On the auditory system: genes, DNA repair and ion channels

Transcription

On the auditory system:
genes, DNA repair and ion channels
A.P. Nagtegaal
PNagtegaal_Book.indd 1
2012-12-05 21:32:31
On the auditory system: genes, DNA repair and ion channels
Thesis, Erasmus University Rotterdam, The Netherlands
ISBN: 978-94-6182-202-4
Author: A.P. Nagtegaal
Cover: Offpage, concept by A.P. Nagtegaal
Layout and printing: Offpage, www.offpage.nl
Financial support for this thesis was kindly provided by:
De J.E. Jurriaanse stichting, ALK-Abelló B.V., GlaxoSmithKline
B.V., Atos Medical B.V., Cochlear Benelux NV, Veenhuis
Medical Audio B.V., Beter Horen B.V., Daleco Pharma B.V., DOS
Medical B.V., de Nederlandse Vereniging voor Keel-NeusOorheelkunde en Heelkunde van het Hoofd-halsgebied.
Copyright © 2012, A.P. Nagtegaal, the Netherlands,
[email protected]
All rights reserved. No part of this thesis may be reproduced
or transmitted in any form or by any means without prior
written permission of the copyrighted owner.
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On the Auditory System: genes, DNA repair and ion channels
Over het auditieve systeem: genen, DNA herstel en ionkanalen
Proefschrift
ter verkrijging van de graad van doctor aan de
Erasmus Universiteit Rotterdam
op gezag van de rector magnificus
Prof.dr. H.G. Schmidt
en volgens besluit van het College voor Promoties.
De openbare verdediging zal plaatsvinden op
woensdag 20 februari 2013 om 15.30 uur
door
Andries Paul Nagtegaal
geboren te Utrecht
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Promotiecommissie
Promotor: Prof.dr. J.G.G. Borst
Overige leden:
Prof.dr. M.A. Frens
Prof.dr. G.T.J. van der Horst
Prof.dr. S. Spijker
2012-12-05 21:32:31
Table of Contents
Chapter 1
Introduction
Chapter 2
An in vivo dynamic clamp study of Ih in the mouse inferior colliculus
25
Chapter 3
Hearing loss in mice with Cockayne syndrome
47
Chapter 4
Accelerated loss of hearing and vision in the DNA-repair deficient
Ercc1δ/– mouse
59
Chapter 5
A novel QTL underlying early-onset, low frequency hearing loss
in BXD recombinant inbred strains
81
Chapter 6
Discussion
107
Chapter 7
Summary / Samenvatting
123
Addendum List of abbreviations
7
133
About the author
135
PhD portfolio
137
Dankwoord
139
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1
Introduction
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Introduction
Our hearing is of utmost importance in life. Not only can it warn us in dangerous situations,
it is also, combined with speech, an important way of communicating with other people.
Unfortunately, hearing loss is a very common disorder, whose prevalence increases with
age. In 2005, according to the World Health Organization, 278 million people suffered from
moderate to profound hearing loss worldwide, accounting for a large socio-economic
burden (1). During life, the hearing organ is continuously subjected to accumulating
damage from within and outside the body, while its regeneration capabilities are limited.
This not only results in increased hearing thresholds, but can also lead to an altered sound
perception and a loss of speech discrimination. A better understanding of the genetics,
environmental influences and normal physiology of the hearing pathway therefore seems
crucial in preventing and treating hearing loss nowadays and in the future.
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Mouse models and hearing loss
The laboratory mouse is extensively used in fundamental auditory research for several
reasons. They are relatively small and have a much shorter life span than humans (2).
The auditory system and genome of mice and men show strong resemblance, making it
easier to extrapolate findings to humans. To date, already a substantial number of human
homologues of hearing loss mutations in mice have been identified (3). Due to genetic
standardization, a large cause of variability can be eliminated, thus greatly enhancing
study power. A large number of these inbred mouse strains suffer from hearing loss,
varying substantially in severity and affected frequencies (4). For example, one of the most
commonly used mouse strains, C57BL/6J, shows signs of progressive hearing loss during
life, beginning at the higher frequencies and eventually affecting the lower frequencies as
well (5). These mice carry a mutation in cadherin 23, also known as otocadherin, leading
to a disrupted organization of inner and outer hair cell stereocilia (6, 7). The C57BL/6J
strain is frequently used as an animal model for age-related hearing loss, presbyacusis,
illustrating the importance of mice within the field of auditory research.
In this thesis, the normal and abnormal physiology of the auditory system in mice is
investigated through various methods, which will be introduced hereafter.
Basic anatomy and physiology of the auditory system
The perception of sounds first starts with the passing of the external ear, which acts as a
direction-dependent filter in sound localization. After that, conduction to the tympanic
membrane takes place through the external auditory canal, which, in humans, has a
resonance peak at approximately 3 kHz to improve speech perception. The tympanic
membrane propagates the sounds via the ossicular chain, passing the air-filled middle
ear, to the cochlea, inducing fluid movements. This process eventually leads to vibrations
of the basilar membrane within the cochlea. The basilar membrane has different
mechanical properties along its baso-apical axis providing each sound frequency with
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Introduction
its place where the vibrations peak. High frequencies peak at the base, low frequencies
at the apex of the cochlea. This phenomenon, the spatial organization of frequencies,
is called tonotopy. It is not restricted to the cochlea, but can be found throughout the
auditory system. The functional, sensory structure of the cochlea, called the organ of
Corti, contains one row of inner and three rows of outer hair cells (Fig. 1). Outer hair cells
regulate sound intensities by varying in length and by using active hair bundle motion
(8, 9); they receive inputs from type II spiral ganglion cells. In addition, outer hair cells are
mainly responsible for the production of oto-acoustic emissions in mammals, the use
of which will be described in more detail later in this chapter. Movements of the basilar
membrane are encoded into action potentials by inner hair cells, which synapse on type
I spiral ganglion cells. These myelinated neurons have a bipolar structure, transporting
information from inner hair cells to the cochlear nucleus (CN), the first station of the
auditory brainstem. The fibers of type I and type II cells form the auditory nerve, which
is part of the vestibulocochlear nerve, the eighth cranial nerve.
Figure 1. Drawing of a cross-section of the cochlea. At the basal turn of the cochlea, the scala
vestibuli is connected to the oval window, while the scala tympani is adjacent to the round
window. Sounds induce a traveling wave of the basilar membrane. The organ of Corti is located
on this basilar membrane in the scala media, which contains the endolymph fluid, consisting of
high potassium and low sodium concentrations. Three rows of outer hair cells regulate sound
intensities, while one row of inner hair cells synapse with cochlear nerve fibers, terminating in
the cochlear nucleus of the auditory brainstem. Reproduced with permission from Willems (78),
Copyright Massachusetts Medical Society.
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Introduction
Cochlear nucleus; from divergence..
11
Type I cochlear nerve fibers branch within the CN and synapse on four different principal
cell types, marking the beginning of several parallel auditory pathways, which converge
again at the level of the inferior colliculus (Fig. 2) (10). These cell types are the bushy
cells of the anterior and the octopus cells of the posterior ventral CN (AVCN and PVCN),
multipolar cells of the VCN and stellate cells of the dorsal CN (DCN). The tonotopicity
of the cochlea is preserved, with low frequencies represented in ventrolateral and high
frequencies in dorsomedial parts of the CN.
Bushy cells (aptly named after their thick dendritic tree) receive large, calyceal synapses
from auditory nerve fibers, transmitting this information to the superior olivary
complex (SOC). This complex consists of three major nuclei, the medial and lateral
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Figure 2. Schematic view of the central auditory pathway. The ascending pathways originate in the
cochlea and eventually end in the auditory cortex of the temporal lobe. Reproduced with permission
from Cummings Otolaryngology Head and Neck surgery, Fig. 128-6 (79), Copyright by Elsevier.
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Introduction
superior olivary nuclei (MSO and LSO) and the medial nucleus of the trapezoid body
(MNTB), surrounded by several smaller diffuse groups of cells, the periolivary nuclei.
The SOC is the first station in the auditory pathway to receive binaural inputs; the
comparison to the inputs from both ears is used in horizontal sound localization. For
lower frequencies, the arrival time at both ears (interaural phase difference) is the main
determinant, encoded in MSO, while higher frequencies are localized based on their
interaural intensity difference in LSO (11). The information from the SOC is passed on to
the IC via the lateral lemniscus (LL).
Octopus cells in the PVCN are broadly tuned, as they receive synapses from many
spiral ganglion cells. They are characterized by their onset response (12), essential
for the coincidence detection of synchronous firing of different auditory nerve fibers
(13). This is achieved by cell properties such as a low membrane resistance (5-10 MΩ),
short time constant (~200 μs) (14) and an unusually large conductance of Ih-channels
(a hyperpolarization-activated cation current, discussed later in this chapter in more
detail) (15). Information from octopus cells is transmitted to nuclei in the contralateral
LL, while also providing collaterals to the periolivary neurons on their way.
Multipolar cells respond to sound stimuli with bursts of action potentials (“chopper”
response) and provide several axonal endings to the periolivary nuclei. Their main target
is the contralateral IC, which is also the ending of fibers arising from stellate cells.
Inferior colliculus; ..to convergence
Both from an anatomical and a physiological point of view, the centerpiece of the
auditory system is the inferior colliculus (IC). Of all subcortical nuclei, it harvests the
largest cell population by far (16) and it contains synapses of almost all ascending
and descending pathways. Ascending information from the IC is directed towards the
medial geniculate body (MGB), situated within the thalamus, from where it is passed on
to the temporal lobe, which houses the primary auditory cortex. Both IC’s are connected
via a commissure and each IC is further divided into several subunits, characterized by
different cell types and connections. The most important subunit is the central nucleus
of the IC (CNIC), which is an essential relay station for ascending information originating
from spiral ganglion cells in the cochlea. From dorsolateral to ventromedial a tonotopic
gradient exists, ranging from low to high frequencies, respectively, organized within
fibrodendritic laminae (17).
The CNIC is capped by a dorsal cortex (DC), whose inputs mainly stem from the
aforementioned CNIC and descending neocortical fibers, although it also receives
sparse ascending information from the lateral lemniscus. Several other, smaller groups
of nuclei surround the CNIC and the DC and are involved in various pathways. The
largest of those nuclei, the lateral nucleus, is involved in the integration of sounds and
information from other sensory modalities, as it receives inputs from the spinal cord and
the somatosensory association cortex.
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Introduction
Audiometry in mice
11
Although several subjective, behavioral tests of hearing function in mice exist (18), we
did not employ them in our studies. We used the Auditory Brainstem Response (ABR)
and otoacoustic emissions (OAE), both well defined, objective means to test hearing
function in mice. The ABR is widely used in human and animal research and consists
of the averaging of EEG’s during short repetitive sound stimuli. By averaging, only the
electrical signal evoked by the sound stimulus remains, while the background noise
gradually reduces to zero. The typical sound evoked response contains different peaks
that can be partly attributed to certain auditory nuclei. These are the cochlea and
auditory nerve (I), the cochlear nucleus (II), the superior olivary complex (III), the lateral
lemniscus (IV) and the inferior colliculus (V) (19). As the number of synapses that need
to be crossed increases, the peaks come progressively later.
The ABR can be easily used to determine hearing level thresholds by gradually decreasing
the Sound Pressure Level (SPL) until no peaks are visible anymore. Secondly, the
conduction speed of the auditory nerve and brainstem can be assessed by measuring
inter-peak time latencies. An increase in inter-peak latency is suggestive of pathology
between the corresponding nuclei (20, 21) and is routinely utilized in humans to detect
vestibular schwannomas (22).
OAEs were discovered about 35 years ago. They are a by-product of the non-linear
properties of the cochlea, in mammals mainly produced by active amplification by the
outer hair cells (23). Depending on the type of stimulation, different types of OAEs exist,
e.g. spontaneous, click-evoked and distortion-product OAEs. In DPOAEs, two tones of
different frequencies (f1 and f2), usually at two different SPLs, are presented to the ear.
Due to the interaction between both tones, the cochlea produces several new tones;
the intensity of one of these distortion products (2f1-f2) can generally be measured
above the background noise level. Although the presence of DPOAEs indicates a good
function of the cochlea and a normal conductive component, the absence of DPOAEs
does not necessarily imply poor cochlear function. A slight conductive hearing loss can
already lead to the absence of DPOAEs, and even in individuals with normal sensory
hearing level thresholds and normal middle ear conduction, emissions cannot always
be detected (24).
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Patch-clamp recordings and dynamic clamp
The methods described above address hearing function at the multicellular level, either
in the cochlea, nuclei in the auditory brainstem or both. In order to tackle questions
on sound processing at the cellular level, extra- or intracellular recordings are required.
Extracellular measurements are relatively easy to perform and provide essential
information on the amount and timing of action potentials, but they lack the ability to
clearly visualize synaptic potentials. In contrast, sub-threshold cellular activity can be
studied with intracellular recordings, making it the gold standard in electrophysiology.
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Introduction
In the late 1970s and early 1980s, the patch-clamp technique was developed by Neher and
Sakmann to study the function and physiology of ion channels (25), building on previous
work by Hodgkin and Huxley in the early 1950s, who were the first to systematically
describe voltage- and time-dependent gating of ion channels. The technique of these
recordings involves the lowering and attachment of a glass patch pipette to the cell
membrane of a cell, resulting in the sealing of the cell to the inside of the pipette.
Negative pressure is then applied to the pipette in order to rupture the cell membrane
patch within the pipette’s lumen, granting the recording electrode direct access to the
intracellular environment. This configuration is called the whole-cell configuration, in
which the net effect of multiple ion channels in the membrane can be studied. Recordings
can be performed in brain slices, in which there are ample possibilities to modify the
composition of the extracellular fluid. In order to study responses to physiological stimuli,
in vivo recordings are essential, requiring measurements in the intact animal brain.
In 1993, the dynamic clamp was first described as a variant of the traditional patch-clamp
technique, which allows users to introduce an artificial conductance in neurons (26). The
setup consists of additional hardware and software, which compute the current of a
simulated ion-channel and inject this current in the cell in real-time. Usually, a HodgkinHuxley model of a channel is used, incorporating gating variables that are both timeand voltage-dependent. As computer processor speed has greatly increased since its
first introduction, possible applications for a dynamic clamp setup have expanded and
now provide scientists with a powerful tool to study the function of simulated fastgating ion-channels, both in vitro and in vivo (27).
The I h -current
The presence of the slow, inward, hyperpolarization-activated current Ih was first described
in sinoatrial node cells in rabbit heart tissue (28) and was later found to be present in the
central nervous system as well (29). It has an unusual activation upon hyperpolarization
(hence its previous designations “funny” and “queer” current) and is carried by cations
(positive ions), mainly potassium and sodium. The physiological function of Ih is related
to the control of excitability, for example setting the resting membrane potential and
controlling dendritic integration and rhythmicity (30, 31). The Ih-channel is a heteromeric
assembly, in which four different subunits, termed hyperpolarization-activated cationnonselective (HCN) channels 1 to 4, are available. These subunits differ in gating time (32),
voltage dependence and their affinity to intra- and extracellular (33) signaling molecules,
providing a basis for the diverse actions of the Ih-current. Throughout the auditory
brainstem, expression of fast-gating HCN1 (~ tens of milliseconds) and slow HCN2
(100 - 1000 ms) is abundant (34). Evidence exists for the presence of Ih in spiral ganglion
cells (35), while in the CN, octopus cells have extraordinary large amounts of Ih (15). Slice
recordings have been performed in the IC, where a correlation was made between firing
type and Ih properties (36), confirmed in vivo more recently (37). However, by comparing
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Introduction
cells with and without evidence for Ih one can only make assumptions regarding causality
and co-expression with other channels cannot be ruled out. No studies have explored
the functional role of Ih in response to sound stimuli yet. Since most of the activity of this
channel is subthreshold, intracellular recordings are obligatory to study its physiological
function. Knock-out studies, although performed (38, 39), have serious disadvantages,
as adaptive network effects cannot be excluded. Therefore, the best way to study the
function of the channel is to make use of a dynamic patch clamp setup. Similar studies
have before been performed in vitro (40, 41), although in these slice studies responses to
physiological stimuli could not be evaluated.
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Age-related hearing loss and DNA repair mechanisms
7
Age-related changes in hearing function, termed presbyacusis, are a product
of accumulated damage during life, resulting from a mixture of noise exposure,
endogenous factors, ageing and of course genetic susceptibility. The classical
presbyacusis categorization by Schuknecht was based on selective atrophy of different
morphological structures in the cochlea or central auditory pathway, supposedly
involving four distinct types of hearing loss (42, 43).
First, a sensory type of presbyacusis was described, characterized by degeneration of
the organ of Corti, mainly at the basal turn of the cochlea, leading to an audiogram with
high-frequency hearing loss. Neural presbyacusis was named as a second type, involving
changes or loss in neural cell populations, either within the cochlea or more central
in the auditory brainstem or cortex. Typically, speech discrimination is poor, without
a profound parallel decline in pure tone thresholds. Thirdly, metabolic presbyacusis
was linked to atrophy of the stria vascularis. The stria maintains an unusually high
endolymphatic potential of +80 mV by active pump mechanisms, yielding a massive
driving force for potassium and, consequently, a high sensitivity of hair cells. An agerelated drop of this potential will affect hair cells at all different frequencies in the
cochlea and will thus lead to a flat audiometric curve. Lastly, mechanical presbyacusis
was distinguished by Schuknecht, in which changes within the basilar membrane impair
the conduction of the traveling wave, e.g. due to stiffening by calcifications. Hearing
loss is located mainly at the higher frequencies, while light microscopy fails to show
consequent morphological changes in cochlea or auditory nerve.
Whether these categories from 1964 actually exist as distinguishable audiometric and
morphological entities has been disputed through the years. Attempts to link a specific
type of hearing loss with localized cochlear pathology have not been very successful,
although changes in all of the above mentioned parts of the ageing cochlea have been
observed in patients with different types of hearing loss (44-46). While the practical and
clinical value of Schuknecht’s classification thus remains unclear, it can still provide a
good framework for research purposes and interventions within the field of age-related
hearing loss (47).
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insight review articles
Introduction
of global genome nucleotide-excision repair and transcription-coupled repair
plex XPC-hHR23B screens first on
pairing53, instead of lesions per se.
storting injury such as cyclobutane
y repaired54. In TCR, the ability of a
- or BER-type) to block RNA
stage I in the figure opposite). The
e displaced to make the injury
this requires at least two
and CSA. The subsequent stages
be identical. The XPB and XPD
nit transcription factor TFIIH open
und the damage (II). XPA probably
amage by probing for abnormal
when absent aborts NER53. The
otein RPA (replication protein A)
diate by binding to the undamaged
equent factors, each with limited
n in toto, still allows very high
ndonuclease duo of the NER team,
pectively cleave 3 and 5 of the
tch only in the damaged strand,
ligonucleotide containing the
replication machinery then
ng the gap (V). In total, 25 or more
. In vivo studies indicate that the
ed in a step-wise fashion from
he site of a lesion. After a single repair
minutes) the entire complex is
Global genome NER
Transcription-coupled repair
NER lesions
(e.g. due to UV damage)
Elongating Pol II-blocking lesions
(e.g. due to UV and oxidative damage)
Genome overall
Transcribed DNA
Elongating
RNA Pol II
XPC-hHR23B
CSB
5'
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TFIIH
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RPA
TFIIH
A
G
III
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ERCC1-XPF
TFIIH
F
A
G
IV
RPA
Replication
factors
V
Figure 3. Nucleotide excision repair (NER) mechanisms, showing both transcription-coupled (TC)
and
global genome
repair pathways.
is involved
in theisfirst steps
of TC-NER,
with
0-fold incidence of sun-induced
skin (GG)
combination
with theCSB
TCR
defect . TTD
a condition
sharinginteracting
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stalled RNA
II. The
endonuclease
ERCC1
with
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symptoms
with
Cockayne complex,
syndrome,
but coupled
with the
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rnal tumours is modestly
andpolymerase
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DNA. The
resulting
filled
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regular
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by
tion is often noted. Thefordisorder
hallmarks
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Mutations
in the XPDReprinted
or
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(54).diseases.
http://dx.doi.org/10.1038/35077232
of seven genes (XPA–XPG).
Cockayne
XPB genes
can giveLtd:
riseNature,
to all three
This puzzle is explained
tation in the CSA or CSB genes, is a
is remarkably dissimilar from xeroderma
osition to cancer is observed, which may
16
that the TCR defect causes Cockayne
articularly sensitive to lesion-induced
ing against tumorigenesis. Physical and
are impaired, resulting in dwarphism and
PNagtegaal_Book.indd
16
ome includes
features of premature
age-
by the fact that, as subunits of TFIIH, XPB and XPD have dual
functions: NER and transcription initiation. Mutations may not only
compromise NER, but also affect transcription, causing developmental delay and reduced expression of the matrix proteins that
causes brittle hair and scaly skin20.
For almost all NER factors, mouse mutants have been generated21.
Overall, the NER defect is accurately preserved, although cancer
predisposition is more pronounced and neurological complications
2012-12-05 21:32:32
Introduction
One of the main concepts of ageing is the free-radical theory, involving life-long accumulated
DNA damage inflicted by free reactive oxygen species (ROS) (48, 49), thus principally
affecting all of Schuknecht’s categories of presbyacusis. These radicals are a product of
normal oxygen metabolism within the cell (and therefore a direct consequence of life),
while their amount can greatly increase under various circumstances (e.g. noise exposure,
ototoxic drugs), a process known as oxidative stress. ROS are highly reactive and give rise
to significant damage to cell structures, including the DNA (50, 51). To defend themselves,
cells have multiple mechanisms of DNA repair: base excision repair, mismatch repair and
nucleotide excision repair (NER) (52). The latter involves a large number of distinct proteins
functioning as a complex to excise and replace the damaged base pairs (Fig. 3). The NER has
two different sub-pathways (53), one of which can be initiated during active gene expression
(transcription-coupled (TC) repair), when RNA polymerase II finds a lesion in the DNA.
Another sub-pathway is global genome (GG) repair, which also handles transcriptionally
silent regions of the genome. Mutations in proteins involved in DNA repair usually result
in premature cell death and / or carcinogenic events (54). Cockayne syndrome B (CS-B) in
humans is a clinically wide-ranged, photosensitive disorder arising from a specific defect in
TC-NER, further characterized by severe dwarfism, microcephaly, psychomotor delay and
sensory loss (retinopathy, hearing loss) (55, 56). Life expectancy is limited, with cachexia
being a frequent cause of death (56). A mouse model with a defect in the CSB protein
resembles the human Cockayne syndrome in some aspects. Crossing Csb deficient mice
with Xpa or Xpc deficient mice dramatically worsens the symptoms (57).
ERCC1 is another protein involved in DNA repair. After forming a complex with XPF, it is
both active in NER and the repair of the very genotoxic interstrand cross-links (58, 59).
Ercc1 or Xpf knock-out mice display a much more severe phenotype than Csb deficient
mice, including multi-organ involvement and death before weaning (58, 60). This is
further illustrated by case-reports of ERCC1 syndrome in humans, which is extremely
rare and generally incompatible with life (61).
Life and its subsequent oxidative metabolism have a large influence on the state of cells
within our body, including the hearing organ. DNA damage is an unavoidable part of
life. Both Ercc1- and Csb-deficient mice have impaired DNA repair and are thus excellent
examples of models to test the contribution of DNA damage to age-related hearing loss.
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Genetic background of age-related hearing loss
and mapping of complex traits
The degree to which people suffer from age-related hearing loss (AHL) is variable (62).
A genetic influence is assumed by many, while evidence is sparse. Data on hearing level
thresholds from members of the Framingham heart study has indeed shown that AHL
has a heritable component, more strongly in women (63). Still, identification of genes or
even gene loci for AHL in humans is greatly hampered by a variety of reasons, including
long lifespan, individual genetic variation and the sheer impossible task to control other
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Introduction
confounding factors (e.g. noise exposure, ototoxic drugs). Studies in mice can therefore
offer valuable information on the genetic basis of AHL. This is highlighted by the
description of up to nine mapped hearing loss loci (6, 64-69) and even the identification
of four causative genes (7, 70-72).
In diseases or conditions with a classic Mendelian inheritance, a trait is controlled by
a single gene locus and the relevant phenotype is frequently a dichotomic variable,
e.g. cystic fibrosis. A complex trait like AHL is characterized by a widely ranged,
continuous phenotype and involves variations in more than one gene, interactions
between genes and interactions with the environment. Analyzing and eventually
mapping a complex trait is therefore not straightforward and the use of a standardized
reference panel of isogenic (mouse) strains can be a powerful tool. The Jackson
Laboratory (http://www.jax.org/), located in Maine, USA, harbors and maintains a large
number of inbred mouse strain panels, including the BXD strains. Derived from the
commonly used and fully sequenced progenitor strains C57BL/6J and DBA/2J, they
initially contained 34 recombinant inbred (RI) strains resulting from at least twenty
B6
X
F1
X
D2
F2
X
X
X
F3
X
X
X
F∞
Figure 4. The generation of BXD recombinant inbred (RI) strains. Homozygosity and a strain unique
mosaic of the parental strains’ genome (C57BL/6J and DBA/2J) are achieved through approximately
twenty consecutive sib matings. The eventual inbred strains are highly recombinant in comparison
with F2 generations and provide an infinite number of genetically identical mice.
