Name: two-photon imaging of microglial processes' attraction toward atp or serotonin in acute brain slices
Uploaded: 2019-01-31T13:30:00
Description: Watch this Scientific Journal Video about Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices at JoVE.com

We thank the Cell and Tissue Imaging Facility of the Institut du Fer &#224; 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 Sant&#233; et de la Recherche M&#233;dicale, the Sorbonne Universit&#233; Sciences, and by grants from Sorbonne Universit&#233;s-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 M&#233;dicale &#34;Equipe FRM DEQ2014039529&#34;, the French Ministry of Research (Agence Nationale pour la Recherche ANR-17-CE16-0008 and the Investissements d&#39;Avenir programme &#34;Bio-Psy Labex&#34; ANR-11-IDEX-0004-02) and a Collaborative Research in Computational Neuroscience program, National Science Foundation/French National Agency for Research (number : 1515686). All the authors are affiliated to research groups which are members of the Paris School of Neuroscience (ENP) and of the Bio-Psy Labex. F.E. is a Ph.D. student affiliated with Sorbonne Universit&#233;, Coll&#232;ge Doctoral, F-75005 Paris, France, and is funded by the Bio-Psy Labex. V.M. is a post-doctoral fellow funded by the Collaborative Research in Computational Neuroscience program, National Science Foundation/French National Agency for Research (number: 1515686). The authors thank Marta Kolodziejczak who participated in the initiation of the project.

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
<ol>
	<li>Prepare pipettes from borosilicate thin-wall glass capillaries with an electrode puller. Adjust the parameters to obtain pipettes with a 4 - 5 &#181;m diameter at their extremity. Figure 2D shows one pipette in brightfield at low magnification.</li>
</ol>
<p class="jov...

<ol>
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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Promote+Learning-Dependent+Synapse+Formation+through+Brain-Derived+Neurotrophic+Factor.">Microglia Promote Learning-Dependent Synapse Formation through Brain-Derived Neurotrophic Factor.</a> Cell. 155 (7), 1596-1609 (2013).</li><li>Wu, Y., Dissing-Olesen, L., Macvicar, B. 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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...

Microglial cells are the brain&#39;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 &#34;scanning&#34; 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 NMDA5,7 in the medium bathing acute brain slices increases the complexity of microglial ramifications, whereas norepinephrine decreases it6. These effects either are directly mediated by microglial receptors (for ATP and norepinephrine) or require an ATP release from neurons (for NMDA). Second, the growth and retraction speed of microglial processes, called motility or &#34;surveillance&#34;, can be affected by extracellular factors8, homeostasis disruptions9,10, or mutations9,10,11. Third, in addition to these isotropic changes of morphology and motility, microglia have the capacity to extend their processes directionally toward a pipette delivering ATP3,5,12,13,14, in culture, in acute brain slices or in vivo, or delivering 5-HT in acute brain slices15. Such oriented growth of microglial processes, also called directional motility, was first described as a response to a local laser lesion3,4. Thus, physiologically, it may be related to the response to injury or required for targeting microglial processes toward synapses or brain regions requiring pruning during development15,16, or in physiological17,18,19 or pathological situations9,18,19,20 in adulthood. The three types of morphological changes rely on different intracellular mechanisms11,13,20, and one given compound does not necessarily modulate all of them (e.g., NMDA, which acts indirectly on microglia, has an effect on morphology but does not induce directional motility5,7). Therefore, when aiming to characterize the effect of a compound, a mutation or a pathology on microglia, it is important to characterize the three components of their morphological plasticity. Here, we describe a method to study the directional growth of microglial processes toward a local source of compound, which is, here, ATP or 5-HT.
There are several models to study microglia processes&#39; attraction: primary cultures in 3D environment6,18,19, acute brain slices6,13,15,&#160;and in vivo imaging3,13. The in vivo approach is the best to preserve the physiological state of microglia. However, intravital imaging of deep regions requires complex surgical procedures and, therefore, it is often limited to superficial cortical layers. The use of microglia primary culture is the easiest technique to test a large number of conditions with a limited number of animals. Nevertheless, it is impossible to obtain the same cell morphology as in vivo, and cells lose their physiological interactions with neurons and astrocytes. Acute brain slices represent a compromise between these two approaches. This model allows researchers to study brain structures which are otherwise difficult to reach and to image with high resolution in vivo, and to investigate slices from neonatal stages, whereas transcranial microscopy is mostly performed at adulthood. Finally, it makes it possible to observe in real-time the effects of local drug application, and to repeat experiments while using a limited number of animals. Nonetheless, an issue with acute brain slices is the limited time (a few hours) during which the cells remain alive, notably for slices from mice older than two weeks, and the potential change of microglia morphology over time21,22.
Here, we describe a protocol to prepare acute brain slices of young and adult Cx3cr1GFP/+ mice up to two months old, with the preservation of microglia morphology and motility for several hours. We, then, describe how to use these slices to study the attraction of microglial processes toward compounds like ATP or 5-HT.

