This work was funded in part by a grant to D.S.G. from the National Cancer Institute (R03CA227312) and by a generous grant from the Lisa Dean Moseley Foundation. Live GBM specimens were obtained with patient consent through the Tissue Procurement Center of the Helen F. Graham Cancer Center and Research Institute. Funding to A.R. was provided by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health (UL1TR003107). Summer undergraduate research fellowships to N.P., A.L., Z.W., and K.S. were provided by the University of Delaware Undergraduate Research Program.

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+L1CAM+stimulates+glioblastoma+cell+motility,+proliferation,+and+invasiveness.">Exosomal L1CAM stimulates glioblastoma cell motility, proliferation, and invasiveness.</a> International Journal of Molecular Sciences. 20 (16), 3982 (2019).</li><li>Stoppini, L., Buchs, P. A., Muller, 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+simple+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> Journal of Neuroscience Methods. 37 (2), 173-182 (1991).</li><li>Ohnishi, T., Matsumura, H., Izumoto, S., Hiraga, S., Hayakawa, T. <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+model+of+glioma+cell+invasion+using+organotypic+brain+slice+culture.">A novel model of glioma cell invasion using organotypic brain slice culture.</a> Cancer Research. 58 (14), 2935-2940 (1998).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Aaberg-Jessen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+primary+glioma-+and+cell+line-derived+spheroids+implanted+into+corticostriatal+slice+cultures.">Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures.</a> International Journal of Clinical and Experimental Pathology. 6 (4), 546-560 (2013).</li><li>Matsumura, H., Ohnishi, T., Kanemura, Y., Maruno, M., Yoshimine, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+glioma+cell+invasion+by+confocal+laser+scanning+microscopy+in+a+novel+brain+slice+model.">Quantitative analysis of glioma cell invasion by confocal laser scanning microscopy in a novel brain slice model.</a> Biochemical and Biophysical Research Communications. 269 (2), 513-520 (2000).</li><li>Ren, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+and+anti-invasion+research+of+glioma+cells+in+an+improved+model+of+organotypic+brain+slice+culture.">Invasion and anti-invasion research of glioma cells in an improved model of organotypic brain slice culture.</a> Tumori. 101 (4), 390-397 (2015).</li><li>Fayzullin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+phenotyping+of+invasive+glioma+cells+ex+vivo+reveals+subtype-specific+movement+patterns+guided+by+tumor+core+signaling.">Time-lapse phenotyping of invasive glioma cells ex vivo reveals subtype-specific movement patterns guided by tumor core signaling.</a> Experimental Cell Research. 349 (2), 199-213 (2016).</li><li>Jensen, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+tumor+stem+cell-based+glioblastoma+invasion+model.">Establishment and characterization of a tumor stem cell-based glioblastoma invasion model.</a> PloS One. 11 (7), e0158746 (2016).</li><li>Marques-Torrejon, M. A., Gangoso, E., Pollard, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+glioblastoma+tumour-host+cell+interactions+using+adult+brain+organotypic+slice+co-culture.">Modelling glioblastoma tumour-host cell interactions using adult brain organotypic slice co-culture.</a> Disease Models &amp; Mechanisms. 11 (2), 031435 (2018).</li><li>Tamura, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+spatiotemporal+dynamics+of+human+glioma+stem+cell+invasion.">Visualization of spatiotemporal dynamics of human glioma stem cell invasion.</a> Molecular Brain. 12 (1), 45 (2019).</li><li>Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+based+on+in+ovo+electroporation+for+chicken+embryonic+central+nervous+system.">Organotypic slice culture based on in ovo electroporation for chicken embryonic central nervous system.</a> Journal of Cellular and Molecular Medicine. 23 (3), 1813-1826 (2019).</li><li>Murrell, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expansion+of+multipotent+stem+cells+from+the+adult+human+brain.">Expansion of multipotent stem cells from the adult human brain.</a> PloS One. 8 (8), e71334 (2013).</li><li>Fotos, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+time-lapse+microscopy+and+high-resolution+tracking+of+cell+migration.">Automated time-lapse microscopy and high-resolution tracking of cell migration.