Laminin and Fibronectin Modulate Inner Ear Spiral
Ganglion Neurite Outgrowth in an In Vitro Alternate
Choice Assay
Amaretta R. Evans,1* Sara Euteneuer,1* Eduardo Chavez,1 Lina M. Mullen,1
Elliot E. Hui,3,4 Sangeeta N. Bhatia,3,4 Allen F. Ryan1,2
1
Department of Surgery/Otolaryngology, University of California San Diego, School of Medicine, and
VA San Diego Health Care Systems, 9500 Gilman Drive, MC 0666, La Jolla, California 92093
2
Department of Neuroscience, University of California San Diego, School of Medicine,
La Jolla, California
3
Department of Bioengineering, University of California San Diego, La Jolla, California
4
Harvard-MIT, Division of Health Sciences and Technology/Electrical Engineering and
Computer Science, Massachusetts Institute of Technology, E19-502D, Cambridge, Massachusetts
Received 22 March 2007; revised 5 May 2007; accepted 11 May 2007
ABSTRACT: Extracellular matrix (ECM) molecules have been shown to function as cues for neurite
guidance in various populations of neurons. Here we
show that laminin (LN) and fibronectin (FN) presented
in stripe micro-patterns can provide guidance cues to
neonatal (P5) inner ear spiral ganglion (SG) neurites.
The response to both ECM molecules was dose-dependent. In a LN versus poly-L-lysine (PLL) assay, neurites
were more often observed on PLL at low coating concentrations (5 and 10 lg/mL), while they were more
often on LN at a high concentration (80 lg/mL). In a FN
versus PLL assay, neurites were more often on PLL
than on FN stripes at high coating concentrations (40
and 80 lg/mL). In a direct competition between LN and
FN, neurites were observed on LN significantly more
often than on FN at both 10 and 40 lg/mL. The data
*Both authors contributed equally to the work.
Correspondence to: A.F. Ryan (afryan@ucsd.edu).
Contract grant sponsor: NIH/NIDCD; contract grant number:
DC00139.
Contract grant sponsor: Medical Research and Rehabilitation
Research and Development Service of the VA.
Contract grant sponsor: German research foundation; contract
grant number: EU 120-1/1.
' 2007 Wiley Periodicals, Inc.
Published online in Wiley InterScience(www.interscience. wiley.com).
DOI 10.1002/dneu.20540
suggest a preference by SG neurites for LN at high concentrations, as well as avoidance of both LN at low and
FN at high concentrations. The results also support a
potential model for neurite guidance in the developing
inner ear in vivo. LN, in the SG and osseus spiral lamina
may promote SG dendrite growth toward the organ of
Corti. Within the organ of Corti, lower concentrations
of LN may slow neurite growth, with FN beneath each
row of hair cells providing a stop or avoidance signal.
This could allow growth cone filopodia increased time to
sample their cellular targets, or direct the fibers upward
toward the hair cells. ' 2007 Wiley Periodicals, Inc. Develop
Neurobiol 00: 000–000, 2007
Keywords: extracellular matrix;
spiral ganglion; pattern assay
neurite
guidance;
INTRODUCTION
Axon and dendrite outgrowth in neurons are influenced by many factors, including the extracellular
environment. Extracellular matrix (ECM) molecules
and their interaction with cellular receptors, primarily
integrins, have been shown to function as neuronal
1
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Evans et al.
survival signals and cues for axon and dendrite guidance in various populations of neurons (Gomez and
Letourneau, 1994; McKerracher et al., 1996).
In the auditory portion of the mammalian inner
ear the cochlea, the first neurons of the auditory pathway, reside in the spiral ganglion (SG). The SG is
centrally located in the modiolus, while the mechanosensory target cells, inner and outer hair cells (HCs),
are located peripherally on the basilar membrane.
