Name: Whole Cell Recording from an Organotypic Slice Preparation of Neocortex (Video) | JoVE Journal
Uploaded: 2011-06-03T12:00:00.000+00:00
Duration: 10 min 29 s
Description: Learn whole cell recording from organotypic neocortical slices using young rat brain tissue. This method enables electrophysiological study of pyramidal neurons after genetic transfection and culture.

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5 minutes

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).

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<li>Guan, D., Lee, J. C. F., Higgs, M., Spain, W. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+roles+of+Kv1+containing+channels+in+neocortical+pyramidal+neurons%5BTitle%5D)+AND+%22J.+Neurophysiol%22%5BJournal%5D)">Functional roles of Kv1 containing channels in neocortical pyramidal neurons.</a> J. Neurophysiol. 97, 1931-1940 (2007).</li>
<li>Guan, D., Tkatch, T., Surmeier, D. J., Armstrong, W. E., Foehring, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kv2+subunits+underlie+slowly+inactivating+potassium+current+in+rat+neocortical+pyramidal+neurons%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Kv2 subunits underlie slowly inactivating potassium current in rat neocortical pyramidal neurons.</a> J Physiol. 581, 941-960 (2007).</li>
<li>Johnson, H. A., Buonomano, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+for+chronic+stimulation+of+cortical+organotypic+cultures+using+implanted+electrodes%5BTitle%5D)+AND+%22Neurosci+Methods%22%5BJournal%5D)">A method for chronic stimulation of cortical organotypic cultures using implanted electrodes.</a> Neurosci Methods. 176, 136-143 (2009).</li>
<li>Johnson, H. A., Buonomano, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+plasticity+of+spontaneous+activity+and+Up+states+in+cortical+organotypic+slices%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Development and plasticity of spontaneous activity and Up states in cortical organotypic slices.</a> J Neurosci. 27, 5915-5925 (2007).</li>
<li>Malin, S. A., Nerbonne, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delayed+rectifier+K%2B+currents%2C+IK%2C+are+encoded+by+Kv2+alpha-subunits+and+regulate+tonic+firing+in+mammalian+sympathetic+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Delayed rectifier K+ currents, IK, are encoded by Kv2 alpha-subunits and regulate tonic firing in mammalian sympathetic neurons.</a> J Neurosci. 22, 10094-10105 (2002).</li>
<li>O'Brien, J. A., Holt, M., Whiteside, G., Lummis, S. C., Hastings, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modifications+to+the+hand-held+Gene+Gun%3A+improvements+for+in+vitro+biolistic+transfection+of+organotypic+neuronal+tissue%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue.</a> J Neurosci Methods. 112, 57-64 (2001).</li>
<li>Stoppini, L., Buchs, P. A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+method+for+organotypic+cultures+of+nervous+tissue%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37, 173-182 (1991).</li>
<li>Villalobos, C., Shakkottai, V. G., Chandy, K. G., Michelhaugh, S. K., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((SKCa+channels+mediate+the+medium+but+not+the+slow+calcium-activated+afterhyperpolarization+in+cortical+neurons%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">SKCa channels mediate the medium but not the slow calcium-activated afterhyperpolarization in cortical neurons.</a> J Neurosci. 24, 3537-3542 (2004).</li>
<li>Walker, P. D., Andrade, R., Quinn, J. P., Bannon, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Real-time+analysis+of+preprotachykinin+promoter+activity+in+single+cortical+neurons%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Real-time analysis of preprotachykinin promoter activity in single cortical neurons.</a> J Neurochem. 75, 882-885 (2000).</li>
<li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li>
<li>O'Brien, J. A., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biolistic+transfection+of+neuronal+cultures+using+a+hand-held+gene+gun%5BTitle%5D)+AND+%22Nat+Proc%22%5BJournal%5D)">Biolistic transfection of neuronal cultures using a hand-held gene gun.</a> Nat Proc. 1, 977-981 (2006).</li>
<li>Joshi, P., Dunaevsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+gun+transfection+of+hippocampal+neurons%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Gene gun transfection of hippocampal neurons.</a> J Vis Exp. , (2006).</li>
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</ol>

No conflicts of interest declared.