</a> Cytotechnology. 51 (1), 7-19 (2006).</li><li>Tufan, A. C., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanatomy. 3 (1), 8-11 (2004).</li><li>Shoin, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryo+assay+as+chemosensitivity+test+for+malignant+glioma.">Chick embryo assay as chemosensitivity test for malignant glioma.</a> Japanese Journal of Cancer Research. 82 (10), 1165-1170 (1991).</li><li>Hagedorn, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proceedings of the National Academy of Sciences. 102 (5), 1643-1648 (2005).</li><li>Balci&#363;niene, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology+of+human+glioblastoma+transplanted+on+chicken+chorioallantoic+membrane.">Histology of human glioblastoma transplanted on chicken chorioallantoic membrane.</a> Medicina. 45 (2), 123-131 (2009).</li><li>De Magalh&#227;es, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+a+new+In+vivo+tumor+spheroid+based+shell-less+chorioallantoic+membrane+3-D+model+in+bioengineering+research.">Applications of a new In vivo tumor spheroid based shell-less chorioallantoic membrane 3-D model in bioengineering research.</a> Journal of Biomedical Science and Engineering. 3 (1), 20-26 (2010).</li><li>Szmidt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+human+glioblastoma+model+cultured+in+ovo.">Morphology of human glioblastoma model cultured in ovo.</a> Journal of Veterinary Research. 56 (2), 261-266 (2012).</li><li>Jaworski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+tumor+morphology+and+structure+from+U87+and+U118+glioma+cells+cultured+on+chicken+embryo+chorioallantoic+membrane.">Comparison of tumor morphology and structure from U87 and U118 glioma cells cultured on chicken embryo chorioallantoic membrane.</a> Journal of Veterinary Research. 57 (4), 593-598 (2013).</li><li>Yuan, Y. J., Xu, K., Wu, W., Luo, Q., Yu, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+chick+embryo+chorioallantoic+membrane+in+neurosurgery+disease.">Application of the chick embryo chorioallantoic membrane in neurosurgery disease.</a> International Journal of Medical Sciences. 11 (12), 1275-1281 (2014).</li><li>Urba&#324;ska, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+silver+nanoparticles+(AgNPs)+on+proliferation+and+apoptosis+of+in+ovo+cultured+glioblastoma+multiforme+(GBM)+cells.">The effect of silver nanoparticles (AgNPs) on proliferation and apoptosis of in ovo cultured glioblastoma multiforme (GBM) cells.</a> Nanoscale Research Letters. 10, 98 (2015).</li><li>DeBord, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chick+chorioallantoic+membrane+(CAM)+as+a+versatile+patient-derived+xenograft+(PDX)+platform+for+precision+medicine+and+preclinical+research.">The chick chorioallantoic membrane (CAM) as a versatile patient-derived xenograft (PDX) platform for precision medicine and preclinical research.</a> American Journal of Cancer Research. 8 (8), 1642-1660 (2018).</li><li>Han, J. M., Jung, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+anticancer+effect+of+a+combination+of+berbamine+and+arcyriaflavin+A+against+glioblastoma+stem-like+cells.">Synergistic anticancer effect of a combination of berbamine and arcyriaflavin A against glioblastoma stem-like cells.</a> Molecules. 27 (22), 7968 (2022).</li><li>Ruiz-Onta&#241;on, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+plasticity+confers+migratory+and+invasive+advantages+to+a+population+of+glioblastoma-initiating+cells+that+infiltrate+peritumoral+tissue.">Cellular plasticity confers migratory and invasive advantages to a population of glioblastoma-initiating cells that infiltrate peritumoral tissue.</a> Stem Cells. 31 (6), 1075-1085 (2013).</li><li>Cage, T. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+patterns+of+human+medulloblastoma+dissemination+in+the+developing+chick+embryo+nervous+system.">Distinct patterns of human medulloblastoma dissemination in the developing chick embryo nervous system.</a> Clinical &amp; Experimental Metastasis. 29 (4), 371-380 (2012).</li><li>Darrigues, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobanked+glioblastoma+patient-derived+organoids+as+a+precision+medicine+model+to+study+inhibition+of+invasion.">Biobanked glioblastoma patient-derived organoids as a precision medicine model to study inhibition of invasion.</a> International Journal of Molecular Sciences. 22 (19), 10720 (2021).</li></ol>

