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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Abstract

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol’s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

Introduction

The advent of mammalian acute brain slice preparations facilitated experiments at the cellular and synaptic level that were previously possible only in invertebrate preparations like Aplysia1. Development of acute hippocampal slices was of particular significance, as it is a structure responsible for working memory and context formation, and has a specialized tri-synaptic circuitry that is amenable to easy physiological manipulation. However, the vast majority of acute brain slices are still prepared from relatively young mice and rats, as it is easier to preserve healthy neurons and circuits, and the slices remain viable for longer periods of ....

Protocol

The protocol is carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Stanford University Institutional Animal Care and Use Committee. Methods are also in accordance with the Policies of the Society for Neuroscience on the Use of Animals and Humans in Neuroscience Research.

NOTE: All mice were maintained in a pathogen-free environment. Wild-type mice on mixed C57Bl/ 6 x SV/ 129J genetic background were used .......

Representative Results

We applied the above protocol to generate hippocampus slices from CamKIIa-Cre+;WT mice on a mixed genetic background C57Bl/ 6 x SV/ 129J, at P > 120. Large numbers of pyramidal cells in the CA1 field (Figure 2A) and subiculum (Figure 2B) appear in low contrast when observed under infrared differential contrast microscopy (IR-DIC), a hallmark of healthy cells in a slice preparation. With this preparation, a high rate of giga-ohm seals (>90%) is routi.......

Discussion

The protocol described here demonstrates that hippocampal slices obtained from adult and aging mice can remain healthy and viable for many hours after cutting. The slices prepared using this protocol are appropriate for patch-clamp recordings, as well as long-lasting field-recordings in the CA1 regions.

There are two critical steps in this protocol. First step is the transcardial perfusion step with an ice-cold solution. Fast clearance of blood is signaled by rapid change of liver color. .......

Acknowledgements

I thank Dr. Carla J. Shatz for advice and support, and Dr. Barbara K. Brott and Michelle K. Drews for critically reading the manuscript. The work is supported by NIH EY02858 and the Mathers Charitable Foundation grants to CJS.

....

Materials

NameCompanyCatalog NumberComments
“60 degree” toolmade in-house
#10 scalpel bladeBard-Parker (Aspen Surgical)371110
1M CaCl2Fluka Analytical21114
95%O2/5%CO2Praxiar or another local supplier
Acepromazine maleate (AceproJect)Henry Schein5700850
AgarFisherBP1423-500
Beakers, measuring cylinders, reagent bottles
Brushes size 00-2Ted PellaCrafts stores are another source of soft brushes, with larger selection and better quality than Ted Pella.
CCD cameraOlympusXM10
Choline bicarbonatePfalz & BauerC21240
Cyanoacrilate glueKrazy glueSingles
Decapitation scissorsFST14130-17
Feather bladesFeatherFA-10
Filter paper #2WhatmanEither rounds or pieces cut from a bigger sheet work well.
ForcepsA. Dumont & FilsInox 3c
Glass bubblers (Robu glass borosillicate microfilter candles) - porosity 3Robuglas.com18103 or 18113Glass bubblers are more expensive than bubbling stones used in aquaria. However, they are easy to clean and sterilize, and can last a long time.
GlucoseSigma-AldrichG8270
HClFisherA144SI-212
Ice buckets
KClSigma-AldrichP4504
Ketamine HCl (KetaVed)VEDCONDC 50989-996-06
KH2PO4Sigma-AldrichP0662
Leica Tissue slicer VT1000SThe cutting settings are 1 mm horizontal blade amplitude, frequency dial at 9, and speed setting at 2
Magnetic stirrers and stir bars
Mg2SO4 x 7H2OSigma-Aldrich230391
MgCl2Sigma-AldrichM9272
MilliQ water machineMilliporeSource for 18 Mohm water
Na-ascorbateSigma-AldrichA4035
Na-pyruvateSigma-AldrichP8574
NaClSigma-AldrichS3014
NaHCO3EMDSX0320-1
Needle 27G1/2
NMDGSigma-AldrichM2004
Paper tape
Peristaltic pumpCole-Parmer#7553-70
Peristaltic pump headCole-ParmerMasterflex #7518-00
Personna bladesPersonna double edgeAmazon
pH meter
Recovery chamberin-house made
Scalpel blade handle size 3Bard-Parker (Aspen Surgical)371030
Scissors angled bladeFST14081-09
Single edge industrial razor blade #9VWR55411
Spatulas
Transfer pipettesSamco Scientific225
Upright microscopeOlympus BX51WI
Xylazine HCl (XylaMed)VetOne510650

References

  1. Glanzman, D. L. The cellular mechanisms of learning in Aplysia: of blind men and elephants. Biological Bulletin. 210 (3), 271-279 (2006).
  2. Aitken, P. G., et al. Preparative methods for brain slices: a discussion.

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Hippocampal SlicesAdultAgingHypoxiaHypothermiaNMDG aCSFTranscardial PerfusionVibrating MicrotomeField recordingsPatch clampNeurodegenerative Diseases

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