Name: preparation and immunostaining of myelinating organotypic cerebellar slice cultures
Uploaded: 2019-03-20T14:00:00
Description: Watch this Scientific Journal Video about Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures at JoVE.com

We thank Dr. Sean Freeman, Dr. Nathalie Sol-Foulon and Dr. Thomas Roux for valuable comments on the manuscript. This work was funded by INSERM, ICM, ARSEP Grants R13123DD, ANR R17127DD (to A.D.) and FRM fellowship, SPF20110421435 (to A.D.), FDT20170437332 (to M.T.).&#160;We thank the CELIS cell culture facility and the icm.Quant imaging platform.

All work involving animals complied with institutional policies and guidelines established by the UPMC, INSERM and the French and European Community Council Directive 86/609/EEC.
1. Preparation of Culture Medium and Culture Inserts (Hands-on Time &#8776; 10&#8211;15 min)
NOTE: Perform this step in a flow culture hood under sterile conditions
<ol>
	<li>Prepare 40 mL of culture medium, which consists of 50% BME, 25% Hank&#8217;s Balanced Salt Soluti...

<ol>
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Here, we detail a protocol to generate an ex vivo model corresponding to the mouse cerebellar organotypic slice cultures, adapted from previously published methods15,16,19 and the subsequent myelin immunostaining of these preparations. This strategy offers the possibility to visualize myelin components with a high-resolution in both healthy and pathological states.
Cerebellar organotypic slice culture...

Neurons are highly polarized cells, which comprise a somato-dendritic compartment that receives inputs from its environment and an axon that ensures the generation and propagation of electrical impulses to other cells. Rapid propagation and timely delivery of information is essential for the proper functioning of the nervous system. In vertebrates, it is facilitated by myelination, which allows increasing axonal conduction velocity1. Myelin is a specialized structure formed by compacted layers of plasma membrane generated by the myelinating glia, namely oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Both in the CNS and the PNS, axoglial interactions drive the formation of specialized axonal domains: the nodes of Ranvier and their surrounding domains, the paranodes, and juxtaparanodes2. The axonal segments insulated by myelin, or internodes, alternate with the nodes of Ranvier, which correspond to small unmyelinated domains enriched in voltage-gated sodium channels (Nav). The high concentration and rapid activation of Nav channels at the nodes of Ranvier allow the regeneration of action potentials, and together with the insulating properties of the myelin sheaths, ensure the efficient and fast saltatory conduction of the nerve impulse along the axon3.
In addition to its role in accelerating the conduction velocity of the nerve impulse, myelinating glia provides metabolic support to the axon, preserving its long-term integrity and participating in its survival4,5. Furthermore, it has become clear in recent years that myelin is dynamically modulated throughout life, thus presumably participating in the regulation and plasticity of various nervous system functions. Adjustments of the distribution, number, length, and thickness of myelin sheaths along axons might thus represent a novel way to finely tune various networks6,7,8. Therefore, the evolutionary acquisition of myelin is a key process for sensory, motor and cognitive functions and the perturbation of the interaction between axons and glia is increasingly considered as contributing to the developmental or acquired neurological diseases9.
Myelin composition has been characterized, with the specific feature of a high proportion of lipids (70%) compared to proteins (30%) in contrast to other cellular membranes10. However, unlike myelin lipids, most of myelin proteins are specific to myelin, including myelin basic protein (MBP), proteolipid protein (PLP), 2&#39;,3&#39;-cyclic nucleotide 3&#39;-phosphodiesterase (CNP), myelin-associated glycoprotein (MAG), myelin-oligodendrocyte glycoprotein (MOG), PMP-22 and P010. Various histological methods to stain myelin exist based on its lipid composition, such as Luxol fast blue11, Sudan Black B12, Baker&#39;s acid hematin method13, as well as silver staining14. Nevertheless, these approaches do not always allow for an adequate contrast and resolution to visualize individual fibers. An alternative approach to detect myelin is through immunohistochemistry directed against myelin proteins. Various antibodies target myelin-specific antigens with a high specificity and can be used routinely to detect myelinated structures. The antibody-antigen interaction can be further revealed using a secondary antibody coupled to a fluorophore directed against the primary antibody and visualized with adequate fluorescence microscopy. Here, we describe an immunochemical protocol to stain myelin on ex vivo cerebellar slices, a model which allows for a good preservation of the nervous tissue architecture. In addition, the organization and size of the Purkinje cells (the sole myelinated neuron of the cerebellum) make them a classical model for electrophysiological studies and they are similarly ideal to perform fixed or live-imaging studies.
The cerebellar slices are generated from P9-P10 mice, a time corresponding to the early onset of Purkinje cell myelination, a process that is mostly achieved by one week ex vivo (6-7 days in vitro, DIV)15. Furthermore, this model is adapted to investigate demyelinating disorders such as multiple sclerosis (MS), as an extensive demyelination can be induced in cerebellar slices using the myelinotoxic compound lysophosphatidylcholine (or lysolecithin, LPC), which is followed by a spontaneous remyelination16,17. Endogenous remyelination takes place from two days after LPC removal from the culture medium and is almost complete a week post treatment.
The completion of this protocol takes approximatively 3 weeks, including half a day for cerebellar slice cultures preparation, a week to obtain fully myelinated slices, followed by 2 days to reach the peak of demyelination and another week for their full remyelination. In addition, immunohistochemistry can be completed in 2 days. The protocol described here is adapted to a standard litter of 6 mice pups and needs to be adapted regarding the number of animals used for the planned experiment.