The ECM molecule laminin (LN), a heterotrimer of
a, b, and c chains, has been identified in the mammalian cochlea in the basement membranes surrounding
the developing SG and underlying the basilar membrane (Cosgrove and Rodgers, 1997; Rodgers et al.,
2001). During postnatal maturation of the organ of
Corti, a change in expression of LN heterotrimer subunits occurs at the same time as the SG dendrites target the HCs (Cosgrove and Rodgers, 1997; Rodgers
et al., 2001), suggesting a possible functional linkage
of LN to postnatal SG differentiation and dendrite targeting. Another major ECM molecule, the dimeric
glycoprotein fibronectin (FN), has also been identified in the mammalian cochlea (Woolf et al., 1992;
Cosgrove and Rodgers, 1997). As with LN, a temporo-spatial expression pattern of FN in the path of
the SG dendrites was observed in mouse, rat and gerbil (Woolf et al., 1992; Cosgrove and Rodgers, 1997),
suggesting a possible function in dendrite targeting.
In addition, Toyama et al. (2005) identified various
isoforms of the integrin ECM receptor family, previously found to be important in the developing brain,
in the perinatal mammalian cochlea. These heterodimeric transmembrane proteins, consisting of one a
and one b chain, bind promiscuously to various ECM
molecules, promoting adherence and functionally
linking them to the actin cytoskeleton and other intracellular effectors.
Homogenous surfaces of LN and FN have been
shown to stimulate rat neonatal SG neurites in cell
culture (Aletsee et al., 2001; our unpublished data).
On homogenous LN surfaces, SG neurites showed a
dose-dependent increase in length and number of
neurites with increasing LN concentrations (Aletsee
et al., 2001). However, in some areas of the inner
ear, such as the osseus spiral lamina, ECMs appear
to be present in more or less continuous surfaces
(Woolf et al., 1992; Rodgers et al., 2001). In the
organ of Corti, the target tissue for the SG dendrites,
they appear to be present in stripes or tracks, and
more than one ECM is present (Woolf et al., 1992;
Cosgrove and Rodgers, 1997), suggesting that the
differential composition of the ECM in the cochlea
could provide guidance cues to SG dendrites. Alternatively, the ECM environment could alter the effect
Developmental Neurobiology. DOI 10.1002/dneu
of a soluble factor on dendrite growth. Indeed, PLL
or LN were found to alter dorsal root ganglion neurite reactions towards nerve growth factor and glial
cell-line-derived neurotrophic factor (Tucker et al.,
2006).
To explore the effects of a complex ECM environment on neurite growth in the cochlea, we evaluated
the response of neonatal SG neurites to alternate
stripes of ECM molecules. We hypothesized that neonatal SG neurites would respond to stripes of the
ECM molecules when presented in alternation with
the in vitro adhesion factor ploy-L-lysine (PLL) by
turning onto the ECM stripe, extending on it and
finally terminating on that stripe. Second, considering
up to 550-lm distance between the SG and the organ
of Corti, we hypothesized that the response to the
ECM molecules would be dose-dependent, consistent
with the concept of neurite targeting by gradients
(Flanagan, 2006). Finally, we hypothesized that SG
neurites would chose one ECM molecule, FN or
LN, over the other when presented in direct stripe
alteration.
METHODS
Preparation of Tissue Culture Plates
To prepare stripe molds, templates were generated by a
photolithographic process to produce parallel stripes of
photoresist compound, 100-lm wide, 100-lm high, and
100-lm apart. Silicone rubber (Sylgard1184 Silicone
Elastomer, Dow Corning, Midland, MI) was then applied
to the templates to generate stripe molds. The 4 3 7 mm2
pieces of the resultant silicone molds were placed groove
side down into 24-well cell culture plates (Costar1, Corning, Acton, MA), as shown in Figure 1(a), after sterilization with 100% ethanol for 20 min and air drying inside a
sterile hood (The Baker Company, Sanford, ME). About
50 lL of the first ECM protein solution was applied to one
side of the mold as shown in Figure 1(b). The ECM protein solution was drawn into the mold’s channels by suction applied from the other side of the mold [Fig. 1(b)] and
the plate was then incubated overnight at 48C. Each well
was then washed twice with phosphate buffered saline
(PBS) with the molds in place as described above. In case
of application of a second ECM protein, an additional
blocking step with 2% bovine serum albumin (378C for
2 h) was performed between the PBS washes. The molds
were removed, followed by an additional wash with PBS
[Fig. 1(c)]. Wells were then filled with 300 lL of PLL solution (5 lg/mL; Sigma-Aldrich, St. Louis, MO) or a second ECM protein solution and incubated at 378C for 1 h.
