Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

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Summary

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.

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Rakotomamonjy, J., Guemez-Gamboa, A. Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures. J. Vis. Exp. (154), e60353, doi:10.3791/60353 (2019).

Abstract

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.

Introduction

In vitro modeling is an essential tool in biomedical research. It allows investigators to study and tightly control specific mechanisms in restricted cell types, or in isolated systems/organs. Organotypic slice culture is a widely used in vitro technique, especially in the field of neuroscience1. The method was first established by Gähwiler, who cultured brain slices using the roller tube technique2, and later modified by Yamamoto et al., who introduced the use of a microporous membrane to perform cortical slice cultures3. Compared to primary cell cultures, organotypic slice cultures present the advantage of preserving the cytoarchitecture of the tissue, as well as native cell-cell connections in the plane of the tissue section.

Organotypic slices have been cultured from many parts of the central nervous system, such as the hippocampus4, cortex5, striatum6, cerebellum4,7, and spinal cord8,9, among others. They have been proven to be a powerful tool in drug discovery studies10. The effects of neuroactive molecules can be assessed in many ways: survival and neurodegeneration using immunostaining and biochemistry assays, neuronal circuit formation, or disruption using electrophysiology and live-imaging.

The goal of this work is to describe a simple method to perform organotypic cerebellar slice culture, which is known to be a relevant model to mimic cerebellar development in vitro. Particularly, we focused on the study of Purkinje cell developmental death. In vivo, Purkinje cells undergo apoptosis during the first postnatal week, peaking at postnatal day 3 (P3)11. The same pattern is observed in cerebellar slice culture, with Purkinje neurons dying by apoptosis when cerebella are taken from animals between P1 and P8, with a peak at P34,12. The use of the organotypic cerebellar slice cultures has allowed to identify several neuroprotective molecules7,13, as well as understanding part of the mechanisms involved in this programmed cell death14,15,16. Here, we describe a protocol based upon the study of Stoppini et al.17 in hippocampus, and adapted to cerebellum by Dusart et al.4 It includes rapid dissection and chopping of postnatal cerebella; slicing culture onto a cell culture insert containing a microporous membrane, with or without neuroprotective treatment; and immunofluorescence staining to assess neuronal survival.

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Protocol

All experiments involving animals were performed in accordance with Northwestern University Animal Studies committee.

1. Preparation prior to organotypic cerebellar slice cultures

  1. In a cell culture hood sprayed with 70% ethanol beforehand, prepare 200 mL of culture medium in a 250 mL bottle-top vacuum filter attached to a sterile bottle receiver. Add 100 mL of Basal Medium Eagle (BME), 50 mL of Hanks' Balanced Salt Solution (HBSS), 50 mL of heat-inactivated horse serum, 1 mL of 200 mM L-Glutamine (final concentration 1 mM), and 5 mL of 200 g/L glucose (final concentration 5 mg/mL). Vacuum-filter the culture medium and keep it at +4 °C for up to a month.
  2. In the sterilized biosafety cabinet, take out a 6-well culture plate from its package. Fill each well with 1 mL of culture medium. If a treatment is tested, add the pharmacological agent to the treated well, and the same volume of vehicle solution for the control well. In the present study, we added 1 µL of sterile 3 M KCl solution to the treated well (30 mM final) and 1 µL of sterile water to the control wells.
  3. Bring inside the cell culture hood the needed number of individually wrapped cell culture inserts with hydrophilic polytetrafluoroethylene (PTFE) membrane (pore size = 0.4 µm). Carefully unwrap them and take out the cell culture inserts with sterile forceps. Drop them one by one in a well of a 6-well plate, avoiding bubbles at the interface between the insert membrane and the cell culture medium. Let the inserts equilibrate in the medium in a 37 °C, 5% CO2 incubator for at least 2 h before culture.
    NOTE: The tissue chopper permanently resides in the cell culture hood.
  4. Prepare two 200 mL beakers, one filled with 150 mL sterile water, and the other filled with 150 mL 70% ethanol. Dip the surgical tools in the 70% ethanol bath and then in water prior to using them.
    NOTE: Do not use surgical tools immediately following contact with ethanol.
  5. Disinfect the double-edged blade in the 70% ethanol beaker. Place it on the blade holder using sterile forceps and hold it into place using the blade clamp wrench. Let it dry until use.
  6. Disinfect the plastic disc provided with the tissue chopper in the 70% ethanol beaker. Spray the cutting table with 70% ethanol prior to placing the disc on it. Let dry until use.

