NORMAL THRESHOLD AND SUPRATHRESHOLD ABR AND ASR RESPONSES TO ACOUSTIC ONSETS IN KCNA1 KNOCKOUT MICE, BUT A REDUCED RESPONSE TO OFFSETS J. R. Ison 1, P. D. Allen 1, J. P. Walton 2, W. J. Bowers 3, & R. D. Frisina 2 1 Brain and Cognitive Sciences, 2 Otolaryngology, & 3 Neurology, University of Rochester ARO Threshold and Suprathreshold ABRThreshold and Suprathreshold Acoustic Startle Reflex and Startle Inhibition by Gaps Temporal acuity in the auditory system - its high rates of firing, its sensitivity to input synchrony, and its fast time constants - is founded on the presence of fast acting potassium channels at the synaptic junctions that are responsible for maintenance and recovery of the resting potentials (Trussell,1999; Oertel, et al. 2000). Cells in regions of the auditory brainstem known to be critical for temporal acuity are heavily invested with certain types of these channels (Grigg et al. 2000). Slice preparations in vitro using both specific neurotoxins to block different K+ channels and knockout mice lacking certain channels have confirmed that the temporal precision in the CN and MNTB depends on the presence of functioning Kv1.1 channels (Kopp-Scheinpflug et al. 2001), and that the upper frequency at which cells in the MNTB are able to follow periodic stimuli is lower in Kv3.1 KO mice compared to wild-type (Macica, et al. 2000). Here we describe several aspects of auditory function in Kv1.1 KO mice. The study was motivated in part by the obvious need to confirm and extend the in vitro findings in an in vivo preparation. In addition the theoretical functions ascribed to potassium channels resemble those in which aged listeners appear deficient, in, e.g., the inability to follow acoustic transients. As part of this work we have begun to characterize the expression of the Shaker and Shaw potassium channels in the auditory brainstem of old CBA mice. Conclusions The young Kv1.1 knock out mice has much the same response to the onsets of tonal stimuli and to noise as the wild type mouse. Hearing thresholds and auditory nerve latencies to tone pips as measured in the ABR are virtually identical, save for a slight improvement in the hearing threshold for the lowest frequency of 3 kHz. Startle reaction amplitudes are comparable in all three genotypes across a range of spectral frequencies of 4 to 32 kHz, and stimulus levels of 60 to 120 dB SPL. "Spontaneous" activity measured in the startle chamber in quiet and in noise was greater in the knockout compared to the wild type mouse. Inhibition of the startle by noise offset or gaps in a noise background are reduced in the knockout mice, though the time constants for the growth of inhibition are not substantially changed. A small but important difference in the effect of noise offset is that the difference between a sharp decrement in the noise and a slow ramp for the same lead time emerges at a later time in the knockout mice, suggesting that neural firing may continue in the absence of stimulation for a longer time in the knockout mouse. The heterozygous +/- mouse was sometimes intermediate, but more resembled the +/+ wild type. Old mice show increased thresholds and increased response latencies on hearing tests, and a severe reduction in responsivity in startle reflex tests. The Kv1.1 knockout mouse shows none of these deficits. Old mice also show deficits in the asymptotic inhibitory effects of gaps in temporal acuity experiments, and particularly the difference in the inhibitory effect of an abrupt noise offset and a ramped noise offset is slower to appear in the older mouse. There are two possible explanations of these common effects, which are not exclusive. First, the reduction in the asymptotic effect of a gap could result because of constant background neural activity in the auditory system, in effect providing the "noise floor" that is known to reduce gap salience. And second, increased variance in neural firing at higher levels of the auditory system may change a coherent neural signal with sharp onset and offset boundaries into a broader band of neural firing with a less decisive offset. This would duplicate the behavioral effect of a ramped noise offset, which also reduces gap salience. The similarities between Kv1.1 knockout mice and old mice (and their dissimilarities) encourages the search for further comparable effects in these mice as well as in other potassium channel knockout mice (and see the adjoining poster on near field evoked potentials to gaps and SAM stimuli in these three genotypes). It should also encourage a more intensive genetic analysis of the possibly changing expression of the various species of potassium channels in young, middle aged, and senescent mice. References: Davis et al. (2001) ARO Abstracts 24:167; Gittelman et al. (2001) ARO Abstracts 24:197. Grigg et al. (2000) Hear Res 140: ; Kopp-Scheinpflug et al. (2001) ARO Abstracts 24:196. Macica, et al. (2000) SFN Abstracts 26:705; Oertel D (2000) PNAS, 97: Schmidt et al. (1999) Brain Res 843:45-60; Trussell