Name: live imaging of the ependymal cilia in the lateral ventricles of the mouse brain
Uploaded: 2015-06-01T15:00:00.000+00:00
Duration: 6 min 58 s
Description: Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

Authors would like to thank Charisse Montgomery for her editing service. A. Alomran&#8217;s work partially fulfills the requirements for a master&#8217;s degree in Pharmacology.This work is funded by The University of Toledo&#8217;s intramural startup fund for W.A.A and NIH grant (DK080640) for S.M.N.

The procedures for animal use were approved by the Institutional Animal Care and Use Committee (IACUC) of The University of Toledo in accordance with the guidelines of the Institutional Animal Care and Use Committee at the National Institutes of Health and the Guide for the Care and Use of Laboratory Animals.
1. Brain Extraction, Sectioning and Tissue Preparation
<ol>
	<li>Sacrifice wild-type mouse strain C57BL/6 by deeply euthanizing with CO2 asphyxiation for 5 min. Assure death by cervical dislocation.</li>
	<li>Clean the mouse head with 70% ethanol.</li>
	<li>Perform craniectomy using sterile scissors and forceps by first pulling the skin off, starting with the top of the head to expose the skull.</li>
	<li>Then, when the skull is exposed, remove the skull by peeling the bone piece-by-piece, starting from the posterior side and moving toward the anterior side. Be cautious not to destroy the brain ventricles.</li>
	<li>Collect the whole brain.</li>
	<li>Place the brain in a 100 mm Petri dish containing DMEM/High-Glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin solution containing 10,000 units/ml of penicillin and 10,000 &#181;g/ml of streptomycin and pre-warmed to 37 &#176;C.</li>
	<li>Slice the brain on the median sagittal plane by hand with a sharp blade, and obtain the first 100-200 mm section from each half using a vibratome.</li>
	<li>Rinse the brain tissue with pre-warmed 37 &#176;C phosphate buffered saline (1x PBS) solution.</li>
	<li>Immediately place the brain section in DMEM/High-Glucose media pre-warmed to 37 &#176;C.</li>
</ol>
2. Live Imaging Configuration and Setup
<ol>
	<li>Place the brain tissue sections in 30 mm glass-bottom culture dishes containing 1 ml of DMEM/High-Glucose media. Adjust the microscope&#8217;s enclosed chamber environment to 37 &#176;C, 95%/5% O2/CO2 content (Figure 1).</li>
	<li>Using a 60X objective oil immersion lens, collect the ependymal cells/cilia images by first placing an oil drop on the 60X objective lens and focusing on the cells with regular DIC transmitted light.</li>
	<li>Then, follow the direction of the DMEM bubble movement as a guide to the location of the motile ependymal cilia as ciliary beatings create a kind of bubble movement in the area. Choose an area containing healthy cells with motile cilia in the brain&#8217;s lateral ventricle using the DIC filter. Once the ependymal cilia are found, adjust the light and focus to obtain a satisfactory image.</li>
	<li>Set the live imaging parameters according to a specific purpose using Metamorph imaging software. In the present demonstration, acquire twenty four-bit images with the camera binning set to 1 x 1 combined with 60X objective and 5-10 msec exposure time.</li>
	<li>Collect the DIC images by opening the microscope aperture to an optimal level in order to have a minimum exposure time. Observe live images stream to the camera to provide fast and immediate image acquisition without delay. Calculate the speed of cilia beating based on the requirement of the minimal exposure times to obtain sufficient image contrast.</li>
</ol>
3. Data Visualization and Analysis
<ol>
	<li>Calculate the number of cilia beatings by counting the number of beatings in 1 min. Do this by decreasing the speed of the video and counting the number of beats using a cell counter or similar tool.</li>
	<li>To calculate the frequency of beatings, multiply the exposure time at which the video is recorded by the number of the frames or time-lapse images acquired to get the number of sec. (Example: exposure time 5 msec 200 frames = 1,000 msec or 1 sec).</li>
	<li>Calculate the number of beatings in one second to obtain the frequency which is expressed as the number of beating per one sec. Do this by dividing the number of cilia beatings over a one-second time interval (Example: cilia beats 50 times in a 200 frames video recorded at exposure time of 5 msec i.e. 5 msec x 200 frames = 1 sec; now divide 50 beats by 1 sec = 50 Hz).</li>
