急性脑组织切片的制备

Thomas  Papouin; Philip G. Haydon

doi:10.21769/BioProtoc.2699

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Peer-reviewed

Obtaining Acute Brain Slices

急性脑组织切片的制备

Thomas Papouin email

PH Philip G. Haydon

发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2699 浏览次数: 23453

评审: Pengpeng LiSalome Calado BotelhoJingli Cao

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参见作者原研究论文

The authors used this protocol in:

Cover of Neuron, featuring study using the protocol.

May 2017

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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Abstract

Obtaining acute brain slices for electrophysiology or amperometric recordings has become a routine procedure in most labs in the field of neuroscience. Yet, protocols describing the step by step process are scarce, in particular for routine acute preparations such as from the mouse hippocampus. Here we provide a detailed protocol for the dissection, extraction and acute slicing of the mouse brain, including tips and list of material required.

Keywords: Acute brain slices (急性脑切片)

Hippocampus (海马)

Dissection (解剖)

Brain extraction (脑提取)

Electrophysiology (电生理学)

Mouse (小鼠)

Background

With the democratization of in vitro electrophysiology and amperometry recording techniques, obtaining acute slices from rodent brains has become a classic and pivotal procedure in neuroscience research. Yet, the know-how required to achieving this procedure is typically passed on verbally, and most labs have developed home-made recipes and adapted the most important steps to their own needs or region of interest, such that there is a lack of protocols describing how to obtain high-quality acute brain slices in a step by step manner. While some protocols can be found describing particularly challenging preparations, such as acute slicing of adult mouse spinal cord (Garre et al., Bioprotocol 2017: https://doi.org/10.21769/BioProtoc.2102), a description of the basic procedure for more routine preparations (e.g., hippocampal slices) is particularly lacking. Aside from the practical aspect, this is a major problem because extracellular field recordings from hippocampal slice have become widely employed, due to their relative simplicity and little equipment-requirement, including by labs without electrophysiology and acute brain slices preparation expertise. Given that the slices’ quality is the limiting factor to obtaining reliable electrophysiological recordings, this poses a major challenge for the reproducibility of results within and across labs. In light of these needs and caveats, we here provide a detailed protocol for the dissection, brain extraction and acute slicing of the mouse hippocampus, including tips and list of material required. It will allow beginner and non-experts to obtain acute hippocampal brain slices of the required quality for follow-up studies such as field recordings, patch-clamp recordings or amperometric recordings (Papouin et al., 2017).

Materials and Reagents

Materials

For the ‘nest beaker’
1. Nylon tights
2. Instant superglue (such as Scotch Super Glue, 3M, catalog number: AD124 )
3. 15 ml tubes (such as VWR, catalog number: 89039-670 US, 525-0450 Europe)
4. Disposable 6 cm diameter plastic Petri dish (such as Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 123TS1 )
For dissection
1. Large kitchen scissors or guillotine
2. Straight fine scissors (such as Fine Science Tools, catalog number: 14060-11 )
3. Curved spatula (such as Fine Science Tools, catalog number: 10092-12 )
4. Scalpel (such as Fine Science Tools, catalog number: 91003-12 )
5. Glass disposable Pasteur pipet (such as Fisher Scientific, FisherBrand, catalog number: 13-678-6A )
6. Dropper bulb (such as Fisher Scientific, FisherBrand, catalog number: 03-448-25 )
7. Plastic container, about 2.5 cm high and 150 ml, such as the lid of a pipet tip box or a large glass Petri dish (Cole-Parmer Instrument, catalog number: EW-34551-06 )
8. Whatman paper (GE Healthcare, Whatman, catalog number: 1001-090 )
9. Disposable Razor blade (such as Personna Double Edge Razor Blades [Amazon, PERSONNA, catalog number: BP9020 ])

Reagents

Glucose (Sigma-Aldrich, catalog number: G7021 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
Sodium phosphate monobasic anhydrous (VWR, catalog number: 470302-666 )
Manufacturer: ALDON, catalog number: SS0756-500GR .
Sodium bicarbonate (NaHCO₃) (Sigma-Aldrich, catalog number: S5761 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
Magnesium chloride solution (1 M) (Sigma-Aldrich, catalog number: 63069 )
Calcium chloride solution (1 M) (Sigma-Aldrich, catalog number: 21115 )
Stock artificial cerebrospinal fluid (ACSF) solution (see Recipes)
Ice-cold Slicing ACSF solution (see Recipes)
Recovery ACSF solution (see Recipes)
Experimental ACSF (see Recipes)

Equipment

250 ml Pyrex beaker (such as VWR, catalog number: 10754-952 )
Straight spring scissors (such as Fine Science Tools, catalog number: 15018-10 )
Curved fine forceps (such as Fine Science Tools, catalog number: 11152-10 )
600 ml Pyrex beaker (such as VWR, catalog number: 10754-956 )
95% O₂/5% CO₂ tank (such as AirGas, catalog number: Z02OX9522000043 )
Vibratome (such as Leica, model: Leica VT 1200 S , catalog number: 14048142066)
Bath heater (such as Thermo Fisher Scientific, Thermo Scientific, model: Precision 180 , catalog number: 51221073)