2. Dissection and Cerebellar Organotypic Slice Cultures

  1. Bring the mouse pup inside the cell culture hood to perform the dissection.
  2. Decapitate the pup quickly using straight operating scissors (Figure 1A).
  3. While holding the pup's head by the nose with straight dressing forceps, cut open the scalp using straight eye scissors, starting from the posterior end, and going lateral to midline.
  4. Proceed identically for the skull.
    NOTE: Make sure to point scissor tips outwards to avoid damaging the cerebellum during dissection.
  5. Take out the brain and transfer it to a 60-mm dish filled with cold HBSS + 5 mg/mL glucose.
  6. Carefully dissect out the cerebellum using sterile straight fine forceps (Figure 1B). Transfer it onto the plastic disc placed on the cutting table of the tissue chopper using sterile curved fine forceps.
  7. Orient the tissue by rotating the cutting table to perform parasagittal sections.
  8. Move the cutting table by pulling the table release knob to the right, so the blade is positioned at the edge of the tissue.
  9. Adjust the slice thickness to 350 µm and the blade speed to medium.
  10. Start the chopper. Once the whole cerebellum has been sliced (Figure 1C), turn off the chopper.
  11. Remove the plastic disc with sterile forceps.
  12. Carefully bring the plastic disc close to a 60-mm dish containing HBSS + 5 mg/mL glucose. Pipette cold HBSS + 5 mg/mL glucose onto the tissue using a sterile transfer pipette to allow harvesting of cerebellar slices in the buffer-filled dish.
  13. Separate the cerebellar slices from each other using a microprobe. Take out good quality sections with the transfer pipette (it is recommended to use tissue sections that are close to the vermis) and drop them off in a pre-equilibrated cell culture insert (Figure 1D).
  14. Position the cerebellar slices on the cell culture insert using the microprobe, then carefully remove the excess HBSS + 5 mg/mL glucose solution with the transfer pipette.
    NOTE: At this stage the tissue is extremely tender and prone to physical damage. The maximum number of cerebellar slices per cell culture insert depends on the age of the donor animal. 6–8 slices can be cultured for a P0–P4-old animal, and 4–6 slices for animals older than P5.
  15. Place the culture dish in the incubator, changing the medium completely every 2–3 days.
  16. To assess Purkinje cell survival, slices can be collected as early as 5 days in vitro. For other purposes, they can be maintained in culture for several weeks (Figure 1F).
    NOTE: In the case of Purkinje cell survival experiments, there is no need to renew the pharmacological treatment when changing cell culture medium (Figure 1F).

3. Immunofluorescence

  1. Take out the 6-well plate from the incubator. Remove cell culture medium, wash the cell culture insert with 1x PBS, and fix the slices with cold 4% paraformaldehyde solution for 1 h, with 1 mL under the cell culture insert and 500 µL on top of the cell culture insert.
  2. Wash the inserts 4x 10 min with 1x PBS, with 1 mL under and 500 µL on top of the insert, on an orbital shaker.
  3. With a brush, transfer the cerebellar slices from 1 cell culture insert to 1 well of a 24-well pre-filled with 1x PBS, 0.25% Triton X-100, and 3% BSA (PBS-TB), 500 µL/well.
  4. Permeabilize and block the slices by incubation in PBS-TB for 1 h at room temperature.
  5. Incubate with primary antibodies diluted in the PBS-TB solution, overnight at + 4 °C, on an orbital shaker, 200 µL/well.
  6. On the following day, perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 µL/well.
  7. Incubate with secondary antibodies diluted in the PBS-TB solution, two hours at room temperature, on an orbital shaker, and protected from light, 200 µL/well.
  8. Perform four washes of 10 min with 1x PBS, on an orbital shaker, 500 µL/well.
  9. Counterstain the slices using Hoechst 33342, diluted in 1x PBS (2 µg/mL), for 10 min at room temperature, protected from light, 500 µL/well.
  10. Mount the cerebellar slices on a microscopy slide with a transfer pipette. Let them air-dry completely, protected from light. Re-humidify them with 1x PBS and apply a coverslip covered with few drops of mounting medium (~80 µL). Avoid making any bubbles.
  11. Once the mounting medium has cured, cerebellar sections are ready to be imaged.