<table>
	<tbody>
		<tr>
			<td>for pipettes preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clark Borosilicate Thin Wall Capillaries</td>
			<td>Harvard Apparatus</td>
			<td>30-0065</td>
			<td>Borosilicate Thin Wall without Filament, 1.5 mm OD, 1.17 mm ID, 75 mm L , Pkg. of 225</td>
		</tr>
		<tr>
			<td>DMZ Universal Puller</td>
			<td>Zeitz Instrumente</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>for solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate (CaCl2)</td>
			<td>Sigma</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Choline Chloride</td>
			<td>Sigma</td>
			<td>C7527</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride solution 1M (MgCl2)</td>
			<td>Sigma</td>
			<td>63020</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride SigmaUltra &#62;99,0% (KCl)</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>MilliQ</td>
			<td></td>
			<td>for all the solutions</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>for slice preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x 200 mL crystalizing dishes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>80 mL Pyrex beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antlia-3C Digital Peristaltic pump</td>
			<td>DD Biolab</td>
			<td>178961</td>
			<td>For mice perfusion and 2-photon chamber perfusion (aCSF)</td>
		</tr>
		<tr>
			<td>Carbogen 5% CO2/95% O2</td>
			<td>Air Liquide France Industrie</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dolethal</td>
			<td>Vetoquinol</td>
			<td>Dolethal 50 mg/mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter papers (Whatman)</td>
			<td>Sigma</td>
			<td>WHA1001042</td>
			<td>Whatman qualitative filter paper, Grade 1 (Pore size: 11&#181;M)</td>
		</tr>
		<tr>
			<td>Fine Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14060-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Food box 10 cm diameter, 8 cm Height</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glue (ethyl cyanoacrylate)</td>
			<td>Loctite</td>
			<td>super glue 3 power flex</td>
			<td></td>
		</tr>
		<tr>
			<td>Hippocampal Tool (spatula)</td>
			<td>Fine Science Tools</td>
			<td>10099-15</td>
			<td>The largest extremity has to be angled at 90 &#176;</td>
		</tr>
		<tr>
			<td>Ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Forceps (curved)</td>
			<td>Moria</td>
			<td>MC31</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens cleaning tissue</td>
			<td>THOR LABS</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh strainer</td>
			<td></td>
			<td></td>
			<td>diameter 7 cm</td>
		</tr>
		<tr>
			<td>Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td>For the slicer</td>
		</tr>
		<tr>
			<td>scalpel blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slice interface holder</td>
			<td></td>
			<td></td>
			<td>home-made, the file for 3D printing is provided in Supplemental Material</td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>14002-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrating slicer</td>
			<td>Thermo Scientific</td>
			<td>720-2709</td>
			<td>Model: HM 650V (Vibrating blade microtome)</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td>Set at 32&#176;C (first recovery step)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>for slice imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#215; 25 0.95 NA water-immersion objective</td>
			<td>Leica Microsystems (Germany)</td>
			<td>HCX Irapo</td>
			<td></td>
		</tr>
		<tr>
			<td>2-photon MP5 upright microscope with resonant scanners (8 kHz) and two HyD Hybrid detectors</td>
			<td>Leica Microsystems (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antlia-3C Digital Peristaltic pump</td>
			<td>DD Biolab</td>
			<td>178961</td>
			<td>For 2-photon chamber perfusion with aCSF</td>
		</tr>
		<tr>
			<td>Carbogen 5% CO2/95% O2</td>
			<td>Air Liquide France Industrie</td>
			<td>I1501L50R2A001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chameleon Ultra2 Ti:sapphire laser</td>
			<td>Coherent (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>disposable transfer pipettes , wide mouth</td>
