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.

<ol>
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B., 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=Neuronal+hyperactivity+recruits+microglial+processes+via+neuronal+NMDA+receptors+and+microglial+P2Y12+receptors+after+status+epilepticus.">Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 34 (32), 10528-10540 (2014).</li><li>Hristovska, I., Pascual, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+Resting+Microglial+Morphology+and+Process+Motility+from+a+Synaptic+Prospect.">Deciphering Resting Microglial Morphology and Process Motility from a Synaptic Prospect.</a> Frontiers in Integrative Neuroscience. 9, 1231 (2016).</li><li>Avignone, E., Lepleux, M., Angibaud, J., N&#228;gerl, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+morphological+dynamics+of+activated+microglia+after+induction+of+status+epilepticus.">Altered morphological dynamics of activated microglia after induction of status epilepticus.</a> Journal of Neuroinflammation. 12, 202 (2015).</li><li>Abiega, O., 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=Neuronal+Hyperactivity+Disturbs+ATP+Microgradients,+Impairs+Microglial+Motility,+and+Reduces+Phagocytic+Receptor+Expression+Triggering+Apoptosis/Microglial+Phagocytosis+Uncoupling.">Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.</a> PLoS Biology. 14 (5), e1002466 (2016).</li><li>Madry, C., 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=Microglial+Ramification,+Surveillance,+and+Interleukin-1&#946;+Release+Are+Regulated+by+the+Two-Pore+Domain+K+Channel+THIK-1.">Microglial Ramification, Surveillance, and Interleukin-1&#946; Release Are Regulated by the Two-Pore Domain K+Channel THIK-1.</a> Neuron. 97 (2), 299-312 (2018).</li><li>Honda, S., 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=Extracellular+ATP+or+ADP+induce+chemotaxis+of+cultured+microglia+through+Gi/o-coupled+P2Y+receptors.">Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 21 (6), 1975-1982 (2001).</li><li>Haynes, S. E., 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=The+P2Y12+receptor+regulates+microglial+activation+by+extracellular+nucleotides.">The P2Y12 receptor regulates microglial activation by extracellular nucleotides.</a> Nature Neuroscience. 9 (12), 1512-1519 (2006).</li><li>Wu, L. -. J., Vadakkan, K. I., Zhuo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-induced+chemotaxis+of+microglial+processes+requires+P2Y+receptor-activated+initiation+of+outward+potassium+currents.">ATP-induced chemotaxis of microglial processes requires P2Y receptor-activated initiation of outward potassium currents.</a> Glia. 55 (8), 810-821 (2007).</li><li>Kolodziejczak, M., 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=Serotonin+Modulates+Developmental+Microglia+via+5-HT+2BReceptors:+Potential+Implication+during+Synaptic+Refinement+of+Retinogeniculate+Projections.">Serotonin Modulates Developmental Microglia via 5-HT 2BReceptors: Potential Implication during Synaptic Refinement of Retinogeniculate Projections.</a> ACS Chemical Neuroscience. 6 (7), 1219-1230 (2015).</li><li>Schafer, D. P., 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+Sculpt+Postnatal+Neural+Circuits+in+an+Activity+and+Complement-Dependent+Manner.">Microglia Sculpt Postnatal Neural Circuits in an Activity and Complement-Dependent Manner.</a> Neuron. 74 (4), 691-705 (2012).</li><li>Pfeiffer, T., Avignone, E., N&#228;gerl, U. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+hippocampal+long-term+potentiation+increases+the+morphological+dynamics+of+microglial+processes+and+prolongs+their+contacts+with+dendritic+spines.">Induction of hippocampal long-term potentiation increases the morphological dynamics of microglial processes and prolongs their contacts with dendritic spines.</a> Scientific Reports. 6, 32422 (2016).</li><li>Parkhurst, C. 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. A., Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia:+Dynamic+Mediators+of+Synapse+Development+and+Plasticity.">Microglia: Dynamic Mediators of Synapse Development and Plasticity.</a> Trends in Immunology. 36 (10), 605-613 (2015).</li><li>Ohsawa, K., 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=P2Y12+receptor-mediated+integrin-beta1+activation+regulates+microglial+process+extension+induced+by+ATP.">P2Y12 receptor-mediated integrin-beta1 activation regulates microglial process extension induced by ATP.</a> Glia. 58 (7), 790-801 (2010).</li><li>Kurpius, D., Wilson, N., Fuller, L., Hoffman, A., Dailey, M. 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The authors have nothing to disclose.

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 ...

two-photon-imaging-microglial-processes-attraction-toward-atp-or

Research

JoVE Journal

Neuroscience

9.7K Views.  Institut National de la Santé et de la Recherche Médicale (INSERM), UMR-S 1270 Paris, France. 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.

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

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 significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

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

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

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 progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

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

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

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

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

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

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

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 live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the 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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

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 analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

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+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute 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

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 methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

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

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

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 ...

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

Summary

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Chapters in this video

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

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

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

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

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

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

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

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

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