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...

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Surgery / transfection / culture:&lt;/p&gt;&lt;ol&gt;&lt;li&gt;Brain Slicer: Campden Vibroslice #MA572 World Precision Instruments, Sarasota, FL, USA&lt;/li&gt;&lt;li&gt;Gene Gun System: Bio-Rad Helios # 165-2431 (Bio-Rad Laboratories, 1000 Alfred Nobel Drive, Hercules, CA 94547)&lt;ul&gt;&lt;li&gt;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&lt;/li&gt;&lt;li&gt;Bio-Rad Helium Regulator (#165-2413)&lt;/li&gt;&lt;li&gt;disposable supplies for Helios from Bio-Rad:&lt;ul&gt;&lt;li&gt;1.6 &amp;#x03BC;m Gold Microcarriers: #165-2264&lt;/li&gt;&lt;li&gt;Tefzel Tubing: #165-2441&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;/ul&gt;&lt;/li&gt;&lt;li&gt;Incubator: Forma Scientific model # 3110 (Thermo-Scientific: (866) 984-3766).&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_step&quot;&gt;Media:&lt;/p&gt;&lt;ol&gt;&lt;li&gt;Horse Serum: Hyclone donor equine #SH 30074. (HyClone, 925 West 1800 South, Logan, UT 84321)&lt;/li&gt;&lt;li&gt;HMEM (Minimal Essential Media plus HBSS and HEPES, no glutamine: Lonza BioWhittaker Catalog #12-137F): GIBCO/INVITROGEN, (800) 955- 6288, Option 1. &lt;/li&gt;&lt;li&gt;HBSS (GIBCO Hanks buffered saline, #24020-117): GIBCO/INVITROGEN, (800) 955- 6288, Option 1.&lt;/li&gt;&lt;li&gt;MEM (GIBCO minimal essential medium, #12360-038), GIBCO/INVITROGEN, (800) 955- 6288, Option 1.&lt;/li&gt;&lt;li&gt;250 mL Millipore 0.2 &amp;#x03BC;m filter: #SC6PU02RE&lt;/li&gt;&lt;li&gt;Plastic Transfer pipettes: Fisher #13-711-20.&lt;/li&gt;&lt;li&gt;50 mL Millipore steriflip 0.22 &amp;#x03BC;m filter (#SCGP00525)&lt;/li&gt;&lt;/ol&gt;&lt;p class=&quot;jove_content&quot;&gt;Items 6-8 obtained from: Fisher Scientific, 1241 Ambassador Blvd, P.O. Box 14989, St. Louis, MO 63132.&lt;/p&gt;&lt;p class=&quot;jove_step&quot;&gt;Recording:&lt;/p&gt;&lt;ol&gt;&lt;li&gt;Pipet glass: Harvard GC150TF-10: Harvard Apparatus, 84 October Hill Road, Holliston, Massachusetts 01746&lt;/li&gt;&lt;li&gt;Sutter P-87 horizontal electrode puller: Sutter Instrument Company, One Digital Drive, Novato, CA 94949&lt;/li&gt;&lt;li&gt;Axon Instruments Multiclamp 700B amplifier: Molecular Devices, Inc. 1311 Orleans Drive, Sunnyvale, CA 94089-1136&lt;/li&gt;&lt;li&gt;PClamp 10 data acquisition software: Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089-1136 &lt;/li&gt;&lt;li&gt;lectrode position is controlled with Sutter R&amp;#8211;-200 manipulators and PC-200 controller: &lt;strong&gt;Sutter Instrument Company&lt;/strong&gt;, One Digital Drive, Novato, CA 94949. &lt;/li&gt;&lt;li&gt;Microscope: Olympus BX-50WI upright microscope with IR-DIC optics&lt;/li&gt;&lt;li&gt;IR-sensitive camera OLY-150 (Olympus) or DAGE-MTI (DAGE-MTI, 01 North R&amp;#8211;ske Avenue, Michigan City, IN 46360).&lt;/li&gt;&lt;/ol&gt;</td></tr></tbody></table>

whole cell recording from an organotypic slice preparation of neocortex

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.