			<td>ThermoFischer scientific</td>
			<td>for example : 232-11</td>
			<td>5.8ml with fin tip, but we cut it (approx 7cm) to have a 4 mm diameter mouth</td>
		</tr>
		<tr>
			<td>emission filter SP680</td>
			<td>Leica Microsystems (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fluorescent cube containing a 525/50 emission filter and a 560 dichroic filter (for fluorescence collection)</td>
			<td>Leica Microsystems (Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass beaker with 50 mL of ACSF to maintain constant perfusion of the slice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating system</td>
			<td>Warner Instrument Corporation</td>
			<td>Automatic Heater Controller TC-324B</td>
			<td>to maintain perfusion solution at 32&#176;C</td>
		</tr>
		<tr>
			<td>perfusion chamber</td>
			<td></td>
			<td></td>
			<td>home-made, the file for 3D printing is provided in Supplemental Material</td>
		</tr>
		<tr>
			<td>slice holder (&#34;harp&#34;)</td>
			<td></td>
			<td></td>
			<td>home made : hairpin made of platinum with the two branches joined by parallel nylon threads</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>for slice stimulation </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A-26209</td>
			<td>to be prepared ex-temporaneously : 1mg/ml (3mM) stock solution prepared the day of the experiment, kept at 4&#176;C (a few hours) and diluted just before use</td>
		</tr>
		<tr>
			<td>Fluorescein (optional)</td>
			<td>Sigma</td>
			<td>F-6377</td>
			<td>use at 1 &#181;M final</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Luigs and Neumann</td>
			<td>SM7</td>
			<td>connected to the micropipette holde</td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td></td>
			<td></td>
			<td>same as for eletrophysiology</td>
		</tr>
		<tr>
			<td>Serotonin hydrochloride</td>
			<td>Sigma</td>
			<td>H-9523</td>
			<td>aliquots of 50mM stock solution in H20 kept at -20&#176;C. 500&#181;M solution prepared the day of the experiment.</td>
		</tr>
		<tr>
			<td>Syringe 5mL (without needle)</td>
			<td>Terumo medical products</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent tubing</td>
			<td>Fischer Scientific</td>
			<td>11750105</td>
			<td>Saint Gobain Performance Plastics&#8482; Tygon&#8482; E-3603 Non-DEHP Tubing</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>for image analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji</td>
			<td></td>
			<td>https://fiji.sc</td>
			<td>Schindelin, J. et al Nat. Methods (2012) doi 10.1038</td>
		</tr>
		<tr>
			<td>Icy</td>
			<td>Institut Pasteur</td>
			<td>http://icy.bioimageanalysis.org</td>
			<td>de Chaumont, F. et al. Nat. Methods (2012)</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CX3CR1-GFP mice</td>
			<td>Jung et al, 2000</td>
			<td></td>
			<td>male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.</td>
		</tr>
		<tr>
			<td>CX3CR1creER-YFP mice</td>
			<td>Parkhurst et al 2013</td>
			<td></td>
			<td>male or females, P3 to 2 months-old ; we have backcrossed these mice on 129sv background.</td>
		</tr>
	</tbody>
</table>

two-photon imaging of microglial processes' attraction toward atp or serotonin in acute brain slices

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 &#34;directional motility&#34;, 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.

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...

Watch this Scientific Journal Video about Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices at JoVE.com

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

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 ...

Research

JoVE Journal

Neuroscience

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Imaging of Intracellular ATP in Organotypic Tissue Slices of the Mouse Brain using the FRET-based Sensor ATeam1.03YEMK

Neuronal activity in the central nervous system (CNS) evokes a high demand on cellular energy provided by the breakdown of adenosine triphosphate (ATP). A ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...