Bassoon and the Synaptic Ribbon Organize Ca2+ Channels and Vesicles to Add Release Sites and Promote Refilling

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DOI: 10.1016/j.neuron.2010.12.020 · Source: OAI
Neuron
Article
Bassoon and the Synaptic Ribbon
Organize Ca
2+
Channels and Vesicles
to Add Release Sites and Promote Refilling
T. Frank,
1,2,3
M.A. Rutherford,
1,10
N. Strenzke,
4,5,10
A. Neef,
3,10
T. Pangr
si
c,
1
D. Khimich,
1
A. Fetjova,
6
E.D. Gundelfinger,
6
M.C. Liberman,
5
B. Harke,
7
K.E. Bryan,
8
A. Lee,
8
A. Egner,
7
D. Riedel,
9,
*
and T. Moser
1,2,3,
*
1
InnerEarLab, Department of Otolaryngology and Center for Molecular Physiology of the Brain, University of Go
¨
ttingen Medical Center,
37099 Go
¨
ttingen, Germany
2
International Max Planck Research School for Neurosciences, Go
¨
ttingen Graduate School for Neurosciences and Molecular Biosciences,
37077 Go
¨
ttingen, Germany
3
Bernstein Center for Computational Neuroscience, 37073 Go
¨
ttingen, Germany
4
Auditory Systems Physiology Group, Department of Otolaryngology and Center for Molecular Physiology of the Brain,
University of Go
¨
ttingen Medical Center, 37099 Go
¨
ttingen, Germany
5
Eaton Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston, MA 02114, USA
6
Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, 39118 Magdeburg, Germany
7
Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, 37077 Go
¨
ttingen, Germany
8
Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA
9
Laboratory of Electron Microscopy, Max Planck Institute for Biophysical Chemistry, 37077 Go
¨
ttingen, Germany
10
These authors contributed equally to the work
*Correspondence: driedel@gwdg.de (D.R.), tmoser@gwdg.de (T.M.)
DOI 10.1016/j.neuron.2010.10.027
SUMMARY
At the presynaptic active zone, Ca
2+
influx triggers
fusion of synaptic vesicles. It is not well understood
how Ca
2+
channel clustering and synaptic vesicle
docking are organized. Here, we studied structure
and function of hair cell ribbon synapses following
genetic disruption of the presyn aptic scaffold protein
Bassoon. Mutant synapses—mostly lacking the
ribbon—showed a reduction in membrane-proximal
vesicles, with ribbonless synapses affected more
than ribbon-occupied synap ses. Ca
2+
channels
were also fewer at mutant synapses and appeared
in abnormally shaped clusters. Ribbon absence
reduced Ca
2+
channel numbers at mutant and wild-
type synapses. Fast and sustained exocytosis was
reduced, notwithstanding normal coupling of the
remaining Ca
2+
channels to exocytosis. In vitro
recordings revealed a slight impairment of vesicle
replenishment. Mechanistic modeling of the in vivo
data independently supported morphological and
functional in vitro findings. We conclud e that
Bassoon and the ribbon (1) create a large number
of release sites by organizing Ca
2+
channels and
vesicles, and (2) promote vesicle replenishment.
INTRODUCTION
Sensory encoding in the auditory and visual system of verte-
brates relies on transformation of graded receptor potentials
into rates of neurotransmitter release at ribbon synapses. The
synaptic ribbon, an electron-dense structure anchored at the
active zone, tethers a halo of synaptic vesicles (Glowatzki
et al., 2008; Nouvian et al., 2006; Sterling and Matthews,
2005). Aside from its major component, RIBEYE/CtBP2 (Khimich
et al., 2005; Schmitz et al., 2000; Zenisek et al., 2004), the ribbon
also contains scaffold proteins such as Bassoon and Piccolo
(Dick et al., 2001; Khimich et al., 2005; tom Dieck et al., 2005).
Genetic disruption of Bassoon perturbs the anchoring of ribbons
to the active zones (AZs) of photoreceptors (Dick et al., 2003) and
cochlear inner hair cells (IHCs) (Khimich et al., 2005). At the IHC
synapse, where the functional effects of Bassoon disruption and
ribbon loss are best studied, fast exocytosis is reduced (Khimich
et al., 2005), and sound encoding by the postsynaptic spiral
ganglion neurons impaired (Buran et al., 2010). Moreover, IHCs
of these Bassoon mouse mutants (Bsn
DEx4/5
) show smaller
Ca
2+
currents. However, matching Ca
2+
currents by reducing
the driving force for Ca
2+
in wild-type IHCs does not equalize
fast exocytosis between wild-type and mutant IHCs. This,
together with an unaltered rate constant of fast exocytosis in
mutant IHCs—indicating a normal vesicular release proba-
bility—led to the previous hypothesis that the defect primarily
reflects a reduction of the readily releasable pool of vesicles
(RRP) due to the loss of the ribbon ( Khimich et al., 2005).
However, the exact structural and functional correlates of the
RRP reduction remained unclear. For example, potential differ-
ences between mutant AZs that still have a ribbon (ribbon occu-
pied) and their ribbonless counterparts have not yet been inves-
tigated. Moreover, it is not known to which degree and by which
mechanism Ca
2+
influx is affected at the level of individual
synapses and how this might contribute to the exocytic deficit.
Several mechanisms may explain the impairment of fast exocy-
tosis in IHCs of Bsn
DEx4/5
mutants. First, mutant AZs may contain
Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc. 1
NEURON 10444
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
fewer vesicular docking sites and/or closely colocalized Ca
2+
channels. Together, they have been suggested to constitute
the numerous release sites of the IHC AZ at which vesicle fusion
is controlled by the Ca
2+
nanodomain of one or few nearby active
Ca
2+
channels (Brandt et al., 2005; Moser et al., 2006; Goutman
and Glowatzki, 2007). Vesicles docked and primed in these
‘slots’ probably constitute the RRP, of which the released frac-
tion but not the release kinetics depends on the number of slots
recruited by a given stimulus (Brandt et al., 2005; Furukawa and
Matsuura, 1978; Wittig and Parsons, 2008). Therefore, fewer
release sites, because of fewer Ca
2+
channels (Neef et al.,
2009) and/or fewer docking sites, could explain impaired fast
exocytosis as a deficit of RRP size. Second, even if the number
of release sites was unchanged, the standing RRP would be
diminished if vesicle occupancy at each of these sites was
reduced in Bsn
DEx4/5
IHCs, e.g., because of impaired replenish-
ment or enhanced undocking of vesicles. Third, the coupling
between Ca
2+
influx and Ca
2+
sensors of the exocytosis
machinery could be altered, such that not all vesicles can
contribute to fast exocytosis, even after proper docking and
biochemical priming. This point subsumes changes in diffusion,
buffering, or homeostasis of [Ca
2+
]
i
, as well as an increased
distance between channels and Ca
2+
sensors, positional priming
(Neher and Sakaba, 2008), as it was reported at the Drosophila
neuromuscular junction after disruption of the presynaptic scaf-
fold protein Bruchpilot (Kittel et al., 2006). Finally, the intrinsic
Ca
2+
sensitivity of exocytosis could be altered.
