The afferent synapse of cochlear hair cells

Paul A Fuchs
y
, Elisabeth Glowatzki
z
and Tobias Moser

Mechanosensory hair cells of the cochlea must serve as both

transducers and presynaptic terminals, precisely releasing
neurotransmitter to encode acoustic signals for the postsynaptic
afferent neuron. Remarkably, each inner hair cell serves as the
sole input for 1030 individual afferent neurons, which requires
extraordinary precision and reliability from the synaptic ribbons
that marshal vesicular release onto each afferent. Recent studies
of hair cell membrane capacitance and postsynaptic currents
suggest that the synaptic ribbon may operate by simultaneous
multi-vesicular release. This mechanism could serve to ensure
the accurate timing of transmission, and further challenges our
understanding of this synaptic nano-machine.
Addresses

The Center for Hearing and Balance, Department of Otolaryngology

Head and Neck Surgery, The John Hopkins University School of
Medicine, Baltimore, Maryland, USA
y
e-mail: pfuchs@bme.jhu.edu
z
e-mail: eglowatz@bme.jhu.edu

Department of Otolaryngology, University of Goettingen, Goettingen,

Germany
e-mail: tmoser@gwdg.de
Current Opinion in Neurobiology 2003, 13:452458
This review comes from a themed issue on
Sensory systems
Edited by Clay Reid and King-Wai Yau
0959-4388/$ see front matter
2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/S0959-4388(03)00098-9
Abbreviations
AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
BK large-conductance, calcium-sensitive, voltage-gated
potassium channel
C
m
membrane capacitance
EPSC excitatory post-synaptic current
GLAST glutamate-aspartate transporter, also known as EAAT1,
excitatory amino acid transporter type 1
IHC inner hair cell
RRP readily releasable pool
VGCC voltage-gated calcium channel
Introduction
The afferent synapse of mammalian cochlear hair cells is
uniquely specialized in form and function. Individual af-
ferent neurons in the peripheral (spiral) ganglion have a
single unbrancheddendrite that is postsynaptic to (usually)
a solitary synaptic active zone of an inner hair cell (IHC).
The active zone is demarcated by a slight thickening
of the plasma membrane, and a collection of synaptic
vesicles associated with the synaptic ribbon (Figure 1).
The ribbon is a spherical or ellipsoidal electron-dense
body, less than a micron in diameter, to which approxi-
mately 100 synaptic vesicles are tethered [1,2]. This
single synaptic structure carries the entire burden of
acoustic signaling for each afferent neuron. Transmitter
release from the hair cell triggers both sound-evoked, and
spontaneous action potentials in afferent neurons (with
spontaneous rates up to 140 per second) throughout the
lifetime of the organism. In addition to this remarkable
endurance, the hair cells afferent synapse also shows high
temporal precision, releasing neurotransmitter to encode
the submillisecond distinctions employed in sound local-
ization (e.g. [3] reviewed in [4]). Somehow, similarly to
retinal photoreceptors and bipolar cells, hair cells perform
these feats of synaptic release without themselves gen-
erating action potentials. What parameters of ribbon
structure and function confer these synaptic capabilities
to hair cells? Here, we focus on insights arising from
recent studies of hair cell synaptic function, but begin
by reviewing ribbon structure and molecular composition.
Ribbon structure and molecular composition
The structure of synaptic ribbons in hair cells has been
described by electron microscopy and more recently
analyzed with 3-D tomography by Lenzi and co-workers
[5,6

]. In frog saccular hair cells the ribbon itself is an

electron-dense sphere averaging 400 nm in diameter.
Electron-lucent vesicles of about 35 nm diameter are
either attached directly to the ribbon with 20 nm la-
ments or concentrated in the immediate surrounding
cytoplasm by as yet unknown means. On the basis of
capacitance measurements of vesicular release (see
below), both directly-attached and nearby vesicles con-
stitute a pool that could sustain at most several seconds of
exocytosis [5]. Presumably then, the ongoing spontaneous
and sustained driven release of hair cells requires
mechanisms for rapid and efcient replenishment of
the readily releasable pool (RRP) of vesicles. During
sustained potassium depolarization of frog saccular hair
cells, synaptic vesicles docked at the plasma membrane
were dramatically reduced in number [6

