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The Journal of Neuroscience, October 1, 2001, 21(19):7630-7641
Delayed Inner Ear Maturation and Neuronal Loss in Postnatal
Igf-1-Deficient Mice
Guadalupe
Camarero1,
Carlos
Avendaño2,
Carmen
Fernández-Moreno3,
Angeles
Villar1,
Julio
Contreras1, 4,
Flora
de
Pablo3,
José G.
Pichel3, and
Isabel
Varela-Nieto1
1 Instituto de Investigaciones Biomédicas Alberto
Sols, Consejo Superior de Investigaciones Científicas (CSIC),
Universidad Autónoma de Madrid (UAM), and
2 Department of Morphology, Facultad de Medicina, UAM,
28029 Madrid, Spain, 3 Department of Cell and Developmental
Biology, Centro de Investigaciones Biológicas, CSIC, 28006 Madrid, Spain, and 4 Department of Anatomy, Facultad
de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid,
Spain
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ABSTRACT |
Insulin-like growth factor-1 (IGF-1) has been shown to play a key
role during embryonic and postnatal development of the CNS, but its
effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5),
P8, and P20 by using stereological methods and immunohistochemistry.
Mutant mice showed reduction in size of the cochlea and cochlear
ganglion. An immature tectorial membrane and a significant decrease in
the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers
nestin and Islet 1/2. Cochlear ganglion and fibers innervating the
sensory cells of the organ of Corti presented decreased levels of
neurofilament and myelin P0 in P20 mouse mutants. In
addition, an abnormal synaptophysin expression in the somata of
cochlear ganglion neurons and sensory hair cells suggested the
persistence of an immature pattern of synapses distribution in the
organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and
maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.
Key words:
cochlear ganglion; insulin-like growth factor 1; IGF-1; myelination; neurogenesis; organ of Corti; synaptogenesis; stereology
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INTRODUCTION |
Insulin-like growth factor-I
(IGF-1), a 70 amino acid protein structurally related to insulin, has
pleiotropic effects during brain embryonic and postnatal development
(D'Ercole et al., 1996 ). IGF-1 and its membrane receptor are expressed
in the developing nervous system in many areas related to projection
neurons during axon growth and synaptogenesis. IGF-1 expression peaks
in the mouse nervous system during the first two postnatal weeks and then declines gradually to low adult levels (Bondy, 1991). Studies disrupting the IGF-1 gene or IGF-type 1 high-affinity receptor confirmed the essential role of IGF-1 in brain development and maturation. IGF-1-deficient animals have severe growth deficiency, generalized organ hypoplasia, and diminished survival (Baker et al.,
1993; Liu et al., 1993; Powell-Braxton et al., 1993; Liu et al., 1998;
Wang et al., 1999a). Their CNS phenotype includes reduced brain size,
hypomyelination, reduced axonal diameters, and loss of selective
neuronal populations (Beck et al., 1995; Cheng et al., 1998; Gao et
al., 1999). Conversely, transgenic animals overexpressing IGF-1 have
increased body and organ weight, and their brains are abnormal,
primarily because of increased myelin (Carson et al., 1993; Ye et al.,
1995; O'Kusky et al., 2000). A detailed study focused on the impact of
IGF-1 deficit on a sensory organ is, however, still lacking.
In vitro studies have strongly suggested that IGF-1 is
required for chicken inner ear organogenesis (León et al., 1995,
1999; Sanz et al., 1999a,b). IGF-1 is also expressed during maturation of the rat auditory system and in adult hair cells (Lee and Cotanche, 1996; Saffer et al., 1996). IGF-1 displays neuroprotective activity (Dore et al., 1997) and, in the rodent inner ear, IGF-1 increases hair
cell regeneration (Oesterle et al., 1997; Staecker and Van de Water,
1998). In addition, a single case of a natural mutation of the
Igf-1 gene in a human showed growth failure and severe sensorineural deafness (Woods et al., 1996, 1997).
The inner ear is a complex sensory organ responsible for sound
detection in vertebrates. The mechanosensory transduction in the organ
of Corti (OC) involves the interaction of specialized sensory hair
cells with the tectorial membrane. The cochlear ganglion contains the
primary neurons that innervate the sensory epithelia of the OC via
afferent dendrites and transduce electrochemical output to the
brainstem. Reciprocally, efferent axonal fibers from the olivocochlear
system innervate sensory cells (Berglund and Ryugo, 1987;
Spoendlin, 1988). The onset and tuning of hearing depend on the correct
relationship between the tectorial membrane and the hair cells and the
proper innervation and synaptogenesis of the sensory epithelium (Romand
et al., 1987; Rueda et al., 1996; Mu et al., 1997). Maturation of the
cochlea during the first two postnatal weeks in rodents involves
cellular differentiation and a massive rearrangement of afferent and
efferent innervation (Rubel, 1978; Knipper et al., 1995).
Here we have studied whether IGF-1 participates in murine postnatal
cochlear development and maturation. We found that mice lacking IGF-1
had a significant reduction in the size of the cochlea and cochlear
ganglion, an immature tectorial membrane, a decrease in the number and
size of auditory neurons, and a general delayed maturation in the
innervation of the OC.
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MATERIALS AND METHODS |
Mice and genotyping. Heterozygous mice with a
targeted disruption of the Igf-1 gene were kindly provided
by Dr. Argiris Efstratiadis at Columbia University (New York, NY) (Liu
et al., 1993). Animals were bred and maintained on a hybrid genetic
background of MF1 and 129/sv mouse strains to increase nullizygous
Igf-1 mutant survival (Liu et al., 1993). Heterozygous
Igf-1 mice (Igf-1+/ )
were bred to generate litters with wild-type Igf-1
(Igf-1+/+),
Igf-1+/ and
Igf-1/ progeny. The
majority of null animals do not survive until adulthood, considering
the mortality before postnatal day 20 (P20) is >80%. We
focused our study on normal Igf-1+/+
and homozygous mutants Igf-1/
animals at P5, P8, and P20. Heterozygous mice were also studied, although they presented a highly variable phenotype and, therefore, these results are not routinely presented. For genotyping by Southern blot analysis, DNA was prepared from the tail tip following standard protocols (Hogan et al., 1994), and probed as previously described (Liu
et al., 1993). All animal handling followed approved institutional protocols.
