J. R. Ison1, P. D. Allen1, J. P. Walton2, W. J. Bowers3, & R. D. Frisina2
1Brain and Cognitive Sciences, 2 Otolaryngology, & 3Neurology, University of Rochester
(Ison@bcs.rochester.edu)
(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.
30
20
0
3 6
12 16
24
32
Frequency (kHz)
P1 latency (ms)
-/- (N=11)
+/- (N=26)
+/+ (N=18)
2.9
2.8
2.7
2.6
2.5
2.4
2.3
6
12
24
Frequency (kHz)
48
C
0.000010
0.000008
90dB
80dB
70dB
60dB
50dB
40dB
0.000006
0.000004
I: Expression of mRNA in old CBA Mice
0
1
2
3
4
5
6
7
8
9
10
11
Time (ms)
0.003
0.002
0.001
0.000
CN - O
CN - Y
IC - O
IC - Y
mRNA Kv3.1 Quantity
Location and Age
0.00075
Kv3.1
0.00050
0.00025
0.00000
CN - O
CN - Y
IC - O
Location and Age
IC - Y
0.000004
0.000002
-0.000000
0
-0.000002
-0.000004
+/+ n = 18
0
1
2
3
4
5
6
7
8
9
10
11
Time (ms)
400
3.0
60
70
80
2.0
1.0
0.8
1.0
0.4
0.8
0.6
0.4
0.2
0.2
+/+
+/-/-
0.0
0 1 2 3
5
10
20
ISI(ms)
0.0
0 20
50
100
150
300
ISI (ms)
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.
E
4.00
0.0
2
3
4
3kHz(+/+)
3kHz(-/-)
3.75
5
Time (ms)
-/- n = 12
90dB
80dB
70dB
60dB
50dB
40dB
2.0
500
400
300
Activity -/-
Activity +/+ & +/-
ASR -/- (N=8)
ASR +/+ (N=15)
ASR +/- (N=19)
100
3.25
16kHz(+/+)
16kHz(-/-)
0
0
3.00
0.4
+/+ n = 12
0
1
1
2
3
4
5
6
7
8
GAP (ms)
9
2
3
4
5
10
3
4
5
10
4
5
10
1.0
0.8
0.6
0.4
+/- n = 16
0
1
2
1.0
0.8
0.6
0.4
-/- n = 8
1
2
3
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.
10 11 12 13 14 15
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.
2.75
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.
2.50
1.0
2
3
4
Time (ms)
5
2.00
References:
32kHz(+/+)
32kHz(-/-)
2.25
0.0
700
200
3.50
3.0
(5) ASR inhibition provided by gaps of
different duration all ending 50 ms before
the startle reaction.
800
600
1.0
0.6
Conclusions
90 100 110 120
(4) ASR inhibition provided by a gap in noise
at different lead times.
0.6
(6) "Physiological Decay Rate" of noise offset.
LT (ms)
Startle Pulse Level (dB SPL)
(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.
+/+ n = 18
90dB
80dB
70dB
60dB
50dB
40dB
DT = 0
DT = LT
0.8
0
200
D
P1 latency (ms)
0.004
0.000006
ABR Potential (V)
Kv1.1+Kv1.2
90dB
80dB
70dB
60dB
50dB
40dB
0.000008
ABR Potential (V)
0.005
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.
ABR Potential (V)
mRNA Kv1. Quantity
0.000010
90 100 110 120
0
ASR (v-units)
mRNA Findings
(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?
80
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.
1.0
Activity
Relative response (%)
-0.000002
70
4 kHz (n = 14)
8 k +/+
16 k +/+
32 k +/+
600
-0.000000
0
-/- n = 12
Activity
60
(C) The amplitude and latency of the wave forms
varied with tone level but these functions did not
vary with genotype.
0.000002
-0.000004
200
0
(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.
3.1
3.0
400
48
B
(3) ASR response across tone frequency
and level, in quiet.
% Relative response
40
600
% Relative Response
+/+ (N=17)
+/- (N=26)
-/- (N=12)
4 k Hz (n = 7)
8 k -/16 k -/32 k -/-
Relative response (%)
Threshold (dB SPL)
50
2: ABR thresholds and suprathreshold
latencies in wild type and Kv1.1 KO mice.
10
ABR Potential (V)
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.
A
Threshold and Suprathreshold Acoustic Startle Reflex and Startle Inhibition by Gaps
% Relative Response
Threshold and Suprathreshold ABR
Introduction
ASR (v-Units)
714
ASR (v-Units)
ARO 2002
NORMAL THRESHOLD AND SUPRATHRESHOLD ABR AND ASR RESPONSES TO ACOUSTIC ONSETS IN KCNA1 KNOCKOUT MICE,
BUT A REDUCED RESPONSE TO OFFSETS
0
10
20
30
40
50
60
Level (dB SPL)
70
80
90
Research supported by NIA, AG09524, and by the Schmitt Program on Integrative Brain Research
Davis et al. (2001) ARO Abstracts 24:167; Gittelman et al. (2001) ARO Abstracts 24:197.
Grigg et al. (2000) Hear Res 140:77-80. ; Kopp-Scheinpflug et al. (2001) ARO Abstracts 24:196.
Macica, et al. (2000) SFN Abstracts 26:705; Oertel D (2000) PNAS, 97:11773-9.
Schmidt et al. (1999) Brain Res 843:45-60; Trussell LO (1999) Ann Rev Phys 61:477-96.
[ARO:2002]