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.

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The authors declare no conflicts of interest.

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.

<table><tbody><tr><td>Agarose&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>A9539</td><td></td></tr><tr><td>BD Microlance 19G</td><td>BD</td><td>301500</td><td>Needles used for in- and outlet of bath</td></tr><tr><td>Bioxide gas</td><td>AGA</td><td>105701</td><td></td></tr><tr><td>Brand pipette bulbs</td><td>Sigma-Aldrich</td><td>Z615927</td><td>Pipette bulbs</td></tr><tr><td>Bunsen burner (Liquid propane burner)</td><td>VWR</td><td>89038-530</td><td></td></tr><tr><td>Cable assembly for heater controllers</td><td>Warner Instruments</td><td>64-0106&amp;nbsp;</td><td>Temperature controller - thermometer part</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka</td><td>21100</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>AGA</td><td>100309</td><td>CO&lt;sub&gt;2&lt;/sub&gt; for incubator</td></tr><tr><td>Cover glass, square Corning</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>13206778</td><td>To attach under bath for live imaging. Seal with glue or petrolium jelly.</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>Delicate forceps</td><td>Finescience</td><td>11063-07</td><td>For dissection</td></tr><tr><td>Diamond scriber pen</td><td>Ted Pella Inc.</td><td>54463</td><td></td></tr><tr><td>Disposable Glass Pasteur Pipettes 230 mm</td><td>VWR</td><td>612-1702</td><td>Glass pipettes</td></tr><tr><td>Double edge stainless steel razor blade</td><td>Electron Microscopy Sciences</td><td>#7200</td><td>Razor blade for vibratome</td></tr><tr><td>Earle&#039;s Balanced Salt Solution (EBSS)</td><td>Gibco-Invitrogen</td><td>24010-043</td><td></td></tr><tr><td>Filter paper circles</td><td>Schleicher &amp;amp; Schuell</td><td>300,220</td><td>Filter paper used for filtration of PFA</td></tr><tr><td>Fun tack</td><td>Loctite</td><td>1270884</td><td>Use to connect/adjust position of in- and outlets in bath</td></tr><tr><td>Hand towel C-Fold 2</td><td>Katrin</td><td>344388</td><td></td></tr><tr><td>Harp, Flat for RC-41 Chamber,</td><td>Warner Instruments</td><td>&amp;nbsp;64-1418</td><td>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><td>HEPES, FW: 260.3</td><td>Sigma</td><td>H-7006</td><td></td></tr><tr><td>Holten LaminAir, Model 1.2</td><td>Heto-Holten</td><td>96004000M</td><td>Laminar flow hood</td></tr><tr><td>Horse serum, heat inactivated</td><td>Gibco-Invitrogen</td><td>26050-088</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td></td></tr><tr><td>LCR Membrane, PTFE,&amp;nbsp;</td><td>Millipore</td><td>FHLC0130</td><td>Confetti&amp;nbsp;</td></tr><tr><td>Leica VT1200</td><td>Leica</td><td>14048142065</td><td>Vibratome</td></tr><tr><td>MEM-Glutamax with HEPES</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>42360024</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>R.P. Normapur</td><td>25 108.295</td><td></td></tr><tr><td>Micro Spoon Heyman Type B</td><td>Electron Microscopy Sciences</td><td>62411-B</td><td>Small, rounded spatula with sharpened end for dissection</td></tr><tr><td>Millex-GP filter unit</td><td>Millipore</td><td>SLGPM33RA</td><td>Syringe filter unit</td></tr><tr><td>Millicell cell culture insert, 30 mm</td><td>Millipore</td><td>PICM03050</td><td>Cell culture inserts</td></tr><tr><td>Minipuls 3 Speed Control Module</td><td>GILSON</td><td>F155001</td><td>Peristaltic pump for live imaging - Control module part (connect to two-cannel head)</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S7907</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S8282</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fluka</td><td>71628</td><td></td></tr><tr><td>Nunclon Delta Surface</td><td>Thermo&amp;nbsp; Fischer Scientific</td><td>140675</td><td>Culture plate</td></tr><tr><td>Nystatin Suspension</td><td>Sigma-Aldrich</td><td>N1638</td><td></td></tr><tr><td>Objective W &quot;Plan-Apochromat&quot; 40x/1.0 DIC&amp;nbsp;</td><td>Zeiss</td><td>441452-9900-000&amp;nbsp;</td><td>Water immersion objective used for live imaging. (WD=2.5mm), VIS-IR</td></tr><tr><td>Parafilm</td><td>VWR</td><td>291-1211</td><td></td></tr><tr><td>Paraformaldehyde, granular</td><td>Electron Microscopy Sciences</td><td>#19208</td><td></td></tr><tr><td>PC-R perfusion chamber</td><td>SiSkiYou</td><td>&amp;nbsp;15280000E</td><td>Bath for live imaging</td></tr><tr><td>Penicillin-Streptomycin, liquid</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Petri dish 140 mm&amp;nbsp;</td><td>Heger</td><td>1075</td><td>Large Petri dish&amp;nbsp;</td></tr><tr><td>Petri dish 92x16 mm&amp;nbsp;</td><td>Sarstedt&amp;nbsp;</td><td>82.1473</td><td>Medium Petri dish&amp;nbsp;</td></tr><tr><td>Petridish 55x14,2 mm</td><td>VWR</td><td>391-0868</td><td>Small Petri dish</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>P4417</td><td>PBS tablets</td></tr><tr><td>R2 Two Channel Head&amp;nbsp;</td><td>GILSON</td><td>F117800</td><td>Peristaltic pump for live imaging - Two channel head part (requires control module)</td></tr><tr><td>Round/Flat Spatulas, Stainless Steel</td><td>VWR</td><td>82027-528</td><td>Large spatula for dissection</td></tr><tr><td>Sand paper</td><td>VWR</td><td>MMMA63119</td><td>Optional, for smoothing broken glass pipettes</td></tr><tr><td>Scissors,&amp;nbsp; 17,5 cm</td><td>Finescience</td><td>14130-17</td><td>Large scissors for dissection</td></tr><tr><td>Scissors, 8,5&amp;nbsp;</td><td>Finescience</td><td>14084-08</td><td>Small, sharp scissors for&amp;nbsp; dissection</td></tr><tr><td>Single edge, gem blade</td><td>Electron Microscopy Sciences</td><td>#71972</td><td>Single edge razor blade</td></tr><tr><td>Single inline solution heater SH-27B</td><td>Warner Instruments</td><td>64-0102</td><td>Temperature controller - heater part</td></tr><tr><td>Steritop-GP Filter unit, 500 ml , 45mm</td><td>Millipore</td><td>SCGPT05RE</td><td>Filter to sterilize solutions</td></tr><tr><td>Super glue precision</td><td>Loctite</td><td>1577386</td><td></td></tr><tr><td>Surgical scalpel blade no. 22</td><td>Swann Morton Ltd.</td><td>209</td><td>Rounded scalpel blade</td></tr><tr><td>Temperature controller TC324B</td><td>Warner Instruments</td><td>64-0100</td><td>Temperature controller for live imaging (requires solution heater and cable assembly)</td></tr><tr><td>Trizma base</td><td>Sigma&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Trizma HCl</td><td>Sigma</td><td>T3253</td><td></td></tr><tr><td>Water jacketed incubator series II</td><td>Forma Scientific</td><td>78653-2882</td><td>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...

