1. Dish Preparation
<ol>
 <li>Dishes used for slice culture are glass-bottomed (with a coverslip as the base) (WillCo dishes). These are placed on lens tissue in a 6 cm tissue culture dish to keep the glass bottom clean.</li>
 <li>On the day before the experiment, add 2 ml 0.1% poly-L-lysine solution to the glass bottomed dish and incubate for 5min at room temperature to allow the poly-L-lysine to coat the glass bottom.</li>
 <li>Remove poly-L-lysine solution and rinse three times in deionised water, and then once in 70% ethanol.</li>
 <li>Leave to dry overnight. Dishes can also be dried by gently heating in a microwave at low power for 30-45 seconds.</li>
</ol>
2. Embryo Electroporation
<ol>
 <li>Incubate eggs at 37 &deg;C to Hamburger-Hamilton (HH) stage 10 (~36 hours) (or other desired stage).</li>
 <li>Before beginning prepare glass needles (we use a Flaming/Brown model p87 microcapillary puller) and break off the tip of the needle using a fine forceps under a dissecting microscope. The end of the needle should be sharp enough to pierce the embryo while not being so narrow that it impedes injection of the DNA solution.</li>
 <li>Window eggs and place electrodes (5mm apart) on either side of the embryo.</li>
 <li>Inject DNA (~0.025 - 0.5 &mu;g/&mu;l in deionised water colored with a small amount of fast green) into the neural tube.</li>
 <li>Apply current - 12-17 V three times, 50ms pulse length with 950 ms between pulses.</li>
 <li>We use low concentrations of DNA and low electroporation voltages to achieve mosaic expression so that we can follow individual cells.</li>
 <li>Cover window in the eggshell with cellotape and make sure it is sealed.</li>
 <li>Allow embryos to recover for 3-4 hours or overnight at 37 &deg;C.</li>
</ol>
3. Collagen and Slice Culture Medium Preparation
<ol>
 <li>Prepare collagen mix and slice culture medium about an hour before slicing.</li>
 <li>To 300 &mu;l type 1 collagen add 100 &mu;l 0.1% acetic acid solution and 100 &mu;l 5 x L15 medium. Vortex thoroughly after each addition. The solution should turn yellow.</li>
 <li>Now add 15-20 &mu;l 7.5% sodium bicarbonate and thoroughly vortex. The solution should become slightly pink, and the volume of sodium bicarbonate required for this may differ. Keep on ice and make up fresh each time.</li>
 <li>To 10 ml neurobasal medium add B-27 supplement and Glutamax, to a 1 x final concentration and 10 &mu;l Gentamicin solution.</li>
 <li>Place medium in a 37 &deg;C incubator buffered with 5% CO2. Leave the top of the container loose to allow it to equilibrate with the CO2.</li>
</ol>
4. Slice Culture
<ol>
 <li>Remove embryos from egg and wash in L15 medium.</li>
 <li>Place in a tissue culture dish with a layer of sylgard at the bottom and pin them out through the surrounding extra-embryonic membranes so that they are stretched taut.</li>
 <li>Using a microknife, slice embryo as straight as possible through region of interest. For spinal cord, slices should be between 1-2 somites thick. Leave slices attached to embryo while you slice other embryos so you don't lose them.</li>
 <li>Prepare a 200 &mu;l micropipette tip by cutting off ~1 mm of the tip and attaching this to a p2 or p10. This tip will be used to transfer the spinal cord slices to the glass bottom dish. A 10 &mu;l tip is not broad enough to pick up slices.</li>
 <li>Coat the inside of the tip with collagen by pipetting 1 &mu;l collagen mix prepared earlier. Leave for 1-2 mins and then rinse with L15 medium. This will prevent the tissue from sticking to the inside of the tip.</li>
 <li>Detach slice from embryo using a microknife and remove from the dish using a p2 or p10 fitted with a 200 &mu;l tip and set to 1 &mu;l. Try to take up as little medium as possible.</li>
 <li>Vortex the collagen mix and drop 5-8 &mu;l of this on to the poly-l-lysine coated dish.</li>
 <li>Immediately put embryo slice into collagen and position into place with a pair of fine forceps. Slices should be positioned so that the side to be imaged is flush with the coverslip. The tissue should adhere to the poly-l-lysine coating on the coverslip.</li>
 <li>Repeat this until you have several slices on the coverslip. We can usually place 6-9 slices on one dish.</li>
 <li>Once all the slices are in place, cover the dish and allow the collagen to set for 20 minutes. Some of the earliest placed collagen may already have started to dry out. To prevent this add a small amount of L15 to these before covering.</li>
 <li>Once set, carefully add 2ml slice culture medium that has been equilibrated in 5% CO2 at 37 &deg;C for at least an hour. Care must be taken to not dislodge the collagen from the coverslip.</li>
 <li>Place in a 37 &deg;/ 5% CO2 incubator and allow slices to recover for at least 3 hours before imaging. If there is no humidity, place dish in a perspex box with a bit of damp tissue paper in one corner.</li>
</ol>
5. Imaging Embryo Slices
<ol>
 <li>We use a DeltaVision Core wide-field microscope fitted with a WeatherStation environmental chamber to image slices. The chamber is constantly maintained at 37 &deg;C, with a CO2 perfusion apparatus to maintain the microscope stage at 5% CO2 / 95% air.</li>
 <li>Imaging is normally carried out using a 40 x / 1.30 NA oil immersion lens and images are captured with a CoolSnap HQ2 cooled CCD camera.</li>
 <li>Z-sections are captured every 1.5 &mu;m through 45 &mu;m tissue. Exposure time should be kept as low as possible. We normally expose for 5-50 ms for each Z-section. Images are captured every 7 minutes and up to 9 slices can be visited using the DeltaVision system's precise point visiting function.</li>
</ol>
6. Representative Results
An example time-lapse sequence of a spinal cord progenitor cell is shown in Fig. 2a and corresponding Movie 1. Imaging was started on a spinal cord slice from a two-day-old (HH stage 12) embryo. This cell was transfected with a construct expressing GFP-&alpha;Tubulin. During this early stage, neural progenitor cells undergo predominantly progenitor-progenitor mode divisions during which the cells divide to generate two further cycling progenitor cells. Fig. 2b and corresponding Movie 2 shows a cell transfected with GFP-GPI (GPI anchored GFP), marking the cell membrane. This cell undergoes a division during which the basal process splits into two and is equally inherited by the daughter cells.
<img alt="Figure 1" src="/files/ftp_upload/3920/3920fig1.jpg" /> 
Figure 1. Slice culture protocol for time-lapse imaging. 
<img alt="Figure 2" src="/files/ftp_upload/3920/3920fig2.jpg" /> 
Figure 2. Cell behavior during normal spinal cord development. (a) Selected frames from Movie 1. Cell expressing GFP-tubulin divides with a cleavage plane that is perpendicular to the apical surface (2 h 20 min), generating two daughter cells that divide once again (24 h 23 min and 25 h 54 min). (b) Selected frames from Movie 2. Cell transfected with GFP-GPI undergoes cell division during which the basal process is split (white arrows) and equally inherited by the daughter cells.
Movie 1. <a href="https://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">Click here to view movie</a>.
Movie 2. <a href="https://www.jove.com/files/ftp_upload/3920/3920movie1.mov" target="_blank">Click here to view movie</a>.

