Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank Sean McDermott for his technical assistance and support, and Maya Epstein for the hand-drawn illustrations shown in Figure 1.

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.
1. Preparation prior to organotypic cerebellar slice cultures
<ol>
	<li>In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks&#39; Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of 200 mM L-Glutamine (final concentration 1 mM), and 5 mL of 200 g/L glucose (final concentration 5 mg/mL). Vacuum-filter the culture medium and keep it at +4 &#176;C for up to a month.</li>
	<li>In the sterilized biosafety cabinet, take out a 6-well culture plate from its package. Fill each well with 1 mL of culture medium. If a treatment is tested, add the pharmacological agent to the treated well, and the same volume of vehicle solution for the control well. In the present study, we added 1 &#181;L of sterile 3 M KCl solution to the treated well (30 mM final) and 1 &#181;L of sterile water to the control wells.</li>
	<li>Bring inside the cell culture hood the needed number of individually wrapped cell culture inserts with hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size = 0.4 &#181;m). Carefully unwrap them and take out the cell culture inserts with sterile forceps. Drop them one by one in a well of a 6-well plate, avoiding bubbles at the interface between the insert membrane and the cell culture medium. Let the inserts equilibrate in the medium in a 37 &#176;C, 5% CO2 incubator for at least 2 h before culture. 
	NOTE: The tissue chopper permanently resides in the cell culture hood.</li>
	<li>Prepare two 200 mL beakers, one filled with 150 mL sterile water, and the other filled with 150 mL 70% ethanol. Dip the surgical tools in the 70% ethanol bath and then in water prior to using them. 
	NOTE: Do not use surgical tools immediately following contact with ethanol.</li>
	<li>Disinfect the double-edged blade in the 70% ethanol beaker. Place it on the blade holder using sterile forceps and hold it into place using the blade clamp wrench. Let it dry until use.</li>
	<li>Disinfect the plastic disc provided with the tissue chopper in the 70% ethanol beaker. Spray the cutting table with 70% ethanol prior to placing the disc on it. Let dry until use.</li>
</ol>
2. Dissection and Cerebellar Organotypic Slice Cultures
<ol>
	<li>Bring the mouse pup inside the cell culture hood to perform the dissection.</li>
	<li>Decapitate the pup quickly using straight operating scissors (Figure 1A).</li>
	<li>While holding the pup&#39;s head by the nose with straight dressing forceps, cut open the scalp using straight eye scissors, starting from the posterior end, and going lateral to midline.</li>
	<li>Proceed identically for the skull. 
	NOTE: Make sure to point scissor tips outwards to avoid damaging the cerebellum during dissection.</li>
	<li>Take out the brain and transfer it to a 60-mm dish filled with cold HBSS + 5 mg/mL glucose.</li>
	<li>Carefully dissect out the cerebellum using sterile straight fine forceps (Figure 1B). Transfer it onto the plastic disc placed on the cutting table of the tissue chopper using sterile curved fine forceps.</li>
	<li>Orient the tissue by rotating the cutting table to perform parasagittal sections.</li>
	<li>Move the cutting table by pulling the table release knob to the right, so the blade is positioned at the edge of the tissue.</li>
	<li>Adjust the slice thickness to 350 &#181;m and the blade speed to medium.</li>
	<li>Start the chopper. Once the whole cerebellum has been sliced (Figure 1C), turn off the chopper.</li>
	<li>Remove the plastic disc with sterile forceps.</li>
	<li>Carefully bring the plastic disc close to a 60-mm dish containing HBSS + 5 mg/mL glucose. Pipette cold HBSS + 5 mg/mL glucose onto the tissue using a sterile transfer pipette to allow harvesting of cerebellar slices in the buffer-filled dish.</li>
	<li>Separate the cerebellar slices from each other using a microprobe. Take out good quality sections with the transfer pipette (it is recommended to use tissue sections that are close to the vermis) and drop them off in a pre-equilibrated cell culture insert (Figure 1D).</li>
