Page 1
What can one synapse tell us about
how the auditory processing works?Ian D. ForsytheMRC toxicology UnitUniversity of Leicester.
• Introduce you to the calyx of Held
• Describe the presynaptic calcium currents.
• Pose some questions about the control of synaptic
transmission
Page 2
The Calyx of Held -Auditory Brainstem
Page 3
The Synapse• Transmission along nerves/axons is
electrical pulses called Action Potentials.
•Transmission between nerve cells is by a chemical messenger called a Transmitter
Via Voltage-gatedion channels
Via Receptor-gatedion channels
EPSP -excitatory postsynaptic potential
Synapticcleft
Page 4
Neurotransmitter subtypes
Ionotropic Metabotropic
Peptides
AMPAR mGluR1-8
catecholamines
GlyR
NMDAR
M1-5 muscarinicnAchRmusc
glutamate
serotonin/5HT
acetylcholine
KainR
glycine
DA1-4
5HT3 5HT1/2/4
nAchRbrain
GABAA GABABGABAGABAC
a1/2/b1-3
purinesP2X P2Y A1/2
SP Enk VP VIP
transmitterIntegral
Ion Channel
G-protein
Coupled-
http://www.iuphar-db.org/index_ic.jsp
Page 5
Most synapses in the CNS are small
Mitochondria
vesicles
Postsynaptic cell
Release zone
From Sanes & Jessell
0.5 µm
Page 6
Mammalian CNS synapse preparations
• Mossy fibre terminal – CA3 Hippocampus– Fast glutamatergic (also peptidergic) – Geiger & Jonas.
• Cerebellar Pinceau – Basket cell, Cerebellum.– Fast Gaba’ergic synapse– Gary Stephens & Brian Robertson.
• Endbulb of Held Auditory brainstem.• Calyx of Held
– Fast glutamatergic synapse– Myself, Gerard Borst and many others.
Page 7
Hans Held
• Worked in Leipzig, Germany in the 19th Century
• Taught Bernard Katz
• Published a Golgi study of the brainstem in 1893
describing two large structures at the end of
axons….. Became known as the endbulb and calyx
of Held.
• The irony is…. He did not subscribe to the synaptic
hypothesis being developed by Sherrington.
Page 8
Confocal images of the calyx of Held
Forsythe, 1994
Page 9
midline
dorsallateral
stimulate
15 µm
The medial nucleus ofthe trapezoid body
View down the
experimental
microscopeRat & mouse
10-14 days old.
Page 10
stimulus intensity (V)
00 2 4 6 8
20
40
60
80
100
120
10
% amplitude
a
bc
20 mV40 ms
current-clamp
0.5 nA40 ms
voltage-clamp
stimulus
MNTB neurone
Calyx of Held
axon
stim.
patch pipette
Action potentialtriggered by synapticresponse
‘All or nothing’ response
The calyx of Held synapse is mediatedby the transmitter glutamate
Experimentalarrangement
A slow EPSC - NMDA receptors
A fast EPSC - AMPA receptors
Page 11
AMPAR-mediated
NMDAR-mediated
Forsythe & Barnes-Davies, 1993
Dual component synaptic currents
Page 12
Fidelity at the Calyx of Held: 4000 stimuli at 200Hz
14 nA
50 mV
5 seconds
50 ms
P15 Rat, 38oC
VC: EPSC
CC: EPSP/AP
RMP= -63 mV
First ‘error’
6.25 sec
50 msMike Postlethwaite
Page 13
mEPSCs at the calyx of Held/MNTB
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
-105-95-85-75-65-55-45-35-25-15
25 degrees
35 degrees
Temperature = 25oC
Temperature = 35oC
N = 55
Tau = 0.64 ms 1 ms10 p
A
N = 220
Tau = 0.34 ms
Amplitude distribution at 25 & 35oC
Amplitude (bin width 10 pA)Num
ber
of
events
(norm
aliz
ed)
25oC 35oC N
Amplitude (pA) -32.7
2.3
-46.0
5.7*
6
Decay Tau (ms) 0.51
0.06
0.32
0.02*
6
Frequency (Hz) 0.10
0.02
0.47
0.2*
6
Postlethwaite et al., J. Physiol 2008
Page 14
NMDAR at the calyx of Held
2 nA
50 ms
+50
-60
5 nA
50 ms
+50
-60-2
-1
0
1
2
31 nA
10 msControl
+ D-AP5
-70 -50 -30
10 30 50
-2
0
2
4
1 nA
10 ms
Control
+ D-AP5
-70 -50 -30
10 30 50
I (nA)
V (mV)
I (nA)
V (mV)
P14
P18
2 nA
50 ms
+50
-60 -2
-1
0
1
2
3
4
1 nA
10 msControl
+ D-AP5
-70 -50 -30
10 30 50
I (nA)
V (mV)
P21
Steinert et al., submitted
Page 15
Billups et al., 2002 Pflugers Arch - Eur J Physiol 444:663–669
Detectingfunctionallyconnected synapses
•Loading: 7mM Fura2-AM
for 5 minutes
•Wash for 30 minutes.
