Table summarizes the procedures used in these studies as well as the age and number of mice used for each procedure. Details for each procedure follow.
The age, genotype, and number of mice used for each procedure
Construction of the GluRδ1 targeting vector and generation of GluRδ1 mutant mice.
We screened a bacterial artificial chromosome library (Research Genetics) containing mouse 129/Sv genomic DNA and obtained overlapping clones with average sizes of 150 kb. A 7-kb GluRδ1 genomic DNA fragment containing exon 11 (transmembrane domains 1 and 2 [TM1 and TM2]) and another 10-kb fragment containing exon 12 (TM3) were isolated, restriction mapped, and sequenced. TL-1 embryonic stem (ES) cells derived from the 129/SvEv strain were electroporated with linearized targeting vector. DNA from the ES cell line was digested with SpeI and analyzed by Southern blotting (Fig. ). One homologously recombined targeted cell line was obtained, and subsequent germ line transmission was achieved. We developed a PCR genotyping assay (30 cycles) with a pair of primers from the deleted region of the GluRδ1 gene (5′ GCAAGCGCTACATGGACTAC 3′ and 5′ GGCACTGTGCAGGGTGGCAG 3′) and a pair of primers from the targeting vector (5′ CCTGAATGAACTGCAGGACG 3′ and 5′ CGCTATGTCCTGATAGCGATC 3′). All mice analyzed were from a mixed background of 129/SvEv and C57BL/6 in the F2 to F6 generations. All mouse use protocols were approved by institutional animal care and use committees.
FIG. 1. Targeted disruption of the GluRδ1 locus. (A) Strategy for targeted deletion of the GluRδ1 gene. At the wild-type GluRδ1 locus, the boxes indicate exons 10 to 12; exon 11 encodes the predicted TM1 and TM2, and exon 12 encodes TM3 (more ...) Western blot analyses.
To confirm the ablation of the GluRδ1 protein in GluRδ1−/−mice, we performed Western blot analysis. Extracts from mouse inner ears, hippocampi, and cerebella containing 50 to 150 μg of protein were separated in a 3 to 8% NuPAGE Tris-acetate polyacrylamide gel (Novex) containing sodium dodecyl sulfate. After transfer, the polyvinylidene fluoride membrane (Immobilon) was treated with primary antibodies (rabbit GluRδ1/2 polyclonal antibody from Chemicon [catalog no. AB1514] and β-actin antibody from Sigma [catalog no. A5441]), horseradish peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech), and SuperSignal (Pierce).
Immunostaining and histologic analysis.
For the evaluation of molecular and morphological changes in GluRδ1−/− mice, mice were anesthetized with Avertin (500 mg/kg of body weight) or ketamine and xylazine (0.72/0.46 mg/30 g of body weight), followed by intracardial perfusions of 0.1 M phosphate-buffered saline and subsequently 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3). Cochleas were postfixed overnight and then decalcified in EDTA for 1 to 3 days. For whole-mount immunolabeling, cochleas were dissected, permeabilized with 1.0% Triton X-100 for 10 min, and incubated overnight in primary antibodies. The next day, the samples were placed in biotin-labeled secondary antibodies, a complex consisting of avidin, biotin, and horseradish peroxidase (ABC kit; Vector Laboratory), and then incubated in peroxidase substrate. For immunostaining of sections, decalcified cochleas were embedded in paraffin and sectioned into 12-μm thicknesses. Slides were deparaffinized. Nonspecific binding of secondary antibody was blocked by incubation with 10% goat serum in phosphate-buffered saline for 30 min at room temperature. Samples were then incubated in primary and secondary antibodies as described above. Samples were observed under a microscope (Olympus BX60). The primary antibodies used were Chemicon AB1514 for GluRδ1/2, Abnova H00002894-A01, specific for GluRδ1 (for hippocampal immunostaining), Santa Cruz SC22926 for Kir3.1, Sigma P6610 for Kv4.1, Santa Cruz SC16053 for the Na+-K+ ATPase β1 subunit (Atp1b1), Santa Cruz SC21547 for the Na+-K+-2Cl− cotransporter (Nkcc1), Chemicon MAB329 for synaptophysin, Sigma V5387 for the vesicular acetylcholine transporter (VAT), Chemicon AB197 for the calcitonin gene-related peptide (CGRP), and Chemicon AB5811P for SNAP25. For the anti-GluRδ1/2 antibody from Chemicon, lots produced before 2002 worked well in our immunostaining and Western blot analyses, but recently produced lots failed in immunostaining. In our hands, the various GluRδ1-specific antibodies (Abnova H00002894-A01 and Abnova H00002894-M01; kindly provided by R. Wenthold) did not result in immunostaining signals that were consistent and different between GluRδ1+/+ and GluRδ1−/− cochlear sections despite numerous attempts with a variety of conditions, including antigen retrieval.
