Name: visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy
Uploaded: 2017-10-23T12:30:00
Description: Watch this Scientific Journal Video about Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy at JoVE.com

We thank Linda Hildegard Bergersen and Magnar Bj&#248;r&#229;s for access to cell lab and equipment, Janelia Molecular Biology Shared Resource staff for plasmid and virus production and Koen Vervaeke for assistance with laser power measurements. This work was funded by the Norwegian Health Association, the Norwegian Research Council and the microscopy equipment was funded by Norbrain.

The procedures described here have been approved by the Norwegian Animal Research Authority. Suppliers and catalog numbers for the consumables and other required equipment are available in the materials list at the end of the document.
1. Preparation of Organotypic Slices
NOTE: This recipe uses two mouse pups at postnatal day 7-9 (p7-9), which yield 24 organotypic slices divided on two six-well culture dishes. Unless stated otherwise, all procedures should be done in ...

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The protocol for making organotypic cultures described here is a modified version18 of the protocol described by De Simoni and Yu (2006)5. The most important changes have been outlined below. Tris buffer is added to the culture medium, which improves the survival of the slices when outside of the incubator during viral transduction and changing of the cell medium. The sterilization procedure for confetti is also changed. While other protocols sterilize confetti by autoclavi...

The brain&#39;s white matter is composed of nerve cell axons wrapped in myelin, a specialized extended plasma membrane formed by oligodendrocytes. Myelin is required for fast and reliable action potential propagation and long-term survival of myelinated axons, and a loss of myelin can cause neurological dysfunction. Despite their importance, the properties of oligodendrocytes are less known compared with neurons and astrocytes. Consequently, fewer tools have been developed for studying oligodendrocytes.
Live imaging of cell organelles such as mitochondria, endoplasmatic reticulum (ER) or different vesicular structures can be useful to study dynamic changes in the organelles over time. Traditionally, imaging of living oligodendrocytes has been performed in monocultures1,2. However, oligodendrocytes in monoculture do not display compact myelin, and organotypic or acute brain slices may, therefore, be a better option when studying localization and movement of organelles. Localization of small organelles and proteins in the myelin sheath can be challenging due to the short distance between the myelinated axon and the surrounding myelin sheath. Thus, light microscopic immunostaining procedures alone do not have the spatial resolution to discriminate between organelles in the myelin sheath and those in the myelinated axon. This can be solved by viral transduction with genes for organelle-targeted fluorescent proteins driven by cell type-specific promoters. The advantages are a cell-specific and sparse expression, which enables accurate assessment of organelle localization and dynamics. Transgenic animals can also be used to achieve such an organelle-targeted cell-specific expression3. However, the production and maintenance of transgenic animals is expensive and usually does not offer the sparse expression that can be achieved by viral methods.
The method described here uses viral transduction of oligodendrocytes with mitochondrial-targeted fluorescent proteins (dsred or green fluorescent protein, GFP) driven by the myelin basic protein promoter (MBP-mito-dsred or MBP-mito-GFP) to visualize oligodendrocyte mitochondria in organotypic brain slices. In addition, expression of another fluorescent protein in the cytoplasm (either GFP used together with mito-dsred or tdtomato used with mito-GFP) is used to enable visualization of cell morphology, including the cytoplasmic compartments of the myelin sheath. The protocol includes the procedure for making organotypic brain slices (a modified version of the protocol described by De Simoni and Yu, 20064,5). We then describe the time-lapse imaging procedure for studying mitochondrial movement. This procedure uses an upright confocal microscope with a continuous exchange of imaging medium, a setup that enables easy application of drugs or other medium changes during imaging. The time-lapse imaging procedure can be performed on any confocal microscope, with some extra equipment for maintaining living slices as described below. The protocol also contains several tips to optimize imaging and reduce phototoxicity.
Lastly, a quick and simple way to visualize unstained myelin by Confocal Reflectance microscopy (CoRe) is described. This can be useful to identify the myelin sheath during live imaging. In recent years, several techniques have been developed to image myelin without any staining required, but most of these require specific equipment and expertise6,7,8. The procedure described here uses the reflective properties of the myelin sheath and is a simplified single-excitation wavelength version of Spectral Confocal Reflectance microscopy (SCoRe, in which several laser wavelengths are combined to visualize myelin)9. CoRe can be done on any confocal microscope that has a 488 nm laser and a 470 - 500 nm bandpass emission filter or a tunable emission filter.

