Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

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 conditions^1,2. They have a highly branched morphology and are constantly extending and retracting their processes^3,4. 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 ATP^5,6 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.

2. Solutions

1. Ensure that only glassware that has been cleaned by an autoclave cycle, followed by rinsing 2x - 3x with ultrapure....

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.......

Materials

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.