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Introduction
generations of consecutive sib matings (Fig. 4). Following advanced intercrosses of the
original RI strains (73), the total number nowadays adds up to 97 (including parental
and F1 strains), comprising the largest panel of mouse RI strains. This has several major
implications (74). First, each of these RI strains can deliver a theoretically infinite number
of nearly homozygous, genetically identical mice. Influence of individual genetic
variation and environmental effects on study outcome is hereby minimized. Secondly,
each RI strain consists of a unique mosaic of chromosomal sections of variable length,
inherited intact from either one of the parental strains. With a high density of single
nucleotide polymorphisms (SNPs) and microsatellite markers, the phenotypic data can
be linked with the genome markers. Owing to the highly recombinant nature of the
strain panel, quantitative mapping of a complex trait to one or more specific regions of
the genome, called quantitative trait loci (QTL), can be performed with high precision
(75). An internet based software package is available at http://www.genenetwork.org/
to aid in complex trait analysis and the mapping of QTLs (76, 77). Obviously, finding a
significant gene locus is not the endpoint and the search for a candidate gene must
follow. Regularly used methods include the search for (missense) mutations in any of the
genes on the locus and the correlation of the phenotypic data with mRNA expression
patterns. Finally, in order to fully confirm a causal link between a mutation and the
hearing loss, the generation of a transgenic mouse is usually required.
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Scope of this thesis
Several studies on the mouse auditory system will be discussed in this thesis. Chapter 2
describes the influence of the hyperpolarization-activated current Ih on basic membrane
properties, current injections and auditory stimuli in cells of the inferior colliculus. Next,
chapter 3 deals with the auditory function in Csb deficient mice, while chapter 4 involves
a study on auditory and visual performance in mice with a mutation in Ercc1. These
proteins are both involved in distinct mechanisms of DNA-repair. Last, the search for
genes involved in early-onset hearing loss in BXD recombinant inbred strains will be
discussed in chapter 5.
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2012-12-05 21:32:33
2
An in vivo dynamic clamp study
of I h in the mouse inferior
colliculus
A.P. Nagtegaal, J.G.G. Borst
J Neurophysiol. 2010 Aug; 104(2): 940-8
2012-12-05 21:32:33
I h in the inferior colliculus
ABSTRACT
Approximately half of the cells in the mouse inferior colliculus have the hyperpolarizationactivated mixed cation current Ih, yet little is known about its functional relevance in
vivo. We therefore studied its contribution to the processing of sound information in
single cells by making in vivo whole-cell recordings from the inferior colliculus (IC)
of young-adult anesthetized C57BL/6 mice. Following pharmacological block of the
endogenous channels, a dynamic clamp approach allowed us to study the responses to
current injections or auditory stimuli in the presence and absence of Ih within the same
neuron, thus avoiding network or developmental effects. The presence of Ih changed
basic cellular properties, including depolarizing the resting membrane potential and
decreasing resting membrane resistance. Sound-evoked excitatory postsynaptic
potentials were smaller but at the same time reached a more positive membrane
potential when Ih was present. With Ih, a subset of cells showed rebound spiking following
hyperpolarizing current injection. Its presence also changed more complex cellular
properties. It decreased temporal summation in response to both hyperpolarizing
and depolarizing repetitive current stimuli, and resulted in small changes in the cycleaveraged membrane potential during sinusoidal amplitude modulated (SAM) tones.
Furthermore, Ih minimally decreased the response to a tone following a depolarization,
an effect that may make a small contribution to forward masking. Our results thus
suggest that previously observed differences in IC cells are a mixture of direct effects
of Ih and indirect effects due to the change in membrane potential or effects due to the
co-expression with other channels.
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INTRODUCTION
The hyperpolarization-activated mixed cation current Ih contributes to various
physiological functions related to the control of excitability, including setting the resting
membrane potential and controlling rhythmicity and dendritic integration (reviewed in
(1, 2)). The HCN channels, which are responsible for the Ih current, are highly expressed
throughout the auditory brainstem (3, 4). The octopus cells of the cochlear nucleus have
especially high levels of Ih; this contributes to the low membrane resistance and short
time constant of these neurons (5, 6). Both the fast-gating Ih channel subunit HCN1 (time
constant in the order of tens of milliseconds) and the slowly gating subunit HCN2 (time
constant 100 -1000 ms) are prominent in the inferior colliculus (IC) (3, 4). Approximately
half of IC neurons show evidence for the presence of Ih, both in vivo, in the form of
a depolarizing sag during hyperpolarizing current steps (7), and in slices, where
immunocytochemical, pharmacological and biophysical evidence for its presence has
been obtained (3, 8). In the IC, on average, cells with Ih had more depolarized membrane
potentials, lower input resistance, increased excitability, more often showed rebound
spiking, and more often showed accommodation or burst-firing on current injection
than cells without evidence for the presence of Ih (7-9). Ih may also be involved in reducing
temporal summation of fast stimuli because Koch and Grothe found more summation
of brief current injections when Ih was pharmacologically blocked (8). Another possible
function of Ih in the auditory system may lie in its contribution to forward masking in
which the detectability of a short probe signal is degraded when it is preceded by a
masking stimulus (10). As the presence of Ih leads to an after hyperpolarization following
a tone (9), the detection threshold of the probe signal may be increased if it falls within
this afterhyperpolarization, thus providing a possible central contribution to forward
masking.
The goal of this study is to investigate the role of Ih in the processing of current
injections and tonal stimuli at the level of the mouse IC. Until now, it is unclear which of
the above features are due to other ion channels which happen to be co-expressed with
Ih (8), an interaction of Ih with for example low-threshold potassium channels (11-13), or
due to a direct effect of Ih. To address these issues, we made in vivo whole-cell patchclamp recordings, and combined this with the dynamic clamp technique (14, 15) to
artificially reintroduce a conductance with Ih kinetics into the cell after pharmacological
block of the endogenous channels. A major advantage of combining these two
techniques is the possibility to test in a single cell the effect of the presence and absence
of Ih on its response to natural stimuli.
1
2
3
4
5
6
7
&
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2012-12-05 21:32:33
MATERIALS AND METHODS
Animals and procedures
All experiments were conducted in accordance with the European Communities Council
Directive (86/609/EEC) and approved by the animal ethics committee of the Erasmus MC.
Patch-clamp recordings were performed under ketamine / xylazine anaesthesia
(60/10 mg/kg, i.p.) in the inferior colliculus of C57BL/6 mice (age: 21 to 35 days), as
described previously (7), with minor modifications. In brief, the skull and dura overlying
the IC were removed. During experiments, Ringer solution (in mM: 135 NaCl, 5.4 KCl,
1 MgCl2, 1.8 CaCl2, 5 HEPES, pH adjustment to 7.2 with NaOH, osmolality: 290 mmol/kg)
was applied to the brain surface to prevent dehydration. Agar (3% agarose in Ringer
solution) was applied to reduce brain pulsations. We used glass pipettes (open pipette
resistance: 4-6 MΩ) filled with internal solution containing (in mM): 126 K-gluconate,
20 KCl, 10 Na2phosphocreatine, 4 MgATP, 0.3 Na2GTP, 0.5 EGTA, 10 Hepes, adjusted
with KOH to pH 7.2, osmolality ~ 310 mmol/kg. The Ih-current blocker 4-(N-ethyl-Nphenylamino)-1,2-dimethyl-6-(methylamino)pyridinum chloride (ZD7288; Tocris),
which acts on an intracellular activation gate (16), was added to the internal solution
in a concentration of 100 µM. We could not monitor the development of the block in
individual cells presumably because it was too fast. Mean access resistance, estimated
from step depolarisations in voltage-clamp mode, was 55 ± 4 MΩ. During currentclamp recordings, on average 30 ± 1 MΩ (n = 19) was compensated. All measurements
involving current injections were corrected off-line for the uncompensated, residual
series resistance, which was estimated as the difference between the voltage clamp
measurement of series resistance and the setting of the bridge balance in current clamp.
Data were acquired with a MultiClamp 700B patch-clamp amplifier and Clampex 9.2
software (MDS Analytical Technologies). The membrane potential (Vm) was sampled at
25 kHz with a Digidata 1322A 16-bit A/D converter; it was corrected for an estimated
–11 mV junction potential. Recordings were only continued if the membrane potential
following break-in was below –60 mV (19 of 27 cells).
Auditory stimulation
Sound stimuli were generated with Tucker Davis Technologies hardware (System 3,
RX6 multifunction processor, PA5 attenuator and ED1 electrostatic speaker driver)
and delivered to the contralateral ear under closed-field conditions. Sound intensities
of frequencies between 1 and 64 kHz were calibrated with an ACO pacific condenser
microphone (type 7017). Recordings were performed in a single-wall sound attenuated
chamber (Gretch-Ken Industries, attenuation ≥ 40 dB above 4 kHz).
Dynamic clamp
Ih-current was calculated by a custom-written LabVIEW (version 8.0, National Instruments)
routine on the basis of a Hodgkin-Huxley-type model of Ih, incorporating both voltageand time-dependent gating variables. Membrane potentials were sampled at 2 kHz.
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2012-12-05 21:32:33
Reversal potential and half-activation potential were obtained from the literature (6, 7)
and confirmed by preliminary voltage-clamp experiments in IC slices (H.P. Theeuwes
and J.G.G. Borst; not shown). The Ih current consisted of a fast and a slow component (7),
presumably reflecting the presence of different subunits within the IC (3, 4); the current
was calculated by the following equation
Ih = (gmax fast ∙ hfast + g max slow ∙ hslow)(Vm – Erev)
1
2
3
4
[1]
5
where hfast and hslow are voltage- and time-dependent gating variables and the reversal
potential Erev = –38 mV. The gating variable h varies between 0 (closed) and 1 (open); hfast
and hslow were obtained by numerical integration (4th-order Runge–Kutta method) of
dh / dt = α ∙ (1 – h) – β ∙ h
6
7
&
[2]
The respective rate constants for activation (αfast and αslow) were determined by (17, 18)
α = a · exp(–b (Vm - V0.5))
[3]
whereas the rate constants for deactivation (βfast and βslow) are given by
β = a · exp(c (Vm - V0.5))
[4]
Two different gating models were used in all experiments. For the fast gating Ih, the
values for the different parameters were as follows: afast = 0.012 ms–1, aslow = 0.0023 ms–1,
b = 0.05 mV–1, c = 0.085 mV–1 and V0.5 = –65 mV. For the slow gating Ih, afast = 0.00045 ms–1,
aslow = 0.003 ms–1, and the other parameters were the same. Because results for both
gating models were similar, only the results of the fast gating models are presented,
except for Figure 7. The total, maximal conductance with Ih kinetics was set to 4 nS;
activation of this conductance generated a depolarizing sag of ~25% during 1 s,
–200 pA, hyperpolarizing constant current steps. Two thirds of the total conductance
was assigned to the first, fast component. The total conductance was based on previous
observations within the IC (7); in this dataset, cells with Ih had a depolarizing sag of 28.5
± 1.5% (n = 52) at the end of a 1 s hyperpolarizing constant current step.
The pipette time constant did not filter the injection of the current substantially,
as the slowest pipette time constant was below the sampling interval and much faster
than the fastest time constant of Ih in the range between –100 and –30 mV (>4 ms). With
a sampling interval of 0.5 ms the driving force will not be updated sufficiently rapidly
during an action potential, but because the contribution of Ih to the total membrane
current during an action potential is very small, we conclude that series resistance and
sampling interval did not limit the quality of the dynamic clamp.
29
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Stimulation protocols
The characteristic frequency (CF), defined as the frequency with the lowest EPSP
threshold, was obtained as described by Tan and Borst (9).
Tonal stimuli were presented at CF, at 30 dB above threshold, with 0.5 ms rise and fall
times, while current stimuli were programmed to evoke an EPSP of similar amplitude as
the tonal response unless stated otherwise. The following protocols were presented to
the cells: 1 s depolarizing current injections, 100 pA above firing threshold; depolarizing
and hyperpolarizing repetitive current injections [400 ms total duration, 80 stimuli
of 5 ms, 10 – 90% rise time = 0.3 ms, decay τ = 3 ms, according to Koch and Grothe
(8)]; a 50 ms tone with and without a preceding 500 ms depolarizing current injection;
sinusoidal amplitude modulated (SAM) tones (400 ms total duration, carrier frequency
at CF, modulation frequency 40 or 80 Hz, modulation depth 100%); up- and downward
frequency-modulated (FM) sweeps (1 – 73 kHz, 120 ms duration, sweep rate 600 kHz/s,
5 ms rise and fall time). All protocols were applied with and without Ih. This sequence
was repeated 5 – 20 times (average = 14), depending on the amount of depolarization
during the experiment (maximally allowed depolarization 10 mV). The minimal interval
between stimuli was 400 ms.
Analysis
Data were analyzed with custom-written Igor procedures (Igor Pro 6.01, WaveMetrics)
running within the NeuroMatic environment (version 1.98; kindly provided by
Dr. J. Rothman, University College London).
Fifteen repetitions of a –200 pA hyperpolarizing current were injected to characterize
the Ih -current. The amount of depolarizing sag was quantified and the sum of two
exponential functions was fitted to both the depolarizing sag and the rebound
depolarization
Vm = Vss + Afast · exp(–t/ τfast) + Aslow · exp(–t / τslow)
[5]
where Vm is membrane potential, Vss is membrane potential at the end of a long current
injection, Afast and Aslow are amplitudes of the two exponential functions at the start of
the step and τfast and τslow are the fast and slow time constants, respectively. Membrane
resistance was calculated at the start and end of the current step.
Firing types were classified based on responses to 1 s constant-current injections
at 100 pA above firing threshold (7). We plotted the reciprocal (Y) of the inter-spike
interval (ISI) against its spike number and a single-exponential was fitted through the
data points. An accommodation index (AI) was calculated by the following equation:
AI = [(Y0 – Y1)/ Y1] · 100%, where Y0 and Y1 are the values of the fit function for the first and
second action potential, respectively. Cells with an AI <4% were classified as sustained,
>4% as accommodating.
30
2012-12-05 21:32:34
The amount of temporal integration during repetitive current injections was
quantified using a summation index, which is defined as the percentage of increase of
the last versus the first PSP-like response (8).
We estimated firing threshold in response to tonal stimuli by taking the point at
which the second time derivative of the membrane potential exceeded three times the
standard deviation of the second derivative of the resting membrane potential (19).
For the analysis of the response to SAM tones, the cycle-averaged membrane
potential was generated by averaging the membrane potential across both repetitions
and modulation periods. Prior to averaging, action potentials were truncated as
described previously (9). The modulated potential (MP) is defined as the maximal peakto-trough amplitude of the cycle-averaged membrane potential (19). Only cells with an
MP >0.5 mV were included for SAM analysis. The vector strength (R), which describes
the synchrony of spiking in relation to the phase of the response (20), was calculated for
cells that fired on average at least one spike per tone presentation. The first 50 ms of the
response were excluded from this analysis to prevent an influence from onset responses.
1
2
3
4
5
6
7
&
Simulations
In all recordings the injected Ih current calculated by the dynamic clamp was recorded.
This current was used in simulations to test the influence of different Ih gating models
on the response to FM-sweeps. As procedures were always repeated in the presence
and absence of Ih, in the experiments in which the variability in individual responses
was sufficiently low the difference in membrane potential that resulted from the current
injection could be used to calculate the membrane conductance with Ohm’s law:
ΔVm(t) = iIh(t)/gm(t)
[6]
where ΔVm(t) is the difference in the average membrane potential in the presence and
absence of Ih, iIh(t) is the injected current and gm(t) is the total membrane conductance.
Although the latter consists of several components that are not easily dissected, it can
be used to predict the response to current injections calculated with different gating
models for Ih. Four different models with the same maximal conductance and steadystate voltage dependence as used in the experiments were tested. Two models were
identical to the ones used in the experiments; as expected, the predicted membrane
potentials always closely matched the measured response for these two models. In
addition, we tested two extremes for Ih gating. In one model, the voltage-dependent
gating was instantaneous, whereas in the other the open probability of Ih was fixed at
the calculated value at the start of the trace. For these four models a dynamic clamp
experiment was simulated using equation [6], where ΔVm(t) is again the change in the
membrane potential compared to the situation in the absence of any injected current,
gm(t) is the previously calculated total membrane conductance, and iIh(t) is the predicted
current, calculated sample by sample according to the four different models.
31
2012-12-05 21:32:34
Statistics
Data are presented as mean ± standard error. If responses were normally distributed, as
tested with a Shapiro-Wilk test, a statistical difference in the mean response between Ih
and no Ih was assessed with a paired t-test, otherwise a paired Wilcoxon test was used.
Significance level was set at p < 0.05.
RESULTS
In vivo dynamic clamp
To study the contribution of Ih to sound processing in the mouse inferior colliculus, we
combined the dynamic clamp technique with in vivo whole-cell recordings in a total of
19 cells. In none of these cells did the size of the depolarizing sag during hyperpolarizing
current injection exceed 7% under control conditions (Fig. 1, top), indicating that
endogenous Ih channels, when present, were effectively blocked by the Ih blocker
ZD7288 (100 µM) in the pipette solution. On addition of the conductance with Ih kinetics
(gh) to the cell using the dynamic clamp, a depolarizing sag appeared (Fig. 1, bottom).
Similar results, but with slower kinetics, were obtained with a different gating model for
Ih. The time course of the depolarizing sag was fitted by a double-exponential function;
the time constants were close to what was predicted based on the H-H model and fell
within the range of gating kinetics observed for native Ih channels within the IC (Table 1)
(7). In the presence of Ih, the membrane resistance calculated at the end of the step was
clearly lower (Table 1). Following the hyperpolarizing current injection, the cells showed
Figure 1. Ih can induce rebound spiking. Response of a cell to 15 repetitions of a 1 s, 200 pA
hyperpolarizing constant-current injection. Average trace is shown as a white overlay. In the
presence of Ih (bottom), the resting membrane potential was more depolarized, a depolarizing
sag was present during the current injection and the cell showed rebound spiking during the
rebound depolarization (‘hump’).
32
2012-12-05 21:32:34
Table 1. The effect of adding gh on different cell properties and responses to current injections
and sound stimuli in vivo.
Cell properties
Vm (mV)
Rm (MΩ)
Spike threshold (mV)
Relative spike threshold (mV)
% depolarizing sag
no Ih
Ih
–66.6 ± 1.5 (13)
–62.7 ± 1.2**
134 ± 11.3 (19)
–50.4 ± 1.5 (10)
1
2
3
78 ± 5.6**
4
–50.1 ± 1.5
14.4 ± 1.8 (10)
10.4 ± 1.3**
2.1 ± 0.4 (19)
24 ± 1.0**
5
τfast depolarizing sag (ms)
-
32.7 ± 0.7 (19)
Amplitude τfast (mV)
6
-
–4.2 ± 0.09 (19)
τslow depolarizing sag (ms)
-
7
Amplitude τslow (mV)
-
250.3 ± 5.8 (19)
&
–2.6 ± 0.07 (19)
Current injections
Depolarizing summation index (%)
146 ± 19 (14)
77 ± 10*
Peak – trough amplitude (mV)
2.04 ± 0.05 (14)
2.10 ± 0.06**
Hyperpolarizing summation index (%)
115 ± 20 (14)
Peak – trough amplitude (mV)
2.06 ± 0.05 (14)
58 ± 6*
2.18 ± 0.06**
Tone response (50 ms)
–54.1 ± 1.6 (13)
–52.3 ± 1.4**
EPSP amplitude (mV)
Max EPSP depolarization (mV)
12.5 ± 1.6 (13)
10.4 ± 1.5**
EPSP full width at half maximum (ms)
41.0 ± 1.9 (13)
37.9 ± 1.8*
Tone response after current injection
–54.0 ± 1.4 (13)
–52.8 ± 1.4**
EPSP amplitude (mV)
Max depolarization (mV)
12.6 ± 1.6 (13)
10.0 ± 1.5**
Max difference with no injection (mV)
0.03 ± 0.36 (13)
–0.42 ± 0.21
Modulated potential (mV)
2.29 ± 0.39 (13)
2.23 ± 0.37
R (vector strength)
0.46 ± 0.13 (4)
0.37 ± 0.10
SAM tones
Values represent averages ± standard errors; the number of cells is between parentheses. * indicates
a significant difference of p < 0.05, ** indicates p < 0.01. Max difference with no injection refers to
the difference in the maximal depolarization in response to a tone with or without a preceding
depolarization.
33
2012-12-05 21:32:35
a rebound depolarization, which had similar kinetics as the depolarizing sag. In 6 out
of 19 cells this rebound depolarization was sufficiently large to trigger spiking (Fig. 1),
whereas in the absence of Ih only one cell showed rebound spiking.
Firing pattern
Fifteen cells were available for analysis of firing pattern during constant-current injections.
Their firing pattern was classified on the basis of a fit to the inter-spike intervals, as
described in Methods. Ten cells were classified as sustained, five as accommodating. The
firing patterns did not change in the presence of Ih, as judged from a lack of a significant
change in the fitting parameters (results not shown).
Repetitive current injections
We tested the effect of repetitive current injections in the presence and absence of Ih
(Fig. 2). The interval between the stimuli was sufficiently short to allow summation. As
a result of summation, the peak depolarization or hyperpolarization reached following
the last current injection was larger than following the first. The percentage increase in
this level, the summation index, was smaller in the presence of Ih for both depolarizing
and hyperpolarizing current injections. Furthermore, the difference between peak and
trough membrane potential of the individual responses was smaller in the absence of Ih
(Table 1).
Figure 2. Ih decreases temporal summation. A: response of a cell to repetitive current injections.
Bottom traces show stimuli. For comparison purposes, resting membrane potentials (–64.9 mV for
no Ih, –61.8 mV for Ih) were equalized. B: the first 7 stimuli are shown at higher time resolution.
C: summation index (% increase of last PSP vs. first PSP) of all cells (n = 14) during depolarizing
repetitive current injections, with the average indicated with a thick black line. Adding Ih resulted
in less summation. D: as C, except hyperpolarizing current injections.
34
2012-12-05 21:32:35
Response to tones
All neurons responded to tones. Their mean characteristic frequency (CF) was 20.9
± 1.4 kHz (range 12.1 – 27.9 kHz) with an average minimum threshold (MT) for evoking
an EPSP of 21 ± 4 dB SPL.
The addition of Ih led to a depolarization of ~4 mV (Table 1). As a result, in the
presence of Ih, a more positive potential was reached in response to a tone 30 dB above
MT, despite a smaller EPSP amplitude (Fig. 3; Table 1; n = 13; excluding 3 cells with an
inhibitory response to tones). EPSPs were also less broad in the presence of Ih, as judged
from the smaller full width at half maximum (Table 1). In the presence of Ih, the amplitude
of the afterhyperpolarization following the tones was slightly larger (-2.10 vs -1.96 mV;
p > 0.05). Ten cells showed spiking in response to tones, at an average threshold of
31 dB SPL; in the other 9 cells all sound-evoked responses were sub-threshold. The firing
threshold during tonal stimuli was slightly more positive in the presence of Ih, while the
amount of depolarization required to reach this threshold was reduced in the presence
of Ih (Table 1). We did not observe a significant effect of Ih on the number of spikes fired
during different tonal stimuli.
1
2
3
4
5
6
7
&
Figure 3. Influence of Ih on the response to a pure tone. A: averaged response of a cell to 16 kHz,
40 dB SPL, 50 ms tones. In the presence of Ih, the cell reached a more depolarized membrane
potential in response to the tone, despite a smaller EPSP amplitude. B: EPSP amplitude values for
all individual cells (circles) and average (squares, connected with a thick black line).
Forward masking
The psychophysical phenomenon of forward masking is defined as the increase in the
hearing threshold of a tone when it is preceded by another tone. Instead of giving
two tones, we replaced the first tone by a current injection to avoid peripheral effects
(e.g. (21) on forward masking. Similar to what was observed without the preceding
current, in the presence of Ih the membrane potential reached a more positive value in
response to a tone 30 dB above MT, while EPSP amplitudes were smaller (Fig. 4, Table 1).
35
2012-12-05 21:32:35
Figure 4. Possible contribution of Ih to forward masking. A: in the absence of Ih, injection of a
depolarizing current immediately before a tone leads to summation with the EPSP (black trace).
Middle panel shows current injection; lower panel shows when tone is presented. B: as A, but in
the presence of Ih. The resulting EPSP is decreased. C: tone-evoked response of A shown at higher
time resolution. Response without the preceding depolarizing current (grey trace) is also shown
in Figure 3A. D: tone-evoked response of B shown at higher time resolution. E: average EPSP
amplitude (n = 13 cells). With Ih injection, the amplitude decreases in the presence of a preceding
current injection, while the amplitude does not change in the absence of Ih. F: average peak
depolarization reached in response to the tone. In the presence of Ih, a more negative membrane
potential is reached when the tone is preceded by a current injection.
In the absence of Ih, the maximal depolarization reached in response to the tone was
similar with and without the preceding current injection. In the presence of Ih, a less
positive membrane potential was reached following the current injection (Table 1).
The observed effect was very small, however, and is therefore unlikely to contribute
significantly to forward masking.
Response to SAM tones
The results of the repetitive current injections (Fig. 2) suggested that Ih channels may
reduce synaptic integration. To test this more directly, we studied the response to
sinusoidal amplitude-modulated (SAM) tones and to frequency-modulated (FM) sweeps.
SAM tones were given at a carrier frequency which was equal to the CF of the cells.
