Evaluation of compounds on NMDA receptors with the Multi-Electrode Array (MEA) technique

1. Evaluation of one compound on
NMDA receptors
September, 2013
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SUMMARY
Introduc*on
Aim
of
the
study
Materials
&
Methods
Prepara*on
of
acute
rat
hippocampal
slices
Slice
perfusion
and
temperature
control
S*mula*on
protocols
Experiments
Determina*on
of
LTP/
Neutral/LTD
protocols
in
the
CA1
region
of
rat
hippocampal
slices
(crossover
point)
Dose-­‐concentra*on
curve
of
Compound
X
on
NMDA-­‐mediated
EPSP
in
the
CA1
region
of
rat
hippocampal
slices
Evalua*on
of
a
range
of
concentra*ons
of
Compound
X
on
the
crossover
point
in
the
CA1
region
of
rat
hippocampal
slices

3. INTRODUCTION
The
aim
of
the
study
is
to
assess
if
NMDA
modulators
could
shiI
the
LTD/LTP
crossover
point.
First,
the
LTD/LTP
crossover
point
is
determined
in
rat
hippocampal
slices.
Next,
the
dose-­‐response
curve
of
the
Compound
X
(a
NMDA
modulator)
is
established
from
recordings
of
NMDA-­‐mediated
EPSP.
Finally,
the
possible
effect
of
the
Compound
X
is
evaluated
on
the
LTD/LTP
crossover
point.
Extracellular
recordings
(EPSP)
are
performed
with
Mul*-­‐Electrode
Arrays
(MEA).
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4. MATERIALS & METHODS
Prepara*on
of
acute
rat
hippocampal
slices
Experiments
are
carried
out
with
Sprague
Dawley
rats
between
3
and
4
weeks
of
age
provided
by
Elevage
Janvier.
Hippocampal
slices
(400
μm
thickness)
are
cut
with
a
MacIIwain
*ssue
chopper
in
a
ice-­‐cold
oxygenated
sucrose
solu*on
(Saccharose
250,
Glucose
11,
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NaHCO3
26,
KCl
2,
NaH2PO4
1.2,
MgCl2
7
and
CaCl2
0.5
in
mM).
Then,
slices
are
incubated
at
room
temperature
for
at
least
1h
in
ACSF
of
the
following
composi*on:
Glucose
11,
NaHCO3
25,
NaCl
126,
KCl
3.5,
NaH2PO4
1.2,
MgCl2
1.3,
CaCl2
2
in
mM.
Slice
perfusion
and
temperature
control
During
experiments,
the
slices
are
con*nuously
perfused
with
the
ACSF
(bubbled
with
95%
O2–5%
CO2)
at
the
rate
of
3
mL/min
with
a
peristal*c
pump
(MEA
chamber
volume:
~1
mL).
Complete
solu*on
exchange
in
the
MEA
chamber
is
achieved
20
s
aIer
the
switch
of
solu*ons.
The
perfusion
liquid
is
con*nuously
pre-­‐heated
at
37°C
just
before
reaching
the
MEA
chamber
with
a
heated-­‐perfusion
cannula
(PH01,
Mul*Channel
Systems,
Reutlingen,
Germany).
The
temperature
of
the
MEA
chamber
is
maintained
at
37
±
0.1°C
with
a
hea*ng
element
located
in
the
MEA
amplifier
headstage.
S*mula*on
protocols
Basal
synap*c
transmission:
The
s*mulus
intensity
is
set
to
300
μA
at
0.033Hz.
Long-­‐Term
Poten*a*on
(LTP)/Neutral/Long-­‐Term
Depression
(LTD)
protocols:
S*mula*on
trains
from
1
to
200
Hz.

5. EXPERIMENTS – PHASE I
Determination of LTP/Neutral/LTD protocols in the CA1 region of rat hippocampal slices (crossover point)
The
effect
of
a
s*mula*on
trains
applied
with
a
wide
range
of
frequencies
(1
to
200
Hz)
were
inves*gated
to
determine
the
LTP/LTD
crossover
point.
Between
1
and
20
H z ,
the
s*mula*ons
train,
induces
Long-­‐Term
Depression
(LTD)
of
evoked-­‐
responses.
At
100
Hz
and
200
Hz,
the
s*mula*ons
train
induces
Long-­‐Term
Poten*a*on
(LTP)
of
evoked-­‐
responses.
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6. EXPERIMENTS – PHASE I
Determination of LTP/Neutral/LTD protocols in the CA1 region of rat hippocampal slices (crossover point)
The
effect
of
a
s*mula*on
trains
applied
with
a
wide
range
of
frequencies
(1
to
200
Hz)
were
inves*gated
to
determine
the
LTP/LTD
crossover
point.
H z
H z
L T P
C ro s s o v e r
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The
LTP/LTD
crossover
point
is
close
to
50
Hz.
Indeed,
a
train
of
s*mula*ons
applied
at
50
Hz
does
not
substan*ally
modifies
the
fEPSP
amplitude
(the
mean
percentage
of
fEPSP
change
aIer
60
minutes
is
of
-­‐2.9%
±5
%).
H z
1
H z
10
H z
2 0
H z
50
1 00
2 00
5 0
0
-­‐5 0
-­‐1 0 0
% o f fE P S P c h a n g e
(a t e n d p o in t)
L TD
p o in t