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		<tr height="36">
			<td height="36" style="height:36px;width:167px;">Agarose&#160;</td>
			<td style="width:139px;">Sigma&#160;</td>
			<td style="width:125px;">A9539</td>
			<td style="width:171px;"></td>
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		<tr height="54">
			<td height="54" style="height:54px;width:167px;">BD Microlance 19G</td>
			<td style="width:139px;">BD</td>
			<td style="width:125px;">301500</td>
			<td style="width:171px;">Needles used for in- and outlet of bath</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">Bioxide gas</td>
			<td style="width:139px;">AGA</td>
			<td style="width:125px;">105701</td>
			<td style="width:171px;"></td>
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		<tr height="31">
			<td height="31" style="height:31px;width:167px;">Brand pipette bulbs</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">Z615927</td>
			<td style="width:171px;">Pipette bulbs</td>
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			<td height="50" style="height:50px;width:167px;">Bunsen burner (Liquid propane burner)</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">89038-530</td>
			<td style="width:171px;"></td>
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			<td height="56" style="height:56px;width:167px;">Cable assembly for heater controllers</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0106&#160;</td>
			<td style="width:171px;">Temperature controller - thermometer part</td>
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		<tr height="32">
			<td height="32" style="height:32px;width:167px;">CaCl2</td>
			<td style="width:139px;">Fluka</td>
			<td style="width:125px;">21100</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:167px;">CO2&#160;</td>
			<td style="width:139px;">AGA</td>
			<td style="width:125px;">100309</td>
			<td style="width:171px;">CO2 for incubator</td>
		</tr>
		<tr height="89">
			<td height="89" style="height:89px;width:167px;">Cover glass, square Corning</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">13206778</td>
			<td style="width:171px;">To attach under bath for live imaging. Seal with glue or petrolium jelly.</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">D-(+)-Glucose</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">G7021</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Delicate forceps</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">11063-07</td>
			<td style="width:171px;">For dissection</td>
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		<tr height="30">
			<td height="30" style="height:30px;width:167px;">Diamond scriber pen</td>
			<td style="width:139px;">Ted Pella Inc.</td>
			<td style="width:125px;">54463</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:167px;">Disposable Glass Pasteur Pipettes 230 mm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">612-1702</td>
			<td style="width:171px;">Glass pipettes</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Double edge stainless steel razor blade</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#7200</td>
			<td style="width:171px;">Razor blade for vibratome</td>
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		<tr height="52">
			<td height="52" style="height:52px;width:167px;">Earle&#39;s Balanced Salt Solution (EBSS)</td>
			<td style="width:139px;">Gibco-Invitrogen</td>
			<td style="width:125px;">24010-043</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Filter paper circles</td>
			<td style="width:139px;">Schleicher &#38; Schuell</td>
			<td style="width:125px;">300,220</td>
			<td style="width:171px;">Filter paper used for filtration of PFA</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Fun tack</td>
			<td style="width:139px;">Loctite</td>
			<td style="width:125px;">1270884</td>
			<td style="width:171px;">Use to connect/adjust position of in- and outlets in bath</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Hand towel C-Fold 2</td>
			<td style="width:139px;">Katrin</td>
			<td style="width:125px;">344388</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:167px;">Harp, Flat for RC-41 Chamber,</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">&#160;64-1418</td>
			<td style="width:171px;">Harp to hold down confetti in bath. Cut off strings before use with organotypic slices. 1.5 mm, 13mm, SHD-41/15</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:167px;">HEPES, FW: 260.3</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">H-7006</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:167px;">Holten LaminAir, Model 1.2</td>
			<td style="width:139px;">Heto-Holten</td>
			<td style="width:125px;">96004000M</td>
			<td style="width:171px;">Laminar flow hood</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:167px;">Horse serum, heat inactivated</td>
			<td style="width:139px;">Gibco-Invitrogen</td>
			<td style="width:125px;">26050-088</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">KCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">P9541</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:167px;">LCR Membrane, PTFE,&#160;</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">FHLC0130</td>
			<td style="width:171px;">Confetti&#160;</td>
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			<td height="36" style="height:36px;width:167px;">Leica VT1200</td>
			<td style="width:139px;">Leica</td>
			<td style="width:125px;">14048142065</td>
			<td style="width:171px;">Vibratome</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:167px;">MEM-Glutamax with HEPES</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">42360024</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">MgCl2</td>
			<td style="width:139px;">R.P. Normapur</td>
			<td style="width:125px;">25 108.295</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Micro Spoon Heyman Type B</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">62411-B</td>
			<td style="width:171px;">Small, rounded spatula with sharpened end for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:167px;">Millex-GP filter unit</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">SLGPM33RA</td>
			<td style="width:171px;">Syringe filter unit</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:167px;">Millicell cell culture insert, 30 mm</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">PICM03050</td>