	<li>Position the cerebellar slices on the cell culture insert using the microprobe, then carefully remove the excess HBSS + 5 mg/mL glucose solution with the transfer pipette. 
	NOTE: At this stage the tissue is extremely tender and prone to physical damage. The maximum number of cerebellar slices per cell culture insert depends on the age of the donor animal. 6&#8211;8 slices can be cultured for a P0&#8211;P4-old animal, and 4&#8211;6 slices for animals older than P5.</li>
	<li>Place the culture dish in the incubator, changing the medium completely every 2&#8211;3 days.</li>
	<li>To assess Purkinje cell survival, slices can be collected as early as 5 days in vitro. For other purposes, they can be maintained in culture for several weeks (Figure 1F). 
	NOTE: In the case of Purkinje cell survival experiments, there is no need to renew the pharmacological treatment when changing cell culture medium (Figure 1F).</li>
</ol>
3. Immunofluorescence
<ol>
	<li>Take out the 6-well plate from the incubator. Remove cell culture medium, wash the cell culture insert with 1x PBS, and fix the slices with cold 4% paraformaldehyde solution for 1 h, with 1 mL under the cell culture insert and 500 &#181;L on top of the cell culture insert.</li>
	<li>Wash the inserts 4x 10 min with 1x PBS, with 1 mL under and 500 &#181;L on top of the insert, on an orbital shaker.</li>
	<li>With a brush, transfer the cerebellar slices from 1 cell culture insert to 1 well of a 24-well pre-filled with 1x PBS, 0.25% Triton X-100, and 3% BSA (PBS-TB), 500 &#181;L/well.</li>
	<li>Permeabilize and block the slices by incubation in PBS-TB for 1 h at room temperature.</li>
	<li>Incubate with primary antibodies diluted in the PBS-TB solution, overnight at + 4 &#176;C, on an orbital shaker, 200 &#181;L/well.</li>
	<li>On the following day, perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Incubate with secondary antibodies diluted in the PBS-TB solution, two hours at room temperature, on an orbital shaker, and protected from light, 200 &#181;L/well.</li>
	<li>Perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 &#181;L/well.</li>
	<li>Counterstain the slices using Hoechst 33342, diluted in 1x PBS (2 &#181;g/mL), for 10 min at room temperature, protected from light, 500 &#181;L/well.</li>
	<li>Mount the cerebellar slices on a microscopy slide with a transfer pipette. Let them air-dry completely, protected from light. Re-humidify them with 1x PBS and apply a coverslip covered with few drops of mounting medium (~80 &#181;L). Avoid making any bubbles.</li>
	<li>Once the mounting medium has cured, cerebellar sections are ready to be imaged.</li>
</ol>
4. Imaging and Quantification of Cell Survival
<ol>
	<li>Turn on the microscope and lasers according to the manufacturer and/or the imaging core facility&#39;s instructions.</li>
	<li>Start the acquisition software.</li>
	<li>Using a 10X objective, locate non-altered cerebellar slices that present 9&#8211;10 lobules (usually cerebellar sections close to the vermis).</li>
	<li>Activate z-stack acquisition. Define the range of the z-stack to include all Purkinje cells present in the cerebellar slice. Set a 2 &#181;m step size and start the acquisition. Save the acquired picture in the format of the acquisition software manufacturer to preserve the associated metadata.</li>
	<li>Open the acquired file in ImageJ using the Bio-formats plugin18.</li>
	<li>Go to Image &#62; Stacks &#62; Z-project&#8230; (Figure 2A).</li>
	<li>Choose Max Intensity in the Projection type dropdown menu and click OK (Figure 2B).</li>
	<li>A flattened 2-D image will be generated. Double click on the Multi-point tool to let the Point Tool window appear. Uncheck the Label points option to ease counted cells visualization. Then, click directly on a soma of a Purkinje cell to begin counting (Figure 1E). The total number of counted cells will be indicated at the bottom of the Point Tool window (Figure 3). 
	NOTE: Usually, at least 3 non-damaged sections containing 9&#8211;10 lobules per cell culture insert can be quantified. To assess the neuroprotective effect of a pharmacological agent, at least 3 independent experiments should be performed.</li>
</ol>

