1 Tübingen Hearing Research Center (THRC), University of Tübingen,
Department of Otolaryngology, Molecular Neurobiology, Elfriede-Aulhorn-Strasse
5, D-72076 Tübingen
2 Center for Molecular Neurobiology Hamburg, University of Hamburg, Falkenried
94, D-20251 Hamburg, Germany
3 European Molecular Biology Laboratory, via Ramarini 32, 00016 Monterotondo,
Italy
* Author for correspondence (e-mail: marlies.knipper{at}uni-tuebingen.de)
Accepted 18 June 2003
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SUMMARY |
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Key words: Bdnf, TrkB, Cochlea, Hearing loss, Mice
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Introduction |
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Cochlea type I sensory neurones, comprising approximately 90% of the
cochlear ganglion, provide afferent innervation to the inner hair cells
(IHCs). The remaining part of the ganglion consists of small-sized type II
sensory neurones, which innervate outer hair cells (OHCs). Initial studies of
knockout animals for Bdnf or TrkB showed a severe reduction of type II sensory
neurones, whereas Nt3 and TrkC mouse mutants lost the majority of type I
neurones (Ernfors et al.,
1995; Schimmang et al.,
1995
). From these results, and from the study of the afferent
innervation patterns using neurofilament antibodies, it was suggested that
survival of specific neurone types and their targets was under the control of
particular neurotrophins and their receptors.
More detailed studies using DiI as a tracer revealed that innervation in
the cochlear sensory epithelium shows a more complex pattern in neurotrophin
and Trk receptor mutants (Fritzsch et al.,
1997a). Bdnf and TrkB mutants showed the most pronounced defects
in the apex, whereas the lack of innervation of TrkC and Nt3 mutants was most
severe at the cochlear base (Bianchi et
al., 1996
; Fritzsch et al.,
1997b
; Fritzsch et al.,
1998
). Moreover, these studies demonstrated that both
neurotrophins and their receptors are responsible for the innervation of OHCs
(Rubel and Fritzsch,
2002
).
Recent data suggest that the spatial gradients of neuronal loss observed in
neurotrophin and Trk receptor mutants is attributable to the spatial-temporal
gradients of neurotrophin expression during embryonic development
(Fariñas et al., 2001).
Based on this study, Bdnf is localised predominantly in the apex of the
cochlea, whereas Nt3 is localised in the base. Additionally, Bdnf expression
has been shown to correlate with the rearrangement of fibres during postnatal
development of the cochlea, and this reorganisation is disturbed in Bdnf
knockout mice (Wiechers et al.,
1999
). However, because Bdnf or TrkB receptor mouse mutants do not
survive past the first postnatal weeks
(Klein et al., 1993
;
Ernfors et al., 1994
;
Jones et al., 1994
;
Korte et al., 1995
), the
consequences of a severe loss of TrkB signalling in the mature cochlea could
not be analysed.
To study the importance of particular signalling pathways used by Trk
receptors for neuronal survival and target innervation, specific point
mutations have recently been introduced in the docking site for the Shc
adapter protein on the TrkB and TrkC receptor
(Minichiello et al., 1998;
Postigo et al., 2002
).
Although in both TrkBshc/shc- and
TrkCshc/shc-mutant mice the RAS/MAPK and
phosphoinositide-3-kinase pathways are similarly affected, neuronal survival
in the peripheral nervous system, including the inner ear, is only modestly
reduced. However, whereas target innervation in
TrkCshc/shc mice is maintained, in
TrkBshc/shc mice several cranial sensory
populations, including vestibular neurones, lose innervation to their
peripheral targets (Postigo et al.,
2002
).
In the present study, we used these animals to create adult mice with a
severe lack of TrkB receptor signalling, by crossing them to
TrkB+/- mutants with a targeted deletion within the
tyrosine kinase domain of the receptor
(Klein et al., 1993). Similar
to TrkB-/- and Bdnf-/- mutants
(Bianchi et al., 1996
;
Fritzsch et al., 1998
), mice
carrying the mutant shc allele and the mutated tyrosine kinase domain of the
TrkB receptor (TrkBshc/-) show a region-specific
absence of outer hair cell innervation in the apical part of the cochlea
during early postnatal development.