The wells were washed twice with Dulbecco’s modified
eagle medium (DMEM), (Gibco by Invitrogen, Carlsbad,
CA) and filled with 170 lL of primary attachment
Developing Cochlear Neurons and ECM Stripes
3
removal of the silicone matrix, experiments comparing LN
versus FN were performed twice, with alternating coating
sequence of the two ECM molecules.
Spiral Ganglion Dissection
The local animal subjects committee of the San Diego VA
Health Care System approved the surgical procedures in accordance with the guidelines laid down by the National
Institute of Health regarding the care and use of animals for
experimental procedures. Neonatal (P5) Sprague–Dawley
rats were deeply anesthetized by intraperitoneal injection of
rodent cocktail (100 mg/mL ketamine, 100 mg/mL xylazine, 10 mg/mL acepromazine) 4 lL/g body weight before
decapitation. The temporal bones were removed and further
dissected similar to the method described by Van de Water
and Ruben (1971). Briefly, the cochlear capsule was opened
and the membranous labyrinth was removed from the modiolus. The spiral lamina containing the SG was carefully
separated from the modiolus and transferred immediately
into primary cell culture medium, where it was then cut
into equal portions of 300–500 lm. Each portion (explant)
was placed into an individual well of a culture plate, and at
least 19 explants were studied under each experimental
condition of stripe composition and ECM dosage.
Cell Culture
Figure 1 Preparation of ECM stripes for cell culture. (A)
Placement of the mold into the cell culture well. (B) Application of ECM solution. (C) Washing and application of a
PLL solution or second ECM solution.
medium, containing DMEM, 10% fetal bovine serum
(Sigma-Aldrich, St. Louis,USA), 25 mM HEPES buffer
(both Gibco by Invitrogen, Carlsbad, USA), and 300 U/
mL Penicillin (Sigma-Aldrich, St. Louis, USA).
The ECM protein solutions for coating contained 5, 10,
20, 40, 80 mg/mL of either FN from human plasma, LN1
from Engelberth-Holms sarcoma (both Sigma-Aldrich, St.
Louis, USA) or bovine serum albumin (BSA), (Chemicon,
Temecula, CA) as a control protein. The conditions studied
were (1) BSA stripes against PLL stripes, (2) LN stripes
against PLL stripes, (3) FN stripes against PLL stripes, and
(4) LN stripes against FN stripes.
Since some binding of a second ECM protein to stripes
of the first ECM molecule could not be excluded after
The explants first were incubated for 24 h at 378C in the primary attachment medium, before the culture medium was
changed to serum-free maintenance media (DMEM
(Gibco), 25 mM Hepes-Buffer (Gibco), 6 mg/mL glucose
(Gibco), 300 U/mL penicillin (Sigma-Aldrich), and 30 lL/
mL N2-supplement (Gibco). For trophic support of SG neuron survival and optimization of neurite outgrowth in the
stripe assays, maintenance medium was supplemented with
10 ng/mL of recombinant BDNF (R&D Systems, Minneapolis, MN). Cultures were kept in a humidified incubator at
5% CO2 and 378C for 72 h.
Immunohistochemistry
After fixation with 4% paraformaldehyde for 20 min at
room temperature (RT) and two washes with PBS (Gibco),
the explants were permeabilized with 5% Triton X-100
(Sigma-Aldrich) for 10 min, washed twice with PBS, and
blocked for nonspecific antibody binding with 5% donkey
serum (Sigma-Aldrich). Neurites were labeled for neurofilament using a polyclonal 200 kDa anti-neurofilament primary antibody (1:400; Sigma-Aldrich) while ECM stripes
were visualized using polyclonal mouse or rabbit antibodies
against LN, FN (both Sigma-Aldrich), or BSA (Fitzgerald
Industries International, Concord, MA) at 1:400 each. After
primary antibody incubation overnight at 48C, followed by
two PBS washes, the neurites and ECM stripes were visualized by 2.5 h of incubation with fluorescein isothiocyanate
(FITC) or Texas-red (TR) conjugated secondary antibodies
(1:100; Jackson Immunoresearch, West Grove, PA) against
Developmental Neurobiology. DOI 10.1002/dneu
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Evans et al.
the species of the respective primary antibody. Specificity
of staining was confirmed by a series of negative control
staining without primary antibodies.
