The hair cell ribbon synapse - Tobias Moser, University of Goettingen
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The hair cell ribbon synapse
Tobias Moser, University of Goettingen
Department of Otolaryngology
Center for Molecular Physiology of the Brain
Bernstein Center for Computational Neuroscience
Goettingen Graduate School of Neuro- and molecular Biosciences
Regis
Nouvian Alexander
Darina Meyer
Andreas Khimich
Brandt
Thomas
Frank
CNS
www.iurc.montp.inserm.fr/cric/audition
Kandel et al., Principles of Neuroscience
Hearing impairment: most frequent human sensory deficit within the US(NIDCD) ~ 28 Mio hard of hearing (increasing due to aging population) ~1:1000 newborns affected by congenital hearing impairment More than 100 genes: Ion channels, structural proteins, transporters, transcription factors… The function of the affected protein in hearing and the mechanism of deafness can be revealed in animal models.
•Introduction into synaptic transmission •Structure and Function •Stimulus-Secretion coupling •Synaptopathic hearing impairment Recent reviews/Books: Fuchs, P., Glowatzki, E., Moser, T. (2003) The afferent synapse of cochlear hair cells. Curr Opin Neurobiol Fuchs, P. A. (2005). Time and intensity coding at the hair cell's ribbon synapse. J Physiol. Sterling, P. & Matthews, G. (2005). Structure and function of ribbon synapses. Trends Neurosci Nouvian, R., Beutner, D., Parsons, T. D., Moser, T. (2006). Structure and Function of the Hair Cell Ribbon Synapse. J Membr Biol. Moser, T., Brandt, A., Lysakowski, A. Hair cell ribbon synapses Cell Tiss Res 2006 Moser, T., Khimich, D., Neef, A. J Physiol 2006 Fuchs, P.A. and Parsons, T.D. Handbook of auditory physiology Classical reviews/Books: Roberts, W. M., Jacobs, R. A. & Hudspeth, A. J. (1991). The hair cell as a presynaptic terminal. Ann N Y Acad Sci Geisler, C.D. From Sound to Synapse
•Introduction into synaptic transmission
Principles of synaptic transmission
Conventional Synapse Ribbon Synapse
Action potentials drive phasic all or none Transmission is governed by graded potentials and
transmission tonic
Goutman and Glowatzki,
PNAS 2007
Spikes per bin
Meinrenken et al., 2003
Strenzke, Buran &
Liberman
K+
Coding of sound and vestibular stimuli
at hair cell ribbon synapses
mechanical stimulus
receptor potential
CNS
spike rate
Ca2+
rate of
transmitter release
Synaptic ribbon
Functional Properties of Hair Cell Ribbon Synapse
Temporally precise sound coding over a wide
range of intensities and long periods of time
Avissar et al, 2007 Palmer and Russell, 1986
Impaired Processing of temporal Structure affects:
Speech Recognition, Sound Localization …..
Indefatigable Sound Coding
Auditory Fatigue
What is the synaptic ribbon?
Electron dense structure decorating
the active zone
„Synaptic nanomachine“
Scaffold, Synaptic organizer
Conveyor belt
Safety belt
Endocytosis machine e.g. Neef et al., J Neurosci 2007
Vesicle stamp
……..
RIBEYE
Otoferlin
Nouvian et al. J Membr Biol 2006 Schmitz et al., Neuron 2000
The number of synaptic contacts per IHC varies
along the cochlea
500 µm
Meyer et al., unpublishedPrinciples of synaptic transmission
„Conventional Synapse“ Ribbon Synapse
Calyx of Held: large synapse of the auditory pathway, 600 AZs
Electroreceptor Hair cell
Meinrenken, C.J., Borst, J.G.G. & Sakmann, B. J Physiol 2003
NMJ
Lenzi & von Gersdorff BioEssays 2001
Photoreceptor
Sterling & Matthews, TINS 200
Harlow .. & McMahan, Nature 2001Principles of synaptic transmission
„Conventional Synapse“ Ribbon Synapse
Calyx of Held: large synapse of the auditory pathway, 600 AZs
Electroreceptor Hair cell
Meinrenken, C.J., Borst, J.G.G. & Sakmann, B. J Physiol 2003
NMJ
Lenzi & von Gersdorff BioEssays 2001
Photoreceptor
Sterling & Matthews, TINS 200
Harlow .. & McMahan, Nature 2001Principles of synaptic transmission
„Conventional Synapse“ Ribbon Synapse