Further analysis of Bdnf-/- and
TrkBshc/- animals revealed that the observed
defect is reversed during maturation of the cochlea, thus leading to normal
innervation of OHCs in the apex but absence of afferent and retardation of
efferent nerve fibres in the basal turn. Although the cellular integrity of
OHCs appeared unaffected in the mutant animals in all cochlear turns, hearing
thresholds of TrkBshc/- mice were significantly
reduced compared with control mice. The present data are consistent with
recent results that suggest that neurotrophins in the mature hearing organ may
have a distribution opposite to that found during development
(Adamson et al., 2002).
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Materials and methods |
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Mice carrying the mutant shc allele of the TrkB receptor
(TrkBshc/shc) have been described in
Minichiello et al., 1998.
Mutant
TrkBshc/- mice were created by crossing TrkB+/- mice with TrkBshc/shc mutants.
Tissue preparation
Cochleae were isolated and dissected as previously described
(Knipper et al., 2000).
Briefly, cochleae were fixed by immersion in 2% paraformaldehyde, 125 mM
sucrose in 100 mM phosphate buffered saline (PBS; pH 7.4), for 2 hours,
followed by overnight incubation in 25% sucrose, 1 mM protease inhibitor
(Pefabloc, Roche) in PBS (pH 7.4). Cochleae of animals older than postnatal
day 10 (P10) were decalcified after fixation for 15 minutes to 2 hours in
Rapid bone decalcifier (#904687, Eurobio, Fisher-Scientific, 61130 Nidderau,
Germany). After overnight incubation, cochleae were embedded in O.C.T.
compound (Miles Laboratories, Elkhart, Ind., USA). Tissues were then
cryosectioned at 10 µm thickness for in situ hybridisation and
immunohistochemistry, mounted on SuperFrost*/plus microscope slides, dried for
1 hour and stored at -20°C before use.
Immunohistochemical staining for fluorescence microscopy
For immunohistochemistry, cochlear sections from different postnatal stages
and adult mice or rats were thawed, permeabilised with 0.1% Triton X-100 for 3
minutes at room temperature, preblocked with 1% bovine serum albumin in PBS
and incubated overnight at 4°C with primary antibodies. The following
primary antibodies were used: anti-NF200 (Sigma N4142); anti-synaptophysin
(The Binding Site #PH510) anti-peripherin (Chemicon #AB1530), anti-prestin
(Weber et al., 2002) and
anti-potassium channel SK2 (Sigma, #P0483). Expression of full-length TrkB in
cochlear neurones was confirmed by antibodies directed against aminoacids
794-808 of the TrkB receptor (#794, Santa Cruz Biotechnology). Primary
antibodies were visualised with either Cy3-conjugated goat anti-rabbit Ig
(0.35 µg/ml; Jackson Immuno Research Laboratories, PA, USA) or with
Alexa-Green conjugated goat-anti-mouse antibodies (1:1500, Molecular Probes,
Leiden, The Netherlands). Sections were rinsed, mounted with Vectashield
(Vector Laboratories, Burlingame, CA, USA) and viewed using an Olympus AX70
microscope equipped with epifluorescence illumination.
Bdnf probe isolation and riboprobe synthesis
Genomic DNA from rat liver was isolated by Easy-DNA kit from Invitrogen,
following the protocol provided by the manufacturer. Polymerase chain reaction
was used to amplify exon 4 of the Bdnf gene
(Timmusk et al., 1993). For
the exon 4-specific probe, a sense primer (5'-cca atc gaa gct caa ccg
aa-3') and an antisense primer (5'-tca ggg tcc aca caa agc
tc-3') corresponding respectively to nucleotide position 1732-1751 and
2059-2078 from genomic fragment B were used. During the PCR reaction, genomic
DNA was first denatured for 4 minutes at 94°C, followed by 30 cycles
consisting of 1 minute at 94°C, 1 minute at 55°C and 1 minute at
72°C. The extension reaction was carried out at 72°C for 10 minutes.
The amplified fragment corresponding to the expected length of 347 nucleotides
was extracted and sequenced. Clones of full-length rat Nt3 and
full-length TrkB were supplied by Regeneron Pharmaceuticals. In situ
hybridisation was performed as described
(Wiechers et al., 1999
).