This carrier frequency was modulated at 40 Hz, resulting in periodic fluctuations of the
membrane potential (Fig. 5). The modulated potential (MP), the peak-to-peak amplitude
of the cycle-averaged response of the cell (19), was lower in the presence of Ih in 8 of
13 cells, although the effects were small and this difference did not reach statistical
36
2012-12-05 21:32:36
1
2
3
4
5
6
7
&
Figure 5. Ih modifies the response to SAM tones. A: averaged, truncated response to a sinusoidal
amplitude modulated (SAM) tone with a modulation frequency of 40 Hz; CF was 27.9 kHz.
B: cycle-averaged membrane potential. A more positive membrane potential was reached in
the presence of Ih. C: as B, except baseline values were equalized, illustrating that peak-to-peak
amplitude (modulated potential; MP) and full width at half maximum were somewhat larger in
the absence of Ih.
significance. The vector strength, which is a measure for the ability of cells to phase-lock
their spikes to the envelope of the SAM tones, was not different in the presence of Ih
(Table 1). Similar results were obtained at a modulation frequency of 80 Hz (not shown).
Response to FM sweeps
Cells responded to up- or downward FM sweeps with a complex response (Fig. 6A and B).
In the presence of Ih, 7 out of 14 cells showed increased IPSPs (Fig. 6A), probably due to
the increased driving force for inhibitory conductances at the more positive membrane
potential in the presence of Ih. In these cells, pure tones evoked IPSPs at the frequencies
predicted by the timing of the inhibitory components within the FM sweep (not shown).
Two types of experiments suggested that Ih gating during the tones did not make
a great contribution to the observed changes in inhibition during the FM sweep.
Firstly, dynamic clamp experiments with an Ih-current that showed slower gating were
similar (Fig. 7A). Secondly, we used the relation between injected currents (Fig. 7B) and
membrane potential changes to get an estimate of membrane conductance (Fig. 7C), as
detailed in the Methods. This estimate was used in simulations to predict the change in
membrane potential with other gating models. Two extreme models were compared.
37
2012-12-05 21:32:36
B
-50
no Ih
Ih
Vm (mV)
A
-60
f (kHz)
-70
73
0
0
100
200
Time (ms)
300
400 0
100
200
Time (ms)
300
400
Figure 6. Response to an FM sweep. A, top: averaged response (11 traces) of a cell to an upward FM
sweep. The tone stimulus is shown in the lower panel. At the onset of the response, an IPSP (arrow)
is apparent in the presence of Ih (grey trace), which is much smaller in control (black trace). This cell
showed an inhibitory response to tones of 6 kHz. B: as A, except FM sweep was downward.
In one the gating was instantaneous, in the other Ih showed no gating. The membrane
potential predictions for these two models were similar (Fig. 7A), despite clear differences
in the injected current (Fig. 7B), suggesting that changes in the open probability during
the FM sweep (Fig. 7D) do not contribute appreciably. A comparison of gh with the total
conductance shows that gh at all times is only a small fraction of the total conductance
(Fig. 7C). This was also the case in the other 13 cells in which FM tones were tested.
DISCUSSION
We made use of the dynamic clamp technique to study the contribution of Ih to
sound processing in single neurons of the mouse IC. The presence of Ih changed basic
membrane properties, causing a more depolarized membrane potential and a decreased
resting membrane resistance. Sound-evoked EPSPs were smaller, but at the same time
reached a more positive membrane potential in the presence of Ih. The addition of Ih
also changed more complex properties of the cells. Its presence decreased temporal
summation in response to repetitive current stimuli, and also induced small changes
in the responses to SAM tones, FM sweeps, and to a tone following a depolarization, an
effect that may contribute to forward masking.
Using dynamic clamp to study Ih in vivo
The effects of Ih have been well characterized in slice recordings (1, 2). Much less is
known, however, about its contribution in vivo. Knock-out studies have revealed the
involvement of Ih in global functions such as memory or gamma oscillations (22-24),
38
2012-12-05 21:32:36
1
2
3
4
5
6
7
&
Figure 7. Effect of different gating models of Ih on response to an upward FM-sweep. A: predicted
and measured membrane potentials with different gating models. Black and red traces are identical
to measured traces shown in Fig. 6A, in the absence and presence of fast-gating Ih, respectively.
Blue trace shows measured membrane potential for slow gating Ih. Brown trace shows predicted
membrane potential for an Ih model with instantaneous gating. Green trace shows predicted
membrane potential for an Ih model with no gating. B: predicted and measured Ih currents with
different gating models. Red and blue trace show injected currents for fast and slow gating model
of Ih, respectively. Brown trace shows predicted current for an Ih model with instantaneous gating;
green trace for an Ih model with no gating. C: solid lines, total membrane conductance calculated
with equation [6], using measured membrane potentials shown in A and measured Ih currents
shown in B. Dashed lines, calculated gh. Red and blue traces indicate fast and slow gating models
of Ih, respectively. D: calculated and predicted open probability of Ih channels using fast (red), slow
(blue), no (green) and instantaneous (brown) gating models. E: tone stimulus.
39
2012-12-05 21:32:37
but little is known about the contribution of Ih to the processing of natural inputs in a
single cell. Our approach was to block endogenous Ih and subsequently insert gh using
a dynamic clamp approach in whole-cell recordings from the mouse IC. This technique
has previously been used to inject synaptic conductances during intracellular in vivo
recordings (15). Its use for studying voltage-dependent ion channels has as an advantage
that both time-dependent gating effects and changes in the driving force can be taken
into account. Changes in driving force are considerable for the mixed cation channel Ih,
which has a reversal potential ~20 mV from the resting potential. Moreover, with this
approach it is possible to study the acute effects in a single cell, thus avoiding possible
compensatory developmental or network effects that can accompany transgenic
studies (25). By interspersing the presentations with and without the dynamic clamp,
very small effects (<1 mV) could be picked up, even when variability between cells was
much larger.
Our Hodgkin-Huxley model of the Ih channel adequately matched the kinetic
properties found within the IC. The time constants of the depolarizing sag in the presence
of Ih were in the same range as reported previously, although somewhat slower than the
fast time constant of the depolarizing sag reported in (7). Its conductance was similar
to previous observations in the mouse IC (7). Although the gating model used was not
based on a detailed biophysical analysis of the properties of Ih in the inferior colliculus,
two lines of evidence suggest that the exact properties of the gating model were not
critical. Firstly, results obtained with a fast and a slow gating model were generally
similar. Secondly, a simulation in which extreme values for the gating of Ih were tested
did not yield clear differences either.
Ih channels are often located at higher density in distal dendrites (26, 27), whereas
we used somatic Ih injections. It has been shown that many of the effects of Ih, including
its effect on the somatic time course of distant synaptic inputs, do not depend on its
exact subcellular distribution (28-30). However, its effect on local temporal summation
in the dendrites will not be adequately mimicked by the somatic dynamic clamp.
Ih can trigger rebound spiking
Although other channels may also contribute to rebound depolarisations (7, 31-33), the
emergence of rebound spiking in the presence of Ih is in agreement with earlier evidence
obtained both in vivo and in slices (7, 8). This effect may contribute to off-responses in
the mouse IC, which may play a role in duration tuning (9, 34).
Lack of evidence for a role of Ih in controlling firing pattern
Although the presence of Ih is associated with certain firing types in the IC (7, 8), the firing
pattern during constant-current injections was not altered in the presence of Ih. This
result is in agreement with an earlier study in slices (8), suggesting that Ih by itself does
not control firing, and that the larger accommodation observed for cells with evidence
for Ih is most likely due to co-expression with a low-threshold potassium channel.
A major role for Ih on firing patterns is not expected based on its relatively low conductance
40
2012-12-05 21:32:37
compared to both synaptic and voltage-dependent currents. A limitation of our study is
that we observed only cells with sustained or accommodating firing types in response
to current injections. We therefore cannot exclude that the effect of Ih on firing types
such as build-up, burst-onset, burst-sustained and accelerating (7, 32, 35) would have
been larger, for example through an interaction with low-threshold K-channels.
1
2
3
4
Ih and excitability
5
The effects of Ih on excitability are complex. Both excitatory (9, 36) and inhibitory effects
(37, 38) have been described. Its effect on excitability depends in part on its interaction
with other voltage-dependent ion channels (11-13) and on the levels and timing of
synaptic activity (37, 39-41). The amplitude of responses to tones decreased in the
presence of Ih. However, the increase in resting Vm compensated for the smaller EPSP size
and the net effect was that a more positive potential was reached in response to tones.
Assuming that the most positive voltage reached determines the effect on excitability
(12), this would indicate that Ih increases excitability, in agreement with our previous
findings (7), although the effect is small and will be counteracted by the slightly more
positive action potential threshold observed in the presence of Ih.
6
7
&
Possible role of Ih in forward masking
Areas more central to the auditory nerve contribute to forward masking (42-44). We
tested the hypothesis that the deactivation of Ih during a depolarization induced by the
masker tone contributes to a decreased response to the probe tone. We replaced the
masker tone by a depolarizing current injection to prevent any peripheral effects. In the
presence of Ih, the EPSP was smaller and a less depolarized membrane potential was
reached when the probe tone was preceded by a depolarization, whereas this was not
observed in the absence of Ih. Due to a non-linear relation between membrane potential
and spike rate, small effects on the membrane potential can already change excitability
in the inferior colliculus (19). Furthermore, the effects of Ih may be cumulative, as it is
abundantly present throughout the auditory brainstem. Nevertheless, the small size of
the observed effects precludes a large contribution to forward masking.
Effect of Ih on integration of sensory inputs
A well studied effect of Ih is that it reduces temporal summation of inputs (reviewed in
(1, 2)). Here we confirmed previous experiments performed in slices of the IC, which
showed that Ih decreases summation of the response to hyper- or depolarizing current
injections (8). The effects for depolarizing and hyperpolarizing current injections
appeared to be similar. The deactivation of Ih during depolarizing current injections (or
EPSPs) effectively creates an outward current, whereas the opposite will happen during
hyperpolarizing current injections (27, 45). It was not yet known whether these effects of
Ih on temporal summation are also relevant for physiological synaptic inputs in vivo. We
observed only small effects on the response to SAM tones. Ih may be relatively ineffective
during synchronized inputs, as evoked by SAM tones, whereas it is expected to have a
41
2012-12-05 21:32:37
stronger effect on desynchronized inputs, such as FM sweeps (40). Furthermore, Ih is
expected to attenuate responses to low-frequency inputs most effectively, whereas the
40 Hz SAM tones have a modulation frequency that is already too high to result in very
large attenuation of fluctuating inputs (23).
In response to FM sweeps, IPSPs became more conspicuous in the presence of Ih,
probably due to a more depolarized membrane potential and a subsequent increase in
driving force for inhibitory conductances (46). This effect may be more pronounced at
a higher driving force for chloride ions, which was relatively small in our experiments
(47). Experimental results with a slower gating model and simulated results with an Ih
channel that lacked gating altogether gave similar results, indicating that changes in
open probability of Ih during the tone did not contribute directly to the responses.
It was previously observed that the presence or absence of Ih is an important predictor
for the electrophysiological properties and responses to sound of IC neurons (7-9).
Here, we find that the more depolarized membrane potentials, more frequent rebound
spiking, lower input resistance and decreased summation during current injections that
have previously been observed for IC cells with Ih (7, 8) are likely due to a direct effect
of Ih. The different firing patterns associated with the presence of Ih or the association with
pauser or chopper responses to tones (7, 9) are most likely due to co-expression of Ih with
other channels. More subtle effects on tone responses that were observed in the present
study may be due to the activation of potassium currents or an increased driving force for
inhibitory inputs as a result of the depolarization caused by the presence of Ih.
ACKNOWLEDGMENTS
We thank C. Donkersloot for LabVIEW programming, A. Rodriguez-Contreras for helpful
discussions and M. Häusser and M. London (UCL) for their comments on an earlier
version of the manuscript.
GRANTS
This work was supported by a Neuro-BSIK grant (BSIK 03053; SenterNovem, The
Netherlands) and the Heinsius-Houbolt fund.
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2012-12-05 21:32:37
2012-12-05 21:32:37
3
Hearing loss in mice with
Cockayne syndrome
A.P. Nagtegaal, I. van der Pluijm, G.T.J. van der Horst,
J.G.G. Borst
To be submitted
2012-12-05 21:32:37
2012-12-05 21:32:38
4
Accelerated loss of hearing
and vision in the DNA-repair
deficient Ercc1 δ/– mouse
A.P. Nagtegaal§, M. Spoor§, Y. Ridwan, N. Zuiderveen
Borgesius, B. van Alphen, I. van der Pluijm,
J.H.J. Hoeijmakers, M.A. Frens, J.G.G. Borst
§
These authors have contributed equally
Mech Ageing Dev. 2012 Feb-Mar; 133(2-3): 59-67
2012-12-05 21:32:38
Hearing and vision loss in Ercc1 δ/– mice
Abstract
Age-related loss of hearing and vision are two very common disabling conditions, but the
underlying mechanisms are still poorly understood. Damage by reactive oxygen species
and other reactive cellular metabolites, which in turn may damage macromolecules such
as DNA, has been implicated in both processes. To investigate whether DNA damage can
contribute to age-related hearing and vision loss, we investigated hearing and vision in
Ercc1δ/– mutant mice, which are deficient in DNA repair of helix-distorting DNA lesions
and interstrand DNA crosslinks. Ercc1δ/– mice showed a progressive, accelerated increase
of hearing level thresholds over time, most likely arising from deteriorating cochlear
function. Ercc1δ/– mutants also displayed a progressive decrease in contrast sensitivity
followed by thinning of the outer nuclear layer of the eyeball. The strong parallels with
normal ageing suggest that unrepaired DNA damage can induce age-related decline of
the auditory and visual system.
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Introduction
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Among the most common impairments associated with ageing are age-related loss of
hearing (presbyacusis) and vision. Presbyacusis is characterized by increased hearing
thresholds, especially at higher frequencies, accompanied by a progressive reduction
of distortion product otoacoustic emissions (DPOAEs) (1-3). This process results from
a lifetime of insults to the auditory system to which the cochlear outer hair cells are
probably most sensitive (4, 5).
Senescent changes in visual performance include declines in visual acuity (the smallest
detail that can be resolved at high contrast levels) and spatial contrast sensitivity (the
ability to detect small increments in shades of gray on a uniform background) (6). Studies
on the effect of age on the spatial contrast sensitivity curve in humans have shown a
fall in high frequency sensitivity at middle age (6-10), leading to intermediate and high
spatial frequency attenuation above 60 years of age (7, 9). Moreover, an age-related
decline in contrast sensitivity was found across all spatial frequencies when measured
under scotopic conditions (11). Recently, an age-related decline in contrast sensitivity
was also revealed in mice (12). The major optic changes during ageing are insufficient
accommodation ability due to hardening of the lens, decrease in pupil size and increase
in density and yellowing of the lens. Within the retina, a loss of photoreceptors, bipolar
cells, ganglion cells, or changes in their connections can contribute to impaired vision (6).
Natural compounds such as cyclopurines produced by free reactive oxygen species (ROS)
(13), malondialdehyde (14, 15) or acetaldehyde (16, 17) can cause DNA damage. Indeed,
damage caused by ROS has been suggested to be one of the driving mechanisms for
age-related hearing and vision loss (18, 19). Even though it is not easy to establish a
causal relationship, ROS-induced DNA damage has been suggested to be implicated in
presbyacusis in several studies (20-23). Mice lacking the Cu/Zn superoxide dismutase
(SOD), a major radical scavenger, exhibit accelerated age-related hearing loss and
cochlear hair cell loss (24, 25).
Further support for the idea that accumulating DNA damage contributes to presbyacusis
and age-related vision loss comes from patients suffering from the rare inherited DNA
repair disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS). Already at
an early age they can show signs of presbyacusis, vision loss and degeneration of the
retina (26-31). However, patients carrying these DNA repair deficiencies are genetically
and phenotypically very heterogeneous, complicating deduction of cause-consequence
relationships. A genetically more defined mouse model of Cockayne syndrome, which
displays several features of the human syndrome, albeit in a relatively mild form (32),
exhibits an age-dependent thinning of the outer nuclear layer (ONL) of the retina due
to spontaneous loss of photoreceptors (33); loss of photoreceptors is also commonly
observed in ageing humans (34-37) and mice (38, 39). Mice with a deletion of the
Ercc1 gene have a progressive neurological phenotype. Already at a young age, they
display among others a reduced optokinetic response (OKR), suggesting that vision is
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reduced (40). The lifespan of this mouse is 2-3 months (41), and the main cause for the
neurological phenotype, including the decreased OKR, is proposed to be a consequence
of the prominent, progressive kidney failure (40).
Here, we investigate in detail the role of unrepaired DNA damage on the onset and
progression of presbyacusis and age-related vision loss in Ercc1δ/– mice of different ages.
The Ercc1δ/– mouse mutant lacks one allele of the excision repair cross-complementing
group 1 (Ercc1; ERCC1 in humans) gene. The protein derived from the other allele shows
reduced activity owing to a seven amino-acid carboxy-terminal truncation (14). This
hypomorph mutation results in severely impaired nucleotide excision repair, interstrand
cross-link repair (14) and double-strand break repair (21). Consequently, spontaneously
occurring DNA damage remains largely unrepaired. As a result the mutant mice display
several features of accelerated segmental ageing, including early cessation of growth
and nuclear abnormalities in liver and kidney, resulting in premature death (14) and
accelerated motor neuron degeneration (42). The maximum lifespan of these mice in
the homogeneous F1 C57BL/6J-FVB/N genetic background used here is approximately
6 months (IvdP and JHJH, unpublished observations), a time frame which is convenient
for analysis of development and progressive decline. Therefore we investigated hearing
and vision in this mutant to assess whether DNA damage exerts any effect on the agerelated functioning of these organs.
Materials and Methods
In this study 60 Ercc1δ/– mutants and 53 wild type littermates in an F1 hybrid Fvb/NC57BL/6J background strain were used. All mice were housed on a 12 h light /12 h dark
cycle with unrestricted access to food and water. Experiments were done during the
light phase. All experiments were approved by the local ethics committee and were
in accordance with the European Communities Council Directive (86/609/EEC). For
the contrast sensitivity experiments 14 Ercc1δ/– mutants and 14 wild type littermates
were used, which were measured longitudinally. For Auditory-evoked Brainstem
Response (ABR) recordings 11 Ercc1δ/– mutants and 10 wild type littermates were
measured longitudinally. For DPOAE measurements, 2 groups of 6 Ercc1δ/– mutants and
6 littermates, were measured, one group at 4 and one group at 12 weeks of age. For
histology 34 Ercc1δ/– mutants and 27 littermates were used.
Auditory experiments
ABR recordings
ABR recordings, which were used to obtain hearing level thresholds, were performed
largely as described previously (43). The mice were anesthetized with a mixture of
ketamine/xylazine (60/10 mg/kg) i.p. and placed in a sound-attenuated box with the ears
at a distance of 4 cm from a frontally placed tweeter loudspeaker (Radio Shack Super
Tweeter 40-1310B). Active needle electrodes were positioned subdermally at the base
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of both pinnae. The reference electrode was placed at the vertex. In addition, a ground
electrode was placed near the sacrum. Presentation of stimuli and averaging of responses
was controlled by custom-made software (EUPHRA = Erasmus University Physiological
Response Averager). Tone pip stimuli (1 ms duration, 0.5 ms cosine-squared ramps,
alternating polarity, repetition rate 80 per second) were generated by a Hewlett Packard
33120A waveform generator. Responses were acquired and averaged by a 32 MHZ DSP
Motorola 56002 board. The sound pressure level (SPL; re 20 µPa) of the stimuli ranged
between -10 and 110 dB. For determining ABR thresholds, 500 responses with artifacts
<30 μV were averaged. Stimuli were calibrated by comparing peak-to-peak values of the
tone pip stimuli (measured with a 1/2” Bruel and Kjaer microphone, type 4192) with a
calibrated tone (Bruel and Kjaer sound calibrator, type 4231, 94 dB) on an oscilloscope
(Tektronix TDS 1002). Hearing level thresholds were measured at 4, 8, 16 and 32 kHz.
Thresholds were defined as the lowest SPL (5 dB resolution) at which a reproducible peak
(usually peak II or IV) was still present in either ear. After finishing recordings the mice
were injected with atipamezole (25 μg s.c.) to facilitate recovery from anesthesia.
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DPOAE
We used the DP 2000, Starkey system to measure 2f1-f2 DPOAEs, with f2/f1 = 1.2. The
intensity of f1 and f2 was set at 65 and 55 dB SPL, respectively. Recordings were done
at f2 = 4, 6, 8, 10, 12 and 16 kHz. First the speaker was calibrated, followed by three
measurements in both ears. The maximum DPOAE amplitude per frequency in each
mouse was included in the analysis. Noise floor values were obtained from recordings
in a dead animal. DPOAEs of wild type animals were not above baseline between 4 and
8 kHz and therefore only results obtained at 10, 12 and 16 kHz are presented.
Visual experiments
Mouse contrast sensitivity function was determined as described previously (12).
This method infers contrast sensitivity by measuring how the magnitude (gain) of
compensatory eye movements varies with different combinations of contrast and
spatial frequency. Animals were prepared for head fixation by attaching two metal nuts
to the skull using a construct made of a micro glass composite.
Stimulus setup
Optokinetic stimuli were created using a modified Electrohome Marquee 9000 CRT
projector (Christie Digital Systems, Cypress CA, USA), which projected stimuli via mirrors
onto three transparent anthracite-colored screens (156 cm x 125 cm) that were placed
in a triangular formation around the recording setup (see (12), for more details). This
created a green monochrome panoramic stimulus fully surrounding the animal. The
stimuli were programmed in C++ and rendered in openGL and consisted of a virtual,
vertically oriented cylinder with a dotted pattern or vertically oriented sine grating on
its wall. Each pixel subtended 4.5 x 4.5 arcminutes. Contrast (C) in the projected sine
gratings was calculated using the Michelson formula: C = (Lmax – Lmin) / (Lmax + Lmin), where
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Lmax and Lmin are the maximum and minimum luminance in a grating, respectively.
The average luminance was kept constant at 17.5 cd/m2 in all stimulus conditions.
At maximum contrast, the minimum and maximum luminance of the stimulus were
0.05 cd/m2 and 35.0 cd/m2, respectively.
Eye movement recordings
Mice were immobilized by placing them in a plastic tube, with the head pedestal
bolted to a restrainer that allowed placing the eye of the mouse in the centre of the
visual stimulus, in front of the eye position recording apparatus. Eye movements were
recorded with an infrared video system (Iscan ETL-200). Images of the eye were captured
at 120 Hz with an infrared sensitive CCD camera (see (12), for more details). To keep the
field of view as free from obstacles as possible, the camera and lens were mounted under
the table surface, and recordings were made with a hot mirror that was transparent to
visible light and reflective to infrared light. The eye was illuminated with two infrared
LEDs at the base of the hot mirror. The camera, mirror and LEDs were all mounted on
an arm that could rotate about the vertical axis over a range of 26.12º (peak to peak).
Eye movement recordings and calibration procedures were similar to those described
previously (44). Trials were randomized, mice were assigned a number and the data
analysis scripts were automated. All data were analyzed after the experiment.
Contrast sensitivity function
Afoveate mammals (like mice) show robust gaze-stabilizing eye movements, such
as the optokinetic reflex (OKR). The OKR prevents the image of the surroundings to
slip across the retina during movement of the visual scene. Contrast sensitivity was
tested by presenting moving visual stimuli to the mice and recording eye movements
evoked by those stimuli (12). Each stimulus was a vertical sine wave grating made up
of a combination of one of seven spatial frequencies (0.03, 0.05, 0.08, 0.17, 0.25, 0.33,
or 0.42 c/º) and one of six contrast values (100%, 75%, 50%, 25%, 10%, 1%). The 42
stimulus combinations were presented in random order. A stimulus was first projected
and kept stationary for one minute, allowing the animal to adjust to changes in the
stimulus. Subsequently, the stimulus started to move with a constant velocity of 1.5º/s.
After moving to one direction for 2 s, it changed direction and moved in the opposite
direction for 2 s. This was repeated six times, yielding ten changes in direction. While the
stimulus was moving, motion of the left eye was recorded.
Recorded eye positions were transformed offline into a velocity signal. Fast phases and
saccades were removed from the eye movement recordings using a velocity threshold
of 3º/s, i.e. twice the stimulus velocity. The first 200 ms after stimulus onset and after
each change in direction were removed as well. Because the stimulus velocity was
constant and eye data in the first 200 ms after the stimulus direction changes were
ignored, average absolute eye velocity could be divided directly by the stimulus velocity
to calculate a gain value for each combination of spatial frequency and contrast. An eye
movement that perfectly follows the visual stimulus has a gain of 1 (45).
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Saccade analysis
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Since changes in the optokinetic reflex may be due to impaired vision, impaired
motor system or a combination of both, we analyzed a second type of eye movement,
saccades, in order to rule out an impaired motor system in the mutant mice. Saccades
are rapid eye movements that are clearly distinct from the optokinetic reflex. Saccades
are made spontaneously and are not directly related to the optokinetic stimulation. The
kinematics of saccades are extremely stereotyped; there is a fixed relation between the
amplitude of a saccade and its peak velocity. This relation is known as the main sequence
(46). Peak velocity increases with amplitude. At larger amplitudes this relation saturates,
but the oculomotor range of the mouse is too small to reach these values. Age-related
changes in the oculomotor system predict that the slope of the main sequence relation
decreases over time. Possible saccade on- and offsets were marked automatically, using
a velocity threshold of 3 times the stimulus peak velocity. The exact on- and offsets were
subsequently marked by hand. To compare the main sequences, regression lines were
fitted for each age and the slope values were used for statistical purposes.
