The authors would like to thank Christine Auste for her support with the video recording and Chalid Ghadban for his excellent technical assistance. Urszula Grabiec was supported by the Roux Programm FKZ 29/18.

Animal experiments were performed in accordance with the Policy of Ethics and the Policy on the Use of Animals in Neuroscience Research as approved by the European Communities Council Directive 2010/63/EU of the European Parliament and of the Council of the European Union on the protection of animals used for scientific purposes.
1. Preparation of Instruments and Culture Media
<ol>
	<li>For preparation of OHSC use the following set of instruments: two small scissors, two curved tweezers, one tweezer with a fine tip, three blades (two of size 11, one of size 15), three scalpel holders, round filter papers (diameter: 35 mm), agar, one razor blade, one unmodified Pasteur pipette, and one modified Pasteur pipette without a tip. Sterilize all materials in an autoclave before usage (Figure 1).</li>
	<li>Weigh 5 g agar and dissolve it in 100 mL distilled water. Sterilize the solution for 20 min at 121.7 &#176;C at 210.8 kPa in an autoclave. Distribute the 3 mL liquid agar solution in 60-mm Petri dishes using a sterile glass pipette, allow it to solidify for 5 h, cover with plastic paraffin film to avoid contamination and store at 4 &#176;C until further use. Agar blocks are needed to stabilize the brains during the slicing procedure.</li>
	<li>Media
	<ol>
		<li>Make 200 mL of the preparation medium (pH 7.35) consisting of 198 mL minimal essential medium (MEM) and 2 mL L-glutamine solution (final concentration 2 mM). Prepare the solution on the day of media preparation and store it at 4 &#176;C.</li>
		<li>Prepare 100 mL of the culture medium consisting of 49 mL MEM, 25 mL Hanks&#39; balanced salt solutions (HBSS), 25 mL (v/v) normal horse serum (NHS), 1 mL L-glutamine solution (final concentration 2 mM), 100 &#181;g insulin, 120 mg glucose, 10 mg streptomycin, 10,000 U penicillin, and 800 &#181;g vitamin C as reported previously. Warm the medium (37 &#176;C), adjust the pH value to pH 7.4 and sterile filter (0.2 &#181;m pore size). Repeat the procedure (warming up, pH adjustment, etc.) every second day before changing the medium. Use the medium at the most for one week when stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Fill one 35-mm Petri dish with preparation medium to store the brain. Place two empty Petri dishes for collecting the tissue on a cooling pack in the working area.</li>
</ol>
2. Preparation and Slicing with a Vibratome
<ol>
	<li>Use brains from 7-9 day old rats or 4-5 day old mice for the OHSC preparation according to Stoppini et al.4 After decapitation of animals, remove the skin from the skull with scissors.</li>
	<li>Introduce the blade of a fine scissor into the foramen magnum and open the skull by cutting along the caudal (back) rostral (front) axis. Make two cuts perpendicular to the first one, so that the scissors point towards the left and right ear, respectively (&#34;T-incision&#34;).</li>
	<li>Open the skull carefully with fine forceps, paying attention not to injure the brain. Use a scalpel (blade size 11) to cut the most rostral part of the frontal pole and the cerebellum.</li>
	<li>By means of a spatula, remove the brain and place it carefully in the Petri dish filled with preparation medium (Figure 2). Position the brain on the specimen holder and fix it with medical cyanoacrylate glue. Use the pieces of agar to assure mechanical stabilization.</li>
