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* These authors contributed equally
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.
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.
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....
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
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.......
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.......
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 ....
Name | Company | Catalog Number | Comments |
for pipettes preparation | |||
Clark Borosilicate Thin Wall Capillaries | Harvard Apparatus | 30-0065 | Borosilicate Thin Wall without Filament, 1.5 mm OD, 1.17 mm ID, 75 mm L , Pkg. of 225 |
DMZ Universal Puller | Zeitz Instrumente | ||
Name | Company | Catalog Number | Comments |
for solutions | |||
Calcium Chloride dihydrate (CaCl2) | Sigma | C5080 | |
Choline Chloride | Sigma | C7527 | |
D-(+)-Glucose | Sigma | G8270 | |
L-Ascorbic acid | Sigma | A5960 | |
Magnesium Chloride solution 1M (MgCl2) | Sigma | 63020 | |
Potassium chloride SigmaUltra >99,0% (KCl) | Sigma | P9333 | |
Sodium bicarbonate (NaHCO3) | Sigma | S5761 | |
Sodium Chloride (NaCl) | Sigma | S5886 | |
Sodium phosphate monobasic | Sigma | S5011 | |
Sodium pyruvate | Sigma | P2256 | |
Ultrapure water | MilliQ | for all the solutions | |
Name | Company | Catalog Number | Comments |
for slice preparation | |||
2x 200 mL crystalizing dishes | |||
80 mL Pyrex beaker | |||
Antlia-3C Digital Peristaltic pump | DD Biolab | 178961 | For mice perfusion and 2-photon chamber perfusion (aCSF) |
Carbogen 5% CO2/95% O2 | Air Liquide France Industrie | ||
Dolethal | Vetoquinol | Dolethal 50 mg/mL | |
Filter papers (Whatman) | Sigma | WHA1001042 | Whatman qualitative filter paper, Grade 1 (Pore size: 11µM) |
Fine Scissors - Sharp | Fine Science Tools | 14060-60 | |
Food box 10 cm diameter, 8 cm Height | |||
glue (ethyl cyanoacrylate) | Loctite | super glue 3 power flex | |
Hippocampal Tool (spatula) | Fine Science Tools | 10099-15 | The largest extremity has to be angled at 90 ° |
Ice | |||
Iris Forceps (curved) | Moria | MC31 | |
Lens cleaning tissue | THOR LABS | ||
Nylon mesh strainer | diameter 7 cm | ||
Razor blades | Electron Microscopy Sciences | 72000 | For the slicer |
scalpel blade | |||
Slice interface holder | home-made, the file for 3D printing is provided in Supplemental Material | ||
Surgical Scissors - Sharp | Fine Science Tools | 14002-14 | |
Vibrating slicer | Thermo Scientific | 720-2709 | Model: HM 650V (Vibrating blade microtome) |
Water bath | Set at 32°C (first recovery step) | ||
Name | Company | Catalog Number | Comments |
for slice imaging | |||
× 25 0.95 NA water-immersion objective | Leica Microsystems (Germany) | HCX Irapo | |
2-photon MP5 upright microscope with resonant scanners (8 kHz) and two HyD Hybrid detectors | Leica Microsystems (Germany) | ||
Antlia-3C Digital Peristaltic pump | DD Biolab | 178961 | For 2-photon chamber perfusion with aCSF |
Carbogen 5% CO2/95% O2 | Air Liquide France Industrie | I1501L50R2A001 | |
Chameleon Ultra2 Ti:sapphire laser | Coherent (Germany) | ||
disposable transfer pipettes , wide mouth | ThermoFischer scientific | for example : 232-11 | 5.8ml with fin tip, but we cut it (approx 7cm) to have a 4 mm diameter mouth |
emission filter SP680 | Leica 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 system | Warner Instrument Corporation | Automatic Heater Controller TC-324B | to maintain perfusion solution at 32°C |
perfusion chamber | home-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 | ||
Name | Company | Catalog Number | Comments |
for slice stimulation | |||
Adenosine 5′-triphosphate disodium salt hydrate (ATP) | Sigma | A-26209 | to 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) | Sigma | F-6377 | use at 1 µM final |
Micromanipulator | Luigs and Neumann | SM7 | connected to the micropipette holde |
Micropipette holder | same as for eletrophysiology | ||
Serotonin hydrochloride | Sigma | H-9523 | aliquots of 50mM stock solution in H20 kept at -20°C. 500µM solution prepared the day of the experiment. |
Syringe 5mL (without needle) | Terumo medical products | SS+05S1 | |
Transparent tubing | Fischer Scientific | 11750105 | Saint Gobain Performance Plastics™ Tygon™ E-3603 Non-DEHP Tubing |
Name | Company | Catalog Number | Comments |
for image analysis | |||
Fiji | https://fiji.sc | Schindelin, J. et al Nat. Methods (2012) doi 10.1038 | |
Icy | Institut Pasteur | http://icy.bioimageanalysis.org | de Chaumont, F. et al. Nat. Methods (2012) |
Name | Company | Catalog Number | Comments |
mice | |||
CX3CR1-GFP mice | Jung et al, 2000 | male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background. | |
CX3CR1creER-YFP mice | Parkhurst et al 2013 | male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background. |
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