Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Summary

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for >24 h.

Abstract

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (>24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 °C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at >24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Introduction

Electrophysiology and functional imaging (calcium, voltage sensitive dyes) are two of the most commonly used experimental techniques in neuroscience. Brain slice preparations and retinal wholemount, which will be examined here, provide a means of examining electrophysiological properties and synaptic connectivity without contamination from anesthetics or muscle relaxants. Brain slices and retinal wholemount maintain their structural integrity, unlike cultures or cell homogenates, allowing the study of specific circuits and brain networks¹. Recordings from isolated tissue have advantages over in vivo recordings as movements associated w....

Protocol

The protocol below describes the preparation of C57BL/6 and C3H/He (retinally degenerate) mouse neuronal tissue, but similar techniques can be applied to other species. All animals were healthy and handled with standard conditions of temperature, humidity, 12 h light/dark cycle, free access to food and water, and without any intended stress stimuli. All experiments were approved and performed in accordance with Western Sydney University Animal Care and Ethics committee and according to the animal use and care guidelines (Animal Research Authority #A9452, #A10396 and #A8967).

1. Brain Slice Preparation

1. Anesthetize the animal by inhalation of isoflura....

Results

Tight regulation of the bacterial load and temperature of the aCSF during incubation is essential to maintain neuronal tissue viability. This can be optimized through irradiation with UVC light and maintaining the temperature of the aCSF at 15 - 16 °C (Figure 1). Furthermore, the aCSF parameters stamp (APS; Figure 1C) provides the experimenter with a record of the environmental conditions (pH and temp.), thus offering a gold standard for parameters when i.......

Discussion

This article describes an incubation method to extend the viability of acute neuronal tissue for imaging and electrophysiological experiments, thereby reducing the animal numbers needed to complete experimental goals. Two main processes govern the deterioration of neuronal tissue over time: i) increased bacteria levels, and the accompanying increase in bacterial endotoxin released, and ii) excitotoxicity which follows the traumatic slicing procedure¹⁰. As acute neuronal tissue is environmentally d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium Chloride	Sigma Aldrich VETEC	V800372
Potassium chloride	Sigma Aldrich	P9333
N-Methyl-D-glucamine	Sigma Aldrich	M2004
D-glucose	Sigma Aldrich	G5767
Sodium bicarbonate	Sigma Aldrich	S6014
Sodium phosphate monobasic	Sigma Aldrich	S2554
Magnesium chloride	Sigma Aldrich	M9272
Calcium chloride	Sigma Aldrich	223506
Papain Dissociation System	Worthington	LK003150
Dimethyl sulfoxide anhydrous	Sigma Aldrich	276855
Pluronic F-127 Low UV absorbance*	Life technologies	P-6867	20% solution in DMSO
Fura-2 AM	Anaspec	84017
Fluo-4 AM	Life Technologies	F23917
Fluo-8 AM	Abcam	Ab142773
Vibrating blade microtome	Leica Biosystem	VT1200 S	Equiped with Leica’s Vibrocheck™ measurement device
Antivibration table	Kinetic Systems Vibraplane	Vibraplane 9100/9200
Fixed Stage Upright Microscope	Olympus	BX51WI
Microscope Objective	Olympus	XLUMPlanFLN 20x/1.00w	20X
Microscope Objective	Olympus	LUMPlanFLN 60x/1.00w	60X
CCD Camera	Andor	Ixon +
High speed wavelength switcher	Sutter instrument	Lambda DG-4
Patch clamp amplifier	Molecular Devices	MultiClamp 700B
Data acquisition system	Molecular Devices	Digidata 1440A	Axon Digidata® System
Borosilicate glass capillaries	Sutter Instrument Co	BF150-86-10
Microelectrode puller	Sutter Instrument Co	P-97	Flaming/Brown type micropipette puller
Recording/perfusion chamber	Warner Instruments	RC-40LP	Low Profile Open Bath Chambers
Software	Molecular Devices	pClamp 10.2
Andor iQ Live Cell Imaging Software	Andor	Andor iQ3
Braincubator	Payo Scientific	BR26021976	www.braincubator.com.au
Peltier-Thermoelectric Cold Plate Cooler	TE Technology	CP-121
Temperature Controller	TE Technology	TC-36-25 RS232