Multi-Electrode Array technique -Evaluation of compounds on NMDA receptors

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Evaluation of one compound on

NMDA receptors

September, 2013


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


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).


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.


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.


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


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



  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).


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



  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).


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



  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.


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


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 )


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


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).


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


  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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Multi-Electrode Array technique -Evaluation of compounds on NMDA receptors

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