synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) ... at the active zone the ribbon may support MVR, occurring through various .... In the WT the average vesicle diameter was 33.6 ± 3 nm (n = 21) and in the. 148 ..... Intracellular Ca2+ buffering can alter release properties depending on the relative.
1
The presynaptic ribbon maintains vesicle populations at the hair cell afferent fiber
2
synapse
3
Lars Becker1, Michael E. Schnee1, Mamiko Niwa1*, Willy Sun2, Stephan Maxeiner3**,
4
Sara Talaei1, Bechara Kachar2, Mark A. Rutherford4, Anthony J. Ricci1,3
5
1 Department of Otolaryngology Stanford University, United States, 2 National Institute
6
of Deafness and Communicative Disorders, United States, 3 Molecular and Cellular
7
Physiology, Stanford University, United States, 4 Department of Otolaryngology,
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Washington University in St. Louis, United States,
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*Present address: Department of Otolaryngology Johns Hopkins University, United
10
States
11
** Present address: Department of Neuroanatomy, Institute for Anatomy and Cell
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Biology, Medical School Saarland University, Germany
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15
16
17
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1
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Abstract: The ribbon is the structural hallmark of cochlear inner hair cell (IHC) afferent
21
synapses, yet its role in information transfer to spiral ganglion neurons (SGNs) remains
22
unclear. We investigated the ribbon’s contribution to IHC synapse formation and
23
function using KO mice lacking RIBEYE. Despite loss of the entire ribbon structure,
24
synapses retained their spatiotemporal development and KO mice had a mild hearing
25
deficit. IHCs of KO had fewer synaptic vesicles and reduced exocytosis in response to
26
brief depolarization; high stimulus level rescued exocytosis in KO. SGNs exhibited a
27
lack of sustained excitatory postsynaptic currents (EPSCs). We observed larger
28
postsynaptic glutamate receptor plaques, potentially in compensation for the reduced
29
EPSC rate in KO. Surprisingly, large amplitude EPSCs were maintained in KO, while a
30
small population of low amplitude slower EPSCs was increased in number. The ribbon
31
facilitates signal transduction at physiological stimulus levels by retaining a larger
32
residency pool of synaptic vesicles.
33
34
35
Key words: auditory hair cell, synaptic transmission, RIBEYE, glutamate receptor,
36
multivesicular release
37
38
39
40
2
41
Introduction
42
The synaptic ribbon is an electron dense structure associated with presynaptic active
43
zones at sensory synapses in the retina, lateral line and inner ear organs. RIBEYE, the
44
major protein associated with the ribbon (Zanazzi and Matthews 2009), consists of a
45
unique amino-terminal A domain that forms the ribbon scaffold and a carboxy-terminal B
46
domain with lysophosphatidic acid acyltransferase activity which is almost identical to
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CtBP2, a transcriptional co-repressor (Schmitz, Konigstorfer et al. 2000). The ribbon is
48
anchored to the presynaptic membrane by bassoon protein in mammalian IHCs
49
(Khimich, Nouvian et al. 2005) as well as retinal photoreceptors and bipolar cells (dick
50
2001). Although it is comprised predominantly of RIBEYE (Zenisek, Horst et al. 2004),
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the ribbon complex may associate with over 30 different synaptic proteins (Uthaiah and
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Hudspeth 2010, Kantardzhieva, Peppi et al. 2012).
53
IHCs respond to sound with graded receptor potentials that drive rapid and precise
54
synaptic release, encoding intensity and timing information (Fuchs 2005, Matthews and
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Fuchs 2010). The ribbon is thought to sustain continuous encoding by tethering a pool
56
of vesicles that replenishes the local pool of docked and primed vesicles upon
57
depletion. Other functions ascribed to the ribbon include: regulating vesicle trafficking
58
from the cytoplasm to the active zone, serving as a vesicle trap to bind freely diffusing
59
vesicles, a location for vesicle formation, a physical barrier for Ca2+ ensuring a high
60
local Ca2+ signal, and as a primary structural component of the synapse anchoring Ca2+
61
channels close to synaptic vesicle release sites (Parsons 1994, Frank, Rutherford et al.
62
2010, Graydon, Cho et al. 2011, Kantardzhieva, Peppi et al. 2012).
3
63
Postsynaptic EPSCs at ribbon synapses range in amplitude by up to 20X. The smallest
64
and largest EPSCs have similar kinetics, which together suggested the existence of a
65
mechanism to synchronize the release of multiple quanta (multivesicular release, MVR;
66
(Glowatzki and Fuchs 2002, Li, Keen et al. 2009, Schnee, Castellano-Munoz et al.
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2013, Rudolph, Tsai et al. 2015). By holding vesicles and voltage-gated Ca2+ channels
68
at the active zone the ribbon may support MVR, occurring through various mechanisms
69
including: i) coordinated fusion of multiple single vesicles, ii) release of large pre-fused
70
vesicles, iii) rapid sequential fusion in which the 1st fusion event triggers fusion of
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additional vesicles, or some combination of these mechanisms (Matthews and Fuchs
72
2010).
73
Previous attempts to study ribbon function used genetic disruption of the anchoring
74
protein Bassoon or acute destruction of the ribbon by fluorophore assisted light
75
inactivation (FALI). These manipulations resulted in smaller vesicle pool size, calcium
76
channel mislocalization, increased auditory thresholds, asynchrony in neural responses
77
and reductions in both tonic and phasic responses (Snellman, Mehta et al. 2011, Jing,
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Rutherford et al. 2013). However, neither of those manipulations were selective for the
79
ribbon and so might be manifestations of accessory protein denaturing.
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Recently, manipulation of the gene encoding CtBP2/RIBEYE allowed for deletion of
81
RIBEYE by removing the A-domain of ctbp2 to eliminate ribbons in mice, enabling a
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more precise examination of ribbon function. This mouse will be referred to as KO and
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the littermate controls as WT throughout the manuscript. Elimination of ribbons in the
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retina reduced phasic and tonic transmission and rendered release sites sensitive to the
85
slow calcium buffer EGTA (Maxeiner, Luo et al. 2016). Here we present analysis of 4
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cochlear synaptic anatomy, hair cell and afferent fiber physiology and hearing function
87
in the RIBEYE knockout mouse. Results are distinct from those in retina and from a
88
RIBEYE deletion in zebrafish (Lv, Stewart et al. 2016) , suggesting a more subtle
89
function for the ribbon than previously assumed.
90
Results:
91
IHC-Spiral Ganglia Neuron (SGN) synapses are formed and maintained despite
92
absence of the ribbon: The inner ear structure of the KO was assessed first for the
93
presence of synaptic ribbons and synapses (Maxeiner, Luo et al. 2016). Validating loss
94
of hair cell ribbons was accomplished using immunohistochemistry for the RIBEYE B-
95
domain (CtBP2). Anti-CtBP2 labelled the ribbons in wildtype (WT) but not in the KO
96
animals (postnatal day 21, P21)(Fig. 1A). Nuclear CtBP2 was still observed in both WT
97
and KO (not shown) because the KO is specific to the RIBEYE A-domain. The nuclear
98
staining served as a usful control for antibody function.
99
Using antibodies to Homer for postsynaptic labeling and Bassoon for presynaptic
100
labeling (Fig. 1B,C) we quantified the number of puncta and whether puncta were
101
juxtaposed as synaptic pairs (Fig. 1I,J). Bassoon and Homer puncta were similar in
102
number between WT and KO (Bassoon: 40 ± 15 in WT, vs. 43 ± 12 in KO, p = 0.76;
103
Homer: 17 ± 3 for both WT and KO; WT: n = 20 mice, KO: n = 19 mice, p = 0.64). The
104
number of paired Homer and Bassoon puncta (Fig. 1D) were similarly comparable:
105
14 ± 2 (n = 20) for WT and 13 ± 2 (n = 19, p = 0.18) for KO IHCs, suggesting that
106
synapses were formed and maintained in comparable numbers regardless of the
107
presence of ribbons. The higher number of Bassoon puncta likely reflects its localization
108
at efferent synapses. Figure 1- Supplemental Fig. 1 presents a lower magnification 5
109
image that illustrates the multiple roles of Bassoon at the auditory periphery. Bassoon is
110
a major component of the presynaptic elements at the auditory periphery including the
111
efferent fibers and the hair cell afferent synapses. Thus it is not surprising that paired
112
Bassoon puncta are much less than total puncta because Bassoon serves multiple
113
synapses. Similarly, when interpreting Bassoon KO data it is not surprising that its
114
phenotype may be more dramatic than simply the RIBEYE effect alone due to number
115
of synapses involved and schaffolding function of Bassoon that leads to structural
116
changes at the synapse.
117
RIBEYE KO in zebrafish revealed a non osmiophilic structure resembling a ribbon that
118
maintained organization of synaptic vesicles in the absence of the RIBEYE protein in
119
hair cells of the lateral line neuromasts (Lv, Stewart et al. 2016). We examined whether
120
such a structure existed in the mammalian cochlea in the IHCs of the KO using
121
transmission electron microscopy (TEM) and electron-tomography. Representative
122
examples of conventional TEM (Fig. 1E, 70 nm sections) and tomographic
123
reconstructions (Fig. 1F,G at 10 nm tomographic sections) are presented. The KO
124
presynaptic synapses consistently showed a small osmiophilic density in contact with
125
the pre-synaptic membrane at the center of the synapse that resembled the arciform
126
density seen at ribbon synapses in the retina. This density may be the Bassoon
127
enriched linker between the membrane and the ribbon. Interestingly, this density is not
128
observed in the Bassoon KO mouse (Frank et al., 2010). Tomographic reconstructions,
129
(Fig. 1G, right) clearly show the presynaptic density, which is smaller than the ribbon
130
and is present also at ribbon synapses in WT IHCs (Fig. 1G, left). We found no spatial
131
organization of vesicles near active zones in KO IHCs, suggestive of the ‘ghost’ ribbons
6
132
observed in the zebrafish lateral-line hair cells (Lv, Stewart et al. 2016). Thus, in the KO
133
mouse, IHCs lack synaptic ribbons and have no obvious compensatory structures. We
134
surmise that the differences observed in the zebrafish model are a function of the KO
135
being incomplete or there being a compensation mechanism in zebrafish, not found in
136
mammal.