None of the authors have any conflicts of interest.

Critical steps in the protocol for the injection of cells into the midbrain (optic tectum) ventricle include not damaging the blood vessels in the chorioallantoic membrane in the egg or surrounding the embryo before and during injection, although the amnion membrane immediately surrounding the embryo can be gently pulled and held to position the head when injecting the cells into the midbrain. The amnion is relatively tough and can be pulled with fine forceps to position the head and hold it steady with one hand, for the injection of cells with the other hand into the optic tectum, which is the large, round structure in the middle of the brain. Generally, the viability of injected embryos ranges from 25% to 75%, depending upon unknown factors, and practically every embryo that survives contains at least a small tumor in the optic tectum. Critical steps in generating viable brain slices include blotting the tissue of excess liquid so that the agarose adheres to the brain during slicing and to keep tissue and slices cold until placed on the membrane insert. As different cell types form spheroids differently (in speed and size), the plated cell density on poly-HEMA plates and the length of time before harvesting spheroids should be optimized for each cell type.
The work here has not been subject to a formal longitudinal study of brain slice viability. Yang et al. used chick embryo brain slice cultures similar to the ones used here and showed good viability of the slices for at least 7 days16. Previous work showed that when OT tissue was kept in suboptimal media, many pyknotic nuclei appeared in the tissue, which did not occur in the slices in the work here. Additionally, when slices degenerate in suboptimal conditions, the blood vessels fragment and appear as rows of laminin-positive spheres (not shown). Thus, although the viability here has not been checked by methods such as electrophysiology or active caspase-3 expression, none of the indicators of cell death that were seen under suboptimal culture conditions appeared here.
The OT has been focused on for in vivo brain tumor experiments because it is the most easily injected region with the largest ventricle. At E5, which is the latest day that the embryo is small enough to remain accessible on top of the yolk, injections must be made into a ventricle, as all brain regions are nothing more than a thin ventricular zone. Nevertheless, these injections result successfully in embedded tumors with cells that invade the brain parenchyma. Sometimes, resulting tumors are found in the forebrain or cerebellum, but this is not common. Ex vivo slices of E15 optic tectum have been primarily used for experiments here, so that the ex vivo co-culture results can be correlated with the in vivo injection experiments. However, forebrain slices are also suitable and have a larger surface area and a very thin ventricle compared to the optic tectum, which might make the forebrain more suitable for ex vivo co-cultures that are not being correlated with in vivo injections.
It has been demonstrated here that in vivo injections, followed by tissue fixation, vibrating tissue slicer sectioning, and immunostaining for laminin and other markers, resulted in high-resolution images of GBM cells and GSCs in brain tissue in close proximity to blood vessels. The ability to determine the interrelationships between tumor cells and blood vessels was greatly facilitated by creating 3D volume renders from z-stacks of confocal optical sections using the confocal software and manufacturer&#39;s instructions. Time-lapse imaging using widefield fluorescence microscopy of GFP, mCherry, and DiD labeled cells was possible; however, migrating cells that were in close proximity to the highly fluorescent spheroids were sometimes obscured by the &#34;glow&#34; from the spheroid. This undesired effect can be somewhat minimized by carefully adjusting the exposure times for collecting widefield images. Time-lapse imaging using confocal z-stacks over time (4D) eliminated the out-of-focus glow from the spheroids and resulted in sharply defined migrating cells with a dark background. This was not described in the protocol, but was carried out similarly to widefield time-lapse imaging, which was performed while brain slices were on the transparent membrane inserts in a 6-well plastic plate. Although confocal time-lapse imaging results in markedly clearer images of individual cells and their behavior, a multi-point time-lapse experiment collecting z-stacks of 10 z-planes/point, at 10 min intervals over a 20 h period, is an extensive use of the scan head galvanometers. As this could significantly decrease the lifespan of the galvanometers, this method is used judiciously.