The availability of a number of techniques such as improved
stimulated emission depletion (STED) microscopy, and fast
confocal imaging of Ca
2+
influx, as well as the generation of
another Bassoon-deficient mouse line (Bsn
gt
) now allowed us
to address these questions. Here, we used in vitro and in vivo
physiology in combination with light and electron microscopy
and computational modeling to study in detail structural and
functional effects of Bassoon disruption at both ribbon-occupied
and ribbonless AZs. Our results indicate that both functional
inactivation of Bassoon and ribbon loss reduce the number of
synaptic Ca
2+
channels. Membrane tethering of vesicles was
improved but not fully normal at ribbon-occupied mutant AZs,
suggesting a partial function of these ribbons. Mutant IHCs
showed a reduction in the number of release sites while main-
taining an intact coupling of Ca
2+
influx to exocytosis. Vesicle
replenishment was slightly impaired in in vitro experiments. We
conclude that the multiprotein complex of the synaptic ribbon
and Bassoon organize Ca
2+
channels and synaptic vesicles at
the AZ, thereby creating a large number of release sites.
RESULTS
The most prominent morphological phenotype of IHCs associ-
ated with the disruption of Bassoon function in mouse mutants
with partial gene deletion (Bsn
DEx4/5
) is the loss of synaptic
ribbons from their AZs (Buran et al., 2010; Khimich et al.,
2005). In IHCs of immunolabeled whole-mounted organs of Corti
from 3-week-old mice, we used confocal microscopy to count
ribbon synapses as juxtaposed spots of presynaptic CtBP2/
RIBEYE (labeling ribbons) and postsynaptic GluR2/3 (labeling
glutamate receptor clusters). Per IHC in Bsn
DEx4/5
, we found on
average 2.5 ribbon-occupied synapses (22% of 1240 synapses,
n = 112 IHCs) instead of 11.9 ribbon-occupied synapses in Bsn
wt
(97% of 1028 synapses, n = 84 IHCs). Consistent with observa-
tions at retinal photoreceptor ribbon synapses (Dick et al., 2003),
we detected expression of the N-terminal Bassoon fragment in
IHCs of Bsn
DEx4/5
mice (Figure S1A, available online) but found
that it was not localized to afferent IHC synapses, arguing
against a residual function at the AZ. This observation and the
absence of an auditory deficit in 8-week-old heterozygous
Bsn
DEx4/5
mice (data not shown) do not support the idea of
a dominant negative effect of the N-terminal Bassoon fragment.
We also observed fewer ribbon-occupied synapses in IHCs of
the newly generated Bassoon-deficient mouse line Bsn
gt
(4.8
versus 9.6 ribbon-occupied synapses per IHC in wild-type),
which, like Bsn
DEx4/5
mice, showed a mild hearing impairment
(threshold increase by 23 dB for click stimuli in four Bsn
gt
mice
compared to three wild-type littermates versus 37 dB increase
in Bsn
DEx4/5
; Pauli-Magnus et al., 2007). A weak Bassoon immu-
nolabeling was observed at a small subset (approximately 10%)
of synapses in Bsn
gt
IHCs (Figure S1B), potentially explaining the
higher number of ribbon-occupied AZs in Bsn
gt
IHCs.
Reduction of Membrane-Proximal Vesicles at Hair Cell
Synapses of Bassoon Mutants
We studied effects of Bassoon disruption and ribbon loss on
synaptic ultrastructure in electron micrographs of 80 nm
sections (Figures 1A and 1B). Membrane-proximal vesicles at
apparently ribbonless Bsn
DEx4/5
AZs showed an altered distribu-
tion. When measuring their lateral position relative to the presyn-
aptically projected center of the postsynaptic density, we
observed a broad and seemingly random distribution of those
vesicles at the AZ (Figure 1C, gray bars). In contrast,
membrane-proximal vesicles at AZs of Bsn
wt
IHCs fell into two
categories: ribbon-associated (red open bars) and non-ribbon-
associated (black open bars). The latter population was indistin-
guishable from membrane-proximal vesicles at ribbonless
Bsn
DEx4/5
AZs (p = 0.27, Kolmogorov-Smirnov test). We then
counted the total number of those vesicles in single 80 nm
sections and observed significantly fewer vesicles at apparently
ribbonless (1.5 ± 0.2 vesicles, 53 AZs) and ribbon-occupied
Bsn
DEx4/5
synapses (2.0 ± 0.4 vesicles/AZ section, 10 AZs)
than at ribbon-occupied Bsn
wt
synapses (4.2 ± 0.4 vesicles/AZ
section, 26 AZs, p < 0.01 for both comparisons).
Because the absence of a synaptic ribbon cannot unequivo-
cally be concluded from not seeing a ribbon in a single 80 nm
synaptic section, we used electron tomography to address
potential differences between ribbon-occupied and ribbonless
AZs in Bsn mutant mice (Figures 1E–1H). We used Bsn
gt
mice
for these experiments because of their larger fraction of
ribbon-occupied AZs. In electron tomography, we counted
vesicles that were tethered to the plasma membrane by filamen-
tous linkers (see Figure 1D for examples; Ferna
´
ndez-Busnadiego
et al., 2010). Indeed, we found a trend toward more membrane-
tethered vesicles when a ribbon was present (6.4 ± 0.8, n = 10
versus 3.7 ± 1.1, n = 6 at ribbonless Bsn
gt
AZs; p = 0.1), probably
reflecting the addition of a ribbon-associated vesicle population.
As in the analysis of 80 nm sections of Bsn
DEx4/5
AZs, vesicle
numbers at ribbon-occupied Bsn
gt
AZs did not reach Bsn
wt
NEURON 10444
Neuron
Bassoon Organizes Hair Cell Active Zones
2 Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc.
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
levels (10.6 ± 0.7, n = 5, p < 0.01). We also observed that, unlike
at Bsn
wt
synapses, ribbons of Bsn
gt
tended to be farther away
from the plasma membrane (Figure 1I). In fact, we found a spec-
trum of ribbon-anchorage phenotypes: from wild-type-like prox-
imity to loosely anchored ribbons (often accompanied by
a second detached ribbon) to complete ribbon absence. It is
tempting to speculate that loosely anchored ribbons may not
fully promote membrane tethering of vesicles. We note that
AB
E1
D
E2
G1 H1
J
IF1
G2 H2F2
membrane-tethered vesicles
presynaptic density
plasma membrane
ribbon-associated vesicles
ribbon
C
3
2
1
0
Vesi
cl
es
/
s
e
c
ti
o
n
n.s.
***
wt
B
s
n
Δ
E
X
4
/
5
C
a
V
β
2
K
O
150
100
50
0
Ri
bb
o
n
d
is
t
a
n
ce
(n
m
)
B
s
n
wt
Bsn
gt
(
1
s
t
rib.
)
Bsn
g
t
(a
ll rib.)
p=0.06
**
0.6
0.4
0.2
0.0
Ve
s
i
cl
e
s /
s
e
ction
6004002000
Distance to PSD center (nm)
Bsn
Δ
Ex4/5
Bsn
wt
ribbon-assoc.
Bsn
wt
non-ribbon-assoc.
Bsn
wt
ribbon-occupied
r
IHC
PSD
aff
SV
100 nm
r
IHC
PSD
aff
SV
Bsn
wt
ribbon-occupied
100 nm
Bsn
ΔEx4/5
ribbon-occupied
100 nm
SV
SC
Bsn
wt
Bsn
gt
SV
SC
Bsn
gt
SV
SC
Bsn
gt
SV
SC
Bsn
gt
ribbon-occupied
100nm
Bsn
gt
ribbon-occupied
100nm
Bsn
gt
ribbonless
100nm
non-tethered vesicles
coated vesicles
Figure 1. Synaptic Ultrastructure and Vesicle Distribution in the Presence and Absence of the Synaptic Ribbon
(A and B) Electron micrographs of single thin sections of Bsn
wt
(A) and Bsn
DEx4/5
ribbon-occupied IHC ribbon synapses (B). The following abbreviations are used:
r, ribbon; SV, synaptic vesicle; PSD, postsynaptic density; aff, afferent bouton.
