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&#039; 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.

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 ...

To fully characterize a microglial phenotype, it's important to address both basal and directional motility. This protocol can be used to test the directional motility of microglia by two-photon imaging in mice brain slices, using customized 3D print interface and perfusion chambers. Demonstrating the procedure will be Fanny Etienne, a Ph.D.student, and Vincenzo Mastriolia, a post-doctoral researcher, both from our laboratories.At least 30 minutes before the dissection, start bubbling 70 milliliters of choline artificial cerebrospinal fluid, or choline aCSF, on ice. Add 150 milliliters of choline aCSF at 32 degrees Celsius, with carbogen. Place a 200 milliliter crystallizing dish with a bar magnet into a sealed food box and add 200 milliliters of aCSF to the dish.Place a 3D printed interface slice holder on top of the dish and remove excess fluid from the crystallizing dish, until only a thin film of solution covering the mesh of the interface slide holder remains. Add a few millimeters of aCSF at the bottom of the food box and start bubbling the fluid with carbogen. Then, close the sealed box while maintaining constant carbogenation to create a humidified, 95%oxygen, 5%carbon dioxide-rich interface chamber.After harvesting, place the brain onto a piece of aCSF-soaked filter paper and dissect out the region of interest, according to the preferred angle of slicing. For coronal slices, position and glue the caudal face of the brain onto a 10 centimeter Petri dish attached to the cutting block of a vibrating slicer, and position the block in the reservoir chamber of the vibrating slicer within a large chamber filled with ice. Fill the dish with ice-cold choline aCSF and use the slicer to obtain 300-micrometer thick brain slices, using a four millimeter diameter disposable transfer pipette after every pass of the blade to collect each slice.Let each slice recover in the 32 degree Celsius choline aCSF for about 10 minutes after it is obtained, before transferring the slices onto pieces of lens cleaning paper, topped with a drop of choline aCSF. Then aspirate the excess choline aCSF and use a spatula to transfer the slices onto the mesh of the interface chamber, allowing the slices to recover in this environment for at least 30 minutes. 30 minutes before starting the recording, connect the peristaltic pump to a customized recording chamber with top and bottom perfusion for optimizing the oxygenation and viability of the tissue slices, and clean the entire perfusion system with 50 milliliters of ultrapure water.At the end of the cleaning cycle, start the perfusion of the recording chamber with 50 milliliters of aCSF in a glass beaker under constant carbogenation, and use a disposable, wide-mouth transfer pipette to transfer the first slice to be imaged to the beaker to remove the lens paper. When the section has sunk to the bottom of the beaker, transfer the section to the recording chamber and position the slice holder onto the slice to minimize movement induced by the perfusion flow. Use bright-field illumination to target the brain region of interest under a five to 10x magnification.Then, use a 25x objective with a 0.35x water immersion lens to adjust the position of the viewing field. Use fluorescence illumination to locate the fluorescent microglial cells and backfill the pipette with 10 microliters of aCSF containing the compound of interest at its final concentration. Point the tip downward with gentle shaking to remove any air bubbles trapped in the tip and mount the filled pipette into a pipette holder connected to a five milliliter syringe with the plunger positioned at the five milliliter position and mounted onto a three-axis micromanipulator.Lower the pipette gently toward the slice, controlling and adjusting the objective at the same time, until the pipette tip lightly touches the surface of the slice. Now tune the laser and switch the microscope to the multiphoton mode. Make sure that chamber is screened from any light source and switch on the non-descanned detectors.Set the gain and use a lookup table with a color-coded upper limit to avoid saturating the pixels in the image. Then, start recording for a total duration of at least 30 minutes, slowly depressing the syringe plunger from the five to one milliliter position, after five minutes, over a period of five seconds, to apply the compound to the section. For image analysis, first perform z-projection and drift correction with ImageJ on the file of interest.Then, open the modified file in Icy and draw a circular, 35-micrometer diameter region of interest, centered over the injection site. Run the movie again with the ROI, to ensure that it is well-positioned. Then, use the region of interest intensity evolution plugin to measure the mean intensity over time in the region of interest, and save the results as a xls file.This protocol allows the responses induced by different compounds, like ATP or serotonin, to be measured. Although the injection of ATP elicits an increase of fluorescence, after most ATP injections, there is an immediate, slight decrease in fluorescence due to tissue distortion that comes back after a few minutes. The size of the region of interest also impacts the quantification, as increasing the diameter reduces the variability among the experiments, but decreases the accuracy and magnitude of the detected response.Assessment of the microglial response at a specific time point can be useful for the statistical comparison of different compounds, or to test the effects of specific antagonists added to the perfusion solution. To quantify the motility in three dimensions, know that you may have to change the z-step interval and the sampling rate of the acquisition to fit within the analysis requirements.

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Research

JoVE Journal

Neuroscience

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 ...

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 ...

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 ...

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 large share of ATP is needed to re-install ion gradients across plasma membranes degraded by electrical signaling of neurons. There is evidence that astrocytes - while not generating fast electrical signals themselves - undergo increased production of ATP in response to neuronal activity and support active neurons by providing energy metabolites to them. The recent development of genetically encoded sensors for different metabolites now enables the study of such metabolic interactions between neurons and astrocytes. Here, we describe a protocol for cell-type specific expression of the ATP-sensitive Fluorescence Resonance Energy Transfer- (FRET-) sensor ATeam1.03YEMK in organotypic tissue slice cultures of the mouse hippocampus and cortex using adeno-associated viral vectors (AAV). Furthermore, we demonstrate how this sensor can be employed for dynamic measurement of changes in cellular ATP levels in neurons and astrocytes upon increases in extracellular potassium and following induction of chemical ischemia (i.e., an inhibition of cellular energy metabolism).

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

We describe a protocol for cell-type specific expression of the genetically encoded FRET-based sensor ATeam1.03YEMK in organotypic slice cultures of the mouse forebrain. Furthermore, we show how to use this sensor for dynamic imaging of cellular ATP levels in neurons and astrocytes.

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 and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

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

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.