137
KO IHCs may upregulate a similar protein to replace RIBEYE loss. CtBP1 is a likely
138
candidate because it is similar to the RIBEYE-B domain, is expressed in hair cells and
139
localizes to the synaptic region (Kantardzhieva, Liberman et al. 2013). Antibody labeling
140
(Fig. 1H,K,L) demonstrates the presence of CtBP1 in both WT and KO animals which
141
overlapped with Homer in WT. A reduction in puncta number was identified in the KO
142
animal due to loss of localization at the afferent synapses (Fig. 1K,L). Thus, the CtBP1
143
localization at the synapse requires the presence of RIBEYE, and CtBP1 does not
144
substitute for RIBEYE in the KO animal.
145
Synaptic vesicles are normal in size but fewer in number:
Vesicle diameters
146
(distance between the outer edges of the bilayer in each vesicle) were measured in the
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reconstructed tomograms. We measured in both X and Y directions with no differences
148
observed. In the WT the average vesicle diameter was 33.6 ± 3 nm (n = 21) and in the
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KO it was 33.8 ± 3 nm (n = 21).
150
Synaptic vesicles were conspicuously fewer in number the KO. Vesicles were counted
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in volumes around the center of the reconstructed synapses (Fig. 1G), where one
152
boundary was the presynaptic membrane. The volumes had XY dimensions of 453 ± 6
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nm for WT (n = 3) and 569 ± 117 nm for KO (n = 11), and Z dimensions of 88 ± 3 and
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95 ± 10 nm for WT and KO, respectively. Total vesicle numbers were 20 ± 2 for WT and 7
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7 ± 3 for KO, significantly greater for WT compared with KO synapses (p < 0.001, Welch
156
correction for unequal variance). Our TEM data is qualitatively similar to that reported in
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the companion paper and supports the conclusion that ribbons are not present in the
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KO.
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Auditory nerve activity is reduced in absence of synaptic ribbons: Previous
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investigation of ribbonless synapses in the KO mouse focused on the retina and did not
161
assess function at the systems level (Maxeiner et al., 2016). To determine how hearing
162
was affected, we measured ABRs and distortion product otoacoustic emissions
163
(DPOAEs). DPOAEs are a direct measure of outer hair cell function and cochlear
164
amplification. DPOAEs are not expected to be affected by absence of RIBEYE and
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serve as a control for developmental acquisition of mature cochlear mechanics. DPOAE
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threshold measurements were not different across frequencies from 4 - 46 kHz (Fig.
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2A). Data are presented in decibels (dB) as referenced to sound pressure level (SPL)
168
with lower values indicating enhanced sensitivity in the mid-cochlea. Normal DPOAEs
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indicate that cochlear amplification was not effected mechanically by the loss of outer
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hair cell ribbons, suggesting that sound-driven activation of IHCs was intact, and that
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any differences in auditory nerve activity should result from changes at the IHC afferent
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synapses.
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Wave 1 of the ABR is a direct measurement of the synchronized firing of SGNs at
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sound onset, triggered by synaptic output from cochlear IHCs. The subsequent waves
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of the ABR reflect the ensuing activation of brainstem nuclei in the ascending auditory
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pathway (Zheng, Henderson et al. 1999). ABR thresholds were elevated by
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approximately 10 dB at all probe frequencies in KO mice (Fig. 2B), except for 48 kHz 8
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where hearing threholds are already relatively high. The variance in our control data
179
ranged from 8-12dB depending on frequency; this dictated the sample size needed to
180
detect a 5% change as statistically different. ABR threshold was identified as the
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stimulus intensity required to evoke a voltage response 5x the RMS noise floor for the
182
measurement. The companion paper found a similar elevation in threshold though it did
183
not reach statistical significance. The difference is likely due to sampling size where we
184
powered our sample sized based on variance found in control measurements. The ABR
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waveform (Fig. 2C: 23 kHz, 60 dB) shows a large reduction in wave 1 amplitude with no
186
effect on the subsequent peaks. This was surprising and suggests considerable
187
plasticity in central processing of data translated from cochlea periphery. KO mice had
188
smaller wave 1 peak amplitudes, for all sound levels that evoked a response (Fig. 2D).
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When wave 1 growth functions were plotted relative to the maximal response amplitude
190
within genotype, the amplitude versus level function for KO scaled up to overlap with
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WT (Fig. 2E). Considering wave 1 amplitude as a fraction of the maximal amplitude, the
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overlap of the normalized wave 1 growth curves suggested that loss of maximum output
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could produce an elevation of threshold in the ABR measurement that does not
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necessarily reflect a sound detection limit or a change in sensitivity of the auditory
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system. Indeed, normal ABR amplitudes beyond wave 1 indicate compensation in the
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cochlear nucleus. Future work should determine if KO mice have increased behavioral
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thresholds.
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In mice lacking Bassoon function, where IHCs lacked most of their membrane-anchored
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ribbons, there was an increase in wave 1 latency (Khimich, Nouvian et al. 2005) and in
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first-spike latency in recordings of SGNs in vivo (Buran, Strenzke et al. 2010). ABR
9
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wave 1 latencies were not different between WT and KO at any stimulus intensity (Fig.
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2F), suggesting a milder phenotype in the KO as compared to Bassoon.
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As the ABR measurements are averaged responses of 260 stimulus repetitions, a
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possible explanation for the reduced ABR wave 1 amplitude in KO is that the response
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reduces over time. If the ribbon is providing a means of maintaining vesicles near the
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synapse, the concomitant loss of vesicles at ribbonless synapses might reduce the
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ongoing response to repetitive stimulation. Comparison of ABR amplitudes where the
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number of repeats was varied from 260 to 1000 showed no difference in amplitudes, nor
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did repeated measurements meant to evoke depletion.Thus, rundown alone is unlikely
210
to account for the decrease in ABR wave 1.
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Characterization of molecular synaptic components indicates an increase in distribution: To begin identifying cellular
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postsynaptic glutamate receptor
213
mechanisms that might underlie the measured reduction in ABR wave 1 amplitude we
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investigated the distribution of presynaptic Ca2+ channels and postsynaptic glutamate
215
receptors as indicators of functional synapses. Figure 3 presents immunohistochemical
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localizations of presynaptic ribbons with anti-CtBP2, presynaptic voltage-gated Ca2+
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channels with anti-CaV1.3, and postsynaptic AMPA-type glutamate receptors with anti-
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GluA3 in P35 mice organ of corti. There was no difference in the number of puncta
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between WT and KO for CaV1.3 (20 ± 6, n = 9 vs. 18 ± 5, n = 13, p = 0.51) or GluA3 (15
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± 3, n = 9 vs. 15 ± 2, n = 13, p = 0.43) (Fig. 4A,B). There was also no difference in the
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number of paired synaptic puncta between genotypes, with values of 14 ± 2 (n = 9) for
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WT and 13 ± 2 (n = 13) for KO (p = 0.13). Thus, the presence of the ribbon did not
223
influence the number of apparently functional synapses per IHC. 10
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Careful inspection of GluA3 immunoreactivity showed that the glutamate receptor
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puncta were larger on average in the KO (Fig. 4D). Puncta volumes were measured for
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presynaptic markers CaV1.3 and Bassoon as well as postsynaptic markers Homer and
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GluA3 (Fig. 4C). Like GluA3, Homer puncta were significantly larger (KS-test,
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p ≤ 0.0001) on average in the KO (Fig. 4D). The volumes of CaV1.3 and Bassoon
229
distributions were very similar among genotypes however the volume of Bassoon was
230
significantly higher in KO. Most WT and KO synapses exhibited the typical elongated
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stripe-like appearance of CaV1.3 puncta. However, we noted a tendency for KO
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synapses to have CaV1.3 clusters that were fragmented into smaller puncta (Fig. 3C,
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arrow; see also Companion Paper). These anatomical data support the hypothesis that
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the ribbon is not a major regulator of synapse formation or stability; however, it does
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perhaps suggest that postsynaptic receptors are sensitive to activity and the increased
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plaque size might reflect a compensation for reduced activity.
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Basolateral IHC properties: We compared the properties of the IHC voltage-gated
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Ca2+ current (Fig. 5), which drives exocytosis. We first blocked outward K+ currents
239
using Cs+ and tetraethylammonium (TEA) in the internal solution to isolate Ca2+ currents
240
in WT and KO IHCs (Fig. 5A,B). Current-voltage plots showed no difference in voltage
241
of half-activation (V1/2) or maximum current amplitude (Fig.5C-E). Half activation
242
voltages were -34 ± 0.3 mV (n = 18, WT) and -33.4 ± 0.3 mV (n = 26, KO) for animals
243
P10-P13. The companion paper found small changes in voltage dependent properties
244
of the calcium channels at older ages and in higher external calcium; differences that
245
might suggest compensatory effects of ribbon loss or later specializations being ribbon
246
dependent. Examples of current elicited with K+ as the major internal cation and without
11
247
TEA are presented in Fig. 5F,G. Recordings were made at P20-24. No differences were
248
observed in maximal current or in V1/2 (Fig. 5H). Zero-current potentials monitored in
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current-clamp mode (Fig. 5I) were not different, with means of -63 ± 10 mV (n = 5, WT)
250
and -65 ± 8 mV (n = 8, KO).
