Review Resources
Reviews:
Safieddine et al., 2012, The auditory hair cell ribbon synapse:
From assembly to function, Ann Rev Neurosci, 35:509-528
Salamanca Study Abroad Program:
Neurobiology of Hearing
Kim et al., 2013, Single Ca2+ channels and exocytosis at
sensory synapses, J Physiol, epub
Note: this is an excellent review describing the differences
between cochlear and retinal ribbon synapses.
Synaptics and the auditory nerve
R. Keith Duncan
University of Michigan
[email protected]
Rabbitt and Brownell, 2011Efferent modulation of hair cell
function, Curr Opinion Otolaryngol Head Neck Surg,19:376381
Outline
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Afferent cochlear innervation
Exocytosis and synaptics
Afferent physiology
Efferent physiology
Type 1 afferents:
• 95% of cochlear afferents
• account for hearing
• each innervates only 1 IHC
• ~20 innervate one IHC
Type 2 afferents:
• less well-described
• en passant innervation of OHCs;
Hair cells form ribbon synapses with
afferent neurons
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Distinct pools of vesicles
• Electron tomography
reconstruction shows 4 pools
Ribbon docked vesicles
(readily releasable pool, 10)
Tethered vesicles
(releasable pool?, 200)
Extrasynaptic docked
vesicles
Cytoplasmic vesicles
(recruited pool?)
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Lenzi & von Gersdorff, 2001
• These pools likely contribute to
distinct kinetics of exocytosis
as stimulus duration increases.
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Distinct phases of exocytosis
The Synaptic Vesicle Cycle Exocytosis/Endocytosis
Capacitance is measured as
a proxy for fusion of vesicles
(exocytosis).
Fusion increases surface
area => capacitance.
Measure using voltage-clamp
depolarizations of various
durations.
Explained further in
supplemental slides.
Approximately 10% of cellular proteins exert a role in membrane traffic and protein targeting.
Molecular Components of
Ribbon Synapses
Vesicle related:
• Synaptotagmin IV, VI-IX (rare types)
• Rab3 and Rab-binding proteins
• Synaptobrevin 1 (VAMP-1)
• Otoferlin…a calcium sensor in hair cells
• Synapsins are not present
• Synaptotagmin I & II are found only in
immature, pre-hearing cochleae. KOs cause
little defect.
Lenzi et al., 1999
Core-complex related:
• Syntaxin 1A
• SNAP-25
• Munc13 is not present (hair cell or retinal
bipolar cell). Ribbon may replace priming role.
Major Open Question Area: what are the roles of various SNARE proteins?
Exocytosis depends on calcium
What do ribbon proteins do?
Tethers have
synapsin-like
function. But no
synapsins present.
Ribeye?
Bassoon anchors
the ribbon?
Organizes Ca2+
channels?
Otoferlin promotes
docking, fusion?
Ca2+ sensor?
VCa
Exocytosis occurs in calcium
nanodomains
Hair cells
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Dependence on Ca2+ is reduced with age
Methods to study post-synaptic
response
Similar amount of fusion but much
less Ca2+ required at P20.
Open questions:
• Increased efficiency?
• change in Ca2+ sensors,
channel types, localization, etc
• Is the Ca2+ change at synapse or
extrasynaptic?
Modeling indicates that by P20, only 1 calcium ion is required to
induce fusion of a single vesicle.
Glowatzki and Fuchs, 2002
Primary Afferents are Glutamatergic
Post-synaptic potentials span a wide range
of amplitudes, not a train of miniEPSCs
Whole-cell voltage-clamp
Vhold = -94 mV
Inward current
Most monophasic
(coordinated release)
Red: Ribeye (Ctbp2)
Green: GluR2/3
Multivesicular release
Ribbons vs Conventional Synapse:
Quantal yet multivesicular
Similar distribution if hair cell bathed in
5 mM K+ or 40 mM.
Frequency of EPSCs increased but
amplitude range unchanged.
True miniEPSC ~40pA
Mean amplitude ~150pA (4 vesicles)
Max amplitude ~800pA (20 vesicles)
Multivesicular release = large ave. EPSC
Bouton has very high input resistance.