	<li>Dissect the tissue horizontally in 350 &#181;m thick OHSC using a sliding vibratome.</li>
	<li>Evaluate the slices optically using a binocular microscope. Discard OHSC of low quality immediately. It is important to take only those slices with intact cytoarchitecture isolated from the middle part of the hippocampus (see Figures 3 and 4) between the dorsal and the ventral hippocampus.</li>
	<li>Separate the hippocampal region and the entorhinal cortex using a scalpel (round blade size 15; Figure 3). The perforant pathway and entorhinal cortex must be preserved.</li>
	<li>Six to eight OHSC are obtained from each brain. Transfer 2-3 slices into one cell culture insert (pore size 0.4 &#181;m) and place it in one well of a 6-well culture dish containing 1 mL culture medium per well.</li>
	<li>Incubate the 6-well dishes at 35 &#176;C in a fully humidified atmosphere with 5% (v/v) CO2 and change the cell culture medium every second day. 
	NOTE: Conduct your experiments at 6 day in vitro (div). Inflammatory reactions associated with the slicing procedure disappear by day 6. At this stage, microglial cells show a ramified morphology again and synaptic connections have matured.</li>
</ol>
3. Evaluation of Tissue Quality
<ol>
	<li>Fixation, labeling and visualization of degenerating neurons in OHSC
	<ol>
		<li>After performing the experiments, incubate the OHSC with 5 &#181;g/mL propidium iodide (PI) for 2 h prior to fixation, in order to stain the nuclei of degenerating neurons. 
		CAUTION: PI is a suspected carcinogen, always wear personal protective equipment (PPE) such as gloves.</li>
		<li>Wash the OHSC with 0.1 M phosphate buffer (pH 7.4) and fix them with a 4% (v/v) solution of paraformaldehyde (PFA) for 24 h. 
		CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.</li>
		<li>Wash the OHSC in inserts with 1 mL PBS and use a rigger brush to separate slices from the membrane.</li>
		<li>Label the OHSC with IB4 dye in a 24-well plate (Figure 5).</li>
	</ol>
	</li>
	<li>Conduction of tumor invasion experiments using fluorescently labeled single cells
	<ol>
		<li>24 h prior to the start of an experiment, label the tumor cells by using the fluorescent dye Carboxyfluorescein diacetate succinimidyl ester (CFDA SE).</li>
		<li>Detach and count the tumor cells using a Neubauer chamber.</li>
		<li>Resuspend the cells in medium, such that 10 &#181;L of suspension contains the desired cell number (normally 50,000 or 100,000 cells).</li>
		<li>Apply 10 &#181;L of the cell suspension onto the slice culture and allow the cells to invade for 3 days.</li>
		<li>At the end of the experiment, fix the slice cultures using 4% (v/v) PFA for 24 h, and label the co-cultures with PI for another 24 h to visualize the cytoarchitecture. 
		CAUTION: PFA is a toxic and suspected carcinogen. Work under fume hood and wear PPE.</li>
		<li>Mount the co-cultures onto a cover slip for further analysis using the mounting media.</li>
	</ol>
	</li>
</ol>
4. Evaluation of OHSC Experiments
Analyze the OHSC with a confocal laser scanning microscope (CLSM). For detection of PI labeled, degenerating neurons or the PI labeled cytoarchitecture use monochromatic light of the wavelength &#955; = 543 nm and an emission band pass filter for wavelengths &#955; = 585-615 nm. For CFDA labeled tumor cells or IB4 microglia, use an excitation wavelength of &#955; = 488 nm. For both experimental types, record a z-stack with 2 &#181;m thick optical slices and used for evaluation.