For the immunostaining of SNAP25, synaptophysin, CGRP, or VAT, each dissected cochlear piece was measured by computerized planimetry and the cochlear location was converted to the frequency that is normally processed at that location (8
). To quantify immunopositive terminals, outlines were traced via a drawing tube using high-numerical-aperture objective lenses (total magnification, ×2,000). During tracing, fine focus was continually adjusted to optimize imaging of each terminal cluster. Traces were digitized, and areas were computed using NIH Image software. For the outer hair cell (OHC) area, all immunopositive terminals were traced and values from each row were averaged within bins corresponding to 100 μm of cochlear length.
For an assessment of histopathology, animals were anesthetized, followed by intracardial perfusion with 2.5% glutaraldehyde and 1.5% paraformaldehyde in phosphate buffer. Temporal bones were extracted, and round and oval windows opened for intralabyrinthine perfusion of fixative. Cochleas were then osmicated (1% OsO4 in dH2O), decalcified (0.1 M EDTA with 0.4% glutaraldehyde), dehydrated in ethanols and propylene oxide, embedded in Araldite resins, and sectioned at 40 μm on a Historange with a carbide steel knife. Sections were mounted on slides and coverslipped.
Laser capture microdissection of cochlear sections and reverse transcriptase PCR (RT-PCR) analysis.
For detailed expression analysis of GluRδ1 in the inner ear, laser capture microdissection was performed using the PixCell II system (Arcturus). We used a previously described method (32
) with some modifications. Briefly, the mice were anesthetized and intracardially perfused with 4% paraformaldehyde in phosphate buffer. Temporal bones were removed, and oval windows were opened for the injection of fixative. Cochleas were then postfixed overnight and decalcified in 0.1 M EDTA for 1 to 3 days. The cochleas were dehydrated in ascending concentrations of alcohol and embedded in paraffin. The embedded cochleas were sectioned into 12-μm thicknesses, and sections were mounted on uncharged slides (six sections on each slide). The sections were deparaffinized in xylene and dried at room temperature. We captured inner hair cells, outer hair cells, spiral ganglion cells, type I and IV fibrocytes, Deiters' cells, Claudius cells, Boettcher cells, inner sulcus cells, marginal cells, and vestibular hair cells from eight slides from each mouse. We pooled all of the cells in each category from different slides into a single tube. As a control, we scraped the whole sections from one slide into a tube. Three GluRδ1+/+
mice and one GluRδ1−/−
mouse were independently analyzed.
We used the Paradise whole-transcript RT reagent system (Arcturus, Mountain View, CA) to purify mRNA from both whole sections and laser-captured cells from cochlear sections. We made cDNA by reverse transcription from the mRNA using the same kit as above. For PCR, we designed four pairs of primers to amplify the cDNA of GluRδ1 (forward, 5′ ACCTCCTGGAATGGGATGAT; reverse, 5′ CCTCAGGCTTCTTGATGAGG), prestin (forward, 5′ AGTGGCTGCCAGCATATAAA; reverse, 5′ CGATGAGTACAGGCCAAACA), p27 (forward, 5′ ATTGGGTCTCAGGCAAACTCT; reverse 5′ GTTCTGTTGGCCCTTTTGTTT), and β-actin (forward, 5′ AATTTCTGAATGGCCCAGGT; reverse, 5′ TGTGCACTTTTATTGGTCTCAA). We used cDNA of P9 whole cochlea as a positive control and mRNA of P9 whole cochlea without reverse transcription as a negative control for PCR.
