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Supplemental Data 1: Further characterization of hearing in CPX KO mice
1. ABR amplitudes and latencies, temperature-sensitivity in CPX I knock-out mice
A detailed analysis of ABR waveforms in response to click stimuli in young and adult
CPX mice demonstrates that both the decrease in ABR peak amplitudes (Fig. S1 A1-A4)
and the increase in latencies (Fig. S1 B1-B2) are evident over the whole intensity range
and progress with age. Unlike in β4Σ1-spectrin knock-out mice, having a defect in action
potential generation and propagation (Lacas-Gervais et al., 2004), the increase in ABR
latencies upon higher stimulus rate of 90 Hz was not different between CPX I-/- mice and
wild-type littermates (data not shown). This absence of auditory fatigue is consistent with
in vivo and in vitro data showing that the main synaptic deficiency in CPX I-/- animals
concerns onset coding, not sustained activity. However, the latency increase that is
normally associated with hypothermia appeared more pronounced in CPX I-/-- than in
CPX I+/+ mice for ABR peaks II-V (Fig. S1 C), yielding steeper temperature-latency
functions and suggesting a stronger susceptibility of synaptic transmission to
temperature- induced changes e.g. in ion currents.
Legend
A1-A4, amplitudes of ABR peaks I-IV in CPX I-/- (grey) and CPX I+/+ (black) mice aged
3-4 (open squares, n(CPX I-/-)=7, n(CPX I+/+)=6)) and 6-10 weeks (filled circles, n(CPX
I-/-)=6, n(CPX I+/+=9)). B1-B2, latencies of ABR waves I-IV in the same animals. C
Dependence of latencies of ABR peaks I-IV in response to 100dB click stimuli on body
temperature in CPX I-/- (grey, n=5) and CPX I+/+ (black, n=6) animals aged 6-14 weeks.
2. Normal hearing upon deletion of complexin isofoms CPX II-IV
Legend
Average ABR thresholds ±
SEM (left) and waveforms
(right) in KO animals for CPX
isoforms II (A, n=4 each) III (B,
n=5, each) and IV (C, n=5 each)
and their respective littermate
controls. We did not observe
significant changes in ABR
thresholds. Individual ABR
traces are shown in pink (KO)
and grey (WT), averaged traces
in red (KO) and black (WT).
There were no significant
differences in ABR thresholds,
amplitudes, or latencies. (D)
Average ABR thresholds ±
SEM (left) and waveforms
(right) in CPX III/IV double KO
animals (individual waveforms
pink, averaged traces red) and in
non-littermate CPX III and CPX
IV WT controls (grey/black
n=10).
Experimental procedures
ABR were evoked by 12 ms tone-bursts or clicks (duration of 0.03 ms) presented at a
stimulation rate of 20 Hz. The difference potential between subdermal needles at the
mastoid and the vertex was amplified 50,000 times by a custom amplifier (JHM
NeuroAmp 401), filtered (0.4 kHz high-pass, 4 kHz low-pass), and averaged (2 x 2000
sweeps) to obtain two mean traces at each intensity. Hearing threshold was determined by
visual inspection as the lowest stimulus intensity that evoked a reproducible response
waveform in both traces. The peak latencies and amplitudes (peak to trough) of peaks IIV were obtained from individual ABR waveforms in response to click stimuli over an
intensity range of 10-80dB. Peak V was not assessed in detail because of its small
amplitude.
To evaluate the temperature-sensitivity of synaptic transmission in the absence of CPX I,
anesthetized mice aged 7-14 weeks were kept at room temperature until a rectal
temperature between 29.5°C and 32°C was reached. The mouse was then placed on the
heat pad under a heating lamp and auditory brainstem responses to 100dB click stimuli
were repeatedly recorded at 4-7 different temperatures until a rectal temperature of 37°C
was reached. The latencies of ABR peaks I-IV at 30, 32, 34, 36 and 38°C were inter/extrapolated from linear fits to the resulting individual temperature-latency functions.
For ASSR, sinusoidally amplitude modulated tones were applied. They consisted of
carrier tones of 12 kHz, which were amplitude modulated at a modulation depth of 100%
and continuously presented for 1 minute at a sound intensity between 0 and 100 dB SPL
(10 dB steps). The electric potentials between mastoid and vertex were filtered digitally
(50 Hz Notch and 60 Hz high-pass), sampled and converted into the frequency domain by
Fast Fourier Transformation (FFT) with a resolution of 0.016 Hz. The amplitude of the
ASSR was determined as the FFT peak at the applied MF and was corrected for the
background noise estimated in the vicinity of the MF (± 10Hz around MF). Before
recording ASSR thresholds, the complete MF range from 100 to 1400 Hz was scanned
(16 sec for each MF) in 29 Hz steps with a suprathreshold intensity stimulus of 80 dB to
detect the individual MF ranges with the best ASSR to noise ratios. Hearing threshold
was defined as the lowest sound intensity at which the peak of the ASSR was greater than
the background noise (see above) by two standard deviations.
For DPOAE, a 24-bit sound card and the ED1/EC1 speaker system (Tucker-Davis) were
used to generate two primary tones with a frequency ratio of f2=1.2xf1. Primary tones
were coupled into the ear canal by a custom-made probe containing an MKE-2
microphone (Sennheiser, Hannover, Germany) and adjusted to an intensity of 60 dB
sound pressure level at the position of the ear drum as mimicked in a mouse ear coupler.
The microphone signal was amplified (DMP3, MIDIMAN) and analyzed by FFT.