<ol>
	<li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Gahwiler, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+monolayer+cultures+of+nervous+tissue.">Organotypic monolayer cultures of nervous tissue.</a> Journal of Neuroscience Methods. 4 (4), 329-342 (1981).</li><li>Yamamoto, N., Kurotani, T., Toyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+connections+between+the+lateral+geniculate+nucleus+and+visual+cortex+in+vitro.">Neural connections between the lateral geniculate nucleus and visual cortex in vitro.</a> Science. 245 (4914), 192-194 (1989).</li><li>Dusart, I., Airaksinen, M. S., Sotelo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purkinje+cell+survival+and+axonal+regeneration+are+age+dependent:+an+in+vitro+study.">Purkinje cell survival and axonal regeneration are age dependent: an in vitro study.</a> Journal of Neuroscience. 17 (10), 3710-3726 (1997).</li><li>Wiegreffe, C., Feldmann, S., Gaessler, S., Britsch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+Confocal+Imaging+of+Migrating+Neurons+in+Organotypic+Slice+Culture+of+Embryonic+Mouse+Brain+Using+In+Utero+Electroporation.">Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation.</a> Journal of Visualized Experiments. 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M., 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=Mifepristone+(RU486)+protects+Purkinje+cells+from+cell+death+in+organotypic+slice+cultures+of+postnatal+rat+and+mouse+cerebellum.">Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7953-7958 (2003).</li><li>Labombarda, F., 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=Neuroprotection+by+steroids+after+neurotrauma+in+organotypic+spinal+cord+cultures:+a+key+role+for+progesterone+receptors+and+steroidal+modulators+of+GABA(A)+receptors.">Neuroprotection by steroids after neurotrauma in organotypic spinal cord cultures: a key role for progesterone receptors and steroidal modulators of GABA(A) receptors.</a> Neuropharmacology. 71, 46-55 (2013).</li><li>Gao, 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=P2X7+receptor-sensitivity+of+astrocytes+and+neurons+in+the+substantia+gelatinosa+of+organotypic+spinal+cord+slices+of+the+mouse+depends+on+the+length+of+the+culture+period.">P2X7 receptor-sensitivity of astrocytes and neurons in the substantia gelatinosa of organotypic spinal cord slices of the mouse depends on the length of the culture period.</a> Neuroscience. , 195-207 (2017).</li><li>Sundstrom, L., Morrison, B., Bradley, M., Pringle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cultures+as+tools+for+functional+screening+in+the+CNS.">Organotypic cultures as tools for functional screening in the CNS.</a> Drug Discovery Today. 10 (14), 993-1000 (2005).</li><li>Jankowski, J., Miething, A., Schilling, K., Baader, S. 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I., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+protein+kinase+C+prevents+Purkinje+cell+death+but+does+not+affect+axonal+regeneration.">Inhibition of protein kinase C prevents Purkinje cell death but does not affect axonal regeneration.</a> Journal of Neuroscience. 22 (9), 3531-3542 (2002).</li><li>Ghoumari, A. M., 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=Neuroprotective+effect+of+mifepristone+involves+neuron+depolarization.">Neuroprotective effect of mifepristone involves neuron depolarization.</a> FASEB Journal. 20 (9), 1377-1386 (2006).</li><li>Repici, M., Wehrle, R., Antoniou, X., Borsello, T., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+N-terminal+kinase+(JNK)+and+p38+play+different+roles+in+age-related+Purkinje+cell+death+in+murine+organotypic+culture.">c-Jun N-terminal kinase (JNK) and p38 play different roles in age-related Purkinje cell death in murine organotypic culture.</a> Cerebellum. 10 (2), 281-290 (2011).</li><li>Rakotomamonjy, J., Ghoumari, A. 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A., Muller, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Linkert, M., 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=Metadata+matters:+access+to+image+data+in+the+real+world.">Metadata matters: access to image data in the real world.</a> Journal of Cell Biology. 189 (5), 777-782 (2010).</li><li>Uesaka, N., 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=Organotypic+coculture+preparation+for+the+study+of+developmental+synapse+elimination+in+mammalian+brain.">Organotypic coculture preparation for the study of developmental synapse elimination in mammalian brain.</a> Journal of Neuroscience. 32 (34), 11657-11670 (2012).</li><li>Falsig, J., 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=Prion+pathogenesis+is+faithfully+reproduced+in+cerebellar+organotypic+slice+cultures.">Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures.</a> PLoS Pathogens. 8 (11), 1002985 (2012).</li><li>Bouslama-Oueghlani, L., Wehrle, R., Sotelo, C., Dusart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+loss+of+the+ability+of+Purkinje+cells+to+regenerate+their+axons+occurs+in+the+absence+of+myelin:+an+in+vitro+model+to+prevent+myelination.">The developmental loss of the ability of Purkinje cells to regenerate their axons occurs in the absence of myelin: an in vitro model to prevent myelination.</a> Journal of Neuroscience. 23 (23), 8318-8329 (2003).</li><li>Apuschkin, M., Ougaard, M., Rekling, J. 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The authors have nothing to disclose.

