Genetic engineering and genotyping of Chrna9L9′T knockin mutant mice.
A HindIII-NotI (the latter derived from the construction of the library) restriction endonuclease fragment of approximately 9,500 bp encoding Chrna9 exons 1–4 (see GenBank accession number NT_039305.7 and A) was obtained from a mouse strain 129S4/SvJae genomic DNA library (kindly provided by Dr Bernhard Bettler, University of Basel) and subcloned into the vector pKO-Select DT (Lexicon Genetics). A 2-kbp neomycin resistance cassette flanked by two loxP sites (loxP-neo-loxP) was inserted in the NcoI restriction site within the intron located between exons 3 and 4 and the Chrna9L9′T mutation (*) introduced via site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis kit (Stratagene) and amplimers A9sense (5′-CTCTGGGAGTGACCATCCTAacGGCCATGACTGTATTTCAGC-3′) and A9antisense (5′-GCTGAAATACAGTCATGGCCgtTAGGATGGTCACTCCCAGAG-3′). This targeting vector was used to electroporate 129S4/SvJae embryonic stem (ES) cells, and homologous recombinants were obtained following gentamicin (G418) selection and Southern blot hybridization analyses. Genomic DNA was purified from G418-resistant ES cell clones, digested with HindIII and KpnI, electrophoresed on 0.8% agarose, and hybridized to a 32P-labled DNA 962-bp SacII-KpnI fragment probe prepared from the DNA fragment shown in A. Based on the sequence of the mouse Chrna9 subunit gene (see GenBank accession number NT_039305.7), wild-type ES cell DNA yielded a fragment of 13,800 bp, whereas ES cells that have undergone homologous recombination yielded a 7,300-bp fragment (see B). Transfection of the linearized targeting vector into murine ES cells resulted in the insertion of the L9′T mutation into the α9 nAChR subunit. The frequency of homologous recombination events in ES cells was 42%. Six independent ES cell lines carrying the mutation were injected into blastocysts to generate germline chimeric males and were then implanted into pseudopregnant females. Chimeric male progeny mice were backcrossed to strain C57BL/6J females, and agouti coat color was used to assess the germline status of the targeted allele. Founder males were backcrossed to wild-type 129S4/SvJae females and heterozygous N1 females mated to cre-expressing transgenic males (FVB/N-Tg(EIIa-cre)C5379Lmgd/J, stock 003314; Jackson Laboratory) to remove the “floxed” neomycin resistance cassette. The excision of the neo cassette and subsequent segregation and loss of the cre transgene were monitored via PCR. The single loxP site footprint and flanking regions of the targeted allele in neo-deleted mice were confirmed by DNA sequencing.
The Chrna9L9′T mutant allele has been maintained in congenic FVB.129P2-Pde6b+ Tyrc-ch/AntJ (stock number 004828; Jackson Laboratory) strain. C57BL/6J mice develop a marked and progressive late-onset hearing loss characterized by cochlear degeneration. The FVB background lacks this hearing loss. Moreover, the wild-type Pde6b allele avoids blindness due to retinal degeneration typical of the FBV strain. All experiments reported in this paper were performed using neo-deleted Chrna9wt/wt, Chrna9wt/L9′T, or Chrna9L9′T/L9′T mutant mice backcrossed with congenic FVB.129P2-Pde6b+ Tyrc-ch/AntJ stock for four to five generations (i.e., N4–N5).
Routine genotyping of Chrna9 mice was performed using tail biopsy tissue DNA samples (Wizard Genomic DNA Purification kit; Promega), amplimers A9LOXP.1 (5′-TAC TGG CTA TCC TCC AGA CAG AGC-3′) and A9LOXP.2 (5′-AGG AGC GAG CAG AGG TCC TAA ATT-3′) (see A), and Failsafe PCR System Kit with buffer D (Epicentre) as described by the manufacturer. PCR cycle parameters were: 95 °C, 0.5 min; 55 °C, 1.0 min; and 72 °C, 2 min for a total of 35 cycles. Reaction products were electrophoresed on 1.5% agarose, stained with ethidium bromide, and photographed. The PCR fragment length for the wild-type Chrna9 allele is 203 bp and 269 bp for the mutant allele (see C). In each breeding pair, the mutant status of the Chrna9L9′′T was assessed by sequencing of the PCR fragment obtained with amplimers L9T5′ (5′-CTCTCTGACTTCATTGAAGACG-3′) and L9T3′ (5′-CCGCACACATACAGGGTTCGAT-3′) (D).