Sections were mounted with Moviol (Sigma) and viewed using an Olympus AX70
microscope.
Auditory brainstem response measurements
Auditory evoked brainstem responses (ABR) were recorded in anaesthetised
mice. Anaesthesia was achieved by the intraperitoneal injection of 65 mg/kg
ketamin hydrochloride (Ketamin 50 Curamed, CuraMED Pharma, Germany), 10.5
mg/kg xylazin hydrochloride (Rompun 290, Bayer Leverkusen, Germany) and 0.33
mg/kg atropine sulphate (Atropinsulfat, Braun, Germany). Recordings were
carried out in a sound-proof chamber (IAC, Type 400-A). A Multi IO Card
(National Instruments MIO 16 E1) was used for the generation of stimuli and
recording of evoked potentials. Tone pips of 3 ms duration (1 ms rise and fall
time, cosine-shaped) were presented at a rate of 60/second with alternating
phase. Clicks with duration of 100 µs of alternating phase were also used.
Sound pressure level was adjusted with a custom-made attenuator and tone pips
were amplified with a custom-made amplifier. Stimuli were delivered to the ear
in a calibrated (Bruel&Kjaer 2610, 4191) closed system by a Beyer DT911
loudspeaker. In the case of tone pips, the sound pressure was calibrated in
situ at all frequencies recorded prior to each measurement taken. As this is
not possible for clicks, sound pressure was calibrated off-line once prior to
the experiments. In this case, 0 dB attenuation corresponds to 112 dB SPL root
means square (rms). To record bio-electrical potentials, subdermal silver wire
electrodes were inserted at the vertex (reference), ventro-lateral to the
measured ear (active) and at the back of the animal (ground). After
amplification (100 dB) and bandpass filtering (0.3-5.0 kHz), electrical
signals were averaged (128 repetitions). ABR responses were recorded for
frequencies between 2.8 kHz and 45.2 kHz at a resolution of 2 steps per
octave. At each frequency ABRs were collected from 20 dB to 100 dB SPL in 5 dB
steps. The software averager included an artefact rejection code (all
waveforms with a peak to peak voltage exceeding a defined voltage were
rejected) to eliminate the ECG and muscle activity. Thresholds were defined as
the sound pressure level where a stimulus correlated response was clearly
identified in the recorded signal. If no response was obtained we used 100 dB
SPL as the threshold value volume for further calculations.
Measurements of distortion product otoacoustic emissions (DPOAE)
Anaesthesia was performed as described for the ABR measurements. For DPOAE
measurements acoustic stimulations were performed by a PC-based system with an
A/D converter board using LabWindows software, which also performed fast
Fourier transformation (FFT) for data analysis. Pure tone sound stimuli were
generated and delivered on two separate channels using dynamic speakers (Beyer
DT48). Sound levels were measured with a probe microphone (Bruel&Kjaer
4135) and a measuring amplifier (Bruel&Kjaer 2610). The speakers and
microphone were coupled together, with the closed field into the ear canal of
the animal, and the sound system calibrated with white noise (50 Hz-20 kHz).
DPOAEs were measured as cubic distortion products at the frequency 2f1-f2. For
each frequency f1 (7, 12 or 18 kHz), the individual best ratio (BR) f2/f1 was
determined using an intermediate sound level (60 dB SPL or higher, if
necessary), varying the ratio from 1.06 to 1.44 in steps of 0.02. This ratio
was used for the following input/output measurement. The sound level of f1 was
kept at a constant 10 dB louder than the f2 level. Sound was initially
presented at the lowest level and increased in steps of 5 dB. Recording
windows were averaged in order to decrease the background noise to at least 10
dB below the level of emission, but not over more than 1000 repetitions.