Fixation and histology. Mice were anesthetized with
CO2 on dry ice (P5) or by intraperitoneal
injection of 0.12 mg/gm ketamine hydrochloride (P20) and perfused
transcardially with 4% paraformaldehyde in 0.1 M
PBS. Heads were removed and processed for celloidin or paraffin
embedding. For celloidin embedding, after removing skull vaults, heads
were post-fixed for 3 d in the same fixative, decalcified in 0.3 M EDTA, pH 6.3, for 3 d (P5) or 7 d
(P20) at room temperature, dehydrated in ethanol for 7 d, and
defatted in ether for 3 d. Specimens were cut along the sagittal
line and sequentially infiltrated in 2, 4, 6, and 12% low-viscosity
nitrocellulose (Celloidin; Fluka, Buchs, Switzerland) dissolved in a
1:1 mixture of ethanol and ether. Celloidin blocks containing the
hemicrania were prepared from fresh 12% solution hardened with
chloroform vapors. Blocks were serially cut in 50-µm-thick sections
using a sliding microtome, following different planes of section for
each side: left hemicrania were sectioned along the sagittal plane and
right hemicrania along the horizontal plane. Sections were collected
and stored in 70% ethanol. For stereological observations, every
second section was mounted and Nissl-stained using 1% cresyl violet.
For some histological studies, additional thinner (25 µm) celloidin
sections were used. For paraffin embedding, temporal bones containing
the inner ear were removed, post-fixed in buffered 4% paraformaldehyde for 2 d, decalcified in 0.3 M EDTA, pH 6.3, dehydrated in ethanol, and embedded in paraffin. Serial 10 µm
sagittal sections were used for TUNEL labeling or immunohistochemical
studies. At least four animals of each genotype were sampled for each assay.
Stereological analysis and statistics. Celloidin sections
were used to obtain quantitative data on morphological parameters of
the cochlea. We estimated the total volume of the cochlea and cochlear
ganglion, the cochlear ganglion total neuron number, and the mean soma
volume of ganglion neurons. No correction for shrinkage was introduced.
All measurements were performed using an interactive computer system
consisting of a high-precision motorized microscope stage, a 0.5 µm
resolution z-axis reader (microcator Heidenhain VZR 401), a
solid-state video camera, and a high-resolution video monitor. The
objectives used were a planachromatic X20 (Splan 20; Olympus, Hamburg,
Germany) and a planapochromatic 100× oil immersion lens with a
numerical aperture of 1.4 (S-Plan Apo 100; Olympus). The
interactive test grids and control of the motorized stage were provided
by the GRID general stereological software package (Olympus)
running on an Amiga 2000 computer.
Every second celloidin section of the whole structure that contained
the entire cochlea or the cochlear ganglion was selected for measuring
their respective volumes (~10 sections per inner ear). An unbiased
estimate of the total volume (V) of the cochlea, including the otic capsule, was obtained by point-counting using a
uniform point grid, applying the principle of Cavalieri (Gundersen et
al., 1988; Blasco et al., 1999). Following this principle, V = t · a(p) ·  Pi, where t is the mean distance
between two sections (t = 0.1 mm), a(p) is
the area associated to each test point [a (p) = 0.002028 mm2], and Pi
is the number of points that fall within the cochlea on each section.
The cochlear ganglion volume was estimated applying the same method and
considering "ganglionar area" all groups of neurons with more than
two juxtaposed neuron bodies.
The absolute number of neurons (N), the mean cell body volume, and the
distribution of individual volumes were obtained simultaneously in each
cochlear ganglion, by means of the optical fractionator (Gundersen et
al., 1988; West et al., 1991), which is a stereological development
that combines the optical disector with a fractionator sampling scheme
and the vertical planar rotator (Jensen and Gundersen, 1993; Tandrup et
al., 1997; Lagares and Avendaño, 2000).
The method used for obtaining the absolute neuron number
(N) consisted in counting the cell nuclei that
appeared within a series of systematically random-sampled volumes
(Q-) and dividing it by the fraction of
the total volume of the ganglion represented by the addition of the
sampled volumes (fT),
N = (Q-) × 1/fT. The fractionator sampling scheme was
applied as follows: (1) every second section was used for counting
(fS = 1/2); (2) the cochlear
ganglion was divided into fixed areas, the first of which was
positioned at random; a stepping motor moved the microscope stage
systematically to other locations at pre-established and fixed
distances. The area of the disector frame covered 352 µm2, which was a fraction
(fd) of the total sampled area
(fd = 1/10). Finally, (3) the
disector height was set at 20 µm, so the fraction of the total
section thickness sampled was fh = 20/t. The mean section thickness for each ganglion was estimated
from the microcator readings of three or four measurements per section,
performed by focusing from top to bottom surfaces of the tissue. The
average t for all ganglia was 48.7 µm. The total sampling
fraction is the product of the fractions at each step of the sampling
procedure, fT = fS × fd × fh. The optical disector (Sterio, 1984)
was applied in each sampled field.
Neuronal nuclei were selected on the basis of cellular pyknosis,
nucleus shape, and defined presence of nucleolus. Cells without a
well-defined nuclear membrane and at least one nucleolus were excluded.
All nuclei whose equator came into focus 10 µm below the section
surface and within the disector frame were counted, as were other
nuclei coming into focus as the focal plane was lowered up to 30 µm
below the surface. Nuclei touching the lowest plane, or either of two
predetermined adjacent sides of the rectangular disector frame, were
not included in the count. The perikaryal volume of the same ganglion
cells that were sampled for obtaining N was estimated by
means of the vertical planar rotator (Jensen and Gundersen 1993;
Avendaño and Dykes, 1996). This procedure consists of positioning
a grid of parallel and equidistant lines orthogonal to a "vertical"
axis drawn through the nucleolus and parallel to the cutting direction.
