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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.
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.
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 ....
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 .......
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.......
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. .......
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.
....Name | Company | Catalog Number | Comments |
“60 degree” tool | made in-house | ||
#10 scalpel blade | Bard-Parker (Aspen Surgical) | 371110 | |
1M CaCl2 | Fluka Analytical | 21114 | |
95%O2/5%CO2 | Praxiar or another local supplier | ||
Acepromazine maleate (AceproJect) | Henry Schein | 5700850 | |
Agar | Fisher | BP1423-500 | |
Beakers, measuring cylinders, reagent bottles | |||
Brushes size 00-2 | Ted Pella | Crafts stores are another source of soft brushes, with larger selection and better quality than Ted Pella. | |
CCD camera | Olympus | XM10 | |
Choline bicarbonate | Pfalz & Bauer | C21240 | |
Cyanoacrilate glue | Krazy glue | Singles | |
Decapitation scissors | FST | 14130-17 | |
Feather blades | Feather | FA-10 | |
Filter paper #2 | Whatman | Either rounds or pieces cut from a bigger sheet work well. | |
Forceps | A. Dumont & Fils | Inox 3c | |
Glass bubblers (Robu glass borosillicate microfilter candles) - porosity 3 | Robuglas.com | 18103 or 18113 | Glass bubblers are more expensive than bubbling stones used in aquaria. However, they are easy to clean and sterilize, and can last a long time. |
Glucose | Sigma-Aldrich | G8270 | |
HCl | Fisher | A144SI-212 | |
Ice buckets | |||
KCl | Sigma-Aldrich | P4504 | |
Ketamine HCl (KetaVed) | VEDCO | NDC 50989-996-06 | |
KH2PO4 | Sigma-Aldrich | P0662 | |
Leica Tissue slicer VT1000S | The cutting settings are 1 mm horizontal blade amplitude, frequency dial at 9, and speed setting at 2 | ||
Magnetic stirrers and stir bars | |||
Mg2SO4 x 7H2O | Sigma-Aldrich | 230391 | |
MgCl2 | Sigma-Aldrich | M9272 | |
MilliQ water machine | Millipore | Source for 18 Mohm water | |
Na-ascorbate | Sigma-Aldrich | A4035 | |
Na-pyruvate | Sigma-Aldrich | P8574 | |
NaCl | Sigma-Aldrich | S3014 | |
NaHCO3 | EMD | SX0320-1 | |
Needle 27G1/2 | |||
NMDG | Sigma-Aldrich | M2004 | |
Paper tape | |||
Peristaltic pump | Cole-Parmer | #7553-70 | |
Peristaltic pump head | Cole-Parmer | Masterflex #7518-00 | |
Personna blades | Personna double edge | Amazon | |
pH meter | |||
Recovery chamber | in-house made | ||
Scalpel blade handle size 3 | Bard-Parker (Aspen Surgical) | 371030 | |
Scissors angled blade | FST | 14081-09 | |
Single edge industrial razor blade #9 | VWR | 55411 | |
Spatulas | |||
Transfer pipettes | Samco Scientific | 225 | |
Upright microscope | Olympus BX51WI | ||
Xylazine HCl (XylaMed) | VetOne | 510650 |
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