Assays of cochlear function.
For auditory brain stem responses (ABR), distortion product otoacoustic emissions (DPOAE), and endolymphatic potential (EP) measurements, mice were anesthetized with xylazine (20 mg/kg intraperitoneally) and ketamine (100 mg/kg intraperitoneally). For ABR, needle electrodes were inserted at the vertex and pinna, with a ground near the tail. ABR potentials were evoked with 5-ms tone pips (0.5-ms rise-fall with a cos2 onset envelope, delivered at a rate of 35/s). The response was amplified (10,000 times), filtered (100 Hz to 3 kHz), and averaged with an analog-to-digital board in a LabVIEW-driven data acquisition system. The sound level was raised in 5-dB steps from 10 dB below threshold to a 90-dB sound pressure level (SPL). At each sound level, 1,024 responses were averaged (with stimulus polarity alternated) using an artifact reject system, whereby response waveforms were discarded if the peak-to-peak amplitude exceeded 15 μV. The threshold was defined by visual inspection of stacked waveforms as the lowest SPL at which coherent responses were detectable at a latency consistent across levels.
The DPOAE at distortion frequency 2f1-f2 was recorded with a custom acoustic assembly consisting of two one-quarter-inch condenser microphones to generate primary tones of different frequencies (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 (National Instruments; catalog no. 6052E), and the maximum level of stimuli for DPOAE was 80 dB SPL. Ear canal sound pressure was amplified and digitally sampled at 4 μs. Fast Fourier transforms were computed from averaged waveforms of ear canal sound pressure, and 2f1-f2 DPOAE amplitude and the surrounding noise floor were extracted. Noise floors ranged from −25 to −5 dB SPL, depending on frequency. Isoresponse contours were interpolated from amplitude-versus-level functions performed in 5-dB steps of primary level.
For EP measurement (on a separate cohort of animals), the bulla was opened, exposing the cochlea, and the bone of the otic capsule over the basal turn was opened using a small knife. The spiral ligament and stria vascularis were left intact. A glass micropipette electrode (20 MΩ) filled with 150 mM KCl was introduced into the opening of the cochlea using a Kopf micropositioner. The signal was amplified 10-fold and read by custom software. The EP was considered valid if (i) there was a rapid rise in voltage (>25 mV per 3-μm advance), (ii) the peak voltage remained stable for 30 s or more, and (iii) the voltage returned to 0 when the electrode was retracted.
For the evaluation of the vulnerability of GluRδ1−/− mice to acoustic injury, mice were exposed, awake, and unrestrained, within cages suspended inside a small reverberant sound exposure box. The exposure stimulus was generated by a custom white-noise source, filtered (Brickwall filter with a 60- dB-octave slope), amplified (Crown power amp), 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 one-quarter-inch Bruel and Kjaer condenser microphone; sound pressure was found to vary by less than 0.5 dB across these measurement positions. Sound pressure was calibrated by positioning the microphone at the approximate position in relation to the animal's head. Mice were exposed for 2 h to the octave band noise (8 to 16 kHz) at 89 dB SPL.
Assays of vestibular function.
For vestibular function in GluRδ1−/− mice, we employed various behavioral and electrophysiological vestibular tests. In the Rotarod test, the rod (San Diego Instruments) rotated at speeds increasing in 5-rpm increments from 0 to 20 rpm and the retention time of the mice was recorded. Mice were tested in four trials each day at the same hours of the day over 4 days. For the swim test, each mouse was placed in a glass aquarium filled with tepid water. The time required for the mouse to surface and to maintain a horizontal bodyline swimming at the surface was recorded.