Electrophysiological recordings from cochlear hair cells.
Mice were sacrificed by decapitation. All experimental protocols were carried out in accordance with the American Veterinary Medical Associations' AVMA Guidelines on Euthanasia
(June 2007). Apical turns of the organ of Corti were excised from mice at postnatal ages P6–P11 for IHCs and P10–P11 for OHCs, and used within 3 h. Day of birth was considered postnatal day 0 (P0). Cochlear preparations were mounted under a Leica DMLFS microscope (Leica Microsystems) and viewed with differential interference contrast (DIC) using a 40× water immersion objective and a Hamamatsu C7500–50 camera with contrast enhancement (Hamamatsu). Methods to record from IHCs and OHCs were essentially as described previously [17
Briefly, hair cells were identified visually with the 40× objective and during recordings, by the size of their capacitance (7 to 12 pF), by their characteristic voltage-dependent Na+
]. Some cells were removed to access IHCs, but mostly, the pipette moved through the tissue under positive pressure. The extracellular solution was as follows (in mM): 155 NaCl, 5.8 KCl, 1.3 CaCl2
, 0.9 MgCl2
, 0.7 NaH2
, 5.6 d
-glucose, and 10 Hepes buffer (pH 7.4). Two different pipette solutions were used. One contained (in mM): 150 KCl, 3.5 MgCl2
, 0.1 CaCl2
, 5 ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′- teraacetic acid (EGTA), 5 Hepes buffer, 2.5 Na2
ATP, pH 7.2 (KCl-EGTA saline). In the other, EGTA was replaced with 10 mM bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA) (KCl-BAPTA saline). The latter solution was used where indicated, in order to record the current through the ACh receptor in isolation from the coupled SK2 current (nAChR-only). In addition, in the latter condition, the SK blocker apamine (5 nM) was added to the recording solution.
Solutions containing ACh or high K+
were applied by a gravity-fed multichannel glass pipette (~150 μm tip diameter), positioned about 300 μm from the recorded cell. All working solutions containing either ACh or elevated K+
or both, were made up in a saline containing low Ca2+
(0.5 mM) and no Mg2+
so as to optimize the experimental conditions for measuring currents flowing through the α9α10 receptors [38
]. sIPSCs were recorded immediately after rupturing into the cell, in the extracellular saline containing 1.3 mM Ca2+
and no Mg2+
. All experiments designed to record synaptic currents, either spontaneous or evoked with high K+
, were done using an extracellular solution containing 1.3 mM Ca2+
and no Mg2+
. sIPSCs were recorded immediately after rupturing the cell.
Glass pipettes, 1.2-mm I.D., had resistances of 5–8 MΩ. Currents in both IHCs and OHCs were recorded in the whole-cell patch-clamp mode with an Axopatch 200B amplifier, low-pass filtered at 2–10 kHz, and digitized at 5–20 kHz with a Digidata 1322A board (Molecular Devices). Recordings were made at room temperature (22–25 °C). Holding potentials were not corrected for liquid junction potentials or for the voltage drop across the uncompensated series resistance. IPSCs were analyzed with Minianalyis (Synaptosoft; Jaejin Software). IPSCs were identified using a search routine for event detection and confirmed by eye. The individual events of each cell were aligned and averaged using Minianalysis. Rise time was used as the criterion to align. For further analysis, Prism 4 (GraphPad Software) was used. The τrise and τdecay were fit to a monoexponential.
Prolonged agonist application responses and concentration-response curves to ACh in IHCs were performed in the nAChR-only condition, normalized to the maximal agonist response, and iteratively fitted with the equation: I/Imax = An/(An + EC50n), where I is the peak inward current evoked by agonist at concentration A; Imax is current evoked by the concentration of agonist eliciting a maximal response; EC50 is the concentration of agonist inducing half-maximal current response, and n is the Hill coefficient.
Auditory brainstem responses and distortion product otoacoustic emissions.