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Results |
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Neuronal loss and hair cell maintenance in adult TrkB mutants
Microscopical examination on serial sections revealed no apparent
morphological changes in the organ of Corti of
TrkBshc/- mice compared with control littermates
(Fig. 2A). To further confirm
the intactness of OHCs, we used antibodies directed against the outer hair
cell motor protein prestin (Weber et al.,
2002). Similar to TrkBshc/+ control
animals, the characteristic staining pattern of prestin localised in the
lateral membrane of the outer hair cell was observed in all cochlear turns of
adult TrkBshc/- mutants
(Fig. 2B). Previous studies had
reported a specific loss of type II sensory neurones, innervating OHCs in TrkB
and Bdnf mutants (Ernfors et al.,
1995
; Schimmang et al.,
1995
). To monitor the presence of type II sensory neurones in
TrkBshc/- mice we used antibodies directed
against peripherin, a 57 kDa type III intermediate filament that has been
defined as a specific marker for these neurones
(Hafidi, 1998
). On serial
sections of P19 and adult control mice distinct small-sized
peripherin-positive neurones were detected
(Fig. 3A,C). No
peripherin-positive neurones could be detected in the cochlear ganglion along
the entire length of the hearing organ in adult
TrkBshc/- mutants
(Fig. 3B). This type of neurone
was also not detected in Bdnf-/- mutants at P19
(Fig. 3D). Therefore, the
absence of type II sensory neurones in TrkBshc/-
mutants confirms the dependence of these neurones on TrkB signalling and
underlines the validity of TrkBshc/- mice as a
model for a severe loss of TrkB function in the cochlea.
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Loss of efferent innervation in Bdnf and TrkB mutants
Upon analysis of the efferent innervation pattern using synaptophysin as a
marker protein (Knipper et al.,
2000), we observed that the loss of afferent type II fibres
projecting to OHCs in basal cochlear turns of
TrkBshc/- mice was associated with a severe
reduction in the number and size of synaptophysin-positive boutons below these
cells (Fig. 5). To further
define this defect on the postsynaptic site of the cell membrane, we studied
the localisation of the apamin-sensitive, small-conductance
Ca2+-activated potassium channel SK2, which has been localised in
OHCs, in a localisation opposite to the efferents
(Fig. 5)
(Oliver et al., 2000
). In the
basal turn of adult TrkBshc/+ animals,
synaptophysin-positive boutons were detected below each hair cell, opposite
SK2 channels and restricted to the area of efferent contact. In adult
TrkBshc/- mice, we observed only a few small
synaptophysin-positive boutons, with either a very weak or no SK2-positive
staining at the outer hair cell contact zone. These findings were also
confirmed in the basal turn of control Bdnf+/+ and
Bdnf-/- mice at P19. These results indicate that the loss
of afferents below OHCs in the basal cochlear turns of Bdnf- and
TrkB-deficient mice may have consequences on the size of the efferent contact
zone on the hair cell, and that the reduced size of the presynaptic efferent
contact zone may subsequently result in a reduction or absence of the
postsynaptic field, characterised by the expression of SK2 ion channels.
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Discussion |
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In the present study, and a previous study
(Postigo et al., 2002), we
observed severe afferent and efferent innervation defects in the vestibular
sensory epithelia of TrkBshc/shc mice, which are
similar to the ones found in TrkB-/- mice that lack the
entire tyrosine kinase domain (Schimmang
et al., 1995
; Fritzsch et al.,
1998
). By contrast, no defects could be observed in cochlear
neurones of TrkBshc/shc mutants. Cochlear
neurones may therefore depend on other signalling domains of the TrkB receptor
(Minichiello et al., 2002
), or
on additional growth factors (Fritzsch et
al., 1999
). By crossing TrkBshc/shc
with TrkB+/- mice we have created
TrkBshc/- mutants with a further reduction in
TrkB signalling but a normal life span. In the vestibular system of
TrkBshc/- mice, we observed similar innervation
defects to the ones observed in TrkBshc/shc
mutants (Postigo et al.,
2002
). Interestingly however, in contrast to
TrkBshc/shc mutants,
TrkBshc/- mice also show a loss of afferent type
II fibres to the OHCs of the adult cochlea, which had so far only been
reported in TrkB-/- mutants during early postnatal
development (Schimmang et al.,
1995
; Fritzsch et al.,
1998
). TrkBshc/- mice thus offered an
interesting model to study the consequences of severely reduced TrkB
signalling in the adult cochlea. The validity of this model was established by
the parallel analysis of postnatal Bdnf-mutant mice, which show an expanded
postnatal lifespan compared with TrkB-/- mutants
(Klein et al., 1993
;
Ernfors et al., 1994
;
Jones et al., 1994
;
Korte et al., 1995
).
