From molecule to memory in the cerebellar neural circuit

Sang Jeong Kim

Department of Physiology

Seoul National University College of Medicine, Korea

Cerebellar cortical circuit

Sensory-motor input

Error signals

Motor output

Two inputs to PC : parallel fiber (PF) climbing fiber (CF)

Purkinje cell (PC)- Main sole output of cerebellar cortex

Linden DJ (2003) Science 301.

Parallel fiber

Climbing fiber

Tone

Shock

LTD = Memory traceTime (min)

Pu

rkin

je C

ell

re

sp

on

se

Pairing

-10 -5 0 5 10 15 20 25 30 35

40

60

80

100

120

140

160

180

LTD

Pairing of PF and CF induces long-term depression (LTD) of PF-PC synapse

Eye-Blink Conditioning: A simple form of associative motor learning

Linden DJ (2003) Science 301.LTD = memory trace -> Tone means Shock

Dendritic spines of cerebellar Purkinje cell

PF-PC synapse

• Excitatory glutamatergic synapse

• Single firing of PF evokes fast excitatory postsynaptic current (fast EPSC) via AMPA receptor (GluR2-containing, Ca2+-impermeable variety).

• Mature PC has no NMDA receptor.

• Pairing of PF and CF induces long-term depression (LTD) which is internalization of AMPA-R in dendritic spines of PC.

• AMPA-R LTD is a memory trace

• PF drives PC up to 100 Hz.

AMPA-R

PresynapticTerminal of PF

Spineof PC

Glutamate

Metabotropic glutamate receptor type1 (mGluR1)

• Single firing of PF : fast EPSC only

• Tetanic burst stimulation of PF

• Spillover of glutamate to perisynaptic region

• mGluR1 in the perisynaptic region of the spine

• Activation of mGluR1 by burst onlymGluR1

AMPA-RSpineof PC

PresynapticTerminal of PF

Glutamate

LTD is mGluR1-dependent: mGluR1 knock-out mice show defects in PF-PC LTD and motor learning

GqPLCb4mGluR1

PresynapticTerminal of PF

Spineof PC

Glutamate

IP3R1

IP3Ca2+

Endoplasmic reticulum

RyR

Ca2+

Ca2+

pump

DAG

Na+, Ca2+

• mGluR1 signal has two limbs

1) PLC pathway

- IP3-mediated Ca release

2) Activation of membrane conductance

- Slow excitatory postsynaptic current (slow EPSC)

TRPC1

mGluR1-mediated signaling in PF-PC synapse

AMPA-R

Tetanic stimulus to PF induces mGluR1-evoked slow EPSC in Purkinje cells

0.5 s

100 pA

2,5,10,15 pulses in 100 Hz, 14 A tetanus at -70 mV

04020318-22

10 M CNQX

The slow EPSC evoked by parallel fiber bursts is mediated by an mGluR1-TRPC1 pathway

Slow EPSC is also blocked by a dominant-negative TRPC1 and a TRPC1 Ab

Kim et al, 2003 nature

LTD of the parallel fiber-evoked mGluR1-mediated slow EPSC by strong depolarization

PF burst(10 pulses, 100 Hz)

Pre

LTD of the parallel fiber-evoked mGluR1-mediated slow EPSC by strong depolarization

PF burst(10 pulses, 100 Hz)

Pre 30 s after depol

LTD of the parallel fiber-evoked mGluR1-mediated slow EPSC by strong depolarization

200 pA0.5 s PF burst

(10 pulses, 100 Hz)

A Pre 30 s after depol

B

1200 s after depol

0

20

40

60

80

100

120 n=11

**Cu

rre

nt

am

pli

tud

e (

%)

Slow EPSC( )

Fast EPSC( )

-300 0 300 600 900 1200 1500

0

20

40

60

80

100

120 depolarization

Slo

w E

PS

C (

%)

1 s2 s5 s

Time (s)

n=11

n=11

n=14

Duration of depolarization (s)

*

**

Slo

w E

PS

C (

%)

1 2 50

20

40

60

80

100

LTD(mGluR1) is blocked by removing external Ca

A

B

DHPG/Glu 200 pA

1 s

Pre 90 s after depolarization

PF burst

Pre 5 min after depolarization

0.5 s

200 pA

Ca-free ACSF

depolarization (n=14)