Mice were anesthetized with xylazine (20 mg/kg i.p.) and ketamine (100 mg/kg i.p.). DPOAEs at 2f1–f2 were recorded with a custom acoustic assembly consisting of two electrostatic drivers (TDT EC-1; Tucker-Davis Technologies) to generate primary tones (f1 and f2 with f2/f1 = 1.2 and f2 level 10 dB < f1 level) and a Knowles miniature microphone (EK3103) to record ear-canal sound pressure. Stimuli were generated digitally, while resultant ear-canal sound pressure was amplified and digitally sampled at 4 μs (16-bit DAQ boards, NI 6052E; National Instruments). Fast Fourier Transforms were computed and averaged over five consecutive waveform traces, and 2f1–f2 DPOAE amplitude and surrounding noise floor were extracted. Iso-response curves were interpolated from plots of amplitude versus sound level, performed in 5-dB steps of f1 level. “Threshold” is defined as the f1 level required to produce a DPOAE with amplitude = 0 dB sound pressure level (SPL). For measurement of ABRs, needle electrodes were inserted at vertex and pinna, with a ground near the tail. ABRs were evoked with 5-ms tone pips (0.5-ms rise-fall, cos2 onset, at 35/s). The response was amplified (10,000×), filtered (100 Hz–3 kHz), and averaged with an A-D board in a LabVIEW-driven data-acquisition system. Sound level was raised in 5-dB steps from 10 dB below threshold to 80 dB SPL. At each level, 1,024 responses were averaged (with stimulus polarity alternated), using an “artifact reject” whereby response waveforms were discarded when peak-to-peak amplitude exceeded 15 μV. Upon visual inspection of stacked waveforms, “threshold” was defined as the lowest SPL level at which any wave could be detected, usually the level step just below that at which the response amplitude exceeded the noise floor (~0.25 μV). For amplitude versus level functions, wave-I peak was identified by visual inspection at each sound level and the peak-to-peak amplitude computed.
Mice were anesthetized with urethane (1.20 g/kg i.p.) and xylazine (20 mg/kg i.p.). A posterior craniotomy and partial cerebellar aspiration were performed to expose the floor of the IVth ventricle. To stimulate the MOC bundle, shocks (monophasic pulses, 150-μs duration, 200/s) were applied through fine silver wires (0.4-mm spacing) placed along the midline, spanning the olivocochlear decussation. Shock threshold for facial twitches was determined, muscle paralysis induced with α-d-tubocurarine (1.25 mg/kg i.p.), and the animal connected to a respirator via a tracheal cannula. Shock levels were raised to 6 dB above twitch threshold. During the MOC suppression assay, f2 level was set to produce a DPOAE 10–15 dB or 20–25 dB greater than the noise floor. To measure MOC effects, repeated measures of baseline DPOAE amplitude were first obtained (n = 56), followed by a series of 70 contiguous periods in which DPOAE amplitudes were measured with simultaneous shocks to the MOC bundle and additional periods during which DPOAE measures continued after the termination of the shock train.
Animals were exposed free-field, in a small reverberant chamber. Acoustic trauma consisted of a 2-h exposure to an 8–16-kHz octave band noise presented at 100 dB SPL (for permanent injury) or a 15-min exposure to the same noise at 94 dB SPL (for temporary injury). For the higher level exposure, animals were anesthetized (ketamine and xylazine, exactly as for the ABR and DPOAE testing), because many mutant animals experienced audiogenic seizures as soon as the high-level noise was turned on. The exposure stimulus was generated by a custom white-noise source, filtered (Brickwall Filter with a 60-dB/octave slope), amplified (Crown power amplifier), and delivered (JBL compression driver) through an exponential horn fitted securely to a hole in the top of a reverberant box. Sound exposure levels were measured at four positions within each cage using a 0.25'' Bruel and Kjaer condenser microphone: sound pressure was found to vary by less than 0.5 dB across these measurement positions.
Statistical analyses of in vitro electrophysiology experiments were carried by the Student t-test in the case of pairwise comparisons or a one-way ANOVA followed by a Dunnet test for multiple comparisons. In the case of in vivo data, a two-way ANOVA was performed. A p < 0.05 was selected as the criterion for statistical significance. Mean values are quoted as means ± the standard error of the mean (S.E.M.).
ACh chloride, strychnine HCl, Na2ATP, BAPTA, and all other reagents were from Sigma Chemical. EGTA and Na2ATP were dissolved at the moment of preparing the intracellular solutions.
All experimental protocols were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals as well as Instituto de Investigaciones en Ingeniería Genética y Biología Molecular (INGEBI), Tufts University, and Massachusetts Eye and Ear Infirmary Institutional Animal Care and Use Committee (IACUC) guidelines, and best practice procedures.