]. In contrast,
vesicles attached to the opposite pole of the synaptic
ribbon were less affected, resulting in a graded loss across
the ribbon, which supports the hypothesis that ribbons
participate in vesicular release. The overall loss of synap-
tic vesicles in the active zone was compensated by an
equivalent increase in nearby cisternal bodies, which are
thought to represent a stage of endocytotic recycling of
vesicular membrane.
Given the intuitive appeal of the ribbon as a kind of
vesicular vending machine, what molecular entities could
452
Current Opinion in Neurobiology 2003, 13:452458 www.current-opinion.com
serve this function? The composition of the electron-
dense proteinaceous synaptic body remains largely
unknown, although several vesicle-associated proteins
have been found at retinal ribbons [7]. For example,
antibodies to a microtubule motor protein, KIF3A, label
ribbons and a subset of associated vesicles in the retina
[8]. It remains to be seen if KIF3A or other putative
ribbon proteins, such as Ribeye [9] (but see Update),
Bassoon and Piccolo [10], and the B16 antigen [11] can
be found in hair cells, but a variety of vesicle and
release-site-associated proteins have been detected
including syntaxin 1, SNAP-25 and the vesicle asso-
ciated membrane protein 1 (VAMP1) [12]. Noteworthy
for their absence are synapsin and some of the synap-
totagmins [13]. Recently, a cysteine-string protein has
been identied in hair cells [14

], raising the possibil-

ity that it regulates interactions among syntaxin-1,
VAMP1 and voltage-gated calcium channels at synaptic
ribbons, as proposed for conventional synapses. Despite
these recent ndings, the molecular basis for the uni-
que synaptic functionality of hair cells remains largely
speculative. Interestingly, OTOF, a gene encoding a
Ca
2
/phospholipid-binding synaptic protein of IHCs
(otoferlin), is mutated in human hereditary deafness
(DFNB9) [15].
Ribbon function
Intracellular recording from hair cells has provided sig-
nicant advances in this eld, such as the capacitance
measurements described below. Voltage-clamp record-
ings from the IHCs have also helped to identify two
important molecular components of the synapse, the
voltage-gated calcium channels that support transmitter
release, and associated potassium channels that modulate
excitability.
Voltage-gated channels at the synapse
Transmitter release from cochlear hair cells is triggered
by calcium inux through dihydropyridine-sensitive, or
L-type, voltage-gated calcium channels (VGCCs); in con-
trast to the N, P, Q and R subtypes that dominate
transmitter release from most neurons [16

18

].
Genetic inactivation of the a
1D
subunit of the L-type
VGCC in mice eliminates 90% of the voltage-gated
calcium current from IHCs, resulting in their eventual
degeneration and deafness [19]. It is worth noting that
non-dihydropyridine-sensitive VGCCs carry a fraction of
the current in vestibular hair cells [20

].
Activation of VGCCs is followed by the opening of
voltage-gated potassium channels. Prominent among
Figure 1
Support
cell (sc)
Afferent bouton
Hair cell
GLAST
GLAST
BKs
VGCCs
AMPARs
sc
Current Opinion in Neurobiology
Ribbon
The hair cells ribbon synapse. The inner hair cells afferent synapse is demarcated by an electron-dense synaptic body, or ribbon, to which small,
clear-core synaptic vesicles are tethered. In the mammalian cochlea the ribbon is around 0.2 microns in diameter, and the synaptic vesicles are
3540 nm. Voltage-gated calcium channels (VGCCs) and BK-type (calcium-sensitive) voltage-gated potassium channels are clustered near the
synaptic ribbons. AMPA receptors are found on the postsynaptic afferent bouton, glutamate transporters (GLAST) are expressed by supporting cells
that surround the inner hair cell and its afferent contacts.
Hair cell transmitter release Fuchs, Glowatzki and Moser 453
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:452458
these are the large-conductance, calcium-sensitive BK
type potassium channels whose cell-specic kinetics pro-
duce tuned receptor potentials in hair cells of non-
mammalian vertebrates [21]. These BK channels may
be specically localized to the active zone [22,23], as
occurs at the neuromuscular junction [24]. mRNA for
both a and b subunits of the BK channel is upregulated in
inner hair cells just before the onset of hearing [25

], and
the functional currents can be detected at this time [26

].
Perfusion of the cochlea with neurotoxins that specically
block BK channels eliminates the afferent compound
action potential (although this toxin could be acting both
on the IHC and the afferent neuron) [27

