Presentacion curso inb 2012

“Ligandos Wnt como neurotrofinas, plasticidad sináptica modulada por ellos”

Dr. Jorge ParodiUniversidad Católica Temuco

Bubnik 2007 TRENDS

Schulte 2010, Phar- Review

Janda et al 2012 Science

Nusse and Varmus 2012 EMBO

Janda et al 2012 Science

Nusse and Varmus 2012 EMBO

Inestrosa and Arenas, 2009, Nat Rev Neurosci

Farias et al., 2009, J Biol Chem

Cuitino et al., 2010, J Neurosci

Technical Support: Juan Godoy and Inés Poblete

In collaboration with Dr. Francisco José Muñoz (Universidad Pompeau Fabra, Barcelona) and Dr. Juan Pablo García-Huidobro (P. Universidad Católica de Chile, CARE)

Condition Reversal potential

Control -53 ± 7 mV

Wnt-5a -56 ± 4 mV

TEA -55 ± 2 mV

-150 -100 -50 0 50 100 150

-500

0

500

1000

1500 Kv 3.2 control SNAP 100 nM SNAP 25 nM SNAP 1 M

mV

pA

-150 -100 -50 0 50 100 150

-500

0

500

1000

1500 Kv 3.2 control Wnt 1:100 Wnt 1.25 Wnt 1:10

mV

pA

-150 -100 -50 0 50 100 150

-500

0

500

1000

1500 Control Kv 3.2 SNAP Wnt-5a 7-NI

mV

pA

-150 -100 -50 0 50 100 150

-500

0

500

1000

1500

mV

pA

Control Kv 3.2 1 nM 10 nM 100 nM 1000 nM

HEK

Neuron

Control sFRP 7-NI Go 69760

200

400

600

800

*

*

* Wnt-5a

Mem

bra

ne

resi

sten

ce (

M)

Wnt-5a

¿Afecta la actividad miniatura?

Control AMPA NMDA0

20

40

60

80

100 Control 15 min

*

*

Am

plit

ud

e m

init

ure

cu

rren

t (p

A)

0

20

40

60

80

100

Formyl-6aaWnt-5a + sFRPWnt-5aControl

**

Am

plit

ud

e m

init

ure

cu

rren

t (p

A)

Control AMPA

Wnt-5a AMPA

Control NMDA

Wnt-5a NMDA

AMPA

NMDA

Control

Control

Wnt-5a

Wnt-5a

0,0

0,5

1,0

1,5

2,0

2,5

3,0

mN

MD

A fre

quen

cy (pA

)

**

Control Wnt-5a Wnt-5a + 7-NI

0

10

20

30

40

50

60

mN

MD

A a

mplit

ude

(pA

)

**

Wnt-5a + 7-NI

Wnt-5aControl

Plasticidad, efectos mas complejos del

ligando Wnt

-20 0 20 40 60 80 100 120 140 1600

1

2

3

4

5

fEPSP s

lope

% o

f C

ontr

ol (R

.U.)

Time (min)

-20 0 20 40 60 80 100 120 1400

2

fV (

%o

f C

on

tro

l)

X Axis Title

-40 -20 0 20 40 60 80 100 120

1,0

1,5

2,0

2,5

3,0

3,5

fEPSP s

lope

% o

f C

ontr

ol (

R.U

.)Time (min)

Control Wnt 5a ACSF

-60 -40 -20 0 20 40 60 80 100 1200

2

4

fEPSP s

lope

% o

f C

ontr

ol (R

.U.)

Time (min)

Wnt 5a 40 min Wnt 5a 20 min Control

-40 -20 0 20 40 600

1

2

3

4

5

Control 4 train TBS sFRP

fEPSP s

lope

(R.U

.)

Time (min)

-40 -20 0 20 40 600

1

2

3

4

5

fEPSP s

lope

(R.U

.)

Control Medium

Time (min)

-40 -20 0 20 40 600

1

2

3

4

5

Time (min)

fEPSP s

lope

(R.U

.)

