Electrophysiological characterisation of metabotropic glu4 receptors

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Electrophysiological characterisation of

metabotropic Glu4 receptors (mGluR4)

Positive Allosteric Modulators (PAM)

with the MEA technique

May, 2012


SUMMARY

Introduction Aim of the study

Materials & Methods Preparation of acute mice hippocampal slices

Slice perfusion and temperature control

Stimulation protocols

Experiments Determination of a dose-concentration curve of LSP1-2111 (mGluR III agonist) at LPP synapses

Evaluation of assumed PAM at LPP synapses


INTRODUCTION

The aim of the study is to assess the effect of four assumed PAM.

Extracellular recordings (EPSP) are performed with Multi-Electrode Arrays (MEA).

Pre-synaptic metabotropic Glu4, receptors are known to be expressed at Lateral Perforant Path (LPP) synapses within the hippocampus (Corti, Neuroscience, 2002). While paired-stimuli with a 50 ms interval are applied, evoked-responses at LPP synapses display a Paired-Pulse Facilitation (PPF, see upper graph), whereas the ones recorded at MPP synapses display a Paired-Pulse Depression (PPD, see lower graph).

2 0 m s

20

0 µ

V

2 0 m s

20

0 µ

V


MATERIALS & METHODS

Preparation of acute mice hippocampal slices Experiments are carried out with C57Black/6 mice between 7 and 10 weeks of age provided by Elevage Janvier.

Hippocampal slices (350 µm thickness) are cut with a MacIIwain tissue chopper in a ice-cold oxygenated sucrose solution (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 composition: 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 continuously perfused with the ACSF (bubbled with 95% O2–5% CO2) at the rate of 3 mL/min with a

peristaltic pump (MEA chamber volume: ~1 mL). Complete solution exchange in the MEA chamber is achieved 20 s after the switch of solutions.

The perfusion liquid is continuously pre-heated at 32°C just before reaching the MEA chamber with a heated-perfusion cannula (PH01,

MultiChannel Systems, Reutlingen, Germany). The temperature of the MEA chamber is maintained at 32 ± 0.1°C with a heating element located in the MEA amplifier headstage.

Stimulation protocols I/O curve: stimuli from 100 µA to 800 µA by 100 µA steps

Basal synaptic transmission: The stimulus intensity is set 40 % of Imax at 0.033Hz.


EVALUATION OF COMPOUNDS AT mGluR III

LSP1-2111 effect at LPP synapses

LSP1-2111 is a potent agonist acting at the orthosteric site of group III mGlu receptors, with a preferential affinity on the mGlu4 receptor.

In the next experiments, the putative mGlur4 PAM were evaluated in the presence of LSP1-2111, at concentrations close to its EC20 (5 µM) and EC80 (20 µM).

5

LSP1-2111 dose-dependently decreases the fEPSP amplitude. The fEPSP inhibition is maximal at 30 and 100 µM (~20% inhibition).

L S P 1 -2 1 1 1 d o s e -r e s p o n s e c u r v e

1 1 0 1 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

E C 2 0 = 3 .7 µ M

E C 8 0 = 1 9 µ M

[L S P 1 -2 1 1 1 ] in µ M

% o

f m

ax

ima

l L

SP

1-2

11

1 e

ffe

ct

0 1 0 2 0 3 0 4 0

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

1 0 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s l ic e s , 1 4 e e lc t r o d e s )

3 0 µ M L S P 1 - 2 1 1 1 (3 m ic e , 3 s l ic e s , 1 4 e le c t r o d e s )

1 0 0 µ M L S P 1 - 2 1 1 1 (3 m ic e , 4 s lic e s , 1 3 e le c t r o d e s )

L S P 1 - 2 1 1 1

3 µ M L S P 1 - 2 1 1 1 (3 m ic e , 4 s l ic e s , 1 4 e le c t r o d e s )



Evaluation of Compound 1 (putative mGluR4 PAM)

The Compound 1 strongly enhances the effect of 5 µM LSP1-2111 (5 µM LSP1-2111 alone decreases the fEPSP amplitude by 14% after a 30-minute exposure whereas fEPSP are decreased by 46% when 10 µM Compound 1 is pre- and co-exposed).

The Compound 1 largely enhances the effect of 20 µM LSP1-2111 (20 µM LSP1-2111 alone decreases the fEPSP amplitude by 24% after a 30-minute exposure whereas fEPSP are decreased by 56% when 10 µM Compound 1 is pre- and co-exposed).

6

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

5 µ M L S P 1 - 2 1 1 1

1 0 µ M C o m p o u n d 1

+ 5 µ M L S P 1 - 2 1 1 11 0 µ M C o m p o u n d 1


1 0 µ M C o m p o u n d 1 + 5 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s lic e s , 1 6 e le c t r o d e s )

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

2 0 µ M L S P 1 - 2 1 1 1


+ 2 0 µ M L S P 1 - 2 1 1 11 0 µ M C o m p o u n d 1

1 0 µ M C o m p o u n d 1 + 2 0 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s l ic e s , 1 7 e le c t r o d e s )





The Compound 2 decreases the fEPSP amplitude, by about 10% after a 40-minute exposure

The effect of 5 µM LSP1-2111 is slightly decreased in the presence of 10 µM Compound 2 (5 µM LSP1-2111 alone decreases the fEPSP amplitude by 14% after a 30-minute exposure whereas fEPSP are decreased by 7% when 10 µM Compound 2 is pre- and co-exposed).