(C) Distribution of membrane-proximal synaptic vesicles in Bsn
wt
and Bsn
DEx4/5
IHCs as a function of distance from the PSD center. The histogram was normal-
ized to the number of sections analyzed in the respective genotype (Bsn
wt
, n = 58 SVs, 18 sections; Bsn
DEx4/5
, n = 74 SVs, 36 sections).
(D) Example slices from single-axis electron tomograms showing membrane-tethered synaptic vesicles. Tethers are marked by arrowheads SC denotes synaptic
cleft. The scale bars represent 40 nm.
(E–H) Single slices from tomograms for Bsn
wt
(E1), Bsn
gt
ribbon-occupied (F1 and G1), and Bsn
gt
ribbonless synapses (H1). (E2–H2, upper) Tomogram-based
model of Bsn
wt
(E2), Bsn
gt
ribbon-occupied (F2 and G2), and Bsn
gt
ribbonless synapses (H2). Vesicles distant from the ribbon and the plasma membrane are
not shown. (Lower) Same models as in upper but only showing membrane-proximal SVs used for analysis (see Results).
(I) Bar plot showing mean minimal distance between ribbon and plasma membrane as measured in electron tomograms of Bsn
wt
AZs (black; n = 5 ribbons/5 AZs),
of just the proximal ribbons at Bsn
gt
AZs (red; n = 10 ribbons/10 AZs), and of all ribbons at Bsn
gt
AZs (l ight red; n = 16 ribbons/10 AZs). The error bars represent
standard error of the mean (SEM).
(J) Bar plot, showing average number of membrane-proximal SVs per thin section for wild-type (black; n = 46 AZs, pooled data from Bsn and Ca
V
b
2
wild-type
littermates) and mutant synapses. Bsn
DEx4/5
(red; n = 67 AZs), but not Ca
V
b
2
knockout synapses (blue; n = 32 AZs), had approximately one-half the numbers of
membrane-proximal SVs. The error bars represent SEM.
NEURON 10444
Neuron
Bassoon Organizes Hair Cell Active Zones
Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc. 3
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
Figure 2. Decreased Immunofluorescence and Altered Shape of Ca
V
1.3 Clusters
(A) Projections of confocal sections of IHCs of apical cochlear coils immunolabeled for synaptic ribbons (CtBP2, red) and Ca
2+
channels (Ca
V
1.3, green) as used
for analysis in (B and C). (Left) Four Bsn
wt
IHCs (n is an abbreviation for nuclei). (Middle) Enlargement of part of the synaptic layer (white box, left) showing coloc-
alization of CtBP2 and Ca
V
1.3. (Right) In the partial gene deletion mutant (Bsn
DEx4/5
), Ca
2+
channels still cluster but few ribbons remain (P28). Arrowheads point to
ribbonless Ca
V
1.3 clusters in wild-type and mutant. Arrow points to a ribbon-occupied Ca
V
1.3 cluster. Asterisk labels a floating ribbon.
(B) Ca
V
1.3 immunofluorescence intensity (mean ± SEM , a.u.) was less at Bsn
DEx4/5
synapses when analyzing only Ca
V
1.3 clusters that colocalized with GluR2
(gray; Bsn
DEx4/5
versus Bsn
wt
, p < 0.0005) or when counting the ten brightest clusters per hair cell (black; Bsn
DEx4/5
versus Bsn
wt
,p<1e
20
). In both Bsn
wt
and Bsn
DEx4/5
, the presence of a ribbon (CtBP2 colocalized: ribbon occupied, red) was associated with greater Ca
V
1.3 intensity when compared to ribbonless
synapses (blue). Bsn
wt
ribbon occupied versus Bsn
wt
ribbonless, p < 0.05; Bsn
DEx4/5
ribbon occupied versus Bsn
DEx4/5
ribbonless, p < 0.005).
(C) Ca
V
1.3 cluster intensity histogram for Bsn
wt
(solid line) and Bsn
DEx4/5
(dotted line). Each distribution is decomposed into ribbon-occupied (red) and ribbonless
(blue) clusters.
NEURON 10444
Neuron
Bassoon Organizes Hair Cell Active Zones
4 Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc.
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
even in the 250 nm tissue sections that were used for tomog-
raphy, the reported vesicle numbers represent underestimates
of the full complement of membrane-proximal vesicles because
synapses were not completely included along one dimension.
However, this error equally affected each synapse type, and
tomograms fully contained the synapse in the other two dimen-
sions. Notably, we found that the electron-dense material lining
the presynaptic plasma membrane (presynaptic density) was
longer and thicker at ribbon-occupied Bsn
wt
AZs than the
spot-like presynaptic densities at Bsn
gt
AZs (regardless of ribbon
presence; Figures 1E2–1H2 and Table S1), which sometimes
harbored more than one density (Figures 1F2 and1 H2).
Finally, we also studied AZs in IHCs of mouse mutants that
contain fewer Ca
2+
channels because of a lack of the b
2
subunit
(Ca
V
b
2
; Neef et al., 2009). Ca
V
b
2
-deficient IHCs display a 70%
reduction of both Ca
2+
influx and RRP exocytosis despite the
presence of synaptic ribbons. Number (Figure 1J, data from
wild-type littermates of Bsn
DEx4/5
and Ca
V
b
2
mutants were
pooled) and distribution (data not shown) of membrane-proximal
vesicles were unaltered in 80 nm sections, suggesting that
proteins of the macromolecular ribbon complex, but not Ca
2+
channels, are required for the formation of vesicle docking sites.
Fewer Ca
2+
Channels and Altered Shape of Ca
2+
Channel Clusters
Voltage-gated Ca
2+
influx is decreased in IHCs of Bsn mutants
(Bsn
DEx4/5
; Khimich et al., 2005). Here, we explored changes of
synaptic Ca
2+
signaling by morphological and functional
imaging. First, we studied synaptic Ca
2+
channel clusters by
confocal and STED microscopy following immunolabeling of
Ca
V
1.3 Ca
2+
channels. Images of Bsn
DEx4/5
and Bsn
wt
organs
of Corti that had been processed for immunohistochemistry in
parallel and following the same protocol were acquired with
identical microscope settings and analyzed for intensity and
shape of Ca
V
1.3 immunofluorescent spots (Figure 2). We esti-
mated the short and long axes of the elliptic fluorescent objects
by fitting 2D Gaussian functions to the background-subtracted
images (see Supplemental Experimental Procedures). The fluo-
rescence integral within this region served as a proxy of the
abundance of synaptic Ca
2+
channels. In Bsn
wt
organs of Corti,
the synaptic location of Ca
V
1.3 clusters (Figure 2A) was readily
confirmed by the colocalization with synaptic ribbons (Brandt
et al., 2005; Meyer et al., 2009) and Bassoon (Figure S1B). In
addition, some lower intensity spot-like immunofluorescence
was present in IHCs (Figure 2). In Bsn
DEx4/5
and Bsn
wt
IHCs,
the synaptic localization of Ca
2+
channel clusters was identified
by costaining for postsynaptic glutamate receptors (GluR2; Fig-
ure S1C). This confirmed that Ca
2+
channels remained clustered
at synapses despite both the disruption of Bassoon and, in most
cases, absence of the ribbon. In comparison to Bsn
wt
, the immu-
nofluorescence of Ca
V
1.3 clusters colocalized with GluR2 was
reduced at Bsn
DEx4/5
synapses (Figure 2B and Figure S1D).