Wnt-5a 2 train TBS Wnt-5a/sFRP sFRP

-20 0 20 40 600

1

2

3

4

5

Time (min)

fEPSP s

lope

(R.U

.) Control 7NI 0.5 M 7NI 1 M

-40 -20 0 20 40 600

1

2

3

4

5 Control 2 train 7NI

Time (min)

fEPSP s

lope

(R.U

.)

-20 0 20 40 600

1

2

3

4

5

Time (min)

fEPSP s

lope

(R.U

.)

Wnt-5a 2 train Wnt-5a 2 train/7-NI

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Control Wnt-5a

20

mV

pu

lse

am

pli

tud

e (A

)

Control Wnt-5a

10 mM Calcium

BAPTA

BAPTA/Wnt-5a

0 mV

0 mV

20 mV

20 mV

BAPTA/Serum

BAPTA/Wnt-5a

Serum

0

200

400

600

800

1000

24 hrs

AT

P c

urre

nt (

nA)

Control Wnt-5a

Acute

0 1 2 3 4

0

20

40

60

80

100

120

Control Wnt-5a

Via

bili

da

d %

of

co

ntr

ol

Days

-200 -150 -100 -50 0 50 100

-1000

-800

-600

-400

-200

0

200

nA

mV

Control Wnt-5a 24 hrs Wnt-5a

-50

-40

-30

-20

-10

0

10

20

72 hrs48 hrs

Mem

bra

ne

pote

ntial

(m

V

Control Wnt-5a

24 hrs

500

nA

Control Prog. Wnt-5a

Control

Prog.

Wnt-5a

Medium

0 5 10 15 20 25

0

20

40

60

80

100

120

Control Medium Prog Wnt-5a

% G

VB

D

Hours of Washed

AgradecimientosDr. Nibaldo InestrosaDr. Fernando Romero, UFRODr. Ataulfo Martinez-Torres UNAMFelipe Serrano

Time (min)

0

0.5

1.0

1.5

2.0Control

ACSF

Case

Depleted

fEPS

P A

mplit

ude

(rela

tive u

nit

s)-10 0 10 20 30

Serum

Serum

Control

0

0.5

1.0

1.5

Facilit

ati

on

In

dex

R2

-R1

/R1

Case

0.5 m

V

50 ms

fEP

SP

A

mp

litu

de

(rela

tive

un

its)

ACSF

00.5

1.0

1.5

2.0

Control Case Depleted

*

0.5 m

V

10 ms

Serum

0

0.5

1.0

1.5

2.0CaseACSF

Depleted

Time (min)

fEPS

P A

mplit

ude

(rela

tive u

nit

s)-

100 10 20 30

CSF

CSF

ACSF 0

0.5

1.0

1.5

2.0

DepletedCase

CSF

fEPSP

Am

plit

ude

(rela

tive u

nit

s) *

0.5

m

V

10 ms

Control

0

0.5

1.0

1.5

CSF

Faci

litati

on Index

R2

-R1

/R1

0.5

m

V

50 ms

1.0

3.5

6.0

8.5 0.025 g/ml0.25 g/ml2.5 g/mlACSF

-10

Time (min)

100 403020 50

fEP

SP

Am

plitu

de(r

ela

tive

units

)

0

Anti-P

0

1.0

3.5

6.0

8.5

*

*

ACSF 2.5 g/ml

0.25 g/ml

0.025 g/ml

fEP

SP

Am

plitu

de(r

ela

tive

units

)

Anti-P

0.5 m

V

10 ms

-100

1.0

2.0

3.0

4.0Anti-P (0.25 g/ml)

ACSFAnti-P + APV (50 M)

Time (min)

fEPS

P A

mplit

ude

(rela

tive u

nit

s)

100 403020 50

ACSF

0

1.0

2.0

3.0

4.0

Anti-P Anti-P + d-APV

*

0.5

m

V

10 ms

fEPS

P A

mplit

ude

(rela

tive u

nit

s)

-20 -10 0 10 20 300

1

2

3

Time (min)

fEP

SP

am

plitu

de

(rela

tive u

nit

s)

KO mouse Control