]. Thus, a
carefully orchestrated interaction between VGCCs and
BK channels is required for effective synaptic transmis-
sion from the mammalian IHC.
Capacitance measurements
The hair cells membrane area changes when vesicles
fuse or pinch off the plasma membrane during exocytosis
and subsequent endocytosis, causing a change in mem-
brane capacitance (C
m
). These changes in membrane
capacitance can be detected by measuring the capacitave
current (I
Cm
C
m
(dV/dt)) during voltage-clamp record-
ings from hair cells [28,29]. There are several advantages
to this technique. Presynaptic C
m
provides a measure of
vesicular cycling that is not subject to postsynaptic lim-
itations of receptor saturation and desensitization. C
m
measurements can be conveniently combined with other
techniques, such as ash photolysis of caged Ca
2
[16

],
to describe exo- and endocytosis. Although capacitance is
a measure of the net change in membrane area, exocytosis
and endocytosis can be studied separately because of
their different time courses. Only at very high cytosolic
Ca
2
concentrations ([Ca
2
]
i
) does endocytosis begin to
overlap exocytosis [16

].
One disadvantage of whole-cell capacitance recordings is
their low sensitivity, which precludes detection of single
vesicle fusion. In addition, C
m
changes under various
experimental conditions are not necessarily synaptic in
origin. So one must ask, how strong is the correlation
between C
m
and synaptic transmission from hair cells?
Voltage-evoked increases in C
m
of hair cells do require
inux through dihydropyridine-sensitive Ca
2
channels
[17

,29]. Moreover, ash photolysis of caged Ca

2
causes
C
m
to rise with a higher order dependence on [Ca
2
]
i
like
that established for synaptic vesicle fusion [16

]. Finally,
the C
m
change evoked by single calcium action potentials
[30

] predicts an amount of vesicular release per active

zone similar to that measured directly by recording action
potential-evoked postsynaptic currents from afferent
dendrites [31

] (see following section on Postsynaptic

effects of release from ribbons).
If we accept that an increase in C
m
represents synaptic
vesicle fusion, then C
m
recordings provide important
insights into the hair cell vesicle pools and the kinetics
of transmitter release. Two kinetic components of exo-
cytosis are observed during voltage-gated Ca
2
inux
[29]. A smaller, faster phase of capacitance increase
saturates within milliseconds and is best resolved when
exogenous Ca
2
chelators are used to prevent the larger,
slower component. Under these conditions, the fast com-
ponent corresponds to the fusion of 58 synaptic vesicles
with a maximal rate of 2,000 vesicles/second at each of
about 25 active zones of an apical IHC of the mouse. This
high initial rate of vesicle fusion clearly sufces to support
the highest ring frequency of the auditory nerve [4].
This readily releasable pool presumably corresponds to
those vesicles docked close to sites of Ca
2
entry at the
active zone. This assumption is supported by the obser-
vation that neither the amplitude nor the time course of
this fast component were affected by a high concentration
of the Ca
2
chelator EGTA.
In contrast, with no added Ca
2
buffers (that is, relying
only on the hair cells intrinsic cytoplasmic buffers), a
sustained large amplitude C
m
increase is observed during
depolarization [17

,28,29]. For the rst second of sti-

mulation, secretory rates of around 8.700 vesicles/sec-
ond, around 8.200 vesicles/second and around 4.000
vesicles/second were estimated for mouse cochlear inner
hair cells, frog saccular hair cells and chicken tall hair
cells, respectively. Secretory rates are provided for the
whole cell rather than per active zone, because it is not
clear how much of this exocytosis actually contributes to
fast synaptic transmission at the hair cell afferent
synapse. The high secretory rate and the lack of satura-
tion clearly distinguish this sustained component of
exocytosis in hair cells from that of retinal bipolar nerve
terminals [32].
When native cytoplasmic buffers were replaced with an
excess of high afnity calcium buffer, the sustained
component of exocytosis in hair cells declined more
than ten-fold (in distinction to the RRP that was unaf-
fected by this treatment). This suggests that the sus-
tained component depends on the delayed rise of Ca
2
at some distance from the active zone. At least two
mechanisms come to mind: a Ca
2
dependent resupply
of vesicles to the RRP, or delayed fusion of outliers,
vesicles that are docked at sites remote from Ca
2
entry
at the active zones [6

