We thank Bernard Foucart for technical assistance. This work was supported by the Belgian Fund for Scientific Research (F.R.S.-FNRS) and by the Queen Elisabeth Fund for Medical Research. Agn&egrave;s Villers is Research Fellow at the Belgian Fund for Scientific Research.

All animal procedures were carried out in accordance with National Institutes of Health regulations for the care and use of animals in research and with the agreement of the local ethics committee.
1. Preparation of Artificial Cerebro-spinal Fluid
The same media is used to dissect, cut and perfuse slices (1 ml/min) during the resting period and the electrophysiological recordings. This media is composed of 124 mM NaCl, 4.4 mM KCl, 26 mM NaHCO3, 1 mM NaH<sub...

We have developed in our laboratory a protocol resulting from the combination of methods developed and used by other laboratories having a big expertise in LTP recordings 11,17. This protocol is adapted to adult mouse hippocampus and can be used in animals of any age and any background genotype. It also allows the analysis of LTP in transgenic mice developing neurodegenerative diseases like Alzheimer's disease 18,19.
Utilization of this protocol for rat hippocampal slice...

Nowadays, there is limited understanding of how complex memories are stored and recalled at the neuronal circuit level. However, a unifying hypothesis of memory storage is available and broadly accepted: memories are stored as changes in the strength of synaptic connections between neurons in the central nervous system. On its own, research on synaptic plasticity has largely benefited from two breakthrough discoveries. (1) In a seminal experiment, Bliss and Lomo 1, using the intact anesthetized rabbit, found that delivery of a brief high-frequency (1 sec, 100 Hz) stimulation to the perforant path of the hippocampus caused a long-lasting (several hours) increase in the related synaptic connections. This fascinating phenomenon was called "Long-Term Potentiation" or LTP by Douglas and Goddard in 1975 2. (2) Later on, it was found that a similar phenomenon could be triggered in brain slices (0.4 mm) artificially maintained alive in vitro. The most widely studied LTP was observed in vitro by delivering one or several tetani to a bundle of axons (the so-called Schaffer collaterals) while recording the resulting field excitatory synaptic potential evoked in the pyramidal neurons of the so-called CA1 region. The mechanisms of LTP induction have largely been revealed. Basically, a Ca2+ influx through the NMDA receptors activates enzymes with two consequences: a phosphorylation of AMPA receptors (which increases their efficiency) and an incorporation of extra AMPA receptors in the postsynaptic membrane 3. By contrast, the mechanisms of the maintenance phase of LTP are largely unknown, notably because it is experimentally much more difficult to maintain a slice healthy for many hours than for 30 to 60 min.
A lot of studies have been dedicated to the understanding of LTP mechanisms and interesting theories have been elaborated over the years 4-11. But until now, the precise molecular mechanisms underlying the stable increase in synaptic strength have not been elucidated. This could be partly due to the difficulty to reproduce previous results in different laboratories using different techniques for the preparation and the maintenance of hippocampal slices. In their methodology paper, Sajikumar et al. 12 stressed the importance of experimental conditions for the preparation of rat hippocampal slices and the recording of stable LTP. In this video we present all the optimization steps developed in our laboratory over the years to be able to record a very stable LTP in mouse hippocampal slices.
This optimization has been made from protocols developed and successfully used by other laboratories which study LTP mechanisms in mice 13 and rats 11. It allows experienced researchers to induce and record a very long lasting LTP in adult mice with a high rate of success. The physiological basis of the induced LTP was carefully checked and demonstrated 14. In this methodology paper, we show that any modifications of experimental conditions, like temperature or oxygenation can have a profound impact on LTP maintenance while the dissection procedure can deeply modify slices excitability. It must also be emphasized that the precise control of all these parameters requires a training of several months for novice students.

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent/Material</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma - Aldrich</td>
			<td>S7653</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma - Aldrich</td>
			<td>S8875</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma - Aldrich</td>
			<td>P9333</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma - Aldrich</td>
			<td>G7528</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Sigma - Aldrich</td>
			<td>S9638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgSO4 1M</td>
			<td>Sigma - Aldrich</td>
			<td>63126</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma - Aldrich</td>
			<td>C4901</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Air Liquide (Belgium)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillaries</td>
			<td>WPI, Inc. (UK)</td>
			<td>TW150-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulating Electrodes</td>
			<td>FHC (USA)</td>
			<td>CE2B30</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical tools</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Filter paper 84 g/m2</td>
			<td>Sartorius</td>
			<td>FT-3-105-110</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Mesh</td>
			<td>Lycra</td>
			<td>15 den</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>UHU</td>
			<td>plus endfest300</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>WPI, Inc. (UK)</td>
			<td>ISO-80</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Interface recording chamber</td>
			<td>FST (Germany)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Peristaltic pumps</td>
			<td>Gilson (USA)</td>
			<td>Minipuls 3</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>University of Edinburgh</td>
			<td><a href="http://www.etcsystem.com" target="_blank">www.etcsystem.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>Mcllwain</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stimulators</td>
			<td>Grass (USA)</td>
			<td>S88X + SIU-V</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Program analysis</td>
			<td>WinLTP</td>
			<td><a href="http://www.winltp.com" target="_blank">www.winltp.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Narishige</td>
			<td>MM-3 and MMO-220A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica Microsystem</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>A/D converter</td>
			<td>National Instruments</td>
			<td>NIPCI-6229 M-series</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

improved preparation and preservation of hippocampal mouse slices for a very stable and reproducible recording of long-term potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This methodology has been used to analyze the properties of long-lasting long-term potentiation induced in acute hippocampal slices from adult C57Bl/6J mice (JANVIER SAS, France) 14. Surprisingly, improvement of the experimental conditions has led to a new way of looking at LTP. We showed that long-lasting increase in synaptic strength did not require the synthesis of new proteins.
Here, we show that LTP induction depends on slices viability and excitability. When dissection of the ...

Watch this Scientific Journal Video about Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation at JoVE.com

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

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JoVE Journal

Neuroscience

26.5K vistas.  University of Mons. En este trabajo se presenta una metodología completa para preparar y conservar In vitro Cortes de hipocampo agudos de ratones adultos. Este protocolo permite la grabación de la potenciación a largo plazo duradera muy estable (LTP) durante más de 8 horas con una tasa de éxito del 95%.