For linear vestibular evoked potential (VsEP) measurements, experimenters were blinded to the genotype during data collection and analysis. Animals were anesthetized (Equithesin, 4 μl/g intraperitoneally), and each skull was prepared with a head mount. A thumbscrew was secured at the midline, and two additional electrodes were placed behind the left and right pinnae with a ground at the ventral neck. The animals were placed supine, and each head was secured to an electromechanical shaker with the naso-occipital axis oriented vertically. Stimuli were linear acceleration pulses (2-ms duration; 16 pulses/s) presented in two directions, normal and inverted. Normal polarity was defined as upward displacement, while inverted stimuli displaced the platform downward. Stimulus amplitude was measured in jerk (i.e., g
/ms, where 1.0g
= 9.8 m/s2
/ms = 9.8 μm/ms3
]) using a calibrated accelerometer attached to the shaker platform. To monitor the jerk component of the stimulus, the output of the accelerometer was differentiated electronically. Stimulus amplitude was recorded in dB re: 1.0g
/ms and ranged from −18 to +6 dB re: 1.0g
/ms, adjusted in 3-dB steps. Electrophysiologic activity was amplified (200,000-fold) and filtered (300 to 3,000 Hz), and VsEPs to normal and inverted stimulus polarities (1,024 points, 10 μs/point, 128 responses per averaged waveform) were recorded. Four waveforms were obtained at each stimulus intensity level, two for normal polarity stimuli and two for the inverted polarity. Averaging of responses to normal and inverted polarities was completed offline to produce the final averaged waveforms used for analysis. Three response parameters were quantified: threshold, peak latencies, and peak-to-peak amplitudes. The threshold measured in dB re: 1.0 g
/ms was defined as the stimulus amplitude midway between that which produced a discernible VsEP and that which failed to produce a response. Thresholds, latencies, and amplitudes were compared among the three groups of animals using one-way analysis of variance (ANOVA) (thresholds) and multivariate ANOVA (latencies and amplitudes) with a significance level of P
less than 0.05.
For the evaluation of the role of GluRδ1 in synaptic transmission and synaptic plasticity, hippocampal slices were prepared from GluRδ1−/− and GluRδ1+/+ male mice without prior knowledge of mouse genotype. Slices were continuously superfused with artificial cerebrospinal fluid containing 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose, with 95% O2 and 5% CO2 at 30 to 31°C (2 ml/min). Schaffer collateral synapses were stimulated with a bipolar tungsten electrode in CA1 stratum radiatum placed 100 to 150 μm from the recording pipette, and field excitatory postsynaptic potentials (fEPSPs) were collected using a MultiClamp 700B amplifier (Molecular Devices). To ensure equivalent activation of postsynaptic neurons in all experiments, stimulation intensities were chosen to evoke an fEPSP with a slope of approximately 1 mV/ms. In long-term potentiation (LTP) experiments, Schaffer collaterals were stimulated at 0.033 Hz before and after the induction of LTP. LTP was induced by a 200-Hz pulse protocol consisting of 10 trains of 200 ms of stimulation at 200 Hz delivered every 5 s at the baseline stimulation intensity. Data were analyzed using Clampfit 9.0 software (Molecular Devices). Results were grouped according to mouse genotype.
Morris water maze test.
For an examination of defects in LTP in GluRδ1−/− mice in vivo, mice of three genotypes were tested in a water maze that consisted of a circular blue plastic tank, 160 cm in diameter and 38 cm deep. The maze was located in a large test room surrounded by external cues that could be used for spatial navigation. The tank was filled to 30 cm with water at 21°C made opaque by the addition of a small quantity of nontoxic white paint (tempera). The platform, a 10-cm square of Plexiglas covered with a rough green plastic scouring pad, was mounted on a solid column 1 cm below the surface such that it could not be seen from water level. Four equally spaced points around the edge of the tank were used as start positions and divided the maze into four quadrants. During the acquisition of the place task, the platform was in the middle of one quadrant, equidistant between the center and the outer wall of the maze. Mice were trained for one block of four trials on each of 10 consecutive days. Within each block of trials, all four start positions were used once each in a pseudorandom sequence. For each trial, a mouse was placed in the water facing the wall at the start position. The time required to find the escape platform was recorded. Any mouse failing to find the platform within 60 s was placed on the platform. Approximately 10 min separated the individual trials in each day's block of tests.