<ol>
	<li>Ulloa, F., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogens+and+the+Control+of+Cell+Proliferation+and+Patterning+in+the+Spinal+Cord.">Morphogens and the Control of Cell Proliferation and Patterning in the Spinal Cord.</a> Cell Cycle. 6, 2640-2649 (2007).</li><li>Briscoe, J., Novitch, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+pathways+linking+progenitor+patterning,+cell+fates+and+neurogenesis+in+the+ventral+neural+tube.">Regulatory pathways linking progenitor patterning, cell fates and neurogenesis in the ventral neural tube.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 363, 57-70 (2008).</li><li>Shiau, C., Das, R., Storey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+effective+assay+for+high+cellular+resolution+time-lapse+imaging+of+sensory+placode+formation+and+morphogenesis.">An effective assay for high cellular resolution time-lapse imaging of sensory placode formation and morphogenesis.</a> BMC Neuroscience. 12, 37 (2011).</li><li>Wilcock, A. C., Swedlow, J. R., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+spindle+orientation+distinguishes+stem+cell+and+terminal+modes+of+neuron+production+in+the+early+spinal+cord.">Mitotic spindle orientation distinguishes stem cell and terminal modes of neuron production in the early spinal cord.</a> Development. 134, 1943-1954 (2007).</li><li>Vilas-Boas, F., Fior, R., Swedlow, J. R., Storey, K. G., Henrique, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+reporter+of+notch+signalling+indicates+regulated+and+random+notch+activation+during+Vertebrate+neurogenesis.">A novel reporter of notch signalling indicates regulated and random notch activation during Vertebrate neurogenesis.</a> BMC Biology. 9, 58 (2011).</li><li>Herman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy.">Fluorescence microscopy.</a> Bios Scientific Publishers. , (1998).</li><li>Conchello, J. -. A., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+microscopy.">Optical sectioning microscopy.</a> Nat. Meth. 2, 920-931 (2005).</li><li>Pawley, J. B., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fundamental Limits in Confocal Microscopy Handbook Of Biological Confocal. , (2006).</li><li>Swedlow, J. R., Hu, K., Andrews, P. D., Roos, D. S., Murray, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+tubulin+content+in+Toxoplasma+gondii:+A+comparison+of+laser-scanning+confocal+and+wide-field+fluorescence+microscopy.">Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy.</a> Proceedings of the National Academy of Sciences. 99, 2014-2019 (2002).</li><li>Murray, J. M., Appleton, P. L., Swedlow, J. R., Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+performance+in+three-dimensional+fluorescence+microscopy.">Evaluating performance in three-dimensional fluorescence microscopy.</a> Journal of Microscopy. 228, 390-405 (2007).</li><li>Biggs, D. S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Deconvolution+Microscopy.">3D Deconvolution Microscopy.</a> Current Protocols in Cytometry. , (2001).</li><li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W., Prasher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263, 802-805 (1994).</li><li>Wiedenmann, J., Oswald, F., Nienhaus, G. 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No conflicts of interest declared.

