Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Summary

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Abstract

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

Introduction

The aim of the presented method is to get high-quality acute brain slices for in vitro electrophysiological experiments, especially when using adult or even old animals.

The acute brain slicing method, as described by Skrede and Westgaard¹ in two elegant sentences, has become one of the foundations of modern neuroscience research and is employed in innumerable variations worldwide. The quality of the slices is reflected in number of living neurons per slice, the period of time during which the cells keep their electrophysiological and morphological properties as well as in the integrity of the tissue. Moreover, the maxim....

Protocol

All experimental procedures described in this protocol were approved by the Hebrew University's Animal Care and Use Committee.

1. Preparing the Solutions and Tools for Slicing

1. Prepare 1 L of standard physiological solution (SPS) containing the ions described in Table 1.
   1. Prepare a stock solution containing the salts at 10 times the final concentration in advance in a storage glass bottle filled with 1,000 ml of deionized water (conductance of 0.055 µS/cm). Add salts (NaCl, KCl, KH₂PO₄, and MgSO₄), and stir until completely dissolved. Store the stock solution i....

Results

Slices prepared in the described manner can be used for various electrophysiological and optogenetic experiments. In Figure 3A and 3C, we show a representative example of a horizontal cerebellar slice and a coronal cerebral cortical slice, respectively, viewed under differential interference (DIC) optics. In the cerebellar slice, several types of cerebellar neurons can be easily recognized by their location and cell body shape, allowing targeted electrophysiological recordings. In

Discussion

We demonstrate a method for preparing acute brain slices from mice in physiological instead of ice-cold temperature.

It has been shown¹¹ that the quality of slices obtained in warm conditions is superior when compared with those prepared with cold conditions, provided that the slicer blade has minimal vertical vibration. Slicing in physiological temperature may prevent physiological artifacts caused by the low temperature, such as those related to changes in metabolic processes^.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pentobarbital	CTS	170066	Concentration: 60 mg/ml in physiological saline.
Big scissors	FST	14001-16	Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation
Iris scissors	Prestige medical	48,148	Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5–2 cm
Fine tip forceps	FST	11254-20
Scalpel	FST	91003-12
Scalpel blade #11	FST	10011-00
Small spatula	Fisher	2350
Filter paper			Any laboratory brand can be used.
Petri dishes	Duroplan	Z231509-1
Glass beakers	SCHOT	10022846
Pasteur pipette	Maple Leaf Brand	14672-029
Super glue	LOCTITE	4091361/1
Slicer	Campden	7000-smz
Ceramic slicing blade	Campden	7550-1-C
Magnetic heater/stirrer			For heating up the SPS for the procedure
Electric kettle			For heating up water for temperature control
Slice recovery chamber + heating unit	Warner instruments	BSC-HT +  BSC-BUW	Home-built models may also be used.
Thermometer			For monitoring SPS temperature during dissection and slicing