This work was supported by the University of Basel, Department of Biomedicine, and the Swiss National Science Foundation (31003A-116624).

1. Setting up Organotypic Cerebellar Slice Cultures
Cerebellar slice cultures are prepared from postnatal day 8 P(8) mouse pups using the static incubation method 10. In our laboratory, we use B6CF1 mice. In some experiments also transgenic mice were used which express EGFP selectively in Purkinje cells. The preparation of slice cultures takes approximately 30 minutes per mouse pup, i.e. 3 hours for a litter of 6 mouse pups. All steps are carried out under sterile conditions in a laminar flow workbench with sterilized surgical instruments.
<ol>
 <li>The cerebellum of P8 mouse pups is dissected from the brain in ice-cold preparation medium (MEM (Gibco Catalog No. 11012) with glutamax 1:100, pH 7.3) using a stereomicroscope.</li>
 <li>The cerebellum is cut into 350 &mu;m thick slices in the sagittal orientation using a McIlwain tissue chopper.</li>
 <li>The slices are placed on Millipore cell culture inserts (approx.6 slices per insert, Millipore PICM03050) and are incubated in 6-well plates with 0.75 ml per well of incubation medium (48.35 ml MEM, 25 ml Eagle Medium, 25 ml horse serum, 1ml glutamax I, 0.65 ml of a sterile 10% glucose solution, pH 7.3) in a humidified atmosphere with 5% CO2 at 37 &deg;C. All tissue culture reagents were from Gibco, Invitrogen.</li>
 <li>Pharmacological treatments are typically started at 3 days in vitro (DIV3) by adding the drug in the desired concentration to the culture medium. Phorbol 12-myristate 13-acetate (PMA) and (RS)-3,5-Dihydroxyphenylglycine (DHPG) were from Tocris, UK. Since many drugs require the use of DMSO or ethanol for preparing a stock solution (e.g. PMA) care should be taken that the total amount of solvent added to the culture medium does not exceed 1% (7.5 &mu;l per well). The drugs are renewed together with the change of the culture medium every 2nd or 3rd day.</li>
</ol>
2. Fixation and Immunostaining of Organotypic Cerebellar Slice Cultures
An important advantage of the cerebellum is that antibodies are available that specifically and brightly stain the principal cerebellar neurons, i.e. Purkinje cells and granule cells. The dendritic morphology of Purkinje cells can be revealed by immunostaining with anti-calbindin D28K.
<ol>
 <li>For fixation of the cultures, the culture medium is removed and 3 ml of cold 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4) are added carefully per well containing the Millicell insert with the cerebellar slices attached to the membrane. Cultures are fixed overnight, then the fixative is removed and the cultures are rinsed with phosphate buffer.</li>
 <li>For immunostaining the following antibodies are used: rabbit anti-calbindin D-28K (Swant, Bellinzona, Switzerland) at 1:1000 for selectively visualizing Purkinje cells and monoclonal anti-NeuN (Chemicon, Millipore) at 1:500 for selectively visualizing granule cells 12. Rabbit anti-GFP was from Abcam, UK. Fluorescent secondary Alexa antibodies were from Molecular Probes, Invitrogen.</li>
 <li>The cultures are permeabilized and blocked by adding 3% normal goat serum + 0.3% TritonX100 in 0.1 M phosphate buffer (blocking solution). Anti-calbindin D28K and anti-NeuN are diluted in blocking solution and the cultures are incubated with the blocking solution containing the antibodies overnight at 4 &deg;C under slight agitation. All dilutions are indicated as % (v/v), Triton X100 is taken from a 10% stock solution in order to facilitate pipetting.</li>
 <li>The cultures are rinsed 3x with 0.1 M phosphate buffer and are then incubated with appropriate second antibodies, e.g. goat anti-rabbit Alexa 568 and goat anti-mouse Alexa 488 diluted 1:500 in 0.1M phosphate buffer + 0.1% TritonX100 for 2 hours at room temperature. The cultures are then rinsed 3x with 0.1M phosphate buffer.</li>
 <li>The stained slices are removed from the culture well with a paintbrush and are mounted on Superfrost plus glass slides and coverslipped with an appropriate mounting medium, e.g. Mowiol. The cultures can now be viewed on a regular microscope with epifluorescence equipment or with a confocal microscope.</li>
</ol>
3. Viewing of Individual Purkinje Cells in Organotypic Slice Cultures
<ol>
 <li>After immunostaining with Calbindin D28k the complete cytoplasm of Purkinje cells is brightly stained including the dendrites and the axon. Due to the dense alignment of Purkinje cells in the Purkinje cell layer, the dendritic arbors of most Purkinje cells are overlapping making it difficult to study the complete arbor of one individual cell. However, due to cell death of some Purkinje cells during the preparation process, in many cultures some areas are present with a reduced density of Purkinje cells. In such areas it is possible to find Purkinje cells with a complete dendritic arbor which does not overlap with other cells. In these cells, the dendritic arbor can be viewed and analyzed in a quality comparable to a Golgi staining. Because in the cerebellum the Purkinje cells are the only cell type expressing Calbindin D28K the cells are also identified as Purkinje cells.</li>