7. EXPERIMENTS – PHASE II
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal
slices
1.5 0.1
μM
Compound
X
1.5 0.3
μM
Compound
X
1.0
0.5
1.5
1.0
0.5
When
applied
at
0.1
μM,
Compound
X
slightly
decreases
the
NMDA
EPSP
amplitude
aIer
about
30
minutes
(the
normalized
fEPSP
amplitude
is
of
0.91±0.02
at
endpoint).
Exposure
to
0.3
μM
Compound
X
also
slightly
decreases
the
NMDA
EPSP
amplitude
aIer
about
15
minutes
(by
about
10%,
the
normalized
fEPSP
amplitude
is
of
0.91
±0.03
at
endpoint).
At
1
μM,
Compound
X
decreases
the
amplitude
of
NMDA-­‐mediated
EPSP
by
about
20
%,
aIer
a
10-­‐
minute
period
(the
normalized
fEPSP
amplitude
is
of
0.78
±0.03
at
endpoint).
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Time (min)
Normalized fEPSP amplitude
0 10 20 30 40 50
1.0
0.5
0.0
2
rats,
4
slices,
17
electrodes
Time (min)
Normalized fEPSP amplitude
0 10 20 30 40 50
0.0
2
rats,
5
slices,
17
electrodes
Time (min)
Normalized fEPSP amplitude
0 10 20 30 40 50
0.0
1
μM
Compound
X
3
rats,
6
slices,
27
electrodes

8. EXPERIMENTS – PHASE II
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal
slices
1 .5 3
μM
C o m p o u n d
X
1 .5 1 0
μM
C o m p o u n d
X
1 .0
0 .5
1 .5 3 0
μM
C o m p o u n d
X
1 .0
0 .5
At
3
μM,
Compound
X
decreases
the
NMDA
EPSP
amplitude
by
about
30
%
(the
normalized
fEPSP
amplitude
is
of
0.70
±0.04
at
endpoint).
About
45
%
of
decrease
of
NMDA
EPSP
amplitude
is
observed
aIer
exposure
to
10
μM
Compound
X
(the
normalized
fEPSP
amplitude
is
of
0.53
±0.03
at
endpoint).
At
30
μM,
Compound
X
decreases
the
amplitude
of
NMDA-­‐mediated
EPSP
by
about
60
%
(the
normalized
fEPSP
amplitude
is
of
0.42
±0.04
at
endpoint).
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T im e (m in )
N o rm a liz e d fE P S P am p litu d e
0 1 0 2 0 3 0 4 0 5 0
1 .0
0 .5
0 .0
3
r a t s ,
8
s lc e s ,
3 9
e le c t ro d e s
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
0 1 0 2 0 3 0 4 0 5 0
0 .0
2
r a t s ,
6
s lic e s ,
3 3
e le c t ro d e s
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
0 1 0 2 0 3 0 4 0 5 0
0 .0
2
r a t s ,
5
s lic e s ,2 3
e le c t ro d e s

9. EXPERIMENTS – PHASE II
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal
slices
1 .5 5 0
μM
C o m p o u n d
X
T im e (m in )
1 .0
0 .5
At
50
μM,
Compound
X
decreases
the
amplitude
of
NMDA-­‐mediated
EPSP
by
about
60
%
(the
normalized
fEPSP
amplitude
is
of
0.44
±0.02
at
endpoint).
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N o rm a liz e d fE P S P am p litu d e
0 1 0 2 0 3 0 4 0 5 0
0 .0
1
r a t ,
2
s lic e s ,
9
e le c t ro d e s