The authors have nothing to disclose

The present protocol describes the preparation of OHSC. This model allows testing of intrinsic capabilities and reactions of brain tissue after the application of physiological and pathological stimuli. Besides analyses of electrophysiological parameters, OHSC can be lesioned and the effects of damage on all cell types can be determined. Treatment with different substances and the detailed description of lesioning processes or healing in the absence of macrophages and lymphocytes is possible.
...

OHSC are a well-characterized in vitro model to study both physiological and pathological properties of neurons, astrocytes and microglia1. It is easy to control the extracellular environment and monitor the cellular and morphological changes after various stimuli. The organization of hippocampal neurons and their connections are well preserved after preparation2,3. Out of several advantages, OHSC allow monitoring of brain injury and tumor invasion without animal surgery. Six to eight OHSC can be obtained from a single rodent brain. OHSC therefore help to significantly reduce the number of animals and allow testing multiple drug concentrations, genetic manipulations or different lesion models in the same animal. In slice-based assays, experimental conditions can be precisely controlled. Additionally, time dependent development of pathological conditions like secondary damages can easily be monitored by time-lapse imaging.
In the given protocol, originally established by Stoppini et al.4, the preparation steps are described and important morphological landmarks for the selection of appropriate slices are highlighted. We recommend the preparation of postnatal day 7-9 rats or postnatal day 4-5 mice. In these periods, OHSC show a robust resistance to mechanical traumas and a high potential for reorganization of neuronal circuits. In contrast, preparations from embryonic or adult rats rapidly change their structure and lose their organotypic morphology during cultivation and are therefore less suitable for studying long-term processes in basic research5,6,7,8,9,10,11. Another critical point for the survival rate of OHSC is the thickness of the slice itself as the diffusion and thus nutrient supply are limited12,13,14.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:412px;">6-Well</td>
			<td style="width:204px;">Falcon</td>
			<td style="width:179px;">35-3046</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agar</td>
			<td>Fluka</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclav</td>
			<td>Systec</td>
			<td>DX-45</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CFDA</td>
			<td>&#160;Thermo Fisher</td>
			<td>V12883</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal laser scanning microscope (CLSM) LSM700</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle&#180;s Minimal Essential Medium&#160;</td>
			<td>Invitrogen</td>
			<td>32360-034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein labeled Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td>Vector Labs</td>
			<td>FL-1101</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescein labeled GSL I &#8211; isolectin B4</td>
			<td>Vector Labs</td>
			<td>FL-1201&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Merk</td>
			<td>1083371000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glutamin</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hank&#180;s Balanced Salt Solution (with Ca2+ and Mg2+)</td>
			<td>Invitrogen</td>
			<td>24020-133</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Hank&#180;s Balanced Salt Solution (without Ca2+&#160; and Mg2+)</td>
			<td>Invitrogen</td>
			<td>14170-138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin</td>
			<td>Sigma Aldrich</td>
			<td>I5500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-ascorbic acid</td>
			<td>Sigma Aldrich</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamin</td>
			<td>Invitrogen</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LN229</td>
			<td>Cell-Lines-Service</td>
			<td>300363</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Medical cyanoacrylate glue (Histoacryl glue)</td>
			<td>B.Braun</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Culture Inserts</td>
			<td>Millipore</td>
			<td>PICMORG50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NMDA N-methyl-D-aspartic acid</td>
			<td>Sigma Aldrich</td>
			<td>M3262</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal Horse Seum</td>
			<td>Invitrogen</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes (all sizes)</td>
			<td>Greiner</td>
			<td>627160/664160/628160</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PFA</td>
			<td>Roth</td>
			<td>0335.1</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Propidium iodid (PI)</td>
			<td>Sigma Aldrich</td>
			<td>81845-25MG</td>
			<td>toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U138</td>
			<td>ATCC</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vibratome</td>
			<td>Leica</td>
			<td>Leica VT 1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic hippocampal slice cultures as a model to study neuroprotection and invasiveness of tumor cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Neuroprotection studies: To determine neuronal damage, the number of PI positive nuclei and IB4 positive microglia in every third optical section of the granule cell layer (GCL) of the dentate gyrus (DG) was counted. For tumor invasion experiments, the maximal intensity z-projection of the stack was used for calculating the area covered by tumor cells, as a measure of invasion and to visualize different invasion patterns (Figure 6)...

Watch this Scientific Journal Video about Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells at JoVE.com

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

organotypic-hippocampal-slice-cultures-as-model-to-study

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JoVE Journal

Neuroscience

12.8K Views.  Martin Luther University Halle-Wittenberg. Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Video: Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

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 ...

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. ...

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 ...

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.

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 ...

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.

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 ...