Design and Methods - Mutations with Hearing Impairment
High-throughput primary screen - Acoustic startle response (ASR) - We will use a clickbox to test for Preyer reflex (ear flick) of all G3 mice. We will carefully observe individual mice for a startle response to an intense auditory stimulus produced by the clickbox. The Preyer reflex, a suprathreshold response, does not measure thresholds and therefore can only be used to search for deaf or severely hearing-impaired mice. Its simplicity, however, allows for very high-throughput testing. Also, because it measures sound perception, it can detect both peripheral and CNS defects missed by screens that rely on only peripheral response, such as distortion product otoacoustic emissions (DPOE).
Automated, combined ASR/PPI/FPS - Developmental work is underway to establish and implement a new automated procedure that will allow the simultaneous assessment of hearing, prepulse inhibition (PPI - see below) and fear-potentiated startle (FPS - see Section 3.8). Acoustic pre-pulse inhibition (PPI) is a logical follow up test for mice that show a startle response. A moderately intense tone "prepulse" is presented 100 msec before an intense startle-eliciting sound, and the intensity of the prepulse stimulus can be varied to obtain an auditory profile. After ASR and PPI testing is completed, ABR thresholds will be obtained for each mouse to elucidate the relationship between PPI and hearing sensitivity. Impaired PPI may indicate CNS defects if the ABR is normal (patients with schizophrenia have impaired PPI). Depending on logistics and throughput, this test would replace the clickbox above, and would also be used in as described below as a follow-up screen for potential mutants in the ABR, or for those with obvious balance defects.
Medium throughput primary screen using the ABR - The ABR measures auditory-evoked activity in the brainstem that is commonly used for prediction of hearing levels in animals and young children. ABRs include several waves; the first represents cochlear and auditory nerve activity and the late waves reflect function within the central auditory pathways [177, 178]. Peak I of the mouse ABR is robust, whereas peak V is most robust in humans and peak V is frequently undetected in the mouse. The ABR is essentially unaffected by the subject's state of arousal or to the effects of the majority of sedative-hypnotic drugs and the commonly used anesthetics, even with doses that make patients comatose and render a "flat" EEG [179]. In mice, ABR thresholds have been used successfully as audiometric thresholds in hearing and genetic research [174, 175, 180-183]. The reliability, sensitivity, non-invasiveness and relative ease of application of ABR makes it a method of choice for assessment of hearing impairment in mice. However, because mice must be anesthetized, the test takes about 15 minutes per mouse and will only be used as a primary screen in a subset (about 1/3) of G3 mice. A description of our ABR testing procedure has been published [171].
Secondary characterization of heritable mutants
ABR - will be used as a follow-up screen for mice with poor startle response or balance defects.
Ear morphology - If hearing impairment is confirmed by ABR and is shown to be heritable, we will examine outer, middle, and especially inner ear structures for pathological abnormalities. These mice will be perfused and their temporal bones will be dissected, fixed, and stained. Whole-mount preps of middle and inner ears will be examined with a dissecting microscope to evaluate gross morphology, cross-section preps of inner ears will be examined with a light microscope to evaluate fine structure, and surface preps of the cochlea will be examined with a scanning electron microscope (SEM) to evaluate hair cell microstructure. Because auditory mutants often have vestibular defects, we will also examine the structure of the inner ear.