EPSP ~ EPSC * Rin
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Stimulus Features Coded by the
Auditory Nerve
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Response to Tone Bursts:
Peristimulus Time Histograms (PSTH)
How does the auditory nerve encode:
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Frequency?
Intensity?
Time?
Pure-tones vs complex stimuli?
Not due to hair bundle adaptation, but
rather dynamics of vesicle trafficking and
fusion.
Tuning Curves and Spectral Response Plots
Response to Frequency: Place Coding
How to generate a tonotopic map
Salient features:
• Iso-response contour or
“tuning curve”
• Threshold (i.e. 26 dB)
• Best Frequency (i.e. 0.8
kHz)
• Frequency Selectivity
(Q10dB = CF / Bandwidth
10 dB above threshold)
0.1
1
4
Frequency (kHz)
Saunders et al., 1996: Auditory nerve responses from chick cochlea
Response to Intensity:
Response to Intensity: RI functions
SPIKE RATE (Spikes/S)
Tone Burst
Threshold Varies with Spontaneous Rate
HSR
• 60% of primary afferents
• > 18 spikes/s; low threshold
• Steep rate-intensity function;
narrow dynamic range (20-30 dB)
• Broadly tuned
MSR
• 25% of afferents
• Between 0.5 and 18 spikes/s
• Sloping-saturation RI function
LSR
• 15% of afferents
• < 0.5 spikes/s; high threshold
• Shallow sloping RI function; large
dynamic range (>60 dB)
• Sharply tuned
INTENSITY (dB)
Why this diversity of RI types?
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Spontaneous Rate:
Multi-modal Distribution
Response to Intensity:
Mechanistic View of Spontaneous Rate
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HSR and MSR/LSR fibers
arise from separate regions
of the inner hair cell
(Liberman, 1982).
Models suggest SR
differences are presynaptic
– differences in calcium
current density (Sumner et
al., 2002).
Patch-clamp of the hair cell
shows non-uniform
distribution of calcium
channels (singles, groups of
2-15, groups of 20-80)
(Rodriguez-Contreras and
Yamoah, 2001).
MSR
LSR
HSR
Liberman, 1978: Auditory nerve responses from cats
Frequency, Place, and Intensity:
Is that it?
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So far…
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Response to Complex Stimuli:
A possible limit to place code theory
F1-3 are formant frequencies for the vowel sound “eh”
We have a mechanism for analyzing frequency content of
sounds (cochlear mechanics).
We can send frequency information to central auditory
systems with labeled lines using a place code.
We can cover a large range of intensities by combining
activity across multiple ANFs from a single hair cell.
Caveat: so far, we’ve only really looked at pure tones.
If place-rate code is all we need for frequency and
intensity discrimination, then spectral content of
complex sounds should not change with intensity.
Intensity coding preserved in phaselocked responses
Phase-locking
The discharges of cochlear nerve fibres to
low-frequency sounds are not random;
they occur at particular times (phase locking).
Evans (1975)
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Response to Time: Phase-locking
Depends on Stimulus Frequency
The ability of the auditory nerve to
synchronize to the sound stimulus is
dependent on the stimulus frequency.
Phasic Responses in High-Frequency
Neurons
In vivo intracellular
recording from a hair cell.
However, there are limits to this coding
modality, due to:
• Capacitance of the hair cell
membrane (LPF cutoff ~ 2 kHz)
• Maximum spike rate of a neuron is
around 1000 kHz.
Auditory neurons show robust phaselocked responses up to 4 kHz.
Phasic Responses in High-Frequency
Neurons Driven at Low-Frequencies
Synchrony within Complex Sounds is
Preserved
ALSR:
Average localized
synchronized rate
function
ALSR accounts for:
(1) firing rate and
(2) synchrony to the
stimulus.
How is frequency coded:
place or timing?
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Place Code
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efferent
Pros: could in principle work at all frequencies
Cons: spectral information by place-alone is compromised
at high sound levels
Temporal Code
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It’s a two-way street
Pros: phase-locking is basically level invariant
Cons: breaks down completely above about 4 kHz
The most important function of place code may be
that it prepares sounds for temporal coding by
dividing the spectra of complex sounds into narrow
bands before their conversion into a temporal code.
Alternatively, if you isolate the activity of lowspont/high-threshold fibers, it is possible to recover
the spectra without fine temporal structure.