4. Imaging and Quantification of Cell Survival

  1. Turn on the microscope and lasers according to the manufacturer and/or the imaging core facility's instructions.
  2. Start the acquisition software.
  3. Using a 10X objective, locate non-altered cerebellar slices that present 9–10 lobules (usually cerebellar sections close to the vermis).
  4. Activate z-stack acquisition. Define the range of the z-stack to include all Purkinje cells present in the cerebellar slice. Set a 2 µm step size and start the acquisition. Save the acquired picture in the format of the acquisition software manufacturer to preserve the associated metadata.
  5. Open the acquired file in ImageJ using the Bio-formats plugin18.
  6. Go to Image > Stacks > Z-project… (Figure 2A).
  7. Choose Max Intensity in the Projection type dropdown menu and click OK (Figure 2B).
  8. A flattened 2-D image will be generated. Double click on the Multi-point tool to let the Point Tool window appear. Uncheck the Label points option to ease counted cells visualization. Then, click directly on a soma of a Purkinje cell to begin counting (Figure 1E). The total number of counted cells will be indicated at the bottom of the Point Tool window (Figure 3).
    NOTE: Usually, at least 3 non-damaged sections containing 9–10 lobules per cell culture insert can be quantified. To assess the neuroprotective effect of a pharmacological agent, at least 3 independent experiments should be performed.

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Representative Results

As shown in Figure 4, this protocol produces organotypic cerebellar slice cultures in which Purkinje cell survival can be assessed following the immunofluorescence and image acquisition steps. Purkinje cells were labeled with a combination of anti-Calbindin D-28K (dilution 1/200) and Alexa594 anti-mouse (dilution 1/300) antibodies. Image stitching was done automatically by the microscope acquisition software (NIS-Elements) to obtain a picture of the whole cerebellar slice. Purkinje cell numbers per slice were entered in the Prism 8 software to generate the chart and perform statistical analysis. At P6, Purkinje cell survival was low in the control, consistent with their known vulnerability window4 (Figure 4A,E). Survival increased as the donor animal grew older and exited this critical period (Figure 4C,E). Cerebellar slices were treated with high KCl concentration to successfully induce their depolarization and survival14 (Figure 4B,D,E).

Figure 1
Figure 1: Illustration of the protocol from dissection to Purkinje cell survival quantification.
The mouse pup is quickly decapitated (A). Following brain dissection, the cerebellum is isolated (B), and then chopped into 350 µm-width slices (C). The cerebellar sections are placed onto a control or treated cell culture insert (D) and maintained in culture for at least 5 days in vitro. The tissue is then fixed in 4% paraformaldehyde, immunostaining is performed, and pictures are acquired with a confocal microscope. Lastly, Purkinje cell number is quantified in each slice using the Multipoint Tool in ImageJ (E). (F) Schematic of the time course of KCl treatment during culture. Please click here to view a larger version of this figure.

Figure 2
Figure 2: ImageJ settings used to flatten the 3D-stack prior to quantification. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Settings used to count Purkinje cell number using the Multipoint tool in ImageJ with an example of a cerebellar slice being quantified. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Purkinje cell survival in organotypic cerebellar slice culture following high KCl treatment.
Representative image of cerebellar slices taken at postnatal day 6 (A, B) or 8 (C, C', D, D'), either untreated (A, C, C') or treated with 30 mM KCl (B, D, D'). (C' and D') Higher magnification view of the white boxed regions in C and D. Slices were kept 5 days in vitro prior to fixation. Purkinje cells are labelled with Calbindin D-28K in red. Scale bar = 500 µm (AD), and 100 µm (C' and D'). (E) Quantification of Purkinje cell survival from P6 and P8 cultures, in control or treated with 30 mM KCl, shows a higher survival with the treatment (Mann-Whitney test, n = 3 to 5 slices). Data are expressed as mean ± SEM. Please click here to view a larger version of this figure.

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Discussion

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

In order to obtain consistent and reproducible results, it is critical to perform the culture as efficiently as possible and to choose healthy cerebellar sections to be cultured. This method is also suitable for long-term culture (up to several months). So, it is essential to use good aseptic technique during the dissection and chopping process to avoid contaminations and the need to supplement cultures with antibiotics.

The cerebellar slice culture model preserves existing cell-cell interactions in the plane of the tissue section, and in the region cultured. This comes with a caveat: connections with targets outside of the cultured region might be severed. The supply in neurotrophic factors provided by afferent fibers might be discontinued as well and impair neuronal survival. This can be partially circumvented by performing organotypic slice co-cultures. It has been shown that climbing fibers can penetrate postnatal cerebellar slice cultures when co-cultured with inferior olive slice cultures14,19. More interestingly, Purkinje cells contacted by climbing fibers survived14.

Here, we focused on the use of organotypic slice culture to search for neuroprotective molecules in the developing cerebellum. However, this method is equally suitable for other conditions such as neurodegeneration20, axonal regeneration21, and monitoring of neuronal circuit activity22,23. The post-culture applications go beyond immunofluorescence. Slices can be used for biochemical or gene expression studies, in order to investigate the biological mechanisms involved in the neuroprotective effect of a given compound. Altogether, this model can lay the groundwork for finding new neuroactive molecules before in vivo testing and be an effective and reliable supplement to in vivo mechanistic studies.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

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

Materials

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

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References

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