Similar to results obtained by neuronal cell counts in TrkB and Bdnf
mutants (Schimmang et al.,
1995; Ernfors et al.,
1995
), we observed a loss of type II cochlear sensory neurones
using a cell-type specific marker for these neurones
(Hafidi, 1998
) in
TrkBshc/- mice. This result thus further confirms
that the primary defect in Bdnf and TrkB mutants is a loss of type II sensory
neurones, and a defect in the innervation of their targets, the OHCs (see
below). However, the morphology and structural integrity of OHCs, as
demonstrated by staining with antibodies against the OHC motor protein
prestin, appears unaffected in TrkBshc/- mutants.
Likewise, only minor or no structural changes of cochlear morphology have been
reported in previous analyses of neurotrophin or Trk receptor mutants
(Schimmang et al., 1995
;
Fritzsch et al., 1997b
).
Dynamics of neurotrophin signalling and innervation in the prenatal
and postnatal cochlea
As the major defect in TrkB and Bdnf mutants appears restricted to the
innervation of OHCs, we have analysed in detail the spatiotemporal pattern of
innervation to these cells during postnatal development along the tonotopic
axis of the cochlea. As previously reported, we observed a lack of afferent
fibres to OHCs in the medial and apical part of the cochlea in Bdnf and TrkB
mutants during the first postnatal week
(Bianchi et al., 1996;
Fritzsch et al., 1998
;
Wiechers et al., 1999
). A
similar defect was also observed in TrkBshc/-
mice, once more confirming the validity of these mice as a model for a severe
reduction of TrkB signalling in the postnatal cochlea.
The early postnatal phenotype of TrkB and Bdnf mutants is most likely
explained by an apical-to-basal gradient of Bdnf expression in the cochlear
sensory epithelia during embryonic development
(Fariñas et al., 2001).
Strikingly, we observed a mirror-image of this phenotype during subsequent
postnatal maturation in the adult cochlea of Bdnf-/- and
TrkBshc/- mutants, respectively. Towards the end
of the third postnatal week, Bdnf mutants showed a complete absence of basal
afferent innervation, whereas the apical part of the cochlea showed a normal
pattern of afferent fibres on OHCs. Likewise, an identical pattern of
innervation was observed in TrkBshc/- adult
mutants. How can the reversal of the phenotype observed in Bdnf and TrkB
mutants during postnatal development be explained?
At birth, a longitudinal expression of Bdnf with highest expression in the
apex (Wheeler et al., 1994;
Wiechers et al., 1999
) or a
homogenous pattern of expression
(Fariñas et al., 2001
)
has been described in the cochlear sensory epithelium. Subsequently, Bdnf has
been detected in OHCs of the apical to midbasal turn, but not of the basal
turn during the first postnatal week
(Wiechers et al., 1999
). After
this timepoint, and in adulthood, no Bdnf expression has been observed in OHCs
(Wheeler et al., 1994
;
Fritzsch et al., 1999
;
Wiechers et al., 1999
). In
summary, these data provide no explanation for the basal loss of afferent
innervation of OHCs observed towards the end of the third postnatal week and
in adulthood.
However, the basal-to-apical gradient of Bdnf and TrkB expression detected
in cochlear neurones in the present report offers an attractive explanation
for the dependence of afferent innervation on Bdnf and TrkB signalling in the
more basal cochlear turns. Specifically, the co-expression of TrkB and Bdnf
suggests an autocrine mechanism that maintains the innervation of the basal
turn during further maturation of the cochlea. This process may be initiated
by the expression of Bdnf in cochlear ganglion neurones during the first
postnatal week (Wiechers et al.,
1999). The gradient of Bdnf expression is first observed at the
end of the second postnatal week (M.K., unpublished observation). Therefore,
the spatial and temporal expression of Bdnf in cochlear neurones during
postnatal development and in adulthood fits perfectly with the gradual
reversal of innervation of OHCs observed in Bdnf and TrkB mutants during the
second and third postnatal weeks. In this context, it is worthwhile mentioning
that Bdnf is not expressed in differentiated neurones that express TrkB during
embryonic development (Fariñas et
al., 2001
). Therefore, the postulated autocrine mechanism for the
maintenance of basal outer hair cell innervation is restricted to the
postnatal and adult phases of life. How can the re-innervation of the medial
and apical turns of the cochlea in Bdnf and TrkB mutants be explained?
