Organotypic Hippocampal Slice Cultures

Summary

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Abstract

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation ¹. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory ². In addition, the well defined anatomy and connectivity of the hippocampus ³ has made it a classical model system to study synaptic transmission and synaptic plasticity⁴.

As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.

Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection ^5,6 or biolistics ⁷. In addition, slices can be easily recovered for biochemical assays ⁸, or transferred to microscopes for imaging ⁹ or electrophysiological experiments ¹⁰.

Protocol

1. Before Starting the Preparation of Hippocampal Slices.

1. Prepare the tissue slicer by placing a piece of Teflon sheet and mounting a new blade.
2. Wipe the tissue culture (TC) hood with 70% ethanol and set the dissecting microscope inside. Sterilize the hood, microscope, tissue slicer and all dissecting instruments for 15 minutes with UV light.
3. Prepare six well TC plates. Add 750 μL slice culture media (SCM) per well and place cell culture inserts in each well. Make sure the membranes are thoroughly wet with no bubbles underneath. Place the plates in incubator at 35°C gassed with 5% CO₂ until needed.
4. Pour....

Discussion

This method is based on the method first described by Stoppini et al. ¹¹ and offers a rapid manner to culture hippocampal slices. The most important aspect of this protocol is to maintain slices sterile; therefore it is critical to use appropriate sterile techniques and to properly disinfect and sterilize all the material in contact with the tissue.

Different serums sources can influence the quality of the slices. We recommend testing several batches first. If contaminat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture inserts	EMD Millipore	PICM03050
6 well plates	BD Biosciences	353046
Tissue Slicer	Stoelting Co.	51425/51415
Microscope	Olympus Corporation	SZX7-ILLD2-100
Hippocampus dissecting tool	F.S.T.	10099-15
Large utility scissors. Perfection	F.S.T.	37500-00/37000-00 Right/ Left handed
Iris Spatula	F.S.T.	10093-13
Straight spatula	F.S.T.	10094-13
Rounded spoon micro spatula	VWR international	57949-039
Dissecting single cutting edge needle	Electron Microscopy Sciences	72946
Dissecting tweezers	Dumont	#2
Small dissecting scissors	F.S.T.	14060-10
MEM Eagle medium	Cellgro	50-019 PB
Horse serum heat inactivated	Invitrogen	26050-88
L-Glutamine (200 mM)	Invitrogen	25030081
CaCl2 (1 M)	Sigma-Aldrich	C3881
MgSO4 (1 M)	Sigma-Aldrich	M2773
Insulin (1 mg/ml), dissolved in HCl 0.01 N	Sigma-Aldrich	I0516
Ascorbic Acid, solution (25%)	Sigma-Aldrich	A4544
D-Glucose	Sigma-Aldrich	G5767
NaHCO3	Sigma-Aldrich	S6014
Hepes	Sigma-Aldrich	H7523
Sucrose	Sigma-Aldrich	S5016
Phenol Red Solution 0.5% in DPBS	Sigma-Aldrich	P0290
KCl	Sigma-Aldrich	P3911
MgCl2 (1 M)	Sigma-Aldrich	M9272