21/05/2015
Neurobiology of Hearing Salamanca, 21st May 2015
The cochlea: inner hair cells & the ribbon synapse Jonathan Ashmore Neuroscience, Physiology and Pharmacology University College London
Information transfer in the mammalian cochlea
1 synapse: 1 fibre
Open Univesity ‘Signals’ Fuchs J Physiol 2005 Meyer et al, 2009
1 21/05/2015
The cochlea must provide the central auditory system with :
the frequency content of the sound; achieved through mechanics and hair cell organisation
the relative phases between the time varying signals, achieved by synaptic properties and multiple fibres?
the amplitudes of the constituent sound components. achieved by synaptic properties and features of IHC?
(.. and it must be correctly wired up!)
How does an inner hair cell respond to a ‘high frequency’ sound?
Russell & Sellick 1983
2 21/05/2015
IHC synapses 1) preserve onset timing over a large range 2) adapt rapidly 3) are ‘indefatigable’
Westerman and Smith, JASA 1987
The dynamic range problem: is intensity segmented?
High sensitivity High spontaneous rate
Lower sensitivity Lower spontaneous rate
Mouse: Taberner & Liberman 2005 Cat: Sachs and Abbas 1974 Liberman 1982 Cat : shows differential distribution of fibre types
3 21/05/2015
The dynamic range problem:
1) Sound intensity is encoded over > 80 dB range 2) Individual nerve fibres only cover 20-30dB 3) => fibre population is ‘stratified’
A pre- or postsynaptic mechanism?
Synaptic technologies
Electrophysiology Patch clamp Extracellular recording from the nerve
Imaging (CLSM, 2PCLSM, STED, TIRF etc) With fluorescent tagged antibodies With calcium indicators With membrane labels
4 21/05/2015
Laser Scanning Confocal Microscopy
scanning mirrors
http://www.uhasselt.be/UH/BIOMED
2 photon laser scanning confocal microscopy
930 nm
465 nm absorption
http://biophotonics.illinois.edu/technology/mpm/
5 21/05/2015
The hair cell synapse
Pre
Post
Baran et al., 2010 Zhing et al, J NSci 2013
Each inner hair cell is associated with multiple synaptic ribbons (10 - 30)
RIBEYE / CtBP2 – ribbon protein GLUR2/3 – postsynaptic receptor Brandt, Khimich & Moser, JNeurosci, 2005
6 21/05/2015
Single and dual recording with patch clamp pipettes
High resolution but low return experiments Measures a single ribbon at a time May select subpopulation of boutons Goutman et al, pnas 2007
Initial IHC synaptic release is multiquantal (not like neuronal synapses)
Glowatzki and Fuchs, Nat Neurosci, 2002
Total vesicles per ribbon ~200 Initial release: 6-8 vesicles per ribbon (mammal) 2-3 vesicles per ribbon (frogs)
7 21/05/2015
Steady release rate is controlled; initial release is multiquantal
IHC pre
Vm (mV) i (pA)
ANF post
Goutman & Glowatzki, pnas 2007
Calcium regulates vesicle fusion at synapses: Normally, synaptotagmin (Syt) is the Ca++ dependent ‘brake’
Syt 1 and 2 not present in IHCs: an alternative calcium sensor? Otoferlin? (Roux et al, 2006; Pangrsic et al, 2010) DFNB9: OTOF codes 6 Calcium binding domains Syt 4? (Johnson et al, 2011)
8 21/05/2015
Measuring vesicles seeing vesicles:
Use membrane labels: FM1-43 or vesicle tags e.g pHluorins
FM1-43
membrane
Ca++
Membrane dye FM1-43 endocytotic uptake Guinea pig: Griesinger et al 2005
Griesinger et al 2005
9 21/05/2015
Fluorescent hotspots = ribbon release sites
2 μm
Because:
Static Correct position Reasonable size ( <1 μm diameter) Fluorescence indicates each spot = about 300 vesicles Can be destained with depolarising stimulus Destaining reduced by Ca channel blocker nifedipine Spots recover fluorescence after destaining
The mouse organ of Corti in a dish: how to record from the hair cells
back
1 mm forward
10 21/05/2015
Mouse hair cells load with FM1-43 by an endocytosis pathway
200 μM streptomycin ( = transduction blocked) : FM1-43 loads myo6 -/- mice ( = disrupted stereocilia) : FM1-43 loads
Whole cell recording under visual control through the bone
Excitation 840nm: back emission 500-650nm forward beam 840nm exciitation & emission
substage detection
11 21/05/2015
FM 1-43 hotspots destain under whole cell patch clamp
C:\Data\2008 Pasteur\080623.mdb\06_c1_HTZ_stim_Ch1.lsm 1.05 1.04
1.03
1.02
1.01 100 % 1
0.99 normalised fluorescence normalised 0.98
0.97 200 ms 0.96
0.95 95% 0 0.5 1 1.5 sec Signal equivalent to ~ 15 vesicles released in 100 ms
- 80 pA
-60mV 0 mV
The signalling pathway: calcium signals in IHCs
Pipette : 140 Cs 0.5 OGB-5N
12 21/05/2015
Multiple calcium signal hotspots
Fluorescence intensity/a.u.
abneural abneural
sec modiolar
sec 500 pA modiolar 5 μm Frame @ 406 ms subtracted Frame rates 68 fps 100 ms step to +10 from -65 mV
Information transfer in the mammalian cochlea
10-20 fibres / IHC
Spiral Ganglion Cells
Open Univesity ‘Signals’ Fuchs J Physiol 2005 Meyer et al, 2009
13 21/05/2015
Thresholds rise after noise exposure and then return to normal ABR = Auditory Brainstem Response (non-invasive measure of whole auditory nerve activity)
Narrow band noise 106 dB Kujawa & Liberman 2009
..but ribbons are lost
‘Hidden hearing loss’
Kujawa & Liberman 2009
14 21/05/2015
IHC synaptic transmission from the cochlea
Conclusions
1) Multiple ribbon synapses per inner hair cell
2) Ribbon synapses are capable of rapid triggered release of neurotransmitter – mechanism unclear
3) Fibres may be organised to segment different sound level ranges. Cellular organisation and mechanism unclear
4) What regulates which synaptic sites survive?
15