Because of the spiral course of the ganglion, the cell bodies revolve
around the vertical axis. This effectively substitutes the use of
various orientations of the cutting planes around the vertical axis, a
requisite to guarantee isotropy in measuring designs that use
"vertical" sections (Gundersen et al., 1988). The rotator allows
estimation of the mean cell volume of the entire population
(VN), as well as plotting a
distribution of neurons on the basis of body size (West, 1999). The
precision of the estimations was evaluated by computing the coefficient of error as described for systematic random samples (Howard and Reed,
1998; Gundersen et al., 1988; Blasco et al., 1999). With this
sampling scheme the coefficient of error was <5% for cochlear and
ganglion volume estimations and 12% for cell number. Individual mean
volume estimates were computed for each ganglion. Cell volume distributions were computed for each size by dividing individual measurements into eight classes using both linear and
logarithmic-converted scales. The population percentage of cells
belonging to each class volume was calculated as the average of the
percentage of cells belonging to that class in different mice.
Student's t test was used to assess significance of the
data on volume and number of cells of the cochlea and cochlear
ganglion. The Kruskal-Wallis test was applied to data on perikaryal volume.
Terminal deoxynucleotidyl transferase-mediated UTP nick end
labeling, 4',6'-diamidino-2-phenylindole, and propidium iodide staining. Terminal deoxynucleotidyl transferase-mediated
UTP nick end labeling (TUNEL) was performed following manufacturer's
instructions (Roche Diagnostics, Mannheim, Germany), adapted to
paraffin sections. Briefly, deparaffinized sections were permeated
in 20 µg/ml of proteinase K, for 15 min at 37°C. Endogenous
peroxidase was inhibited by treatment with 3.0%
H2O2 (Sigma, St. Louis, MO)
in methanol. Sections were then incubated with the terminal
deoxynucleotidyl transferase enzyme diluted 1:20 (v/v) in the
terminal deoxynucleotidyl-transferase buffer containing
biotin-16-deoxy-UTP, for 1 hr at 37°C. Unspecific binding was then
blocked by treatment with 3% BSA (Sigma) for 30 min. TUNEL signal was
visualized with an antibody coupled to peroxidase (Converted-Pod, 1:5
dilution; Roche Diagnostics), for 30 min at 37°C. Signal development
was performed with 0.5 mg/ml diaminobenzidine tetrahydrochloride
(Sigma) and 0.01% H2O2 and observed with an Olympus M021 light microscope. For
4',6'-diamidino-2-phenylindole (DAPI) staining, paraffin sections were
mounted using 4 µg/ml DAPI (Sigma) to visualize pyknotic nuclei.
Alternatively, sections were stained with propidium iodide (10 µg/ml)
(Sigma) and observed with a Zeiss Axioscope fluorescence microscope or
an MCR 1924 confocal microscope (Bio-Rad, Hemel Hempstead,
Hertfordshire, UK).
Immunohistochemistry. Paraffin sections (10 µm) of
temporal bones from at least four
Igf-1+/+ and
Igf-1/ mice were used. After
deparaffination and rehydration in PBS, sections were incubated with a
blocking solution of 0.1% Triton X-100 in PBS, containing either 1%
BSA or 10% sheep serum. Samples were quenched in 0.6-1.5%
H2O2 and left overnight at
4°C in blocking solution containing different primary antibodies.
Sources, dilution, and cell specificity of the antibodies used in this
study were: anti-proliferative cell nuclear antigen (PCNA) mouse
monoclonal (Concepta, Barcelona, Spain) (1:500); anti-caspase-3
activated mouse polyclonal (Srinivasan et al., 1998) (1:100);
anti-Islet 1/2, mouse monoclonal (Developmental Studies Hybridoma Bank)
(1:200); anti-Nestin 128, rabbit polyclonal (Lendahl et al., 1990)
(1:500); anti-Neurofilament 200 kDa, mouse monoclonal (Diagnostic,
Mannheim, Germany) (1:50); anti-synaptophysin, rabbit polyclonal (Dako, Copenhagen, Denmark) (1:100); anti-GFAP, rabbit polyclonal (Dako) (1:100); anti-Myelin P0, mouse monoclonal
(Cao et al., 1996) (1:500); and anti-vimentin VIM3B4 mouse monoclonal
(Colucci-Guyon et al., 1994) (Profer Immuno-Diagnostika, Heidelberg,
Germany) (1:100). These molecules are markers for proliferative cells,
apoptotic cells, young neurons, precursor cells, neurons, synapses,
glial cells, myelin and immature neurons, respectively. Anti-IGF-I
rabbit polyclonal UB3-189 (1:100) was from National Pituitary Agency (Baltimore, MD). After incubation with EnVision+ anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Dako, Copenhagen, Denmark) for 30 min, peroxidase was reacted with 0.66 mg/ml
diaminobenzidine tetrahydrochloride (DAB) (Sigma) and 0.02% H2O2 (Sigma).
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RESULTS |
IGF-1 is required for normal postnatal development of
cochlear structures
To assess the effect of IGF-1 deficiency on the general morphology
of the cochlea, we examined serial inner ear sections of normal
Igf-1+/+ and mutant
Igf-1/ mice at P5 and P20 (Fig.
1). P5
Igf-1/ animals showed a thicker
cartilaginous otic capsule and a more disperse distribution of the
fibers from the auditory branch of the eighth cranial nerve than the
normal littermates (Fig. 1A,B). At P20, mutant
cochleas displayed no major alteration in general gross anatomy,
but a significant reduction in size was evident (Fig.
1D) when compared with normal mice (Fig.
1C). Figure 1, E and F, shows positive
immunolabel for IGF-1 in the cochlea and cochlear ganglion of normal
mice at P20. IGF-1 is strongly expressed in a subpopulation of neurons
of the cochlear ganglion. Other structures in the cochlea,
such as stria vascularis, spiral limbus, and support cells of the
OC, presented also IGF-1 staining. A similar expression
pattern was observed at P5 (data not shown). The lack of expression of
IGF-1 in the cochlear neurons of null mice is shown in the inset
(Fig. 1G) and is compared with positive wild-type neurons
(Fig. 1F, arrowheads).