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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 ...

visualization-live-imaging-oligodendrocyte-organelles-organotypic

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Neuroscience

10.8K Views.  University of Oslo. Myelinating oligodendrocyten bevorderen snelle actiepotentiaal propagatie en neuronale overleving. Hier beschreven is een protocol voor Oligodendrocyt-specifieke expressie van fluorescente proteïnen in organotypic hersenen segmenten met latere time-lapse beeldvorming. Verder is wordt een eenvoudige procedure voor het visualiseren van onbevlekt myeline gepresenteerd.

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

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter lesions in inflammatory demyelinating disorders like multiple sclerosis (MS). In these disorders, mainly CD8+ T-cells of putative specificity for myelin- and oligodendrocyte-related antigens are found, so that neuronal apoptosis in grey matter lesions may be a collateral effect of these cells. Different types of animal models are established to study the underlying mechanisms of the mentioned pathophysiological processes. However, although they mimic some aspects of MS, it is impossible to dissect the exact mechanism and time course of &#8216;&#8216;collateral&#8217;&#8217; neuronal cell death. To address this course, here we show a protocol to study the mechanisms and time response of neuronal damage following an oligodendrocyte-directed CD8+ T cell attack. To target only the myelin sheath and the oligodendrocytes, in vitro activated oligodendrocyte-specific CD8+ T-cells are transferred into acutely isolated brain slices. After a defined incubation period, myelin and neuronal damage can be analysed in different regions of interest. Potential applications and limitations of this model will be discussed.

An Ex vivo Model of an Oligodendrocyte-directed T-Cell Attack in Acute Brain Slices

To address mechanisms of demyelination and neuronal apoptosis in cortical lesions of inflammatory demyelinating disorders, different animal models are used. We here describe an ex vivo approach by using oligodendrocyte-specific CD8+ T-cells on brain slices, resulting in oligodendroglial and neuronal death. Potential applications and limitations of the model are discussed.

Death of oligodendrocytes accompanied by destruction of neurons and axons are typical histopathological findings in cortical and subcortical grey matter ...

Immunology and Infection

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 ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

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 ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Longitudinal In Vivo Imaging of Retinal Cells with Tailored Adeno-Associated Virus Serotypes via Confocal Scanning Laser Ophthalmoscopy

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding and treatment of ocular diseases. This study introduces an optimized adeno-associated virus (AAV) approach, utilizing specific serotypes and promoters to achieve optimal transfection efficiency in targeted retinal cells, including retinal ganglion cells (RGCs) and M&#252;ller glia. Leveraging the precision of confocal scanning laser ophthalmoscopy (CSLO), this work presents a non-invasive method for in vivo imaging that captures the longitudinal expression of AAV-mediated green fluorescent protein (GFP). This approach eliminates the need for terminal procedures, preserving the continuity of observation and the well-being of the subject. Furthermore, the GFP signal can be traced in AAV-infected RGCs along the visual pathway to the superior colliculus (SC) and lateral geniculate nucleus (LGN), enabling the potential for direct visual pathway mapping. These findings provide a detailed protocol and demonstrate the application of this powerful tool for real-time studies of retinal cell behavior, disease pathogenesis, and the efficacy of gene therapy interventions, offering valuable insights into the living retina and its connections.

This protocol describes the use of adeno-associated virus (AAV) vectors for cell-specific labeling and in vivo imaging using a confocal scanning laser ophthalmoscope (CSLO). This method enables the investigation of different retinal cell types and their contributions to retinal function and disease.

The dynamic nature of retinal cellular processes necessitates advancements in gene delivery and live monitoring techniques to enhance the understanding ...

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

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