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.

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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 ...

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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Neuroscience

18.5K Views.  University of Basel. 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.

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Organotypic Slice Cultures 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 ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Analyzing Dendritic Morphology in Columns and Layers

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and columns to facilitate brain wiring during development and information processing in developed animals. Postsynaptic neurons elaborate dendrites in type-specific patterns in specific layers to synapse with appropriate presynaptic terminals. The fly medulla neuropil is composed of 10 layers and about 750 columns; each column is innervated by dendrites of over 38 types of medulla neurons, which match with the axonal terminals of some 7 types of afferents in a type-specific fashion. This report details the procedures to image and analyze dendrites of medulla neurons. The workflow includes three sections: (i) the dual-view imaging section combines two confocal image stacks collected at orthogonal orientations into a high-resolution 3D image of dendrites; (ii) the dendrite tracing and registration section traces dendritic arbors in 3D and registers dendritic traces to the reference column array; (iii) the dendritic analysis section analyzes dendritic patterns with respect to columns and layers, including layer-specific termination and planar projection direction of dendritic arbors, and derives estimates of dendritic branching and termination frequencies. The protocols utilize custom plugins built on the open-source MIPAV (Medical Imaging Processing, Analysis, and Visualization) platform and custom toolboxes in the matrix laboratory language. Together, these protocols provide a complete workflow to analyze the dendritic routing of Drosophila medulla neurons in layers and columns, to identify cell types, and to determine defects in mutants.

Here, we show how to analyze dendritic routing of Drosophila medulla neurons in columns and layers. The workflow includes a dual-view imaging technique to improve the image quality and computational tools for tracing, registering dendritic arbors to the reference column array and for analyzing the dendritic structures in 3D space.

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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 ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Cerebellar Purkinje Cells: DNA Damage Response in Organotypic Cultures

Visualizing the DNA Damage Response in Purkinje Cells Using Cerebellar Organotypic Cultures

Cerebellar Purkinje cells (PCs) exhibit a unique interplay of high metabolic rates, specific chromatin architecture, and extensive transcriptional activity, making them particularly vulnerable to DNA damage. This necessitates an efficient DNA damage response (DDR) to prevent cerebellar degeneration, often initiated by PC dysfunction or loss. A notable example is the genome instability syndrome, ataxia-telangiectasia (A-T), marked by progressive PC depletion and cerebellar deterioration. Investigating DDR mechanisms in PCs is vital for elucidating the pathways leading to their degeneration in such disorders. However, the complexity of isolating and cultivating PCs in vitro has long hindered research efforts. Murine cerebellar organotypic (slice) cultures offer a feasible alternative, closely mimicking the in vivo tissue environment. Yet, this model is constrained to DDR indicators amenable to microscopic imaging. We have refined the organotypic culture protocol, demonstrating that fluorescent imaging of protein-bound poly(ADP-ribose) (PAR) chains, a rapid and early DDR indicator, effectively reveals DDR dynamics in PCs within these cultures, in response to genotoxic stress.

Author Spotlight: Deciphering the Role of ATM in Ataxia-Telangiectasia and the Associated Cerebellar Degeneration

Cerebellar Purkinje cells (PCs) are particularly sensitive to deficiencies in the DNA damage response. A protocol is presented for the visual evaluation of the dynamics of PC response to DNA damage, which involves staining protein-bound poly(ADP-ribose) chains within cerebellar organotypic cultures.