251
Release is slower and smaller for naturalistic stimuli in absence of the ribbon: We
252
measured presynaptic exocytosis with the two-sine wave method for tracking vesicular
253
fusion by monitoring real time changes in cellular membrane capacitance (Cm) from
254
IHCs (Santos-Sacchi 2004, Schnee, Castellano-Munoz et al. 2011). The two-sine
255
method allows continuous monitoring of Cm during the depolarizing stimulus as
256
compared to a before and after monitoring of capacitance changes, which is particularly
257
helpful during smaller, longer depolarizations (Schnee, Santos-Sacchi et al. 2011).
258
Typically, small depolarizations lead to relatively slow, linear increases in Cm during
259
depolarization. In contrast, larger depolarizations induce an abrupt departure from
260
linearity termed the ‘superlinear’ response (Schnee et al. 2011). Both types of
261
responses were present in recordings from IHCs of WT and KO mice alike (Fig. 6A,B).
262
From Cm measurements, we estimated the sizes of synaptic vesicle pools, their rates of
263
release and the Ca2+-dependence of release. In response to strong depolarizations, we
264
found no difference in maximal release between genotypes for either the linear or
265
superlinear release components (Fig. 6C). The linear component was 52 ± 24 fF (n =
266
15, WT) compared to 42 ± 22 fF (n = 15, KO); and the superlinear component of release
267
was 223 ± 79 fF (n = 17, WT) compared to 253 ± 116 fF (n = 17, KO). Likewise, no
268
differences in release rates (Fig. 6D) were observed for the linear component: 101 ± 72
269
fF/s (n = 15, WT) compared to 75 ± 50 fF/s (n = 14, KO) or for the superlinear 12
270
component: 256 ± 164 fF/s (n = 17, WT) and 253 ± 117 fF/s (n = 18, KO). In addition,
271
the Ca2+-dependence of release for the linear component was not different (0.9 ± 0.03
272
fF/pC (n = 37, WT) as compared to 0.93 ± 0.03 fF/pC (n = 34, KO). The voltage at which
273
the superlinear component of release first appears was also not different between mice
274
(Fig. 6E). Fitting the data with a simple Gaussian function gave values of -40 ± 9 mV for
275
WT and -41 ± 3 mV for KO for the center values. However the width of the functions
276
were different, with values of 7.6 ± 2 for WT compared to 14.3 ± 3 for KO (values p
-
299
39 mV) resulted in no difference in release latency. The time constants of endocytosis
300
were not significantly different (6.0 ± 4s, n = 13, WT and 7 ± 5 s, n = 13, KO). Together
301
these data suggest that fewer vesicles were available for release at ribbonless
302
synapses, but that with stronger depolarizations or longer time courses of depolarization
303
the recruitment of additional vesicles masked this immediate difference in release
304
latency and amplitude.
305
The ribbon may be a barrier to Ca2+ diffusion, creating a restricted space for sharp Ca2+
306
nanodomains beneath the ribbon (Roberts 1994, Graydon, Cho et al. 2011).
307
Intracellular Ca2+ buffering can alter release properties depending on the relative
308
distribution of Ca2+ channels with release sites. In retina the KO increases buffering
309
efficacy (Maxeiner et al. 2016), suggesting looser coupling between Ca2+ channels and
310
release sites. To investigate the effects of ribbon absence on stimulus-secretion
311
coupling in p11 to p13 IHCs, we compared between genotypes the effects on release
312
properties of changing concentration of the slow Ca2+ buffer EGTA (Fig. 7). Examples of
313
release obtained with either 0.1 or 5 mM EGTA are presented in Fig. 7A (control
314
experiments all use 1 mM EGTA). Although increasing buffer concentration reduced
315
release and lowering buffer increased release, the relative changes were comparable
14
316
between WT and KO mice for release measured at 1 sec (Fig. 7B). Data previously
317
demonstrated that increasing Ca2+ buffering increased the time to superlinear release
318
(Schnee, Santos-Sacchi et al. 2011). Fig. 7C demonstrates that this effect is retained in
319
the KO and that there are no major differences between WT and KO modulation by
320
Ca2+ buffering. These results contrast with findings from retinal bipolar cells and in the
321
zebrafish KO (Maxeiner, Luo et al. 2016); see Discussion). Similarly, release rates
322
measured for steps to -45 mV were not significantly different for the initial release
323
components (Fig. 7D). We further investigated the first component of release, typically
324
thought to represent vesicles in early release pools, not requiring trafficking. Comparing
325
release at two voltages again showed that the buffer greatly altered release properties
326
but not in a differential way between genotypes. However, it should be noted that the
327
difference in early release observed in Fig. 6 with 1 mM EGTA was lost with 0.1 mM
328
EGTA. Thus, it is possible that a sensitivity exists that is below a clear detection limit for
329
our capacitance measurements.
330
EPSCs are more heterogeneous in size and kinetics in the absence of the ribbon:
331
Cm measurements are a powerful tool for investigating whole-cell exocytosis but they
332
lack sensitivity (1 fF reflects fusion of ~20 vesicles), they reflect information from all
333
synapses and they can be quite variable. To obtain a more refined view of activity from
334
individual synapses we recorded postsynaptically, from the afferent fiber bouton. Hair
335
cells bathed in artificial perilymph lack the standing inward current through
336
mechanoelectric transduction channels in hair bundles, causing the resting membrane
337
potential to be relatively hyperpolarized (Farris, Wells et al. 2006, Johnson, Kennedy et
338
al. 2012). To depolarize hair cells and increase the rate of vesicle fusion, we applied a
15
339
high potassium (40 mM K+) solution to depolarize the hair cell. K+ application to the hair
340
cell in current-clamp mode depolarized hair cells by an average of 32 ± 5 mV (n = 8;
341
Fig. 8A). There was no difference in depolarization between genotype thus allowing
342
conclusions regarding postsynaptic responses to be ascribed to presynaptic effects.
343
Afferent boutons of type I auditory nerve fibers were recorded ex vivo at ages of P17-
344
P21. Due to the approach vector of the recording pipette, recordings were made from
345
boutons located on the side of the IHC facing the ganglion (modiolar face) rather than
346
the pillar cell side. The start of the high K+ response was defined when the mean EPSC
347
rate exceeded the rate in normal external solution by 2 standard deviations. Examples
348
of postsynaptic responses to K+ applications are provided in Figure 8B-D. Two types of
349
responses were observed, sustained responses (Fig. 8B,C) and transient responses
350
(Fig. 8D). WT responses were mainly of the sustained type (7 of 8) while the KO
351
responses had a smaller proportion of sustained responses (7 of 11). EPSC rates are
352
plotted for transient (Fig. 8E) and sustained responses (Fig. 8F) for both WT and KO.
353
EPSC rates in 40 mM K+ perilymph were quantified over one second windows at 100
354
ms intervals and the means are displayed as thicker lines (Fig. 8F). The peak release
355
rates varied considerably across fiber populations (0-120 s-1) and there was a tendency
356
for the KO neurons to reach a lower peak EPSC rate on average: 57 ± 30 s-1, (n = 11,
357
WT) compared with 44 ± 31 s-1, (n = 18, KO) (Fig. 8G).
358
The time to peak EPSC rate was less variable than absolute rates (Fig. 8H). The KO
359
had one outlier that steadily increased throughout the K+ application, removal of this
360
point from the statistical analysis resulted in the KO reaching peak EPSC rate sooner (9
361
± 10 sec, n = 10) than WT (21 ± 12 sec, n = 9; p = 0.037). Observing the sustained 16
362
release group shows that both WT and KO reduced EPSC rate over time but that the
363
KO reduced more completely than WT (Fig. 8G). 8/11 KO reduced to less than 5% of
364
peak as compared to 3/8 WT. Integrating the rates over time (Fig. 8I) confirmed an
365
increase in total EPSCs in WT (1.8 ± 1.1 x 103) compared to KO (0.81 ± 0.66 x 103, p =
366
0.021).
367
EPSC amplitude distributions were generated for each recorded fiber. Frequency
368
histograms of EPSC amplitudes could be fit with either a double Gaussian function or a
369
Gaussian and gamma function where the smallest peak was always fit by a Gaussian
370
function (e.g. Fig. 8J,K). The area under the smaller peak was calculated and plotted as
371
a percentage of the total in Fig. 8L, where the KO showed a statistically larger (p =
372
0.034) percentage of small events with 5.5 ± 5.6% (n = 12) as compared to 1 ± 0.7% (n
373
= 7). The increase in number of small events might suggest less coordination in release
374
because there are fewer vesicles filling release sites. It might also suggest that smaller
375
responses are visible because of an increased in receptor plaque size as well. Only
376
fibers with more than 100 EPSCs were included for this analysis. No difference was
377
found in the proportion of simple events for each unit, where a simple event is described
378
as having a single rise and decay phase (Fig. 8M). We also compared the median
379
amplitudes (Fig. 8N). Although there was a tendency for EPSCs in the KO to be greater
380
in amplitude 212 ± 46 pA (n = 13) compared to 173 ± 36 pA (n = 7), the values were not
381
statistically different. A difference here would support the argument of a postsynaptic
382
compensatory increase in glutamate receptor plaque size. The coefficient of variation
383
(CV) for the amplitude distributions were different on average (p = 0.017; Fig. 8O). The
384
control values are similar to those obtained by (Li, Keen et al. 2009, Grant, Yi et al.