In experiments colabeling for Ca
V
1.3 and the synaptic ribbon
marker RIBEYE/CtBP2, we were able to separate ribbon-occu-
pied AZs from ribbonless AZs in Bsn
DEx4/5
and Bsn
wt
mice.
Because of the absence of an additional synaptic marker at rib-
bonless Bsn
DEx4/5
AZs, and to exclude nonsynaptic Ca
V
1.3
immunofluorescent spots from analysis, we considered only
the ten brightest spots in each cell for both genotypes. This
approach was justified by knowledge of cochlear location (ten
synapses per cell in apical turn; Meyer et al., 2009) and the
observation that 92.2% and 89.4% of the ten brightest Ca
2+
channel clusters were juxtaposed to GluR2 immunofluorescent
spots in Bsn
DEx4/5
and Bsn
wt
IHCs, respectively. In confocal
images, Ca
V
1.3 immunofluorescence was reduced by 42% at
Bsn
DEx4/5
AZs (Figure 2B; p < 1e20). As quantified in Figures
2B and 2C and Table 1, the Ca
V
1.3 immunofluorescence
decreased in the order: ribbon-occupied Bsn
wt
> ribbonless
Bsn
wt
> ribbon-occupied Bsn
DEx4/5
> ribbonless Bsn
DEx4/5
.
Ca
V
1.3 channel clusters of ribbon-occupied Bsn
DEx4/5
AZs
were also more similar to Bsn
wt
AZs in shape than those of rib-
bonless Bsn
DEx4/5
AZs. The altered shape of the latter was
evident in a smaller long-to-short axis ratio (standard STED;
Figures 2D and 2E and Table 1).
To resolve a potential substructure within Ca
V
1.3 clusters, we
used a custom-built STED microscope (STED*; lateral point
spread function [PSF] less than 100 nm at a tissue depth of
15 to 25 mm; yellow range in Figure 2E). Ca
2+
channel clusters
of Bsn
wt
AZs typically displayed one to three stripes of Ca
V
1.3
immunofluorescence (Figure 2F). Parallel confocal observation
of the associated CtBP2/RIBEYE immunofluorescence sug-
gested that these synapses featured one ribbon regardless of
the number of stripes, although two closely-spaced ribbons
may fall within the confocal PSF and thus may not be resolved
as individual ribbons. In contrast, Bsn
DEx4/5
AZs showed
Ca
V
1.3 immunofluorescence spots rather than stripes (Figure 2F;
full width at half maximum of long and short axes: 120 ± 7.9 nm
and 95 ± 5.5 nm, n = 13) with ribbon-occupied AZs typically
harboring more spots than ribbonless AZs. Ca
V
1.3 immunofluo-
rescent stripes and spots were reminiscent of the patterns of
presynaptic density observed in electron tomography (Figures
1E2–1H2). In summary, the abundance of synaptic Ca
2+
chan-
nels and the cluster shape are altered upon Bassoon disruption,
which might reflect the loss of a direct Bassoon action on Ca
2+
channel clustering or of the Bassoon-mediated ribbon
anchorage. To test for a potential role of Bassoon in the direct
synaptic anchoring of Ca
2+
channels, we determined whether
Bassoon and the Ca
V
1.3 channel interacted in a heterologous
expression system. We did not find evidence that Bassoon coim-
munoprecipitated or colocalized with Ca
v
1.3 in transfected
HEK293T cells (Figure S4). Therefore, the role of Bassoon in
(D) Single Z sections of CtBP2 (confocal) and Ca
V
1.3 (standard STED) for size analysis in (E) of Ca
V
1.3 clusters at Bsn
wt
(left) and Bsn
DEx4/5
AZs (right).
(E) Compared to Bsn
wt
(filled circles: individuals, gray; mean, black) and ribbon-occupied Bsn
DEx4/5
synapses (red open circles; mean, dark red), the ribbonless
Ca
V
1.3 clusters (blue open circles; mean, dark blue) fell closer to unity (dashed line). Apparent sizes of 40 nm beads mount ed above and below the organ of Corti
illustrate the PSF range for the two STED microscopes (black, Leica STED; yellow, custom STED*). The error bars represent SEM.
(F) STED* microscopy revealed ribbon-occupied Bsn
wt
Ca
V
1.3 clusters as one or more elongated stripes, not observed at Bsn
DEx4/5
synapses.
NEURON 10444
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Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
recruiting Ca
2+
channels to the AZ may not involve a direct asso-
ciation of the two proteins.
Reduced Synaptic Ca
2+
Influx Results from Fewer
Channels and Lower Open Probability
To study synaptic Ca
2+
influx in Bsn
DEx4/5
IHCs, we performed
whole-cell patch-clamp recordings of Ca
2+
current (I
Ca
) and
confocal imaging of presynaptic Ca
2+
microdomains (Frank
et al., 2009). We found a reduction of peak whole-cell I
Ca
ampli-
tude in Bsn
DEx4/5
IHCs of 3-week-old mice (Figure 3, Table 1, Fig-
ure S2, and Table S1). It ranged between 62% (ruptured-patch,
5 mM [Ca
2+
]
e
; Figure 3A and Table 1) and 69% (perforated-patch,
10 mM [Ca
2+
]
e
; Figure S2 and Table S1)ofBsn
wt
amplitude. The
difference from Bsn
wt
was alleviated in the presence of the dihy-
dropyridine agonist BayK8644 (77%, ruptured-patch, 10 mM
[Ca
2+
]
e
; Figure 3B and Table 1), suggesting that Ca
2+
channel
open probability is reduced in Bsn
DEx4/5
IHCs in the absence of
BayK8644. Moreover, we found that Ca
2+
current activation
was slowed in Bsn
DEx4/5
IHCs (Figure 3C and Figure S2), while it
was indistinguishable from wild-type IHCs in the presence of
BayK8644 (Figure 3D). Finally, the Ca
2+
currents inactivated
slightly more in Bsn
DEx4/5
IHCs (Figure 3E and Table S1).