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Figure 1.
Cochlear anatomy of the mouse genotypes
analyzed and IGF-1 expression. Nissl staining of mid-modiolar celloidin
sections of the cochlea at P5 (A, B) or P20 (C,
D) in Igf-1+/+ (A,
C) and Igf-1/ (B,
D) mice. An increased thickness of the otic capsule cartilage
and a dispersion of the fibers in the auditory branch of the eighth
cranial nerve of Igf-1/ at P5
(star) were observed in all animals studied. Note
the reduction in size of
Igf-1/ P20 cochlea
compared with the Igf-1+/+ P20
controls. IGF-1 immunohistochemical expression in the cochlea
(E) and cochlear ganglia
(F) of P20
Igf-1+/+ mice.
Asterisk in E indicates the area shown in
F; arrowheads in F point
to the subpopulation of IGF-1-positive neurons. The
inset shows a magnification of the cochlear ganglion of
Igf-1/ P20 animals
(G). C, Cochlear duct;
O, otic capsule; OC, organ of Corti;
CG, cochlear ganglion; VIII, eighth
cranial nerve; TM, tectorial membrane;
SL, spiral limbus; SV, stria vascularis.
Scale bars: A-D, 650 µm; E, 600 µm;
F, 55 µm; G, 10 µm.
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The most typical phenotypic characteristic of the Igf-1 null
mutation is general growth retardation (Liu et al., 1993; Baker et al.,
1993; Powell-Braxton et al., 1993). Therefore, the volume of the
cochlea was calculated by a quantitative stereological analysis (Table
1). The body weight of
Igf-1/ animals used in
this study was reduced between 50 (P5) and 60% (P20) relative to that
of the Igf-1+/+ littermates, as
previously published (Baker et al., 1993). In contrast,
Igf-1/ mice showed a
nonsignificant reduction 9% in cochlear volume at P5 when compared
with Igf-1+/+ but a very significant
34% reduction (compare 1.41 ± 0.15 with 2.13 ± 0.16 mm3) at P20 (Table 1, Fig. 1). This
suggests that growth retardation in the cochlea was less marked and
appeared later than the whole body dwarfism.
A detailed analysis of Igf-1/
cochleas evidenced additional morphological alterations when compared
with normal Igf-1+/+ controls (Fig.
2). The anchorage of the tectorial
membrane to the sensory hair cells of the OC in mutant P20 mice is
altered with respect to normal mice. During early postnatal development of the mouse inner ear, the tectorial membrane remains firmly attached
to the sensory epithelium until the marginal pillars, a glycoprotein
structure secreted by Deiters' cells, disappear around P14. It is also
by the end of the second postnatal week when the mouse cochlear
microarchitecture is considered fully developed and ready for normal
auditory function (Rueda et al., 1996). Microscopic examination of the
P20 inner ear from Igf-1/ mice
indicated that in all sections, analyzed from the base to the apex, of
the cochlea (20 sections from each of the five animals studied), the
tectorial membrane remained physically attached to the underlying OC,
by a membrane-like structure that may be a remnant of marginal
pillars (Fig. 2B,D). Attachment was also observed in
mice of both genotypes at P5, in accordance with their immature state
(data not shown). The OC was normal, as judged from the morphological
studies performed (Fig. 2). These results indicate that the absence of
IGF-1 causes a delay in cochlear development at the level of tectorial
membrane maturation.
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Figure 2.
Altered morphology of the OC tectorial
membrane in Igf-1/ P20 mice. Nissl staining
of celloidin-embedded cross sections of the OC of P20
Igf-1+/+ (A, C) and
Igf-1/ (B, D)
animals. A and B show basal turns,
whereas C and D show apical turns of the
cochlea. Physical attachment of the tectorial membrane to the hair
cells was noticed in all sections of P20
Igf-1/ (B, D,
arrows). BM, Basilar membrane;
DC, Deiters' cells; IHC, inner hair
cells; OHC, outer hair cells; TM,
tectorial membrane. Scale bars, 30 µm.
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Reduced total volume, neuronal cell loss, and decreased neuronal
cell size in the cochlear ganglion of
Igf-1/ mice
Multiple cellular abnormalities were observed in
Igf-1/ cochleas at P5 and, more
dramatic, at P20 stages. At P5, normal and mutant ganglia presented
similar size and cellular densities (Fig. 3A,B), although
Igf-1/ mice showed a subtype of
small cells, with more intense chromatin Nissl staining and
non-neuronal appearance (Fig. 3B). At P20, when the sense of
hearing is completely functional, ganglia from Igf-1/ mice showed a severely
affected morphology compared with their Igf-1+/+ littermates. The formers
were reduced in size, with fewer neurons and wider intercellular spaces
(Fig. 3C,D). PCNA is a DNA-binding protein required for
progression through the S phase of the cell cycle (Celis and Celis,
1985). Figure 3, E and F, shows absence of PCNA
expression indicating that cochlear neurons were postmitotic at P5 in
both null and normal mice (Fig. 3E,F), whereas
cerebellar neurons were actively proliferating at the same age (Fig.
3G).
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Figure 3.
Altered cochlear ganglion morphology in
Igf-1/ mice. Nissl staining of the ganglion
in the cochlear basal turn in
Igf-1+/+ (A, C) and
Igf-1/ (B, D) mice
at P5 (A, B) and P20 (C, D). At P5, the
cochlear ganglion and its neurons show similar morphology in both
genotypes (arrowheads), except that
Igf-1/ mouse also show a subtype
of abnormally small, strongly chromaffinic cells (open
arrow). At P20, ganglion cells are noticeably reduced both in
size and number in Igf-1/ mice.