Although the chick embryo system is very suitable for both in vivo injection and ex vivo co-culture experiments that investigate GBM cell behavior, there are several limitations to this model system. As with any xenograft system, the environment in which human cells are implanted is not the human brain, but GBM cell behavior appears to mimic that in rodent models and in human patients. After performing in vivo injection experiments on E5, tumors are normally allowed to form for 10 days, until E15. This clearly is not enough time to study all aspects of tumorigenesis and cell invasion. However, it has been demonstrated here that solid tumors form in the brain parenchyma, cells interact and rearrange themselves within the tumor, and significant brain invasion occurs both along blood vessels and diffusely within this relatively short time period. Another limitation to the in vivo chick embryo system is that it is not suitable for drug or other treatments because of the large yolk and extraembryonic circulatory system that operates during chick embryo development. Topical liquid drug treatments would result in a highly variable and unknown concentration in the brain due to diffusion away from the embryo into the much larger yolk mass. Similarly, intravenous injection of drugs into the very delicate extraembryonic circulatory system would leak or diffuse out of the blood vessels and also result in unknown concentrations in the brain. This is one of the main reasons that the ex vivo slice culture method was adopted-so that not only could cell behavior be observed and tracked via time-lapse microscopy, but also so that treatments that have been successful in altering GBM cell behavior in a dish4 could be tested in a more relevant brain tissue environment.
The development of the chick embryo orthotopic brain tumor model system is seen as a significant addition to the systems and tools available for the study of GBM tumor formation and invasive cell behavior. Fertilized chicken eggs are likely to be readily available in most areas, they are inexpensive compared to rodents, there are no animal care costs, the embryos are very resilient and resistant to infection (i.e., most work is done on a bench top), the embryos are highly manipulable and can be grown in shell-less culture19, and chick embryos are not considered vertebrate animals and so do not require IACUC approval by NIH guidelines (institutional requirements may vary). Thus, these multiple advantages make the chick embryo system very attractive if one confines their questions and experiments to those that fall within its limitations. Multiple GBM cell studies have been performed by others using the chick embryo, but these almost exclusively have utilized the chorioallantoic membrane (CAM) of the embryo20,21,22,23,24,25,26,27,28,29 and limb bud30, and not the brain. There has also been a report implanting medulloblastoma into the chick brain on E231. Undoubtedly, the use of the chick embryo as an orthotopic xenograft model system, as described here, should yield results that are much more meaningful to human GBM tumor biology than studies using the CAM.
Although these studies have only begun to fully utilize the chick embryo brain tumor model system for studies of human GBM cell and GSC behavior, it is hoped that others will extend the uses and find further potential applications. One could imagine that not only will this system uncover mechanisms that regulate GBM tumor formation and cell behavior, but will also allow preclinical testing of specific drugs and substances on specific patients&#39; cells. For instance, if brain slice cultures were set up in advance, then tumor cells, pieces from surgical tumor resections, or patient-derived GBM organoids32 could be placed directly in ex vivo co-culture, and various treatments could be assessed in a matter of days. Similarly, dissociated patient cells could be injected directly into E5 midbrains in ovo to assess their ability to form tumors and invade brain parenchyma. Thus, it is hoped that the descriptions of the methods and representative results here will facilitate and encourage increased use of this highly underutilized system for brain cancer research.

