Protocol for compounds evaluation on the ltp ltd threshold

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Protocol for compounds

evaluation on the LTP/LTD

threshold

September, 2013


SUMMARY

Introduction Aim of the study

Materials & Methods Preparation of acute rat hippocampal slices

Slice perfusion and temperature control

Stimulation protocols

Experiments Determination of LTP/ Neutral/LTD protocols in the CA1 region of rat hippocampal slices (crossover point)


INTRODUCTION

The objective of this protocol is to determine the “cross-over point” point between LTD and LTP, providing the ability to investigate the potency of compounds to shift this point.

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.

Stimulation protocols Basal synaptic transmission: The stimulus intensity is set to 300 µA at 0.033Hz.

Long-Term Potentiation (LTP)/Neutral/Long-Term Depression (LTD) protocols: Stimulation trains from 1 to 200 Hz.


EXPERIMENTS

Plots of LTD – LTP profiles that are used to determine the cross-over point

Between 1 and 20 Hz, the stimulations train, induces Long-Term Depression (LTD) of evoked-responses. The 50 Hz stimulation train does not modify the evoked-response amplitudes.

At 100 Hz and 200 Hz, the stimulations train induces Long-Term Potentiation (LTP) of evoked-responses.

The effect of a stimulation trains applied with a wide range of frequencies (1 to 200 Hz) were investigated to determine the LTP/LTD crossover point.


EXPERIMENTS

Determination of LTP/Neutral/LTD cross-over point in the CA1 region of rat hippocampal slices

The LTP/LTD crossover point is close to 50 Hz. Indeed, a train of stimulations applied at 50 Hz does not substantially modifies the fEPSP amplitude (the mean percentage of fEPSP change after 60 minutes is of -2.9% ±5 %).

Bar chart of LTD / LTP amplitudes expressed as a function of the stimulation frequency

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The LTP/LTD crossover point is close to 50 Hz. Stimulations below 50 Hz trigger LTD, whereas stimulations above 50 Hz trigger LTP.

Compounds such as NMDA modulators could be evaluated in order to determine if they can shift the crossover point, and then have the potency to “balance” the mechanisms of synaptic plasticity and long-term memory.

CONCLUSION

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Protocol for compounds evaluation on the ltp ltd threshold

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