Calyx of Held: large synapse of the auditory pathway, 600 AZs
Electroreceptor Hair cell
Meinrenken, C.J., Borst, J.G.G. & Sakmann, B. J Physiol 2003
NMJ
Lenzi & von Gersdorff BioEssays 2001
Photoreceptor
Sterling & Matthews, TINS 200Principles of synaptic transmission
Neuromuscular Junction
Calyx of Held Ribbon SynapsePrinciples of synaptic transmission
„the synaptic vesicle cycle“
Südhof, Ann Rev Neurosci (2004)
•Synaptic vesicle – quantal hypothesis
of synaptic transmission
•Active Zone of transmitter release
•Stimulus-Secretion couplingPrinciples of synaptic transmission
•SNARE proteins/hypothesis
•SNARE regulators
•Ca2+ sensor of exocytosis
•Cytomatrix of the Active Zone
•Ca2+ channel
Rizo und Südhof, Nature
Reviews Neuroscience 3, 641-
653 (2002),
Wojzic and Brose Neuron 2007Conclusions I Coding of acoustic and vestibular stimuli tales place at specialized synapses: ribbon synapses The hair cell synapse shows astonishing capabilities in temporally precise coding of a large range of receptor potentials over long periods of stimulation. The ribbon is a primarily morphologically defined structure at sensory synapses of inner ear and retina with mostly unknown function. Ribbon synapses display rather large active zones (somewhere between CNS and NMJ active zones). Transmitter release at ribbon synapses is governed by graded potentials rather than by action potentials. The molecular dissection has only begun and reveals important similarities but also differences to well studied CNS synapses.
•Structure and Function
Morphological and Molecular Analysis
transmission electron microscopy: Smith and confocal analysis
Sjostrand…………………… GluR2/3/RIBEYE
Synapse reconstruction from serial sections:
Schnee…Furness, Ricci, Neuron 2005
Quantitative 4Pi microscopy of
fluorescently labeled ribbons
electron tomography:
Lenzi et al., J Neurosci 1999, Neuron 2002
Freeze fracture (Roberts et al., 1990)
Immunoelectron microscopy (Matsubara et al., 1996) Khimich et al., Nature 2005Morphological and Molecular Analysis:
EM-tomography
Calculated Vesicle capacitance: 37 aF
0Ca2+, 2 EGTA 45 K+
Lenzi…Roberts, J Neurosci 1999, Neuron 2002Morphological and Molecular Analysis
Shape/size of the
ribbon depend on:
•species
•organ
•tonotopic position
•spontaneous rate of nerve fiber
•age
~10-50docked vesicles (first to go?)
~40-400 ribbon associated vesicles
Details in Review by Nouvian et al., J Membr Biol 2006Structure – Function I
Larger ribbons at synapses with low spont. fibers
Merchan-Perez & Liberman, J Comp Neurol 1996
Nerve fibers differ in spontaneous rate and threshold: see Ian Russell‘s Talk
Backfilled high spont. fibers preferentially contact the pillar side of Cat IHCs
Liberman, Science 1982Morphological and Molecular Analysis
Synaptic connectivity
Amphibian and reptile HC
Mammalian Cochlear Avian short HC
Inner HC Type II Vestibular HC,
Bouton-type ending
Functional consequences?
Type I and II
Type I Vestibular HC
Vestibular HC,
Calyx-type ending
Dimorphic endings
Details in Moser et al., Cell Tiss Res 2006Conclusions II Ribbons/dense bodies range in number per hair cell (5-60), size (80-400 nm) and shape (plate – sphere) among organs, species, develeopmental stages and tonotopic position Serial reconstruction from EM and EM tomography yield the most reliable estimates for ribbon and vesicles, high resolution Light microscopy is good for number and size of ribbons in large samples Docked vesicle counts range between ~10 and 50 Total ribbon associated vesicles between 40 and 400 Hair cells can talk to several nerve fibers, each nerve fiber may sample just one or more active zones/hair cells. None of the classical fast Ca2+ sensors is present
Functional Analysis
Auditory brainstem response synchronous activation of the nuclei along the ascendant auditory pathway
K+ How to study function/dysfunction
of the Hair Cell Ribbon Synapse?