<table>
	<tbody>
		<tr>
			<td>BME medium</td>
			<td>ThermoFisher Scientific</td>
			<td>41010026</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (10X HBSS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14180046</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Horse Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>26050088</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;Streptomycin (10.000 IU/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gey&#8217;s Balanced Salt Solution</td>
			<td>Sigma Aldrich</td>
			<td>G9779-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose Solution (45%)</td>
			<td>Sigma Aldrich</td>
			<td>G8769-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysophosphatidylcholine (LPC)</td>
			<td>Sigma Aldrich</td>
			<td>L4129-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol (100% ethanol)</td>
			<td>VWR Chemicals</td>
			<td>20821.330</td>
			<td>Cooled at -20&#176;C</td>
		</tr>
		<tr>
			<td>Triton&#174; X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Normal Goat Serum (NGS)</td>
			<td>ThermoFisher Scientific</td>
			<td>500622</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>EuroMEDEX</td>
			<td>ET330-A</td>
			<td>pH 7.4</td>
		</tr>
		<tr>
			<td>Anti-GFP Antibody (Polyclonal, Chicken)</td>
			<td>Merck Millipore</td>
			<td>06-896</td>
			<td>Dilution 1/300</td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein (MBP) Antibody (Polyclonal, Chicken)</td>
			<td>Merck Millipore</td>
			<td>AB9348</td>
			<td>Dilution 1/150</td>
		</tr>
		<tr>
			<td>Anti-Myelin Basic Protein (MBP) Antibody (Monoclonal, Mouse IgG2b)</td>
			<td>Merck Millipore</td>
			<td>NE1019</td>
			<td>Dilution 1/200</td>
		</tr>
		<tr>
			<td>Anti-PLP Antibody (Rat, Hybridoma)</td>
			<td>Gift from Dr. K. Ikenaka; Okasaki, Japan</td>
			<td></td>
			<td>Dilution 1/5 to 1/10</td>
		</tr>
		<tr>
			<td>Anti-Sodium Channel, Pan Antibody (Monoclonal, Mouse IgG1, clone K58/35)</td>
			<td>Sigma Aldrich</td>
			<td>S8809</td>
			<td>Dilution 1/150</td>
		</tr>
		<tr>
			<td>Anti-Caspr Antibody (Polyclonal, Rabbit)</td>
			<td>Abcam</td>
			<td>ab34151</td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Goat Secondary Antibodies conjugated to Alexa Fluor 488, 594, 647 or 405</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td>Dilution 1/500</td>
		</tr>
		<tr>
			<td>Fluoromount</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue chopper</td>
			<td>McIlwain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Large scissors</td>
			<td>F.S.T</td>
			<td>14101-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Small scissors</td>
			<td>F.S.T</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine-straight forceps</td>
			<td>F.S.T</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved-fine forceps</td>
			<td>F.S.T</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dishes (60-mm and 100-mm)</td>
			<td>TPP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-, 6-well culture plates</td>
			<td>TPP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell culture inserts (0,4 &#181;m, 30-mm diameter)</td>
			<td>Merck Millipore</td>
			<td>PICM0RG50</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td></td>
			<td></td>
			<td>37&#176;C, 5% CO2</td>
		</tr>
		<tr>
			<td>Fine-end pipette tips</td>
			<td>Dutscher</td>
			<td>134000CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-bore pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>2079G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe</td>
			<td>Terumo Europe</td>
			<td></td>
			<td>20 or 50 mL</td>
		</tr>
		<tr>
			<td>Sterile syringe filters</td>
			<td>Terumo Europe</td>
			<td></td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Swann-Morton</td>
			<td>0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>RS France</td>
			<td></td>
			<td>76 x 26 x 1.1 mm</td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>RS France</td>
			<td></td>
			<td>22 x 22 mm</td>
		</tr>
		<tr>
			<td>Kimtech Sciences Tissue Wipers</td>
			<td>Kimberly-Clark Professional</td>
			<td>5511</td>
			<td></td>
		</tr>
		<tr>
			<td>Binocular microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and immunostaining of myelinating organotypic cerebellar slice cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Examples of representative myelin immunostainings in organotypic cerebellar slices obtained from P9-P10 C57black6 wild-type (WT) (Figure 2A), as well as PLP-GFP transgenic mice (Figure 2B), together with Purkinje cells staining. Cerebellar slices myelinate from the white matter tracks region of the slices towards the periphery of the folia and myelination of the Purkinje cells is mostly achieved after 6 to 7 DIV. At 7 DIV, the in...