The effect of 20 µM LSP1-2111 is also decreased in the presence of 10 µM Compound 2 (20 µM LSP1-2111 alone decreases the fEPSP amplitude by 28% after a 30-minute exposure whereas fEPSP are decreased by 9% when 10 µM Compound 2 is pre- and co-exposed).

7

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

5 µ M L S P 1 - 2 1 1 1

C o m p o u n d 2



1 0 µ M C o m p o u n d 2 + 5 µ M L S P 1 -2 1 1 1 (4 m ic e , 4 s lic e s , 2 4 e le c t r o d e s )

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

2 0 µ M L S P 1 - 2 1 1 1








The Compound 3 very strongly enhances the effect of 5 µM LSP1-2111 (5 µM LSP1-2111 alone decreases the fEPSP amplitude by 13% after a 30-minute exposure whereas fEPSP are decreased by 66% when 2 µM Compound 3 is pre- and co-exposed).

As well, the Compound 3 very strongly enhances the effect of 20 µM LSP1-2111 (20 µM LSP1-2111 alone decreases the fEPSP amplitude by 23% after a 30-minute exposure whereas fEPSP are decreased by 66% when 2 µM Compound 3 is pre- and co-exposed).

8

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

5 µ M L S P 1 - 2 1 1 1

2 µ M C o m p o u n d 3

+ 5 µ M L S P 1 - 2 1 1 12 µ M C o m p o u n d 3


2 µ M C o m p o u n d 3 + 5 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s l ic e s , 1 9 e le c t r o d e s )

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

2 0 µ M L S P 1 - 2 1 1 1

2 µ M C o m p o u n d 3

+ 2 0 µ M L S P 1 - 2 1 1 12 µ M C o m p o u n d 3

2 µ M C o m p o u n d 3 + 2 0 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s lic e s , 1 6 e le c t r o d e s )





The Compound 4 does not modify the effect of 5 µM LSP1-2111 (5 µM LSP1-2111 alone decreases the fEPSP amplitude by 13% after a 30-minute exposure and fEPSP are decreased by 13% when 10 µM Compound 4 is pre- and co-exposed).

As well, the Compound 4 does not modify the effect of 20 µM LSP1-2111 (20 µM LSP1-2111 alone decreases the fEPSP amplitude by 23% after a 30-minute exposure and fEPSP are decreased by 18% when 10 µM Compound 4 is pre- and co-exposed).

9

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

5 µ M L S P 1 - 2 1 1 1




1 0 µ M C o m p o u n d 4 + 5 µ M L S P 1 - 2 1 1 1 (4 m ic e , 4 s lic e s , 1 7 e le c t r o d e s )

0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0

0 .2 5

0 .5 0

0 .7 5

1 .0 0

1 .2 5

1 .5 0

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de

2 0 µ M L S P 1 - 2 1 1 1






CONCLUSION

10

5 µM

LS P 1 -2

1 1 1

1 0 µM

Co m

p o u n d 1 +

5 µ

M L

S P 1 -21 1 1

2 0 µM

LS P 1 -2

1 1 1

Co m

p o u n d 1 +

20 µ

M L

S P 1 -21 1 1

5 µM

LS P 1 -2

1 1 1

1 0 µM

Co m

p o u n d 2 +

5 µ

M L

S P 1 -21 1 1

2 0 µM

LS P 1 -2

1 1 1

1 0 µM

Co m

p o u n d 2 +

20 µ

M L

S P 1 -21 1 1

5 µM

LS P 1 -2

1 1 1

2 µM

Co m

p o u n d 3 +

5 µ

M L

S P 1 -21 1 1

2 0 µM

LS P 1 -2

1 1 1

2 µM

Co m

p o u n d 3 +

20 µ

M L

S P 1 -21 1 1

5 µM

LS P 1 -2

1 1 1

1 0 µM

Co m

p o u n d 4 +

5 µ

M L

S P 1 -21 1 1

2 0 µM

LS P 1 -2

1 1 1

1 0 µM

Co m

p o u n d 4 +

20 µ

M L

S P 1 -21 1 1

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0%

of

fEP

SP

de

cre

as

e

(aft

er

LS

P1

-21

11

ex

po

su

re)


CONCLUSION

11

10 µM Compound 1 strongly enhances the effect of both 5 and 20 µM LSP1-2111. That increase is not maximal when Compound 1 is co-exposed with LSP1-2111 at its ~EC20 (5µM). Thus, Compound 1 behaves as a mGluR4 PAM.

Compound 2 at 10 µM decreases the effect of both 5 and 20 µM LSP1-2111. Since that compound also decreases the fEPSP amplitude when applied alone, this might indicate partial agonist properties at mGluR4.

Compound 3 at 2 µM very strongly enhances the effect of both 5 and 20 µM LSP1-2111. That increase seems already maximal when Compound 3 is co-exposed with LSP1-2111 at its ~EC20 (5µM). Thus, Compound 3 behaves as a very potent mGluR4 PAM.

Compound 4 at 10 µM does not modify the LSP1-2111 effect, neither at its ~EC20 (5µM), nor at its ~EC80 (20 µM). Thus, Compound 4 is devoid of effect at mGluR4.

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Electrophysiological characterisation of metabotropic glu4 receptors

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