]. Some evidence suggests that

outliers do contribute. In particular, the resupply of
vesicles to the RRP appears to be too slow to account
for the reduction in sustained exocytosis produced by
strong calcium buffering. The maximum rate of recovery
(the resupply rate) of the RRP in mouse IHCs was an
order of magnitude smaller than the rate of exocytosis
during ongoing stimulation [29]. Thus, the loss of this
component could not produce the tenfold reduction in
exocytosis seen with strong calcium buffering. On the
other hand, the contribution from outlier vesicles could
454 Sensory systems
Current Opinion in Neurobiology 2003, 13:452458 www.current-opinion.com
be quite large, as suggested by the rapid C
m
rise pro-
duced by photolysis of caged calcium, rather than that
produced by inux through voltage-gated channels. The
global rise of cytosolic [Ca
2
] produced by buffer photo-
lysis caused a rapid rise in C
m
that exceeded the RRP
size by about two orders of magnitude [16

]. Therefore,
hair cells seem to contain a greater number of fusion-
competent vesicles than those that can be released
during brief calcium transients at the active zone, and
these may be recruited for release during sustained
elevations in calcium or by the global calcium signals
created by buffer photolysis.
Postsynaptic effects of release from ribbons
Individual spiral ganglion neurons make a single bouton
ending on a single IHC in the adult mammalian cochlea.
Thus, an intracellular recording from an afferent bouton
should reveal the entire input provided by its presynaptic
ribbon. Voltage-clamp recordings from afferent boutons
in the cochlea of one to two week old rats showed that the
majority of spontaneous excitatory postsynaptic currents
(EPSCs) had a uniformand rapid time course, lasting only
12 ms at room temperature [31

], consistent with the

demands of acoustic timing. EPSCs were carried by non-
selective cation channels, and were sensitive to a-amino-
3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
receptor antagonists, conrming previous in vivo studies
[33] and immuno-EMshowing the glutamatergic receptor
subunits (GluR)2/3 and 4 at this synapse [34]. However,
EPSC amplitudes were much larger and more variable
than would be expected from the spontaneous release of
single vesicles. EPSCs recorded at 90 mV ranged more
than 20-fold in amplitude, up to a maximum of 800 pA
(around 9 nS), with an average of 150 pA in most record-
ings. Thus, spontaneous EPSCs at the ribbon synapse
were quite different from glutamatergic minis seen
elsewhere, which would be distributed normally about
a mean amplitude of 3040 pA (around 0.4 nS) in these
conditions. Interestingly, although the amplitude histo-
gram of ribbon EPSCs was skewed to larger values, a
prominent modal peak at around 30 pA was present in
most recordings.
Thus, one possibility is that the modal peak represents
single vesicles, with larger EPSCs resulting from multi-
vesicular release, as suggested by earlier in vivo studies
[35]. How might coordinated multivesicular release
occur? In cerebellar Purkinje cells, it has been hypothe-
sized that large amplitude minis might result from the
release of calcium from presynaptic calcium stores [36].
Indeed, ryanodine-sensitive (ryanodine affects calcium-
induced calcium release from internal stores) calcium
transients have been observed during depolarization of
mouse inner hair cells [37

], although their signicance

for transmitter release remains unknown. Another possi-
bility is suggested by the intriguing way that synaptic
vesicles are tethered to the electron dense body at ribbons
in hair cells and retinal cells. It has been suggested that
this arrangement facilitates a process of compound (inter-
vesicular or homotypic) fusion to cause larger EPSCs
[38

]. Preliminary results from a quantal analysis of repe-

titive C
m
increases are compatible with such an hypoth-
esis of compound fusion, at least at the immature synapse
(D Khimich, T Moser, unpublished data). These and
other possibilities await further study.
Does an hypothesis of multivesicular release accord with
measurements of presynaptic C
m
? Certainly the max-
imum release rates seen in IHCs could result from the
simultaneous release of many vesicles at each synapse. A
direct comparison of presynaptic C
m
and EPSCs can be
made during release produced by calcium action poten-
tials in neonatal IHCs. A single action potential caused
an average C
m
increase equivalent to the release of 40
vesicles from each of a hair cells about 25 synaptic
ribbons [30