Watch this Scientific Journal Video about Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures at JoVE.com

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...

This method allows to study the basic mechanism of myelination and remyelination at the cellular and tissular level. This technique is at the crossroad between primary cultures and in vivo approaches. It is a fast and accessible model with the main advantage of preserving the tissue architecture.Demonstrating the procedure will be Melina Thetiot, a postdoctoral researcher and Remi Ronzano, a PhD student from my laboratory. To begin, insert a small scissors gently into the foramen magnum and cut the skull by making one lateral incision towards the side, and then cut all around the head skull. To retrieve the dorsal part of the skull, use a fine, straight forceps to carefully lift the dorsal part of the skull.Then carefully introduce the forceps between the ventral skull and the brain. Gently flip out the brain and cut the optic and trigeminal nerves with small scissors. Next, turn the head or the dorsal part of the skull upside down just above a 60 millimeter cell culture dish containing ice cold dissection medium to help the brain to drop by gravity.Using fine forceps, orientate the brain with the dorsal side facing up and the ventral side lying down. Under the binocular microscope, use the fine, straight forceps to immobilize the brain on the forebrain side. Then, separate the hind brain from the rest of the brain.Cut the cerebellar peduncles underneath the cerebellum to separate the cerebellum from the rest of the hind brain. Once the cerebellum is isolated, use fine, straight forceps to carefully tear away the meninges. Hold the cerebellum gently with the fine, curved forceps and place it with the dorsal side up onto the plastic platform, perpendicularly to the chopper razor blade.Next, with a sterile, thin end pipette tip attached to a one milliliter pipette, aspirate any access of dissection medium around the cerebellum. Then, with a tissue chopper, slice 300 micrometer thick saggital sections of the cerebellum. Next, add a drop of dissection medium onto the sliced cerebellum gently.Then, with a wide boar pipette tip attached to the one milliliter pipette, slowly aspirate the sliced cerebellum and transfer it back into the 60 millimeter cell culture dish containing ice cold dissection medium. Next, use two fine, straight forceps to separate individual slices. Then, with a wide boar pipette tip attached to the one milliliter pipette, transfer up to four selected slices from the vermis, along with some dissection medium onto one culture insert of a six well plate.Remove any access of dissection medium around the slices using a thin end pipette tip. For demyelination, remove all culture medium below the culture inserts after six days in vitro, and replace it with one milliliter per well of the pre-warmed, fresh culture medium containing 0.5 milligram per milliliter LPC. Incubate for 15 to 17 hours at 37 degrees Celsius in 5%carbon dioxide environment.After incubation, wash the inserts by placing them in a 25 milliliter Petri dish containing one milliliter of pre-warmed culture medium. Then, immediately transfer the culture inserts in a new, six well plate, containing fresh, pre-warmed culture medium. To begin immunohistochemistry, first use forceps to lift culture inserts and remove the culture medium beneath them.Then, add two milliliters of 4%PFA in 1x PBS, ph 7.4, onto the membrane inserts to fix the cerebellar slices. After 30 minutes, wash the slices three times with two milliliters of 1x PBS for 10 minutes each wash. Next, under the binocular microscope, using a 25x to 30x magnification, use a scalpel or a brush to gently detach the slices from the membrane inserts.Then, use a brush to transfer the floating slices into the wells of a four well plate, containing 1x PBS to limit antibody consumption. Then, aspirate the PBS from each well and incubate the slices in pre-cooled 100%ethanol at minus 20 degrees Celsius for 15 to 20 minutes. After incubation, aspirate the 100%ethanol and wash the slices briefly with 1x PBS.Then, wash them two times at room temperature with 1x PBS for 10 minutes each wash. To block non-specific antibody fixation sites, aspirate the PBS and incubate the slices in a solution containing 1x PBS, 5%NGS, and 0.3%non ionic detergent at room temperature for 30 to 45 minutes. Next, add primary antibodies diluted in the blocking solution and incubate the slices at four degrees Celsius overnight.After overnight incubation, wash the slices three times with 1x PBS for 10 minutes each wash. Then add the secondary antibodies, diluted in the blocking solution, at one to 500 dilution ratio and incubate the slices at room temperature in the dark for three hours. After incubation with the secondary antibody, wash the slices three times in 1x PBS in the dark for 10 minutes each wash.Next, under a binocular microscope, place 100 microleters of 1x PBS on the slide and with a brush, transfer the slices into the PBS and flatten them on the slide. Remove any access of PBS. Finally, place a drop of mounting medium directly onto the glass cover slip and gently cover the slices.Myelin immunostainings in organotypic cerebellar slices, obtained from post natal days 9 to 10 C57 black six wild type mice were observed at 11 days in vitro with nodes of ronviae enriched in voltage gated sodium channels, flanked by the paranodal axoglial junction domain. Same results were observed for PLP-GFP transgenic mice. Myelination of the parkengy cells is mostly achieved after one week in culture.LPC treatment fully demyelinates the slices, which remyelinate spontaneously and are fully myelinated six days post demyelination. This procedure should take 25 minute maximum. It is really the most critical point.Organotypic slice culture are suitable for live imaging study of myelination dynamics. It can also be used for targeting drug screening experiments. This technique allows an easy, quantitative approach to study myelination and remyelination processes.Experimenters are to follow the basic safety procedures applying to animal welfare, excessive and sharp myelination.

