1
Supplementary Table 1: Supporting Morphological Results
WTEM
MUTEM
WTLM
MUTLM
numIHCs
109
110
80
60
numafferent synapses
250
210
909
701
fractionribbon-containing synapses
(%)
lengthpostsynaptic density (nm)
42.4
0.4
91.3
12.4
746 ± 26
823 ± 57
-
-
(n = 42)
(n = 20)
230 ± 9
-
255
-
-
332
-
-
200
-
-
-
-
3.0 ± 0.2
1.9 ± 0.3
-
-
(n = 33)
(n = 18)
-
-
heightribbon/4Pi RIBEYE spot (nm)
(n = 42)
lengthribbon/4Pi RIBEYE spot (nm)
278 ± 18
(n=9)
widthribbon/4Pi RIBEYE spot (nm)
55 ± 8
(n=9)
numribbon-associated SV in 2D
14.2 ± 0.5
(n = 33)
numdocked SV in 2D
diameterSV
(nm)
distanceribbon-SV
(nm)
30.0 ± 0.3
27.8 ± 0.3
(n = 206, cv =
15.2)
(n = 202, cv =
13.7)
26.2 ± 0.6
-
-
-
-
260 ± 4 (n = 201)
220 ± 20
(n = 195)
sizesmall RIBEYE spots, 4Pi
-
(n = 15)
(nm)
sizebig RIBEYE spots, 4Pi
(nm)
(nm2)
surface
numribbon-associated SV
3D
numdocked SV 3D
RRPsynapse (vesicles/synapse)
763 ± 12
1273 ± 70
(n = 38)
(n = 28)
-
ellipsoid:~3.82e5
-
~125
-
sphere:~3.79e5
ellipsoid:~203
-
~16
-
ellipsoid:~30
-
53-64 ribbon-
~58ribbon-contain.(1)
-
-
~2.38e5
-
containing (10)
~14ribbon-deficient(11)
(conversion factor: Cm/SV)
(28 aF)
(24 aF)
RRPIHC
15-18
5
(fF)
Table presents morphological findings obtained on WT and mutant IHCs of 8week-old mice. In the electron microscopy of 7 WT (WTEM) and 6 mutant
(MUTEM) mice synapses (numsynapses) were identified as contacts of IHC
(numIHCs) and dendrite displaying pre- and postsynaptic densities in ultrathin
sections. One cochlea of each of the 7 WT and 6 mutant mice was processed.
In our confocal analysis of 7 WT (WTLM) and 5 mutant (MUTLM) mice numIHCs
2
and numsynapses represent the total counts of CtBP2-positive IHC nuclei and
GluR immunofluorescent spots, respectively, which we observed in animated
3D reconstructions of organs of Corti (see Supplementary Movies 1a and 1b).
The number of RIBEYE-juxtaposed GluR spots was related to numsynapses to
yield the fraction of ribbon containing synapses. RIBEYE-juxtaposed GluR
spots were identified, when we found signals to be in contact by visual
inspection. Counting revealed a strong reduction of ribbon-containing
synapses in electron microscopy (along with abundance of floating ribbons
and accumulation of cisterns) and light microscopy, allowing 100% correct
genotype prediction. We captured many more synapses per investigated IHC
and also obtained a much larger fraction of ribbon containing synapses in
both WT and mutant IHCs in the confocal analysis. We suspect an
overestimation of mutant ribbon-containing synapses due to insufficient
lightmicroscopical separation of some close but not synaptically related ribbon
and GluR signals in our confocal analysis (e.g. floating ribbons of Fig. 1c). On
the other hand, we probably missed a significant number of anchored ribbons
in electron microscopy mainly because, depending on its orientation with
respect to the cutting axis, the ribbon may span only a single section and the
series of thin sections was often not complete. Therefore, electron microscopy
and confocal measurements provide lower and upper estimates of the fraction
of ribbon-containing synapses.