•Stimulate with short train.
•Image at 350/380 nm.
•Connected MNTB
neurones ‘light up’.
Page 16
Presynaptic Lucifer yellow fill
Page 17
0.3nA20mV
5ms
Post.
Pre
A. AP triggered B. Calcium dependence
PrePost
Simultaneous pre- and post-synaptic recording
Page 18
The calyx of Held
10 ms
1 ms
• Calyx occupies around half of the soma.
• Only one forms on each MNTB cell body.
• It forms up to 700-1500 release sites.
• P-type calcium channels mediate exocytosis.
• Exoytosis requires [Ca2+]i rise to 10-20mM.
• Endogenous Ca2+ buffering is low (Ca2+ BP).
• Mitochondria are a major non-mobile presynaptic
sequestration mechanism.
• Quantal amplitude is around 50 pA (-60 mV) 70pS.
• EPSC 3nA to over 20 nA in amplitude.
• Fast kinetics (mEPSC tau 0.31ms 350C).
• Dominated by GluRDo AMPAR subunits.
• Exhibits multiple forms of short-term plasticity,
including autoreceptor depression.
– other forms include Facilitation, depression,
depletion and desensitisation.
Fluo3 loaded into calyx of Held
calcium transients induced by two
1 ms step depolarizations
Schneggenburger & Forsythe 2006Billups & Forsythe 2002
Page 19
Which calcium channels mediate transmitter release?
• Measure the EPSC following application of different calcium channel antagonists:
» Dihydropyridines (nimodipine) blocks L- type
» Conotoxin GVIA (Ctx GVIA) blocks N-type
» Agatoxin IVA (AgaIVA) blocks P/Q type
» Cadmium [Cd2+]o blocks ALL calcium channels
• Patch the presynaptic terminal and study the calcium current following block of other voltage-gated channels:
» Tetrodotoxin (TTX)……..
» Tetraethylammonium (TEA)
» Caesium [Cs+]i
• Compare this to channels on different parts of the neurone.
Page 20
Presynaptic calcium current is P-typeE
PS
C a
mp
litu
de
(-n
A) i ii iii iv
v0
2
4
6
8
10
0 5 10 15 20 25 30
time (mins)
AgaTx-IVA (200 nM)ConoTx-GVIA (2 µM)Nimodipine (10 µM)
2nA3ms
0.2nA3ms
Forsythe et al., 1998
Page 21
P-type presynaptic calcium channels
time (min)
Calcium Current (-nA)
5 nM200 nM
50 µM Cd2+
0
0.5
1
1.5
2
0 10 20 30 40 50
-Agatoxin-IVA
Forsythe et al., 1998
Page 22
Voltage-gated Ca2+ currents
Takahashi et al., Science 1996
Page 23
Different Ca2+ currents in Bushy cell somata
and terminals……
0
10
20
30
40
50
60
L N R P
Soma – in cochlear nucleus Terminal - in the MNTB
0
20
40
60
80
100
L N R P
Calcium Channel Subtypes
a1C a1B a1E a1Aa1C a1B a1E a1A
N.B: Developmental shift from N to P/Q type channels in young animals around hearing onset
Doughty et al., 1998
Page 24
Calcium sequestration at the calyx of Held
20ms
1nA
Synaptic current
20ms
1nA
Calcium current
200ms
10µM
Presynaptic calcium
200ms
0.05 AU
Mitochondrial calcium
Postsynaptic Presynaptic
Rhod-2
FuraFF
Page 25
Calcium, Transmitter Release and Mitochondria
mitochondria
• Ca2+ triggers transmitter release.