To test whether the observed reduction in I
Ca
was caused by
changes in channel number (N
Ca
), unitary current (i
Ca
), or open
Table 1. Summary of IHC Physiology in Bsn Wild-Type and Mutant Mice
Parameter Bsn
wt
Bsn
DEx4/5
p value
Ca
V
1.3 Immunofluorescence
Intensity, confocal (a.u.) Ribbonless:
3.1e5 ± 3e4
(n = 16)
Ribbon-occupied:
4.0e5 ± 1e4
(n = 219)
Ribbonless:
2.1e5 ± 1e4
(n = 196)
Ribbon-occupied:
2.9e5 ± 3e4
(n = 46)
Bsn
wt
, ribbonless versus
ribbon-occupied, p < 0.05;
Bsn
DEx4/5
, ribbonless versus
ribbon-occupied, p < 0.01;
wild-type versus mutant,
p < 0.001
FWHM, STED,
long:short (nm)
Ribbon-occupied:
345.9 ± 12:
230.0 ± 6 (n = 68)
Ribbonless:
300.3 ± 15:
251.4 ± 14
(n = 45)
Ribbon-occupied:
355.6 ± 22:
251.8 ± 15 (n = 16)
Bsn
wt
versus Bsn
DEx4/5
,
ribbonless, p < 0.001; Bsn
wt
versus Bsn
DEx4/5
, ribbon-
occupied, n.s.; Bsn
DEx4/5
,
ribbonless versus ribbon-
occupied, p < 0.05
Whole-Cell Ca
2+
Current
Peak I
Ca
(pA): 5Ca
2+
/BayK 179.5 ± 9.6 (N = 31) 111.1 ± 6.2 (N = 38) p < 0.001 (W)
Peak I
Ca
(pA): 10Ca
2+
/+BayK 417.5 ± 29.0 (N = 29) 321.0 ± 33.9 (N = 19) p < 0.05 (W)
N
Ca
: 10Ca
2+
/+BayK 1574 ± 92 (N = 27) 1227 ± 111 (N = 22) p < 0.01 (W)
Synaptic Ca
2+
Microdomains
DF
avg
(a.u.): 5 Ca
2+
/BayK 85.5 ± 9.1
(n = 74/N = 30)
30.5 ± 2.0
(n = 112/N = 45)
p < 0.001 (W)
DF
avg
(a.u.): 5 Ca
2+
/+BayK 89.5 ± 9.0
(n = 53/N = 21)
46.9 ± 5.2
(n = 52/N = 20)
p < 0.001 (W)
DF
avg
(a.u.): ±BayK p = 0.14 (W) p < 0.01 (W)
Exocytosis
DC
m,20 ms
(fF) pp 13.2 ± 1.1 (N = 38) 7.9 ± 0.9 (N = 37) p < 0.001 (W)
Q
Ca,20ms
(pC) pp 4.4 ± 0.3 (N = 38) 3.0 ± 0.2 (N = 37) p < 0.001 (W)
DC
m,20 ms
(fF) rp 8.1 ± 1.0 (N = 17) 4.7 ± 0.8 (N = 16) p < 0.01 (W)
Q
Ca,20ms
(pC) rp 3.3 ± 0.2 (N = 17) 2.6 ± 0.1 (N = 16) p < 0.05 (T)
DC
m,100 ms
(fF) pp 39.6 ± 5.2 (N = 41) 21.9 ± 4.3 (N = 40) p < 0.001 (W)
Q
Ca,100ms
(pC) pp 20.4 ± 1.4 (N = 41) 13.2 ± 0.9 (N = 40) p < 0.001 (W)
DC
m,100 ms
(fF) rp 35.1 ± 5.3 (N = 13) 19.5 ± 5.8 (N = 11) p < 0.01 (W)
DC
m,100 ms
(fF) rp 15.5 ± 1.0 (N = 13) 11.2 ± 0.9 (N = 11) p < 0.01 (T)
sustained DC
m
(DC
m,100
DC
m,20
; fF)
26.8 ± 4.6 (N = 38) 15.1 ± 4.4 (N = 35) p < 0.001 (W)
n denotes number of synapses (Ca
V
1.3 immunofluorescence and synaptic Ca
2+
microdomains) and N number of IHCs (whole-cell Ca
2+
current, Ca
2+
imaging, and capacitance measurements). n.s. denotes not significant. For immunofluorescence, Bsn mutant data were separated into ribbonless and
ribbon-occupied synapses. Statistical comparisons were made with an independent two-sample t test (T) or a Mann-Whitney-Wilcoxon (W) test
(Experimental Procedures). a.u., arbitrary units; FWHM, full width at half-maximum; I
Ca
, whole-cell Ca
2+
current; N
Ca
, number of Ca
2+
channels; DF
avg
,
average Ca
2+
microdomain amplitude; DC
m
, exocytic membrane capacitance changes; pp, perforated-patch configuration; Q
Ca
,Ca
2+
current integral;
rp, ruptured-patch configuration. Sustained DC
m
was calculated cell-wise, by subtracting the average DC
m
response to 20 ms from the average DC
m
response to 100 ms. Data are presented as mean ± SEM.
NEURON 10444
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6 Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc.
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
probability (p
open
), we performed a nonstationary fluctuation
analysis on Ca
2+
tail-currents ([BayK8644]
e
=5mM; Brandt
et al., 2005). In line with the observed reduction in I
Ca
amplitude,
both variance and mean were reduced in Bsn
DEx4/5
IHCs (Figures
3F and 3G). The analysis indicated a 20% decrease in the
number of functional Ca
2+
channels but statistically indistin-
guishable single-channel currents and maximal open probabili-
ties in the presence of BayK8644 (Table 1 and Table S1). We
note that due to uncertainties associated with the channel prop-
erty estimates from fluctuation analysis (Tables 1 and Table S1),
which also deviate from those obtained from single-channel
recordings in immature IHCs (Zampini et al., 2010), emphasis
is on comparison between the genotypes rather than on absolute
values (see also Supplemental Experimental Procedures).
Synaptic Ca
2+
microdomains, primarily reflecting Ca
2+
influx
at the AZ (Frank et al., 2009), were visualized with the low-
affinity Ca
2+
indicator Fluo-5N (400 mM, K
d
=95m M) in conjunc-
tion with the slow Ca
2+
chelator EGTA (2 mM). The Ca
2+
micro-
domain amplitude (DF) measured under these conditions
probably reflects a linear summation of the Ca
2+
influx contrib-
uted by the individual synaptic Ca
2+
channels (Frank et al.,
2009). Consistent with the finding of Ca
V
1.3 channel clusters
in immunohistochemistry, we readily observed Ca
2+
microdo-
mains also in Bsn
DEx4/5
IHCs (Figure 4A). However, their
average amplitude (DF
avg
; Table 1), measured at 7mVin
spot-detection experiments at the center of the Ca
2+
microdo-
mains, was reduced to 36% of control (Figures 4B and 4C),
exceeding the reduction of whole-cell I
Ca
(to 60%–70%;
A
C
F
E1 E2
G
D
B
− BayK
− BayK
+ BayK
+ BayK
-150
-100
-50
I
Ca
(pA)
-80 -60 -40 -20 20 40
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
-400
-200
I
Ca
(pA)
-80 -60 -40 -20 20 40
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
300
200
100
0
τ
activation
(μs)
-40 -20 0 20
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
*
*
**
***
***
***
1.0
0.5
0.0
τ
ac
t
iv
a
tion
(ms)
-40 -20 0 20
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
-1.0
-0.5
0.0
<
I
Ca
>
(
n
A
)
200
0
<VAR>
(
pA²
)
1 ms
Bsn
wt
Bsn
ΔEx4/5
150
100
50
0
<V
a
r
>
(
p
A
²
)
8006004002000
<I
Ca
> (pA)
Bsn
wt
Bsn
ΔEx4/5
-1.0
-0.5
0.0
N
o
rma
lized I
Ca
50 ms
Bsn
wt
Bsn
ΔEx4/5
-1.0
-0.5
0.0
Nor
ma
li
z
e
d
I
Ca
100 ms
Bsn
wt
Bsn
ΔEx4/5
Figure 3. Biophysical Properties of Voltage-
Dependent Whole-Cell Ca
2+
Current (I
Ca
)
(A) Average steady-state I
Ca
-V for Bsn
wt
and
Bsn
DEx4/5
IHCs in 5 mM [Ca
2+
]
e
(n [Bsn
wt
]=31
IHCs, n [Bsn
DEx4/5
] = 38 IHCs). Note the reduction
of max. I
Ca
to 60% of wild-type level in Bsn
DEx4/5
IHCs (Table 1).