We present here a novel time-lapse imaging assay to monitor cell behavior in chicken embryonic slice culture. This assay enables high resolution imaging of living tissue for up to 70 hours, although time frames between 24-48 hours are easier to capture. The use of a high NA oil objective and relatively short intervals between time-points enables image acquisition at high resolution, as well as allowing us to monitor cell behavior that often occurs rapidly, and can be easily missed during conventional time-lapse imaging using confocal microscopy.
The major advantage of this assay is the ability to image cells over long periods of time. The use of wide-field6 instead of confocal laser scanning7 microscopy is critical for this. Confocal microscopy has traditionally offered several advantages over wide-field microscopy, including the ability to take optical sections and eliminate out of focus information directly7, but the use of a pinhole leads to a loss of light to the detector8, excluding a significant amount of information, and necessitating longer exposure times. Wide-field microscopy, on the other hand, uses full field illumination and all the light passing through the objective is sent to the detector. This coupled with a high quantum efficiency cooled coupled device (CCD) camera ensures imaging with a high signal to noise ratio compared to confocal microscopy9, with very fast exposure times. In our application, we must image for long periods, record 3D stacks and ultimately resolve small&#x2014;near-diffraction limited structures. Although confocal microscopy prevents out-of-focus light from ever reaching the detector and thus significantly reduces background in thick, densely-labeled samples7, 8, it only achieves a usable signal-to-noise ratio with much larger light input than wide-field microscopy10. For very light-sensitive sparsely-labeled samples like our electroporated spinal cord slices, therefore, wide-field microscopy performs better than confocal microscopy. When combined with image restoration by deconvolution, which removes out of focus information and improves contrast, wide-field is then particularly good for detection of small, dim objects11. While not appropriate for every tissue imaging experiment, this approach has been very effective for our live cell imaging application.
The choice of fluorescent protein is an important factor that can influence cell survival. We find that the best results are obtained from constructs that use green fluorescent protein (GFP)12 as a marker. We are currently assessing a range of red fluorescent proteins that can be effectively used in combination with GFP for dual channel time-lapses. There are a variety of such proteins available13. Although many proteins are stable as fusions with a fluorescent protein, some proteins may be rendered unstable by fusion, resulting in reduced cell viability. In such cases, the use of constructs containing an internal ribosome entry site (IRES) is useful to separate the protein of interest from the fluorescent protein. 
When imaging spinal cord slices, care must be taken to minimize exposure to fluorescent light. The microscope eyepieces must be used only with transmitted light (brightfield) to locate and position slices. Fluorescent images should always be acquired using the microscope software using minimal exposure times. These should be kept in the range of 5-50 ms and the use of neutral density filters should be experimented with. Although longer exposure times may initially produce clearer images, the phototoxic effects of fluorescent light exposure may lead to cell death. The first 5-10 &mu;m of tissue that is closest to the coverslip should not be imaged as this region contains tissue that may have been damaged during slicing. 
Although the examples presented here are of embryos electroporated at HH stage 10 and imaged 6-7 hours later, this method can be used for embryos up to HH stage 18. At later stages, the spinal cord tissue is much larger and so is difficult to slice by hand. In these cases, embedding the embryos in low melting point agarose and sectioning with a vibratome may yield better results. Even though we present this assay as a method for imaging cells in the developing spinal cord, this approach has also been modified to image other embryonic tissues, including the sensory placodes3.