17
385
2010). Recordings with a larger proportion of smaller events had larger CV. When small
386
events were removed from the histogram, the CVs were not statistically different
387
between KO and WT. The postsynaptic responses in KO also had a greater proportion
388
of EPSCs > 350 pA (Fig. 8J,K, 9D): 1.3 ± 1.3% in WT compared to 8.0 ± 10% for KO
389
EPSCs (t-test for unequal variances, p = 0.04). The change in amplitude was not
390
accompanied by a change in kinetics. The increased amplitude might reflect the
391
increase in postsynaptic glutamate receptor plaque (Fig. 3,4).
392
Examples of EPSCs are presented in Fig. 9A to show the variation in amplitudes and to
393
identify a smaller population of EPSCs with slower kinetics (see inset). As the ribbon is
394
proposed to be involved with multivesicular release (MVR), and the kinetic similarities
395
between large and small EPSCs are a signature of MVR, we investigated the decay of
396
EPSCs in auditory nerve fiber recordings from WT and KO animals. Fitting the EPSC
397
decay with single exponentials provided time constants (Fig. 9B). Calculating the CVs of
398
EPSC decay time constants (Fig. 9C) revealed more variability in KO but no significant
399
difference compared to WT. The skewness (Fig. 9F) of the decay time constant
400
distributions was significantly larger for the KO (0.46 ± 0.2, n = 13) compared to WT
401
(0.29 ± 0.07, n=7; p = 0.04); the KO mean EPSC decay time was greater than the
402
median decay time. As a population 0.44% of WT EPSCs were < 100 pA in amplitude
403
with decay time constants > 1 ms while 1.9% of KO events fell in that category. Plotting
404
the decay time constant against amplitude for each EPSC in all fibers (Fig. 9D) (WT: n =
405
7 recordings, 15,564 EPSCs; KO: n = 13 recordings, 17,210 EPSCs) identifies a
406
population of small slow EPSCs in the KO that are present in greater proportion than in
407
WT (upper left quadrant). The mean EPSC decay time constants plotted as a function of
18
408
EPSC amplitude for each recording (bin of 10 pA) illustrates that all the KO fibers had a
409
proportion of EPSCs with slow time courses while the WT have only 2 fibers with these
410
slower events (Fig. 9E). An example of the smaller, slower EPSC is provided in the
411
inset of Fig. 9A. The distributions of EPSC durations for all EPSCs < 100 pA (Fig. 9G,H)
412
demonstrates the shift toward slower longer duration EPSCs in KO fibers (p > 0.001,
413
using the Kolmogorov-Smirnov test). The cumulative probability functions of EPSC
414
duration for all fibers by genotype (thick red and blue lines) illustrates the larger
415
proportion of slow EPSCs in the KO fibers (Fig. 9H). These data may also support the
416
argument for a larger postsynaptic glutamate receptor plaque, where diffusion of
417
glutamate, coupled with the lower glutamate concentration might serve to slow the
418
decay kinetics.
419
Discussion:
420
The KO mouse provides an unprecedented opportunity to investigate the functional
421
relevance of synaptic ribbons in driving information transfer at the primary auditory
422
synapse. Synapse formation and stability appeared normal based on synapse counts
423
and protein localization, despite complete absence of the ribbon (Figs. 1, 3-4). No
424
evidence was found for partial or defective ribbons structures, nor did we detect
425
compensation by other proteins in the absence of RIBEYE (Fig. 1). Also, cochlear
426
mechanics appeared normal as assessed by DPOAE (Fig. 2). Thus, this mouse
427
provides an excellent model to investigate the ribbon’s role in synaptic transmission.
428
Whole animal function: ABR responses reported a 10 dB threshold shift and about a
429
50% reduction in ABR amplitude with no significant effect on the shape of the
430
waveforms or on the delay (Fig. 2). These data agree qualitatively with the companion 19
431
paper data, the lack of significance likely being a function of the sample size. The minor
432
auditory deficit may not be expected to produce a change in behavioral sound detection
433
threshold as the ensuing ABR peaks, representing brainstem nuclei, were unaffected.
434
Alternatively, central compensation might occur that mask peripheral deficits. These
435
data suggest that the ribbon is not essential for setting hearing threshold, and that its
436
role in auditory function is more nuanced than for example otoferlin which when absent
437
or altered leads to deafness with threshold elevations of more than 100 dB (Roux,
438
Safieddine et al. 2006, Pangrsic, Lasarow et al. 2010). Further work is needed to better
439
understand ribbon function at the systems level. Additional testing to better investigate
440
auditory processing such as sound localization experiments may provide additional
441
insight into how the ribbon might serve systems level function.
442
Synapse formation: Synapse formation is surprisingly normal with appropriate number
443
of synapses and synaptic markers present (Figs. 1,4). Little change in Ca2+ channel
444
distribution or biophysics were observed in the KO (Fig. 5) relative to Bassoon
445
disruption, suggesting that Ca2+ channel distribution or regulation does not depend
446
critically on the ribbon (Frank, Rutherford et al. 2010). Our companion paper identifies
447
minor changes in Ca2+ channel distribution and voltage sensitivity at later ages; whether
448
these are a function of ribbon absence or activity dependent modulation remains to be
449
explored. The lack of effect on calcium channel properties and distributions at early
450
ages supports the hypothesis of a compensatory mechanism occurring later during
451
synapse maturation. Given the reports of late development changes in ribbon size and
452
glutamate receptor plaque sizes, activity driven shaping of the synapses is a reasonable
453
expectation (Fernandez, Jeffers et al. 2015). Postsynaptically, both Homer and the
20
454
glutamate receptors had larger volumes in the KO animal than in WT (Fig. 4). This
455
change also may be an indication of activity dependent synaptic shaping where the
456
reduced activity in the KO animal results in a postsynaptic up regulation of receptors.
457
The increase in the population of larger amplitude EPSC’s in KO animals further
458
supports this conclusion (Figs 8I,J, 9D).
459
Comparison to other systems: In zebrafish a frameshift mutation was used to
460
eliminate ribeye from ribbon synapses. This work revealed ghost like ribbons where
461
vesicles were organized about invisible ribbon-like structures (Lv C et al.,2016). Neither
462
our investigations, nor the companion paper nor the original work investigating the
463
retina (Maxeiner et al., 2015) found comparable ghosts in this mouse. We would
464
conclude that either the frame shift approach did not account for all ribeye or there is a
465
strong compensatory mechanism in zebrafish that is not found or is much weaker in
466
mammal. We investigated CTBP1 as a potential compensatory molecule but as Fig. 1
467
shows there is no labeling near synapses in the KO mouse. The presence of ribbons in
468
photoreceptors of zebrafish support the idea that there are fundamental differences
469
between zebrafish and mammalian systems. Additionally, work in zebrafish suggests a
470
tight reciprocal relationship between calcium channel numbers, calcium current
471
amplitude and ribbon size (Lv, Stewart et al. 2016). As no changes in calcium channel
472
distribution or numbers were observed in the KO animal despite the loss of the ribbon,
473
this relationship does not appear to exist in mammal or is greatly reduced, again
474
supporting fundamental differences between model organisms.
475
Mechanism of ABR amplitude reduction: The decrease in ABR amplitude is likely a
476
function of the reduced frequency of EPSCs leading to fewer action potentials 21
477
generated (Fig. 8F). In addition, the increase in proportion of EPSCs with lower
478
amplitudes and longer durations (Fig. 9H) is expected to increase the likelihood of spike
479
generation failure (Rutherford, Chapochnikov et al. 2012). As larger EPSCs are better
480
phase-locked than smaller EPSCs (Li, Cho et al. 2014) and present works finds a 5-
481
10% increase in smaller EPSCs one might predict a lower vector strength and reduced
482
phase locking. Our companion paper in fact does observe these differences in single
483
unit firing. Similarly the increased population of smaller EPSCs is reminiscent of
484
observations in young animals when compared to older animals and so might reflect a
485
developmental delay in the KO animal. Fewer fibers fire together at sound onset,
486
reducing auditory nerve synchrony and wave 1 amplitude (Fig 2C,D). Measurements of
487
exocytosis from IHCs in response to small depolarizations, clearly showed a reduction
488
in release and an increase in time to release in the KO (Fig. 6E,F). This suggests a
489
decrease in vesicles available at the synapse for immediate release (supported by TEM
490
counts). The reduction in available vesicles for release as well as the population of
491
reduced amplitude EPSCs might also predict a spike timing deficit as described in the
492
companion paper. However, the more rapid response in WT was masked by stronger
493
stimulation (Fig. 6F), indicating that vesicle replenishment remained robust despite the
494
absence of the ribbon-associated synaptic vesicle pool. Given the robust nature of
495
vesicle trafficking, the primary role for the ribbon maybe in ensuring a population of
496
vesicles is present for the timing of onset responses, whereas vesicle trafficking can
497
serve to replenish vesicle populations during sustained release. A similar result was
498
reported by (Maxeiner, Luo et al., 2016) in retina.