Immunohistochemistry
For isolation of the eyes animals were euthanized by CO2 inhalation. Eyes were marked
on the nasal side with Alcian blue (5% Alcian blue in 96% ethanol), dissected and
subsequently fixed in 4% paraformaldehyde in 0.1 M phosphate buffer and embedded
in paraffin. Five μm thick, transverse paraffin sections were cut. For determination of
retinal surface area, digital images of the whole retina were taken using a microscope
equipped with a high-resolution camera (BX40 microscope, ColorViewIllu camera
and the CellA program). The CellA program was used to make digital images of the
same sections used for quantification of the apoptotic cells in the outer nuclear layer
(ONL). The program Sigmascan Pro5 was used to measure the surface area of the ONL.
Apoptotic cells were visualized using the TdT-mediated dUTP Nick-End Labeling (TUNEL)
method according to the specifications of the manufacturer of the kit (Apoptag Plus
Peroxidase In Situ Apoptosis Detection Kit, Chemicon). For quantification, the number of
TUNEL-positive cells in the ONL was counted using a BX40 microscope with a minimum
of 5 transverse cut sections per mouse. To standardize the chosen area of the retina,
the sections closest to the eye nerve were selected for immunohistochemistry and
quantification. The number of apoptotic cells and retinal surface area in 5 sections per
animal were averaged and animals were averaged per group accordingly.
Statistics
The effect of the Ercc1δ/– mutation on contrast sensitivity, hearing thresholds and
DPOAEs was analyzed using a repeated measures ANOVA with three factors. For contrast
sensitivity we used genotype as between subjects factor, and contrast and spatial
frequency as within subject factor. Post hoc, groups were compared at each contrast by
averaging OKR gains over spatial frequencies. Differences between groups were analyzed
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for significance with a Student’s t-test. For the effect of the Ercc1δ/– mutation on hearing
thresholds and DPOAEs we used genotype and age as between, and frequency as within
subjects factors. Post hoc, groups were compared with two-way ANOVAs and Bonferroni
tests. Comparison of main sequence slopes was done by Monte Carlo bootstrapping
each main sequence (N = 1000), and comparing them by two-tailed Student’s t-tests.
Results
Progressive hearing loss in Ercc1δ/– mice
To objectively assess whether presbyacusis develops in Ercc1δ/– mice, hearing level thresholds
were measured longitudinally using the auditory brainstem response (ABR). Auditory
thresholds in Ercc1δ/– mice were already elevated at 5 weeks of age, the first measurement
point. Importantly, auditory function further deteriorated in the following 9 weeks (Fig. 1A),
suggesting that the defect at 5 weeks of age is part of a progressive degenerative process. We
performed a three-way repeated measures ANOVA with genotype and age as between, and
frequency as within subjects factors. All three main effects and their pair-wise interactions
had a significant effect on hearing level thresholds (all p < 0.001). To clarify the origin of
these significant changes, we performed a two-way repeated measures ANOVA. The Ercc1δ/–
mice showed a significant effect of age (p < 0.001) and a significant interaction of age and
frequency (p < 0.001). In contrast, there was no effect of age in the wild type mice (p = 0.30;
Fig. 1A). This shows that only the Ercc1δ/– mice have an age-dependent increase in hearing
thresholds and, importantly, that this increase is not the same for all frequencies. Age had
a significant effect for 8 kHz (p < 0.05), 16 kHz and 32 kHz (both p < 0.001) but not for 4 kHz
(p > 0.05; Bonferroni post hoc test). Taken together, these findings show that the Ercc1δ/– mice
have an age-dependent increase in hearing thresholds especially at higher frequencies,
while their wild type littermates are not affected.
Progressive hearing loss in Ercc1δ/– mice is caused by hair cell loss
Further studies were performed to determine the site of origin of the hearing loss within
the auditory pathway. The conduction speed of the auditory response was used as a test
for possible retrocochlear pathology. We identified peaks I to V in the ABR and compared
the inter-peak latencies of the most prominent peaks, II and IV, at ages 4 and 12 weeks
between wild type and Ercc1δ/– mice at 4 and 16 kHz. No statistically significant latency
differences were observed between wild type and Ercc1δ/– mice both with and without
correction for difference in minimum hearing level threshold (p > 0.05). II-IV inter-peak
latencies at 4 kHz were 1.89 ± 0.06 (mean ± SEM) and 1.82 ± 0.11 ms for wild type and
1.91 ± 0.03 and 1.71 ± 0.08 ms for Ercc1δ/– mice at 4 and 12 weeks, respectively. At 16 kHz,
II-IV intervals were 1.99 ± 0.04 and 1.83 ± 0.10 ms for wild type and 2.03 ± 0.08 and 1.82 ±
0.09 ms for Ercc1δ/– mice at 4 and 12 weeks, respectively (results not shown). This suggests
that the observed differences in hearing level thresholds are not due to retrocochlear
deficits in the mutants.
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(a)
ABR threshold longitudinal
ABR threshold (dB SPL)
100
80
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Wildtype 5 wks
Ercc1δ/– 5 wks
Wildtype 14 wks
Ercc1δ/– 14 wks
60
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32
Frequency (kHz)
(b)
4 wk old DPOAEs
30
DPOAE (dB)
25
20
Wildtype
Ercc1δ/–
Noise floor
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0
-5
10
12
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Frequency (kHz)
(c)
12 wk old DPOAEs
30
DPOAE (dB)
25
20
Wildtype
Ercc1δ/–
Noise floor
15
10
5
0
-5
10
12
Frequency (kHz)
16
Figure 1. The combination of increased ABR hearing thresholds and reduced DPOAEs suggests
outer hair cell loss in the cochlea of Ercc1δ/– mice. A: Ercc1δ/– mice show hearing loss, especially
at higher frequencies, from 5 to 14 weeks of age. Filled symbols represent wild type and open
symbols Ercc1δ/– mice. Circles and triangles represent 5 and 14 week old animals, respectively.
B: 4 week old Ercc1δ/– mice have similar DPOAEs as their wild type littermates. C: 12 week old Ercc1δ/–
mice have reduced DPOAEs at higher frequencies compared to their littermates, suggesting outer
hair cell loss. Data are shown as means ± SEM.
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Otoacoustic emissions are a sensitive assay of cochlear function, especially of the outer
hair cells. We evoked DPOAEs at 4 and 12 weeks of age in Ercc1δ/– mice and their agematched littermates (Fig. 1B, C). We performed a three-way repeated measures ANOVA
with genotype and age as between, and frequency as within subjects factors, which
revealed that all main effects and their interactions were significant (all p < 0.05).
Two-way ANOVA testing indicated no effect of genotype (p = 0.27) at 4 weeks (Fig. 1B),
while a highly significant effect size (p < 0.0001) was observed at 12 weeks (Fig. 1C).
Together, the lack of a significant difference in ABR inter-peak latencies and the
decreased DPOAEs in 12-week-old Ercc1δ/– mice indicate a cochlear origin of the hearing
loss, probably including outer hair cell damage.
Progressive decline of vision in Ercc1δ/– mice
To assess visual performance of Ercc1δ/– mice over their life span we measured in a
longitudinal study the optokinetic reflex (OKR) gains at several different spatial
(a)
(b)
100% contrast
OKR Gain
1
0.5
(c)
0.2
0.4
25% contrast
0
(f)
1
50% contrast
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75% contrast
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10% contrast
(g)
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1% contrast
OKR Gain
Wildtype
Ercc1δ/–
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0.2
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Spatial Frequency (c/º)
0
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0.2
0.4
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0.4
Figure 2. Normal contrast sensitivity in Ercc1δ/– mice and their littermates at 6 weeks of age.
A: the color reflects the OKR gains at 42 different combinations of spatial frequency and contrast.
Measured points are connected through linear interpolation. B-G: six cross sections of A and B are
plotted, one for each contrast. Even at low contrast values, Ercc1δ/– mice have similar OKR gains as
their wild type littermates, indicating normal vision. All data represented as means ± SEM. Filled
circles represent wild type and open circles Ercc1δ/– mice.
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2012-12-05 21:32:39
frequencies and contrast values. At 6 weeks of age, all stimuli evoked OKR gains
that were similar in both genotypes (Fig. 2). The only exception was at 75% contrast,
where the gain was slightly lower in Ercc1δ/– (p < 0.05). Both groups showed a similar
optimum for all contrasts, which was reached at 0.17 - 0.25 c/º. At 10 weeks of age,
OKR gains were lower in Ercc1δ/– mutants (Fig. 3). Ercc1δ/– mice performed worse than
their wild type littermates at all contrast levels (p < 0.05), except at 100% contrast.
Gains were lower and the window in which responses occurred narrowed, i.e. the
highest (0.42 c/º) and lowest (0.03 c/º) spatial frequencies did not elicit any response
in mutant mice at contrasts below 100%. At 14 weeks of age, all OKR gains were
strongly reduced in Ercc1δ/– mutants (p < 0.001) (Fig. 4). The response window
narrowed further, i.e. there was almost no response below 0.08 c/º or above 0.25 c/º.
Below 50% contrast, hardly any response was observed. Moreover, even within the
optimal stimulus window (100% contrast and 0.17 - 0.25 c/º), the evoked OKR gain
was lower in the Ercc1δ/– mice.
1
2
3
4
5
6
7
&
(a)
(b)
100% contrast
OKR Gain
1
(c)
0.5
0.5
0
1
(d)
0.2
25% contrast
0
0
0.4
(f)
1
50% contrast
1
0.5
0
0
(e)
75% contrast
1
0.2
0.4
10% contrast
0
(g)
1
0.2
0.4
1% contrast
OKR Gain
Wildtype
Ercc1δ/–
0.5
0.5
0
0.5
0
0
0.2
0.4
0
0
0.2
0.4
0
0.2
0.4
Figure 3. Reduced contrast sensitivity in Ercc1δ/– mice compared to their littermates at 10 weeks
of age. A: the color reflects the OKR gains at 42 different combinations of spatial frequency and
contrast. Measured points are connected through linear interpolation. B-G: six cross sections of
A and B are plotted, one for each contrast. Compared to 6 week old animals, Ercc1δ/– mice show
slightly reduced OKR gains at all contrast values. All data represented as means ± SEM. Filled circles
represent wild type and open circles Ercc1δ/– mice.
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2012-12-05 21:32:40
(a)
(b)
100% contrast
OKR Gain
1
0.5
(c)
0.2
0.4
25% contrast
0
(f)
1
50% contrast
1
0
0
0
1
(d)
0.5
0.5
0
(e)
75% contrast
1
0.2
0
0.4
10% contrast
(g)
1
0.2
0.4
1% contrast
OKR Gain
Wildtype
Ercc1δ/–
0.5
0.5
0.5
0
0
0
0
0.2
0.4
0
0.2
0.4
0
0.2
0.4
Figure 4. Severely reduced contrast sensitivity in Ercc1δ/– mice compared to their littermates
at 14 weeks of age. A: the color reflects the OKR gains at 42 different combinations of spatial
frequency and contrast. Measured points are connected through linear interpolation. B-G: six cross
sections of A and B are plotted, one for each contrast. Visual performance is severely reduced in
Ercc1δ/– mice, even at 100% contrast. All data represented as means ± SEM. Filled circles represent
wild type mice, open circles Ercc1δ/– mice.
At all ages, the mutant mice made smaller saccades compared to the wild type mice
(Fig. 5), probably due to a smaller oculomotor range. However, the main sequence was
not different between mutants and wild types, nor did it change over time in either
group, indicating an intact oculomotor system (all p > 0.05).
Progressive reduction of outer nuclear layer in Ercc1δ/– mice
One explanation of the loss of visual performance could be loss of photoreceptor cells
in the retina. Therefore we determined the surface area of the outer nuclear layer (ONL)
in cross sections of the eye at 4, 9, 18 and 25 weeks of age in Ercc1δ/– mice and their agematched littermates (Fig. 6A). We performed a two-way ANOVA and found a significant
effect for both age and genotype (p < 0.0001) and for their interaction (p < 0.005).
Bonferroni post hoc tests revealed that only at 25 weeks of age there was a significant
genotype effect (p < 0.001). This shows an age-dependent reduction of the ONL in
Ercc1δ/– mice suggestive of loss of cells, most likely photoreceptors. To further investigate
70
2012-12-05 21:32:40
1
2
3
4
5
6
7
&
Figure 5. Saccade peak velocity as function of amplitude. A, B: representative examples of saccade
peak velocity recorded from a wild type (A) and a mutant mouse (B) at three different ages. Each dot
represents a single saccade. C, D: average peak velocity of saccades recorded from 14 wild type (C) and
12 mutant mice (D) at three different ages. There is no evidence for degeneration of the oculomotor
system, as the slope remains unaltered with increasing age. Data represented as means ± SEM.
ONL Surface Area
(a)
Surface Area ONL (mm2)
0.25
0.20
0.15
0.10
0.05
0
4
9
18
25
Age (weeks)
(b)
ONL TUNEL Positive Nuclei
TUNEL Nuclei (#/mm2)
250
200
Wildtype
Ercc1δ/–
150
100
50
0
4
9
18
25
Age (weeks)
Figure 6. Age-dependent loss of cells through apoptosis in the ONL in Ercc1δ/– mice. A: agedependent reduction of ONL surface in cross sections of Ercc1δ/– eyes compared to those of their
littermates. B: Age-dependent increase of TUNEL-positive cells normalized to ONL surface in Ercc1δ/–
mice. Black columns represent wild type mice, open columns Ercc1δ/– mice. All data represented as
means ± SEM.
71
2012-12-05 21:32:43
the cause of the reduction of the ONL we determined the number of TUNEL-positive
cells, i.e. cells in apoptosis, in the ONL at 4, 9, 18 and 25 weeks of age (Fig. 6B). Two-way
ANOVA analyses revealed a significant effect of age, genotype and their interaction
(p < 0.0001). These results strongly suggest that the progressive reduction of the ONL
was caused by loss of cells through apoptosis.
Discussion
Our findings demonstrate that the DNA repair defect in Ercc1δ/– mouse results in
accelerated age-related decline of both vision and hearing compared to wild type
littermates. This indicates that accumulation of spontaneous DNA damage, which
normally would have been repaired by nucleotide excision and cross link repair, triggers
accelerated loss of hearing and vision abilities. The most likely cause for these deficits
is that helix-distorting and interstrand crosslink DNA damage within the cochlea and
retina is responsible for the observed age-dependent deterioration. However, cochleaand retina-specific mutants would be needed to fully exclude a possible contributing
role of for example liver or kidney failure (14, 40, 41, 47-49).
Presbyacusis
We found that Ercc1δ/– mice display a progressive increase of hearing thresholds in
combination with reduced DPOAEs. Thresholds were already elevated at the youngest
age we tested. This might be due to very rapid deterioration or due to abnormal
development, a distinction which is not always easily made for the neurological
abnormalities that are associated with DNA repair disorders (50). The unchanged ABR
inter-peak latencies suggest a lack of major defects in the central auditory pathway,
even though more subtle changes cannot be excluded (51). These findings thus indicate
a cochlear origin of the hearing loss. The hearing loss is unlikely to be an indirect
consequence of renal or liver failure (14, 40, 41, 47), since encephalopathy following
renal or liver failure is generally not associated with hearing loss (52, 53), whereas high
bilirubin resulting from liver failure targets central auditory structures, but spares the
cochlea (54, 55). The phenotype of the Ercc1δ/– mice, i.e. increased hearing threshold,
reduced DPOAE and normal ABR inter peak latencies, is similar to what is seen in human
presbyacusis (1-5, 56, 57).
Considerable evidence for a causative role of mitochondrial DNA (mtDNA) damage in
presbyacusis has been presented (58-60). However, the complex nucleotide excision
repair pathway involving at least 30 proteins (61) is restricted to the nucleus and
the Ercc1δ/– mutations are not expected to directly affect mtDNA repair. Hence, our
results indicate that, in addition to mtDNA damage, nuclear DNA damage can also
play a causative role in presbyacusis. Moreover, since nuclear DNA encodes the vast
majority of mitochondrial proteins, an indirect effect on mitochondrial function can
be anticipated.
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2012-12-05 21:32:43
One of the options emerging from the above scenario for the etiology of presbyacusis
is the possibility for prevention of hearing loss by anti-oxidants to reduce ROS levels,
which may be one of the causative factors for DNA damage. Several such studies have
been reported in mice (57, 62-64), rats (65) and humans (66, 67) with overall promising
results. Our findings lend support to the idea that a possible preventive effect of antioxidants in presbyacusis would act by preventing cochlear DNA damage.
1
2
3
4
5
6
7
&
Age-related vision loss
We measured OKR gains to infer the contrast sensitivity and visual acuity of mice. To
this end we presented visual stimuli with different spatial frequencies and contrasts.
In 6-week-old mice there were only subtle differences in contrast sensitivity between
wild types and mutants, as similar OKR gains were recorded for all stimuli in our study.
However, at 14 weeks of age, contrast sensitivity had decreased to the point where
mutant mice were only able to observe a small set of contrast-spatial frequency
combinations. The overall decrease in contrast sensitivity occurring in the Ercc1δ/–
during a period of only 8 weeks followed a similar pattern as was reported for normal
ageing in C57BL/6J mice (12), for which the ability to perceive low contrasts and high
spatial frequencies disappeared first. A much more severe phenotype was previously
observed in an Ercc1 null mutant, in which the very severe, lifespan-limiting liver
degeneration of Ercc1 null mice was rescued by an Ercc1-liver-specific transgene. In
this model the OKR response was already very poor at 4 weeks, with little change at
8 weeks (40). These mice have a life span of only 2 - 3 months (41) as compared to
4 - 6 months for the milder Ercc1δ/– mice, suggesting that the more severe phenotype
of the null mutant is most likely caused by the more complete genetic deletion. Apart
from confirming the contrast sensitivity in another Ercc1 deficient mouse model, our
study used a different contrast sensitivity measurement than Lawrence et al. (40) and
recorded the actual eye movements instead of observing head tracking. This enables
measurement of the optokinetic response and therefore vision more accurately (12). In
any behavioural response, pathology of the motor system can play a role, as was also
suggested by Lawrence et al (40). However, by also analyzing saccadic eye movements,
and showing that their dynamics were intact (Fig 5), we demonstrated that both the
neural and the muscle part of the oculomotor system were unaffected by the mutation.
We conclude that the decrease in OKR gain most likely resulted from age-related
deterioration of the visual system.
In humans, OKR gains decline with age, at high frequencies as well as at high velocities,
owing to an impaired sensorimotor system (68, 69). In contrast, this effect was not
found in mice (70, 71). However, it could be that the mice were still too young (about
15 months) to reveal an age-dependent effect. Studies in humans on the effect of age
on the spatial contrast sensitivity function curve have shown a decline in high frequency
sensitivity at middle age, leading to intermediate and high spatial frequency attenuation
with increasing age due to optical and neural degeneration (7, 9, 10, 72-76).
73
2012-12-05 21:32:43
Our histology data showed increased, age-related loss of photoreceptors in the mutants.
However, it is unlikely that this fully explains the observed vision loss, as the effects of
age on vision were already apparent at 10 weeks while the reduction of the ONL did not
reach significance until 25 weeks. Indeed, Samuel et al (77) showed recently that other,
more subtle factors in the mouse retina are mainly responsible for the deterioration
of vision in ageing mice, and similar mechanisms may play a role in the Erccδ/– mutant.
Our data thus agree with and extend previous experiments on an Ercc1 null mutant, for
which the loss of contrast sensitivity was not matched by histological abnormalities in
the retina (40).
Although photoreceptor loss or thinning of the ONL is commonly seen in ageing in
humans (34-37), mice (38, 39) and mouse models of accelerated ageing (33, 78, 79), it
is not clear how relevant this phenomenon is to age-related vision loss. Nevertheless,
since optic changes cannot fully explain age-related vision loss, it is likely that loss or
reduced function of photoreceptors does contribute to age-related vision loss (6).
In conclusion, the Ercc1δ/– mutant shows accelerated age-related loss of hearing and
vision with characteristics very similar to human presbyacusis and age-related vision
loss. This suggests a causative role of DNA damage in the etiology of presbyacusis and
age-related vision loss. Our results also suggest a possible causative role for (functional)
loss of outer hair cells and photoreceptors, although a contribution of other cell types
cannot be excluded. Additional research using conditional knockouts will help resolve
the relative contribution of different cell types to these processes.
Disclosure statement
The research was supported in part by Top Institute Pharma, a public-private partnership
consortium. The authors declare there is no conflict of interest.
Acknowledgments
We thank Dr. Efstathios B. Papachristos for help with statistical analysis and Dr. Ype
Elgersma for helpful comments on an earlier version of this manuscript. MS is supported
by a HFSP grant. BvA was supported by a NWO-ALW grant. APN was supported by
the Heinsius-Houbolt fund. JHJH and YR were supported by a Top Instititute Pharma
grant, Markage (FP7-Health-2008-200880), LifeSpan (LSHG-CT-2007-036894), National
Institute of Health (NIH)/National Institute of Ageing (NIA) (1PO1 AG-17242-02) and the
European Research Council (ERC advanced scientist grant to JHJH).
74
2012-12-05 21:32:43
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66. Takumida M, Anniko M. Radical scavengers
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67. Durga J, Verhoef P, Anteunis LJ, Schouten E,
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70. Stahl JS. Eye movements of the murine
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71. Stahl JS, James RA, Oommen BS, Hoebeek FE,
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72. Wang YZ. Effects of aging on shape
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on spatial frequency perception in human
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74. Owsley C, Sekuler R, Boldt C. Aging and
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75. Nameda N, Kawara T, Ohzu H. Human visual
spatio-temporal frequency performance
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76. Nomura H, Ando F, Niino N, Shimokata H,
Miyake Y. Age-related change in contrast
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77. Samuel MA, Zhang Y, Meister M, Sanes
JR. Age-related alterations in neurons of
the mouse retina. J Neurosci. 2011 Nov
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78. Shoji M, Okada M, Ohta A, Higuchi K,
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2012-12-05 21:32:44
2012-12-05 21:32:44
5
A novel QTL underlying
early-onset, low frequency
hearing loss in BXD recombinant
inbred strains
A.P. Nagtegaal§, S. Spijker§, T.T.H. Crins, Neuro-Bsik
Mouse Phenomics consortium1, J.G.G. Borst
§ These authors have contributed equally
1
See appendix
Genes Brain Behav. 2012 Sep 18 [Epub ahead of print]
2012-12-05 21:32:44
Novel gene locus for early-onset hearing loss
ABSTRACT
The DBA/2J inbred strain of mice has been used extensively in hearing research as it suffers
from early-onset, progressive hearing loss. Initially, it mostly affects high frequencies,
but already at 2–3 months hearing loss becomes broad. In search for hearing loss genes
other than cadherin 23 (otocadherin) and fascin-2, which make a large contribution to
the high frequency deficits, we used a large set of the genetic reference population of
BXD recombinant inbred strains. For frequencies 4, 8, 16 and 32 kHz, auditory brainstem
response (ABR) hearing thresholds were longitudinally determined from 2–3 up to 12
weeks of age. Apart from a significant, broad quantitative trait locus (QTL) for high
frequency hearing loss on chromosome 11 containing the fascin-2 gene, we found a
novel, small QTL for low frequency hearing loss on chromosome 18, from hereon called
ahl9. Real-time quantitative PCR of organs of Corti, isolated from a subset of strains,
showed that a limited number of genes at the QTL was expressed in the organ of Corti.
Of those genes, several showed significant expression differences based on the parental
line contributing to the allele. Our results may aid in the future identification of genes
involved in low frequency, early-onset hearing loss.
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INTRODUCTION
1
2
3
4
5
6
7
&
The genetic basis of age-related hearing loss (AHL) has been well studied in the mouse
(1, 2). Longitudinal measurements of hearing level are relatively easy and the contribution
of genetic and environmental factors can be assessed more readily than in humans. Many
mouse strains develop age-related hearing loss (3), including the C57BL/6J mouse, which
is often used as the background strain in transgenic studies. In this strain, the first AHL
locus (ahl) was described and mapped to a site on chromosome 10 (4). Additional studies
in inbred mouse strains identified more quantitative trait loci (QTLs) for age-related
hearing loss, including ahl2 (5), ahl3 (6, 7), ahl4 (8), ahl5 & 6 (9), and, more recently, ahl8,
which is located on the distal end of chromosome 11 (10). In addition, Phl1 and Phl2 were
identified as QTLs for progressive hearing loss (11). To date, the responsible genes were
only identified for ahl and ahl8. For ahl, the responsible gene is cadherin 23, also called
otocadherin, which is required for the normal organization of hair cell stereocilia (12).
For ahl8, a mutation in the fascin-2 gene, which also encodes a component of stereocilia,
contributes to the early-onset hearing loss in DBA/2J mice (13).