This reduction, despite the presence of enlarged intercellular spaces
(arrows), leads to a considerable decrease in the
ganglion cross-sectional area. E and F
show negative PCNA expression in the cochlear ganglion cells of P5
Igf-1+/+ (E)
and Igf-1/
(F) mice. The inset
(G) shows a positive control of PCNA-positive
cerebellum cells from the same section of P20
Igf-1/ mouse shown in
F. Scale bar, 30 µm.
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To further characterize these phenotypic differences, we performed
measurements of the cochlear ganglion, as detailed in Materials and
Methods. The comparison of P5
Igf-1+/+ with
Igf-1/ ganglia revealed no
significant differences in the three parameters evaluated, ganglion
volume, number of neurons, and neuronal cell volume (Table
2). In P20
Igf-1/ mice, however, all three
morphological parameters presented significant reductions with respect
to normal mice: 27% in ganglion volume (0.035 vs 0.048 mm3), 22% in neuron number (13,400 vs
16,600; ~3400 ganglion neurons were lost at P20 from an initial total
calculated of 16,800 in Igf-1+/+ at
P5), and 31% in neuronal cell volume (380 vs 547 µm3). Comparison of P20
Igf-1+/+ with P5 ganglion cells of
either genotype did not show significant changes during this time
interval (Table 2). Data on
Igf-1+/ indicated a moderate
decrease in cochlear ganglion volume (10%) and in neuronal size
(12%).
Computerized treatment of the data obtained in the stereological
analysis allowed us to study the distribution of the different proportions of neuronal soma sizes. Figure
4 shows the ganglion cellular
distribution by cell volumes. By P5,
Igf-1+/+ and
Igf-1/ cochlear neurons showed
identical proportions of the different cell volumes represented, with
most cells (>80%) measuring between 300 and 800 µm3 (Fig. 4). Although the mean neuron
cell volume was ~500 µm3 (Table 2),
the modal neuronal cell volume was ~425
µm3. These data indicate that smaller
cells were present in higher proportions. At P20, the
Igf-1+/+ cochlear ganglion contained
a similar distribution of neuronal soma sizes, but with an increase in
the proportion of cells with larger volumes, as indicated by the
increase of the mean cell volume to 547 µm3 (Table 2). A new subclass of cells
appeared with a volume >950 µm3, which
was not observed in P5 Igf-1+/+ mice
(Fig. 4). A logarithmic representation showed that neuronal volume
distribution of the P20 Igf-1/
ganglion was almost symmetric around the most abundant cell size value,
350-400 µm3 (data not shown; mean, 380 µm3). This result reflects a significant
reduction in the number of large ganglion neurons in P20
Igf-1/ mice compared with their
Igf-1+/+ littermates. The former did
not display cells larger than 800 µm3,
which represented 10% of the ganglion neurons in normal animals. In
contrast, 75% of the Igf-1/
neurons were smaller than 400 µm3,
whereas in Igf-1+/+ animals they
only represented 40%. Taken together, these data demonstrate that the
absence of IGF-1 during postnatal maturation of the inner ear produces
a decrease in the number of cochlear ganglion neurons and reduces their
mean soma volume.
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Figure 4.
Different perikaryal volume distributions of
cochlear ganglion neurons in Igf-1+/+ and
Igf-1/ mice. Linear plots of data
obtained by application of stereological methods (see Materials and
Methods) are represented for P5 (top) and P20
(bottom) mice. Error bars represent the SD among animals for
each volume class. Whereas the distribution is monomodal in all cases,
note the leftward displacement of the curve in the mutant mice at P20,
with a marked decrease of cells in the largest size classes. See Table
2 for statistical analysis.
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To analyze whether neuronal cell loss was attributable to apoptosis, we
performed TUNEL labeling and activated caspase-3 detection in
Igf-1+/+ and
Igf-1/ inner ear paraffin
sections at P5, P8, and P20. Normal
Igf-1+/+ mice were negative for
apoptotic nuclei (Fig.
5A,E)
and activated caspase-3 (Fig. 5B,F,J) across the ages
studied (5, 8, and 20 d). In contrast, in the
Igf-1/ mice cochleas a
significant number of cells were positive with both TUNEL labeling
(Fig. 5C,G,K) and anti-activated caspase-3 immunostaining (Fig. 5D,H,L). Cells positive for
activated caspase-3 decreased in number from P5 to P20, which indicates
that most affected cells have disappeared at P20. These data suggest
that in the absence of IGF-1 cochlear neurons suffer caspase-3-mediated cell death. The number of apoptotic TUNEL-positive cells decreased from
base to apex at P5 and P8 but it increased at P20 (data not shown). Because the structural maturation of the postnatal cochlea proceeds from basal to apical regions, these data suggest that IGF-1
deficit affects more the survival of cells at a more advanced stage of
maturation. In parallel, sections of the inner ear from Igf-1+/+ and
Igf-1/ mice were stained with
propidium iodide or DAPI. P5 samples from both genotypes showed no
differences, whereas P20 Igf-1/,
but not Igf-1+/+ ganglion cells
presented strong staining and nuclear anomalies (data not shown)
that may be part of the apoptotic program.
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Figure 5.
Apoptotic cell death in the cochlear ganglion of
Igf-1/ mice. TUNEL labeling (A,
C, E, G, K) and detection of activated caspase-3 expression
(B, D, F, H, J, L) were performed on paraffin sections from
normal (Igf-1+/+) and mutant
(Igf-1/) mice at postnatal days
5, 8, and 20. The area analyzed is shown in a schematic drawing of the
cochlea in which the square indicates basal turn cochlear
ganglia (I). Note the increase in apoptotic nuclei
(C, G, K) and intense activated caspase-3
immunostaining (D, H, L) in the mutant mice.
Arrowheads point to apoptotic neurons, whereas
arrows point to dying glial cells. The sections correspond
to basal turns of the cochlea. Scale bar, 30 µm.
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Delayed neural differentiation of postnatal
Igf-1 null cochlear ganglion
To further characterize the cellular abnormalities evidenced by
the stereological analysis in the
Igf-1/ cochlea, a panel of
differentiation and maturation markers of neurons and glia was tested
by immunostaining of sections from normal and mutant cochlear ganglia.