mechanical stimulus
Optical measurements
h∗v
Ca2+ dyes
Membrane dye
h∗v
Patch-clamp recordings receptor potential
from the postsynaptic fiber
Patch-clamp measurements of membrane
current (Im) and capacitance (Cm) at the IHC
depolarization
spiking rate
Ca2+
rate of
•classical in vivo transmitter release
auditory nerve fiber recordings
•auditory evoked potentials Ca2+
Ca2+
UV-photolysis
of photolabile Ca2+-chelatorKinetics of transmitter release - FM1-43 Imaging -Function of single presynaptic active zones can be studied -population behavior of vesicles Griesinger et al., J Neurosci 2002 -sensitivity for detecting single exocytic event? Griesinger et al., Nature 2005 -specificity for reporting synaptic vesicle exocytosis indicated by Ca2+ dependence should be further tested in mutants and genetic vesicle labels may be useful
Kinetics of transmitter release-postsynaptic recordings
Highly synchronized release of several vesicles: ‘multivesicular’
Glowatzki and Fuchs, Nat Neurosci 2002
Neef et al., submitted
paired pre- and postsynaptic recordings
Keen and Hudspeth, PNAS 2006Membrane Capacitance Measurements
Ca2+
Exocytosis Endocytosis
80 100 ms
Δ Cm (fF)
Cm (pF) F360/F390
60 1.0
40
50 ms
20
0 10 ms 0.8
0
Im (pA)
8.2
-100 8.0
0.2 0.4 0.6 0.8 1.0 10 s
s
Moser and Beutner, PNAS 2000
-whole-cell capacitance measurements:
exo- and endocytosis at all synapses and throughout the cell
low sensitivity, but truly presynaptic measurement
specificity for synaptic vesicle exocytosis is indicated by Ca2+ dependence and work in mutants
-use cell-attached capacitance measurements for exploring single fusion and fission eventsThe Readily Releasable vesicle Pool of IHCs
20
15
fF
10
5
0
250 0 10 20 30 40 50
ms
200
Δ C m (fF)
RRP:
150 •low sensitivity to EGTA
100 0.1 mM EGTA •saturates within msec
5 mM EGTA •depends on the presence of the ribbon
50 •probably docked vesicles
0 •mediates synchronous transmission
Sustained component:
0 500 1000 •Highly sensitive to EGTA
duration of depolarization (ms) •Ca2+-dependent vesicle supply and synaptic release
(Moser and Beutner, 2000; Spassova et al., 2004; Edmonds et al., 2004;
Keen & Hudspeth et al., 2006)
•Depends on the on the presence of the ribbon?
(Khimich et al., 2005)
Moser and Beutner, PNAS 2000
Beutner et al., Neuron 2001
parallel fusion of vesicles docked
remote from Ca2+ channels: outliers
(Moser & Beutner 2000; Beutner et al., 2001; Lenzi et al., 2002)Size of RRP and rate of sustained exocytosis
co-vary with the number of synaptic contacts
60
12 synapses
Capacitance increase (fF)
50
40
8 synapses
30
20
10 apical (avg. DTA: 303 µm, n=14)
basal (avg. DTA: 1383 µm, n=11)
0
0 50 100 150 200
Depolarization (ms)
Exocytosis varies by a factor of ~ 1.4
Ca2+ current varies only by a factor of ~ 1.1
Meyer et al., unpublishedThe synaptic ribbon (and/or Bassoon) is required for stabilizing
the large RRP of hair cell active zones and normal hearing
RRP ~ 29-34 SV
12-21 docked SV
~90-150 total ribbon-associated SV
RRP ~ 8 SV
Khimich et al., Nature 2005Structure – Function II
Correlating structural and functional vesicle
populations
Nouvian et al., J Membr Biol 2006Conclusions III Various techniques have now been applied for functional studies and have yielded quite consistent results. Presynaptic capacitance recordings are straight forward, avoid complications due to postsynaptic receptors properties etc. and have provided a basic description of functional vesicle pools. However, they are of limited sensitivity and still of uncertain specificity for synaptic release. Postsynaptic recordings are tough, but essential for characterizing the postsynaptic properties. In combination with presynaptic patch-clamp they will clarify the whole synapse’s response and some presynaptic issues: e.g. mode of release.
Imaging requires more specific indicators, but has great potential for mechanistic studies in more “in vivo” conditions. Combining cellular approaches and systems tests provides a powerful set-up to study normal and impaired synaptic function. The RRP most likely reflects exocytosis of vesicles that are docked close to Ca2+ channels. The RRP is large and supports synchronous release of several vesicles. Thereby, it probably helps to reduce the jitter of the postsynaptic spiking. The hair cell ribbon synapse is capable of massive sustained transmission, probably involving a Ca2+ dependent vesicle re- supply. The RRP size is operationally defined by the hair cells depolarization.
Stimulus-secretion coupling
Fusion of a vesicle is controlled by Ca2+ from
one or few, adjacent channels several Ca2+-channels
single channel characteristics matters channel population matters
Squid giant synapse (e.g. Augustine et al.) Immature calyx of Held? (e.g. Sakmann et al.)
NMJ, ciliary ganglion (e.g. Stanley et al.)
Mature calyx of Held? (e.g. Wong et al.)
Ca2+ Nanodomain Ca2+ MicrodomainYou can also read
