG, encoding threonine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2628405&req=5

pbio-1000018-g002: Genetic Engineering and Genotyping of Chrna9L9'T Mutant Mice(A) Gene structures and restriction maps for the targeting construct, wild-type, and recombinant alleles (before and after removal of the neomycin resistance cassette) are shown. Black boxes represent the exons of the Chrna9 gene. The introduction of the mutation is shown by an asterisk (*).(B) An autoradiograph of a genomic Southern blot using DNA prepared from wild-type (left lane) and successfully targeted ES cells (right lane) and hybridized to a 32P-labled DNA probe prepared from the 962-bp SacII-KpnI fragment shown. Wild-type (WT) ES cell DNA yield a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yield a 7,300-bp fragment.(C) Routine genotyping of neo-deleted Chrna9L9′T mutant mice performed by PCR from isolated tail biopsy tissue genomic DNA samples and amplimers flanking the lox P footprint (white box). A representative gel indicating the results for wild-type, heterozygous, and homozygous knockin mice is shown. The PCR fragment length for the wild-type Chrna9 allele is 203 bp, and 269 bp for the mutant allele. MW, molecular weight.(D) A PCR fragment using amplimers flanking the L9′T mutation (Figure 1A) was obtained from genomic DNA isolated from tail biopsies and sequenced to check the integrity of the mutation in each breeding pair. A representative electropherogram indicating the results for wild-type and homozygous knockin mice is shown. The wild-type sequence at nucleotide position 210, corresponding to the codon at position 9′ in the second transmembrane domain, is TTG, encoding leucine; the mutant sequence is ACG, encoding threonine.

Mentions: The targeting construct and generation of Chrna9L9′T mutant mice are described in the Materials and Methods section and Figure 2. Both Chrna9wt/L9′T and Chrna9L9′T/L9′T mutant mice were viable through adulthood, fertile, showed the expected gender and Mendelian genotype ratios, and exhibited no overt behavioral phenotype. Gross morphology of the cochlear duct was normal as seen in light microscopic evaluation of plastic sections of osmium-stained cochleae (Figure S1). As assessed by quantitative real-time PCR (RT-PCR), the expression level of cochlear nAChR α9 subunit mRNA was similar in Chrna9wt/wt and Chrna9L9′T/L9′T mice (Table S1). In addition, the expression levels of genes encoding proteins known to localize to the olivocochlear (OC) synaptic complex (for example, the nAChR α10 subunit and SK2 channel) were not changed in mutant mice (Table S1). Lastly, the electrophysiology of IHCs and OHCs was normal, as indicated by similar voltage-dependent K+ currents (Figure S2). Hair cell membrane area measured by capacitance was identical across phenotypes (Chrna9wt/wt: 8.3 ± 0.2 pF, n = 19 animals, 41 IHCs; Chrna9wt/L9′T: 8.4 ± 0.1 pF; n = 14 animals, 41 IHCs; Chrna9 L9′T /L9′T: 8.6 ± 0.1 pF, n = 26 animals, 79 IHCs, measured at P9–P11; Chrna9wt/wt: 8.0 ± 0.4 pF, n = 5 animals, 13 OHCs; and Chrna9 L9′T /L9′T: 8.1 ± 0.2 pF, n = 8 animals, 19 OHCs, measured at P10–P11).


A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection.

Taranda J, Maison SF, Ballestero JA, Katz E, Savino J, Vetter DE, Boulter J, Liberman MC, Fuchs PA, Elgoyhen AB - PLoS Biol. (2009)

Genetic Engineering and Genotyping of Chrna9L9'T Mutant Mice(A) Gene structures and restriction maps for the targeting construct, wild-type, and recombinant alleles (before and after removal of the neomycin resistance cassette) are shown. Black boxes represent the exons of the Chrna9 gene. The introduction of the mutation is shown by an asterisk (*).(B) An autoradiograph of a genomic Southern blot using DNA prepared from wild-type (left lane) and successfully targeted ES cells (right lane) and hybridized to a 32P-labled DNA probe prepared from the 962-bp SacII-KpnI fragment shown. Wild-type (WT) ES cell DNA yield a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yield a 7,300-bp fragment.(C) Routine genotyping of neo-deleted Chrna9L9′T mutant mice performed by PCR from isolated tail biopsy tissue genomic DNA samples and amplimers flanking the lox P footprint (white box). A representative gel indicating the results for wild-type, heterozygous, and homozygous knockin mice is shown. The PCR fragment length for the wild-type Chrna9 allele is 203 bp, and 269 bp for the mutant allele. MW, molecular weight.(D) A PCR fragment using amplimers flanking the L9′T mutation (Figure 1A) was obtained from genomic DNA isolated from tail biopsies and sequenced to check the integrity of the mutation in each breeding pair. A representative electropherogram indicating the results for wild-type and homozygous knockin mice is shown. The wild-type sequence at nucleotide position 210, corresponding to the codon at position 9′ in the second transmembrane domain, is TTG, encoding leucine; the mutant sequence is ACG, encoding threonine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2628405&req=5