Data Analysis
As noted above, at least 19 SG explants in stripe assays
were evaluated for each experimental condition. (The few
explants that failed to extend any neurites were excluded
from analysis). Digital images were obtained on an inverted
fluorescence microscope (Olympus IX70, Center Valley,
PA) equipped with appropriate excitation and emission filters for FITC and TR. For publication in this manuscript,
images were optimized to achieve uniform brightness and
contrast using Adobe Photoshop (Adobe Systems, San Jose,
CA).
The number of neurites terminating on each stripe substrate was counted for each individual explant. For each experimental condition, the neurite termination data of these
explants showed normal distributions (in a KolmogorowSmirnow test) and homogenous variances. Therefore the
individual explant data, grouped by experimental condition,
were compared by ANOVA followed by Tukey post-hoc
tests and paired t tests using Statistica 5.1 (StatSoft, Tulsa,
OK). Results were considered to be significant when the
likelihood for a Type 1 error was less then 5% (p < 0.05).
Significance level was corrected according to the Bonferroni method when multipe tests were applied. The data presented in the text and figures are the means and standard
deviations of individual explant data for each experimental
condition. Although the PLL control concentration was
constant at 5 lg/mL for all experiments, PLL data were
treated as paired data with the respective protein stripe data,
rather than as an isolated control group within the ANOVA,
because it was assumed that the alternating ECM protein
might influence the PLL data.
RESULTS
SG Neurites on Stripes of BSA
Versus PLL
Before investigating the effects of the ECM proteins
LN and FN, experiments were performed with BSA
to control for possible mechanical or nonspecific protein boundary effects between stripes, due to the
stripe generation process. As stated in the methods
section, BSA was applied through the channels in the
silicone matrix, which was then removed and the
BSA stripes overlaid with PLL. This procedure
involves the risk of a slight height difference between
the stripes, possibly influencing neurite outgrowth, as
well as a nonspecific protein difference. However,
there was no significant difference between the number of neurites per explant ending on BSA compared
with the number of neurites ending on PLL at 20, 40,
Developmental Neurobiology. DOI 10.1002/dneu
Figure 2 Equivalent termination of neonatal SG neurites
on alternating stripes of BSA (20 to 80 lg/mL) versus PLL
(5 lg/mL). Bars represent the mean number of neurites terminating on each substrate, per explant (error bar ¼ 1 SD).
or 80 lg/mL BSA (two-way ANOVA p > 0.8; Fig. 2).
In addition, no obvious pursuit for growth along a particular type of stripe was detected, when the entire neurite was evaluated as opposed to the growth cone only
[Fig. 3(a)]. Nevertheless, there was an overall effect of
BSA coating concentration (two-way ANOVA p ¼
0.001; Fig. 2). The number of neurites terminating on
PLL as well as on BSA decreased slightly with the
increase in BSA coating concentration.
SG Neurites on Stripes of LN or FN
Versus PLL
The growth behavior of neurites from neonatal SG
neurons on stripes of LN opposed to PLL and stripes
of FN versus PLL was not uniform, but dependent on
the ECM molecule used in the experiment (LN–PLL,
FN–PLL, three-way hierarchic ANOVA: p < 0.0001,
Fig. 4). Neonatal SG neurons extended fewer neurites
in the FN versus PLL experiment than in the LN
versus PLL experiment. The ECM coating concentration used in the experiment had an overall effect on
neurite outgrowth as well (three-way hierarchic
ANOVA, concentration effect: p < 0.021). However,
no over-all stripe effect of ECM molecule versus
PLL was detected (three-way hierarchic ANOVA:
p > 0.05), reflecting the fact that neurites showed different growth patterns for the different ECM molecules in the two PLL experimental series. While
more neurites ended on PLL in the FN versus PLL
experiments, more neurites ended on PLL than on LN
at some concentrations and LN rather than PLL at
others. This leads to two important interaction effects
observed within the ANOVA. There was a strong
interaction effect between the type of experiment
(LN–PLL, FN–PLL) as well as the stripe type (ECM
Developing Cochlear Neurons and ECM Stripes
5
Figure 3 Representative examples for SG explants grown on various ECM stripe patterns after
72 h in culture. Stripe width ¼ 100 lm. (A) Growth on BSA (80 lg/mL) versus PLL (5 lg/mL).
