Exaggerated Startle Reflex to Low Frequency Tone Bursts in C57BL/6J Mice with Progressive Hearing Loss: A Longitudinal Aging Study Jim Ison, Peter Rivoli, Jason Moore, and Paul Allen Brain and Cognitive Sciences, University of Rochester, Rochester NY USA ARO 2005 #430 Methods Subjects: C57BL/6J mice (n = 12 survivors of 16) were tested fortnightly from 10 weeks to 52 weeks of age (ASR), from 6 to 54 weeks (ABR). Startle stimuli and recording: The mice were held in a wire cage mounted to a platform over an accelerometer (See Figure 1). Startle eliciting stimuli (ES) were delivered from overhead: 15 ms duration + 5 ms rt/ft; 0, 60, 70, 80, 90, 100, 110 dB; 3, 6, 12, and 24 kHz; 8 trials each condition; at 15 s intervals. Overview The C57BL/6J mouse cochlea progressively degenerates beginning at about 2 months of age. Some data suggest a small initial functional deficit across the spectrum (and see Figure 2), but the major loss in sensitivity appears first for high frequency hearing then gradually spreads to lower frequencies (see Figure 2 again, and also Figure 3). In a prior between-group study (JARO, 2003) the loss of high frequency hearing (ABR thresholds) was associated with a progressive increase in the amplitude of the acoustic startle reflex (ASR) to low-frequency tone bursts even as the ASR to high-frequency tones declined. Here we replicate these findings in a 10-month long longitudinal design that allows better tracking of the within-subject association of ABR measures of hearing sensitivity with increased low-frequency ASR. This experiment confirms the prior data in its showing that individual differences in hyper-reflexia are related to the degree of hearing loss, and extends them in showing that the effect is present in every mouse. The strength and uniformity of hyper-reflexia across mice suggests the utility of this animal model for studying the functional effects of the neural reorganization that accompanies hearing loss in C57 mice. Loss of ABR thresholds from 6 to 54 weeks of age, across frequency Initial loss in 110 dB ASR in all frequencies, then gains at 3 and 6 kHz beyond 23 weeks Initial ASR correlates with later ASR: and ABR threshold losses correlate with the residuals Supported by NIA Grant #AG09524 Figure 1: The mouse is shown in the test cage located directly under the ES speaker. The pedestal fits onto a stiff platform to which an accelerometer is attached. ABR stimuli and recording: Beginning at 6 or 8 weeks of age, and continuing to 54 weeks, fortnightly ABR tests (and DPOAE tests, not reported here) were interspersed with ASR tests. Measurements were made from Avertin anaesthetized mice using needle electrodes at the vertex and mastoid, and hind limb. Tone-pip stimuli (5ms; 3, 6, 12, 24, 32, 48 kHz; dB SPL) were generated and ABR waveforms measured (11/s, duplicates of 100 reps) using TDT System II and 3 DSP workstations in conjunction with BioSig. Stimulus level was reduced by 10 dB then the threshold (visible 4.5 complex) bracketed in 5 dB steps. Figure 2: At Week 6 ABR thresholds (3 to 48 kHz in order) were 51; 27; 8; 25; and 26 dB SPL. The overall loss of sensitivity appears to result from two linear processes, the first having a slope of less than 1 dB/week, the second a slope of 3 dB/week or more; however the calculated slope of the second process is in part an artifact of its onset being variable across mice (see individual mice, Figure 5) Loss of 90 dB ABR P1/N1 amplitude from 6 to 28 weeks of age, across frequency Figure 3: Note parallel changes in P1/N1 amplitude, compared to threshold changes. Neither ABR thresholds nor amplitudes correlated with the early ASR across mice. (Inset is an example of the replicated 12 kHz 90 dB ABR waveform at 6 weeks of age in S#973) Figure 4: At 10 weeks of age the most vigorous ASR is for 12 kHz, as might be expected from the ABR thresholds and amplitudes. The ASR to 3 and 6 kHz, relative to 24 kHz, is better than their relative ABR levels. The ASR first declined across all frequencies, similar to the Figure 2 ABR, but then increased beyond Week 23 for 3 kHz and 6 kHz stimuli (similar effects were obtained for lower ES levels). Figure 5: Data from four mice - note amplitude gain from early to late ASR, and also that 24, 32, and 48 kHz ABR losses always preceded ASR gain, while ASR gain always preceded 12 kHz ABR losses. Figure 6: In A, the late (>23 wk) ASR levels (3 & 6 k, 100 & 110 dB) correlate with the early (10-12 wk) ASR level. This accounts for about 50% of the late ASR variance between mice. Next, in B, the overall early (10-30 wk) high frequency ABR thresholds (24-48 kHz) account for 36% of the residual variance in the late ASR left over from A. Finally, in C, the late (> 30 wk) 12 kHz ABR threshold accounts for about 36% of the variance in the residual late ASR variance left over from B. These data support the hypothesis that low-frequency ASR hyperreflexia results from hearing loss at higher frequencies.