A deeper understanding of CNS computations is
needed!
afferent
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Division of Labor in Cochlear Hair Cells
OHC efferent innervation
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Efferents from medial olivocochlear system (bilaterally) synapse
onto several OHCs.
MOC Inhibition:
Two channel hypothesis
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Stimulation of MOC fibers reduces
cochlear sensitivity
MOC efferents
MOC efferents release ACh.
Ca2+ influx activates SK/BK.
K+ efflux hyperpolarizes hair cell.
Hyperpolarization would enhance
the drive on transduction but shift
operating range of the cochlear
amplifier.
K+, SK
Ca2+,
AChR
ACh
Wersinger and Fuchs, 2011
Division of Labor in Cochlear Hair Cells:
IHC efferent innervation
LOC fibers regulate afferent sensitivity
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Less well understood
What neurotransmitters are
released?
What effects?
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Efferents from lateral olivocochlear system synapse onto hair cell
early in development and then onto the Type I afferent bouton.
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When activated, the LOC elicits
either slow excitation or suppression
of cochlear output.
Putative roles include protection from
excitotoxicity from noise, gain
control, and binaural adjustment of
sensitivity for sound localization
cues.
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Open questions
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Supplementary material
What underlies the diverse dynamic ranges of spont.
rates in Type I afferents?
What is the functional role of Type II afferents?
Are complex sound spectra encoded by fine temporal
structure or high-threshold fibers at loud sound
levels?
What roles do other neurotransmitters have in
efferent system (GABA, CGRP, etc)?
Does the LOC enable ‘Gain control’ of the auditory
periphery?
Time-resolved Membrane
Capacitance Measurements
Itotal = IR + IC
where IR is current through the resistive arm
and IC is current through the capacitor
Ra
Rseal
Cm
1
Vm  Er   Cm dVm
Rm
dt
Now, under voltage-clamp, stimulate with a
sine wave. Capacitive current will vary as
cosine.
Note 90o phase shift between IR and IC allowing
each I to be monitored or determined
separately using a lock-in amplifier
The amplitude of the Cm current is dependent
upon the cell surface area. (~ 1 mF/cm2).
Vesicles fuse at calcium nanodomains
Current
Voltage
Rm
I (t ) 
Time
IR
IC
Rapid calcium buffering prevents the outward current
The calcium sensor for
vesicle fusion is nearly
saturated.
Fusion depends on the
activity of 1-2 channels.
Ribbon likely enhances
localization of docked
vesicles with Ca2+ channels.
Brandt et al., 2005
Addition of
BAPTA to
cytoplasm
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Blocking potassium channels to isolate the hair cell’s
calcium current
Dihydropyridine-sensitive calcium channels –
(α1D - CaV1.3) in hair cells
Fuchs et al., 1990
AMPA receptors mediate rapid glutamatergic
signaling from OHCs to type II afferents
Amplitude histograms: Type I and
Type II EPSCs
Type I
Type II
Glowatzki and Fuchs 2002
Synaptic signals are small and infrequent in Type II fibers
Avg. Amplitude
Type I
Type II
21 cells
4,083 EPSCs
28 cells
9371 EPSCs
~150 pA
26.3 pA ± 13.7
0.43 ± 0.10 ms
1.11 ± 0.37 ms
1.07 ±0.22 ms
3.12 ±1.21 ms
1.5 ± 1.1 Hz
0.12 ± 0.13
10-90% Rise Time
Time constant decay
Frequency in 5.8 mM K
Frequency in 40 mM K
27.2 ± 22.1 Hz
5.13 ± 4.8 Hz
Pre-synaptic cell
membrane mV
~ -63 mV (IHC)
~ -65 mV (OHC)
Pre-synaptic cell
membrane mV in 40mM K
~ -35mV
~ -37 mV
Type I afferents
Type II afferents
large caliber
One IHC
Glutamate
Sensitive
Hearing
small caliber
Many OHCs
Glutamate/ATP
Insensitive
Pain(?)
Functional dichotomy parallel to somatic sense: discriminative touch versus
pain/temperature.
Pathogenesis of hyperacusis, tinnitus related to neuropathic pain?
Glowatzki and Fuchs, ‘02
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