			<td style="width:171px;">Cell culture inserts</td>
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		<tr height="90">
			<td height="90" style="height:90px;width:167px;">Minipuls 3 Speed Control Module</td>
			<td style="width:139px;">GILSON</td>
			<td style="width:125px;">F155001</td>
			<td style="width:171px;">Peristaltic pump for live imaging - Control module part (connect to two-cannel head)</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">Na2HPO4</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S7907</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:167px;">NaCl</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S9888</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">NaH2PO4</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">S8282</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">NaHCO3</td>
			<td style="width:139px;">Fluka</td>
			<td style="width:125px;">71628</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:167px;">Nunclon Delta Surface</td>
			<td style="width:139px;">Thermo&#160; Fischer Scientific</td>
			<td style="width:125px;">140675</td>
			<td style="width:171px;">Culture plate</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:167px;">Nystatin Suspension</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:125px;">N1638</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="93">
			<td height="93" style="height:93px;width:167px;">Objective W &#34;Plan-Apochromat&#34; 40x/1.0 DIC&#160;</td>
			<td style="width:139px;">Zeiss</td>
			<td style="width:125px;">441452-9900-000&#160;</td>
			<td style="width:171px;">Water immersion objective used for live imaging. (WD=2.5mm), VIS-IR</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Parafilm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">291-1211</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Paraformaldehyde, granular</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#19208</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:167px;">PC-R perfusion chamber</td>
			<td style="width:139px;">SiSkiYou</td>
			<td style="width:125px;">&#160;15280000E</td>
			<td style="width:171px;">Bath for live imaging</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Penicillin-Streptomycin, liquid</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:125px;">15070-063</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:167px;">Petri dish 140 mm&#160;</td>
			<td style="width:139px;">Heger</td>
			<td style="width:125px;">1075</td>
			<td style="width:171px;">Large Petri dish&#160;</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:167px;">Petri dish 92x16 mm&#160;</td>
			<td style="width:139px;">Sarstedt&#160;</td>
			<td style="width:125px;">82.1473</td>
			<td style="width:171px;">Medium Petri dish&#160;</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:167px;">Petridish 55x14,2 mm</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">391-0868</td>
			<td style="width:171px;">Small Petri dish</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Phosphate buffered saline (PBS)</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">P4417</td>
			<td style="width:171px;">PBS tablets</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">R2 Two Channel Head&#160;</td>
			<td style="width:139px;">GILSON</td>
			<td style="width:125px;">F117800</td>
			<td style="width:171px;">Peristaltic pump for live imaging - Two channel head part (requires control module)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:167px;">Round/Flat Spatulas, Stainless Steel</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">82027-528</td>
			<td style="width:171px;">Large spatula for dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Sand paper</td>
			<td style="width:139px;">VWR</td>
			<td style="width:125px;">MMMA63119</td>
			<td style="width:171px;">Optional, for smoothing broken glass pipettes</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Scissors,&#160; 17,5 cm</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">14130-17</td>
			<td style="width:171px;">Large scissors for dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Scissors, 8,5&#160;</td>
			<td style="width:139px;">Finescience</td>
			<td style="width:125px;">14084-08</td>
			<td style="width:171px;">Small, sharp scissors for&#160; dissection</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:70px;width:167px;">Single edge, gem blade</td>
			<td style="width:139px;">Electron Microscopy Sciences</td>
			<td style="width:125px;">#71972</td>
			<td style="width:171px;">Single edge razor blade</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;width:167px;">Single inline solution heater SH-27B</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0102</td>
			<td style="width:171px;">Temperature controller - heater part</td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:167px;">Steritop-GP Filter unit, 500 ml , 45mm</td>
			<td style="width:139px;">Millipore</td>
			<td style="width:125px;">SCGPT05RE</td>
			<td style="width:171px;">Filter to sterilize solutions</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:167px;">Super glue precision</td>
			<td style="width:139px;">Loctite</td>
			<td style="width:125px;">1577386</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:167px;">Surgical scalpel blade no. 22</td>
			<td style="width:139px;">Swann Morton Ltd.</td>
			<td style="width:125px;">209</td>
			<td style="width:171px;">Rounded scalpel blade</td>
		</tr>
		<tr height="113">
			<td height="113" style="height:113px;width:167px;">Temperature controller TC324B</td>
			<td style="width:139px;">Warner Instruments</td>
			<td style="width:125px;">64-0100</td>
			<td style="width:171px;">Temperature controller for live imaging (requires solution heater and cable assembly)</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:167px;">Trizma base</td>
			<td style="width:139px;">Sigma&#160;</td>
			<td style="width:125px;">T1503</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:167px;">Trizma HCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:125px;">T3253</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:167px;">Water jacketed incubator series II</td>
			<td style="width:139px;">Forma Scientific</td>
			<td style="width:125px;">78653-2882</td>
			<td style="width:171px;">Incubator</td>
		</tr>
	</tbody>
</table>