There was no tendency for neurites to be found preferentially on either substrate. (B) Growth on LN
(10 lg/mL) versus PLL (5 lg/mL). In this explant, neurites more often terminated upon and lay
along PLL stripes. (C) Growth on LN (80 lg/mL) versus PLL (5 lg/mL). Neurites were preferentially observed on LN stripes. (D) Growth on FN (10 lg/mL) versus PLL (5 lg/mL). Neurites were
distributed equally between FN and PLL stripes. (E) Growth on FN (80 lg/mL) versus PLL (5 lg/
mL). Neurites were most often observed on PLL. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
or PLL) with the coating concentration of the respective ECM (ANOVA: p < 0.004 and p < 0.0001).
LN Versus PLL. At low LN concentrations (5 and
10 lg/mL), more neurites ended on PLL than on LN
(post hoc paired t test, p < 0.01), while at the highest
LN concentration of 80 lg/mL the observed trend
reversed, with more neurites ending on LN than on
PLL [post hoc paired t-test, p < 0.001, Fig. 4(b,c)]. In
addition, neurites from many (but not all) explants
clearly were observed lying along the PLL stripes
(stripe ‘‘tracking’’) when PLL was opposed to 5 or
10 lg/mL LN stripes [Fig. 3(b)], while at 80 lg/mL
extending neurites from many explants were clearly
found along LN stripes [Fig. 3(c)]. At intermediate
LN concentrations, examples of differential distribution on stripes were observed, but neurites from some
explants were found on LN while those from others
were found on PLL.
FN Versus PLL. Neurites extending from neonatal
SG neurons on stripes of FN versus PLL preferentially terminated on PLL at 40 and 80 lg/mL FN
(paired t-test: p < 0.001 and p < 0.01), while at lower
Developmental Neurobiology. DOI 10.1002/dneu
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Evans et al.
Figure 5 Stripe ‘‘tracking’’ by neurites was influenced by
the angle at which the initial segment approached a stripe
border: stripe tracking was most prevalent when the initial
portion of the neurite was oriented at a shallow angle (A)
and least prevalent when the initial portion was oriented head
on (B). Stripe width 100 lm. [Color figure can be viewed in
the online issue, which is available at www.interscience.
wiley.com.]
Figure 4 Dose-dependent, preferential termination of
neonatal SG neurites on alternating stripes of (A) LN (5 to
80 lg/mL) or (B) FN (5 to 80 lg/mL) versus PLL (5 lg/
mL); mean of neurites/explant and SD, post-hoc paired
t-test, *p < 0.05, **p < 0.001, ***p < 0.0001.
FN concentrations, comparable numbers of neurites
terminated on both substrates [Figs. 3(d,e) and 4]. In
addition, at 40 and 80 lg/mL FN, for some explants
the neurites clearly appeared to lie off of the FN
stripes, and to be found along PLL stripes, along their
lengths [Fig. 3(e)].
Stripe Restriction Depends Upon Initial
Neurite Angle
As noted above, neurites were observed to lie along
one type of stripe much of their length in many
experiments. For the neurites showing such obvious
stripe restriction, the initial angle of the neurite relative to the stripe border seemed to be of importance.
When it occurred, stripe restriction was most prevalent for neurites in which the orientation of the initial
portion of the neurite toward the stripes was at a shallow angle and least prevalent when the initial portion
of the neurite was at right angles to the stripes (Figs.
3 and 5).