	<li>Calculate the ciliary beating angle by evaluating the path taken by the ependymal cilia during both the power and recovery strokes. Perform this according to a previously described method, with minor modifications 11. The precise movement of individual cilia is observed during the complete beat cycle.</li>
	<li>On an acetate sheet placed over the monitor, draw a horizontal line along the ependymal edge and a vertical line through the midline position of the cilia at the start of the power stroke.</li>
	<li>Plot the precise position of the cilium frame by frame as it moves forward during the power stroke. In a similar manner, plot the movement of the cilia during the recovery stroke.</li>
	<li>Calculate the ciliary beating angle from the maximum deviation of the cilium from the midline during the power stroke as well as the recovery stroke.</li>
</ol>
4. Calcium Signal Recording
<ol>
	<li>After slicing the brain, briefly rinse the brain slice with 1x PBS or Dulbecco&#8217;s PBS (pH 7.0). Prepare fresh Fluo-2 to avoid fluorescence quenching and to obtain good signal-to-noise ratio.</li>
	<li>Prepare 1 mM stock solution of Fluo-2 solution in dimethylsulfoxide (DMSO), mix and vortex the solution for at least 5 min to ensure that Fluo-2 is homogenously dissolved in DMSO.</li>
	<li>Dilute the Fluo-2 stock solution in 500 ml of DMEM/High-Glucose supplemented with 2% B27 pre-warmed to 37 &#176;C to a final concentration of 20 mg/ml. 
	NOTE: B-27 is an optimized serum-free supplement containing vitamin A,&#160;antioxidant cocktail and&#160;insulin used to support short- or long-term viability of hippocampal and other CNS neurons.</li>
	<li>Immediately incubate the brain slice with 20 mg/ml Fluo-2 for 30 min at 37 &#176;C in a glass-bottom plate.</li>
	<li>To determine the optimal loading concentration of calcium fluorophore Fluo-2 and to avoid calcium dye cell toxicity, challenge cells with ATP, and check the cell viability by determining the time course and peak magnitude of calcium signals in response to ATP.</li>
	<li>Record the video for calcium oscillation at a capture rate of 5 msec for a minimum of 1 sec (200 frames per sec), with excitation and emission wavelengths of 488 nm and 515 nm, respectively (Movie 2).</li>
	<li>To distinguish the Fluo-2 calcium signal from autofluorescence or movement artifacts, ensure that the intensities emitted at 515 nm is monitored separately. 
	NOTE: Fluo-2 is not the calcium fluoremetric suitable to quantify intracellular calcium. However, it is an excellent dynamic calcium dye to detect fast calcium changes, such as calcium oscillations. For more accurate calcium quantification, Fura-2 is recommended. However, the dynamic changes of Fura-2 are limited by its ratiometric nature of the dye.</li>
	<li>Follow the formulas provided by the manufacturer to calculate the exact free intracellular calcium values. Example [Ca2+] = Kd &#215; (R&#8211;Rmin)/(Rmax &#8211; R). Where Kd is the dissociation constant of the dye from the released calcium, R is the measured fluorescence at 488, and Rmin and Rmax are the fluorescence ratios at minimum and maximum ion concentration 12.</li>
</ol>
5. Immunofluorescence Microscopy
<ol>
	<li>Fix the brain sections with phosphate buffered saline solution containing 4% paraformaldehyde (PFA) and 2% sucrose for 10 min. Alternatively, fix the whole brain with 4% PFA and then section into 50 mm sections using a cryostat.</li>
	<li>Wash the tissue three times with 1x PBS for 5 min each time.</li>
	<li>Incubate the brain slice with a solution of 0.1% Triton-X in 1x PBS for 5 min, then rinse three times with 1x PBS for 5 min each time.</li>
	<li>Incubate the brain slice with mouse primary antibody, antiacetylated a-tubulin, used at a dilution of 1:5,000 in 10% FBS in 1x PBS for one hour at room temperature (RT) or overnight at 4 &#176;C.</li>
	<li>Wash the tissue three times with 1x PBS for 5 min each time.</li>
	<li>Incubate the brain slice in secondary antibody, fluorescein anti-mouse IgG at a dilution of 1:500 in 10% FBS in 1x PBS solution for 1 hr at RT.</li>
	<li>Before observation under a fluorescent microscope, counterstain the section with 4&#39;,6-diamidino-2-phenylindole (DAPI) for 5 min to stain the nucleus (or DNA) 13. To minimize photobleaching, image the sections immediately with minimum exposure time possible.</li>
</ol>

