Name: whole cell recording from an organotypic slice preparation of neocortex
Uploaded: 2011-06-03T12:00:00
Description: Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

The authors would like to thank Mayumi Sakuraba and Rebecca Foehring for outstanding technical assistance. In addition, we would like to thank Drs. Rodrigo Andrade for assistance in implementing the organotypic slice culture and biolistic transfection protocols and Dr. Jeanne Nerbonne for providing us with cDNA constructs for transfection. This work was supported by NIH grant: NS044163 from NINDS (to RCF).

1. Preparations Before the Day of Slicing
We find it is more efficient to autoclave the surgical instruments and prepare solutions prior to the day of slicing.
<ol>
 <li>Autoclave instruments. (The surgery and slicing are performed under semi-sterile conditions). Autoclave the following packages, individually wrapped in autoclave paper:
 <ul class="none">
 <li>Surgery package: spatula, #22 scalpel blade handle, scissors,forceps</li>
 <li>Slicing packa...

<ol>
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We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex (4, 9-11). Because of the lack of specific pharmacological agents for these channels, we use a genetic approach for manipulating channel expression (1,14,15,17-19). We utilize an organotypic culture preparation (2,3;5-8;12,13,15-22) modified from the approach of Stoppini et al. (16), in order to maintain cell morphology and the laminar pattern of cortex. We isolate acute neocor...

Surgery / transfection / culture:
<ol>
	<li>Brain Slicer: Campden Vibroslice #MA572 World Precision Instruments, 	Sarasota, FL, USA</li>
	<li>Gene Gun System: Bio-Rad Helios # 165-2431 (Bio-Rad Laboratories, 1000 	Alfred Nobel Drive, Hercules, CA 94547)
		<ul>
			<li>Includes: Gene gun, helium hose assembly with regulator, tubing prep station (#165-2418), syringe kit, Tefzel tubing, tubing cutter, optimization kit (#165-2424), tubing cutter</li>
			<li>Bio-Rad Helium Regulator (#165-2413)</li>
			<li>disposable supplies for Helios from Bio-Rad:
				<ul>
					<li>1.6 &mu;m Gold Microcarriers: #165-2264</li>
					<li>Tefzel Tubing: #165-2441</li>
				</ul>
			</li>
		</ul>
	</li>
	<li>Incubator: Forma Scientific model # 3110 (Thermo-Scientific: (866) 984-	3766).</li>
</ol>
Media:
<ol>
	<li>Horse Serum: Hyclone donor equine #SH 30074. (HyClone, 925 West 1800 	South, Logan, UT 84321)</li>
	<li>HMEM (Minimal Essential Media plus HBSS and HEPES, no glutamine: Lonza 	BioWhittaker Catalog #12-137F): GIBCO/INVITROGEN, 	(800) 955- 6288, 	Option 1. </li>
	<li>HBSS (GIBCO Hanks buffered saline, #24020-117): GIBCO/INVITROGEN, 	(800) 955- 6288, Option 1.</li>
	<li>MEM (GIBCO minimal essential medium, #12360-038), GIBCO/INVITROGEN, 	(800) 955- 6288, Option 1.</li>
	<li>250 mL Millipore 0.2 &mu;m filter: #SC6PU02RE</li>
	<li>Plastic Transfer pipettes: Fisher #13-711-20.</li>
	<li>50 mL Millipore steriflip 0.22 &mu;m filter (#SCGP00525)</li>
</ol>
Items 6-8 obtained from: Fisher Scientific, 1241 Ambassador Blvd, P.O. 	Box 14989, St. Louis, MO 63132.
Recording:
<ol>
	<li>Pipet glass: Harvard GC150TF-10: Harvard Apparatus, 84 October Hill Road, 	Holliston, Massachusetts 01746</li>
	<li>Sutter P-87 horizontal electrode puller: Sutter Instrument Company, One 	Digital Drive, Novato, CA 94949</li>
	<li>Axon Instruments Multiclamp 700B amplifier: Molecular Devices, Inc. 1311 Orleans Drive, Sunnyvale, CA 94089-1136</li>
	<li>PClamp 10 data acquisition software: Molecular Devices, Inc., 1311 Orleans 	Drive, Sunnyvale, CA 94089-1136 </li>
	<li>lectrode position is controlled with Sutter ROE-200 manipulators and PC-200 controller: Sutter Instrument Company, One Digital Drive, Novato, CA 	94949. </li>
	<li>Microscope: Olympus BX-50WI upright microscope with IR-DIC optics</li>
	<li>IR-sensitive camera OLY-150 (Olympus) or DAGE-MTI (DAGE-MTI, 01 North Roeske Avenue, Michigan City, IN 46360).</li>
</ol>

whole cell recording from an organotypic slice preparation of neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

Watch this Scientific Journal Video about Whole Cell Recording from an Organotypic Slice Preparation of Neocortex at JoVE.com

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

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