(B) As in (A) but in 10 mM [Ca
2+
]
e
and presence
of 5 mM BayK8644 (n [Bsn
wt
] = 29 IHCs,
n[Bsn
DEx4/5
] = 19 IHCs). Note the smaller differ-
ence in max. I
Ca
between the two genotypes
(Bsn
DEx4/5
: 80% of Bsn
wt
level; Table 1).
(C) Average time-constant of I
Ca
activation in 5 mM
[Ca
2+
]
e
as a function of membrane voltage (V
m
),
derived from single exponential fits to the initial
3.5 ms of I
Ca
(see Supple mental Experimental
Procedures;n[Bsn
wt
]: % 30 IHCs, n [Bsn
DEx4/5
]:
% 35 IHCs). Asterisks indicate V
m
at which differ-
ences between genotypes were statistically signif-
icant (a = 0.05; Bonferroni correction). Average
series resistance (R
S
) was 6.0 ± 2.3 MU for Bsn
wt
IHCs, and 6.1 ± 2.3 MU for Bsn
DEx4/5
IHCs
(mean ± SD), respectively.
(D) Same as (C) but in 10 mM [Ca
2+
]
e
and 5 mM
extracellular BayK8644 (n [Bsn
wt
]: % 29 IHCs,
n[Bsn
DEx4/5
]: % 19 IHCs). Average R
S
was 4.7 ±
3.1 MU for Bsn
wt
IHCs, and 5.3 ± 3.3 MU for
Bsn
DEx4/5
IHCs (mean ± SD), respectively.
The error bars in (A–D) represent SEM.
(E1and E2) Average paired-pulse I
Ca
traces (depo-
larization to V
m
of maximum I
Ca
; [Ca
2+
]
e
= 10 mM)
illustrate stronger inactivation in Bsn
DEx4/5
IHCs,
being more evident for longer (100 ms; E2) than
for shorter depolarizations (20 ms; E1).
(F) Example mean Ca
2+
tail-currents (lower) used
for nonstationary fluctuation analysis (Tables 1
and Table S1), elicited by repolarizing IHCs
from +57 mV to 68 mV, and corresponding
mean trial-to-trial variance (upper). [Ca
2+
]
e
=
10 mM, [BayK8644]
e
=5mM.
(G) Grand average (lines with filled circles) of vari-
ance versus mean relationships (n [Bsn
wt
]=27
IHCs, n [Bsn
DEx4/5
] = 22 IHCs). Filled areas depict
SD of grand average of variance. Broken lines
represent grand average of parabolic fits (Supple-
mental Experimental Procedures).
NEURON 10444
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Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
Figure 3) and Ca
V
1.3 immunofluorescence (to 58%; Figure 2).
Augmenting influx through Ca
V
1.3 Ca
2+
channels (5 mM
BayK8644) alleviated the amplitude reduction of synaptic Ca
2+
influx in Bsn
DEx4/5
IHCs (to 52% of control; Figure 4D and Table 1)
and increased amplitude variability among the Bsn
DEx4/5
, but not
the Bsn
wt
synapses (Figures 4E and 4F). Kinetics (Figures 4C
and 4D, Figure S3, and Table S1), voltage dependence (Fig-
ure 4G, Figure S3, and Table S1), and spatial extent (Figure 4H
and Table S1) of the Ca
2+
microdomains in Bsn
DEx4/5
IHCs were
similar to control. There was, however, a tendency toward faster
kinetics and more negative activation of Bsn
DEx4/5
Ca
2+
microdo-
mains (Table S1 ). While the former may reflect differences in Ca
2+
A
B
1000
500
0
Δ
F(a
.
u
.
)
2 μm
20 ms
− BayK
+ BayK
20 ms
C
D
E
F
H
G
I1
20 ms
20 ms
400
200
0
Δ
F
(a
.
u.
)
2 μm
I2
20 ms 20 ms
1.0
0.5
0.0
norm. ΔF/F
0
-60 -40 -20 0
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
200
100
0
ΔF(a
.
u.)
Bsn
wt
Bsn
ΔEx4/5
200
100
0
Δ
F(
a.
u.
)
Bsn
wt
Bsn
ΔEx4/5
1.0
0.5
0.0
Cumulative frequen
cy
3002001000
ΔF
avg.
(a.u.)
– BayK (CV=0.91)
+BayK(CV=0.73)
Bsn
wt
1.0
0.5
0.0
C
u
mu
l
a
t
ive freq
ue
n
c
y
150100500
ΔF
avg.
(a.u.)
–BayK(CV=0.70)
+ BayK (CV=0.79)
Bsn
ΔEx4/5
Bsn
Δ
Ex4/5
Bsn
wt
Bsn
Δ
Ex4/5
Bsn
wt
100
50
0
ΔF(a.
u.
)
-200
0
I
C
a
(
p
A
)
Bsn
wt
Bsn
ΔEx4/5
100
50
0
Δ
F
(
a
.
u
.
)
Bsn
wt
ribbon-occupied
(n=92)
Bsn
wt
ribbonless
(n=12)
100
50
0
ΔF(a.
u
.
)
Bsn
ΔEx4/5
ribbon-occupied
(n=46)
Bsn
ΔEx4/5
ribbonless
(n=50)
Figure 4. Reduced Presynaptic Ca
2+
Influx
(A) Exemplary localized Ca
2+
influx sites in optical
sections through the basal part of IHCs (Experi-
mental Procedures). Resting fluorescence (F
0
)
was subtracted (DF images). Ca
2+
microdomains
at AZs are present in Bsn
DEx4/5
IHCs, albeit of
smaller amplitude.
(B) Exemplary spot-detection responses during
depolarization to 7 mV (bar, lower; all reported
responses were from the Ca
2+
microdomain
center) and simultaneously acquired whole-cell
I
Ca
(upper). Note the out-of-proportion reduction
of synaptic Ca
2+
influx.
(C) Grand average of spot-detection responses
from Ca
2+
microdomains (n [Bsn
wt
] = 74 AZs/30
IHCs, n [Bsn
DEx4/5
] = 112 AZs/45 IHCs); shaded
areas indicate SD.
(D) Same as (C) but in presence of 5 mM BayK8644
(n [Bsn
wt
] = 53 AZs/21 IHCs, n [Bsn
DEx4/5
] = 52 AZs/
20 IHCs). Note the peak at the end of the stimula-
tion, corresponding to tail current-mediated Ca
2+
influx (prolonged due to BayK8644).
(E and F) Cumulative frequency distributions of
Bsn
wt
(E) and Bsn
DEx4/5
(F) Ca
2+
microdomain
amplitudes (averaged over the second half of the
stimulus) in either absence (black/gray) or presence
(red/light red) of 5 mM BayK8644. CV denotes coef-
ficient of variation (SD/mean).
(G) Normalized steady-state fluorescence-voltage
relationships (n [Bsn
wt
] = 19 AZs, n [Bsn
DEx4/5
]=
27 AZs). Relative fluorescence changes were aver-
aged over the last 14.6 ms of the 20 ms stimulus
and normalized to the peak response of the given
Ca
2+
microdomain. Shaded areas depict SEM.
(H) Representative line scans across the Ca
2+
microdomain center (5 mM [Ca
2+
]
e
). Bar indicates
period of depolarization to 7 mV.
(I) Grand average of Bsn
wt
(I1) and Bsn
DEx4/5
(I2)
spot-detection responses, sorted according to
the presence/absence of a colocalized ribbon
(Experimental Procedures). n (Bsn
wt
) = 104 AZs/
32 IHCs, n (Bsn
DEx4/5
) = 96 AZs/37 IHCs.
buffering and/or diffusion, the latter
may indicate an altered gating of synaptic
Ca
2+
channels in the absence of Bassoon
and/or the ribbon.
