First published online August 18, 2003
doi: 10.1242/10.1242/dev.00676
Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss
Thomas Schimmang2,
Justin Tan1,
Marcus Müller1,
Ulrike Zimmermann1,
Karin Rohbock1,
Iris Köpschall1,
Annette Limberger1,
Liliana Minichiello3 and
Marlies Knipper1,*
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
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Fig. 1. Innervation of vestibular (A,B) and cochlear (C) sensory epithelia in TrkB-
and Bdnf-mutant mice. Cochlear sections through the labyrinth of newborn (P0)
and adult animals were labelled with anti-NF200 antibody to visualise afferent
innervations. Arrows point to fibres; filled arrowheads mark hair cells (A,B).
(A) TrkBshc/shc mice maintain innervation of the
sacculus at birth but show a severe reduction of NF200-positive fibres
opposite the sensory epithelia in adults compared with
TrkBshc/+ controls. In
TrkBshc-/- mutants innervation is
already lost at birth. (B) In the utriculus, both
TrkBshc/shc and
TrkBshc/- mutants lack afferent innervation at
adulthood. (C) In the first postnatal week (P0-P8) neurofilament-positive
projections are detected in the cochlea opposite all three rows of outer hair
cells (OHC) in TrkBshc/+ controls, but not in
TrkBshc/-, Bdnf-/- or
TrkB-/- mutants. Scale bars: 20 µm.
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Fig. 2. Normal morphology and prestin expression in hair cells of TrkB-mutant mice.
(A) Representative micrographs (DIC) showing the organ of Corti of adult
control TrkBshc/+ and
TrkBshc/- mutant mice. The inner (IHC) and outer
hair cells (OHC) are indicated by arrows. (B) Sections were stained with
anti-prestin antibodies. No significant differences were noted between either
the intensity or expression pattern of prestin in OHCs of
TrkBshc/+ and
TrkBshc/- mice. Scale bars: 20 µm.
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Fig. 3. Loss of peripherin-positive type II afferent neurones in the cochleae of
TrkB and Bdnf mutants. Cochleae were isolated, sectioned and stained with
antibodies against peripherin as described under Materials and methods.
Representative micrographs illustrating the presence of type II spiral
ganglion neurones (SG) in the midbasal cochlear turn of the organ of Corti in
adult TrkBshc/+ (A) and P19
Bdnf+/+ (C) control mice; no peripherin-positive neurones
were detected in TrkBshc/- (B) and
Bdnf-/- mutants (D). Scale bars: 50 µm.
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Fig. 4. Spatial reversal of innervation patterns during postnatal development in
TrkB and Bdnf mutants. Representative images of sections through the cochlear
turns at the indicated levels (apical/medial/midbasal/basal) of animals with
the indicated genotypes, labelled with anti-NF200 antibodies as described
under Materials and methods. Arrows point to the level of the three rows of
outer hair cells (OHC) and the single row of inner hair cells (IHC). The open
arrows mark the position of afferent fibres innervating OHCs. Note the absence
of NF200-positive fibres below OHCs in the apical and medial cochlear turns of
TrkBshc/-, Bdnf-/- or
TrkB-/- mutants compared with
TrkBshc/+ control animals during the first
postnatal week (A). Sections through basal and midbasal cochlear turns of
adult TrkBshc/- and P19
Bdnf-/- mice lack afferent innervation to OHCs, compared
with their respective age-matched controls, shown for
TrkBshc/+ (B). Scale bars: 20 µm.
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Fig. 5. Reduced size of efferent synapses in the absence of Bdnf and TrkB
signalling. Cochleae of adult TrkBshc/+,
TrkBshc/-, Bdnf+/+ and
Bdnf-/- mice at P19 were isolated, sectioned, and
double-immunostained with anti-synaptophysin and anti-SK2 antibodies as
described under Materials and methods. (A) Representative micrographs
illustrating synaptophysin (Syn; green) and SK2 (red) staining at the outer
hair cell (OHC) level of the basal cochlear turn in mice with the indicated
genotypes. Note the severe reduction of synaptophysin-positive boutons and of
SK2 staining opposite the synaptic bouton in
TrkBshc/- and Bdnf-/- mutant
mice compared with their respective controls. (B) Higher magnification view of
a representative OHC from A. Area outlined by dotted line represents the
position of the OHC body. Scale bars: 10 µm in A; 5 µm in B.
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Fig. 6. Expression of Bdnf, Nt3 and TrkB mRNA in the spiral
ganglion of adult rats. (A) TrkB, Bdnf and Nt3 riboprobes
specifically hybridise with spiral ganglia of the midbasal cochlea turn of P35
rat cochlea. Note the expression of Nt3 in the IHCs (inset). (B) The
expression of Bdnf, Nt3 and TrkB was analysed in cochlear
neurones along the tonotopic axis at the indicated levels
(apical/medial/midbasal/basal) in rats. Bdnf and TrkB are
expressed with increasing levels towards the basal part of the cochlea,
whereas Nt3 mRNA shows an opposite gradient of expression, with the
highest amounts of mRNA detected in the apex. SG, spiral ganglion. Scale bars:
10 µm in inset in A; 20 µm in B.
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Fig. 7. Auditory brainstem response thresholds (A) and distortion product
otoacoustic emissions (B) in adult TrkBshc/-
mutant and TrkBshc/+ control mice. ABR thresholds
were elevated at all frequencies tested, and DPOAEs were absent in
TrkBshc/- mutants compared with controls.
Amplitude of DPOAE determined at 2xf2-f1 (f1=8.86 kHz; f2=10.99 kHz;
delta I1-I2=10 dB). DPOAE, distortion product otoacoustic emission; SPL, sound
pressure level; ABR, auditory brainstem response.
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© The Company of Biologists Ltd 2003