Cerebellar slice culture is a powerful tool to study programmed Purkinje cell death during postnatal development. This technique can be used to rapidly screen candidate molecules for their neuroprotective potential. The main advantage is that the setup is simple and very cost effective, and only requires a modest investment in equipment (a vibratome can cost up to 3 times more than a tissue chopper). Moreover, 10 to 15 healthy slices can be generated from one mouse pup, allowing for different assays to be performed in pa...

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience1. The method was first established by G&#228;hwiler, who cultured brain slices using the roller tube technique2, and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures3. Compared to primary cell cultures, organotypic slice cultures present the advantage of preserving the cytoarchitecture of the tissue, as well as native cell-cell connections in the plane of the tissue section.
Organotypic slices have been cultured from many parts of the central nervous system, such as the hippocampus4, cortex5, striatum6, cerebellum4,7, and spinal cord8,9, among others. They have been proven to be a powerful tool in drug discovery studies10. The effects of neuroactive molecules can be assessed in many ways: survival and neurodegeneration using immunostaining and biochemistry assays, neuronal circuit formation, or disruption using electrophysiology and live-imaging.
The goal of this work is to describe a simple method to perform organotypic cerebellar slice culture, which is known to be a relevant model to mimic cerebellar development in vitro. Particularly, we focused on the study of Purkinje cell developmental death. In vivo, Purkinje cells undergo apoptosis during the first postnatal week, peaking at postnatal day 3 (P3)11. The same pattern is observed in cerebellar slice culture, with Purkinje neurons dying by apoptosis when cerebella are taken from animals between P1 and P8, with a peak at P34,12. The use of the organotypic cerebellar slice cultures has allowed to identify several neuroprotective molecules7,13, as well as understanding part of the mechanisms involved in this programmed cell death14,15,16. Here, we describe a protocol based upon the study of Stoppini et al.17 in hippocampus, and adapted to cerebellum by Dusart et al.4 It includes rapid dissection and chopping of postnatal cerebella; slicing culture onto a cell culture insert containing a microporous membrane, with or without neuroprotective treatment; and immunofluorescence staining to assess neuronal survival.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 594 Donkey anti-Mouse IgG secondary antibody</td>
			<td>ThermoFisher scientific</td>
			<td>A21203</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal Medium Eagle (BME)</td>
			<td>ThermoFisher scientific</td>
			<td>21010046</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety cabinet Class II, Type A2</td>
			<td>NuAire</td>
			<td>NU-540-400</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Millipore Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Calbindin D-28K antibody (CB-955)</td>
			<td>Abcam</td>
			<td>ab82812</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NuAire</td>
			<td>NU-5700</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Costar Flat Bottom 6-well Cell Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips, 22 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12-545-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Dressing forceps, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8949</td>
			<td></td>
		</tr>
		<tr>
			<td>Double edge blades</td>
			<td>Fisher Scientific</td>
			<td>50949411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Labs, Inc</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-127</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 100X</td>
			<td>ThermoFisher scientific</td>
			<td>25030149</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose solution</td>
			<td>ThermoFisher scientific</td>
			<td>A2494001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks' Balanced Salt Solution</td>
			<td>ThermoFisher scientific</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Fisher Scientific</td>
			<td>H21492</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum, heat inactivated, New Zealand origin</td>
			<td>ThermoFisher scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain Tissue Chopper</td>
			<td>Fisher Scientific</td>
			<td>NC9914528</td>
			<td></td>
		</tr>
		<tr>
			<td>Microprobes</td>
			<td>Fisher Scientific</td>
			<td>08-850</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore Sigma</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane, 250 mL</td>
			<td>ThermoFisher scientific</td>
			<td>168-0045</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R confocal laser microscope system</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS-Elements Imaging Software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Acros Organics</td>
			<td>41678-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20D</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>ThermoFisher scientific</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissors, straight</td>
			<td>Harvard Apparatus</td>
			<td>72-8403</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker Belly Dancer</td>
			<td>IBI Scientific</td>
			<td>BDRLS0003</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Dish, 60 mm w/ grip ring</td>
			<td>Fisher Scientific</td>
			<td>FB012921</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 well</td>
			<td>Falcon/Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes, sterile</td>
			<td>ThermoFisher scientific</td>
			<td>13-711-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher scientific</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