• No evidence for presynaptic calcium stores.
• Caffeine
• Ryanodine
• Thapsigargin
• Ca2+ Mopped up:• Buffering proteins
• Extrusion by pumps
• Into other compartments
• MITOCHONDRIA (22%)
Page 26
Cytoplasmic and Mitochondrial Calcium
ControlRotenone
0.5 1.0 1.5 2.0
40
30
20
10
0
Fura FF
Time (sec)
1 2 3 4
1.2
1.0
0.8
0
0.6
0.4
0.2
Rhod-2
RelativeCa2+ mito.
Time (sec)
Cytoplasmic[Ca2+] (µM)
4 x 2 ms
@ 100Hz
Page 27
Modulation by mGluRs
Glutamate can act presynaptically as an autoreceptor,
modulating its own release.
This may be observed as reduced EPSC amplitude.
• Originally observed in Barnes-Davies & Forsythe 1995 (J. Physiol)
• Demonstrated to be of minor significance by von Gersdorff et al 1998.
• Re-examined by and shown to be present but masked in older animals.
Billups et al., 2005.
Gp III mGluR agonists
Or glutamate
Or synaptic stimulation
But remove the agonist and it quickly reverts to control amplitude.
Page 28
In young animals group III mGluRs suppress transmitter release
This is mediated by suppression of the presynaptic P-type calcium currents
Barnes-Davies & Forsythe J. Physiol, 1995.
Takahashi et al., Science, 1996.
Page 29
Summary• The calyx of Held is a glutamatergic synapse which forms
on principal neurons in the MNTB.
•It is part of an inverting relay in the auditory pathway
subserving sound source localization.
• Presynaptic recording shows that P-type Ca2+ channels
dominate exocytosis at the mature calyx.
• Mitochondria are the major presynaptic calcium store.
• Short term forms of modulation act at presynaptic P-type
Ca2+ channels to regulate release of transmitter.
Page 30
Modulation of synaptic transmission
Presynaptic – changes in transmitter release
Facilitation - Ca2+ dependent G-protein-dependent.
Depression – Ca2+/calmodulin dependent.
Post-tetanic potentiation
Auto-receptor activation.
Postsynaptic – changes in glutamate receptors
Desensitization of the AMPA receptors.
Short-term
Long-term
No-one has discovered anything like long-term potentiation (LTP) or
long-term depression (LTD)!
Why?
Your turn……. Ask questions…… form a hypothesis…….
Page 31
What use is giant synapse?
Perhaps it is big because it is primitive?
Why is it so much bigger than nearly all other synapses?
What information does it convey?
Why would an ‘ordinary’ synapse not do as good a job
What are it’s physiological limitations?
It uses the fastest glutamate receptors in the brain.
Each target neuron has only one giant synaptic input.
It acts as a relay (in contrast to nearly every other synapse).
It is 30 times larger than it needs to be to trigger an AP in the target
But it still only triggers one AP in the target neuron
It can fire APs at over 1000Hz
Page 32
Security at the Calyx of Held: 4000 stimuli at 200Hz
14 nA
50 mV
5 seconds
50 ms
P15 Rat, 38oC
VC: EPSC
CC: EPSP/AP
RMP= -63 mV
First ‘error’
6.25 sec
50 msMike Postlethwaite
Page 33
20 mV40 ms
current-clamp
voltage-clamp
stimulus
MNTB neurone
Calyx of Held
axon
stim.
patch pipette
Action potentialtriggered by synapticresponse
The calyx of Held is a secure glutamatergic synapse
Experimentalarrangement
AMPAR-mediated EPSC
NMDAR-mediated EPSC
Control
2 nA
5 ms
P14, 350C
+40mV
0mV
-50mV
How to cope with a calyx?
•Large EPSCs (300 nS): expensive!
•Huge safety-factor: secure AP (30-fold).
Supercharges membrane cap..
Answer: Potassium channels.
•Controlling target neuron excitability is an
integral part of regulating synaptic efficacy and
information transmission
.