Nestin, the intermediate filament characteristic of progenitor cells,
showed differential expression at stages P5 and P20. At P5, there was
no staining in normal Igf-1+/+ mice
(Fig. 6A), but most
Igf-1/ ganglion neurons showed
intense expression (Fig. 6B). At P20, none of the
genotypes expressed nestin in ganglion neurons; however, nestin was
detected in a population of small non-neuronal cells in
Igf-1+/+ animals (Fig.
6C), but not in the mutants (Fig. 6D). The
scattered pattern of nestin expression detected in
Igf-1+/+ ganglia may correspond to
glial cell precursors (Lendahl et al., 1990). The expression pattern of
the transcription factor Islet 1/2 in the cochlear ganglion of mutant
mice also points to delayed or failed maturation of PCNA-negative
postmitotic cells (Fig. 3E-H). Islet 1/2 was present
at P5 in both genotypes (Fig. 6E,F), but
disappeared in normal P20 Igf-1+/+
mice (Fig. 6G). In contrast, P20
Igf-1/ ganglion neurons still
expressed this transcription factor (Fig. 6H).
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Figure 6.
Delayed differentiation of postnatal cochlear
ganglion in Igf-1/ mice.
Immunohistochemical analysis of paraffin sections of the cochlear
ganglion at midmodiolar levels using nestin
(A-D), Islet-1/2 (E-H),
synaptophysin (I-L), myelin P0
(M-P), and GFAP (Q-T)
antibodies. Left panels (A,
E, I, M, Q)
correspond to P5 Igf-1+/+ samples,
middle-left panels (B, F, J, N,
R) to P5 Igf-1/,
middle-right panels (C, G, K, O,
S) are from P20
Igf-1+/+, and right
panels (D, H, L, P, T)
from P20 Igf-1/ mice.
E-H correspond to basal turns of the cochlear ganglion,
and the remaining sections in the figure are from apical turns. Scale
bars: A-L, 40 µm; M-T, 30 µm.
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Immunostaining of cochlear ganglion with synaptophysin evidenced
expression of this synaptic marker in almost all neurons at P5 and P20
in both genotypes (Fig. 6I-L). However, the normal P20 Igf-1+/+ showed a compact and
localized synaptophysin staining pattern, whereas in P20
Igf-1/ neurons the immunolabeled
material covered most of the cell surface in a diffuse, less mature
pattern (Fig. 6L). This distinct pattern in the P20
mutant ganglion may reflect altered synaptic refinement in the absence
of IGF-1 action. Neurofilament expression was observed at P5, with no
differences between genotypes, whereas at P20
Igf-1/ animals showed less
staining than Igf-1+/+ littermates (data
not shown).
In most of the bipolar neurons of the postnatal vertebrate cochlear
nerve, myelin envelops the axon, the perikaryon, and the dendrite
(Toesca, 1996). Because previous studies have reported altered
myelination in Igf-1 mutant mice, we used myelin
P0 and GFAP antibodies to detect possible
alterations in myelination and Schwann cells. At P5, there were no
differences in the expression pattern of both markers between
genotypes. Myelin P0 immunoreactivity surrounded
most ganglion neurons (Fig. 6M,N), confirming
previous data obtained in rats (Toesca, 1996; Knipper et al., 1998),
whereas GFAP was undetectable (Fig. 6Q,R). In contrast, at
P20 the levels of both antigens were lower in the mutants (Fig.
6P,T) than in the normal mice (Fig.
6O,S), indicating a deficit in myelination during postnatal
cochlear ganglion development of
Igf-1/ mice.
Altered innervation, synaptogenesis, and myelination in the sensory
cells of the organ of Corti in Igf-1 mutant mice
The synaptogenesis and innervation of the OC sensory cells was
also altered in Igf-1/ animals
(Fig. 7). In rats, anti-synaptophysin
antibody labels the synaptic vesicles of the efferent fibers and
follows a dynamic expression pattern as synaptic maturation progresses
beyond early postnatal stages (Knipper et al., 1995, 1996). In P5 mice,
synaptophysin labeled nerve terminals with a similar pattern in both
genotypes (Fig. 7A,B). Positive synaptophysin staining
appeared with low intensity in nerve fibers from the cochlear ganglion.
Staining increased considerably as fibers continue to innervate the
base of the sensory cells, forming a diffuse "cup-like" shape
almost covering the basal half of the cell bodies. The strongest but most diffuse staining was localized at the base of the inner hair cells
in Igf-1+/+ animals (Fig.
7A). As synaptogenesis progresses, in P20 hair cells the
area of synaptophysin expression was reduced, remaining circumscribed
to the basal part of the sensory cells and to discrete points located
more distally from the base. In contrast, P20
Igf-1/ mice showed stronger,
less localized staining (Fig. 7C,D). In these mice,
immunopositive presumptive nerve terminals appeared to project synapses
up to the supranuclear level of the inner and outer hair cells,
resembling the immature situation observed at P5. Neurofilament protein
(NF-200 kDa) labeling of the bundle of fibers between the cochlear
ganglion and the sensory epithelium presented the same strong
expression pattern in both genotypes at P5 (Fig.
7E,F). At P20,
Igf-1+/+ fibers showed considerable
neurofilament staining (Fig. 7G), which was notably reduced
in Igf-1/, indicating deficits
in the density of innervation to the hair cells (Fig.
7H), and correlating with the defective cochlear
neuron survival. The loss in cochlear ganglion neurons produced by
IGF-1 deficit also affected the cochlear nerve. In P20 normal mice the nerve showed a continuous fiber-like distribution of neurofilament staining, whereas, on the contrary, P20
Igf-1/ animals showed less
neurofilament staining in a discontinuous patchy distribution and
increased levels of vimentin (data not shown). Myelin
P0 immunostaining of fibers projecting from the cochlear ganglion to the sensory cells at P5 was similar in normal and
mutant animals (Fig. 7I,J), whereas the increase in
expression in P20 Igf-1+/+ cochleas
(Fig. 7K) was not observed in the
Igf-1/ counterparts (Fig.
7L). Taken together, these data show that the
differentiation and maturation of the entire cochlear innervation system is affected by IGF-1 deficit.