visualization and live imaging of oligodendrocyte organelles in organotypic brain slices using adeno-associated virus and confocal microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Organotypic brain slices that were cultured and transduced as described above showed a sparse distribution of cortical oligodendrocytes expressing mito_dsred and GFP. Immunostaining with antibodies against Olig2 and MBP confirmed that the expression was specific to oligodendrocytes (Figure 1).
For live imaging, transduced oligodendrocytes were recognized by their characteristic morphology of several...

Watch this Scientific Journal Video about Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy at JoVE.com

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

The overall goal of this viral transduction and time-lapse recording is to visualize movement of oligodendrocyte mitochondria in organotypic brain slices.This method can help answer key questions in the oligodendrocyte field.Such as how the oligodendrocytes and their organelles respond to changes in the local environment.The main advantage of this technique is that it gives detailed information about oligodendrocyte structure and organelle dynamics in a preparation where all the different brain cells are present and oligodendrocytes produce compact myelin.Transduce oligodendrocytes in slice cultures at day seven in vitro.First draw up 1.3 microliters of diluted AAV per slice.Making sure the outside of the pipette tip is dry, hold pipette above the cortex as close as possible without touching the slice with the pipette tip and slowly push solution out of the pipette.When the solution touches the slice, move the pipette tip over the cortex to spread the solution over the whole cortex area.Once all the slices in one dish have been transduced, return the dish to the incubator and then repeat the process on the other dishes.Examine the slices for expression of fluorescent markers and image the slices when the expression levels of the fluorescent markers are sufficient and the slices look healthy.Begin setting up the imaging bath by using modeling clay to attach syringe needles bent at a 45

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Research

JoVE Journal

Neuroscience

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Regioselective Biolistic Targeting in Organotypic Brain Slices Using a Modified Gene Gun

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various heterologous genes could thus be investigated easily while maintaining many aspects of in vivo biology. There has been increasing interest to transfect terminally differentiated neurons for which conventional transfection methods have been fraught with difficulties such as low yields and significant losses in viability. Biolistic transfection can circumvent many of these difficulties yet only recently has this technique been modified so that it is amenable for use in mammalian tissues.
New modifications to the accelerator chamber have enhanced the gene gun&#39;s firing accuracy and increased its depths of penetration while also allowing the use of lower gas pressure (50 psi) without loss of transfection efficiency as well as permitting a focused regioselective spread of the particles to within 3 mm. In addition, this technique is straight forward and faster to perform than tedious microinjections. Both transient and stable expression are possible with nanoparticle bombardment where episomal expression can be detected within 24 hr and the cell survival was shown to be better than, or at least equal to, conventional methods. This technique has however one crucial advantage: it permits the transfection to be localized within a single restrained radius thus enabling the user to anatomically isolate the heterologous gene&#39;s effects. Here we present an in-depth protocol to prepare viable adult organotypic slices and submit them to regioselective transfection using an improved gene gun.

Recent improvements in organotypic brain slice preparations have permitted their exploitation for biotechnological applications. Organotypic slices maintain local structural characteristics of in vivo biology, including functional synaptic connections. Here we present a regioselective biolistic delivery method to label and genetically manipulate these slices.

Transfection of DNA has been invaluable for biological sciences and with recent advances to organotypic brain slice preparations, the effect of various ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

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 ...