presentacion curso inb 2012
TRANSCRIPT
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“Ligandos Wnt como neurotrofinas, plasticidad sináptica modulada por ellos”
Dr. Jorge ParodiUniversidad Católica Temuco
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Bubnik 2007 TRENDS
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Schulte 2010, Phar- Review
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Janda et al 2012 Science
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Nusse and Varmus 2012 EMBO
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Janda et al 2012 Science
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Nusse and Varmus 2012 EMBO
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Inestrosa and Arenas, 2009, Nat Rev Neurosci
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Farias et al., 2009, J Biol Chem
Cuitino et al., 2010, J Neurosci
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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)
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Condition Reversal potential
Control -53 ± 7 mV
Wnt-5a -56 ± 4 mV
TEA -55 ± 2 mV
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-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
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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?
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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
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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
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Plasticidad, efectos mas complejos del
ligando Wnt
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-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
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-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
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-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)
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-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
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-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
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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
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BAPTA
BAPTA/Wnt-5a
0 mV
0 mV
20 mV
20 mV
BAPTA/Serum
BAPTA/Wnt-5a
Serum
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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
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-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
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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
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AgradecimientosDr. Nibaldo InestrosaDr. Fernando Romero, UFRODr. Ataulfo Martinez-Torres UNAMFelipe Serrano
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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
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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
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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
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-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)
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-20 -10 0 10 20 300
1
2
3
Time (min)
fEP
SP
am
plitu
de
(rela
tive u
nit
s)
KO mouse Control