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Figure 7.
Differential expression of neural and glial
markers in the innervation of the organ of Corti. Immunohistochemical
staining of midmodiolar paraffin sections of basal turns of the cochlea
with synaptophysin (A-D), neurofilament 200 kDa
(E-H), and myelin P0
(I-L) antibodies. Left panels
(A, E, I) correspond to P5
Igf-1+/+ samples, middle-left
panels (B, F, J) to P5
Igf-1/, middle-right
panels (C, G, K) to P20
Igf-1+/+, and right
panels (D, H, L) to P20
Igf-1/ mice. At P5 (A,
B), synaptophysin immunoreactivity appears diffuse and displays
a "cup-like" shape surrounding the IHCs (arrowheads)
and OHCs (arrows). At P20 (C, D), the
synaptophysin staining pattern is better defined at the base of IHC and
nerve fibers in the Igf-1+/+ controls
than in Igf-1/ mutants. NF-200K
and myelin P0 immunostainings do not evidence major
differences at P5 but are clearly less intense in P20
Igf-1/ mice (H, L)
than in Igf-1+/+ controls (G,
K, arrow). Scale bars: A-D, 20 µm;
E-L, 30 µm.
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DISCUSSION |
The present study demonstrates that IGF-1 absence impairs normal
postnatal development of mice cochlear structures. At the whole organ
level, we found that the cochleas of P5
Igf-1/ mice were similar to those of
Igf-1+/+. But, from P5 to P20,
Igf-1/ cochlear volume increased by
14%, whereas normal mice cochlear volume increased by 57%. Many, if
not all, cochlear structures appeared to be affected. This suggests
that the absence of IGF-1 is compensated by alternative factors during
embryonic and early postnatal cochlear development, but cochlear growth
is IGF-1-dependent at later stages. Igf-1 nullizygous mice
display postnatal general growth retardation with organ-specific size
alterations (Liu et al., 1993). For example, adult brain is less
affected by the growth deficit (~30%) than whole body (~60%)
(Cheng et al., 1998). To our knowledge, no organ has been studied early
enough to know if there is normal growth before P5 as we report for the
cochlea (Powell-Braxton et al., 1993; Beck et al., 1995; Wang et al., 1999a).
At the cellular level, the actions of IGF-1 appeared to be diverse.
Cochlear ganglia cellular content was reduced by 22%, and the mean
size of the sensory neurons of the cochlear ganglia was also reduced in
the knock-out mice at P20. Cochlear ganglion neurons suffered a
mean size reduction in the range of that described for other cell
types. Thus, chondrocytes from bone growth plates and testicular Leydig
Igf-1/ cells show size reduction,
attributed to their delayed developmental stage (Baker et al., 1996;
Wang et al., 1999b). Proliferating mutant uterine cells also present a
20-40% decrease in cell size and augmented DNA content, associated to
slow progression through the cell cycle (Adesanya et al., 1999). In a
mirror image, IGF-1 P7 transgenic mice present increased cell size in
the hippocampal dentate gyrus (O'Kusky et al., 2000). Cell size is
regulated by a complex net of transduction pathways (Conlon and Raff,
1999), where the participation of an insulin/IGF receptor substrate has been shown even in Drosophila, in which mutant cells were
fewer and smaller than normal (Bohni et al., 1999).
Transient IGF-1 gene expression has been reported during the maturation
of the mice auditory sensory relay system (Bondy, 1991). Here,
we show the local expression of IGF-1 in cochlear structures including
a subpopulation of cochlear ganglion neurons, stria vascularis, spiral
limbus, and support cells of the OC. Therefore, the alterations
observed in inner ear structures may be attributable to the local
deficit of IGF-1 actions superimposed to the lack of circulating IGF-1
(Trejo et al., 2001).
In our study, both mutant and normal cochlear ganglia have similar
numbers of postmitotic neurons at P5, but
Igf-1/ mice showed a considerable
loss of ganglion neurons from P5 to P20, a time frame in which IGF-1
action appears to be critical for survival of cochlear neurons. This
cell loss was attributable to apoptosis accompanied by an increased
number of neurons expressing activated caspase-3, a protease implicated
in neural cell death as deduced from the caspase-3 mutant mice that
present decreased apoptosis in the brain (Kuida et al., 1996).
Apoptosis caused by IGF-1 deficit was more intense in cells of the
basal turn of the cochlea, the more mature region, whose development
proceeds from base to apex (Mikaelian and Ruben, 1964). Therefore,
IGF-1 should be included in the list of previously described trophic factors required for mouse auditory neurons survival (Fekete, 1999;
Bussoli and Steel, 2001), which all together must cooperate postnatally
in modulating the number of neurons in the cochlear ganglion.
Our results also demonstrate that IGF-1 action is required for timely
differentiation and maturation of the cochlea. Two major morphological
alterations were observed in the P20 Igf-1 mutants. The
first was the presence of a thicker cartilaginous otic capsule in the
Igf-1/ cochlea, confirming studies
that reported retarded ossification (Baker et al., 1993). The second
was a firmer anchoring of the tectorial membrane to the OC by the
abnormal permanence of the marginal pillars, a transitory laminar
structure that disappears at ~P14 in normal mice. A fully functional
tectorial membrane is required for an appropriate gain and timing of
cochlear feedback (Legan et al., 2000). The immature permanence of the
marginal pillars in P20 limit relative mechanical motion between the
tectorial membrane and the hair cells, thus creating higher thresholds
than in the normal cochlea (Romand et al., 1987; Rueda et al., 1996). Permanent attachment and severe distortion of the tectorial membrane were reported in hypothyroid rats (Uziel et al., 1983; Prieto et al.,
1990). Because it has been reported that adult IGF-1 mutant animals
might hear loud noises (Cheng et al., 1998), the evaluation of the
impact of the abnormalities shown in the present study on frequency
thresholds will need future electrophysiological analysis.