In a second set of experiments, we
studied Ca
2+
signaling at ribbonless and
ribbon-occupied AZs in separation with a fluorescent
RIBEYE-binding peptide to identify ribbon-occupied AZs (Frank
et al., 2009; Zenisek et al., 2004) in both Bsn
DEx4/5
and Bsn
wt
IHCs. While Ca
2+
microdomains at ribbon-occupied AZs had
larger amplitudes than ribbonless synapses in Bsn
wt
IHCs,
there was no significant difference between ribbonless and
ribbon-occupied AZs in Bsn
DEx4/5
IHCs (Figure 4I). The latter
finding was unexpected given their difference in Ca
V
1.3 immu-
nofluorescence but could reflect limited sensitivity of functional
Ca
2+
imaging, precluding detection of very dim Ca
2+
signals at
Bsn
DEx4/5
ribbonless synapses. In summary, the reduced ampli-
tude of Ca
2+
microdomains and its partial alleviation upon the
NEURON 10444
Neuron
Bassoon Organizes Hair Cell Active Zones
8 Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc.
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
BayK8644-mediated increase in open probability led us to
conclude that Bsn
DEx4/5
synapses contain fewer Ca
2+
channels
with a lower open probability. The reduction of synaptic Ca
2+
influx beyond the decrease observed in whole-cell I
Ca
indicates
a higher proportion of extrasynaptic channels in Bsn
DEx4/5
IHCs.
Reduced RRP and Sustained Exocytosis but Intact Ca
2+
Influx-Exocytosis coupling
How does the reduction of Ca
2+
channels and membrane-prox-
imal vesicles—as well as a potential mislocalization of these two
elements—affect hair cell exocytosis? We addressed this ques-
tion in Bsn
DEx4/5
IHCs by measuring exocytic membrane capac-
itance changes (DC
m
) in response to short (20 ms, D C
m, 20 ms
)
and longer (100 ms, DC
m, 100 ms
) depolarizations to the maximum
Ca
2+
current potential in native buffering conditions (perforated-
patch configuration; Figure 5). Based on previous work (Gout-
man and Glowatzki, 2007; Li et al., 2009; Meyer et al., 2009;
Neef et al., 2009; Rutherford and Roberts, 2006; Schnee et al.,
2005), we interpret DC
m, 20 ms
as fast (synchronous) exocytosis,
representing release of a standing RRP, and the difference
between DC
m, 100 ms
and DC
m, 20 ms
as sustained exocytosis, re-
flecting vesicle supply to the RRP and subsequent fusion. In this
set of experiments, DC
m, 20 ms
was reduced to 60% (Figure 5A
and Table 1) and sustained exocytosis to 56% (Figure 5B and
Table 1). These results are consistent with a model in which
RRP size and sustained exocytosis rate are related to the
number of physical docking and release sites at the AZ (reduc-
tion of membrane-proximal vesicles: 50%, Figure 1J). To test
whether the intrinsic Ca
2+
dependence of exocytosis differed
between genotypes, we used flash photolysis of caged Ca
2+
,
but found comparable time constants of the fast component of
the DC
m, flash
for elevations of [Ca
2+
]
i
to 25–37 mM(Bsn
DEx4/5
:
2.4 ± 0.4 ms, mean postflash [Ca
2+
]
i
: 29.0 ± 1.9 mM, n = 6 versus
Bsn
wt
: 2.6 ± 1.1 ms, mean postflash [Ca
2+
]
i
: 31.5 ± 2.5, n = 4; Fig-
ure 5C and Table S1) suggesting an unaltered biochemical Ca
2+
sensitivity of exocytosis. Notably, despite the lack of ribbons
from most synapses in Bsn
DEx4/5
IHCs the amplitude of their
flash-evoked C
m
rise was statistically indistinguishable from
Bsn
wt
.
AB C
DE1E2F
-500
0
I
Ca
(pA)
50
0
ΔC
m
(f
F
)
100 ms
Bsn
wt
Bsn
ΔEx4/5
-200
0
I
Ca
(p
A)
50
0
Δ
C
m
(
fF
)
100 ms
Bsn
wt
Bsn
ΔEx4/5
Bsn
wt
Bsn
ΔEx4/5
1.0
0.5
0.0
C
m,flash
(pF)
0.20.0
Time (s)
Δ
2
1
0
Δ
C
m,
P
P
/
ΔC
m
,
EG
T
A
20 ms 100 ms
Bsn
wt
Bsn
ΔEx4/5
Bsn
wt
10
5
0
Δ
C
m,20
m
s
(
f
F
)
420
Q
Ca
(pC)
-32mV
Bsn
ΔEx4/5
-27mV
-22mV
-12mV
+18mV
3210
synaptic Q
Ca
(pC)
-32mV
-27mV
-22mV
-12mV
+18mV
1.0
0.5
0.0
G
/
G
max
-50 0
V
m
(mV)
Bsn
wt
Bsn
ΔEx4/5
-32mV
-27mV
+18mV
-22mV
-12mV
Figure 5. Reduced Exocytosis but Normal Ca
2+
Influx-Exocytosis Coupling
(A) Representative membrane capacitance changes (DC
m
) and Ca
2+
currents (I
Ca
) in response to 20 ms depolarizations to peak-I
Ca
V
m
.
(B) Same as (A), but in response to 100 ms depolarizations. See Table 1 for pooled data (A and B).
(C) Average DC
m
responses recorded during flash photolysis of caged Ca
2+
. Dataset comprises IHCs (n [Bsn
wt
]=4,n[Bsn
DEx4/5
] = 6) with comparable postflash
[Ca
2+
]
i
, (range: 25–37 m M; Table S1); 0 ms indicates time of UV flash delivery.
(D) Normalized conductance (G)-voltage relationships for both genotypes.
(E1) Summary of exocytic DC
m
responses to 20 ms depolarizations to the five test potentials depicted in (D) plotted versus the corresponding mean Ca
2+
current
integrals (Q
Ca
). Pulses were applied to 21 Bsn
wt
IHCs and 23 Bsn
DEx4/5
IHCs in random order at intervals of > 30 s. Note the larger responses at +18 mV compared
to 27 mV, despite similar Q
Ca
, coinciding with a larger p
open
of Ca
2+
channels (see D). (E2) To compare the relation between p
open
and the efficiency of synaptic
vesicle release in the two genotypes, we applied 3 transformations to the plot shown in (E1): (1) assuming a certain extrasynaptic N
Ca
(Brandt et al., 2005), we
estimated the fraction of synaptic Ca
2+
channels (out of total N
Ca
; Table 1), and multiplied Q
Ca
by the respective ratio (<1) to estimate ‘synaptic Q
Ca
.’ (2) We then
doubled the mutant DC
m
data to account for the halving of membrane-proximal synaptic vesicles seen at mutant AZs (Figure 1). (3) Last, we accounted for the
apparently reduced number of synaptic Ca
2+
channels at mutant AZs by multiplying mutant Q
Ca
by 1/0.52 (assuming that the Ca
2+
microdomain amplitude in the
presence of BayK8644 presents the most reliable reflection of synaptic N
Ca
; Table 1).
(F) Ratio of exocytic responses (20 ms and 100 ms depolarizations to peak-I
Ca
V
m
) between perforated-patch (endogenous Ca
2+
buffers) and ruptured-patch
([EGTA]
i
= 5 mM) configurations for Bsn
wt
and Bsn
DEx4/5
IHCs (Table 1).