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 ...

The overall goal of this procedure is to obtain electrical recordings from parametal neurons in organotypic slices of neocortex. This is accomplished by first cutting acute slices from rats at postnatal ages P eight to P 10. The second step of the procedure is to affect Bic transfection of CD NA.The third step of the procedure is to culture the slices for three to seven days.The final step of the procedure is to obtain electrical recordings from transfected cells using current or voltage clamp. Ultimately, results can be obtained that show parametal cells in organotypic slices have similar properties to those from acute slices of the same age. Transfection of GFP does not alter these properties, allowing the study of neurons after genetic manipulations of ion channels through whole cell current or voltage clamp recordings.The chief advantage of this technique is as opposed to say acute slices, is that cells can be maintained in a normal environment for long enough to be able to genetically manipulate ion channels. The method can help answer key questions in the ion channel field, such as the functional roles of specific ion channel types, Prepare the laminar flow hood, vacuum flow hood, surgical supplies and solutions as specified in the accompanying manuscript. Wear gloves for all subsequent steps after filtering the prepared 250 milliliters cutting solution aliquot 40 milliliters of it into a 50 milliliter beaker.Place the cutting chamber, 40 milliliter aliquot, and the remaining 210 milliliters of the cutting solution in the freezer at minus 20 degrees Celsius under the laminar flow hood. Place 1.1 milliliters of culture media into three diagonal wells of a six well plate. Repeat this for a second.Six well plate. Place the six well plates into an incubator at 37 degrees Celsius for at least one hour. Next, use sterile scissors to shorten four transfer pipettes to be used for transferring slices.Another three pipettes are kept intact when the solutions have become slushy mixtures. Remove the 40 milliliter aliquot of cutting solution from the freezer and put it under the vacuum hood. Bubble it with 95%oxygen, 5%carbon dioxide.Then remove the 210 milliliters of cutting solution from the freezer and put it under the laminar flow hood bubble. It also with 95%oxygen, 5%carbon dioxide. Next, remove the wash media from the refrigerator and bubble it with 100%oxygen under the laminar flow hood.Place two milliliters of culture media in a Petri dish. Place the remaining culture media in the refrigerator for changing media during the week to prepare for the slicing of the brain. Pour about 10 milliliters of cold cutting solution from the 50 milliliter beaker into a 25 milliliter beaker under the vacuum hood.Following anesthesia and decapitation, remove the brain of a postnatal day, eight to 10 rat. Wash the brain in the beaker containing 30 milliliters of cold cutting solution for about 30 seconds. Then carefully transfer the brain to the 25 milliliter beaker.Change gloves and saturate the gloves with ethanol. Then carry the brain in the cold cutting solution to the laminar flow hood. The next step is to orient and block the brain by removing the cerebellum and the frontal pole and making a partial mid sagittal cut glue the blocked brain on the stage with the frontal cortex up.Make additional scalpel cuts as needed to restrict the size of the slices. Then place the stage with the brain into the sterilized metal slicing chamber. Attach to the vibrating tissue slicer.Fill the bath with ice cold bubbling cutting solution. Pour 30 milliliters of the remaining cutting solution into a 50 milliliter beaker and bubble with 95%oxygen, 5%carbon dioxide. This beaker will be used to collect the slices.Cut 250 micron coronal slices of the frontal parietal cortex. Place at least six slices into the 50 milliliter peaker containing cutting solution. Next place about two milliliters of wash media in a 35 millimeter Petri dish.Using a cut pipette, transfer the slices from the cutting solution to the Petri dish containing wash media. Wash the slices three times using fresh pipettes to add media and to aspirate the waste solutions after washing, transfer the slices to the 35 millimeter Petri dish containing culture media. Next, remove the tops from the packaging for the mesh inserts, but leave the inserts in the packages.Then use forceps to lift one mesh insert from its package using a cut transfer pipette. Transfer one slice from