The BXD strains are a panel of recombinant inbred strains derived from the parental
strains C57BL/6J and DBA/2J (from hereon called B6 and D2, respectively). D2 is a widely
used mouse strain in which hearing loss starts much earlier than in B6. In addition, low
frequencies are also affected in D2, in contrast to the high frequency hearing loss of B6
(14). The BXD strains have been used to identify QTLs for the early-onset hearing loss of
D2 (10, 14). At frequencies above 8 kHz, up to 75% of total variance can be explained by
mutations in cadherin 23 (ahl), fascin-2 (ahl8) and their interaction in (B6.CAST-Cdh23Ahl+
x D2) x D2 backcross mice. However, at 8 kHz, approximately two thirds of the variance
remains unexplained, while up to half of the variance in auditory brainstem response
(ABR) to clicks (2-8 kHz range) could not be attributed to ahl or ahl8 (10). In addition,
preliminary evidence has suggested the presence of an additional third locus in D2
(15). In this study, we therefore performed a search for the existence of additional QTLs
underlying early-onset hearing loss in BXD strains, incorporating recordings at lower
frequencies and making use of the advanced intercross BXD mouse strains that have
recently become available (16).
MATERIAL AND METHODS
Animals
Parental (C57BL/6J, DBA/2J) and 33 BXD lines were received from The Jackson
Laboratory (http://www.jax.org), or from Oak Ridge Laboratory (BXD43, BXD62, BXD65,
BXD68, BXD69, BXD73, BXD75, BXD87, BXD90), and were bred in the facility of the
Neuro-Bsik consortium of the VU University Amsterdam. Hearing level thresholds were
longitudinally assessed at either 2 or 3 weeks (to minimize the burden of anesthesia at a
very young age) and subsequently at 4, 6 and 12 weeks of age under ketamine / xylazine
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2012-12-05 21:32:44
anesthesia (60/10 mg/kg i.p.) in both male and female mice. The average number of
mice per strain was 7 (range 2 – 13), generally derived from 2 different litters. To facilitate
recovery from anesthesia, mice were injected with atipamezole (25 μg s.c.) at the end of
the experiments. Recordings were performed during the light phase. Mice were housed
in same-sex groups after weaning, from 3 to 4 weeks onwards. The experiments were
approved by the Erasmus University Animal welfare committee (DEC).
Auditory brainstem response
The auditory brainstem response (ABR) was used to obtain hearing level thresholds as
described previously (17). Briefly, hearing level thresholds were measured in response
to 1 ms tone pips at 4, 8, 16 and 32 kHz at intensities ranging from -10 to 110 dB sound
pressure level (SPL; re 20 µPa) at 5 dB resolution. At each sound intensity tested, 500
brainstem evoked responses with artifacts below 30 µV were averaged; the minimum
threshold was defined as the lowest SPL at which a reproducible peak could be identified.
If no response was elicited at 110 dB, the threshold was classified as 115 dB.
Heritability calculation
As a quantitative trait for early-onset hearing loss, we used the difference between the
ABR threshold at 12 weeks and the lowest threshold measured at any age. For each
frequency, this maximum hearing loss (MHL) was calculated per mouse and subsequently
averaged per strain. Narrow-sense heritability (h2) of the MHL was calculated as the
ratio of the within- and between-strain variance: (F - 1) / (F – 1 + 2k), in which F is the
between-groups divided by within-groups mean squares ratio, which can be retrieved
from a normal one-way ANOVA (18) and k is a function of the replicate number of mice
per strain, defined by: (N – ((Σ n2) / N )) / (S – 1), in which N is the total number of mice,
n is the number of mice for each strain and S is the number of different strains (19), as
implemented previously (20, 21).
QTL linkage mapping
Linkage mapping of MHL values of BXD plus parental strains to genotypes was performed
by WebQTL scripts (http://www.genenetwork.org/), which uses a set of 3795 single
nucleotide polymorphism (SNP) and microsatellite markers (22, 23). For each part of the
genome a likelihood ratio statistic (LRS) was calculated by Haley–Knott interval mapping
(24). Suggestive and significant LRS values were determined by a permutation test (25),
consisting of 1000 permutations in which trait values were randomly reassigned across
all strains, followed by a comparison of the permuted and original outcome data to assess
significance. A significant LRS value was defined as a 5% probability of falsely rejecting
the null hypothesis that there is no linkage anywhere in the genome. A suggestive
threshold represents the LRS value which yields, on average, one false-positive QTL per
genome scan (p = 0.63). Candidate genes were analyzed within the 1-LOD (= 4.61 LRS)
drop-off region with respect to the maximum LRS, which theoretically corresponds
to a 97% confidence interval (26). Pearson correlation coefficients and respective
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2012-12-05 21:32:44
probabilities were calculated across strain means to approximate genetic correlations.
Linear regression was used to quantify independent contributions of the gene loci to
the observed maximum hearing loss. For the analysis of SNPs and (non)-synonymous
mutations in genes at the QTL peak, the SNP browsers from the Phenome database of
Jackson Laboratories (http://phenome.jax.org/db/q?rtn=snp/ret1) and GeneNetwork
(http://www.genenetwork.org/webqtl/main.py?FormID=snpBrowser) were used, the
latter of which harbors data from multiple sequencing projects, including the Wellcome
Trust Sanger Institute and the UCLA genome browser.
1
2
3
4
5
6
7
&
Expression analysis of candidate genes
Real-time quantitative PCR (qPCR) for genes on the chromosome 18 and 10 loci (Tables
1, 2, S3) was carried out on cochlea tissue of selected strains at 12 weeks of age (BXD11,
n = 7; BXD13, n = 8; BXD32, n = 6; BXD73, n = 5; B6, n = 12; D2, n = 10). RNA extraction
(tissue pooled from 2 mice) and cDNA synthesis (300 ng RNA equivalent in a 25 µl
reaction) was performed as described previously (21, 27). PCR measurements (10 μl; ABI
PRISM 7900 Applied Biosystems, Foster City, CA, USA) were performed with transcriptspecific primers (300 nM; Table S4) on cDNA corresponding to ~1.2 ng total DNAse-I
treated RNA. Cycle of threshold (Ct) values were used to calculate the relative level of gene
expression (log2 scale) normalized to the geometric mean of the replicated reference
controls GAPDH and b-actin, as described before (27). Control genes, such as Cdh23 (28),
Otof (29) and the cochlea-specific subunit of the acetylcholine receptor Chnra9 were
measured twice. Only primer sets for which a single product could be detected were
used, as determined by dissociation analysis of the end-product. Presence or absence
of gene expression in cochlea was determined using a pooled cDNA sample of cochlea
versus total brain cDNA. The gene was called ‘not expressed’ when primers detected
a true product in the total sample in a replicate PCR but not in the cochlea sample, or
when the Ct value of the cochlea sample was within 2 cycles of the water control.
Statistical analysis
Quantitative measurements were analyzed by Pearson correlation to the trait and by
ANOVA for the factor allele (i.e., B6 or D2). Non-parametric ranks are indicated for strain
averages carrying the B6 or D2 allele. All data are presented as average ± standard error
(SEM).
RESULTS
The hearing of a total number of 35 (including parental) strains was evaluated in
longitudinal recordings from a postnatal age of either 2 or 3 weeks up to the last
measurement at 12 weeks of age (Fig. S1 and Table S1). Maximum hearing loss (MHL)
varied greatly among strains and frequencies (Fig. 1). Pearson correlations between
MHLs at 4, 8 and 16 kHz were high, ranging from r = 0.77 to 0.89. A negative correlation
(r ≈ -0.24) between MHL at 32 kHz and each of the other tested frequencies was found.
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2012-12-05 21:32:44
80
60
4 kHz
Pearson's r
8 kHz: 0.89
16 kHz: 0.77
32 kHz: -0.23
8 kHz
Pearson's r
16 kHz: 0.89
32 kHz: -0.24
20
80
16 kHz
69
90
68
65
13
1
19
75
28
31
29
15
87
62
39
14
9
34
B6
11
40
38
23
43
D2
73
18
8
32
27
6
12
2
21
42
0
13
69
65
75
90
1
68
28
19
62
87
29
31
15
39
9
8
14
34
11
B6
38
40
73
27
42
32
2
D2
18
43
12
23
21
6
Maximum hearing loss (dB SPL)
40
32 kHz
Pearson's r
32 kHz: -0.25
60
40
20
73
29
21
D2
2
34
1
69
9
6
75
19
B6
15
28
14
18
32
27
12
43
38
8
42
31
68
90
13
11
39
23
87
65
40
62
69
90
75
65
28
14
29
62
1
87
68
31
19
B6
13
39
15
40
18
9
34
D2
11
23
6
43
27
73
21
12
8
38
2
32
42
0
Figure 1. Strain means of maximum hearing loss (MHL) at four different frequencies. MHL was
defined as the difference between the ABR threshold at 12 weeks and the lowest threshold
measured at any time point. Insets contain Pearson’s correlation coefficients between MHL data of
the tested sound frequencies.
At 32 kHz, most BXD strains already showed high thresholds at a young age (Fig. S1).
Even though these mice would typically become deaf at this frequency before or at an
age of 12 weeks, the MHL was nevertheless low. This ceiling effect was responsible for
the negative correlation with the MHL at other frequencies.
Heritability and correlations with previously published phenotypes
Narrow-sense heritability was high: 0.30 at 4 kHz, 0.45 at 8 kHz, 0.56 at 16 kHz and 0.21
at 32 kHz. Our data correlated well with other auditory studies on BXD mice within the
database of WebQTL; not only with spiral ganglion cell density measurements in different
baso-apical sections of the cochlea (r from -0.49 to -0.78, p ≤ 0.03 for several baso-apical
sections at 4, 8 and 16 kHz) (14), but also with post-mortem MRI measurements on total
volume of the lateral lemniscus (r = -0.70, p = 0.02 at 16 kHz; r ~ -0.85, p < 0.001 at 4 and
8 kHz) (30). Higher hearing loss values were correlated with lower cell count or lateral
lemniscus volume.
Quantitative trait mapping
A whole genome scan, which was performed separately for each of the four frequencies,
yielded two significant peaks of interest (Fig. 2), in each case contributed by the D2
allele. First of all, we found a novel QTL at chromosome 18, which will be called ahl9. It
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2012-12-05 21:32:44
1
2
3
4
5
6
7
&
100 Mb
20
significant LRS
LRS
15
suggestive LRS
10
5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
X
Chromosome
Figure 2. Complete genome linkage analysis for early-onset hearing loss. All four frequencies
are shown in different colors (red = 4 kHz; blue = 8 kHz; green = 16 kHz; black = 32 kHz), with
approximate suggestive and significance levels indicated by horizontal, dashed lines. Two QTLs
at chromosome 11 and 18 reached significance (LRS > 18). These QTLs, highlighted by circles, are
determined by the presence of the D2 allele.
06
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was significant at 4 kHz (LRS = 20.9), but not at higher frequencies, and contains only
a small number of genes (Fig. 3 and Table 1). Detailed analysis of the region indicated
a small, non-significant peak upstream of ahl9 (Fig. 3). Because there was considerable
recombination surrounding the 1-LOD interval, with 60% of the BXD lines analyzed
having the D allele, we assumed that this extended region represents a separate QTL.
20
10
**
*
16
5
12
*
0
8
55
60
65
Mb
70
75
80
74
75
Mb
76
Figure 3. Significant locus at chromosome 18, ahl9. A: magnified view of the significant QTL at
chromosome 18 (peak at LRS = 20.9; additive allele effect of D2 = 7.5) for early-onset, low frequency
hearing loss (data from 4 kHz). Horizontal, dashed lines indicate suggestive (*; LRS = 10.7) and
significance (**; LRS = 18.8) levels. B: zoomed-in view of A showing an overview of the genes
present on ahl9, the significant QTL for early-onset hearing loss at 4 kHz.
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2012-12-05 21:32:44
Secondly, a locus was mapped to the distal end of chromosome 11, confirming the
recently published fascin-2 gene (13). This locus was only significant at 16 kHz (LRS =
20.3 at 16 kHz), and produced peaks of smaller magnitude for 8 and 4 kHz (LRS = 11.7
and 7.6, respectively). Furthermore, we found a QTL at chromosome 10. It barely missed
significance at 4 kHz (LRS = 17.1) and, similar to the locus on chromosome 18, harbors
only a small number of genes (Fig. S2 and Table S3). Exclusion of parental strains did not
alter significance. Pair-scan analysis revealed no significant epistatic interactions among
the significant and suggestive loci at chromosome 18 and 10, respectively, or between
the newly found loci and the known locus of the fascin-2 gene. Composite interval
mapping, which allows controlling for variation caused by the fascin-2 locus, revealed
a small drop in LRS value to 17.3, just below significance, of the locus on chromosome
18 at 4 kHz. Vice versa, the LRS value of the fascin-2 locus at 16 kHz also drops below
significance, to 16.9, after controlling for the new locus at chromosome 18.
Table 1. Genes at ahl9, the significant QTL on chromosome 18.
Gene symbol
Name
Start
(Mb)
Length
(kb)
75.13
# coding
SNPs
Expressed
S / NS
Ccdc11
Coiled-coil domain containing 11
74.44
Myo5b
Myosin 5b
74.60
0/0
328.86 0 / 0 (19 / 6)
N
Y
1700120E14Rik RIKEN cDNA 1700120E14 gene
74.66
35.45
0/0
N
Acaa2
Acetyl-Coenzyme A
acyltransferase 2
74.94
27.00
0/0
Y
Lipg
Lipase, endothelial
75.10
21.94
0/0
N
9030625G05Rik RIKEN cDNA 9060325G05 gene
75.13
8.78
0/0
Y
Rpl17
Ribosomal protein L17
75.16
2.91
0/0
Y
BC031181
cDNA sequence BC031181
75.17
4.03
0/0
Y
Dym
Dymeclin
75.18
268.20
0 / 0 (2 / 0)
Y
2010010A06Rik RIKEN cDNA 2010010A06 gene
75.45
8.66
0/0
N
Smad7
MAD Homolog 7 (Drosophila)
75.53
28.57
0/0
Y
Gm672 (Ctif )
Gene model 672 / CBP80/
20-dependent translation
initiation factor
75.59
266.48
0 / 0 (1 / 0)
Y
2900040J22Rik
RIKEN cDNA 2900040J22 gene
75.63
1.31
0/0
N
1700034B16Rik
RIKEN cDNA 1700034B16 gene
75.94
1.27
0/0
N
Displayed are genes within the 1-LOD drop-off region (except top and bottom gene) with their gene
symbol, description, start site, length and number of synonymous (S) and non-synonymous (NS) coding
SNPs that are similar in normal hearing strains B6 and CBA/J, but differ from D2. Those SNPs that differ
between B6 and D2 are listed between parentheses (for a full list of SNPs, see Table S2). ‘Expressed’
indicates whether expression could be measured above background in cochlea by qPCR.
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2012-12-05 21:32:44
At 4 kHz, the new QTL showed a higher correlation with maximum hearing loss
(r = 0.67) than the known mutation in the fascin-2 gene (r = 0.44). Calculation of the
partial correlation indicated that the QTL at chromosome 18 could explain 39.1% of the
variance in MHL at 4 kHz that remained after accounting for the effects of fascin-2.
Additional analysis using absolute hearing levels instead of MHL is given as Supplemental
Information.
1
2
3
4
5
6
7
&
Candidate gene analysis
First, the QTL was checked for (non)-synonymous mutations. A list of genes and
number of SNPs is shown in Table 1. We looked for the presence or absence of these
SNPs in inbred mouse strains without early-onset hearing loss (3), in particular the
CBA/J, DBA/1J and DBA/2HaSmnJ strains. Several coding mutations that were present
in D2 and absent in B6 were also present in strains with good hearing function and
were therefore discarded. Mutations absent in B6 and the above mentioned normal
hearing strains, and hence possibly related to hearing loss, were present in the 3’ UTR
of Ctif and in the 5’ UTR of Smad7 (Table S2), all of which could possibly interfere
with mRNA stability. No additions or changes in stop codons were found in any of
the genes.
Second, as mutations could possibly interfere with regulation of gene expression, we
analyzed SNPs in intronic regions. Intronic SNPs were present in all genes listed, except
for 1700120E14Rik, Lipg, Rpl17, 2010010A06Rik and 2900040J22Rik. The analysis of SNP
differences between B6 and D2 that were also present in the good hearing strain CBA/J
greatly reduced the list of hearing-loss related intronic SNPs, although this analysis
does not take into account that predisposing alleles at other loci may be required for
the hearing loss. A full list of coding (non-synonymous and synonymous) as well as
non-coding SNPs is available in Table S2, which also includes the suggestive locus at
chromosome 10.
Cochlear expression of candidate genes
We quantitatively measured the cochlear expression of the genes found at the QTL
of interest in a selected number of strains, including parental strains (B6 and D2) and
strains with divergent MHL values (BXD 11, 13, 32 and 73). Only a subset of these
genes is expressed in the cochlea (Figs. 4 and S3; Tables 1 and S2) with considerable
variation by strain (ANOVA; Table 2). First, we determined whether the presence of either
the B6 or the D2 allele would influence the relative expression of a gene. A significant
contribution (p < 0.01) of the parental allele in BXD strains was observed for Acaa2,
BC031181, and Smad7, and a trend (p = 0.011) for Ctif (Table 2). We next analyzed the
cochlear gene expression for Spearman rank correlation with MHL at 4 kHz (Figs. 5 and
S3). Only the expression of BC031181 showed significant correlation to the hearing loss
trait (Table 2). However, several other genes approached significance (p < 0.1), including
Dym, Ctif, Myo5b and Smad7. In the case of Dym, Ctif, and Myo5b, MHL at 4 kHz was also
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2012-12-05 21:32:44
H
L
Chrna9
3.62E-01 (0.9)
2.17E-03 (11.8)
4.38E-02 (4.9)
5.96E-01 (0.7)
5.32E-10 (19.5)
2.80E-04 (6.7)
1.05E-02 (7.7)
1.84E-04 (6.3)
4.54E-01 (4.3)
1.38E-04 (17.3)
3.21E-01 (1.0)
5.55E-06 (26.4)
2.72E-09 (54.6)
4.02E-03 (9.2)
3.44E-07 (35.4)
1.65E-02 (6.2)
4.37E-03 (9.05)
1.74E-06 (30.0)
1.09E-04 (17.9)
B6 or D2
p-value (F1,46)
4.3
4.3
4.3
5.0
2.7
2.7
2.7
2.0
2.0
2.0
5.0
5.0
2.0
4.0
6.0
2.0
2.0
3x D2
5.0
6.5
4.5
5.0
5.0
3 x B6
Average ranks
B6 or D2 allele
-0.31
-0.54
-0.54
-0.83
-0.77
-0.83
-0.94
-0.71
0.26
-0.73
-0.77
Corr.
0.703
0.222
0.222
0.063
-0.59
-0.67
-0.36
-0.80
-0.82
-0.64
0.063
0.084
-0.95
-0.40
0.62
-0.81
-0.68
Corr.
0.220
0.146
0.489
0.056
0.047
NA
NA
NA
20.7
20.7
20.7
20.7
0.168
20.7
0.427
20.7
20.9
19.5
LRS
score
0.004
0.186
0.051
0.141
p-value
Pearson product MHL at
MHL at 4 kHz
4 kHz
0.035
0.110
0.562
0.103
0.084
p-value
Spearman rank
MHL at 4 kHz
Displayed are genes (see Table 1) used in the qPCR analysis across six strains with divergent hearing loss, their gene symbol, the average expression level, the
ANOVA results (a = 0.001) across strains, and the presence of the B6 or the D2 allele (* without parental lines; a = 0.01), the mean of ranks when strain averages
were used for the analysis of having the B6 or D2 allele, the Spearman rank and Pearson product-moment correlation (and p-value) with MHL at 4 kHz and the LRS
score of this trait. XH, Relative expression level (log2) across strains (Expression) ≥ 10; H, 10 > Expression ≥ 8; M, 8 > Expression ≥ 5; L, 5 > Expression. Significant
(bold; ANOVA, p < 0.001; correlation, p < 0.05) and trends for (italics; correlation, 0.05 < p < 0.1) differences are highlighted. Note that no significant results were
obtained in any analysis for Cdh23, a gene on chromosome 10 outside the suggestive QTL for MHL.
M
Cdh23
Otof
Control
3.05E-04 (17.8)
5.86E-15 (41.3)
M-H
Smad7
H
H
Dym
Gm672 (Ctif )
1.31E-05 (8.5)
BC031181
1.06E-01 (2.8)
3.38E-01 (1.0)
4.11E-04 (16.8)
2.19E-08 (14.8)
XH
Rpl17
3.81E-02 (2.6)
1.89E-01 (1.8)
4.23E-05 (7.7)
L
6.98E-03 (8.7)
1.14E-04 (6.7)
XH
M-H
Acaa2
1.93E-02 (6.3)
B6 or D2*
p-value (F1,24)
2.52E-03 (4.4)
Strain p-value
(F5,42)
ANOVA
9030625G05Rik
M
Expression
level
Myo5b
Chr18
Gene symbol
Table 2. Gene expression analysis of genes at the significant QTL on chromosome 18 and controls.
90
2012-12-05 21:32:44
***
Rpl17
BC0311
Dym
Smad7
***
***
**
***
**
1
2
3
4
5
6
7
&
Ctif / GM672
**
10
8
6
4
Otof
Chrna9
B6 allele
***
**
D2 allele
13
11
B6
73
32
D2
Cdh23
14
13
11
B6
73
32
D2
PCR controls
13
11
B6
73
32
D2
13
11
B6
73
32
D2
0
13
11
B6
73
32
D2
2
13
11
B6
73
32
D2
Normalized gene expression (log2)
12
9030625G05Rik
13
11
B6
73
32
D2
**
*
13
11
B6
73
32
D2
Acaa2
Myo5b
14
13
11
B6
73
32
D2
Chromosome 18
13
11
B6
73
32
D2
12
10
8
6
4
0
13
11
B6
73
32
D2
2
Figure 4. Gene expression of genes present within the 1-LOD interval of ahl9 and controls. Relative
gene expression levels for genes at the significant ahl9 locus at chromosome 18 are shown. The
bars represent different BXD strains in the sequence (from left to right): BXD13, BXD11, B6, BXD73,
BXD32 and D2. Shading indicates allele inheritance from either B6 (dark gray) or D2 (light gray).
Significant differences (ANOVA) between strains (hatched line) or for the presence of the B6 or D2
allele (black lines; values without parental strains) are indicated (see Table 2). Cadherin 23 (Cdh23;
chromosome 10), which is expressed by all BXD strains and is located outside of the suggestive
QTL, was used as a PCR control. Otof and Chrna9 (chromosome 5) were used as controls for hair cell
quantity. * p < 0.01; ** p < 0.001; *** p < 0.0001
significantly correlated with whole-brain gene expression. However, only in the case of
Dym the correlation was positive, whereas for Ctif and Myo5b it was negative (Table S5;
http://dx.doi.org/10.1111/j.1601-183X.2012.00845.x). Gene expression of Otof, Chrna9
and Cdh23 was used as a control for hair cell quantity. Otof and Chrna9 showed variable
expression between strains (Fig. 4), but the expression of these two genes and of Cdh23
was not significantly correlated with MHL (Table 2).
SNPs in intronic sequences could contribute to altered promotor function, and hence
altered levels of gene expression. Therefore, intronic SNPs not present in normal hearing
strains at the chromosome 18 locus were further selected for having SNPs in the first
exon. These were present in Myo5b (16x), Acaa2 (2x), BC031181 (3x), Dym (1x) and Ctif (6x).
DISCUSSION
In this study on early-onset hearing loss in BXD recombinant inbred strains, we found a
new significant QTL on chromosome 18, designated ahl9. Using expression studies we
showed that the majority of genes on this QTL are expressed within the cochlea, and
that some of these genes were differentially expressed depending on the parental strain
the allele was derived from, and/or were correlated with MHL at 4 kHz.
91
2012-12-05 21:32:45
r = -0.94
12.0
BC031181
B6 allele
D2 allele
11.0
Normalized gene expression level (log2)
10.0
9.0
0
5
r = -0.77
8.0
10
15
20
25
30
Myo5b
7.0
10.0
6.0
9.0
5.0
0
5
10
20
25
30
Smad7
r = -0.77
10.0
15
8.0
9.5
8.0
8.5
7.0
0
5
10
15
0
10.5
9.0
20
MHL 4 kHz
25
30
7.5
Dym
r = -0.83
11.0
5
r = -0.83
0
5
10
15
20
25
30
Gm672 (Ctif)
10
15
20
25
30
Figure 5. Low frequency hearing loss correlations with gene expression. Spearman rank correlations
(r) between MHL at 4 kHz and genes at the ahl9 locus that are significant (p < 0.05; BC031181) or
show a trend (p < 0.1; Myo5b, Dym, Smad7, Ctif (Gm762); see Table 2). The parental origin of the
allele is indicated for each sample.
Validity of the approach
We confirmed the significant, high frequency QTL on chromosome 11, called ahl8 (10),
for which a mutation in fascin-2 is responsible (13). Moreover, our data showed high
correlations with previous data on spiral ganglion cell density in BXD strains (14). The
inclusion of 4 kHz tones in our study largely explains why previous studies did not
find the QTL on chromosome 18. The fascin-2 locus explains only a modest fraction of
variance in the maximum hearing loss at 4 kHz, and after accounting for these effects,
ahl9 on chromosome 18 explained about 40% of the remaining variance. Additional
differences are that our study comprises a larger number of BXD strains, including
several advanced intercrosses, and incorporates recordings at earlier ages than were
used in detecting ahl8 (10).