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No conflicts of interest declared.

Described here is a protocol for the preparation of mouse brain tissue for both live-imaging and fluorescence microscopy that provides a rapid and sensitive close observation of the ependymal cilia within the brain ventricles. This technique is not restricted to the lateral ventricle only; it could be utilized to observe the cilia in other brain ventricles. This imaging technique provides a live stream that resembles the movement of the CSF by ciliary beating in an ex vivo setting. Moreover, it enables analysis ...

Cilia are sensory microtubule-based organelles that extend from the cell surface to the extracellular environment. Depending on their microtubule organization, cilia can be categorized into two types - &#8220;9+0&#8221; or &#8220;9+2&#8221;. Functionally, based on their motility, these can be classed as motile or non-motile cilia 1. Primary cilia is a term commonly used to denote &#8220;9+0&#8221; non-motile cilia. These have nine parallel doublet microtubules (denoted by &#8216;9&#8217;) and a central pair of microtubules is absent within the central sheath (denoted by &#8216;0&#8217;). However, some &#8220;9+0&#8221; cilia, such as nodal cilia, which regulate embryo laterality are motile 2. On the other hand, motile cilia are characterized, in addition to the nine parallel microtubule doublets, by an additional central pair of microtubule doublets and associated with dynein motor proteins to facilitate motility. In addition, some &#8220;9+2&#8221; cilia such as olfactory cilia are non-motile 3. Ependymal cells lining the brain ventricles and the central canal of the spinal cord are characterized by motile cilia that propel the cerebrospinal fluid (CSF) along the brain ventricles 4.
The overall goal of this method is to facilitate studying the motile cilia dynamics and structural abnormalities. The brain&#8217;s health and development heavily depend on efficient circulation of CSF within the brain ventricles. For instance, normal CSF flow and fluid balance require normal beating and functional ependymal cilia 5,6, which in turn play critical roles in regulating the directional movement of neuronal cells and stem cell migration 7. As such, abnormal ependymal cilia function or structure can lead to abnormal CSF flow, which is associated with hydrocephalus, a medical condition in which there is an abnormal accumulation of CSF in the ventricles of the brain. This may consequently cause increased intracranial pressure and progressive enlargement of the head, convulsion, tunnel vision, and mental disability 8.
The advantages of this technique over existing methods is that it allowed for the first time to report three distinct ependymal cell types: I, II, and III, based on their unique ciliary beating frequency and beating angle. These ependymal cells are localized within certain regions in the brain ventricles. Furthermore, the effects of age and pharmacological agents such as alcohol and cilostazol on altering the ependymal cell types or their localizations can be demonstrated, which was not possible before this classification of ependymal cells. Cilostazol is an inhibitor of phosphodiesterase-3, an enzyme that metabolizes cAMP to AMP and it also regulates intracellular calcium 9. Using high-speed fluorescence imaging analysis allows imaging and quantifying of the unique intracellular calcium oscillation properties of the ependymal cells. For instance, both alcohol and cilostazol significantly altered the ependymal ciliary beating frequency as well as the intracellular calcium oscillations properties, which in turn, could lead to a change in the cerebrospinal fluid volume replacement by ependymal cilia 10. In summary, this technique was key to provide the first evidence of three distinct types of ependymal cells with different calcium oscillation properties.
In the following section, a detailed step-by-step overview of the procedure is provided, paying close attention to tissue preparation and handling.

