Name: prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging
Uploaded: 2017-02-15T15:00:00
Description: Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

We thank Colin Symons and James Wright for technical assistance. This work was supported by seed funding grant (WSU) to Y.B. and the innovation office at Western Sydney University. M.C. is supported by ARC fellowship (DECRA).

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 Auth...

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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...

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 with the heartbeat and respiration are eliminated. Moreover, direct visualization allows specific classes of cells to be targeted, and local application of pharmacological tools 2,3.
Patch-clamp recordings and calcium dye-loading in retinal wholemount is complicated by the existence of the Inner Limiting Membrane (ILM), which covers the Retinal Ganglion Cell (RGC) layer and prevents direct access to the cells. Typically, this membrane is scraped away with a glass pipette to allow direct application of a patch pipette and formation of a gigaohm seal on a single cell. In addition, bath-applied calcium&#160;dyes do not cross the ILM and must either be injected beneath this membrane4, retrogradely transported following injection at the optic nerve5 or electroporated through the tissue6. Furthermore, when utilizing a rodent model of retinitis pigmentosa, the rd/rd mouse, the ILM is thicker and more impenetrable. Here, we use a technique to remove the ILM with enzymatic digestion7, to allow both ubiquitous calcium&#160;dye-loading, and direct access to retinal ganglion cells for patch-clamp recordings8.
Successful recordings from either brain slices or retinal wholemount depend on dissection and incubation of viable neuronal tissue. Typically, tissue is extracted on the morning of the experiment and incubated in artificial cerebrospinal fluid (aCSF) until it is used for recordings. Usually, tissue remains viable for 6 - 8 h, with significant degradation following this time window. However, both brain slices and wholemount retinae preparations usually produce more tissue than can be recorded from within this short time period. Consequently, tissue is often discarded at the end of the day and the dissection is completed again on subsequent days. This means another animal is utilized and ~2 h of setup and dissection/staining repeated. The following protocol describes a method for extending the life of neuronal tissue for more than 24 h, meaning fewer animals are utilized, and more experimental time is available. Tissue viability was assessed through recording electrophysiological properties and calcium dynamics, and these properties were indistinguishable between &#60;4 h and &#62;24 h postdissection.
These results indicate that not only are single cell properties intact and functional after prolonged incubation, but network activity, as assessed by calcium-imaging and electrophysiological recordings, is unchanged &#62;24 h postdissection. Moreover, we show that calcium dyes can remain in cells for prolonged periods without causing any detrimental effects. Application of this protocol demonstrates that the functional activity of neurons in acute neuronal tissue can be maintained for long periods, once the external environment is highly regulated. Moreover, as tissue viability varies greatly between laboratories due to different incubation protocols, this method establishes a gold standard for the ideal parameters that should be applied to reduce variability in the health of acute neuronal tissue.

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			<td height="19" style="height:19px;width:315px;">Pluronic F-127 Low UV absorbance*</td>
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			<td style="width:225px;">20% solution in DMSO&#160;</td>
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			<td height="20" style="height:20px;width:315px;">Fluo-8 AM</td>
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			<td style="width:135px;">Ab142773</td>
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			<td height="60" style="height:60px;width:315px;">Vibrating blade microtome&#160;</td>
			<td style="width:159px;">Leica Biosystem</td>
			<td style="width:135px;">VT1200 S</td>
			<td style="width:225px;">Equiped with Leica&#8217;s Vibrocheck&#8482; measurement device&#160;</td>
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			<td height="40" style="height:40px;width:315px;">Antivibration table</td>
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			<td style="width:135px;">Vibraplane 9100/9200&#160;</td>
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			<td height="20" style="height:20px;width:315px;">Fixed Stage Upright Microscope</td>
			<td style="width:159px;">Olympus</td>
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			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">XLUMPlanFLN 20x/1.00w</td>
			<td style="width:225px;">20X&#160;</td>
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			<td height="40" style="height:40px;width:315px;">Microscope Objective</td>
			<td style="width:159px;">Olympus</td>
			<td style="width:135px;">LUMPlanFLN 60x/1.00w</td>
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			<td style="width:159px;">Andor</td>
			<td style="width:135px;">Ixon +</td>
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			<td height="20" style="height:20px;width:315px;">Patch clamp amplifier</td>
			<td style="width:159px;">Molecular Devices</td>
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			<td style="width:225px;">Flaming/Brown type micropipette puller</td>
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			<td style="width:159px;">Warner Instruments</td>
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			<td height="20" style="height:20px;width:315px;">Braincubator</td>
			<td style="width:159px;">Payo Scientific</td>
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			<td style="width:225px;">www.braincubator.com.au</td>
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			<td height="20" style="height:20px;">Peltier-Thermoelectric Cold Plate Cooler</td>
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			<td>CP-121</td>
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			<td height="20" style="height:20px;">Temperature Controller</td>
			<td>TE Technology</td>
			<td>TC-36-25 RS232</td>
			<td></td>
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prolonged incubation of acute neuronal tissue for electrophysiology and calcium-imaging

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&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;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 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;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.

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 &#176;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 w...

Watch this Scientific Journal Video about Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging at JoVE.com

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

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 &#62;24 h.

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

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