 <li>An alternative method is to use cultures derived from B6;FVB-Tg(Pcp2-EGFP)146.244Yuza/J mice (here called Pcp2-EGFP mice, available from Jackson laboratories) which express EGFP under the Purkinje cell specific L7 promoter 11. In such slice cultures living Purkinje cells can be visualized by expression of EGFP. It is also possible to follow the dendritic morphology of a single cell over time. Alternatively, after fixation, EGFP-expressing Purkinje cells can be immunostained with an anti-GFP antibody. Both methods are suitable for staining dendrites, cell bodies and axons of Purkinje cells in organotypic cultures. Because the L7 promoter in the Pcp2-EGFP mice is not expressed in every Purkinje cell and there are variations in the frequency of expressing Purkinje cells between mice (Kapfhammer, data not shown) slice cultures derived from these mice make it easier to visualize and measure Purkinje cells which have dendritic trees which overlap with other Purkinje cell dendritic trees. Such dendritic trees can be viewed individually when the neighboring Purkinje cells with overlapping dendritic trees do not express GFP (Figure 1A, B).</li>
</ol>
4. Measurement of Purkinje Cell Dendritic Tree Size and Branch Points
<ol>
 <li>Dendritic tree size. With the fluorescent labeling of Purkinje cells the size of the dendritic tree of a given cell can easily be measured if the tree is not overlapping with other cells. Because the Calbindin immunostaining labels all Purkinje cells, it can be difficult to identify cells with non-overlapping dendritic trees. In this case the use of the Pcp2-EGFP is recommended to simplify the identification of sufficient Purkinje cells with non-overlapping dendritic trees. Once a Purkinje cell with a non-overlapping dendritic tree is identified, it is viewed with the 20x lens and an image is recorded with a digital camera. This image can then be analyzed with an image analysis program. We use Image Pro plus, which allows outlining the dendritic tree of the cell with a single mouse click using the magic wand tool in measuring mode (Figure 2A). The program then computes the area covered by the dendritic tree and exports it to MS Excel. Statistical analysis of the data is done with GraphPad Prism software (see below).</li>
 <li>Number of branch points. Due to the highly branched and fine morphology of the Purkinje cell dendritic tree branch points need to be counted manually. The 20x image file of the Purkinje cells is zoomed in such that it covers most of the screen using Adobe Photoshop. Then every branch point is counted and marked with a bright dot (Figure 2B).</li>
</ol>
5. Representative Results
Monitoring the development of the Purkinje cell dendritic tree during the culture period 
Organotypic slice cultures derived from Pcp2-EGFP mice which express EGFP specifically in Purkinje cells allow to study the morphology of individual cells during several days in culture. This way the growth and development of the dendritic tree during the culture period can nicely be documented. Living identified Purkinje cells were photographed every 2nd or 3rd day during the culture period with the 10x objective. This low power objective was chosen to avoid and minimize phototoxic damage to the cells due to the illumination with fluorescent light. Figure 3 shows two examples of Purkinje cells photographed in living cultures. The first cell was followed from 2 days in vitro until 7 days in vitro (Figure 3, A-C). It is evident that the dendritic tree of this cell during this time period grows several new branches and expands in size. In the second example, a Purkinje cell was followed from 4 to 11 days in vitro (Figure 3, D-F). The small dendritic tree present at DIV4 expands substantially during this time period. Both examples show that most of the dendritic tree of Purkinje cells present at the end of the culture period did indeed develop in the culture.
Development of the Purkinje cell dendritic tree is inhibited by PKC or mGluR1 stimulation 
Organotypic cerebellar slice cultures were cultured for 11 days. Starting at the 2nd day in vitro, some of the cultures were treated with either PMA (50 nM), a phorbol ester stimulating PKC or DHPG (10 &mu;M), a mGluR1 agonist, until the cultures were fixed. Both compounds have been shown previously to inhibit and limit Purkinje cell dendritic growth 7, 8, 9, 5. In the untreated control slices Purkinje cells developed a typical large and highly branched dendritic tree which was visualized either by anti-Calbindin immunostaining (Figure 4A) or in cultures with EGFP-expressing Purkinje cells, by immunostaining with anti-GFP (Figure 4D). In cultures treated with PMA the morphology of the dendritic tree was profoundly altered. The dendrites appeared thickened and had only few short side branches. Many Purkinje cells, unlike in control cultures, were no longer unipolar, with one primary dendrite emanating from the cell body, but developed two or even more primary dendrites (Figure 4B, E). The territory covered by the dendritic tree was markedly reduced (see below). A similar situation was present in DHPG-treated cultures. The dendritic tree of the Purkinje cells was greatly reduced in size and the branching was markedly reduced (Figure 4C, F). However, there were also some qualitative differences compared to PMA-treated cultures. There was no thickening of the dendritic branches, and the primary dendrites carried many very short secondary dendrites (Figure 4C, F) suggesting that the signaling events going on in PMA and DHPG-treated Purkinje cells may be similar, but not identical.