22
499
Postsynaptic recordings suggest that the KO reaches peak release sooner than WT and
500
to a similar peak rate, implying that vesicle numbers are comparable for immediate
501
release. In contrast, TEM suggests fewer vesicles available at the synapse and the
502
small increase in population of small EPSCs also suggests a reduction in filled release
503
sites. Thus, it is possible that the ribbon acts as a barrier for vesicle interaction with the
504
release site while simultaneously serving to increase the total vesicles number
505
available. Interestingly, this idea was first suggested by (Furukawa and Matsuura 1978)
506
and later supported by (Schnee, Castellano-Munoz et al. 2013) as a possible
507
mechanism for explaining neural adaptation. However, the companion paper suggests
508
modifications at the synapse that might also be in accord with the postsynaptic
509
measurements here as well, i.e. more vesicles at the membrane. If there is an increase
510
in presynaptic density area and more vesicles per active zone, it might be predicted that
511
faster immediate release occurs but sustainability might be reduced, EPSC data also
512
supports this idea. To resolve these possibililities we need a better understanding of the
513
molecular underpinnings of the active zone and number of available release sites.
514
Synaptic vesicle replenishment for sustained release: Our data would argue against
515
a role for the ribbon in vesicle formation and replenishment. Total exocytosis, from both
516
the linear and superlinear components, was not reduced in the KO, implying a similar
517
total number of vesicles that could be recruited for release (Figs. 6A,B and 7A). In
518
contrast to zebrafish lateral line (Lv, Stewart et al. 2016), we measured no change in
519
vesicle size between KO and WT, indirectly supporting our view that the ribbon is not
520
regulating or involved in vesicle formation, a result corroborated by the companion
23
521
paper. Thus, no change in vesicle size, or maximal release supports the conclusion that
522
the ribbon is not involved in vesicle formation.
523
Ca2+ influx and coupling to exocytosis: The ribbon may act as a barrier to Ca2+
524
diffusion, creating a higher level of local [Ca2+] to synchronize vesicle fusion of multiple
525
vesicles (Graydon, Cho et al. 2011). In this case, the ribbon’s presence would reduce
526
the sensitivity of exocytosis to exogenous Ca2+ buffers (by elevating concentrations of
527
Ca2+). Likewise in retina from KO mice, EGTA reduced spontaneous release in the KO
528
but not in WT (Maxeiner, Luo et al., 2016). In contrast, our data shows that buffering
529
efficacy was not altered in the KO (Fig. 7), suggesting the ribbon does not play a major
530
role in regulation of intracellular Ca2+ concentration at IHC active zones. The similar
531
response between WT and KO might indicate a small effect though at a level below a
532
clear detection limit for capacitance measurements. It is possible that the geometric
533
differences and synaptic architecture between retina and cochlea alter the sensitivity to
534
Ca2+ buffers or that the effect noted in retina is indirect, for example EGTA-AM might
535
alter the presynaptic cell’s resting potential. Thus, our results are not fundamentally
536
different from (Maxeiner, Luo et al., 2016), both supporting the idea that the ribbon
537
serves to consolidate vesicles near to release sites as suggested by (von Gersdorff,
538
Vardi et al. 1996).
539
Ribbon and Multiquantal release: The underlying mechanism for multiquantal release
540
remains an open question but might be driven by simultaneous multiquantal or
541
multivesicular release (Glowatzki and Fuchs 2002). The majority of EPSCs measured in
542
the KO remain large and uniformly fast in onset and decay times and the EPSC
543
amplitude is not reduced suggesting that the ribbon does not play a pivotal role in 24
544
multivesicular release. Thus, mechanisms that involve sequential fusion of vesicles
545
along the ribbon and then release or rapid sequential vesicle release that piggyback
546
onto a release site from the ribbon are unlikely to be valid as these should be
547
dramatically altered by the loss of the ribbon. One remaining possibility is the
548
synchronous release of vesicles from multiple release sites. Although the ribbon is not
549
required for this mechanism, it may indirectly modulate this mechanism by influencing
550
the number of docked vesicles available for release. The increase in number of smaller
551
slower EPSCs likely arising from fewer docked vesicles available for synchronous
552
release may support a ribbon role; however, the small relative contribution of this
553
vesicle pool indicates a minor role. The phenomenon of large EPSC amplitude
554
variability may still be explained by a univesicular mechanism including a dynamic
555
fusion pore, which presumably would not require a ribbon (Chapochnikov, Takago et al.
556
2014). Where identified, the slow footlike response described by (Chapochnikov,
557
Takago et al. 2014) was not different between genotypes.
558
Comparison to Bassoon KO: In mice, the effects of KO are subtle compared with
559
disruption of Bassoon protein. Loss-of-function mutation in Bassoon led to a reduction in
560
the number of IHC ribbons associated with presynaptic elements, ABR threshold
561
elevation of 30 dB, longer ABR wave 1 latency and first-spike latency, and fewer Ca2+
562
channels at active zones (Khimich, Nouvian et al. 2005, Buran, Strenzke et al. 2010,
563
Frank, Rutherford et al. 2010, Jing, Rutherford et al. 2013). Thus, the effects of loss of
564
Bassoon protein (and membrane-anchored ribbons) were greater than loss of RIBEYE
565
protein, consistent with a more critical role for Bassoon in scaffolding synaptic proteins.
566
Also given Bassoon’s presence at efferent synaptic endings in the organ of Corti, it is
25
567
not surprising that the functional loss of Bassoon has a greater phenotype than that of
568
RIBEYE (Supplemental Fig. 1).
569
Summary: A central tenet for ribbon function is maintaining a large pool of vesicles
570
near the synapse. The ribbon may serve as a conveyor belt to both maintain and to
571
traffic vesicles to release sites (Parsons 1994, Nouvian, Beutner et al. 2006). Our data
572
suggests the ribbon increases the time a vesicle remains in the synaptic region. Robust
573
vesicle trafficking and replenishment either via recycling or transporting new vesicles to
574
the synaptic zone provides vesicles to the synaptic zone, but the time vesicles remain
575
near to release sites may be shortened in the absence of the ribbon serving to maintain
576
them close to the synapses. A model for this basic idea was proposed (Graydon, Zhang
577
et al. 2014) that reproduced data from bipolar cell synapses. The addition of a more
578
robust trafficking mechanism may allow this model to also reproduce hair cell synapses.
579
The increase in variance, whether measured as CV, skewness or simply standard
580
deviation in measured parameters such as time to release, release rate and EPSC
581
amplitude was greater for KO. The increased variance may simply reflect a nonuniform
582
population of vesicles near the synapse in the absence of the ribbon. Thus, the ribbon
583
may serve as a biological buffer, maintaining a uniform population of vesicles near
584
release sites.
585
Materials and Methods
586
Animals
587
Mice of both sexes from a mixed genetic background (C57bl6, 129/Ola) were bred from
588
heterozygous breeding pairs and genotyped by PCR for the knockout of the A-domain
589
in Ctbp2 (Maxeiner et al., 2016) while wild-type littermates served as controls. The 26
590
Administrative Panel on Laboratory Animal Care at Stanford University (APLAC
591
#14345) and Animal Care and use Committee and Washington University in St. Louis
592
approved all animal procedures. Experiments were performed blinded to genotype.
593
Auditory measurements:
594
distortion products of optoacoustic emissions (DPOAEs) in mice of postnatal day 21 as
595
previously described (Oghalai, 2004). Mice were anesthetized by administering
596
100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally and their body temperature
597
held constantly at 37°C (FHC, DC-temperature controller and heating pad) until they
598
fully recovered.
599
Sound stimuli were generated digitally by a self-written software (MATLAB 2009b,
600
MathWorks), controlling a custom-built acoustic system using a digital-to-analog
601
converter (National Instruments, NI BNC-2090A), a sound amplifier (TDT, SA1), and
602
two high frequency speakers (TDT, MF1). The speakers were connected to an ear bar
603
and calibrated in the ear canal prior to each experiment using a probe-tip microphone
604
(microphone type 4182, NEXUS conditioning amplifier, Brüel and Kjær).
605
Auditory Brainstem Responses (ABRs) were recorded by placing three needle
606
electrodes subcutaneously at the vertex, below the left ear, and a ground electrode
607
close to the tail. The signals were amplified 10,000 times using a biological amplifier
608
(Warner Instruments, DP-311) digitized at 10 kHz, and digitally band-pass filtered from
609
300 to 3,000 Hz. The stimulus for eliciting the ABR was a 5 ms sine wave tone pip with
610
cos2 envelope rise and fall times of 0.5 ms. The repetition time was 50 ms, and 260
611
trials were averaged.
We measured auditory brainstem responses (ABRs),
27
612
At each frequency, the peak to peak voltages of ABR signals at stimulus intensities
613
ranging from 10 to 80 dB sound pressure levels (SPL) were measured in 10 dB steps
614
and fitted and interpolated to find threshold five standard deviations above the noise
615
floor. For statistical purposes we defined threshold as 80 dB SPL, if no ABR was
616
detected.
617
Distortion Products of Otoacoustic Emission (DPOAE): For stimulation of DPOAEs
618
two sine wave tones of equal intensity (l1=l2) and 1-second duration were presented to
619
the ear. The tones ranged from 20 to 80 dB SPL attenuated in 10 dB increments and
620
the two frequencies (f2 = 1.2f1) ranged from f2 with 4 to 46 kHz. The acoustic signal was
621
detected by the microphone in the ear bar was sampled at 200 kHz and the magnitude
622
of the cubic distortion product (2f1-f2) determined by FFT. Averaging 20 adjacent
623
frequency bins surrounding the distortion product frequency determined the noise floor.
624
DPOAE thresholds were reached when the signal was greater than three standard
625
deviations above the noise floor.