<table><tbody><tr><td>DMEM/HIGH GLUCOSE</td><td>Cellgro Mediatech Inc.</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30088-03</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo Scientific</td><td>SV30010</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Scientific</td><td>SH30256-01</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710-SP</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S-2395</td><td></td></tr><tr><td>Triton-X</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Fluo-2&amp;nbsp;</td><td>TEF Labs</td><td>#0200</td><td></td></tr><tr><td>DMSO</td><td></td><td></td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>17504044</td><td></td></tr><tr><td>VECTASHIELD HardSet Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1500</td><td></td></tr><tr><td>Anti-acetylated a-tubulin antibody</td><td>Sigma-Aldrich</td><td>T7451&amp;nbsp;</td><td>clone 6-11B1</td></tr><tr><td>FITC Anti-mouse antibody</td><td>Vector Labs</td><td>FI-2000</td><td></td></tr><tr><td>Cell Culture plate</td><td>VWR Vista Vision</td><td>30-2041</td><td></td></tr><tr><td>Cover Slip (18 x 18)</td><td>VWR Vista Vision</td><td>16004.326</td><td></td></tr><tr><td>Vibratome</td><td>Leica Biosystems</td><td>Leica VT1200S</td><td></td></tr><tr><td>Cryostat</td><td>Leica Biosystems</td><td>Leica CM1860</td><td></td></tr><tr><td>Inverted Fluorescence Microscope</td><td>Nikon&amp;nbsp;</td><td>Nikon TE2000</td><td>60X oil&amp;nbsp;</td></tr><tr><td>Microscope cover glass 24 x 60 mm&lt;sup&gt;2&lt;/sup&gt;</td><td>VWR Vista Vision</td><td>16004-312</td><td></td></tr><tr><td>Mounting Medium with DAPI</td><td>Vector Laboratories</td><td>H-1500</td><td></td></tr><tr><td>DAPI filter cube</td><td>Chroma Technology</td><td></td><td></td></tr></tbody></table>

live imaging of the ependymal cilia in the lateral ventricles of the mouse brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Measuring ependymal cilia function in live mouse brain
The method described in this protocol is used to monitor ependymal cilia function and structure in the fresh tissue dissected from the mouse brain as well as to monitor and study cilia beating frequency. The steps followed to accomplish a complete experiment are depicted in a schematic flowchart (Figure 1). It is highly recommended that the experiment is conducted within a short time frame in order to keep the motile cilia...

Watch this Scientific Journal Video about Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain at JoVE.com

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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Neuroscience

13.7K Views.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Video: Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.

Using MRI scans (human), 3D imaging software, and immunohistological analysis, we document changes to the brain&#8217;s lateral ventricles. Longitudinal 3D mapping of lateral ventricle volume changes and characterization of periventricular cellular changes that occur in the human brain due to aging or disease are then modeled in mice.

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

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 ...

Visualizing Neuronal and Glial Cell Dynamics in the Developing Mouse Brain

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. This process is crucial for proper brain function, and its dysregulation can result in neurodevelopmental and psychiatric disorders after birth. In fact, many genes responsible for these diseases have been found to be involved in this process, and therefore, revealing how these mutations affect cellular dynamics is important for understanding the pathogenesis of these diseases. This protocol introduces a technique for time-lapse imaging of migrating neurons and glial progenitors in brain slices obtained from mouse embryos. Cells are labeled with fluorescent proteins using in utero electroporation, which visualizes individual cells migrating from the ventricular zone with a high signal-to-noise ratio. Moreover, this in vivo gene transfer system enables us to easily perform gain-of-function or loss-of-function experiments on the given genes by co-electroporation of their expression or knockdown/knockout vectors. Using this protocol, the migratory behavior and migration speed of individual cells, information that is never obtained from fixed brains, can be analyzed.

Author Spotlight: Exploring Cell Migration and Gene Roles in the Developing Brain

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain surface. Many genes are involved in this process. This protocol introduces the technique for the time-lapse imaging of migrating neurons and glial progenitors.

During the development of the cerebral cortex, neurons and glial cells originate in the ventricular zone lining the ventricle and migrate toward the brain ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

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Chapters in this video

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Time-Lapse Imaging of Migrating Neurons and Glial Progenitors in Embryonic Mouse Brain Slices