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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

Abstract

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Introduction

Microglial cells are the brain's resident macrophages and play a role in both physiological and pathological conditions1,2. They have a highly branched morphology and are constantly extending and retracting their processes3,4. This "scanning" behavior is believed to be related and necessary to the survey of their surroundings. The morphological plasticity of microglia is expressed in three modes. First, some compounds rapidly modulate microglial morphology: the addition of ATP5,6 or....

Protocol

All experiments were approved by the local ethical committee (Darwin Committee, agreements #1170 and #10921).

1. Preparation of Glass Micropipettes for the Local Application of Compounds

  1. Prepare pipettes from borosilicate thin-wall glass capillaries with an electrode puller. Adjust the parameters to obtain pipettes with a 4 - 5 µm diameter at their extremity. Figure 2D shows one pipette in brightfield at low magnification.

Representative Results

This protocol describes a method to induce, observe, and quantify the oriented growth of microglial processes toward a locally applied compound, for example, ATP or 5-HT, in acute brain slices from young or adult (at least up to two-month-old) mice. Among the factors that contribute to maintaining brain slices from adult animals in a healthy state for several hours is the use of two tools designed to optimize cell survival at two steps of the protocol. First, the interface slice holder in.......

Discussion

By maintaining, unlike in dissociated or organotypic slice culture, a structural integrity with limited network adjustments, acute brain slices allow researchers to study microglia in their physiological environment. However, one of the major limitations is the fact that the slicing procedure creates injuries that can rapidly compromise the viability of neurons, particularly in the adult brain. As microglia are particularly reactive to cell damage, it is important to limit neuronal cell death as much as possible to prese.......

Acknowledgements

We thank the Cell and Tissue Imaging Facility of the Institut du Fer à Moulin, where all image acquisition and analysis have been performed. This work has been supported in part by the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Sorbonne Université Sciences, and by grants from Sorbonne Universités-Pierre et Marie Curie University (Emergence-UPMC program 2011/2014), the Fondation pour la Recherche sur le Cerveau, the Fondation de France, the Fondation pour la Recherche Médicale "Equipe FRM DEQ2014039529", the French Ministry of Research (Agence Nationale ....