LO (1999) Ann Rev Phys 61: [ARO:2002] Research supported by NIA, AG09524, and by the Schmitt Program on Integrative Brain Research I: Expression of mRNA in old CBA Mice We are beginning the examination of changes in expression of Shaker (Kv1) and Shaw (Kv3) genes in young and old CBA mice, using “real-time” quantitative RT-PCR. We measured mRNA expression of the Kv3.1 channel gene and the joint expression of Kv1.1 and Kv1.2 channel genes, in the CN and IC of 2-3 month old and 24 month old mice (n=8,8). These analyses revealed no significant differences in the Kv3.1 channel gene at either site, but a decline in expression of about 25% in the Kv1.1 and 1.2 complex in the CN of the old mice, but none in the IC. While expression of mRNA should not be equated with functional channel proteins (Schmidt et al. 1999), these data provide a strong rationale for beginning an investigation of age-related changes in protein expression of both the Shaker and the Shaw families of genes. They suggest also that a comparison of auditory behavior in old mice with those of K+ channel KO mice may reveal features of presbycusis that relate to changing K+ channel gene expression as well as those that do not. 2: ABR thresholds and suprathreshold latencies in wild type and Kv1.1 KO mice. (A) Hearing thresholds: The +/+, +/-, and -/- genotypes show great similarity in thresholds across the spectrum. There is one potentially interesting difference, that the -/- mice have a significantly lower threshold for 3 kHz tone pip. (B) P1 latency for 90 dB tone pips (CN8 firing). Latencies increased with low tonal frequency, reflecting in part increased travel time across the basilar membrane. Davis et al. (2001) found in a slice preparation of the spiral ganglion that the high frequency basal region had narrow APs and fast latencies (15 ms) while those from the apex had wide APs and slow latencies (54 ms), the differences ascribed the relative proportions of potassium channel species. Here we found no difference in the base to apex latency change across genotypes, suggesting the Kv1.1 channel is not critical for rapid basal cochlear activation. (C) The amplitude and latency of the wave forms varied with tone level but these functions did not vary with genotype. (D) The wave forms for the 3 kHz tone pips across intensity (note expanded time scale) show an early rounded peak at about 3 ms at 90, then slowing at 80 and 70 dB SPL for the +/+ genotype. In the -/- genotype this increasingly delayed peak continued down to 40 dB. At this frequency the shaped 1 ms tone pips have a maximum of 3 cycles, 330 μS apart. Is it possible that the low-threshold rapid rectification provided by the Kv1.1 channel prevents summation across these peaks in wild type mice? (E) Latencies for 3, 16 and 32 kHz all vary with level, with 32 and 16 faster than 3 kHz, but genotype did not affect the P1 latency. (3) ASR response across tone frequency and level, in quiet. These functions in the +/+ and -/- mice are characteristic of young mice, in showing relatively strong responses at 8 and 16 kHz, poor responses at 4 and 32 kHz, at the extremes of their hearing range. There was no difference between the responses, but background activity, measured over 100 ms periods without a preceding tone, were higher in the -/- genotype. The source of this slight hyperactivity is unknown. At this age the -/- mice show no visible seizures. (4) ASR inhibition provided by a gap in noise at different lead times. These functions show the inhibitory effect of a 10 ms gap in noise presented at different lead times out to 300 ms prior to the startle response (the insert shows the first 30 ms). All mice show the rapid development of inhibition peaking at about 15 ms, and its slow decay. The amount of inhibition varied with genotype: +/+ > +/- > -/-, but the rate of development of inhibition was the same. (5) ASR inhibition provided by gaps of different duration all ending 50 ms before the startle reaction. These functions show that the KO mice with the -/- genotype show less asymptotic inhibition than the +/+ mice, with the +/- intermediate. The time constant for the development of inhibition appears to be the same across genotypes. The -/- KO mice again showed more background activity than the others. (6) "Physiological Decay Rate" of noise offset. In all of these conditions noise offset occurs just before the startle stimulus, at intervals of 1 to 10 ms, either with an abrupt offset or with a decay ramp time that matches the lead time. The inhibitory effect of the offset is graded with genotype and smallest in -/-: there is a strong indication that these KO mice do not respond rapidly to the early lead times when the decay time is 0 ms. The time constants for the +/+, +/-, and -/- mice were.96,.91, and 1.85, while asymptotic plateaus were not different. This suggests that neural excitation persists for a longer duration in the -/- null mutant KO mice after an abrupt offset of a background noise. A B C D mRNA Findings Introduction E