Expression data
Fourteen out of 25 genes tested were expressed in the organ of Corti. Many of the
analyzed genes showed strain variability; however, only a few genes showed expression
patterns related to the presence of the parental allele. Expression patterns in other
cochlear structures relevant to hearing was not investigated. Notably, genes with a profile
92
2012-12-05 21:32:45
dependent on the allele of origin were all at the peak of the locus, but were interspersed
with genes that showed expression independent of the parental allele. Most likely, this
was not due to experimental error, as the parent-independently expressed genes showed
high expression in our samples. From this, we may conclude that genes at this locus have
a differentiated set of enhancers that regulate gene expression levels independently from
each other. All parent-dependently expressed genes showed a high correlation with MHL
at 4 kHz. The correlation of Otof expression with MHL was considerably lower than that
of genes at the loci, and not significant. Moreover, only expression of Smad7 and Chrna9
correlated positively with that of Otof. Thus, despite the limited set of strains in this
analysis and possible strain differences in hair cell numbers, as suggested by differences in
Otof expression, our gene expression analysis indicated several candidate genes on ahl9.
1
2
3
4
5
6
7
&
Candidate genes and pathways involved in hearing loss
The novel QTLs on chromosome 18 and 10 contained only few genes. Several nonsynonymous mutations are present in different genes on the loci. Only two of them were
specific for D2 only, whereas most were also present in several normal hearing strains.
This procedure is a first crude analysis, which reduced the number of candidate SNPs at
ahl9 substantially. However, this analysis does not take into account that predisposing
alleles at other loci may be required for manifestation of the hearing loss effect. In
addition, several of the exon SNPs were found to be synonymous or had mutations in
the 5’ or 3’ UTR of the mRNA. All of these could be related to changing the stability of the
mRNA and hence could lead to differences in measured expression levels. None of the
genes on the new loci have previously been associated with hearing loss or had strong
homology to known proteins involved in hearing loss, albeit that several members of
the myosin family are involved in hearing loss (31, 32). Myosin 5b is involved in receptor
trafficking, especially in polarized epithelial cells (33-35), suggesting that it may also
play a role in the trafficking of proteins and vesicles in (polarized) cochlear hair cells.
It is also expressed in spiral ganglion neurons (36). Mutations in myosin 5b cause
microvillus inclusion disease, which is characterized by lack of microvilli on the surface
of enterocytes and occurrence of intracellular vacuolar structures containing microvilli
(37). However, decreased hearing has not been reported in these patients.
Several genes on the newly found significant and suggestive loci are involved in fatty acid
and lipid metabolism and sterol regulation, including Acaa2 and Lipg on chromosome 18,
and Zdhhc17 and Osbpl8 on chromosome 10. Caloric restriction can reduce the advancement
of age-related hearing loss (38). One of the proposed mechanisms lies in the accumulation
of reactive oxygen species due to fatty acid metabolism (39), which may accumulate over
time and cause auditory sensory cell damage, eventually resulting in apoptosis of auditory
neurons and hair cells. In humans, NADPH-oxidase activity, as measured by the amount
of plasma superoxide anion radicals, is correlated mainly with low frequency hearing
thresholds (40). Reducing oxidative stress by using lipoic acid as a dietary supplement has
been shown to reduce hearing loss in D2 mice in an age-related manner (41). Similarly, oral
93
2012-12-05 21:32:45
treatment of pravastatin, an inhibitor of the rate-limiting enzyme of cholesterol synthesis,
which reduced serum cholesterol levels, protected against noise-induced cochlear injury
(42). Dietary restriction can protect against hearing loss via antioxidant proteins named
sirtuins, more specifically Sirt3 in the mouse cochlea (43).
Our genetical genomics approach in the BXD strains suggests the involvement of a
selected set of candidate genes in low frequency, early-onset hearing loss. The list of
most likely candidates comprises only 6 genes that showed (correlated) expression
differences, and in case of ahl9, a limited set of possible causative SNPs: Myo5b, Acaa2,
BC031181, Dym, Smad7 and Ctif, although it should be noted that the low expression
of several genes in the organs of Corti might have hampered this analysis. Future
studies that identify the causative genes at these new QTLs will help to understand the
mechanisms underlying low frequency, early-onset hearing loss.
ACKNOWLEDGMENTS
We would like to thank Maarten Loos for his help with calculating heritability and primer
design; Rolinka van der Loo and Iris van Zutphen for coordinating animal deliveries.
Madi Salehi and Annie Offenberg performed part of the ABR measurements. The authors
declare no conflict of interest.
GRANTS
This work was supported by a Neuro-Bsik grant (BSIK 03053; SenterNovem, The
Netherlands) and the Heinsius Houbolt fund.
APPENDIX
The Neuro-Bsik Mouse Phenomics consortium is composed of the laboratories of
A.B. Brussaard, J.G.G. Borst, Y. Elgersma, N. Galjart, G.T.J. van der Horst, C.N. Levelt,
C.M. Pennartz, A.B. Smit, B.M. Spruijt, M. Verhage and C.I. de Zeeuw and companies
Noldus Information Technologies B.V. (http://www.noldus.com/) and Synaptologics B.V.
(http://www.synaptologics.com/).
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SUPPLEMENTAL INFORMATION
1
2
3
4
5
6
7
&
Absolute ABR hearing thresholds at all five time points for all four frequencies were
separately entered into a search at GeneNetwork.org, which yielded two significant
QTLs. Input of 8 kHz hearing thresholds at 4 weeks gave a significant QTL located on
chromosome 10 (91.4 – 94.0 Mb; LRS = 19.3). Secondly, a rather broad QTL was mapped
to chromosome 3 (40.4 – 50.9 Mb; peak LRS = 21.4), related to hearing thresholds in
response to 32 kHz tone pips at 3 weeks of age. For both QTLs, presence of the D2 allele
increased trait values. These results are given for completeness, but are suggestive at
best, since absolute hearing thresholds were not our primary outcome measure.
120
4 kHz
8 kHz
16 kHz
32 kHz
100
80
ABR threshold (dB SPL)
60
40
20
0
120
100
80
60
40
20
0
2
3
4
6
12
2
age (weeks)
3
4
6
12
B6
D2
BXD1
BXD2
BXD6
BXD8
BXD9
BXD11
BXD12
BXD13
BXD14
BXD15
BXD18
BXD19
BXD21
BXD23
BXD27
BXD28
BXD29
BXD31
BXD32
BXD34
BXD38
BXD39
BXD40
BXD42
BXD43
BXD62
BXD65
BXD68
BXD69
BXD73
BXD75
BXD87
BXD90
Figure S1. Average ABR thresholds as a function of time per sound frequency for all tested strains.
For some strains, no data are available at either 2 or 3 weeks of age, because data collection was
more sparse at these early ages. Additional information about the ABR measurements in the
different strains is provided in Table S1.
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Bb
s1
0
1
8
O
sb
pl
N
ap
1l
17
00
02
0G
17
C
R
sr
ik
Zd p2
hh
c1
7
10
Er
td
70
9e
15
E2
f7
A8
30
06
1P
03
R
ik
**
92
30
10
2K
24
R
ik
N
av
3
20
D
B
Chromosome 10
A
31
10
04
3J
17
R
ik
LRS
20
10
**
*
16
4 kHz
4 kHz
8 kHz
16 kHz
12
5
*
8
0
4
108
Mb
112
116
109
110
111
Mb
Figure S2. Highly suggestive locus at chromosome 10. A: suggestive QTL at chromosome 10, highest
peak at 4 kHz (LRS = 17.1; additive allele effect = 6.8). Frequencies 8 and 16 kHz show peaks with
decreasing magnitude (LRS = 16.3 and 14.0, respectively). Approximate suggestive (*; LRS = 10.7) and
significant (**; LRS = 18.8) values are indicated by horizontal, dashed lines. Presence of the D2 allele
increases trait values. This locus has a higher correlation with MHL values at 4 kHz (r = 0.62) than the
known mutation in the fascin-2 gene (r = 0.44). Calculation of the partial correlation showed that the
locus at chromosome 10 could explain 28.4% in the variation of the MHL at 4 kHz after accounting
for the effects of fascin-2. B: zoomed-in view of the genes present at the locus on chromosome 10.
A
3110043J17Rik
Nav3
Csrp2
***
14
12
Bbs10
Osbpl8
***
***
***
*
8
6
4
10.0
Zdhhc17
r = -0.81
10.0
9.0
9.0
8.0
8.0
7.0
0
5
10
15
20
MHL 4 kHz
25
30
7.0
Osbpl8
r = -0.81
0
5
13
11
B6
73
32
D2
13
11
B6
73
32
D2
13
11
B6
73
32
D2
13
11
B6
73
32
D2
0
13
11
B6
73
32
D2
2
13
11
B6
73
32
D2
Normalized gene expression (log2)
10
B
Zdhhc1
***
***
B6 allele
D2 allele
10
15
20
MHL 4 kHz
25
30
Figure S3. Gene expression analysis for genes at the suggestive locus on chromosome 10. A: relative
gene expression levels are shown. The bars represent different BXD strains in the sequence (from left
to right): BXD13, BXD11, B6, BXD73, BXD32 and D2. Shading indicates allele inheritance from either
B6 (dark gray) or D2 (light gray). Significant differences (ANOVA) between strains (hatched line) or
for the presence of the B6 or D2 allele (black lines; values without parental strains) are indicated (see
Table S3). * p < 0.01; ** p < 0.001; *** p < 0.0001. B: Spearman rank correlations (r) of MHL at 4 kHz
with gene expression of Zdhhc17 and Osbpl8, (p < 0.1; see Table S3). The parental origin of the allele
is indicated for each sample.
98
2012-12-05 21:32:45
2
5
11
8
10
7
9
6
7
3
4
4
6
8
11
8
13
5
3
8
7
11
8
11
8
13
5
4
4
5
6
4
8
7
6
C57BL/6J
DBA/2J
BXD1
BXD2
BXD6
BXD8
BXD9
BXD11
BXD12
BXD13
BXD14
BXD15
BXD18
BXD19
BXD21
BXD23
BXD27
BXD28
BXD29
BXD31
BXD32
BXD34
BXD38
BXD39
BXD40
BXD42
BXD43
BXD62
BXD65
BXD68
BXD69
BXD73
BXD75
BXD87
BXD90
2
2
8
3
5
4
5
5
4
0
2
2
1
5
7
6
4
3
2
2
2
3
3
5
4
7
2
2
2
4
1
2
7
3
4
# males
0
3
3
5
5
3
4
1
3
3
2
2
5
3
4
2
9
2
1
6
5
8
5
6
4
6
3
2
2
1
5
2
1
4
2
# females
15,00
28,00
2,73
26,88
34,00
12,14
11,67
14,17
29,29
1,67
12,50
10,00
28,33
5,00
32,73
31,88
21,15
4,00
8,33
9,38
25,71
12,73
15,63
10,45
18,75
23,08
29,00
6,25
2,50
3,00
1,67
18,75
2,50
6,43
2,50
4 kHz
17,50
31,00
2,27
47,50
44,00
32,86
10,56
22,50
45,71
1,67
10,00
7,50
32,50
3,13
50,00
27,50
35,38
5,00
6,67
5,00
35,00
12,73
25,63
9,55
24,38
58,46
29,00
8,75
1,25
1,00
0,83
31,25
3,75
8,57
0,83
17,50
38,00
9,09
64,38
44,00
56,43
27,22
40,83
52,86
18,33
7,50
21,25
25,83
13,13
51,36
41,25
50,00
7,00
8,33
11,88
67,14
31,82
63,13
20,00
23,13
72,69
50,00
8,75
2,50
11,00
1,67
51,25
1,88
10,00
1,67
16 kHz
MHL value
8 kHz
25,00
15,00
21,82
16,25
23,00
33,57
22,78
39,17
29,29
38,33
27,50
25,00
28,33
23,75
13,64
47,50
29,23
26,00
13,33
34,38
28,57
21,36
30,00
43,18
53,13
33,85
30,00
56,25
51,25
36,00
22,50
1,25
23,13
50,71
38,33
32 kHz
40,00
67,00
53,18
48,75
67,00
57,86
56,67
87,50
42,14
40,00
38,75
47,50
50,83
47,50
58,64
81,88
58,85
57,00
66,67
53,13
55,00
49,55
38,13
62,27
46,25
47,69
54,00
48,75
41,25
44,00
47,50
48,75
41,25
40,71
40,83
4 kHz
22,50
55,00
35,00
36,88
53,50
42,14
37,22
71,67
31,43
20,00
22,50
28,75
30,83
29,38
43,18
72,50
46,54
37,00
48,33
37,50
45,00
31,36
19,38
49,09
26,25
31,15
33,00
28,75
21,25
25,00
28,33
31,25
23,13
20,71
20,83
8 kHz
20,00
61,00
26,82
32,50
46,50
30,00
38,33
50,83
35,00
20,00
15,00
25,00
21,67
25,00
58,64
50,00
40,38
29,00
40,00
28,75
37,86
26,82
11,88
34,55
17,50
22,31
27,00
16,25
13,75
17,00
22,50
25,00
15,00
10,71
10,83
16 kHz
52,50
100,00
78,18
98,75
91,50
80,71
88,33
75,83
83,57
56,67
63,75
70,00
80,83
77,50
101,36
65,00
83,08
77,00
91,67
63,75
86,43
86,36
81,25
70,00
51,88
81,15
83,00
40,00
46,25
58,00
57,50
113,75
63,75
40,00
40,00
32 kHz
Average minimum ABR threshold (dB SPL)
55,00
95,00
55,91
75,63
101,00
70,00
68,33
101,67
71,43
41,67
51,25
57,50
79,17
52,50
91,36
113,75
80,00
61,00
75,00
62,50
80,71
62,27
53,75
72,73
65,00
70,77
83,00
55,00
43,75
47,00
49,17
67,50
43,75
47,14
43,33
4 kHz
40,00
86,00
37,27
84,38
97,50
75,00
47,78
94,17
77,14
21,67
32,50
36,25
63,33
32,50
93,18
100,00
81,92
42,00
55,00
42,50
80,00
44,09
45,00
58,64
50,63
89,62
62,00
37,50
22,50
26,00
29,17
62,50
26,88
29,29
21,67
8 kHz
37,50
99,00
35,91
96,88
90,50
86,43
65,56
91,67
87,86
38,33
22,50
46,25
47,50
38,13
110,00
91,25
90,38
36,00
48,33
40,63
105,00
58,64
75,00
54,55
40,63
95,00
77,00
25,00
16,25
28,00
24,17
76,25
16,88
20,71
12,50
16 kHz
77,50
115,00
100,00
115,00
114,50
114,29
111,11
115,00
112,86
95,00
91,25
95,00
109,17
101,25
115,00
112,50
112,31
103,00
105,00
98,13
115,00
107,73
111,25
113,18
105,00
115,00
113,00
96,25
97,50
94,00
80,00
115,00
86,88
90,71
78,33
32 kHz
Average ABR threshold at 12 weeks (dB SPL)
The number of mice used in our study, including distribution of sexes, is given for each strain. Secondly, the average best hearing threshold and hearing threshold
at the endpoint (12 weeks) are provided per frequency. The difference between these two gives the maximum hearing loss (MHL) used in our analysis.
n
strain
Table S1. Extended overview of ABR threshold data.
1
2
3
4
5
6
7
&
99
2012-12-05 21:32:45
Table S2. Analysis of SNPs at the significant and suggestive QTLs on chromosome 18 and 10,
respectively.
Present
in good
Location
hearing
SNP
SNP Reference Type strains
Location
SNP
SNP Reference
74803807
A
T
NSC
Y
74608969
G
74842206
74860294
74860326
74861137
74861143
74861160
74861161
74861162
74861174
74861227
74861251
74861263
74874490
74876722
74882112
74895804
74895831
74901791
74901816
74904412
74918844
74918898
74920555
74920585
74930990
C
T
G
T
G
G
G
T
C
A
C
A
A
G
A
C
G
C
T
A
C
T
G
G
C
T
C
A
C
A
A
A
C
G
G
T
G
G
A
C
T
A
T
C
G
T
C
A
C
T
SC
NSC
NSC
SC
SC
NSC
SC
NSC
NSC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
SC
3’ UTR
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
74608975
74608986
74612987
74612988
74618188
74618204
74621204
74621206
74621207
74623438
74651764
74715993
74715994
74717611
74735025
74799541
74799664
74799893
74799896
74801457
74805463
74805472
74847829
74862519
74882600
74882601
74882602
74902474
74921118
74922479
74925153
74925154
Acaa2
Type
Present
in good
hearing
strains
T
Nonsplice Site
N
G
C
C
C
A
T
T
A
G
G
T
T
A
A
T
C
T
C
C
G
A
C
G
G
A
A
A
T
T
A
T
T
T
G
A
T
G
A
C
C
A
A
A
A
T
G
C
T
C
T
T
C
G
T
C
A
G
G
G
C
A
G
G
G
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
74941936
74941938
C
C
G
G
Nonsplice Site
Nonsplice Site
N
N
9030625G05Rik
75134725
75135079
75135080
75137725
G
A
A
T
T
G
C
G
NA
NA
NA
NA
N
N
N
N
BC031181
75166225
75166329
75166357
G
T
T
C
A
G
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
75181316
75208162
75209885
75215144
75216690
75225879
75230808
75230835
75230845
75230917
75233760
75233763
75234202
75234207
75237689
75237704
75237968
A
C
G
C
A
A
T
G
A
T
C
T
T
T
A
T
A
T
G
T
T
T
T
C
T
G
C
T
A
G
G
G
A
T
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Gene
Exon SNPs
(coding
& 5’/3’ UTR)
Chromosome
18
Myo5b
Dym
Intronic
75222783
75239893
A
A
G
G
SC
SC
Y
Y
100
2012-12-05 21:32:46
respectively. Continued.
Gene
Present
in good
Location
hearing
SNP
Exon SNPs
(coding
& 5’/3’ UTR)
Location
SNP
SNP Reference
Type
Present
in good
hearing
strains
1
2
3
4
5
6
7
&
Intronic
75242334
75242335
75243195
75243198
75243203
75246106
75273439
75273442
75273556
75290903
75290905
75290907
75290909
75295461
75296677
75299232
75302352
75302354
75302382
75302389
75302686
75309734
75309738
75310423
75347219
75347222
75348163
75351820
75356752
75371458
75387802
75387803
75388753
75442249
75445334
G
T
C
T
C
A
C
G
A
C
C
T
T
T
A
C
T
C
C
G
G
C
C
A
T
C
C
G
A
T
G
G
G
T
T
T
C
T
C
T
C
T
T
T
A
A
A
A
C
G
A
C
T
A
A
C
G
G
T
A
T
T
A
G
C
A
A
C
C
A
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Smad7
75527308
C
A
5’ UTR
N
75540303
75544394
75544395
75544396
75544397
75545171
75548631
A
G
G
G
G
G
T
G
C
C
C
C
A
G
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
Gm672 (Ctif )
75770334
75592514
A
C
G
T
SC
3’ UTR
Y
N
75608920
75608938
75610474
75614115
75614857
75614859
75643485
75644466
75644468
75645554
75646454
75648017
75658864
75684873
75685798
75746183
75746184
75746186
75769111
75769134
A
T
C
A
G
G
T
T
T
T
A
A
T
G
A
A
C
A
G
A
G
A
T
G
C
C
C
G
C
A
C
G
C
A
G
C
A
C
A
G
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
101
2012-12-05 21:32:46
respectively. Continued.
Gene
Present
in good
Location
hearing
SNP
Exon SNPs
(coding
& 5’/3’ UTR)
Chromosome
10
Location
SNP
SNP Reference
Type
Present
in good
hearing
strains
Intronic
75773098
75773189
75773195
75780504
75787769
75813041
75841135
75841139
75841143
75851256
75851265
C
A
G
G
G
C
T
C
C
T
C
A
G
A
A
A
T
C
A
T
C
T
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
Nonsplice Site
N
N
N
N
N
N
N
N
N
N
N
Nav3
109353471
G
T
Nonsplice Site
Y
9230102K24Rik
25 SNPs
Nonsplice Site
Y
193 SNPs
1 SNP
Nonsplice Site
Nonsplice Site
Y
N
268 SNPs
4 SNPs
Nonsplice Site
Nonsplice Site
Y
N
E2f7
110191420
110191454
110211735
110223314
T
A
G
G
C
G
A
A
NSC
SC
SC
3’ UTR
N
N
N
N
1700020G17Rik
67 SNPs
Nonsplice Site
Y
Zdhhc17
Csrp2
110380616
110380871
110381274
110387725
110392195
C
C
C
A
A
T
T
T
G
G
3’ UTR
3’ UTR
3’ UTR
SC
SC
N
N
N
N
N
14 SNPs
Nonsplice Site
Y
Osbpl8
110601936
110601948
110641865
110706431
110709268
110709274
110712068
110730196
110730591
110730595
110731131
110731302
110731308
110731959
110732355
110732391
110733737
110733747
110733756
110733811
110734227
C
G
G
A
T
A
A
G
C
A
G
G
A
T
T
A
G
T
G
C
G
G
C
C
G
C
C
G
A
G
C
A
A
T
A
A
G
T
G
T
T
A
5’ UTR
5’ UTR
NSC
SC
SC
SC
SC
SC
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
3’ UTR
Y
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
774 SNPs
Nonsplice Site
Y
Genes on ahl9 (chromosome 18) and chromosome 10 were analyzed for SNPs in exons (left) and
introns (right). Indicated is the location (bp) of the SNP, the sequence of the SNP (D2) and the reference
population (B6), the type of SNP (SC, synonymous coding; NSC, non-synonymous coding; 5’ UTR, exon:
5’ UTR; 3’ UTR, exon: 3’ UTR; NA, sequence not annotated in Genome browser; Nonsplice Site, intron
not at a splice site) and whether the SNP was also present in normal hearing strains, CBA/J, DBA/1J,
DBA/2HaSmnJ. Note that at chromosome 10 for all genes, except Nav3, the sequence of B6 and good
hearing strains was mostly similar and therefore only the total number of SNPs is indicated.
102
2012-12-05 21:32:47
2.53E-01 (1.4)
4.42E-09 (17.0)
4.84E-05 (7.4)
2.96E-08 (14.5)
2.01E-02 (3.0)
M
M-H
H
M-H
L-M
Nav3
Csrp2
Zdhhc17
Osbpl8
Bbs10
8.85E-03 (8.1)
2.83E-05 (26.5)
1.39E-02 (7.0)
9.54E-06 (31.9)
1.88E-01 (1.8)
2.13E-01 (1.6)
B6 or D2*
p-value (F1,24)
ANOVA
5.37E-02 (3.9)
7.93E-11 (70.4)
3.173E-06 (28.1)
2.75E-11 (76.8)
1.91E-01 (1.8)
3.90E-03 (9.3)
B6 or D2
p-value (F1,46)
4.0
4.5
4.5
4.5
4.5
3.0
3 x B6
2.5
1.5
1.5
1.5
1.5
4.5
3x D2
Average ranks
B6 or D2 allele
0.067
-0.81
0.944
0.067
-0.81
0.03
0.142
0.142
0.401
p-value
-0.66
-0.66
0.37
Corr.
Spearman rank
MHL at 4 kHz
0.039
0.613
-0.83
-0.26
0.020
0.059
-0.88
0.677
-0.80
0.100
p-value
-0.22
0.73
Corr.
Pearson product
MHL at 4 kHz
11.45
14.81
17.05
17.05
14.51
14.51
LRS
score
MHL at
4 kHz
Displayed are genes (see Table 1) used in the qPCR analysis across 6 strains with divergent hearing loss, their gene symbol, the average expression level, the ANOVA
results (a = 0.001) across strains, and the presence of the B6 or the D2 allele (* without parental lines; a = 0.01), the mean of ranks when strain averages were used
for the analysis of having the B6 or D2 allele, the Spearman rank and Pearson product-moment correlation (and p-value) with MHL at 4 kHz and the LRS score of this
trait. XH, Relative expression level (log2) across strains (Expression) ≥ 10; H, 10 > Expression ≥ 8; M, 8 > Expression ≥ 5; L, 5 > Expression. Significant (bold; ANOVA,
p < 0.001; correlation, p < 0.05) and trends for (italics; correlation, 0.05 < p < 0.1) differences are highlighted.
2.98E-08 (15.1)
Strain p-value
(F5,42)
L
Expression
level
3110043J17Rik
Chr10
Gene symbol
Table S3. Gene expression analysis of genes at the suggestive QTL on chromosome 10.
1
2
3
4
5
6
7
&
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2012-12-05 21:32:47
Table S4. Primer sequences used for expression analysis of genes at ahl9 (chromosome 18) and
chromosome 10. Genes outside the 1-LOD interval are shown in gray.
