Auditory Function, Startle Sensitivity & PPI
ABRs and DPOAEs will be recorded from anesthetized mice in specialized sound testing enclosures. For ABRs, the ear canal and hearing apparatus will be presented with 5-millisec tone pips and the response amplified (10,000 times), filtered (0.1–3 kHz), and averaged with an analog-to-digital board in a PC-based data-acquisition system. Sound level will be raised in 5 dB steps from 0 to 90 dB sound pressure level (decibels SPL). At each level, 1024 responses will be averaged (with stimulus polarity alternated) after “artefact rejection”. Threshold will be determined by visual inspection. The DPOAE in response to two primary tones of frequency f1 and f2 will be recorded at (2 x f1)–f2, with f2/f1= 1.2 and the f2 level 10 dB lower than the f1 level. Measurements will be performed using an ER-10C (Etymotics Research) acoustic system consisting of two sound sources and one microphone. Ear-canal sound pressure will be amplified and digitally sampled at 4-microsec intervals (16-bit DAQ boards, NI 6052E; National Instruments). Fast Fourier transforms (FFTs) will be computed from averaged waveforms of ear-canal sound pressure, and the DPOAE amplitude will be extracted at (2 x f1)–f2. The background level of ambient noise will be also measured, defined as the average of six points in the FFT on either side of the (2 x f1)–f2 frequency and surrounding noise background. Iso-response curves will be performed in 5 dB steps of f1 level. Threshold will be defined as the f1 level required to produce a DPOAE at zero dB SPL.
VESTIBULAR EVOKED POTENTIAL (VSEPS) RECORDINGS.
Vestibular evoked potential (VsEPs) recordings will be performed in anesthetized animals. Stimuli will be linear acceleration ramps, 2 ms duration, applied in the earth-vertical axis at 16 per second with alternating stimulus polarity. Electrophysiological activity will be amplified (10,000×), filtered (300 to 3000 Hz), and digitized (125,000 Hz), and 512 responses will be averaged at each stimulus level. We will collect an intensity series in 5 dB steps encompassing stimulus levels above and below threshold.
Several studies have demonstrated that pups removed from the nest emit distress calls at ~65 kHz or ~95 kHz, and that male mice vocalize to females at ~80 kHz. From postnatal day 3 pups the call development over their lifespan can be followed. We expect that the pup calls will drop off around weaning time (postnatal day 21) and that the adult calls will appear soon after this drop-off (postnatal day 45). We expect that the drop-off rate and post-natal age will be similar for both males and females.
SOUND PREFERENCE TEST:
Animals are placed in the center of an open arena (Noldus Phenotyper 4500) with up to three corners containing semi-opaque shelters of nesting materials. The arena is placed in a sound-attenuated chamber. A small microphone is positioned on top of each shelter, through which the sound stimulus is played. The behavior of mice is recorded for three hours with an infrared camera mounted on top of the arena, during which mice explore and choose to stay in a shelter (or shelters). The final nesting place typically stabilizes by the last half hour, indicating environmental preference (Jouhaneau & Bagady, 1984). The behavior is tracked and analyzed using Ethovision XT 8.5 (Noldus Information Technology, Netherlands).
To test for the level of startle response to various noise stimuli, animals can experience various acoustic stimuli ranging from 20 to 120 dB presented in a random order with a variable inter-trial interval e.g. average 15 sec (10-20sec). The duration of the noise stimulus is 40 msec. Responses are recorded for 150 msec from startle onset and are sampled every msec.
The phenomenon of pre-pulse inhibition is one where the startle response to a loud stimulus is lessened on repeated exposure to a quieter stimulus presented approx 10-100msec prior to the loud stimulus. This test can determine the sensory motor processing ability of an animal and is sensitive to antipsychotic compounds and manipulations of the CNS that have similarities to schizophrenia. Here is an example of a test procedure:
Animals are placed in the startle chambers for a 5 min session of white noise (70 dB) habituation. After this acclimation period the test session is automatically started. The session begins with a habituation block of 6 presentations of the startle stimulus alone, followed by 10 PPI blocks of 6 different types of trials. Trial types are: null (no stimuli), startle (120 dB), startle plus prepulse (Variable dB over background noise i.e. 74, 78 or 82 dB) and prepulse alone. Trial types are presented at random within each block. Each trial begins with a 50 ms null period during which baseline movements are recorded. There is a subsequent 20 ms period during which prepulse stimuli are presented and responses to the prepulse measured. After further 100 ms the startle stimuli are presented for 40 ms and responses recorded for 100 ms from startle onset. Responses are sampled every ms. The inter-trial interval is variable with an average of 15 s (range from 10 to 20 s). The percent prepulse inhibition will provide a measure of sensorimotor gating performance.