SG Neurites on Stripes of FN Versus LN
After investigating the effects of the individual ECM
proteins LN and FN on SGN neurite extension by
Developmental Neurobiology. DOI 10.1002/dneu
comparison against PLL-control, we then examined
the effects of direct competition between LN and
FN, by alternating stripes of the two ECM molecules, since the two ECM molecules occur in nonidentical patterns in the perinatal organ of Corti
(Woolf et al., 1992; Cosgrove and Rodgers, 1997;
Rodgers et al., 2001). By immunofluoresence staining of the stripes, little deposition of the second
ECM protein onto the stripes of the first ECM
protein was observed (see Methods section). However, we repeated each experiment with the reverse
sequence of ECM protein application, to control
for any potential deposition or other effect of
application order. ECM coating concentrations of
10 and 40 lg/mL were chosen for the competitive
experiment, since in ECM/PLL assays neurites were
found preferentially off of LN at 10 lg/mL but
were not differentially distributed at 40 lg/mL, while
FN showed no effect at 10 and neurites were
observed more often off of FN at 40 lg/mL, when
opposed to PLL. Based on these individual studies,
one might then expect neurites to be observed off of
LN at 10 lg/mL, and off of FN at 40 lg/mL.
Significantly more neurites terminated on LN than
on FN in all experimental conditions, (Fig. 6). The
coating concentration (10 and 40 lg/mL) did not
influence this result. However, when LN was coated
as the first stripe, increasing the coating concentrations from 10 to 40 lg/mL for both ECM proteins
produced a stronger difference in neurite distribution
(two-way ANOVA: p < 0.0001 for interaction
between order and concentration).
Developing Cochlear Neurons and ECM Stripes
7
Stripe Assays to Investigate Functional
Guidance Cues in SG Neurite Outgrowth
Figure 6 Termination of neonatal SG neurites on alternating stripes of LN versus FN with LN coated first (A) or
FN coated first (B); mean and standard deviation, post-hoc
paired t-test, *p < 0.05, ***p < 0.0001.
DISCUSSION
The data presented in this study suggest that single or
paired ECM molecules, presented in patterns, are sufficient to guide the neurites of neonatal SG neurons
in vitro, indicating a potential role in dendrite guidance in vivo. We found that neonatal SG neurites
respond to stripes of LN and FN when presented in
alternation with the adhesion factor PLL. As hypothesized, the response to both ECM molecules was dosedependent. In the LN versus PLL assay, neurites were
preferentially found off of LN at low concentrations
(5 and 10 lg/mL) and preferentially on LN at high
concentration (80 lg/mL). In the FN versus PLL
assay, neurites were most often found off of FN
stripes, but only at high FN coating concentrations
(40 and 80 lg/mL). In the direct competition between
FN and LN, neurites were found preferentially on LN
at low (10 lg/mL) as well as at high (40 lg/mL) concentrations. These results suggest that SG neurites
actively preferred or avoided certain substrates,
although in the absence of kinetic information we
report only the final positions of the neurites.
Previous studies have used stripe patterns of LN to
investigate in vitro neurite guidance in various neuron
populations (Hammerback et al., 1985; Vielmetter
et al., 1990; Gomez and Letourneau, 1994). In the later
two studies, the stripe generation methods described
were virtually identical to those used in the present
study, using a mold system to apply the first ECM
protein and then applying the second ECM to the
entire dish after mold removal and blocking with hemoglobin or BSA (Gomez and Letourneau, 1994).
The molds generated stripes with clear borders. We
detected minimal binding of the second applied ECM
on the surface coated with the first ECM molecule by
immunofluorescence (data not shown). Concordantly,
Gomez and Letourneau (1994) detected less than 6%
binding of the second ECM molecule to the surface
coated with the first ECM at ECM concentrations of
50 lg/mL. However, we repeated competition experiments of LN versus FN with alternate coating sequences, and observed the same results (see Fig. 6),
suggesting that limited overcoating by the second
ECM molecule was not confounding the results.