626
Tissue Preparation, Staining and Confocal Microscopy: For staining of pre- and
627
postsynaptic markers we used antibodies against Homer (Synaptic Systems, 160003,
628
1:800 dilution), Bassoon (AbCam, ab82958, 1:500 dilution), CtBP1 (BD-Biosciences,
629
612042, 1:600 dilution), CtBP2/RIBEYE (Santa Cruz Biotechnology, sc-5966, 1:300
630
dilution; BD Transduction Lab, 612044, 1:400), CaV1.3 (Alomone Labs, ACC-005, 1:75),
631
GluA3 (Santa Cruz Biotechnology, sc-7612, 1:200 dilution). Cochlea of P22 or P35 mice
632
were isolated in HBSS solution or artificial perilymph, a small hole was introduced at the
633
apex, cochlea were gently perfused through round and oval windows with freshly
634
prepared 4 % (w/v) PFA (Electron Microscopy Sciences) in PBS until the fixative
28
635
became visible at the apex. Organs were incubated in the same fixative solution for 30
636
min at room temperature. Whole mount organs of Corti were micro dissected, the
637
tectorial membrane removed, the tissue permeabilized for 30 min in 0.5 % (v/v)
638
Triton X-100/PBS, blocked for 2 h in 4 % (w/v) BSA/PBS at room temperature. Staining
639
with all primary antibodies was performed overnight at 4°C in the same blocking buffer.
640
For staining with antibodies against GluA2 and CaV1.3 the protocol was slightly varied:
641
fixation was 20 min at 4°C, followed by decalcification in 10 % EDTA (Sigma) for 2 h,
642
permeabilization and blocking was carried out in a blocking buffer containing 16%
643
donkey serum and 0.3 % Triton X-100. After washing all species appropriate secondary
644
antibodies (Alexa Fluor 488, 546 or 555, 647: 1:600 dilution in 4 % (w/v) BSA/PBS)
645
were applied for 1 h at room temperature. After washing, the organs of Corti were
646
mounted hair bundles facing upward in ProLong Gold© Antifade (Life Technologies).
647
Samples were imaged using confocal microscopy (LSM700 or LSM880, Zeiss) on 1.4
648
NA oil immersion objectives for confocal imaging, and 1.2 NA water immersion objective
649
for Airyscan imaging. The optimal voxel size was adjusted to the green channel
650
(confocal x/y/z: 100/100/350 nm; Airyscan x/y/z: 33/33/222 nm). RIBEYE WT and KO
651
samples were prepared in parallel and images were collected with identical settings.
652
All images were taken in the apical region, between 200-1000 µm from the apex, of the
653
organ of Corti.
654
Image analysis
655
For
656
colocalization, we used the Imaris software package (Version 8.4, Bitplane). Puncta
657
were initially detected using the spot algorithm allowing for different spot sizes and
three-dimensional
analysis
of
synaptic
29
punctum
volume,
intensity
and
658
elongated point spread function in z-direction (initial seed size x-y/z: CtBP1, Ctbp2,
659
Homer, 0.44/1.13 µm; Bassoon 0.37/1.13 µm; GluA3 0.7/1.4 µm; CaV1.3 0.56/1.4 µm),
660
followed by co-localizing the spots with a threshold lower than 1. All counts were
661
normalized to the number of Inner hair cells in a region of interest.
662
To estimate the size of synaptic puncta we used the surface algorithm in Imaris. For all
663
stainings, the surface detail was set to 0.15 µm, background subtraction to 0.562 µm,
664
and touching object size was 0.750 µm (Bassoon puncta were split at 0.36 µm).
665
TEM and Tomography
666
Temporal bones of P21 mice (WT: n = 5, KO: n = 5) were isolated in HBSS, a small
667
hole was introduced at the apex of the cochlea, gently perfused through round and oval
668
windows with freshly prepared 4 % (w/v) PFA,
669
Microscopy Sciences) in a buffer containing (in mM) 135 NaCl, 2.4 KCl, 2.4 CaCl2, 4.8
670
MgCl2, 30 HEPES, pH 7.4 until the fixative became visible at the apex. Temporal bones
671
were soaked for 4 hours in the same buffer and shipped in PBS. Cochleae were
672
microdissected out from fixed adult mouse temporal bones and slowly equilibrated with
673
30% glycerol as cryoprotection. Tissues were then plunge frozen in liquid ethane
674
at -180C using a Leica Biosystems grid plunger. Frozen samples were freeze
675
substituted using Leica Biosystems AFS in 1.5% uranyl acetate in absolute methanol
676
at -90C for 2 days and infiltrated with HM20 Lowicryl resin (Electron Microscopy
677
Sciences) over 2 days at -45C. Polymerization of the resin was carried out under UV
678
light for 3 days at temperatures between -45C and 0C. Ultrathin sections at 100 nm
679
were cut using a Leica ultramicrotome and collected onto hexagonal 300-mesh Cu grids
680
(Electron Microscopy Sciences). 30
2 % (w/v) Glutaraldehyde (Electron
681
Samples were imaged using a 200kV JEOL 2100 with a Gatan Orius 832 CCD camera.
682
Single images were acquired using DigitalMicrograph (Gatan). Tilt series were acquired
683
using SerialEM (Mastronarde 2005) at 1 tilt increment from -60 to +60 and
684
tomographic reconstruction of tilt series and segmentation of tomograms was done with
685
IMOD software suite (Kremer, Mastronarde et al. 1996). Fiji was used to generate
686
projection images from tomograms and for cropping, rotating and adjusting the
687
brightness and contrast of images.
688
Hair cell recordings:
689
IHCs from the apical coil of the organ of Corti were dissected from p10-13 Ctbp2
690
littermates of either sex, placed on a plastic coverslip and secured with two strands of
691
dental floss. Cells were visualized with a water immersion lens (100x) attached to a
692
BX51 Olympus upright microscope. Patch-clamp recordings were obtained at room
693
temperature (21-23 C) in an external solution containing in (mM) NaCl 140, KCl, 5.4,
694
CaCl2, 2.8, MgCl2 1, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 10, D-
695
glucose 6, creatine, ascorbate, Na pyruvate 2, pH 7.4 adjusted with NaOH. 100 nM
696
apamin was included to block the sK K+ channel. Cesium at 3 mM was used externally
697
to reduce inward leak current. Pipette solution contained (mM) CsCl 90, TEA,
698
tetraethylammonium chloride 13, 3 NaATP, MgCl2 4, creatine phosphate 5, ethylene
699
glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), HEPES 10, pH
700
adjusted with CsOH to 7.2.
701
Two-Sine Method
702
Hair cells were voltage-clamped with an Axon Multiclamp 700B (Axon Instruments-
703
Molecular Devices, Sunnyvale, CA.) and data recorded at 200-300 kHz with a National 31
704
Instrument USB-6356 D/A acquisition board and JClamp software (SciSoft). Vesicle
705
fusion was determined by monitoring membrane capacitance as a correlate of surface
706
area change. Capacitance was measured with a dual sinusoidal, FFT-based method
707
(Santos-Sacchi 2004). In this RC analysis method, two voltage frequencies, f1 and f2
708
(twice the f1 frequency) are summed and the real and imaginary components of the
709
current response are used to determine the magnitudes of the three model components.
710
The time resolution of the Cm measurement is the period of f1, which we varied from
711
0.16 to 0.32 ms, corresponding to 6250 to 3125 Hz. Stimulation protocols were
712
performed after 10 minutes in the whole-cell mode to allow Ca2+ current run up and full
713
exchange of the internal solution into the cell (Schnee, Castellano-Munoz et al. 2011). A
714
dual sinusoid of 40 mV amplitude was delivered superimposed to the desired voltage
715
step. The mean IHC Cm was 10 ± 1 pF N = 25 and uncompensated series resistance of
716
10 ± 3 M. All voltages were corrected for a 4 mV junction potential.
717
Postsynaptic Recordings
718
Whole-cell voltage-clamp recordings were made at terminal endings of SGNs from P17-
719
21 animals (Grant, Yi et al. 2010). Cochlea were acutely dissected from the temporal
720
bone in chilled (~ 4°C) standard extracellular solution containing 1 mM curare. The
721
apical turn was excised after removing stria vascularis and tectorial membrane, and
722
placed under two dental floss fibers on a recording chamber filled with the standard
723
extracellular solution at room temperature. Recording pipettes were made from 1.5 mm,
724
thick-wall borosilicate glass (WPI). Pipettes were pulled using a multistep horizontal
725
puller (Sutter) and fire polished while applying a pressure to the back end of pipettes to
726
an impedance of 6-8 MΩ (Johnson, Forge et al. 2008). When whole-cell recording was 32
727
achieved from a bouton, 5 µM TTX was perfused into the chamber, and EPSCs were
728
recorded in the standard solution (5.8 mM K+) for at least 3 min, then in 40 mM KCl for
729
1-5 min (depending on the duration of synaptic activity on each nerve).
730
external solution contained (in mM): 5.8 KCl, 140 NaCl, 0.9 MgCl2, 1.3 CaCl2, 0.7
731
NaH2PO4, 5.6 D-glucose, 10 HEPES, 2 Ascorbate, 2 Na-Pyruvate, 2 Creatine
732
Monohydrate, Osmolarity was 305 mosmls, pH = 7.4 (NaOH). Intracellular solutions
733
contained (in mM): 135 KCl, 3.5 MgCl2, 0.1 CaCl2, 5.0 EGTA, 5.0 HEPES, 2.5 Na2-ATP,
734
pH = 7.2 (KOH), osm ~290 mosmls.
735
Data Analysis:
736
Data were collected blinded to genotype for the experimentalist. Genotype was revealed
737
during analysis phase to ensure appropriate n values were obtained between groups.