The most attractive model may be an apical-to-basal gradient of expression
of Nt3 in the postnatal inner ear. Indeed, such a gradient with the highest
expression in the apical part has been described for the cochlear sensory
epithelium at birth (Pirvola et al.,
1992; Wheeler et al.,
1994
; Fritzsch et al.,
1997b
). However, recently this gradient could not be reconfirmed
(Fariñas et al., 2001
).
In the present analysis we have detected an apical-to-basal gradient of Nt3
expression in cochlear neurones, which develops postnatally in the opposite
direction to the Bdnf gradient described above. The increased expression of
Nt3 in cochlear neurones in the apical turn thus offers an attractive
explanation for the re-innervation of OHCs in this part of the cochlea in Bdnf
and TrkB mutants. At present we are analysing whether this expression is
accompanied by the expression of its high-affinity receptor TrkC. Similar to
the situation in the base, co-expression of the neurotrophin and its
high-affinity receptor may initiate an autocrine mechanism. Thus, in the
apical turns of the cochleae of TrkB and Bdnf mutants, a presumptive
Bdnf-independent, Nt3-dependent process of re-innervation of OHCs may be
maintained.
At present, it is unclear which neurones are involved in the process of
reinnervation. In theory, one would postulate the involvement of two classes
of type II sensory neurones, which naturally may provide embryonal and adult
innervation to OHCs. In support of this, 'embryonal' type II neurones have
been shown to be severely reduced in the absence of Bdnf, as assessed using
cell counts in early postnatal Bdnf and TrkB mutants
(Ernfors et al., 1995;
Schimmang et al., 1995
). In
the present study, using peripherin as a marker, we also found no evidence for
the presence of type II sensory neurones in adult
TrkBshc/- mutants. However, we cannot rule out
the existence of type II neurones that have failed to express peripherin in
the Bdnf and TrkB mutants analysed in the present study. Moreover, the
presence of type II sensory neurones in Bdnf-/- mutants
has been postulated based on the expression of the neurofilament marker RT97
(Bianchi et al., 1996
).
Therefore, two populations of these neurones may exist, which may be involved
in initiating the process of embryonal innervation and postnatal
re-innervation of OHCs.
It may be considered that neurones innervating OHCs in the apical region,
which provide two times more synaptic contacts to OHCs than basally localised
neurones of this type (Rubel and Fritzsch,
2002), are possibly Nt3-dependent, type I sensory neurones
(Ernfors et al., 1995
) sending
out collaterals to the OHCs. As such, it has been suggested that initially
individual ganglion cells innervate both IHCs and OHCs during development
(Echteler, 1992
;
Pujol et al., 1998
). In the
mammalian cochlea, an extensive process of afferent and efferent fibre
reorganisation takes place during the first and second postnatal week
(Wiechers et al., 1999
).
Therefore, the possibility of an early innervation of OHCs by type I neurones,
and a Bdnf-dependent phenotypic switch of type I to type II neurones in more
basal and less apical turns during postnatal development, should be further
investigated. Finally, as shown previously in Nt3 mutants
(Fritzsch et al., 1997b
;
Coppola et al., 2001
),
tangential extensions of fibres running underneath the cochlea may also
contribute to the innervation patterns observed.
The relevance of the neuronal expression of Bdnf described in the present
article is also highlighted by recent results on the electrophysiological
behaviour of postnatal cochlear neurones
(Adamson et al., 2002). In
these experiments it was shown that exposure to Bdnf caused all neurones,
regardless of their original cochlear position, to display characteristics of
the basal neurones. Conversely, Nt3 caused all neurones to show the properties
of apical-cochlear neurones. These data fit perfectly with the opposing
basal-to-apical gradients of Bdnf and Nt3 expression observed in the postnatal
cochlea in the present study.
