Name: slice it hot: acute adult brain slicing in physiological temperature
Uploaded: 2014-10-30T14:00:00
Description: Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

We would like to acknowledge the significant contribution Dr. Shiwei Huang (Australian National University) in validating the method. Furthermore, we would like to thank Ms. Kasia Pietrajtis for helpful comments regarding Golgi cells and Mr. Vitaly Lerner for the cortex experimental data. This work was supported by PITN-GA-2009-238686 (CEREBNET), FP7-ICT (REALNET), ELSC and ISF.

All experimental procedures described in this protocol were approved by the Hebrew University&#39;s Animal Care and Use Committee.
1. Preparing the Solutions and Tools for Slicing
<ol>
	<li>Prepare 1 L of standard physiological solution (SPS) containing the ions described in Table 1.
	<ol>
		<li>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 &...

<ol>
	<li>Skrede, K., Westgaard, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transverse+hippocampal+slice:+a+well-defined+cortical+structure+maintained+in+vitro.">The transverse hippocampal slice: a well-defined cortical structure maintained in vitro.</a> Brain Research. 35, 589-593 (1971).</li><li>Aghajanian, G., Rasmussen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+studies+in+the+facial+nucleus+illustrating+a+simple+new+method+for+obtaining+viable+motoneurons+in+adult+rat+brain+slices.">Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices.</a> Synapse. 3, 331-338 (1989).</li><li>Gueritaud, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+of+rat+ocular+motoneurons+recorded+in+vitro.">Electrical activity of rat ocular motoneurons recorded in vitro.</a> Neuroscience. 24, 837-852 (1988).</li><li>Lipton, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+best+of+brain+slices:+comparing+preparative+methods.">Making the best of brain slices: comparing preparative methods.</a> Journal of Neuroscience Methods. 59, 151-156 (1995).</li><li>Richerson, G., Messer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+composition+of+experimental+solutions+on+neuronal+survival+during+rat+brain+slicing.">Effect of composition of experimental solutions on neuronal survival during rat brain slicing.</a> Experimental Neurology. 131, 133-143 (1995).</li><li>Brahma, B., Forman, R., Stewart, E., Nicholson, C., Rice, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+inhibits+edema+in+brain+slices.">Ascorbate inhibits edema in brain slices.</a> Journal of Neurochemistry. 74, 1263-1270 (2000).</li><li>Moyer, J. 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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...

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 maximal duration for stable recordings depends on the quality of the slices. Thus, along the decades, the original slicing method has been further developed by individual research groups to enhance slice recovery after cutting2-10, often by complex modifications of the composition of cutting or recovery solutions (such as adding ascorbate, thiourea or even H2O2) as well as intra-cardiac pre-perfusion of the animal with cooled physiological solutions.
As has been recently shown11, physiological temperature during slicing seems to be more beneficial than cooling to neuronal health; the improvement is most striking when working with adult (2-8 month) rodents. Avoiding dramatic temperature changes prevents artifacts due to temperature-dependent processes in the cells, such as plasticity13 and ion-channels kinetics13,14. Such changes could influence membrane voltage and intracellular calcium signaling, spike threshold, and spike shape.
The &#8220;hot&#8221; acute slice preparation method presented here is a general procedure for obtaining high-quality acute brain slices from any brain region, including the cerebellum, the cortex and hippocampus, brainstem nuclei16 as well as the olfactory bulb, both in rats and mice.
Notably, the physiological temperature slicing procedure requires that the cutting blade vibrates nearly perfectly horizontally and is without any structural defects. Such precision might not be attainable with older slicer models; in such cases, we recommend performing the slice preparation in freezing-cold conditions as the low temperature seems to make the tissue more resistant to mechanical damage, even if at the cost of metabolic aberrations.

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		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Name</td>
			<td style="width:73px;">Company</td>
			<td style="width:69px;">Catalog #</td>
			<td style="width:292px;">Comments</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">Pentobarbital</td>
			<td style="width:73px;">CTS</td>
			<td style="width:69px;">170066</td>
			<td style="width:292px;">Concentration: 60 mg / ml&#160; in physiological saline.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Big scissors&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">14001-16</td>
			<td style="width:292px;">Any large scissors or a guillotine with sufficiently sharp edges can be used for decapitation&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Iris scissors&#160;</td>
			<td style="width:73px;">Prestige medical</td>
			<td style="width:69px;">48,148</td>
			<td style="width:292px;">Any fine tip scissors can be used, provided the scissor blades are not longer than 1.5 - 2 cm</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Fine tip forceps&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">11254-20</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel&#160;</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">91003-12</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Scalpel blade #11</td>
			<td style="width:73px;">FST</td>
			<td style="width:69px;">10011-00</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Small spatula&#160;</td>
			<td style="width:73px;">Fisher&#160;</td>
			<td style="width:69px;">2350</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Filter paper</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">Any laboratory brand can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Petri dishes</td>
			<td style="width:73px;">Duroplan</td>
			<td style="width:69px;">Z231509-1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Glass beakers&#160;</td>
			<td style="width:73px;">SCHOT</td>
			<td style="width:69px;">10022846</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Pasteur pipette&#160;</td>
			<td style="width:73px;">Maple Leaf Brand</td>
			<td style="width:69px;">14672-029</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Super glue&#160;</td>
			<td style="width:73px;">LOCTITE</td>
			<td style="width:69px;">4091361/1</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Slicer</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7000-smz</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Ceramic slicing blade</td>
			<td style="width:73px;">Campden</td>
			<td style="width:69px;">7550-1-C</td>
			<td style="width:292px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Magnetic heater/stirrer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up the SPS for the procedure</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:140px;">Electric kettle</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For heating up water for temperature control</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:56px;width:140px;">Slice recovery chamber + heating unit</td>
			<td style="width:73px;">Warner instruments</td>
			<td style="width:69px;">&#160;BSC-HT +&#160; BSC-BUW</td>
			<td style="width:292px;">Home-built models may also be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:140px;">Thermometer</td>
			<td style="width:73px;"></td>
			<td style="width:69px;"></td>
			<td style="width:292px;">For monitoring SPS temperature during dissection and slicing</td>
		</tr>
	</tbody>
</table>

slice it hot: acute adult brain slicing in physiological temperature

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.

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 <stron...

Watch this Scientific Journal Video about Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature at JoVE.com

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

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.

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

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