738
Although gender was noted, no gender differences were observed between groups and
739
so data is presented combined. Data analysis was largely performed using unpaired
740
two-tailed t-tests unless otherwise stated. When non normal distributions were
741
observed, as in the EPSC histograms we selected different comparators as described
742
with the data. Similarly when large n values would bias comparisons, such as with
743
analyzing immunohistochemistry data, we moved to alternative more appropriate tests
744
as described in the text. Data is presented as mean ± standard deviation unless
745
otherwise stated. Sample size is presented in legends or in text. For physiology
746
experiments sample size reflects cell numbers which also indicates animal numbers as
747
we did not record from more than one cell in a preparation. For immunohistochemistry,
748
data is presented for animal numbers as well as image, cell and puncta numbers.
33
Standard
749
The voltage against capacitance plots were arbitrarily fit with an exponential equation of
750
the form: Y = Y0 + A1 exp(-x(x-x0)/t) where: Y0 is the baseline change in capacitance and is
751
near 0, A1 is the asymptote for the maximal change in capacitance, x0 is the voltage
752
where changes in capacitance measurements were first noted and t reflects the voltage-
753
dependence of the release (Cm/Vm). The equation for the Gaussian fit used in Fig.
754
6E is: Y = Y0 + (A/(w*√(π/2)))*exp(-2*((x-xc)/w)^2) where Y0 is baseline, typically 0, A is the
755
area under the curve, w is the width and xc is the peak of the Gaussian.
756
Acknowledgments
757
This work was supported by National Institutes of Health, National Institute on Deafness
758
and Other Communication Disorders (NIDCD), Grants R01DC014712 to M.A.R.; M.A.R.
759
was also supported by an International Project Grant from Action on Hearing Loss.
760
NIDCD; F32 4F32 DC013721 grant from NIDCD to MN, Intramural Research Program
761
(Z01-DC000002) and NIDCD Advanced Imaging Core ZIC DC000081) to W.S. and
762
B.K.: RO1DC009913 to AJR, core grant P30 44992. We thank Autefeh Sajjadi for her
763
help with genotyping animals and basic husbandry. We thank Bill Roberts for comments
764
on the manuscript.
765
The authors declare no competing financial interests
766
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767
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892
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904
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905
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909
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912
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913 914
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915
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917 918
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919
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920 921
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922
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923
41
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Zheng, X. Y., D. Henderson, S. L. McFadden, D. L. Ding and R. J. Salvi (1999).
925
"Auditory nerve fiber responses following chronic cochlear de-efferentation." J Comp
926
Neurol 406(1): 72-86.
927
928
Figure Legends
929
Figure 1. Number of synapses stays unaffected in ribbon less organs of corti.
930
A-D Max intensity projections of confocal z-sections of inner hair cells (IHCs) in apical
931
cochlea turns of P22 KO and wildtype littermates, immunolabeled for CtBP2/RIBEYE
932
(A), Homer (B), Bassoon (C) and Overlay (D). Virtually no punctated extra nuclear
933
CtBP2 staining can be seen in KO mice that is colocalized with other synaptic proteins,
934
while Bassoon and Homer appear generally unaffected. (E) 100 nm thick TEM sections
935
of pre- and postsynaptic densities in WT and KO mice at P28 confirm the absence of
936
electron dense ribbon structures in KO mice. (For E-G) Black arrows point to ribbon
937
structure, white arrows point to synaptic vesicles and white arrowheads point to the
938
remaining density resembling the arciform density. (F) Single slice from a 10 nm thick
939
tomographic section. (G) Reconstruction of complete synapses from data obtained used
940
to create E,F. Presynaptic density in cyan and postsynaptic density in green.Tethered
941
synaptic vesicles modeled in yellow, electron dense structure attributed to synaptic
942
ribbons in magenta. (H) Confocal max intensity projection of IHCs immunolabeled for
943
CtBP1 (green) and Homer (magenta). (I) Quantification of RIBEYE, Homer and
944
Bassoon puncta in the organ of corti normalized to the number of IHCs in analyzed
945
regions of interest, showing complete loss of synaptic RIBEYE staining in KO. The
42
946
amount of Homer and Bassoon staining stays unaffected (p > 0.05). WT in blue, n = 20
947
mice, quantified puncta: RIBEYE = 3796, Bassoon = 10215, Homer = 4340; KO in red,
948
n = 19 mice, n puncta: RIBEYE = 33, Bassoon = 10825, Homer = 4201. (J) Paired
949
synaptic puncta per IHC is lost in absence of RIBEYE staining (p ≤ 0.001) between WT
950
and KO. The number of synapses defined by colocated postsynaptic Homer and
951
presynaptic Bassoon puncta (p = 0.18) is unaffected in KO. (K) The number of extra
952
nuclear CtBP1 puncta is greatly reduced (p = 0.003) in KO mice. (WT in blue, n = 9
953
mice, n puncta: CtBP1 = 19929, Homer 4940; KO in red, n = 7 mice, n puncta: CtBP1 =
954
11960, Homer = 5033). (L) Remaining CtBP1 puncta show significantly reduced
955
(p ≤ 0.001) pairing with postsynaptic Homer staining. Scale Bars: A-D, H: 5 µm; E, F:
956
100 nm. Boxes represent standard deviations of the mean. Significance levels of two
957
tailed unpaired t-tests: n.s., not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
958
959
Figure 1 Supplemental Figure 1: Immunohisotchemistry from WT (A) and KO (B) for
960
Bassoon (green) CTBP2 (red) and myosin VIIa (Blue). Low power images show
961
Bassoon is present in efferents of outer hair cells as well as presynaptically in IHCs.
962
Bassoon localization is much broader than RIBEYE’s and is minimally effected in the
963
mutant animal.
964
965
Figure 2 Hearing in RIBEYE knockout mice. A Distortion products of otoacoustic
966
emissions (DPOAE) show no significant differences in the tested frequency range (4-
967
46 kHz in half octave increments) between KO (red, n = 30) and WT littermates (blue,
43
968
n = 22), suggesting normal outer hair cell function. (B) Auditory brainstem response
969
(ABR) measurements in the tested frequency range (4-46 kHz in half octave
970
increments) reveal a small (≈ 10 dB SPL), but significant threshold shift in KO (red, n =
971
28) over their WT littermates (blue, n = 22) at P21-P22. (C) Mean ABR waveforms to
972
5 ms tone pips at 23 kHz at 60 dB SPL from KO (red, n = 30) and wild type littermates
973
(blue, n = 24) reveal a decrease in amplitude of the first ABR wave in KO. (D)
974
Quantification of peak to peak amplitude (wave I: p1-n1) differences between KO (red)
975
and WT littermates (blue) at 23 kHz were significant over most sound pressure levels
976
(20 dB SPL: p > 0.05; 30 & 60 dB SPL: p ≤ 0.01; 40-60 dB SPL: p ≤ 0.001) (E)
977
Amplitudes of first peak amplitudes normalized to their respective values at 60 dB SPL.
978
Amplitudes scale with their maximum value in WT and KO. (F) First peak latency (p1) is
979
not significantly altered in KO mice. For wave I amplitude and latency (D-F) analysis the
980
n-number varied between sound pressure levels by a clearly detectable first wave and
981
speaker calibration, n of WT/KO: 20 dB, 11/7; 30 dB, 21/11; 40 dB, 23/22; 50 dB, 23/26;
982
60 dB, 23/28; 70 dB, 5/6. Boxes represent standard deviations of the mean; whiskers
983
and shaded line in (C) represent standard errors of the mean. Significance levels of two
984
tailed unpaired t-tests: n.s., not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
985
986
Figure 3. Ribbonless synapses contain clusters of presynaptic voltage-gated Ca2+
987
channels juxtaposed to postsynaptic glutamate receptors. A-D Max intensity
988
projections of airyscan z-sections of IHCs in apical cochlea turns of P35 KO and WT
989
littermates, immunolabeled for CtBP2/RIBEYE (A), GluA3 (B), CaV1.3 (C) and overlay
990
between calcium channels and glutamate receptors (D). Inner hair cell afferent 44
991
synapses from WT have a synaptic ribbon: a cytoplasmic presynaptic density comprised
992
mainly of RIBEYE protein, recognized by anti-CtBP2 (green). Co-labelled are the
993
closely associated presynaptic voltage-gated Ca2+ channels (CaV1.3, gray) and
994
postsynaptic AMPA receptors (GluA3, magenta). Inner hair cell afferent synapses from
995
KO lacked synaptic ribbons. The AMPA receptors in KO mice exhibited a ring-like
996
morphology, and the CaV1.3 channels clustered in the form of round spots or elongated
997
stripes, both similar to WT synapses. Scale bar: 2 µm
998
Figure 4. Changed size distribution but unaffected synapse number and
999
organization in ribbonless synapses (A) Quantification of RIBEYE, GluA3 and
1000
CaV1.3 puncta in the organ of corti normalized to the number of IHCs in analyzed
1001
regions of interest. The number of GluA3 receptor and CaV1.3 puncta is unaffected (p >
1002
0.05). WT in blue, n = 9 images from 3 mice, 95 cells quantified puncta: RIBEYE =
1003
1456, GluA3 = 1459, CaV1.3 = 1897 KO in red, n = 13 images from 3 mice, 248 cells, n
1004
puncta: RIBEYE = 28, GluA3 = 2149, CaV1.3 = 2681. (B) Colocalization of synaptic
1005
puncta per IHC. In absence of RIBEYE, the number of synapses, defined by
1006
colocalization of postsynaptic GluA3 and presynaptic CaV1.3, is unaffected (p = 0.13) in
1007
KO. (C) Volume of presynaptic CaV1.3, Bassoon and postsynaptic Homer and GluA3 in
1008
WT (blue, n synapses: CaV1.3 = 1370; Bassoon = 1917; Homer = 5219; GluA3 =
1009
1611) and KO (red, n synapses: CaV1.3 = 2132; Bassoon = 1817; Homer = 4990;
1010
GluA3 = 2334) mice. (D) Normalized cumulative distributions of volumes show
1011
increased sizes of postsynaptic GluA3 and Homer immunoreactivity. (C) Boxes
1012
represent standard deviations of the mean. Significance levels of two tailed unpaired t-
1013
tests (A/B) or two sample Kolmogorov-Smirnov test (C): D represents the maximum
45
1014
distance of the Kolmogorov-Smirnov distribution functions. n.s., not significant; * p ≤
1015
0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.