Time (min)

Fast current Slow current

Cu

rre

nt

am

plit

ud

e (

%)

-6 -4 -2 0 2 4 620

40

60

80

100

120

-10 -5 0 5 10 15 200

20

40

60

80

100

120

Fast EPSCSlow EPSC

depolarization (n=11)

Time (min)Cu

rre

nt

am

plit

ud

e (

%)

Fast EPSCSlow EPSC

Ca-free

Ca-free

A dynamin blocker, dynasore inhibited LTD of mGluR1

DMSO Dynasore0

20

40

60

80

100

% o

f Ba

selin

e

0 10 20 300

20

40

60

80

100

120

No

rma

lize

d S

low

EP

SC

(%)

Time (min)

DMSODynasore

Before depolarization

30 min after depolarization

100 pA500 ms

Physiological CF bursts produce LTD of mGluR1-mediated dendritic Ca transients

1 nA

100 %1 s

CF burst x 50

20 s 50 %

1st burst 2nd burst 50th burst

20 ms 20 mV

1.2 s

dF/F

CF burst x 50

Pre-drug +CPCCOEt

100 %1 s

1 m

PF burst

LTD(mGluR1) blocks subsequent LTD of AMPA-Rs

B

C

A

5 stim. @ 100 Hz

0 mV for 75 ms

PF

PC

X 30, 2 sec interval

Control (n=9) After LTD(mGluR1) (n=8)

0.1 s

100 pA 100 pA

0.1 s

No

rma

lize

d E

PS

C (

%)

pairing

Time (min)-10 -5 0 5 10 15 20 25 30 35

40

60

80

100

120

140

160

180

Time (min)

No

rma

lize

d E

PS

C (

%)

pairing

-10 -5 0 5 10 15 20 25 30 35

40

60

80

100

120

140

160

180

LTD of mGluR1 in a Ischemia Model: Oxygen-Glucose Deprivation in Organotypic Slice Culture

OGD(100min)Control

DIC

PI staining

Total mGluR1a

Surface mGluR1a

Con 1hr afte

r

OGD 6hr afte

r

OGD 24hr afte

r

OGD 48hr afte

r

OGD

Actin

0

20

40

60

80

100

120

con 1hr 6hr 24hr 48hr

Su

rface

exp

ressio

n o

f m

Glu

R1

a

Control OGD(100min)0

100

200

300

400

500

600

% o

f P

I u

pta

ke

0

20

40

60

80

100

120

con 1hr 6hr 24hr 48hr

Tota

l exp

ressio

n o

f m

Glu

R1

a

Internalization of mGluR1 as expression mechanism of LTD of mGluR1

AMPA 수용체 (AMPAR)

Spine

Internalization ??

LTD of mGluR1

mGluR1

mGluR1DepolarizationCFHypoxia

Higher axial resolution Monitoring of tagged mGluR1 distribution

Greater sample penetration Dendritic and axonal mobility in the intact brain such as spinal cord

Single-photon Multi-photon

OGB-1, Zeiss LSM 510

Coincidence detector for AMPA-R LTDCoincidence detector for AMPA-R LTD

LTD of mGluR1/TRPCLTD of mGluR1/TRPC

Mechanisms of mGluR1 internalizationMechanisms of mGluR1 internalization

Peptide delivery to Purkinje cellPeptide delivery to Purkinje cell

Vestibulo-ocular reflexVestibulo-ocular reflex

RegulationRegulation

FunctionFunction

Apply to BehaviorApply to Behavior

Tool DevelopmentTool Development

ControlControl

Identification mGluR1/TRPC signalingmGluR1/TRPC signaling

Acknowledgements Lab of Neuronal information storage, Seoul National University College of Medicine (http://brain.snu.ac.kr)

Johns Hopkins University: David Linden Lab, Paul Worley Lab

Sang Jeong KimJun KimHong Goo ChaeYunju JinYon Wha Hong Hae Young KimLyan ChoiWon Sok ChangSung Soo ChangJi Young KimSung Won HurChang Hee Kim