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Research

JoVE Journal

Neuroscience

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

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 ...

Derivation of Enriched Oligodendrocyte Cultures and Oligodendrocyte/Neuron Myelinating Co-cultures from Post-natal Murine Tissues

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications for understanding the pathogenesis of demyelinating diseases such as Multiple Sclerosis (MS). Cellular development is commonly studied with primary cell culture models. Primary cell culture facilitates the evaluation of a given cell type by providing a controlled environment, free of the extraneous variables that are present in vivo. While OL cultures derived from rats have provided a vast amount of insight into OL biology, similar efforts at establishing OL cultures from mice has been met with major obstacles. Developing methods to culture murine primary OLs is imperative in order to take advantage of the available transgenic mouse lines. 
Multiple methods for extraction of OPCs from rodent tissue have been described, ranging from neurosphere derivation, differential adhesion purification and immunopurification 1-3. While many methods offer success, most require extensive culture times and/or costly equipment/reagents. To circumvent this, purifying OPCs from murine tissue with an adaptation of the method originally described by McCarthy &amp; 
de Vellis 2 is preferred. This method involves physically separating OPCs from a mixed glial culture derived from neonatal rodent cortices. The result is a purified OPC population that can be differentiated into an OL-enriched culture. This approach is appealing due to its relatively short culture time and the unnecessary requirement for growth factors or immunopanning antibodies. 
While exploring the mechanisms of OL development in a purified culture is informative, it does not provide the most physiologically relevant environment for assessing myelin sheath formation. Co-culturing OLs with neurons would lend insight into the molecular underpinnings regulating OL-mediated myelination of axons. For many OL/neuron co-culture studies, dorsal root ganglion neurons (DRGNs) have proven to be the neuron type of choice. They are ideal for co-culture with OLs due to their ease of extraction, minimal amount of contaminating cells, and formation of dense neurite beds. While studies using rat/mouse myelinating xenocultures have been published 4-6, a method for the derivation of such OL/DRGN myelinating co-cultures from post-natal murine tissue has not been described. Here we present detailed methods on how to effectively produce such cultures, along with examples of expected results. These methods are useful for addressing questions relevant to OL development/myelinating function, and are useful tools in the field of neuroscience.

This article describes methods to derive enriched populations of murine oligodendrocyte precursor cells (OPCs) in primary culture, which differentiate to produce mature oligodendrocytes (OLs). In addition, this report describes techniques to produce murine myelinating co-cultures by seeding mouse OPCs onto a neurite bed of mouse dorsal root ganglion neurons (DRGNs).

Identifying the molecular mechanisms underlying OL development is not only critical to furthering our knowledge of OL biology, but also has implications ...

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 ...

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 ...