In order to relate our functional results to synapse morphology we
semiquantitatively analysed 42 WT and 20 mutant synapses in single ultrathin
electron microscopy sections and performed 4Pi microscopy on RIBEYE
stained apical organs of Corti from WT and mutant mice (Fig. 1i-j). From
electron microscopic observations we conclude that the average ribbon of
mature mouse IHC takes an ellipsoid shape. Only ribbons with a
perpendicular orientation with respect to a sharply delimited postsynaptic
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density were analysed. The maximal extensions along the vertical and
horizontal axes were taken as height and ‘width’ estimates. Due to random 2D
orientation of ribbons in the sections ‘width’ estimates ranged between 25 and
354 nm (data not shown). For a rough approximation of ribbon surface from
the electron microscopy data we averaged the lowest 20% of this distribution
to yield an apparent width (“widthribbon“) and the highest 20% groups for an
apparent length (“lengthribbon”). The 4Pi estimates of the three principal axes
were obtained by approximating the major peak of the WT RIBEYE size
distribution by a model that assumed an ellipsoid shape and random
orientation of the ribbon with respect to the optical axis (Fig. 1k,
Supplementary Methods).
The number of ribbon-associated synaptic vesicles in 2D sections
included all vesicles within 30 nm distance from the ribbon. Docked synaptic
vesicles touched the presynaptic membrane and were dominated by ribbonassociated synaptic vesicles (~ 2/3) in WT synapses. The total number of
docked synaptic vesicles in EM was roughly approximated based on the 2D
count of 3 docked synaptic vesicles, assuming a hexagonal packing with a
55% density along the ribbons length plus 3 synaptic vesicles at each end of
the ribbon. Although we cannot rule out that our fixation paradigm caused
transmitter release and subsequent synaptic vesicles depletion, we do not
favour this hypothesis, because different from strongly stimulated ribbons1,2
our WT IHC ribbons were densely populated with synaptic vesicles also at
their base. In fact, our EM estimate of docked synaptic vesicles probably
represents an upper estimate rather than an underestimate, since the 2D
estimate of ribbon-associated synaptic vesicles lumped together slim and
wide ribbon cross-sections. Synaptic vesicle diameters were measured from
the lipid bilayer’s centres and synaptic vesicle capacitances calculated
assuming a specific membrane capacitance of 10 fF/µm 2 (ref. 3). We did not
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correct for shrinkage due chemical fixation. Because of shrinkage we may
have underestimated the vesicle size and hence further overestimated the
number of docked and ribbon-associated vesicles. For a rough approximation
of the number of ribbon-associated synaptic vesicles we calculated the
surface around the ribbon that is available for vesicle packing based on EM
and 4Pi ribbon shape estimates, the average distance of the vesicle from the
ribbon (distanceribbon-SV) and the outer synaptic vesicle radius (17.5 nm). We
assumed hexagonal packing of synaptic vesicles (1039 nm 2 hexagons) within
this surface at a density of 55%1,2. To estimate the number of ribbonassociated vesicles from the 4Pi data with the least assumptions we also
calculated a spherical packing surface around the mean thickness of the small
RIBEYE spots, which gave nearly identical results. The 4Pi estimate of
docked synaptic vesicles number relied on the assumption that docked
ribbon-associated synaptic vesicles made up ~ 10% of all ribbon-associated
vesicles as described for frog saccular hair cells1,2. We multiplied this estimate
by 1.5 to roughly account for outlying vesicles (see above). The RRP of
synaptic vesicles per synapse (RRPsynapse ) was estimated from exocytic Cm
of Fig. 4, converted into vesicles numbers and related to the average number
of synapse per IHC (Results section). For mutant IHCs we considered the
remaining ribbon-containing synapse to hold as many synaptic vesicles as the
average wt synapse (58 synaptic vesicles). The other synaptic vesicles were
attributed to the ~ 11 ribbon-deficient synapses.
1.
2.
3.
Lenzi, D., Runyeon, J. W., Crum, J., Ellisman, M. H. & Roberts, W. M.
Synaptic vesicle populations in saccular hair cells reconstructed by electron
tomography. J. Neurosci. 19, 119-132 (1999).
Lenzi, D., Crum, J., Ellisman, M. H. & Roberts, W. M. Depolarization
redistributes synaptic membrane and creates a gradient of vesicles on the
synaptic body at a ribbon synapse. Neuron 36, 649-59. (2002).
Breckenridge, L. J. & Almers, W. Final steps in exocytosis observed in a cell
with giant secretory granules. Proc Natl Acad Sci U S A 84, 1945-9 (1987).
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