What can one synapse tell us about how the auditory ...

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

The Calyx of Held -Auditory Brainstem

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

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

Most synapses in the CNS are small

Mitochondria

vesicles

Postsynaptic cell

Release zone

From Sanes & Jessell

0.5 µm

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.

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.

Confocal images of the calyx of Held

Forsythe, 1994

midline

dorsallateral

stimulate

15 µm

The medial nucleus ofthe trapezoid body

View down the

experimental

microscopeRat & mouse

10-14 days old.

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

AMPAR-mediated

NMDAR-mediated

Forsythe & Barnes-Davies, 1993

Dual component synaptic currents

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

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

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

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’.

Presynaptic Lucifer yellow fill

0.3nA20mV

5ms

Post.

Pre

A. AP triggered B. Calcium dependence

PrePost

Simultaneous pre- and post-synaptic recording

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

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.

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

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

Voltage-gated Ca2+ currents

Takahashi et al., Science 1996

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

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

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%)

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

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.

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.

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.

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…….

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

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

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

.