A delayed developmental pattern was also observed in the cochlear
ganglia of IGF-1 deficient mouse. Differentiation of
Igf-1/ cochlea was evaluated with the
markers nestin and Islet-1/2. Nestin is a marker for neural precursor
cells (Lendahl et al., 1990), whereas Islet transcription factors play
important roles in neuronal differentiation (Tsuchida et al., 1994).
Cochlear ganglion cells are postmitotic but cells that have recently
withdrawn from the cell cycle can still be nestin positive
(Vicario-Abejón et al., 1995). Therefore, P5
Igf-1/ nestin-positive cells
supported the presence of neuroblasts, delayed in developmental stage
compared with P5 Igf-1+/+ counterparts.
Altered Islet 1/2 expression confirmed the delayed developmental pattern.
Our results indicate that absence of IGF-1 causes increased apoptosis,
a decrease in neuronal size, and abnormal neuronal differentiation.
Damage of cochlear ganglion neurons may be deleterious to hair cells
and vice versa (Ryan, 2000), leaving open the possibility that the
alterations described in Igf-1/
cochlear neurons could lead to damage in hair cells. In fact, P20
Igf-1/ mutant animals maintained
innervation to the cochlear sensory cells, but their synapses were
altered, either because of a delay in differentiation or refining, or
because they tried to compensate the deficit of innervation because of
neuronal loss. The cochlear nerve showed a dispersed fiber phenotype in
the center of the cochlea, associated with decreased neurofilament
protein expression in nerve fibers. Mice with mutations in
neurofilament genes have reduced axonal diameters, a phenotype
previously described in Igf-1 mutant nerves (Hirokawa, and
Takeda, 1998; Gao et al., 1999). Synaptophysin expression in normal
cochlear ganglion and OC was similar to that reported in the rat inner
ear (Knipper et al., 1995, 1996). The alterations observed in
Igf-1/ cochlea might reflect
transient growth of immature synaptic processes. Therefore, the local
presence of IGF-1 would be required during this critical period for
neuronal connections. It has been reported that a coordinated process
of axonal growth and synaptogenesis takes place at P9 in rodents,
probably as a consequence of competition for the sensory epithelium
(Pujol, 1986). In the dentate gyrus, IGF-1 overexpression produces
increases in the total number of synapses (O'Kusky et al., 2000), thus
confirming that IGF-1 supports synaptogenesis during mice postnatal development.
Myelination in Igf-1/ cochlear fibers
was also delayed or altered, as reflected by the expression of
myelination markers. The myelination of the cochlear ganglia sensory
neurons occurs postnatally (Schwartz et al., 1983). At P20, The
reduction in the level of myelin P0 was dramatic.
Previous studies on hypothyroid rats also show decreased myelin
P0 mRNA levels in the cochlear ganglion and hair
cell innervation (Knipper et al., 1998). Interestingly, myelin protein
P0 is an autoantigen in human autoimmune inner ear disease (Cao et al., 1996). The innervation of the OC has been
proposed as critical for neuronal survival and postnatal development of
cochlear sensitivity (Ernfors et al., 1995; Bruce et al., 2000). These
results are in agreement with earlier studies on mice with a different
Igf-1 targeted mutation, showing that Igf-1 gene
disruption produces loss of certain neuronal subpopulations, a general
decrease in axonal diameters, selective hypomyelination, and reduced
nerve conduction velocities in vivo (Beck et al., 1995;
Cheng et al., 1998; Gao et al., 1999). Furthermore, selective overexpression of IGF-1 in brain causes an increase in neuron number,
total brain myelin, and regional density of myelinated axons (Behringer
et al., 1990; Carson et al., 1993; Ye et al., 1995).
In summary, we have shown that IGF-1-deficient mice had a size
reduction of the cochlea and cochlear ganglion, an immature tectorial
membrane, and a significant decrease in the number and size of auditory
neurons. Analysis of key markers demonstrated that lack of IGF-1
produces a general delay in differentiation of the cochlear ganglion
cells during postnatal development. Cochlear ganglion fibers presented
decreased myelination, abnormal synaptogenesis, and deficient
innervation of the sensory cells in the OC. This cochlear phenotype
provides basis to understand one of the mechanisms leading to
sensorineural deafness, as found in the human patient with a homozygous
Igf-1 gene deletion (Woods et al., 1996, 1997). Our findings
further support the inclusion of IGF-1 among the essential neurotrophic
factors to be considered as molecules with potential therapeutic value.
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FOOTNOTES |
Received Feb. 26, 2001; revised June 25, 2001; accepted July 17, 2001.
This work was supported by Dirección General de
Investigación y Desarrollo (Spain) Grants PM96.0075 and
PM99-0111 (I.V.-N.) and PM97.0143 (F.d.P.) and Autonomous Community of
Madrid grant CAM 08.5/0023/98 (C.A.). The fellowships to G.C. and
C.F.-M. were awarded by the Ministerio de Educación y Cultura
(Spain). We thank A. Efstratiadis for kindly providing the
Igf-1 heterozygous mice and a DNA probe for genotyping,
R. McKay for anti-nestin antibody, J.-M. Tomasi and M. H. Ryder
for anti-myelin P0 antibody, and C. Gamallo for
anti-synaptophysin antibody. The monoclonal antibody Islet-1/2 was
obtained from the Developmental Studies Hybridoma Bank under the
auspices of the National Institute of Child Health and Human
Development and maintained by the University of Iowa, Department of
Biological Sciences (Iowa City, IA). We also thank Vanessa Coronado for
technical assistance. We are grateful to C. Vicario-Abejón,
E. J. de la Rosa, and J. Rueda for comments on this manuscript.
J.G.P. and I.V.-N. contributed equally to this work.
Correspondence should be addressed to Isabel Varela-Nieto, Instituto de
Investigaciones Biomédicas Alberto Sols, Consejo Superior de
Investigaciones Científicas, Universidad Autónoma de
Madrid, Arturo Duperier 4, 28029 Madrid, Spain. E-mail:
ivarela{at}iib.uam.es.
J. G. Pichel's present address: Unidad de Investigación,
Hospital de Mérida, Mérida, Badajoz, Spain.
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TOP
ABSTRACT
INTRODUCTION