]. Likewise, spontaneous action potentials

produced an average EPSC burst in single afferent
boutons equivalent to the release of 47 vesicles, a mea-
surement formed on the basis of the estimated single
vesicle response from the amplitude histograms [31

].
The close correspondence between these two results
helps to justify the very different assumptions under-
lying each method, and supports the hypothesis of multi-
vesicular release.
Excitatory amino acid transporters
Glutamate released from the IHC may be taken up by
specic transporters in the surrounding supporting cells.
Among the ve subtypes of Na

-dependent glutamate
transporters, immunohistology shows EAAT1 (excitatory
amino acid transporter type 1, also known as GLAST)
expression in supporting cells surrounding cochlear hair
cells [39,40,41

], although others are found in afferent

neurons [42

]. Functional evidence for the presence of

glutamate transporters has been obtained recently by
voltage-clamp recording from inner phalangeal cells,
which surround inner hair cells in the rat cochlea [43].
These currents were not observed in a GLAST knockout
mouse (D Bergles, E Glowatzki, unpublished data).
Emphasizing the functional signicance of glutamate
transport, the GLAST knockout mouse suffers noise-
induced damage to afferent terminals and associated
hearing loss [44]. Afferent terminal swelling can be pro-
duced in the guinea-pig cochlea by perfusion with a
glutamate transporter blocker [42

], this treatment also

suppresses the tone-evoked compound action potential.
These deleterious changes can be prevented if an AMPA
receptor blocker is also applied.
Conclusions
Several important steps have been taken in the study of
the hair cell ribbon synapse over the past year. A rm
description of the structural and functional basis of
ribbon release will soon emerge from the combination
Hair cell transmitter release Fuchs, Glowatzki and Moser 455
www.current-opinion.com Current Opinion in Neurobiology 2003, 13:452458
of capacitance measurements with highly detailed ultra-
structural studies, as exemplied by the analogy between
slower components of release and outlier vesicles. Post-
synaptic recordings from afferent boutons have nearly
settled some questions, such as the functional identica-
tion of postsynaptic glutamate receptors, but at the same
time they have raised new questions concerning the
mechanisms of release.
Studies of hair cells not only extend the frontiers of
synaptic physiology but also have a profound impact
on our understanding of inner ear disease. Some inher-
ited deafness is known to result from mutations in vol-
tage-gated ion channels whose normal role must be to
shape the receptor potentials that drive transmitter
release (e.g. [45]). A growing body of evidence suggests
not only that glutamate excitotoxicity itself can disrupt
cochlear function but it also may contribute to common
pathologies such as the hearing loss that follows loud
sound exposure [46

]. Understanding the hair cell ribbon

synapse will not only help to complete our knowledge of
inner ear function but it may also inspire newtherapeutic
strategies aimed at ameliorating or even preventing
cochlear pathogenesis.
Update
The role of synaptic ribbons in transmitter release by
hair cells has received additional conrmation in recent
work by Zenisek and colleagues [47

]. These authors
used calcium imaging methods and immunohistology to
draw a compelling correspondence between sites of
voltage-gated calcium inux and the synaptic ribbons
of retinal bipolar cells and saccular hair cells. A uor-
escent calcium indicator dye (Fluo-3 or Fluo 5F) was
injected into hair cells along with a high concentration of
calcium buffer (EGTA). Calcium evoked uorescence
was visualized with evanescent wave uorescence micro-
scopy that only captures light within 100 nm of the
plasma membrane. Combined with the high concentra-
tion of calcium buffer that limits repeated binding of
calcium to indicator dye, this technique enabled the
authors to visualize near-membrane calcium hotspots
that were a fraction of a micrometer in diameter, and
whose brightness varied directly with calcium channel
gating. The density of calcium hotspots in bipolar cells
and hair cells corresponded closely to the pattern of
uorescent puncta labeled with an antibody to the
synaptic ribbon protein Ribeye. Thus, voltage-gated
calcium channels may be expressed specically at trans-
mitter release sites demarcated by synaptic ribbons, as
suggested in earlier studies on hair cells of frog [48,49]
and chicken [50].
Acknowledgements
Work in the authors laboratories is supported by the National Institute of
Deafness and Communication Disorders at the National Institutes of Health
(P Fuchs and E Glowatzki), and by the Deutsche Forschungsgemeinschaft
and the Max Planck Gesellschaft (T Moser).
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458 Sensory systems
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