pbio-1000018-g002: Genetic Engineering and Genotyping of Chrna9L9'T Mutant Mice(A) Gene structures and restriction maps for the targeting construct, wild-type, and recombinant alleles (before and after removal of the neomycin resistance cassette) are shown. Black boxes represent the exons of the Chrna9 gene. The introduction of the mutation is shown by an asterisk (*).(B) An autoradiograph of a genomic Southern blot using DNA prepared from wild-type (left lane) and successfully targeted ES cells (right lane) and hybridized to a 32P-labled DNA probe prepared from the 962-bp SacII-KpnI fragment shown. Wild-type (WT) ES cell DNA yield a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yield a 7,300-bp fragment.(C) Routine genotyping of neo-deleted Chrna9L9′T mutant mice performed by PCR from isolated tail biopsy tissue genomic DNA samples and amplimers flanking the lox P footprint (white box). A representative gel indicating the results for wild-type, heterozygous, and homozygous knockin mice is shown. The PCR fragment length for the wild-type Chrna9 allele is 203 bp, and 269 bp for the mutant allele. MW, molecular weight.(D) A PCR fragment using amplimers flanking the L9′T mutation (Figure 1A) was obtained from genomic DNA isolated from tail biopsies and sequenced to check the integrity of the mutation in each breeding pair. A representative electropherogram indicating the results for wild-type and homozygous knockin mice is shown. The wild-type sequence at nucleotide position 210, corresponding to the codon at position 9′ in the second transmembrane domain, is TTG, encoding leucine; the mutant sequence is ACG, encoding threonine.
Mentions: The targeting construct and generation of Chrna9L9′T mutant mice are described in the Materials and Methods section and Figure 2. Both Chrna9wt/L9′T and Chrna9L9′T/L9′T mutant mice were viable through adulthood, fertile, showed the expected gender and Mendelian genotype ratios, and exhibited no overt behavioral phenotype. Gross morphology of the cochlear duct was normal as seen in light microscopic evaluation of plastic sections of osmium-stained cochleae (Figure S1). As assessed by quantitative real-time PCR (RT-PCR), the expression level of cochlear nAChR α9 subunit mRNA was similar in Chrna9wt/wt and Chrna9L9′T/L9′T mice (Table S1). In addition, the expression levels of genes encoding proteins known to localize to the olivocochlear (OC) synaptic complex (for example, the nAChR α10 subunit and SK2 channel) were not changed in mutant mice (Table S1). Lastly, the electrophysiology of IHCs and OHCs was normal, as indicated by similar voltage-dependent K+ currents (Figure S2). Hair cell membrane area measured by capacitance was identical across phenotypes (Chrna9wt/wt: 8.3 ± 0.2 pF, n = 19 animals, 41 IHCs; Chrna9wt/L9′T: 8.4 ± 0.1 pF; n = 14 animals, 41 IHCs; Chrna9 L9′T /L9′T: 8.6 ± 0.1 pF, n = 26 animals, 79 IHCs, measured at P9–P11; Chrna9wt/wt: 8.0 ± 0.4 pF, n = 5 animals, 13 OHCs; and Chrna9 L9′T /L9′T: 8.1 ± 0.2 pF, n = 8 animals, 19 OHCs, measured at P10–P11).

Bottom Line: The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells.One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear.The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

ABSTRACT
The transduction of sound in the auditory periphery, the cochlea, is inhibited by efferent cholinergic neurons projecting from the brainstem and synapsing directly on mechanosensory hair cells. One fundamental question in auditory neuroscience is what role(s) this feedback plays in our ability to hear. In the present study, we have engineered a genetically modified mouse model in which the magnitude and duration of efferent cholinergic effects are increased, and we assess the consequences of this manipulation on cochlear function. We generated the Chrna9L9'T line of knockin mice with a threonine for leucine change (L9'T) at position 9' of the second transmembrane domain of the alpha9 nicotinic cholinergic subunit, rendering alpha9-containing receptors that were hypersensitive to acetylcholine and had slower desensitization kinetics. The Chrna9L9'T allele produced a 3-fold prolongation of efferent synaptic currents in vitro. In vivo, Chrna9L9'T mice had baseline elevation of cochlear thresholds and efferent-mediated inhibition of cochlear responses was dramatically enhanced and lengthened: both effects were reversed by strychnine blockade of the alpha9alpha10 hair cell nicotinic receptor. Importantly, relative to their wild-type littermates, Chrna9(L9'T/L9'T) mice showed less permanent hearing loss following exposure to intense noise. Thus, a point mutation designed to alter alpha9alpha10 receptor gating has provided an animal model in which not only is efferent inhibition more powerful, but also one in which sound-induced hearing loss can be restrained, indicating the ability of efferent feedback to ameliorate sound trauma.

Show MeSH
Related in: MedlinePlus