In vitro studies of cancer cell behaviors are often used to dissect potential mechanisms that operate during the more complex behavior that is observed during tumor formation and cell invasion in in vivo xenograft models. For example, with glioblastoma (GBM), in vitro studies have uncovered mechanisms of how L1CAM potentially operates during tumor formation and brain invasion in a novel chick embryo xenograft brain tumor model1,2,3,4,5. Although in vitro and in vivo experiments complement each other in useful ways, they leave a substantial gap in how the results can be correlated. For instance, mechanistic analyses of GBM cell motility on a dish is a highly artificial situation, and in vivo xenograft models can only reveal static time point or endpoint analyses of tumor formation and cell behavior. In vivo studies using either rodents or chick embryos do not easily lend themselves to monitoring cell behavior while the cells invade brain tissue in these xenograft models. Nevertheless, the chick embryo xenograft model has demonstrated that the adhesion protein L1CAM plays a stimulatory role in the invasive ability of human T98G GBM cells2,5.
A suitable solution to this problem can be reached by bridging both in vivo and in vitro methods using an organotypic brain slice culture model, referred to as an ex vivo model. In this ex vivo model, live brain tissue can be maintained at a thickness of several hundred microns for up to a few weeks, making it possible to implant cancer cells, observe their behavior in actual tissue over time, and then perform a more detailed marker analysis at the endpoint of the experiment.
A popular organotypic slice culture method has been to culture a several hundred micron-thick brain slice on top of a translucent or transparent porous membrane, leaving the tissue exposed to air, yet allowing nutrient media to sustain the tissue from below the membrane (refer to Stoppini et al.6). Different variations of this method have been used for different studies, including using different media or different membrane inserts. Different membrane inserts include a 30 mm diameter, porous (0.4 &#181;m) membrane insert in a 35 mm culture dish6, and cell culture inserts (0.4 &#181;m) for 6-well plates7. Different media include 50% MEM/HEPES + 25% heat-inactivated horse serum + 25% Hanks balanced salt solution (HBSS)8, 50% reduced serum media + 25% horse serum + 25% HBSS9, as well as others. If a translucent or transparent membrane is used along with fluorescently labeled GBM cells, then such cultures can be imaged from below using an inverted widefield or confocal fluorescence microscope10,11,12,13,14,15.
While many in vivo orthotopic brain tumor xenograft and ex vivo organotypic brain slice culture models have been established using rodents, as cited above, the chick embryo (Gallus gallus) has been underutilized for these purposes. However, the chick embryo has been demonstrated to be capable of being used as an in vivo orthotopic xenograft model for the study of both human and rat glioma invasion1,2,5. Xenografted cells into chick embryo brains have exhibited invasion patterns similar to those observed in rodent models, further supporting the use of chick embryos as an in vivo model for GBM tumor cell analysis. Chick embryos also are inexpensive, can be more easily maintained than rodents (i.e., in their egg shells in a lab incubator), and are much easier to work with, making them an attractive option for short-term in vivo GBM studies. A recent article has described the use of chick embryo brain slice cultures for the formation and growth of axons during normal brain development where the slices were viable for at least 7 days16. However, the use of such chick embryo brain slice cultures for ex vivo analysis of GBM cell behavior in a tissue environment is lacking. In this article, both the transplantation of human GBM cells and GBM stem cells (GSCs) into the early chick embryo brain in vivo, as well as the introduction of GBM cells onto live chick embryo brain slice cultures ex vivo, are described. Some representative examples of the resulting tumors and cell invasion patterns obtained from these preparations are also provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 cm x 1 cm square hole paper punch</td>
			<td>Birabira</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm biopsy punch pen</td>
			<td>Robbins Instruments</td>
			<td>20335</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well insert plate (Corning Transwell)</td>
			<td>Millipore Sigma</td>
			<td>CLS3450</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#34; Disposable Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Corning Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher BioReagents</td>
			<td>BP1423-500</td>
			<td>for embedding fixed brains</td>
		</tr>
		<tr>
			<td>Alexafluor 488-conjugated GAM IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-605-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexafluor 647-conjugated GAM IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-545-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>ReynoldsWrap</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A-9518</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-integrin alpha-6 monoclonal antibody GOH3</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-19622</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-L1CAM monoclonal antibody UJ127</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-53386</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-laminin monoclonal antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3H11</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-nestin monoclonal antibody 10c2</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23927</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sox2 monoclonal antibody E-4</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365823</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 supplement without vitamin A</td>
			<td>GIBCO</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>bisbenzimide (Hoechst 33258)</td>
			<td>Sigma-Aldrich</td>
			<td>B2883</td>
			<td>nuclear stain</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Forma</td>
			<td></td>
			<td>standard humidified CO2 incubator</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon Instruments</td>
			<td>C2si+</td>
			<td>With custom-made cell incubator chamber</td>
		</tr>
		<tr>
			<td>Confocal microscope objective lenses</td>
			<td>Nikon Instruments</td>
			<td></td>
			<td>Plan Apo lenses, except S Plan Fluor ELWD 20x 0.45 NA objective lens for confocal time-lapse imaging</td>
		</tr>
		<tr>
			<td>Confocal microscope software</td>
			<td>Nikon Instruments</td>
			<td>NIS Elements</td>
			<td>Version 5.2</td>
		</tr>
		<tr>
			<td>Curved foreceps</td>
			<td>World Precision Intruments</td>
			<td>504478</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved spatula</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>Krazy Glue</td>
			<td>KG-585 12R</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Millipore Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD far red fluorescent dye</td>
			<td>Invitrogen</td>
			<td>V22887</td>
			<td>Vybrant DiD</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D5671</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma Aldrich</td>