<table border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>WillCo Dish</td>
 <td>WillCo Wells</td>
 <td>GWst-3522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>poly-l-lysine</td>
 <td>Sigma</td>
 <td>P8970</td>
 <td>0.1% in Tris</td>
 </tr>
 <tr>
 <td>Borosilicate glass capillaries</td>
 <td>Harvard Instruments</td>
 <td>GC100-10</td>
 <td>1.0mm outer diameter, 0.58mm inner diameter</td>
 </tr>
 <tr>
 <td>Electrodes (Genetrodes, in ovo, L-shaped</td>
 <td>BTX</td>
 <td>10-001850-01</td>
 <td>5mm gold plated</td>
 </tr>
 <tr>
 <td>ECM 830 square pulse generator</td>
 <td>BTX</td>
 <td>45-0002</td>
 <td>for in ovo electroopration</td>
 </tr>
 <tr>
 <td>Fast Green</td>
 <td>Sigma</td>
 <td>F7258</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Type I rat collagen</td>
 <td>Sigma</td>
 <td>C3857</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-15 medium (powder)</td>
 <td>Sigma</td>
 <td>&nbsp;</td>
 <td>2.76 g/L in distilled water for 5x solution</td>
 </tr>
 <tr>
 <td>Sodium bicarbonate</td>
 <td>Sigma</td>
 <td>S8761</td>
 <td>7.5% solution</td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen</td>
 <td>12348-017</td>
 <td>without phenol red</td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>A1286001</td>
 <td>100x solution</td>
 </tr>
 <tr>
 <td>B-27 supplement</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gentamicin</td>
 <td>Invitrogen</td>
 <td>15710049</td>
 <td>10 mg/ml</td>
 </tr>
 <tr>
 <td>L15 medium (liquid 1x)</td>
 <td>Invitrogen</td>
 <td>11415-049</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microknife</td>
 <td>Altomed</td>
 <td>A10102</td>
 <td>15 degree incline</td>
 </tr>
 <tr>
 <td>Stainless steel headless pins</td>
 <td>Watkins and Doncaster</td>
 <td>E6871</td>
 <td>A1 size</td>
 </tr>
 <tr>
 <td>Sylgard 184 elastomer</td>
 <td>VWR</td>
 <td>634165S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DeltaVision Core microscope system</td>
 <td>Applied Precision</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Coolsnap HQ2 CCD camera</td>
 <td>Photometrics</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

high-resolution live imaging of cell behavior in the developing neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Watch this Scientific Journal Video about High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium at JoVE.com

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

high-resolution-live-imaging-cell-behavior-developing

Research

JoVE Journal

Neuroscience

13.3K 次观看.  University of Dundee, Dundee, UK. 实时成像胚胎组织挑战过很长一段时间。在这里，我们提出了一个小鸡脊髓细胞和亚细胞的变化，具有高时空分辨率的长期监测分析。这种技术可以适应其他地区的神经系统和胚胎发育。

视频: High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

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 ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...