In our stripe assay experiments we used organotypic explants of the inner ear’s SG. Besides neurons,
the explants contained supporting cells, including
fibroblasts and Schwann cells, which reside between
the neurons in vivo. Both cell types are known to provide guidance cues to advancing neurites and growth
cones, and therefore might have influenced the
observed neurite patterns in our experiments. However, these supporting cells grew out in a homogenous, radial way from the explants (see Fig. 3). We
did not see a clear alignment of cells to either FN or
LN stripes, as Thompson and Buettner (2006)
reported for LN in neonatal rat sciatic nerve Schwann
cells. This suggests that the response of SG neurites
to ECM patterns may have been mediated directly,
rather than via Schwann cells or fibroblasts, although
this possibility must still be considered.
SG Neurites Respond to ECM Stripes
in a Dose-Dependent Manner When
Opposed to PLL
In the FN versus PLL assay, neurites appeared to
avoid FN stripes at high FN coating concentrations
(40 and 80 lg/mL). This threshold effect suggests
that FN would have to be present in relatively high
concentrations to influence SG neurite projections
in vivo. Cytochemical localization in the developing
organ of Corti, while not quantitative, shows intense
Developmental Neurobiology. DOI 10.1002/dneu
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Evans et al.
labeling around the dendrites of the SG neurons, and
in tracks beneath the HCs of the organ of Corti
(Woolf et al., 1992). This suggests that FN is present
in relatively high concentrations during the late stages
of sensory epithelial innervation, when neurites are
remodeling and when initial synapses are forming
with the HCs (e.g. Hafidi et al., 1990; Pujol et al.,
1998). While it has been speculated that FN may
serve as a substrate for directing neurite growth in the
developing organ of Corti (Woolf et al., 1992), our
observation of apparent FN avoidance suggests that
this ECM molecules may function to channel neurites
toward their sensory cell targets. Alternatively, FN
may serve to reduce the projection rate of SG dendrites as they near their HC targets. Kuhn et al.
(1995) has shown that chick dorsal root ganglion
(DRG) neurites slow down as they approach a bead
coated with FN. FN beneath the HCs could thus serve
as a stop signal for projecting SG dendrites, giving
the growth cone filopodia more time to explore the
region and to find their HC targets.
In the LN versus PLL assay, neurites appeared to
avoid LN at low concentrations (5 and 10 lg/mL)
while they apparently preferred LN at a high concentration (80 lg/mL). This suggests that SG neurites
can respond to lower levels of LN in comparison with
FN. Moreover, the differential response across concentration suggests that SG neurites could respond to
a gradient of LN, by turning in the direction of
increasing concentration. Currently there is no evidence for LN gradients in the developing cochlea.
However, a decreased concentration of LN could
serve to slow or terminate SG neurite projection,
since Aletsee et al. (2001) have indirectly shown that
the rate of SG neurite extension on LN is dose-dependent. In their experiments, neurite length observed
after 72 h in cell culture increased with increasing
LN concentrations. Additionally, Dolda and Bellamkonda (2006) recently found that neurites of chicken
DRG respond to anisotrophic LN-1 scaffolds with
higher extension rates than to homogenous LN-1. In
the inner ear, Rodgers et al. (2001) detected strong
immunoreactivity for LNs in the projection path of
developing SG dendrites through the osseus spiral
lamina and basal lamina of the organ of Corti, but
only weak labeling in the organ itself.
SG Neurites Appear to Prefer LN Over FN
When stripes of LN and FN directly opposed each
other, SG neurites apparently preferred LN over FN
at two coating concentrations (10 and 40 lg/mL).
These results are consistent with Vielmetter et al.’s
Developmental Neurobiology. DOI 10.1002/dneu
(1990) observation in goldfish retinal axons, with
axons preferentially extending on LN (20 lg/mL)
than on FN (50 lg/mL). They found LN to be the
most permissive substrate for neurite outgrowth
amongst several tested substrates. Also, Kuhn et al.
(1995) observed that growth cones of chick DRG
fibers extending on FN change their direction towards
LN-coated beats.
