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

Summary

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%.

Abstract

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.

Introduction

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) incr....

Protocol

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 NaH₂PO₄, 2.5 mM CaCl₂, 1.3 mM MgSO₄ and 10 mM D-glucose.

1. Weigh the different components for 2 L of ....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

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