What can one tell us about how the auditory processing works?

Ian D. Forsythe MRC toxicology Unit University 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/ is electrical pulses called Action Potentials. Via Voltage-gated ion channels Synaptic cleft

EPSP -excitatory postsynaptic potential

•Transmission between nerve cells is by a chemical messenger called a Transmitter Via Receptor-gated ion channels Neurotransmitter subtypes

Integral transmitter G-protein Coupled- Ion Channel Ionotropic Metabotropic

nAchRmusc nAchRbrain acetylcholine M1-5 muscarinic

NMDAR AMPAR KainR glutamate mGluR1-8

GABAC GABAA GABA GABAB

5HT3 /5HT 5HT1/2/4

catecholamines DA1-4 a1/2/b1- Peptides SP Enk VP3 VIP GlyR glycine

P2X purines P2Y A1/2 http://www.iuphar-db.org/index_ic.jsp Most in the CNS are small

Mitochondria

vesicles

Release zone

Postsynaptic cell

0.5 µm From Sanes & Jessell 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 The medial nucleus of the

dorsal lateral midline

stimulate

15 µm

View down the Rat & mouse experimental 10-14 days old. microscope The calyx of Held synapse is mediated by the transmitter glutamate Experimental patch arrangement pipette

MNTB neurone stim. current-clamp Calyx of Held triggered by synaptic response

20 mV ‘All or nothing’ response 40 ms % amplitude b 120 c stimulus 100 80

60 voltage-clamp 40 A slow EPSC - NMDA receptors 20 0.5 nA a 40 ms A fast EPSC - AMPA receptors 0 0 2 4 6 8 10 stimulus intensity (V) Dual component synaptic currents

NMDAR-mediated

AMPAR-mediated

Forsythe & Barnes-Davies, 1993 Fidelity at the Calyx of Held: 4000 stimuli at 200Hz

50 ms

VC: EPSC

First ‘error’ 6.25 sec

14 nA 50 mV CC: EPSP/AP 5 seconds RMP= -63 mV

50 ms P15 Rat, 38oC Mike Postlethwaite mEPSCs at the calyx of Held/MNTB

o Temperature = 25oC Amplitude distribution at 25 & 35 C

0.4

0.35 25 degrees

0.3 35 degrees

0.25

0.2

0.15

0.1 N = 55 0.05 0 10 pA 10 1 ms Tau = 0.64 ms -15 -25 -35 -45 -55 -65 -75 -85 -95 -105

Number of of Number events (normalized) Amplitude (bin width 10 pA) Temperature = 35oC 25oC 35oC N Amplitude (pA) -32.7 -46.0 6 2.3 5.7* Decay Tau (ms) 0.51 0.32 6 N = 220 0.06 0.02* Tau = 0.34 ms Frequency (Hz) 0.10 0.47 6 0.02 0.2* Postlethwaite et al., J. Physiol 2008 NMDAR at the calyx of Held

+50 Control 1 nA I (nA) 10 ms 4

+ D-AP5 P14 2 -70 -50 -30 0 10 30 50 2 nA V (mV) 50 ms -2 -60 +50 3 I (nA) 1 nA Control 10 ms 2

P18 + D-AP5 1 -70 -50 -30 0 5 nA 10 30 50 50 ms -1 V (mV) -60 -2

+50 4 I (nA) Control 1 nA 3 10 ms 2 P21 + D-AP5 1 -70 -50 -30 0 2 nA 10 30 50 -1 V (mV) 50 ms -60 -2 Steinert et al., submitted Detecting functionally connected 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’.

Billups et al., 2002 Pflugers Arch - Eur J Physiol 444:663–669 Presynaptic Lucifer yellow fill Simultaneous pre- and post- Post synaptic recording Pre

A. AP triggered B. Calcium dependence

Post.

0.3nA 20mV

5ms

Pre The calyx of Held • Calyx occupies around half of the . • Only one forms on each MNTB cell body. • It forms up to 700-1500 release sites. • P-type calcium channels mediate exocytosis.

2+ • Exoytosis requires [Ca ]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. Fluo3 loaded into calyx of Held • Exhibits multiple forms of short-term plasticity, calcium transients induced by two including autoreceptor depression. 1 ms step 1 ms – other forms include Facilitation, depression, 10 ms depletion and desensitisation. Billups & Forsythe 2002 Schneggenburger & Forsythe 2006 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 2+ » Cadmium [Cd ]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-type

AgaTx-IVA (200 nM) 10 ConoTx-GVIA (2 µM) Nimodipine (10 µM) i ii iii iv

) 8

A

n

-

(

e 6 d

u 2 nA t

i 3 ms

l p

m 4

a

C S

P 2 0.2 nA E 3 ms v 0 0 5 10 15 20 25 30 time (mins) Forsythe et al., 1998 P-type presynaptic calcium channels

Calcium Current (-nA)

5 nM 2 200 nM -Agatoxin-IVA 50 µM Cd2+ 1.5

1

0.5

0 0 10 20 30 40 50 time (min) Forsythe et al., 1998 Voltage-gated Ca2+ currents

Takahashi et al., Science 1996 Different Ca2+ currents in Bushy cell somata and terminals……

Soma – in Terminal - in the MNTB

60 100 50 80 40 60 30

20 40

10 20

0 0 L N R P L N R P

a1C a1B a1E a1A a1C a1B a1E a1A Calcium Channel Subtypes 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

Synaptic current Calcium current

1nA 1nA Presynaptic 20ms 20ms Postsynaptic Fura FF Mitochondrial calcium Presynaptic calcium

10µM 200ms Rhod-2 0.05 AU 200ms mitochondria Calcium, Transmitter Release and 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

Fura FF Rhod-2 Cytoplasmic Relative [Ca2+] (µM) Ca2+ mito. 40 1.2 30 1.0 Control 0.8 20 Rotenone 0.6 0.4 10 0.2

0 0 0.5 1.0 1.5 2.0 1 2 3 4 Time (sec) Time (sec) 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

Barnes-Davies & Forsythe J. Physiol, 1995.

This is mediated by suppression of the presynaptic P-type calcium currents

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

Short-term Presynaptic – changes in transmitter release Facilitation - Ca2+ dependent G-protein-dependent. Depression – Ca2+/ dependent. Post-tetanic potentiation Auto-receptor activation. Postsynaptic – changes in glutamate receptors Desensitization of the AMPA receptors. 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

50 ms

VC: EPSC

First ‘error’ 6.25 sec

14 nA 50 mV CC: EPSP/AP 5 seconds RMP= -63 mV

50 ms P15 Rat, 38oC Mike Postlethwaite The calyx of Held is a secure glutamatergic synapse

current-clamp patch Experimental pipette arrangement Action potential triggered by synaptic MNTB neurone stim. response axon Calyx of Held 20 mV 40 ms How to cope with a calyx? Control stimulus •Large EPSCs (300 nS): expensive!

•Huge safety-factor: secure AP (30-fold). voltage-clamp NMDAR-mediated EPSC Supercharges membrane cap.. +40mV 0mV Answer: Potassium channels. -50mV 2 nA •Controlling target neuron excitability is an 5 ms integral part of regulating synaptic efficacy and information transmission P14, 350C AMPAR-mediated EPSC .