Stripe ‘‘Tracking’’ of Neurites
We often observed stripe ‘‘tracking’’ behavior, with
neurites apparently following PLL stripes in opposition to high concentrations of FN and low concentrations of LN, and following LN at high concentrations
(Figs. 3 and 5). This stripe tracking appeared to
depend upon the angle at which neurites approached
the stripes, with a higher probability of tracking if the
approach was made at a shallow angle. This could
reflect the signal presented to the growth cone when
approaching a stripe. At a perpendicular angle of
approach, filopodia could deliver symmetrical signals
to the growth cone, resulting in stripe crossing. At a
shallow angle, the signal received by the growth cone
could be highly asymmetric, resulting in turning to
maximize the preferred signal. It should be noted that
we did not see any evidence of sharp turns near borders in most neurites. Of course, this could reflect
remodeling of the neurite after the crossing. Indeed,
using live videomicroscopy, Gomez and Letourneau
(1994) noted that the angle of DRG neurite approaching to a LN/FN stripe border measured in post-fixation imaging did not reliably reflect the actual angle
at which the neurite’s growth cone had encountered
the border.
Even when approach angle was taken into consideration, the stripe tracking of neurites observed in this
study was not necessarily homogenous for all neurites
extending from one explant, or from different
explants at one concentration of an ECM molecule.
Especially in experiments with intermediate LN concentrations opposed to PLL, neurites from a single
explant showed PLL tracking alongside LN tracking,
or different explants in the same experimental condition showed opposite tracking preferences. These
data suggest that SG neurons are a heterogeneous
population. Indeed, there are two subtypes of SG neurons, the Type I and Type II SG neurons (e.g. Reid
et al., 2004), which have different target cells and
functions. Type I cells form afferent synapses with
the inner HCs, while Type II cells form afferent synapses with the outer HCs in the organ of Corti (Reid
et al., 2004). However, less than 5% of neurons in the
Developing Cochlear Neurons and ECM Stripes
SG are Type II neurons, and we did not see any subpopulation that fit this proportion.
Another factor potentially influencing the likelihood of stripe tracking might be the initial orientation
of the explant to the stripes. SG explants are often
‘‘polar’’ in organotypic cell culture, with neurites
preferably growing out of one side of the explant [see
Fig. 3(e,c)]. We did not control for the orientation of
the explant towards the stripes. Thus variable positioning of explants within one experimental condition
might have contributed to the inhomogenous tracking
behavior of neurites from different explants in this
group. However, as orientation was to our knowledge
random for all explants, it should not have biased
tracking toward any particular striped molecule.
Finally, another explanation for the somewhat inhomogeneous response of neurites from different
explants could involve the origin of the explants along
the length of the cochlea. The neurons in the different
turns of the cochlea are known to vary in their characteristics, including their response to growth factors
(e.g. Davis, 2003). Since we used explants from
throughout the cochlea in our experiments, different
turn origin of the explants might account for variable
tracking behavior of different explants.
CONCLUSIONS
Our results are consistent with the idea that patterns of
LN and FN help to direct SG neurite outgrowth during
the innervation of the inner ear, as has been proposed
at other sites (e.g. Woolf et al., 1992; Rodgers et al.,
2001). Moreover, our observations suggest a potential
model for neurite guidance. High concentrations of
LN in the SG and osseus spiral lamina may promote
SG dendrite growth through this structure and toward
the organ of Corti. Within the organ of Corti, lower
concentrations of LN may provide a reduced signal
and slow neurite growth. FN beneath each row of HCs
may provide a stronger stop or avoidance signal. This
could allow growth cone filopodia increased time to
sample their cellular targets, or might even direct the
fibers upward toward the HCs. Gradients of LN could
also provide directional signals.
Further studies focusing on the ECM microenvironment and receptor distribution within the organ of
Corti are needed to elucidate the impact of the ECM
on neurite guidance and synapse formation. Moreover, the sensory epithelium of the cochlea is a threedimensional structure. Patterning of substrates in 3D
has been used by many groups to extend observations
obtained in 2D (e.g. Yu et al., 1999) and could be
applied to SG neurons as well. In addition, ECM
9
molecules act in concert with other guidance cues
including soluble growth factors (Ryan et al., 2006).
Evaluating neurite responses to combined cues will
be required to more fully understand developmental
events in the inner ear.
The authors thank Dr. Richard Lieber, University of
California San Diego, Department of Orthopedic Surgery,
for advice on statistics. We thank Kwang Pak, University of
California San Diego, Department of Surgery/Otolaryngology, for excellent technical assistance.
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