Multi-Electrode Array technique -Evaluation of compounds on NMDA receptors
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Transcript of Multi-Electrode Array technique -Evaluation of compounds on NMDA receptors
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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
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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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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, 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.
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EXPERIMENTS – PHASE I Determination of LTP/Neutral/LTD protocols in the CA1 region of rat hippocampal slices (crossover point)
Be tween 1 a nd 2 0 H z , t h e s*mula*ons train, induces Long-‐Term Depress ion ( LTD) o f evoked-‐responses.
At 100 Hz and 200 Hz , the s*mula*ons train induces Long-‐Term Poten*a*on (LTP) of evoked-‐responses.
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.
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EXPERIMENTS – PHASE I Determination of LTP/Neutral/LTD protocols in the CA1 region of rat hippocampal slices (crossover point)
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 %).
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.
1 H z
1 0 Hz
2 0 Hz
5 0 Hz
1 00 H z
2 00 H z
-‐1 0 0
-‐5 0
0
5 0
% of fE
PSP
change
(at endpoin
t)
LTD
LTP
C ro s so v e r
p o in t
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EXPERIMENTS – PHASE II
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).
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal slices
Time (min)
Normalized
fEPSP am
plitu
de
0 10 20 30 40 500.0
0.5
1.0
1.5 0.1 µM Compound X
2 rats, 4 slices, 17 electrodesTime (min)
Normalized
fEPSP am
plitu
de0 10 20 30 40 50
0.0
0.5
1.0
1.5 0.3 µM Compound X
2 rats, 5 slices, 17 electrodesTime (min)
Normalized
fEPSP am
plitu
de
0 10 20 30 40 500.0
0.5
1.0
1.5 1 µM Compound X
3 rats, 6 slices, 27 electrodes
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EXPERIMENTS – PHASE II
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).
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal slices
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
0 1 0 2 0 3 0 4 0 5 00 .0
0 .5
1 .0
1 .5 3 µ M C o m p o u n d X
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 )Norm
alize
d fEPSP
am
plitu
de
0 1 0 2 0 3 0 4 0 5 00 .0
0 .5
1 .0
1 .5 1 0 µ M C o m p o u n d X
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 )
Norm
alize
d fEPSP
am
plitu
de
0 1 0 2 0 3 0 4 0 5 00 .0
0 .5
1 .0
1 .5 3 0 µ M C o m p o u n d X
2 r a t s , 5 s l ic e s ,2 3 e le c t ro d e s
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EXPERIMENTS – PHASE II
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).
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal slices
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
0 1 0 2 0 3 0 4 0 5 00 .0
0 .5
1 .0
1 .5 5 0 µ M C o m p o u n d X
1 r a t , 2 s l ic e s , 9 e le c t ro d e s
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EXPERIMENTS – PHASE II
Compound X dose-‐dependently decreases the amplitude of NMDA-‐mediated EPSP, with an IC50 of 3.6 µM.
The top of the concentra*on-‐response curve seems reached with 30-‐50 µM Compound X.
Evaluation of a dose-concentration curve of Compound X on NMDA-mediated EPSP in the CA1 region of rat hippocampal slices
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
0 1 0 2 0 3 0 4 0 5 00 .0
0 .5
1 .0
1 .5 C o m p o u n d X
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
Lo g [C om p o u n d X ] (M )
% of base
line fEPSP
after 40' e
xposu
re
-‐7 -‐6 -‐5 -‐40 .0
0 .2
0 .4
0 .6
0 .8
1 .0
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EXPERIMENTS – PHASE III Evaluation of Compound X on LTP induced by a 100 Hz train of stimulations
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).
0 2 0 4 0 6 0 8 0 1 0 00 .0
0 .5
1 .0
1 .5
2 .0
2 .5
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
C o n t ro l (9 r a t s , 1 4 s l ic e s , 6 4 e le c t ro d e s )
0 .3 µ M C o m p o u n d X (9 r a t s , 1 5 s l ic e s , 7 7 e le c t ro d e s )
0 .3 µ M C o m p o u n d X
1 0 0 H z
0 2 0 4 0 6 0 8 0 1 0 00 .0
0 .5
1 .0
1 .5
2 .0
2 .5
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
1 µM C om p o u n d X (9 r a t s , 1 5 s l ic e s , 7 7 e le c t ro d e s )
C o n t ro l (9 r a t s , 1 4 s l ic e s , 6 4 e le c t ro d e s )
1 µ M C o m p o u n d X
1 0 0 H z
0 2 0 4 0 6 0 8 0 1 0 00 .0
0 .5
1 .0
1 .5
2 .0
2 .5
T im e (m in )
Norm
alize
d fEPSP
am
plitu
de
C o n t ro l (9 r a t s , 1 4 s l ic e s , 6 4 e le c t ro d e s )
3 µ M C om p o u n d X (9 r a t s , 1 8 s l ic e s , 9 1 e le c t ro d e s )
3 µ M C o m p o u n d X
1 0 0 H z
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).
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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 on trol
0 .3 µM
Com
p ou nd X
1 µM
Com
p ou nd X
3 µM
Com
p ou nd X
0
1 0
2 0
3 0
4 0
5 0% of fE
PSP
change
(mean ove
r period after HFS
) * *n s
n s
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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.
CONCLUSION
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