10. EXPERIMENTS – PHASE II
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal
1 .0
0 .8
0 .6
0 .4
0 .2
Compound
X
dose-­‐dependently
decreases
the
amplitude
of
NMDA-­‐mediated
EPSP,
with
an
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IC50
of
3.6
μM.
The
top
of
the
concentra*on-­‐response
curve
seems
reached
with
30-­‐50
μM
Compound
X.
slices
1 .5 C o m p o u n d
X
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
0 1 0 2 0 3 0 4 0 5 0
1 .0
0 .5
0 .0
0 .1
μ M
0 .3
μ M
1
μ M
3
μ M
1 0
μ M
3 0
μ M
5 0
μ M
L o g [C om p o u n d X ] (M )
% o f b a s e lin e fE P S P
a fte r 4 0 ' e x p o su re
-­‐7 -­‐6 -­‐5 -­‐4
0 .0

11. EXPERIMENTS – PHASE III
Evaluation of Compound X on LTP induced by a 100 Hz train of stimulations
2 .5
2 .0
1 .5
1 .0
0 .5
Compound
X
has
been
evaluated
at
3
different
concentra*ons
(0.3
μM,
1
μM,
3
μM)
on
LTP
induced
by
a
100
Hz
train
of
s*mula*ons
(with
control
slices
recorded
in
parallel).
1 0 0
H z
0 .3
μM
C o m p o u n d
X
2 .5
2 .0
1 .5
1 .0
0 .5
1 0 0
H z
1
μM
C o m p o u n d
X
C o n t ro l
(9
r a t s ,
1 4
s lic e s ,
6 4
e le c t ro d e s )
2 .5
2 .0
1 .5
1 .0
0 .5
1 0 0
H z
3
μM
C o m p o u n d
X
In
the
presence
of
0.3
μM
Compound
X
the
LTP
amplitude
is
slightly
lower
than
the
one
recorded
in
control
condi*ons:
the
poten*a*on
is
of
12
±
6%
at
endpoint,
versus
20
±
6%
in
control
condi*ons.
The
LTP
amplitude
is
slightly
lower
in
the
presence
of
1
μM
Compound
X
than
in
control
condi*ons
(the
poten*a*on
is
of
14±
6%
at
endpoint,
versus
20
±
6%
in
control
condi*ons).
In
the
presence
of
3
μM
Compound
X
the
LTP
amplitude
is
significantly
decreased
when
compared
to
control
condi*ons
(the
poten*a*on
is
of
7
±
4%
at
endpoint,
versus
20
±
6%
in
control
condi*ons).
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0 2 0 4 0 6 0 8 0 1 0 0
0 .0
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
C o n t ro l
(9
r a t s ,
1 4
s lic e s ,
6 4
e le c t ro d e s )
0 .3
μM
C o m p o u n d
X
(9
r a t s ,
1 5
s lic e s ,
7 7
e le c t ro d e s )
0 2 0 4 0 6 0 8 0 1 0 0
0 .0
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
1
μM
C om p o u n d
X
(9
r a t s ,
1 5
s lic e s ,
7 7
e le c t ro d e s )
0 2 0 4 0 6 0 8 0 1 0 0
0 .0
T im e (m in )
N o rm a liz e d fE P S P am p litu d e
C o n t ro l
(9
r a t s ,
1 4
s lic e s ,
6 4
e le c t ro d e s )
3
μM
C om p o u n d
X
(9
r a t s ,
1 8
s lic e s ,
9 1
e le c t ro d e s )

12. EXPERIMENTS – PHASE III
Evaluation of Compound X on LTP induced by a 100 Hz train of stimulations
Compound
X
slightly
decreases
the
LTP
amplitude
at
0.3
and
1
μM,
that
effect
remains
however
not
significant
(p=
0.3005
and
p=
0.2656,
respec*vely).
3
μM
Compound
X
significantly
decreases
the
LTP
amplitude
(p=
0.0040).
C o n tr o l
X
C o m p o u n d
μ M
0 .3
X
C o m p o u n d
μ M
1
C o m p o u n d
μ M
3
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X
5 0
4 0
3 0
2 0
1 0
0
% o f fE P S P c h a n g e
(m e a n o v e r p e r io d a fte r H F S )
* * n s
n s

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CONCLUSION
Phase
I
The
LTP/LTD
crossover
point
is
close
to
50
Hz.
S*mula*ons
below
50
Hz
trigger
a
LTD
of
the
evoked-­‐responses,
whereas
s*mula*ons
above
50
Hz
trigger
a
LTP
of
the
evoked-­‐
responses.
Phase
II
Compound
X
dose-­‐dependently
decreases
the
NMDA
EPSP
amplitude.
The
IC50
of
Compound
X
is
3.6
μM,
and
the
top
of
the
concentra*on-­‐response
curve
seems
reached
at
30-­‐50
μM.
Phase
III
Compound
X
at
0.3,
1
and
3
μM
decreases
the
LTP
amplitude,
however
its
effect
is
significant
only
at
3
μM.

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