Quantitative evaluation of Purkinje cell dendritic tree size and branch points For quantitative evaluation, the size of the Purkinje cell dendritic tree and the number of branch points per Purkinje cell is measured as described above. Results from at least three independent experiments are pooled together, and a minimum of 20 cells need to be analyzed. The mean of the area covered by the dendritic trees for the control experiments is determined and set as 100%, and the values for the other experiments are expressed as percent values accordingly. Statistical analysis of the data is done with GraphPad Prism software. Because we do not assume that all values determined are part of a Gaussian distribution, we use statistical tests that do not assume such a distribution of the measured values. For comparing multiple conditions and testing for statistical significance, we use Kruskal-Wallis test followed by an appropriate procedure for post hoc tests for rank-based statistics, as, e.g., implemented in the Dunn's post test in GraphPad Prism. Results are typically presented as bar graphs. An example of such a statistical evaluation is given in Figure 5. Both the analysis of the dendritic tree size (Figure 5A) and the number of branch points per Purkinje cell (Figure 5B) show clear differences between the control condition and DHPG or PMA treatment. Both parameters were different with a statistical significance of p&lt;0.001 according to the Kruskal-Wallis test.
<img alt="Figure 1" src="/files/ftp_upload/3637/3637fig1.jpg" /> 
Figure 1. EGFP-labeled Purkinje cell. In Pcp2-EGFP mice not all Purkinje cells express EGFP. Therefore, dendritic trees of some Purkinje cells can be viewed individually when they express EGFP (EGFP channel in A) and the neighboring Purkinje cells with overlapping dendritic trees do not (negative in EGFP channel shown in A, positive in the anti-Calbindin staining shown in the red channel in B). Scale bar = 50 &mu;m.
<img alt="Figure 2" src="/files/ftp_upload/3637/3637fig2.jpg" /> 
Figure 2. Measurement of dendritic parameters of Purkinje cells. (A). The size of the dendritic tree can easily be measured by tracing the outline of the Purkinje cell with a single mouse click in the Image analysis software Image Pro plus using the magic wand tool. The magic wand tool is used such that the entire dendritic tree including the cell soma and all dendritic branches are completely encircled by the line. (B). The number of branch points is counted manually in images of Purkinje cells. Every branch point is marked with a yellow dot. Scale bar = 50 &mu;m.
<img alt="Figure 3" src="/files/ftp_upload/3637/3637fig3.jpg" /> 
Figure 3. Monitoring the development of the Purkinje cell dendritic tree. Identified Purkinje cells were photographed repeatedly to monitor the growth of the dendritic tree. (A-C): Dendritic tree of a Purkinje cell growing and developing from day in vitro (DIV) 2 until DIV7. There is continuous growth and branching of the dendrites during this culture period. (D-F): Dendritic tree of a Purkinje cell growing and developing from DIV4 until DIV11. The dendritic tree expands substantially during this culture period. Scale bar = 50 &mu;m.
<img alt="Figure 4" src="/files/ftp_upload/3637/3637fig4.jpg" /> 
Figure 4. Development of the Purkinje cell dendritic tree is inhibited by PKC or mGluR1 stimulation. (A), (D): Untreated control cells with a well developed dendritic tree. (B), (E): After 9 days of PMA treatment dendrites appear thickened and the dendritic tree is strongly reduced in sized. Cells often lose their polarization and become bipolar (E). (C), (F): After 9 days of DHPG treatment the dendritic tree is greatly reduced in size. Unlike in the PMA treatment, many very fine and short side branches are present on the primary and secondary dendrites. Cells often lose their polarization and become bipolar (F). Purkinje cells were visualized by anti-Calbindin immunostaining in (A - C) and by EGFP-Expression in cultures derived from Pcp2-EGFP mice in (D - F). Scale bar = 50 &mu;m.
<img alt="Figure 5" src="/files/ftp_upload/3637/3637fig5.jpg" /> 
Figure 5. Quantitative evaluation of Purkinje cell dendritic tree size and branching. Quantitative measurements show that both dendritic tree size (upper bar graph) and the number of branch points (lower bar graph) are greatly reduced after PMA or DHPG treatment. Differences between control dendritic trees and dendritic trees of Purkinje cells from treated cultures were significant with p&lt;0.001.