the culture media to the mesh. Insert and position the slice in the middle of the membrane using an intact transfer pipette.Remove any excess culture media.Now. Place the mesh insert with its slice in one of the fluid-filled wells of the six well plate. Be careful to prevent formation of air bubbles under the mesh after placing one slice in each of the fluid filled wells.Place both six well plates back in the incubator and incubate at 37 degrees Celsius for about one hour. In the meantime, clean the work area under the laminar flow hood in preparation for transfection. The next step is to load the BioRad Helios gene gun, leaving the first position free.Load a cartridge containing CD NA coated bullets into every other position of the cartridge holder of the gene gun. Then place the cartridge holder into the gene gun. First, clear the gun by firing a barrel with no bullets.Then advance the barrel to a cartridge containing CDNA.Bullets. Take the six well plates from the incubator and place them under the laminar flow hood holding the gun vertically just above the top of the six well plate shoot a slice at 100 PSI then advance the barrel and clear it again by firing a blank. Shoot the remaining slices with one shot per slice.After shooting, return the six well plates to the incubator and incubate the slices at 37 degrees Celsius for two to seven days with 5%carbon dioxide during the incubation period. Replace the media every other day as follows, working under the laminar flow hood, add new media to the three unoccupied wells. Then replace the six well plate in the incubator for at least one hour after an hour return the six well plate to the laminar hood and use ethanol cleaned curved forceps to move each mesh insert and its slice to a new well aspirate the old media and return the plate to the incubator.After several days of incubation, the slices are now ready for whole cell recordings. Remove a six well plate from the incubator under the laminar flow hood. Use ethanol cleaned curved forceps to remove one mesh insert and its slice.Then replace the six well plate in the incubator. Next, transfer the Petri dish with a mesh insert and slice to the recording area, applying tension with forceps or a flexible piece of metal. Use a number 11 scalpel to cut the mesh away from around the slice if necessary, finish the final cuts with the scissors.Then use forceps to transfer the slice and the attached mesh to the recording chamber. On the stage of an upright microscope, bathe the slice in carbogen related artificial cerebral spinal fluid under IR DIC optics. Visualize the parametal neurons in layers two, three, or five under epi fluorescence and the fitz filter.Locate the green transfected cells under IR DIC microscopy. Look for the CDNA bullets, which show up as small black dots inside the cells. Select a transfected cell for whole cell recording.Next, advance a micro electrode to the selected cell and use gentle suction to attain a giga ohm seal. Then apply additional suction to break into the cell for whole cell.Recording. The cell is now ready for either current or voltage clamp recordings.This figure shows a fluorescent image of cells transfected with CDNA for GFP. The following recordings are taken from cells transfected with CDNA for GFP. Shown here is an action potential in a layer three parametal cell in response to a supra threshold 10 millisecond current injection.This figure shows repetitive firing from a layer five parametal neuron in response to a two second 400 pico amp current injection. This figure shows a voltage clamp record from a layer five parametal neuron. Tetrodatoxin was used to block the voltage gated sodium current.The traces represent outward potassium currents elicited by a family of 500 millisecond voltage steps from a holding potential of minus 70 millivolts. Once master, the slight preparation and transfection can be done in about two hours, including preparation time. When attempting this Procedure.It's important to maintain sterile conditions and to restrict the thickness and dimensions of the slice to prevent drying. Thanks for watching. Good luck with your experiment.Thank you.

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Learn whole cell recording from organotypic neocortical slices using young rat brain tissue. This method enables electrophysiological study of pyramidal neurons after genetic transfection and culture.

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

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