purkinje cell survival in organotypic cerebellar slice cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

As shown in Figure 4, this protocol produces organotypic cerebellar slice cultures in which Purkinje cell survival can be assessed following the immunofluorescence and image acquisition steps. Purkinje cells were labeled with a combination of anti-Calbindin D-28K (dilution 1/200) and Alexa594 anti-mouse (dilution 1/300) antibodies. Image stitching was done automatically by the microscope acquisition software (NIS-Elements) to obtain a picture of the whole cerebellar slice. Purkinje cell numb...

Watch this Scientific Journal Video about Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures at JoVE.com

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...

purkinje-cell-survival-in-organotypic-cerebellar-slice-cultures

Research

JoVE Journal

Neuroscience

7.8K Views.  Northwestern University, Feinberg School of Medicine. Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

Video: Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Selective Depletion of Microglia from Cerebellar Granule Cell Cultures Using L-leucine Methyl Ester

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating innate immunity. Primary rodent cell culture models have greatly advanced our understanding of neuronal-glial interactions, but only recently have methods to specifically eliminate microglia from mixed cultures been utilized. One such technique &#8211; described here &#8211; is the use of L-leucine methyl ester, a lysomotropic agent that is internalized by macrophages and microglia, wherein it causes lysosomal disruption and subsequent apoptosis13,14. Experiments using L-leucine methyl ester have the power to identify the contribution of microglia to the surrounding cellular environment under diverse culture conditions. Using a protocol optimized in our laboratory, we describe how to eliminate microglia from P5 rodent cerebellar granule cell culture. This approach allows one to assess the relative impact of microglia on experimental data, as well as determine whether microglia are playing a neuroprotective or neurotoxic role in culture models of neurological conditions, such as stroke, Alzheimer&#8217;s or Parkinson&#8217;s disease.

Microglia can influence neurons and other glia in culture by various non-cell autonomous mechanisms. Here, we present a protocol to selectively deplete microglia from primary neuronal cultures. This method has the potential to elucidate the role of microglial-neuronal interactions, with implications for neurodegenerative conditions where neuroinflammation is a hallmark feature.

Microglia, the resident immunocompetent cells of the CNS, play multifaceted roles in modulating and controlling neuronal function, as well as mediating ...