Forward
Reverse
Expressed
Cochlea
Ccdc11
GCACAGATCAAACGAGAGGA
CTGTACTCCCGCTGCATAGA
N
Myo5b
Gene symbol
Chromosome 18
AACATGAAAATGCCCTCTTGAAG
GCACAGGATTTGGTTGTTAAGGTA
Y
1700120E14Rik
TCTCTCATCCCTGCAGACCAA
TCTCTCATCCCTGCAGACCAA
N
Acaa2
CAGAAGGCCCTGGATCTTGA
GGCGCCTCCACTCACATT
Y
Lipg
CTCCCCATGCTTCTGTGATTG
GCGGCTAGCAAGCTTCACAT
N
9030625G05Rik
GGTCAGAGGCGAATGAGAGAA
GGCTCCACCACAGGATCACTT
Y
Rpl17
AGTCATTGAACACATCCAGGTGAA
GTTCGTCGGCGCATCTTAG
Y
BC031181
ATATACCCTGTGGTCAGTCGCATA
GGATTGCTGGACGGCTTTT
Y
Dym
AGGCAAGCTGTCTCGTCATTTAA
TGGGAACGGACTGCTGTCTT
Y
CTGCCTCCATTAGCAAGGACA
AGGAAAGGCCCAGGGAGTT
N
CCAAGAGCCCTCCCTGGATA
AAGCCATTCCCCTGAGGTAGAT
Y
Gm672 (Ctif )
GCTGGAAGATGGAGATGGTATCA
GGGAGGACCTTCTCAATGTCATT
Y
2900040J22Rik
ACTTTTGAACCCACAGCACTGA
AGGAAACAGAGACCACCAGCAT
N
1700034B16Rik
CAGTCCAGGCCCACTCTAATCTT
GAGAGTGCTTCCTTGGTTCTCAA
N
TTCCTCCTTCTGCTACTTTGCAAT
GCATGAAGGGCCAAGCATAT
Y
Nav3
CTGCTCTTCCGATACCGAATCT
CTGCTCTTCCGATACCGAATCT
Y
A830061P03Rik
GCACACGTGGCAATTAGCTATG
GCCCATTGGGCATTTGATAA
N
2010010A06Rik
Smad7
Chromosome 10
3110043J17Rik
9230102K24Rik
E2f7
1700020G17Rik
Csrp2
Zdhhc17
AGTCGCCTCTTCCTCCGATAC
TGGGAAACCCTCTGTAGACAATG
N
CCAGCCTGGCTCTGATAAAGAA
TTCCGGCCTCGCTCTTC
N
TCCAGCACCGGCTATGAAAG
TCATCTAGCTGGGCAATCTCTCT
Y
TGGTTTGCAGGAAAAATTTAGACA
CTTCATCATGAATCGCCACTGT
Y
ATGTACGGCAACCAGACAAAGA
TGGCAGCCCAATGAAGAAGT
Y
Osbpl8
CCTGGCGTGCTTCTGATCTAT
TCCGACCCACTGACCATTTT
Y
D10Ertd709e
TCCTGTCTCAGTCCTCCGATTT
GCCGGTAATCTCAGGGTTGAA
N
GCTGGCTCGGAGTTTGTTCT
CTGAGATGCCTGAAACTGTGTTTC
Y
Bbs10
Controls
GAPDH
TGCACCACCAACTGCTTAGC
GGCATGGACTGTGGTCATGA
Y
b-actin
GCTCCTCCTGAGCGCAAG
CATCTGCTGGAAGGTGGACA
Y
Cdh23
CGACTACCTTTCTTCACCAATCACTT
AGAACCCACTGGCGTGTCTT
Y
AAGGAGCAACTTCGACAACATGA
GCGCTTTCCATCTCTTCCTTCT
Y
AGGCCGGACATTGTCCTCTA
CGGCTCTGAAGACTCGTCATC
Y
Otof
Chrna9
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6
Discussion
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Discussion
In this thesis, several topics regarding the auditory system in mice were addressed. We
evaluated the role of a specific ion-channel, the Ih-current, in shaping cell responses to
current injections and sound stimuli in an in vivo dynamic clamp setup. Secondly, DNA
repair mechanisms were found to be involved in maintaining a good cochlear function.
This came to light by studying two groups of mice, each with a different defect in proteins
involved in DNA repair. Lastly, a new significant gene locus was found to contribute to
early-onset, low frequency hearing loss in BXD strains. Cochlear expression of the genes
on the locus was evaluated by qPCR. Each of these topics will be discussed below.
1
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3
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5
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7
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The role of the I h -current within physiology and
pathophysiology of the auditory system
Since its discovery in the mid-70s (1), the Ih-channel continues to draw attention from
electrophysiologists. Its unusual feature of activation upon hyperpolarization and the
subsequent question which role this so-called ‘funny’ current plays within cells and
synapses are probably responsible for this. As the channel is abundantly expressed in heart
tissue and various brain structures, its role should be linked to the structure it resides in.
Many different functions including pacemaker activity in the heart (2), stabilization of the
membrane potential and its link to epileptogenesis (3, 4), control of dendritic excitability
(5, 6), coincidence detection (7) and more recently the strength of a synapse (8), have
been ascribed to these channels. Strikingly, global HCN1 knockouts are generally healthy,
although they do have impaired motor learning and memory deficits (9). In the auditory
system, Ih-channels are mainly expressed in the auditory brainstem (10), throughout which
specific subtypes, kinetics and magnitude of the current may vary (11). Ih is expressed in
the cochlea (12), but appears not to be responsible for mechano-transduction in hair
cells, as this mechanism is intact after blocking the Ih -current and even remains unaltered
in mice lacking both HCN1 and HCN2 (13). It does play a role in shaping EPSPs at the
first afferent synapse at the inner hair cell (14) and has a role in the functioning of the
vestibular organ, the other part of the inner ear (15). Despite its abundant presence in the
auditory system, hearing thresholds in HCN1 knock-outs appear not to be very different
from control animals, as judged by the Preyer’s reflex (16). This test is obviously not the
gold standard in the assessment of hearing function, but reports of ABR thresholds in
HCN1 knock-outs are still lacking. An important question is therefore why the Ih -channel
is expressed in the auditory brainstem? What is its involvement in the shaping and timing
of sounds, or the detection of interaural time and intensity differences? Previous patchclamp recordings in the inferior colliculus (17, 18) tried to tackle this subject on a general
level: is it possible to predict the outcome of a cell, based on a model of its excitatory and
inhibitory inputs and cellular properties (e.g. membrane potential, time constant and
resistance, expression of specific ion channels)? This mission proved to be too hard, as
many factors are unknown or simply cannot be put into a model. However, an important
distinction in (sound-evoked) responses was found between cells that show signs of the
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Discussion
presence of Ih and those that do not. Chapter two describes a study intended to follow
up on this question.
Several observations were in line of expectations, amongst others the rise in resting
membrane potential by Ih, a decrease in membrane resistance and the ability to trigger
rebound spiking in a subset of cells. It is worth noting that rebound spiking can also be
calcium-induced, more specifically involving the low-threshold T-type channels (19). A
bit more unexpected was the inability of Ih to determine the firing pattern of a cell. In
hindsight this is not so remarkable, as the sodium currents during firing are far larger
than the Ih -currents, rendering the latter incapable of playing a significant role. This is
nicely illustrated in figure 7 of chapter 2, which depicts various gating models of Ih and
their effect on a FM-sweep. The question which channel controls accommodating and
burst firing patterns remains unanswered. In order to generate such a firing pattern,
the current has to be activated upon depolarization and should include a net efflux
of positive or an influx of negative ions. One or several members of the large family
of potassium channels would match this description. In particular, sodium-activated
potassium channels (KNa) may be a good candidate for controlling firing patterns (20).
These channels are able to respond with hyperpolarizations in response to rises in
sodium concentration, perfectly matching the necessary specifications to do the job. In
the rat neocortex, one type of sodium-activated potassium channel determines burstfiring behavior (21). A slower type of adaptation of firing rate was seen in the visual
cortex of the ferret (22). The sodium-activated potassium channel has a fast (Slick) and
slow (Slack) activating subunit, both of which are expressed in the inferior colliculus
(23, 24). In this way, the slow type of KNa-channels might control the accommodating
firing type, while the fast variant can explain the burst firing behavior in inferior
colliculus cells. Their possible co-expression with HCN channels would then explain the
correlation between signs of the presence of Ih and the accommodating and burst type
firing patterns. To our knowledge, no attempt has been made to explore the role of Slick
and Slack in the shaping of firing patterns in the inferior colliculus. Ideally, in vitro patchclamp recordings would be performed in order to have full control of the extracellular
environment, although specific blockers are unfortunately lacking at the moment.
Another interesting feature and role within the auditory system is the interplay of Ih and
other ion channels, in particular members of the family of potassium channels. HCN1
and Kv1.1 are frequently co-localized on neurons in the ventral cochlear nucleus (25).
Magnitudes of Ih and low-voltage-activated potassium currents intimately co-vary in
VCN slice recordings, thereby balancing each other near resting membrane potential
(16). The interaction between Ih and a low-threshold potassium current was studied
in a single compartment model of bushy cells from the same VCN, providing two
interesting findings (26). First, modulating Ih (and the resting membrane potential as a
consequence) gave a more adequate regulation of the activation level of low-threshold
potassium currents than by modulating that specific current directly. Secondly, Ih and
low-threshold potassium currents decrease the width of EPSP responses, but individually
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Discussion
both change resting membrane potential either closer or farther away from firing
threshold, respectively. A combination of these two channels resulted in an even smaller
width of EPSPs, thereby aiding in coincidence detection, while at the same time resting
membrane potential roughly stayed the same. In a slice study in the medial superior
olive, the interaction between Ih and low-voltage-activated potassium channels helped
to maintain a nearly uniform shape of synaptic responses during repetitive stimulation,
which could not be achieved by one of these channels alone (27).
This complex interplay of different currents has not been fully unraveled yet. It could
very well explain why the direct effects of Ih on current injections and various sound
stimuli seemed limited in our recordings, while the channel is abundantly expressed
throughout the auditory system nonetheless.
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3
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5
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Is Ih involved in tinnitus?
A common, sometimes very disabling and largely untreatable condition called tinnitus
might be linked to the Ih-channel. It is defined as the perception of sounds (not speech) in
the absence of an external source. Tinnitus is generally generated in regions more central
of the cochlea and auditory nerve (28, 29), matching the functional expression pattern
of the channel. Note, however, that although tinnitus originates from central auditory
nuclei, changes in cochlear function are widely believed to be the cause (30). Tinnitus
could be generated by an abnormal spontaneous excitatory activity, a lack of inhibitory
control on such an activity or a combination of both. As the Ih-channel has strong ties
to excitability of cells, a possible link to tinnitus is easily made. On a general level, the
subject of controlling excitability has already been tackled in the discussion of chapter 2.
In the extreme case, an epileptic attack can occur. The Ih-channel might play a pivotal role
in the process of epileptogenesis as well, although this is still subject of further studies
(31). In the auditory system, blocking Ih by ZD7288 is able to reduce excitability in SOC
neurons (32). Furthermore, excitability in the superior olivary complex is increased by
cAMP, principally by shifting the activation curve of Ih-channels (33). Various substances
that regulate intracellular cAMP, like serotonin and noradrenalin (34), could therefore
influence the perception of tinnitus. This is in line with patients reporting that one’s mood
and external factors like stress can greatly enhance complaints of tinnitus. In congenitally
deaf mice, the Ih current is larger in the MNTB, but not in the AVCN, compared to controls.
To some extent, by reducing temporal summation, Ih even counteracts its own effects
of increasing excitability in the MNTB (35). Another study on sensory deprivation shows
ambiguous results as well. After cochlear ablation before hearing onset, Ih currents were
increased in the LSO, but decreased in MNTB (36). Nevertheless, considerably more
evidence exists for a role of decreased GABAergic inputs in the genesis of tinnitus (37-41).
In conclusion, it is difficult to give a definite answer to the question whether Ih is involved
in tinnitus. Although it is very likely that the development of tinnitus is more dependent
on decreased GABAergic inhibition and not so much on Ih itself, it is plausible that the
channel might play a role in regulation of the networks surrounding tinnitus.
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Discussion
Is Ih involved in the ageing central auditory system?
The ageing auditory system is mainly characterized by increased hearing level thresholds
in the cochlea. As a result, the central auditory system undergoes degenerative changes,
amongst others a change in tonotopic organization (42, 43). Primary lesions of the
central auditory pathway do exist but are still poorly understood (14), partly owing to a
lack of focus on this subject, partly owing to the difficulty to exclude secondary cochlear
effects. It is known that the ageing inferior colliculus in rats shows decreased levels of
GABAergic inhibition (44). Expression of HCN channels changes during maturation of the
hippocampus (22). Moreover, in the sinoatrial node of the heart, Ih expression gradually
decreases during life (45). Apparently, the presence of Ih channels is a dynamic temporal
process and this changing presence might also be involved in the ageing auditory
system. Following sensory deprivation, in some areas Ih is upregulated, whilst in others
downregulation takes place (36). This could be a mechanism to enhance processing
of remaining inputs from decreased cochlear function. More data regarding Ih and the
ageing central auditory system are lacking and thus remain an area of interest in future
research. The ageing cochlea on the other hand, has been subject of more studies and
was addressed in chapters 3 and 4.
Ageing of the peripheral auditory system:
inevitable or (partly) preventable?
The quest for eternal youth is probably as old as mankind. The alchemists are well known
for pursuing this goal in centuries past, while modern-day alchemists widely promote
their anti-ageing cosmetic products on billboards, television and in magazines. The
reversal or even the slowing of ageing has thus always had our interest. The auditory
system is specifically prone to the consequences and damages of ageing, as it is
constantly put under external stress from birth onwards and has limited regeneration
capabilities. Despite many years of study, the exact mechanism of ageing is still not well
understood. Most people support the “free-radical theory” of ageing, first proposed by
Harman half a century ago (46). This theory postulates that the life expectancy of an
organism can be explained by the rate of (oxidative) metabolism and the subsequent
formation of free reactive oxygen species (ROS) (47, 48). Although ROS can have a
function in triggering specific signaling pathways (49, 50), their main (adverse) action
is inflicting damage to various cell compounds, including DNA. As a result, various cell
functions will become suboptimal and eventually cell death could be the consequence
when the DNA is uncontrollably damaged. Most DNA damages are resolved by DNA
repair mechanisms, thereby slowing down the (supposed) process of ageing. In this
light, mice with defects in DNA maintenance have become a model to study the events
during ageing. In chapters 3 and 4, we provide evidence for the importance of intact
DNA repair mechanisms in the preservation of the function of the cochlea. The exact
mechanism behind the hearing loss in the Csbm/m and Ercc1δ/– mice was however not
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Discussion
studied, but a first step would be to perform hair cell counts and immunostainings to
test the theory of apoptosis as a driving force in their phenotype.
Several signs of degeneration associated with hearing loss have been reported in
different parts of the cochlea, including vascular changes in the stria (51), calcium
homeostasis (52), induction of apoptosis pathways and damaged collagen (53-55).
The underlying mechanisms are, similar to general ageing, still not elucidated, yet an
increasing amount of evidence is able to link presbyacusis with oxidative stress and DNA
damage (56-60). It is interesting to note that noise-induced hearing loss also has strong
ties to ROS (61, 62). Whether ROS and DNA damage are the main driving force behind
presbyacusis remains to be seen, but they can play a role in most pathways underlying
age-related cochlear decline. Therefore, they are the most obvious target in attempts to
prevent or slow down presbyacusis. Proposed methods usually include changes in diet,
i.e. extra intake of radical scavengers and / or dietary restriction.
The increased intake of antioxidants has been studied with various substances, amongst
others vitamin C, acetyl-L-carnitine, n-acetyl-L-cysteine, in both age- and noise-related
hearing loss with (63-66) and without (67, 68) success. In the Csbm/m mouse model,
we undertook a study ourselves to evaluate the effects of proline and mannitol, both
antioxidants, on hearing loss (unpublished results, D. Slump, APN, JGGB). No influence
of these compounds on hearing thresholds was observed. A lack of effect does not
necessarily mean that the compound was ineffective, as dosage and frequency of
intake might influence results, or maybe the antioxidants do exhibit an effect when the
phenotype is more severe. Another interfering factor in this study was the observation
that control mice had higher hearing thresholds than previously observed. The nature of
this is still unclear, but could be related to a spontaneous mutation in one of the many
hearing loss genes. Therefore, these results need to be interpreted with caution.
The endogenous ROS defense mechanisms have been subject of studies as well, in
mice with deletion of glutathione peroxidase or Cu/Zn superoxide dismutase (SOD).
Apparently, reducing antioxidant activity (to 50% in heterozygous deletions) does not
affect hearing function (69, 70), while complete removal leads to accelerated hearing
loss (70, 71) or increased vulnerability to noise-induced hearing loss (61). Interestingly,
overexpression of SOD activity did not provide much protection from age-related
hearing loss either (70, 72). This implicates that the cochlea needs some intrinsic
antioxidant activity to minimize hearing loss but that there is not much to gain by extra
intake of exogenous antioxidants. Studies in humans have shown effects of antioxidant
intake, although results cannot be extrapolated to any population yet (73, 74).
Although several studies in the past have indicated that calorie restriction has the ability
to expand lifespan (75, 76), nowadays this is much disputed and the books are certainly
not closed on this subject yet (77, 78). Regarding age-related hearing loss, the influence
of calorie restriction is also subject to debate. Various studies have shown that calorie
restriction can delay signs of age-related hearing loss in several strains of mice (79-81)
and rats (64). However, in a long-term study in rhesus monkeys, no clear effects were
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Discussion
seen on hearing thresholds after years of dietary restriction (82). As rhesus monkeys are
phylogenetically closer to humans, this casts some doubts on the translation of rodent
results to humans.
A recent promise in preventing presbyacusis is less general and more directed at a
specific cell type. The use of T-type calcium channel blockers is apparently able to delay
the loss of spiral ganglion neurons during ageing (83). The general applicability of using
that type of medication is still very limited at the moment.
To summarize, age-related hearing loss is still inevitable today. Whether it is partly
preventable remains to be seen. Dietary restriction is not really a recommendable
option, as it is yet unknown whether this would really benefit humans and it would also
seriously affect quality of life for most people. Obviously, a reasonable restriction in the
amount of calorie intake is advisable though, but not primarily for preserving cochlear
function. There is also at least some doubt whether extra intake of radical scavengers, on
top of those within our regular diet and our built-in defense mechanisms against ROS,
will make a difference. The high prevalence and morbidity of presbyacusis do warrant
further investigations into this matter. Large, long-term, placebo-controlled trials with
enhancement of diet should shed more light on whether the events of presbyacusis (or
general ageing) can be slowed down. For the moment, living healthy and minimizing
ototoxic exposure (including noise) are still the best ways to maintain hearing function
as good as possible.
Genetic susceptibility to hearing loss
Hearing loss and its susceptibility have a large genetic component (84). It is a complex
trait, meaning that multiple genes, environmental factors and their interactions each
make a contribution to the hearing loss (85). Any causal genes may be difficult to detect
in human population studies, as individual contributions can be small. This is further
hampered by the sheer impossibility to control confounding factors, our relatively long
lifespan and individual genetic differences. Despite these challenges, a large number
of genes has already been confirmed to be involved in hearing loss (86), a database of
which can be readily accessed at the website hereditaryhearingloss.org (87). Even the
advancement of genome-wide association studies has provided several novel loci of
interest on age-related hearing loss in humans (88, 89).
In finding new genes, the detection of a gene locus is usually the first step in genomewide association studies, both in humans and mice. Every novel gene locus, including
ahl9, needs confirmation of its involvement in the phenotype studied by identifying a
causal gene. Regularly employed methods include segregation studies by backcrossing
in order to refine the locus, the search for mutations in the genes on a locus, the
generation of knock-outs and rescues by transgenes, immunohistochemistry and gene
expression profiles. Although the mouse and human genome share great similarities
(90), the extrapolation of results from mice to humans cannot be taken for granted and
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Discussion
should be verified. This can be done by identifying individuals suffering from hearing
loss who have a mutation in the human homologue gene of interest.
Many more associated gene loci than causal genes have been identified. This
discrepancy could be due to the appearance of false-positives or the difficulties related
to detecting causal genes with a small effect size. With regard to age-related hearing
loss, the balance is actually quite positive, as 4 out of 8 ahl loci (including ahl9) were
able to yield causal genes.
The first of those was cadherin 23 (or otocadherin), confirmed as the causal gene on ahl.
Mutations in cadherin 23 are the cause of Usher syndrome type ID and nonsyndromic
autosomal recessive deafness DFNB12 in humans (91, 92) and a hearing loss (amongst
others) phenotype in mice (93). Three different hearing loss loci map to cadherin 23 in
mice: waltzer (v), modifier of deafwaddler (mdfw) and age-related hearing loss (ahl). The
last two actually map to the same position on chromosome 10. To identify the specific
mutation responsible for ahl in the B6 strain, backcrosses were initially employed to
refine the original locus to a smaller region. Two sequence changes were found after
comparing B6 and the good hearing strain CBA/J (94). One of those, the 753G->A
polymorphism, showed nearly perfect correlation with the occurrence of hearing loss
in a large number of different inbred strains. The mutation affects the splicing of exon 7
and leads to in-frame skipping.
For ahl8, all of its genes were checked for (non-synonymous) mutations unique to the
D2 strain, yielding only one result in the fascin-2 gene (95). The original amino acid
was found to be highly conserved throughout several different species, suggesting
that it might be part of an important gene. The fascin-2 protein was located as an
actin crosslinker in hair-cell stereocilia by immunogold electron microscopy. The most
convincing evidence was given by showing that the hearing loss in the D2 strain can be
decreased by replacing the fascin-2 locus by the B6 allele. As known previously (96), ahl8
requires ahl in order to result in a phenotype.
Ahl5 in Black Swiss mice was refined by generating congenic lines and linking those to
the hearing loss phenotype (97). On the delimited locus, sequencing analysis of coding
exons revealed one mutation in Gipc3, a guanine to adenine mutation, resulting in
hearing loss. The hearing deficit (as well as the susceptibility to audiogenic seizures)
in Black Swiss mice with a homozygous adenine mutation could be prevented in
backcrosses with transgenic mice expressing Gipc3. Immunostainings localized Gipc3
to (inner and outer) hair cells and spiral ganglion neurons. Mutations linked to hearing
loss in humans were also investigated and could be confirmed (DFNB15 and DFNB95).
Lastly, in finding the responsible gene on ahl4, congenic lines involving the A/J inbred
strain were analyzed to narrow down the region of the locus (98). On the refined
locus, only one SNP was found to be unique to the A/J strain, located in an exon of
citrate synthase. Citrate synthase plays a role in mitochondria, where it is the first and
rate-limiting enzyme of the citric acid cycle (99). A previously known mitochondrial –
nuclear DNA interaction (100) was also observed. First, in B6 x A/J crosses, hearing loss
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Discussion
worsened significantly in offspring from A/J females compared to A/J males. Secondly,
the inheritance of the A/J mitochondrial DNA led to a slightly more severe type of
hearing loss than expected in the presence of ahl4. Mitochondrial dysfunction and
the generation of excess ROS have been implicated in hearing loss (58, 101) and thus
provide a legitimate background for the causal gene.
The large database of hearing loss genes has not yet resulted in many new treatment
options in patient care. Patients can receive more appropriate counseling about the
origin of their hearing loss, what to expect in the future and whether their children are at
risk. However, currently the most important impact of these causal genes lies elsewhere,
as they have been able to provide much more insight into the mechanisms underlying
hearing loss (102-104). Our knowledge on the pathophysiology of this process has
gradually grown through the years, though it is, to date, still far from fully understood.
The combination of studies from different fields, ranging from electrophysiology to
genetics, will in term fill out most of the lacunas in our current knowledge on hearing
loss. Maybe, in a not so distant future, hearing loss may be preventable or even reversed
as a result.
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underlying deafness to hair-bundle
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Summary
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Hearing is a complex sensory modality, important in survival, communication and
leisure. Research on the auditory system encompasses various approaches and
techniques from distinct scientific fields, combining genetic, histological, behavioral
and electrophysiological aspects. Despite its importance, the auditory pathway is quite
vulnerable, already before birth. Hearing loss is indeed a common feature in patients,
both as syndromic and non-syndromic variants. Especially the cochlea is susceptible to
damage, as it is constantly subject to internal and external stress (e.g. loud noise, middle
ear infections) and has limited regeneration capabilities. In order to achieve a better
preservation of hearing function or maybe even a reversal of hearing loss, a decent
understanding of the system is essential. Several topics on the mouse auditory system
are presented in this thesis to contribute to the present knowledge of hearing loss.
The role of the hyperpolarization-activated ion-channel Ih within the inferior colliculus
was subject of the first study, described in chapter 2. After blocking the endogenous Ihchannels, we could compare responses with and without Ih in the same cell by using the
dynamic clamp technique. Adding Ih resulted in a depolarization of the resting membrane
potential, a decrease in membrane resistance and the appearance of a characteristic
depolarizing sag upon injection of a hyperpolarizing current. In a subset of cells, Ih was
even able to trigger rebound spiking. The spiking pattern of a cell was unaltered after
adding Ih. Although sound-evoked EPSPs were smaller in size in the presence of Ih, they did
reach a slightly more positive membrane potential. Temporal summation was decreased
in response to both depolarizing and hyperpolarizing repetitive current injections and Ih
had a small effect on the cycle-averaged membrane potential during sinusoidal amplitude
modulated tones. Lastly, Ih was able to decrease the response to a tone following a
depolarization, by which it might make a small contribution to the phenomenon of forward
masking. In conclusion, these results suggest that previously observed differences in cells
with and without Ih are a mixture of direct effects of the channel itself and indirect effects
due to either the change in membrane potential or the co-expression with other channels.
DNA is the blueprint for all functional proteins in an organism and thus DNA maintenance
is an important means in the defense against ageing. Several mechanisms exist to
repair mistakes made during replication or to reverse damage inflicted by, for example,
free reactive oxygen species or UV-radiation. These so called DNA repair mechanisms
are a combination of proteins collaborating in different steps. One of these proteins,
Cockayne syndrome B, or CSB, was deficient in the mice studied in chapter 3. Hearing
level thresholds were determined at different ages, both in separate groups and in
longitudinal measurements. Csb deficient mice showed a progressive deterioration
of hearing function, especially at the higher frequencies. Furthermore, otoacoustic
emissions, usually a good indicator of outer hair cell function, were absent in older Csb
deficient mice. Combined, these findings indicate a cochlear origin of the hearing loss,
underscoring the importance of functional DNA repair in the safekeeping of hearing.