			<td>D8437</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate buffered saline (PBS)</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>egg incubator</td>
			<td>Humidaire</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>electrical tape (10 mil thick/254 &#181;m)</td>
			<td>Scotch</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast green FCF dye</td>
			<td>Avocado Research Chemicals</td>
			<td>16520</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini Bio-products</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>filter paper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Dynarex</td>
			<td>3353</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries for microinjection</td>
			<td>World Precision Instruments</td>
			<td>TW100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP228-1</td>
			<td>for mounting media</td>
		</tr>
		<tr>
			<td>GSCs (human glioblastoma stem cells)</td>
			<td>Not applicable</td>
			<td></td>
			<td>Isolated from patient GBM specimens in Galileo laboratory in GSC media and then transduced with a GFP encoding lentiviral vector.&#160; Cells used were between passage 10 and 30.</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher Scientific</td>
			<td>BP2524-100</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Sigma Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum (HI)</td>
			<td>Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF-2</td>
			<td>BioVision</td>
			<td>4037-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#945;</td>
			<td>BioVision</td>
			<td>4339-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted phase contrast microscope</td>
			<td>Nikon Instruments</td>
			<td>TMS</td>
			<td>for routine viewing of cultured cells</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P284</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory film</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labquake Shaker</td>
			<td>LabIndustries</td>
			<td>T400-110</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glut:Pen:Strep</td>
			<td>Gemini Bio-products</td>
			<td>400-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melt agarose</td>
			<td>Fisher Scientific</td>
			<td>BP1360</td>
			<td>for embedding live brains</td>
		</tr>
		<tr>
			<td>Matrix</td>
			<td>Corning Matrigel</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>GIBCO</td>
			<td>11150-059</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM</td>
			<td>Corning</td>
			<td>10-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Metal vibratome block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips (20, 200, 1,000 &#181;L)</td>
			<td>Fisherbrand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettors (20, 200, 1,000 &#181;L)</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverglass (no. 1.5 thickness)</td>
			<td>Fisherbrand</td>
			<td>12544A</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4 + H2O</td>
			<td>Fisher Scientific</td>
			<td>S369</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fisher Scientific</td>
			<td>BP328</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Millipore Sigma</td>
			<td>526-M</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>Sigma Aldrich</td>
			<td>P3130</td>
			<td>for mounting media</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Parafilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pencil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plain Microscope slides</td>
			<td>Fisherbrand</td>
			<td>12-550-A3</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic 35 mm Petri dish</td>
			<td>Becton Dickinson</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>pneumatic picopump</td>
			<td>World Precision Intruments</td>
			<td>PV830</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(2-hydroxyethyl methacrylate)&#160; (poly-HEMA)</td>
			<td>Sigma Aldrich</td>
			<td>P-3932</td>
			<td></td>
		</tr>
		<tr>
			<td>razor blade- double edge</td>
			<td>PACE</td>
			<td></td>
			<td>for cutting fixed brain slices</td>
		</tr>
		<tr>
			<td>sapphire knife</td>
			<td>Delaware Diamond Knives</td>
			<td></td>
			<td>for cutting live brain slices</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>TruMed</td>
			<td>1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium cacodylate buffer 0.2 M pH 7.4</td>
			<td>Electron Microscopy Sciences</td>
			<td>11652</td>
			<td></td>
		</tr>
		<tr>
			<td>Specimen chamber for vibratome</td>
			<td>custom-made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Dissecting Microscope</td>
			<td>Nikon Instruments</td>
			<td>SMZ1500</td>
			<td>Equipped with epifluorescence</td>
		</tr>
		<tr>
			<td>straight foreceps</td>
			<td>World Precision Intruments</td>
			<td>500233</td>
			<td></td>
		</tr>
		<tr>
			<td>straight scissors</td>
			<td>Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Mallinckrodt</td>
			<td>7723</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse fluorescence microscope (widefield fluorescence)</td>
			<td>Nikon Instruments</td>
			<td>TE2000-E</td>
			<td>With custom-made cell incubator chamber (see Fotos et al., 2006)</td>
		</tr>
		<tr>
			<td>Tissue culture dish polystyrene 100 mm</td>
			<td>Thermo Fisher Scientific</td>
			<td>130182</td>
			<td>for cell culturing</td>
		</tr>
		<tr>
			<td>Tissue culture dish polystyrene 60 mm</td>
			<td>Becton Dickinson</td>
			<td>353004</td>
			<td>for cell culturing</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>American Central Scientific Co.</td>
			<td>FFP011</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.25%) + 2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>U-118 MG human GBM cell line</td>
			<td>ATCC</td>
			<td>HTB-15</td>
			<td>Cells were transduced with a lentiviral vector encoding the entire ectodomain sequence of the L1CAM adhesion protein and then with lentiviral vector pUltra-hot encoding mCherry.&#160; Passage numbers are unknown.</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Cole-Parmer</td>
			<td>EW-07532-40</td>
			<td>&#34;Air Cadet&#34;</td>
		</tr>
		<tr>
			<td>Vibrating tissue slicer</td>
			<td>Vibratome</td>
			<td>3000</td>
			<td>for cutting live and fixed brain slices</td>
		</tr>
	</tbody>
</table>

using the chick embryo brain as a model for in vivo and ex vivo analyses of human glioblastoma cell behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Watch this Scientific Journal Video about Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior at JoVE.com

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

using-chick-embryo-brain-as-model-for-vivo-ex-vivo-analyses-human

Research

JoVE Journal

Neuroscience

3.4K Views.  University of Delaware. Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis. 

Video: Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

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

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

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

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

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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 ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

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 ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.