<ol>
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Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by Swiss authorities. The authors have nothing to disclose.

The methods presented here allow to study Purkinje cell dendritic development in organotypic cerebellar slice cultures and to quantitatively evaluate Purkinje cell dendritic expansion by measuring dendritic tree size and the number of dendritic branch points. Of course, a more extensive and sophisticated quantitative analysis of Purkinje cell dendrites is possible, e.g. by determining total dendritic length, performing a Sholl analysis or determining the fractal dimension of the dendritic tree. For this type of analysis ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>
			 Tissue culture Inserts </td>
			<td>
			 Millipore </td>
			<td>
			 PICM 03050 PICM ORG50 </td>
			<td>
			 The PICMORG50 inserts have a low rim and allow viewing of live cultures at a microscope </td>
		</tr>
		<tr>
			<td>
			 MEM </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 11012044 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Glutamax 1 </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 35050038 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Basal Medum Eagle </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 41010026 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Horse serum </td>
			<td>
			 Gibco, Invitrogen </td>
			<td>
			 26050070 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Phorbol 12-myristate 13-acetate (PMA) </td>
			<td>
			 Tocris </td>
			<td>
			 1201 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 (RS)-3,5-Dihydroxy-phenylglycine (DHPG) </td>
			<td>
			 Tocris </td>
			<td>
			 0342 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Rabbit anti-calbindin D-28K </td>
			<td>
			 Swant </td>
			<td>
			 CB38 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Anti NeuN, clone A60 </td>
			<td>
			 Chemicon, Millipore </td>
			<td>
			 MAB377 </td>
			<td>
			 </td>
		</tr>
		<tr>
			<td>
			 Rabbit anti-GFP </td>
			<td>
			 Abcam </td>
			<td>
			 Ab290 </td>
			<td>
			 </td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures at JoVE.com

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

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 ...

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JoVE Journal

Neuroscience

18.5K vistas.  University of Basel. Se presenta un protocolo que permite ver y para evaluar cuantitativamente la morfología del árbol dendrítico de las células de Purkinje individuales cultivadas en cultivos de cortes organotípicos cerebelosos. Este protocolo tiene como objetivo promover estudios sobre los mecanismos de desarrollo de Purkinje células dendríticas.

Video: The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

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 ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

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 ...

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 ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.