Defects in efferent innervation and hearing loss
Hair cells are the targets of olivocochlear fibres that control efferent
inhibitory feedback from the brain. Efferent feedback to OHCs is predominantly
provided by the release of acetylcholine (ACh) from olivocochlear nerve
fibres, which activates the characteristic high Ca2+ conductance
heterooligomeric complex of ACh receptors (AChRs)
(Elgoyhen et al., 1994;
Elgoyhen et al., 2001
). The
Ca2+ flux through AChRs in turn, activates the small-conductance
Ca2+-activated potassium channel SK2. The fast coupling of both is
reflected by their co-localisation (Oliver
et al., 2000
; Knipper and
Zenner, 2003
). Although current concepts indicate an autonomous
morphological differentiation of hair cells independent from ganglion neurones
(Sobkowicz, 1992; van de Water et al.,
1992
; Silos-Santiago et al.,
1997
; Ma et al.,
2000
), which acquire their basic physiological properties in the
absence of innervation (He and Dallos,
1997
; Rüsch et al.,
1998
; Rubel and Fritzsch,
2002
), data in the present study indicate that distinct steps of
the final functional phenotype of OHCs is influenced by the innervating
fibres.
The loss of afferent type II fibres below OHCs in Bdnf and TrkBshc/- mutants is accompanied by a significant reduction in size of efferent synaptic endings and postsynaptic fields on OHCs, in which SK2 channels are inserted. As we observed no defects in the innervation of IHCs, the alteration of the OHC phenotype may directly be related with the observed hearing loss of 10-30 dB in TrkBshc/- mice across all frequencies tested. This hearing loss and the lack of DPOAEs suggest a common mechanism that is impaired in the mutant animals. As no DPOAEs were detectable, the defect may reside at the level of the OHC and may be caused by the reduction of efferent input. However, at present it remains unclear whether this defect is caused by structural or physiological changes of the OHC.
Previous analysis has shown that the neurotrophin receptor mutants lacking
TrkB and TrkC develop central cochlear nuclei of a reduced size
(Schimmang et al., 1997). The
reduction of these nuclei may be explained by a direct trophic role of
neurotrophins within these nuclei. Alternatively, a lack of trophic support of
central auditory neurones by the peripheral auditory neurones may be
responsible for the observed phenotype. In this context, it is also tempting
to speculate that Bdnf expression, which is involved in several
plasticity-driven processes (Berardi et
al., 2000
; Schinder and Poo,
2000
), may also influence the tonotopic map present in the central
part of the auditory system. Expression of Trk receptors and neurotrophins has
been described in central auditory neurones
(Hafidi et al., 1996
;
Hafidi, 1998
), and their
involvement in activity-dependent trophic interactions with the peripheral
neurones has been suggested (Rubel and
Fritzsch, 2002
). The postnatal gradient of Bdnf expression in the
cochlear ganglion may thus set up, and later reinforce or maintain, the
tonotopic map in the central cochlear nuclei. Therefore, a central component
may also be involved in the hearing loss of
TrkBshc/- mice.
Finally, the observation that Bdnf and TrkB are co-expressed in cochlear
neurones reinforces the idea of an autocrine mode to maintain functional
activity of these neurones (Hansen et al.,
2001). Treatment of cochlear neurones with neurotrophins has been
shown to stimulate their survival and alter their functional activity
(Hegarty et al., 1997
;
Adamson et al., 2002
).
Moreover, depolarisation of spiral ganglion neurones together with
neurotrophins promotes survival in an additive manner in vitro
(Hegarty et al., 1997
). Our
present results suggest that the interplay of an autocrine neurotrophic
mechanism and neuronal activity may also maintain the survival and correct
functioning of auditory neurones in vivo, thus ensuring the intactness of the
sensory organ. In this context, it is worthwhile mentioning that the
expression of Bdnf described in the present article may also give a functional
explanation for the positive activity of neurotrophins in animal models of
hearing loss. Application of Bdnf and Nt3 to the inner ear has been shown to
ameliorate hearing loss in several of these models
(Ernfors et al., 1996
;
Duan et al., 2002
;
Shinohara et al., 2002
). Our
results suggest that the underlying mechanism is the stimulation of cochlear
neurones by neurotrophins, which may lead to a functional re-stimulation of
neuronal activity. Further experiments will show if re-innervation processes,
like the ones observed in the present study, can also be re-initiated in
postnatal animals and adults. In summary, our results may provide an important
step for understanding the basis of treatment of hearing loss by
neurotrophins, and may eventually allow us to improve therapies by targeting
specific neurotrophins to subsets of cochlear neurones.
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ACKNOWLEDGMENTS |
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