The error bars in (C, E, and F) represent SEM.
NEURON 10444
Neuron
Bassoon Organizes Hair Cell Active Zones
Neuron 68, 1–15, November 18, 2010 ª2010 Elsevier Inc. 9
Please cite this article in press as: Frank et al., Bassoon and the Synaptic Ribbon Organize Ca
2+
Channels and Vesicles to Add Release Sites and
Promote Refilling, Neuron (2010), doi:10.1016/j.neuron.2010.10.027
The observation that the reduction of Ca
2+
-influx-triggered
exocytosis did not exceed the reduction in the number of
membrane-proximal and -tethered vesicles (Figure 1) suggests
that the remaining docking sites are equipped with nearby Ca
2+
channels (reduction of synaptic Ca
2+
channels: 50%, Table 1;
estimated from Bsn
DEx4/5
versus Bsn
wt
synaptic Ca
2+
microdo-
main amplitude in the presence of BayK8644). Yet, a looser
coupling between Ca
2+
channels and vesicle docking sites
than implied for the Ca
2+
nanodomain regime suggested for
wild-type IHC AZs could not be excluded (Brandt et al., 2005;
Goutman and Glowatzki, 2007; Moser et al., 2006 ). Therefore,
we studied the sensitivity of exocytosis to the slow Ca
2+
chelator EGTA (Figure 5F). Consistent with the preservation of
nanodomain-controlled vesicle fusion in Bsn
DEx4/5
IHCs, their
DC
m, 20 ms
in the presence of 5 mM [EGTA]
i
was reduced to
58% of control levels (Table 1)—closely resembling the reduction
in the presence of endogenous Ca
2+
buffers (see above). Addi-
tionally, we probed RRP exocytosis as a function of Ca
2+
influx
at different membrane potentials (Figures 5D and 5E). Changing
the membrane potential manipulates open probability and
single-channel current in opposite directions. Thus, exocytosis
can be tested for the same absolute Ca
2+
influx through either
few open channels with high single-channel current (mild depo-
larizations) or more open channels with low single-channel
current (strong depolarizations). If exocytosis of a given vesicle
was under control of a population of several Ca
2+
channels
(Ca
2+
microdomain control), exocytosis should be identical for
the same Ca
2+
current independent of the membrane potential.
In case of a Ca
2+
nanodomain control, more exocytosis is
expected for more open Ca
2+
channels, i.e., at more depolarized
potentials (hysteresis; Zucker and Fogelson, 1986). This was
indeed observed in Bsn
wt
IHCs (Figure 5E1 and Figure S5), as
described before (Brandt et al., 2005), but also in Bsn
DEx4/5
IHCs (Figure 5E1 and Figure S5), further arguing that Ca
2+
nano-
domain control of exocytosis is maintained at mutant AZs. As
a further consistency check, we scaled the exocytosis-Ca
2+
current integral relationship of Bsn
DEx4/5
IHCs by experimentally
derived factors to normalize the data to the lower number of
membrane-proximal vesicles and synaptic Ca
2+
channels. This
resulted largely in an overlap with the wild-type data (Figure 5E2).
In summary, the data indicate that the coupling of Ca
2+
channels
to release sites remains intact despite Bassoon disruption but
that the rates of initial and sustained exocytosis are reduced to
a similar extent as the number of membrane-proximal vesicles.
In Vitro and In Vivo Analysis of Synaptic Vesicle
Replenishment
Traditionally, the synaptic ribbon has been assigned a conveyor
belt and/or attractor function (Holt et al., 2004; Sterling and
Matthews, 2005 ), according to which it is responsible for rapid
supply of vesicles to the RRP and enables high rates of tonic
neurotransmitter release (Gomis et al., 1999; Johnson et al.,
2008; Moser and Beutner, 2000; Rutherford and Roberts,
2006; Schnee et al., 2005; Spassova et al., 2004). Hence, we
tested whether the rate of RRP refilling was reduced in the
absence of the ribbon and functional Bassoon protein. Here,
we explored vesicle replenishment in vitro by measuring relative
DC
m
in paired-pulse protocols, with the stimuli (20 ms or
100 ms long depolarizations) being separated by various
time intervals (98, 198, and 398 ms; Figure 6). The ratio of Ca
2+
current integrals was close to one in both genotypes (marginally
smaller in Bsn
DEx4/5
IHCs; Figures 6C and 6D and Table S1) indi-
cating that the Ca
2+
signals that drive exocytosis were mostly
comparable between both pulses. For 20 ms stimuli at short
inter-pulse-intervals (IPI: 98 ms) we observed stronger depres-
sion of the exocytic response in Bsn
DEx4/5
IHCs, indicating
a slower recovery of the RRP at Bsn
DEx4/5
synapses (p < 0.01).
For longer recovery times (IPI: 198, 398 ms), the difference
did not reach statistical significance. While both Bsn
wt
and
Bsn
DEx4/5
IHCs showed depression for short stimuli, Bsn
wt
IHCs exhibited a tendency toward facilitation for long depolariza-
tions (100 ms). In contrast, Bsn
DEx4/5
IHCs also showed depres-
sion when challenged with long stimuli (p < 0.01 for 98, 198, and
398 ms IPI).
**
****
**
*
**
**
*
**
20 ms 100 ms
A
CD
B
1.0
0.5
0.0
Δ
C
m
2
/ ΔC
m1
0.1
2 4 6 8
1
2 4 6 8
10
2
Inter-pulse-interval (s)
1.0
0.9
Q
2
/Q
1
20 ms
Bsn
wt
Bsn
Δ Ex4/5
1.0
0.5
0.0
Δ
C
m2
/
ΔC
m1
0.1
2 4 6 8
1
2 4 6 8
10
2
Inter-pulse-interval (s)
1.0
0.9
Q
2
/Q
1
100 ms
Bsn
wt
Bsn
Δ Ex4/5
-500
0
I
Ca
(pA)
40
20
0
Δ
C
m
(fF)
100 ms
Bsn
wt
(98 ms)
Bsn
Δ Ex4/5
(98 ms)
-400
-200
0
I
Ca
(p
A)
100
50
0
ΔC
m
(
f
F
)
100 ms
Bsn
wt
(98 ms)
Bsn
Δ Ex4/5
(98 ms)
Figure 6. Slowed Vesicle Replenishment
Kinetics
(A) Example DC
m
responses and Ca
2+
currents
(I
Ca
) from Bsn
wt
and Bsn
DEx4/5
IHCs upon two
20 ms depolarizations to maximum I
Ca
potential,
separated by 98 ms.
(B) Same as (A) but with 100 ms depolarizations.
(C) Summary of paired-pulse DC
m
recordings
following 20 ms depolarizations. The graph shows
the ratio of response magnitudes between the
second and the first pulse (DC
m2
/DC
m1
) for
different inter-pulse-intervals. Note the depression
in both genotypes, which is, however, more
pronounced in Bsn
DEx4/5
IHCs (p < 0.01 for IPI of
98 ms; n [Bsn
wt
]: 23 to 32 IHCs; n [Bsn
DEx4/5
]: 20
to 32 IHCs).
(D) Same as (C) but for 100 ms depolarizations.
Note the slight facilitation in Bsn
wt
IHCs, but
consistent depression in Bsn
DEx4/5
IHCs for short
IPIs, respectively (p < 0.01 for IPI of 98, 198, and
398 ms; n [Bsn
wt
] = 22 to 35