Investigation of one compound effect on main neuronal properties in a native network final

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Investigation of one compound

potential effects on main

neuronal properties in a native

neuronal network

December, 2011


SUMMARY

Introduction Aim of the study

Materials & Methods Preparation of acute rat hippocampal slices

Slice perfusion and temperature control

Stimulation protocols

Experiments Evaluation of Compound A on I/O properties

Evaluation of Compound A on Paired-Pulse Facilitation

Evaluation of Compound A on basal synaptic transmission

Evaluation of Compound A on Long-Term Potentiation

Evaluation of Compound A on Long-Term Depression

Evaluation of Compound A on Paired-Pulse Inhibition


INTRODUCTION

The aim of the study is to evaluate one compound effect on the main neuronal properties in a native neuronal network, such as the hippocampal slice.

For this aim, the effect of the compound will be investigated in several protocols:

Input/Output (I/O) curve (overall effect on excitability)

Paired-Pulse Facilitation (PPF; effect on short-term synaptic plasticity)

Basal synaptic transmission (balance between excitatory and inhibitory transmission)

Long-Term Potention (LTP) and Long-Term Depression (LTD) (long term synaptic plasticity mechanisms)

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


MATERIALS & METHODS

Preparation 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 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 37°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 37 ± 0.1°C with a heating element located in the MEA amplifier headstage.


MATERIALS & METHODS

Stimulation protocols • Input/Output (I/O) curve: stimulation intensities between 100 and 800 μA, by 100 μA steps.

• Paired-Pulse Facilitation (PPF): Two pulses with a decreasing inter-stimulus interval (e.g. 300 ms, 200 ms, 100 ms, 50 ms, 25 ms) are applied at Schaeffer collaterals. Both stimuli are of equal intensity and settled at 40 % of the maximal amplitude responses.

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

• Long-Term Potentiation (LTP): two 1 s trains of stimulations at 100 Hz, applied at 20 s intervals.

• Long-Term Depression (LTD): 900 stimulations at 1 Hz.

• Paired-Pulse Inhibition (PPI): Two pulses with a 20 ms inter-pulse interval are applied every minute with an intensity evoking PPI of the second PS


EXPERIMENTS

Evaluation of Compound A on I/O properties

No difference is observed between the I/O curve determine in control conditions and in the presence of compound A.

I/ O c u r v e

0 2 0 0 4 0 0 6 0 0 8 0 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

C o n t r o l

C o m p o u n d A

S t im u lu s In te n s ity (µ A )

fEP

SP

am

pli

tud

e (

µV

)


EXPERIMENTS

Evaluation of Compound A on PPF characteristics

No significant difference is observed for the PPF profile between control conditions and in the presence of compound A.

P a ire d P u ls e F a c ilita t io n

0 1 0 0 2 0 0 3 0 0

1 .0

1 .2

1 .4

1 .6

1 .8

2 .0

C o n t r o l

C o m p o u n d A

In t e r -s t im u lu s In te r v a l (m s )

fEP

SP

2/

fEP

SP

1


EXPERIMENTS

Evaluation of Compound A on basal synaptic transmission

Compound A slightly decreases the basal synaptic transmission. At endpoint, the fEPSP amplitude is decreased by about 12%.

B a s a l s y n a p t ic t r a n s m is s io n

0 1 0 2 0 3 0 4 0

0 .4

0 .6

0 .8

1 .0

1 .2

C o m p o u n d A

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de


EXPERIMENTS

Evaluation of Compound A on LTP

In control conditions, HFS induces a strong and durable potentiation of evoked-responses, which stabilized around 55%.

Compound A decreases the basal synaptic transmission by about 10%, as observed previously. However, HFS-induced potentiation remains very close to the one in control conditions (the potentiation is of 56 ± 7% in the presence of Compound A versus 57 ± 5% in control conditions, at endpoint).

L T P S t im S C

1 0 3 0 5 0 7 0 9 0

1 .0

1 .5

2 .0

2 .5

C o m p o u n d A

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de


EXPERIMENTS

Evaluation of Compound A on LTD

Compound A slightly decreases the basal synaptic transmission.

However, LFS-induced LTD does not substantially differ in hippocampal slices exposed to Compound A and in control conditions (the depression is of 57 ± 1% in the presence of Compound A versus 64 ± 4% in control conditions, at endpoint.

L T D S t im S C

1 0 3 0 5 0 7 0 9 0

0 .0

0 .4

0 .8

1 .2

C o m p o u n d A

T im e (m in )

No

rma

liz

ed

fE

PS

P a

mp

litu

de


EXPERIMENTS

Evaluation of Compound A on PPI

Compound A does not modify the paired-pulse inhibition (PS ratio remains steady over the 30 minute exposure).

P a ir e d P u ls e In h ib it io n

0 1 0 2 0 3 0 4 0 5 0

0 .0

0 .5

1 .0

1 .5

2 .0

P e a k 2

N o r m a liz e d R a t io P 2 / P 1

P e a k 1

C o m p o u n d A

T im e (m in )


Input/Output properties are not modified by Compound A

Paired-pulse facilitation is not modified by Compound A

Compound A slightly decreases the fEPSP amplitude, by about 10%.

The LTP amplitude is not modified in the presence of Compound A, thereby indicating that this compound does not modify NMDA receptors function and downstream mechanisms involved in LTP establishment and maintain.

The LTD amplitude is not modified in the presence of Compound A. This further confirms that Compound A does not modify NMDA receptors function and downstream pathways involved in the establishment of LTD and its maintain.

The PPI ratio is not modified in the presence of Compound A, thereby indicating that Compound A does not change the balance between excitatory and GABAA-mediated inhibitory transmission

CONCLUSION

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Investigation of one compound effect on main neuronal properties in a native network final

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