Materials

NameCompanyCatalog NumberComments
for pipettes preparation
Clark Borosilicate Thin Wall CapillariesHarvard Apparatus30-0065Borosilicate Thin Wall without Filament, 1.5 mm OD, 1.17 mm ID, 75 mm L , Pkg. of 225
DMZ Universal PullerZeitz Instrumente
NameCompanyCatalog NumberComments
for solutions
Calcium Chloride dihydrate (CaCl2)SigmaC5080
Choline ChlorideSigmaC7527
D-(+)-GlucoseSigmaG8270
L-Ascorbic acidSigmaA5960
Magnesium Chloride solution 1M (MgCl2)Sigma63020
Potassium chloride SigmaUltra >99,0% (KCl)SigmaP9333
Sodium bicarbonate (NaHCO3)SigmaS5761
Sodium Chloride (NaCl)SigmaS5886
Sodium phosphate monobasicSigmaS5011
Sodium pyruvateSigmaP2256
Ultrapure waterMilliQfor all the solutions
NameCompanyCatalog NumberComments
for slice preparation
2x 200 mL crystalizing dishes
80 mL Pyrex beaker
Antlia-3C Digital Peristaltic pumpDD Biolab178961For mice perfusion and 2-photon chamber perfusion (aCSF)
Carbogen 5% CO2/95% O2Air Liquide France Industrie
DolethalVetoquinolDolethal 50 mg/mL
Filter papers (Whatman)SigmaWHA1001042Whatman qualitative filter paper, Grade 1 (Pore size: 11µM)
Fine Scissors - SharpFine Science Tools14060-60
Food box 10 cm diameter, 8 cm Height
glue (ethyl cyanoacrylate)Loctitesuper glue 3 power flex
Hippocampal Tool (spatula)Fine Science Tools10099-15The largest extremity has to be angled at 90 °
Ice
Iris Forceps (curved)MoriaMC31
Lens cleaning tissueTHOR LABS
Nylon mesh strainerdiameter 7 cm
Razor bladesElectron Microscopy Sciences72000For the slicer
scalpel blade
Slice interface holderhome-made, the file for 3D printing is provided in Supplemental Material
Surgical Scissors - SharpFine Science Tools14002-14
Vibrating slicerThermo Scientific720-2709Model: HM 650V (Vibrating blade microtome)
Water bathSet at 32°C (first recovery step)
NameCompanyCatalog NumberComments
for slice imaging
× 25 0.95 NA water-immersion objectiveLeica Microsystems (Germany)HCX Irapo
2-photon MP5 upright microscope with resonant scanners (8 kHz) and two HyD Hybrid detectorsLeica Microsystems (Germany)
Antlia-3C Digital Peristaltic pumpDD Biolab178961For 2-photon chamber perfusion with aCSF
Carbogen 5% CO2/95% O2Air Liquide France IndustrieI1501L50R2A001
Chameleon Ultra2 Ti:sapphire laserCoherent (Germany)
disposable transfer pipettes , wide mouthThermoFischer scientificfor example : 232-115.8ml with fin tip, but we cut it (approx 7cm) to have a 4 mm diameter mouth
emission filter SP680Leica Microsystems (Germany)
fluorescent cube containing a 525/50 emission filter and a 560 dichroic filter (for fluorescence collection)Leica Microsystems (Germany)
glass beaker with 50 mL of ACSF to maintain constant perfusion of the slice
Heating systemWarner Instrument CorporationAutomatic Heater Controller TC-324Bto maintain perfusion solution at 32°C
perfusion chamberhome-made, the file for 3D printing is provided in Supplemental Material
slice holder ("harp")home made : hairpin made of platinum with the two branches joined by parallel nylon threads
NameCompanyCatalog NumberComments
for slice stimulation
Adenosine 5′-triphosphate disodium salt hydrate (ATP)SigmaA-26209to be prepared ex-temporaneously : 1mg/ml (3mM) stock solution prepared the day of the experiment, kept at 4°C (a few hours) and diluted just before use
Fluorescein (optional)SigmaF-6377use at 1 µM final
MicromanipulatorLuigs and NeumannSM7connected to the micropipette holde
Micropipette holdersame as for eletrophysiology
Serotonin hydrochlorideSigmaH-9523aliquots of 50mM stock solution in H20 kept at -20°C. 500µM solution prepared the day of the experiment.
Syringe 5mL (without needle)Terumo medical productsSS+05S1
Transparent tubingFischer Scientific11750105Saint Gobain Performance Plastics™ Tygon™ E-3603 Non-DEHP Tubing
NameCompanyCatalog NumberComments
for image analysis
Fijihttps://fiji.scSchindelin, J. et al Nat. Methods (2012) doi 10.1038
IcyInstitut Pasteurhttp://icy.bioimageanalysis.orgde Chaumont, F. et al. Nat. Methods (2012)
NameCompanyCatalog NumberComments
mice
CX3CR1-GFP miceJung et al, 2000male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.
CX3CR1creER-YFP miceParkhurst et al 2013male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.

References

  1. Salter, M. W., Stevens, B. Microglia emerge as central players in brain disease. Nature Publishing Group. 23 (9), 1018-1027 (2017).
  2. Tay, T. L., Savage, J., Hui, C. W., Bisht, K., Tremblay, M. -. &. #. 2. 0. 0. ;.

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Two photon ImagingMicroglial ProcessesATPSerotoninAcute Brain SlicesDirectional Motility3D Print InterfacePerfusion ChambersCholine ACSFVibrating SlicerInterface ChamberPeristaltic PumpRecording Chamber

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