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Next, chapter 4 elaborates on the effects of a deficient ERCC1 protein on both hearing and
visual performance. Like CSB, ERCC1 is one of the many proteins involved in DNA repair.
Complete knock-out mice die prematurely at the age of 3 weeks due to hepatic failure,
making them unsuitable for our experiments. Therefore, we used mice with a hypomorphic
Ercc1 mutation, which results in reduced activity of the protein and an expanded lifespan
of up to approximately 6 months. Hearing level thresholds were increased from an early
age onwards, progressing over time. Both low and high frequencies were affected, although
effects were most prominent at the higher frequencies. No evidence for retrocochlear
pathology was found, as the conduction speed of the auditory pathway was unaltered.
Otoacoustic emissions were reduced in older Ercc1-deficient mice, suggesting a cochlear
origin of the observed hearing loss. Visual performance was addressed by recording the
optokinetic reflex in response to stimuli of different spatial frequencies and contrast values.
The mutant mice had reduced gains of the optokinetic reflex, especially at an older age
and low contrast values, while the oculomotor system appeared to be intact. Furthermore,
ageing Ercc1 deficient mice showed a decrease in surface of the outer nuclear layer of the
eyeball, which contains the photoreceptors known as rods and cones. The number of TUNEL
positive cells, a marker for apoptosis, in this concerning layer increased drastically with age.
As the observed visual decline preceded the thinning of the outer nuclear layer, degenerative
changes were not restricted to the photoreceptors and probably existed throughout the
visual system. Overall, from this study we can conclude that DNA maintenance plays an
important role in the preservation of both auditory and visual performance during life.
As mentioned in the introductory remarks, hearing loss and even the susceptibility to hearing
loss have a large genetic component. Hearing loss is a complex trait, usually controlled by
more than one gene, interactions between genes and interactions with the environment.
As a result, the contribution of individual genes to hearing loss is often small and difficult to
detect owing to the many confounding factors. The BXD panel of recombinant inbred strains
(a cross between C57BL/6J and DBA/2J) offers a unique possibility to bypass this obstacle
and study quantitative contributions of gene loci to variations in hearing loss between the
strains. Chapter 5 describes a study intended to elucidate which gene loci underlie the
early-onset hearing loss in DBA/2J mice. To this end, hearing level thresholds at four different
frequencies were measured longitudinally up to the age of 12 weeks in mice from a large
number of different BXD recombinant inbred strains. Two new, relatively small QTLs were
found to control early-onset, low frequency hearing loss. Both gene loci, a new significant
locus at chromosome 18, designated ahl9, and a highly suggestive locus at chromosome 10,
contained only a small number of genes. Furthermore, we could confirm the known fascin-2
locus at chromosome 11 with a significant QTL. Mutations absent in strains without earlyonset hearing loss were detected in two genes, while no additions or changes in stop codons
were found in any of the genes. Quantitative PCR of tissue isolated from the organs of Corti
from a subset of strains revealed that only a limited number of genes was expressed in the
cochlea. Several of these showed significant expression differences based on the parental
origin of the allele. A causal gene could not be identified yet.
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Samenvat ting
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Het gehoor is een complexe sensorische modaliteit, belangrijk binnen overleving,
communicatie en ontspanning. Onderzoek betreffende het auditieve systeem behelst
verscheidene benaderingen en technieken uit verschillende wetenschappelijke
velden, waarbij genetische, histologische en elektrofysiologische aspecten worden
verenigd. Ondanks het belang van het gehoor, is dat toch bijzonder kwetsbaar, zelfs
al voor de geboorte. Gehoorverlies komt immers frequent voor bij patiënten, zowel
syndromaal als niet-syndromaal. Helaas verandert dit niet gedurende het leven. Vooral
het slakkenhuis is gevoelig voor schade, aangezien dat constant blootgesteld wordt
aan belastende factoren van binnen en buiten het organisme (bijvoorbeeld lawaai en
middenoorontstekingen) en slechts beperkte regeneratieve vermogens bezit. In het
streven om het gehoor beter te preserveren of misschien zelfs gehoorverlies ongedaan
te maken, is een goed begrip van het gehele systeem essentieel. In dit proefschift werden
enkele onderwerpen betreffende het auditieve systeem in de muis gepresenteerd in
een poging bij te dragen aan de kennis omtrent gehoorverlies.
De rol die het door hyperpolarisatie geactiveerde ionkanaal Ih speelt in de colliculus
inferior was onderwerp van de eerste studie, zoals beschreven in hoofdstuk 2. Na
blokkade van de reeds aanwezige Ih kanalen, werd de respons op verschillende
stroominjecties en geluiden gemeten met en zonder toegevoegde Ih in dezelfde cel,
middels een “dynamic clamp” techniek. Toevoeging van het Ih kanaal resulteerde in een
depolarisatie van de rustpotentiaal, een vermindering van de membraan weerstand
en een karakteristieke depolariserende “sag” als reactie op een hyperpolariserende
stroominjectie. In een subgroep van cellen bleek Ih in staat om “rebound” vuren te
induceren. Het vuurpatroon van een cel werd echter niet veranderd door Ih. Hoewel de
respons op geluiden kleiner was in aanwezigheid van Ih, leverde dit wel een sterkere
depolarisatie op. Temporele summatie werd verminderd door Ih bij zowel depolariserende
als hyperpolariserende repeterende stroominjecties en Ih had een klein effect op de
gemiddelde membraanpotentiaal als reactie op sinusvormig amplitude gemoduleerde
tonen. Als laatste bleek Ih in staat om de respons op een geluid na een depolariserende
stroominjectie te verminderen, waarmee mogelijk een kleine bijdrage wordt geleverd
aan het fenomeen van voorwaartse maskering. Concluderend kan gesteld worden dat
de in het verleden vastgestelde verschillen tussen cellen met en zonder Ih een gevolg
zijn van directe effecten van het kanaal zelf en indirecte effecten door een verandering
van rustpotentiaal of de co-expressie met andere ionkanalen.
Het DNA is de blauwdruk van alle functionele eiwitten in een organisme en hiermee
is onderhoud aan het DNA een belangrijk middel in de strijd tegen veroudering.
Verscheidene mechanismen zijn aanwezig om mogelijke fouten die gemaakt worden
tijdens de replicatie te repareren en exogene schade, hetgeen verricht kan worden door
bijvoorbeeld vrije zuurstof radicalen en UV-licht, ongedaan te maken. Deze zogenaamde
DNA herstel mechanismen bestaan uit een verzameling van interacterende eiwitten die
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2012-12-05 21:32:49
in verschillende stappen de klus klaren. Eén van deze eiwitten, Cockayne syndroom B,
of CSB, was deficiënt in de bestudeerde muizengroep in hoofdstuk 3. Gehoordrempels
werden bepaald op verschillende leeftijden, onder andere met longitudinale metingen.
Csb deficiënte muizen lieten een progressief gehoorverlies zien, meer prominent in de
hogere fequenties. Bovendien waren de DPOAEs, over het algemeen een goede indicator
van de functie van de buitenste haarcellen, afwezig in de oudere Csb deficiënte muis.
Deze bevindingen zijn indicatief voor een cochleaire origine van het gehoorverlies en
onderstrepen het belang van een operatief DNA herstel in het preserveren van het gehoor.
Het volgende hoofdstuk, 4, bespreekt de gevolgen van een deficient ERCC1 eiwit
voor zowel het auditieve als visuele functioneren. ERCC1 is eveneens een eiwit dat
betrokken is binnen het DNA herstel. Muizen met een compleet uitgeschakelde versie
van dit gen sterven zeer vroeg op de leeftijd van 3 weken als gevolg van leverfalen,
waardoor deze muizen niet geschikt waren voor onze experimenten. Daarom maakten
wij gebruik van muizen met een hypomorfe mutatie in het Ercc1 gen, waardoor de
activiteit van het eiwit verminderd is en de levensduur verlengd wordt tot ongeveer
6 maanden. De hoordrempels waren reeds op vroege leeftijd al verhoogd, hetgeen
progressief slechter werd met de tijd. Zowel de lage als hoge frequenties waren
aangedaan, hoewel de effecten wel het meest zichtbaar waren in de hogere frequenties.
Er werden geen aanwijzingen gevonden voor retro-cochleaire pathologie, aangezien
de geleidingssnelheid van de verwerking in de auditieve hersenstam onveranderd
was. Wel waren de DPOAEs verminderd, hetgeen een cochleaire origine van het
gevonden gehoorverlies suggereert. Het visuele functioneren werd gemeten middels
de optokinetische reflex in respons op stimuli met verschillende spatiële frequenties
en contrastwaardes. De muis mutanten hadden een verminderde “gain” van deze
optokinetische reflex. Dit werd vooral gevonden bij oudere muizen en bij stimuli met
lage contrastwaardes, terwijl het oculomotor systeem intact bleek te zijn. Verder lieten
Ercc1 deficiente muizen ook een afname van het oppervlak van de buitenste laag
kernen van de oogbol zien, een laag welke de kegels en staven bevat. Het aantal cellen
in die laag dat positief was voor TUNEL, een marker voor apoptose, nam fors toe met
de leeftijd. Aangezien de achteruitgang van het zicht eerder optrad dan de afname in
fotoreceptoren, zullen degeneratieve veranderingen niet gelimiteerd zijn tot alleen de
fotoreceptoren, maar zich waarschijnlijk door het gehele visuele systeem bevinden. Al
met al kan gesteld worden dat het DNA onderhoud een belangrijke rol speelt in het
preserveren van het auditieve en visuele functioneren gedurende het leven.
Zoals reeds vermeld in de inleidende opmerkingen, hebben gehoorverlies en de
vatbaarheid hiervoor een belangrijke genetische component. Gehoorverlies is een
complexe eigenschap, waarvan de ernst bepaald wordt door meerdere genen, de
omgeving en een interactie hiertussen. Hierdoor is de bijdrage van een individueel gen
aan het gehoorverlies vaak klein en lastig te detecteren door een veelheid aan storende
factoren. Het paneel aan BXD recombinante muizen inteeltlijnen (een kruising tussen
moederlijnen C57BL/6J en DBA/2J) biedt een unieke mogelijk om veel van die storende
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2012-12-05 21:32:49
factoren uit te schakelen en kwantitatieve bijdrages van verschillende genloci aan het
gehoorverlies te onderzoeken. Hoofdstuk 5 beschrijft een studie met als doel op te
helderen welke genen betrokken zijn bij het vroegtijdige gehoorverlies van DBA/2J
muizen. Voor dit doeleinde werden de hoordrempels op vier verschillende frequenties
in een groot aantal BXD lijnen gemeten in de tijd, tot een leeftijd van 12 weken. Er
werden twee nieuwe, vrij smalle gen loci gevonden die een bijdrage leverden aan laag
frequent, vroegtijdig gehoorverlies: een significant locus op chromosoom 18, vanaf nu
ahl9 genoemd, en een zeer suggestief locus op chromosoom 10. Verder werd het reeds
bekende fascine-2 gen op chromosoom 11 eveneens bevestigd met een significant QTL.
In twee verschillende genen werden mutaties gevonden die afwezig waren in muizen
stammen zonder vroegtijdig gehoorverlies, terwijl er geen toevoegingen of wijzigingen
in de stop codons van de genen gevonden zijn. Kwantitatieve PCR, verricht op weefsel
verkregen uit de slakkenhuizen van een geselecteerd aantal BXD-lijnen, onthulde dat
er slechts een beperkt aantal genen van de loci daadwerkelijk tot expressie kwam in
het slakkenhuis. Enkele van deze genen lieten significante verschillen in expressie zien,
afhankelijk van welke moederlijn het allel afkomstig was. Een evident kandidaat gen
kon echter nog niet aangewezen worden.
1
2
3
4
5
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7
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&
Addendum
List of abbreviations
About the author
Portfolio
Dankwoord
2012-12-05 21:32:49
2012-12-05 21:32:49
Addendum
List of abbreviations
ABR
A(R)HL
AVCN
BXD
B6
CF
CN
CNIC
CSB
D2
DNA
DPOAE
EEG
EPSP
ERCC1
FM
GABA
GG
HCN
IC
Ih
IPSP
LL
LOD
LRS
LSO
MGB
MHL
MNTB
MP
MSO
MT
NER
OKR
ONL
PVCN
qPCR
QTL
RI
1
2
3
4
5
6
7
&
auditory brainstem response
age-related hearing loss
anterior ventral cochlear nucleus
B6 x D2 (recombinant inbred strain)
C57BL/6J
characteristic frequency
cochlear nucleus
central nucleus of the inferior colliculus
Cockayne syndrome group B
DBA/2J
deoxyribonucleic acid
distortion product otoacoustic emissions
electroencephalography
excitatory postsynaptic potential
excision repair cross-complementing group 1
frequency modulation
gamma-aminobutyric acid
global genome (repair)
hyperpolarization activated cyclic nucleotide-gated channel
inferior colliculus
hyperpolarization-activated current
inhibitory postsynaptic potential
lateral lemniscus
log of the odds
likelihood ratio statistic
lateral superior olive
medial geniculate body
maximum hearing loss
medial nucleus of the trapezoid body
modulated potential
medial superior olive
minimum threshold
nucleotide excision repair
optokinetic reflex
outer nuclear layer (of the retina)
posterior ventral cochlear nucleus
quantitative polymerase chain reaction
quantitative trait locus
recombinant inbred
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2012-12-05 21:32:49
Addendum
mRNA
ROS
SAM
SOC
SNHL
SNP
SPL
TC
Vm
XP
messenger ribonucleic acid
reactive oxygen species
sinusoidal amplitude modulation
superior olivary complex
sensorineural hearing loss
single nucleotide polymorphism
sound pressure level
transcription-coupled (repair)
membrane potential
xeroderma pigmentosa
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2012-12-05 21:32:49
Addendum
About the author
1
2
3
4
5
6
7
&
De auteur van dit proefschrift werd geboren op
25 september 1980 in Utrecht en heeft de eerste
zes jaar van zijn leven in Maarssen gewoond.
Vervolgens verhuisde hij naar Gouda, waar hij
in 1998 het gymnasium diploma behaalde aan
de scholengemeenschap “De Goudse Waarden”.
Datzelfde jaar begon hij de studie Geneeskunde
aan de Erasmus Universiteit te Rotterdam. Aan
het einde hiervan werd enige tijd aan onderzoek
en audiometrie op de afdeling audiologie
gespendeerd, gevolgd door een oudste co-schap
KNO. Hierna werd hij aangenomen door het toenmalige hoofd van de Rotterdamse
kliniek, professor L. Feenstra. De eerste drie maanden werden besteed als ANIOS in
het Sophia kinderziekenhuis, waarna het promotieonderzoek startte in februari 2005,
onder leiding van professor J.G.G. Borst. Drie en een half jaar werd gewerkt op het
lab van de afdeling Neurowetenschappen, waarbij de experimenten eindigden in
augustus 2008. Het analyseren van de verkregen gegevens en het schrijven van de
manuscripten werd in de loop van 2012 afgerond. Parallel hieraan heeft hij, onder
supervisie van professor R.J. Baatenburg de Jong, de opleiding tot KNO-arts gevolgd,
welke in 2013 voltooid zal worden.
135
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2012-12-05 21:32:49
Addendum
PhD portfolio
1
2
3
4
5
6
7
&
Courses:
- Artikel 9
- Desiderius school (teamwork, communication, hospital management, health
law, medical ethics, evidence-based medicine)
- Advanced imaging techniques for medical doctors, 2009
- Course for vestibular assistants, 2009
- ENT residency program:
o Course on endoscopy of the upper airway, Groningen, 2009
o Course on nasal surgery, Utrecht, 2010
o Course on ear surgery, Leuven, 2010
o Course on paranasal sinus surgery, Amsterdam, 2010
o Around the nose course, Nijmegen, 2012
- Course on emergency medicine, HAGA hospital, 2011
Teaching activities at Erasmus medical school:
- VO: Ear and hearing physiology
- VO: Anatomy of the brain
- VO: Clinical examination of the Ear, Nose and Throat
- VO: Intercellular communication
- VO: Bronchoscopy
- EARP extra-curricular training on anatomy of the head and neck area
- Supervision of MLO and HLO students
Oral presentations:
- NeuroBsik symposium; March 2006, March 2007; May 2008
- Several labtalks
Biannual Dutch ENT meeting:
- April 2008: Hearing loss in CSB-deficient mice
- April 2011: A study of the Ih-channel in the mouse inferior colliculus
- November 2012: A new QTL underlying early-onset, low frequency hearing loss
in BXD recombinant inbred strains
Poster presentation:
- ENP meeting, June 2008: The function of the Ih-channel in the mouse inferior
colliculus: an in vivo dynamic clamp study
137
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2012-12-05 21:32:50
Addendum
Dankwoord
1
2
3
4
5
6
7
&
Gerard Borst: een goudeerlijke wetenschapper met een vlijmscherp analytisch vermogen,
bij wie kwaliteit boven kwantiteit gaat. Hoewel de experimenten aanvankelijk niet altijd
liepen zoals de bedoeling was – het geluk was soms ook wel een beetje ver te zoeken –
zijn we toch na enige jaren gekomen tot een proefschrift waar we allebei tevreden mee
kunnen zijn. De latere fases van analyses en schrijven zijn eigenlijk altijd soepel gelopen.
Ik ben er trots op onder jou te kunnen promoveren.
Rob Baatenburg de Jong: ondanks het feit dat je lange tijd weinig resultaat (lees:
publicaties) van mijn kant zag, heb ik het idee gehad toch altijd je vertrouwen hierin
te hebben. Dit stukje rust heb ik erg gewaardeerd. Daarnaast wil ik je bedanken voor
een fijne en kwalitatief goede opleidingstijd. In het bijzonder wil ik jouw voortdurende
initiatieven prijzen om zowel de kwaliteit als de werksfeer op de afdeling te verbeteren.
Dit werpt namelijk zeker zijn vruchten af.
Prof.dr. L. Feenstra: omdat u ruim 8 jaar geleden “het” wel zag zitten in mij, sta ik nu hier.
Dat zegt genoeg, mijn dank is groot.
Sabine Spijker: ik ben blij dat we samen een artikel hebben kunnen schrijven. Jouw
enthousiasme en kennis van zaken hebben er toe bijgedragen dat ons artikel stukken
verbeterd is en mede daardoor bij het eerste tijdschrift geaccepteerd. Dank ook dat je
zitting hebt willen nemen in mijn leescommissie. Beide goede voorbeelden van het feit
dat 010 en 020 prima samen kunnen werken.
Bert van der Horst: jouw inmenging in mijn promotie heeft een gunstige invloed gehad.
In een tijd dat het licht aan het einde van de tunnel verre van zichtbaar was, kwam je
met de Csb muis langs waarmee ineens mijn eerste artikel heel dichtbij leek. Het is een
beetje ironisch dat dit manuscript uiteindelijk als enige nog niet tot publicatie heeft
mogen leiden, maar ik hoop dat dit er wel op korte termijn van komt. Ook mijn dank
voor het zitting nemen in de leescommissie en het kritisch lezen van het manuscript.
Maarten Frens, Hans van der Steen, prof.dr.ir. J.H.M. Frijns: dank voor het plaatsnemen
in mijn promotiecommissie. Een extra bedankje voor Maarten voor het fungeren als
secretaris van de leescommissie.
Marcel van der Heijden / Bas Meenderink: helden van Matlab en kenners van het
auditieve systeem. En gezellig bij een biertje op de vrijdagmiddag. Met dit alles heb ik
mijn voordeel mogen doen. Succes in jullie verdere carrière, dat gaat zeker lukken.
139
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Addendum
Hans van den Burg: dat je handig bent, dat weten we nu wel. Belangrijker vind ik dat er
altijd tijd was voor een gesprek, meestal om te lachen, soms ook serieus. Jammer dat je
met pensioen bent. Dat kamertje op de 12e met soldeerbout, schroefjes en moertjes is
saai zonder jou.
Michael Brocaar / Teun van Immerzeel: jullie hebben mij in de begindagen erg geholpen
bij het beoordelen van de BERA’s, dan wel het onder de knie krijgen en begrijpen van
EUPHRA. Deze kennis is uiteindelijk gebruikt voor drie van mijn vier manuscripten en
heeft dus een grote invloed gehad op mijn promotie.
Nils Zuiderveen Borgesius: inderdaad, onze schrijfstijl is wat anders, maar dat heeft de
pret zeker niet mogen drukken. We hebben een mooi artikel samen gemaakt. Succes in
het koude Noorwegen.
Joel Shapiro: klein van stuk, maar groot van daden (en vooral ook woorden). De eerste
dag dat ik begon werd ik door Gerard meegenomen naar de kamer van Bas Koekkoek.
In het voorportaal stelde jij je voor, uitkijkend boven dat karakteristieke brilletje van je.
Toen is de onderzoekspret langzaamaan begonnen, van zwarte bunker, pizza en bier
drinken tot de eerste tracheotomie.
Het Borst lab:
Ron Habets: eerste promovendus van Gerard in Rotterdam en daarmee grondlegger van
veel gebruiken en recepten in het lab.
Hilco Theeuwes: altijd de vrolijke noot op de twaalfde en daarbuiten. Ik ben erg blij dat
je je verdiende opleidingsplek bij de chirurgie gekregen hebt.
Heiko Locher: ik weet niet hoeveel potjes 501 we gespeeld hebben, ik denk dat de
eindstand uiteindelijk op 587 – 585 in jouw voordeel ligt. Erg leuk dat je ook tot de KNO
bent toegetreden.
Silviu Rusu: initially, you’ve experienced lots of bad luck with the neuron cultures. I’m
glad you were able to catch up thanks to the slice experiments and are also approaching
your thesis defense soon.
Jeannette Lorteije: we moesten een opstelling delen. En ja, jij was net iets slordiger
daarin dan ik. Toch hebben we er allebei mooie publicaties aan over gehouden, het
heeft dus goed gewerkt.
Adrian Rodrigues-Contreras: thanks for the helpful suggestions and discussions on my
experiments.
John Soria van Hoeve: of nee, toch eigenlijk niet.
Kees Donkersloot: ik begreep niet altijd je antwoord als ik wat vroeg, maar je hebt me
– waar kon – geholpen. Het programmeren van de dynamic clamp in Labview heb jij
gedaan. Mijn dank daarvoor, dat is mijn eerste artikel geworden. Een artikel waar ik nog
steeds trots op ben.
140
2012-12-05 21:32:50
Addendum
Ru verdient een aparte, eervolle vermelding. Een Duitser met humor, die luistert naar
death metal, houdt van koekjes bakken en de kerstmarkt bezoeken. Ja. Het bestaat.
We hebben erg fijn samengewerkt en ik heb altijd goed mijn wetenschappelijke ei (en
andere eieren) bij je kwijt gekund. Ik wens je veel succes met je verdere carrière, waarvan
ik overtuigd ben dat dit gaat lukken. Ik kijk uit naar jouw promotie binnenkort.
1
2
3
4
5
6
7
&
Assistenten KNO: van 2004 tot en met 2013 hebben we aaneengesloten een leuke tijd
gehad met elkaar. Dat moet vooral zo blijven.
Liane Tan, Tannetje: het is nu lastig voor te stellen, maar we moesten echt even aan
elkaar wennen in het begin. Misschien had dat ook wel meer met mij te maken dan met
jou. Jij bent mijn voorganger geweest bij Gerard en in die tijd (en ook later nog) heb je
mij op weg geholpen, zowel binnen de neurowetenschappen als de KNO. Daarnaast
werd ik af en toe voorzien van wat broodnodig vers fruit en allerlei wijze raad. Het kan
dan ook niet anders dan dat jij naast me staat tijdens mijn promotie.
Tom Crins: mijn “opvolger” op de neurowetenschappen en misschien zelfs wel voorlezer
van een stelling straks. Binnen korte tijd konden we het goed vinden met elkaar en
hebben we frequent KNO, onderzoek, muziek en andere zaken besproken onder het
genot van hectoliters Doppio koffie. Ik hoop dat jij ook snel een promotie tegemoet
kan zien.
Sam Schoenmakers en Lindy Santegoets: ik ben de laatste van de drie musketiers en
dat heb ik meer dan eens moeten horen. We hebben een vreselijk leuke (promotie)tijd
gehad, waarin we werkelijk om alles konden lachen en ieder feest hebben afgelopen.
Werd een van ons uitgenodigd voor een feestje, dan waren we er alle drie. Werd geen
van ons uitgenodigd voor een feestje, dan waren we er ook alle drie. Als ik weg zou
komen met vier paranimfen, hadden jullie er uiteraard bij gezeten! Nou jullie nog je
specialisatie afmaken…
Pa, ma, broer, zus: ik ben bang dat ik binnen ons gezin de enige zal zijn die promoveert.
Toch zijn doorzettingsvermogen en nieuwsgierigheid – noodzakelijk om te kunnen
promoveren – waardes die we allen hebben meegekregen. Dank voor jullie altijd
aanwezige steun.
Lieve Wendy, dank je voor alles.
141
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2012-12-05 21:32:50

On the auditory system: genes, DNA repair and ion channels

Transcription

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