1016
Figure 5: No differences were observed in calcium or potassium currents
1017
between KO and WT animals. Ca2+ currents were obtained from mice ranging in age
1018
from P10-P12 for WT (A, blue) and P10-13 KO (B, red), with time course of stimulus
1019
shown above, are comparable in all measured properties. Voltage-current plots,
1020
presented in (C) as mean ± SD show no difference (n provided in figure). Summaries of
1021
peak current (D) and voltage of half activation (V1/2, E) are not statistically different (n=
1022
18 and 26 for WT and KO, p = 0.13 for peak current and 0.326 for V1/2). Potassium
1023
currents, obtained from responses to 50 ms voltage pulses ranging from -124 mV to 16
1024
mV in 10 mV increments, from a holding potential of -84 mV are presented in (F) for WT
1025
and (G) for KO mice at P24. A summary of the half activation voltage as estimated from
1026
a Boltzmann fit to the tail current voltage-current (H) found no statistical difference (n =
1027
6 and 8 for WT and KO, respectively with p = 0.084). Current-clamp measurements
1028
found no difference in resting potential (I) (n = 5 and 8 for WT and KO, respectively, p =
1029
0.69).
1030
1031
Figure 6. Capacitance responses recorded from RIBEYE knockout mice show
1032
little difference between phenotypes. Example records of Cm responses (top),
1033
showing two release components and Ca2+ currents (below) recorded from a WT (blue)
1034
(A) and KO (red) (B) in response to 1s depolarizations from -84 mV to -49 and -44 mV.
1035
Dashed line indicates pre-stimulus baseline Cm. Black solid line indicates the 1st linear
1036
component of release and cyan line the 2nd, superlinear component. (C) Summary plot 46
1037
of 1st and 2nd component magnitudes. No statistical difference was found between
1038
groups. For the linear component n = 15 for WT and 15 for KO, p = 0.23. For 2nd,
1039
superlinear component n = 17 for both groups and p = 0.38. Filled stars indicate mean
1040
for this and all subsequent figures. (D) Summary plot of 1st and 2nd component rates
1041
obtained by linear fits to data points, as indicated by lines in (A,B). No statistical
1042
difference was found between groups where, n = 16 for WT amd 13 for KO, with p =
1043
0.266. For the second component rate, n = 17 for both, with p = 0.085. (D). (E) presents
1044
a population histogram for the voltage at which the superlinear response is first
1045
observed. Fits to Gaussian distributions show no change in the mean value but an
1046
increase in the width of the plot for the KO. (F) Example of delay in onset of capacitance
1047
change with small depolarizations for a KO cell. Plots of Cm verses voltage responses
1048
measured at 0.1 (G), 0.5 seconds (H) 1 sec (I) or 3 sec (J) show a significant difference
1049
for release at 0.1s and -44 mV (p=0.03), error bars represent SEM. Fitting the
1050
relationships with simple exponential functions reveal differences in the shapes of the
1051
curves but not in maximum release for the 0.1 and 0.5 plots but not the 1 and 3 sec time
1052
points. N provided in figure for each group. (K) Shows time to release is significantly
1053
longer for KO compared to WT for depolarizations eliciting less than 50% of the
1054
maximal calcium current; n = 32 for WT and 26 for KO, p = 0.0012.
1055
Figure 7. Changing internal Ca2+ buffering does not distinguish phenotypes. (A)
1056
Example records of Cm changes in response to 1s depolarizations from -49 mV in 0.1
1057
EGTA and -39 mV in 1 and 5 mM EGTA for WT (blue) and KO (red). (B) Plot of the
1058
change in Cm in response to 1s voltage steps of -49, -44 and -29 mV for 0.1 EGTA and
1059
-49,-44,-39 and -34 mV for 5 EGTA, n provided in figure. (C) The time to the 2nd
47
1060
component increases significantly with elevated Ca2+ buffer (p value indicated in figure)
1061
but is similar between phenotypes; for 0.1 EGTA n = 7 for WT and 6 KO , p = 0.93,
1062
steps to -29 mV for WT and KO, for 1 EGTA n = 15 for WT and13 for KO , with p = -
1063
0.13, steps to -36 + 8 mV WT and -37 + 9 mV KO and for 5 EGTA n = 3 for WT and 4
1064
for KO, with p = 0.67, steps to -39 + 5 mV WT and -35 + 2 mV KO. (D) The rate of
1065
release to the -49 mV step declines equally between phenotypes with increasing
1066
[EGTA] for 0.1 EGTA, n = 7 for WT and 6 for KO, p = 0.323, for 1 EGTA n = 20 for WT
1067
and 22 for KO, p = 0.99 and for 5 EGTA n = 7 for WT and 6 for KO, p = 0.235. (E,F)
1068
Provide box plots of the first component of release for depolarizations to -49 mV (E) or -
1069
44 mV (F). Effects of buffers were similar between the two groups.
1070
Figure 8: Subtle differences exist between WT and KO mice postsynaptic
1071
properties. Postsynaptic bouton recordings were obtained from WT or KO in control
1072
and during application of 40 mM K+ to the bath. Hair cell recordings demonstrate similar
1073
depolarizations in WT and KO, suggesting that any differences in response properties
1074
may be synapticaly mediated. (A). Cells rapidly recovered following washout of K+. The
1075
solid line below the trace indicates the duration of the application.
1076
Postsynaptic recordings from afferent boutons in response to 40 mM K+ external
1077
solution application are presented in (B) for WT and (C) for KO with a sustained
1078
response and (D) for KO with a transient response. Recordings were made in the
1079
presence of 5 µM tetrodotoxin (TTX). Frequency responses were generated using a 1
1080
second window sliding at 100 ms intervals (E,F). Populations were defined as transient
1081
or sustained by whether firing persisted after 20 seconds. Number of each response
1082
type is presented as fraction in each panel. Maximum frequency responses are 48
1083
presented in box plot format with mean value shown as start (G); n = 10 for WT and 18
1084
for KO, p = 0.313. The time to peak is presented in (H) where onset of K+ application
1085
response was defined as the time point when the frequency exceeded the spontaneous
1086
frequency +3x SD for two overlapping time windows (200 ms). The time to peak was
1087
statistically different when the one outlier was removed (time point at 181 sec); n = 8 for
1088
WT and 10 for KO. Time to peak only includes fibers whose peak frequency reached 30
1089
Hz because those lower had too few points to make an accurate measure. (I)
1090
Integrating the frequency response over 50 seconds shows that WT release in a more
1091
sustainable manner than the KO, n = 8 for WT and 10 for KO, p = 0.021. Representative
1092
probability density functions for EPSC amplitudes are presented for WT number of
1093
events = 6583 WT and 1551 KO in (J) and KO in (K). Solid lines represent fits to either
1094
double Gaussian functions or Gaussian and gamma functions (smaller peak always fit
1095
with Gaussian function). The curve fitting the small events were integrated to measure
1096
the % of events under the small peak (shown next to each example). Only nerves that
1097
had at least 100 events are included to ensure robustness of the fitting. (L) presents a
1098
box plot of the WT and KO percent of area contributed by the small peak. Comparisons
1099
using a t-test for unequal variance (Welch test) suggest a significant, p = 0.032, n = 7
1100
for WT and 13 for KO. (M) presents a box plot comparing the percentages of simple
1101
events, defined as single rise and decay times plots are not significantly different, n = 7
1102
for WT and 13 for KO, p = 0.3. (N) provides box plots of the median amplitude, where
1103
no significant difference was observed, n = 7 for WT and 13 for KO for each, p = 0.105,
1104
while (O) are box plots of the coefficient of variation for the distributions in (N). Plots
1105
were different, p = 0.017, when equal variance was not assumed.
49
1106
Figure 9: KO synapses have a larger population of small slow EPSCs. 7 WT
1107
including 15,564 events and 13 KO fibers including 17210 events were analyzed. (A)
1108
shows examples of EPSCs that range in amplitude and kinetics. The inset highlights
1109
one of the small slower EPSCs identified in KO fibers. Median decay times (B) and the
1110
Coefficient of Variation for the distribution in decay times (C) are presented as box plots.
1111
Neither population show statistical significance. (D) plots the skewness of the
1112
distributions of decay times and demonstrates that the KO decay time is significantly
1113
greater (away from 0), suggesting that the mean is larger than median, largely because
1114
they have a long tail of large decay time. (E), plots the EPSC decay time constant for
1115
every fiber (each fiber a different color) for both WT and KO. (F), summarizes the data
1116
from C as average decay time constant with EPSCs binned over 10 pA. (G), pooling
1117
fibers for WT and KO recordings allowed for the generation of a probability density
1118
function for event duration. These distributions are significantly different by KS test
1119
(p
