We wish to thank Drs. Meera Saxena and Jonathan Mathias for helpful comments on the manuscript. We thank Drs. Michael Parsons, Pamela Raymond, and Rachel Wong for providing transgenic lines. Experiments on animals were performed in accordance with the guidelines and regulations set forth by the MCG IACUC approved protocol 07-12-003*B. This work was supported by NIH R21 MH083614 and a March of Dimes Basil O Conner Starter Scholar Award to J.S.M.

<ol>
	<li>K&#246;ster, R. W., Fraser, S. E. <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+microscopy+of+brain+development.">Time-lapse microscopy of brain development.</a> Methods Cell Biol. 76, 207-235 (2004).</li><li>Mumm, J. S., Williams, P. R., Godinho, L., Koerber, A., Pittman, A. J., Roeser, T., Chien, C. B., Baier, H., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+dendritic+targeting+of+laminated+afferents+by+zebrafish+retinal+ganglion+cells.">In vivo imaging reveals dendritic targeting of laminated afferents by zebrafish retinal ganglion cells.</a> Neuron. 52, 609-621 (2006).</li><li>Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+targeted+cell+ablation+in+zebrafish:+a+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies.</a> Dev Dyn. 236, 1025-1035 (2007).</li><li>Ren, J. Q., McCarthy, W. R., Zhang, H., Adolph, A. R., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+visual+responses+of+wild-type+and+hypopigmented+zebrafish.">Behavioral visual responses of wild-type and hypopigmented zebrafish.</a> Vision Res. 42, 293-299 (2002).</li><li>Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+ablation+of+beta+cells+in+the+embryonic+zebrafish+pancreas+using+E.+Coli+nitroreductase.">Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. Coli nitroreductase.</a> Mech Dev. 124, 218-229 (2007).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2010).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Vis Exp. , (2009).</li></ol>

J.S.M. has been awarded a patent on the use of the nitroreductase-based system of cell ablation in zebrafish (Mumm J.S., and Schroeter, E.H. Targeted and Regional Ablation Cellular Ablation in Zebrafish" US Patent 7514595. 7 Apr. 2009). This technique is used as the general platform for a tools and services biotechnology company, Luminomics, Inc., that J.S.M. founded and remains a stakeholder in.

In this video, an overview of techniques we use for multicolor confocal time-lapse imaging and targeted cell ablation is provided. We employ time-lapse imaging to monitor the behavior of multiple cell types of interest in vivo, while targeted cell ablation is used to study neural circuit function and cell-specific neuronal regeneration paradigms. The examples shown highlight several advantages that can be gleaned from these approaches. Perhaps most significantly, time-lapse imaging provides an internally controlled paradigm; any and all phenomena observed can be related back to previous states. This allows direct rather than inferred comparisons to be made across experimental time points, making relative changes easier to quantify and reducing experimental 'noise' associated with variations within a population. An advantage of multicolor imaging is the ability to visualize changes in interacting and/or neighboring cell types. For instance, using a transgenic line which labels M&uuml;ller glia, we are now characterizing the response of these potential injury-induced stem cells to the loss of discrete retinal cell subpopulations. We have also used this approach to define novel mechanisms of neural circuit formation 2. Finally, in addition to studies of cellular regeneration, we have recently begun using the NTR-based ablation system to investigate the functional role of neuronal subtypes thus discrete neural circuits using various behavioral paradigms and electrophysiological assays. The unique advantage of employing the zebrafish for such studies is that, due to their regenerative capacity, induced deficits are temporary. Thus, by correlating with time-lapse imaging assays we can typify reinnervation mechanisms that lead to, as well as the extent of reinnervation required for, functional recovery.
The methods provided can be adapted to many different questions. For instance, similar analyses of ablation and regeneration can be applied to any transgenically definable cellular subtype. Collectively, such studies have potential to expand our understanding of regeneration at the level of individual cell types and within discrete stem cell niches.

1. Reagents:
Danieau's solution:
<ul>
<li>sodium chloride (Fisher, S271 1)</li>
<li>potassium chloride (Fisher, P217 500)</li>
<li>HEPES (Fisher, BP310500)</li>
<li>magnesium sulfate (Fisher, M65 500)</li>
<li>calcium nitrate (Fisher, C109500)</li>
<li>Penicillin-Streptomycin (Fisher, ICN1670049)</li>
</ul>
Other reagents:
<ul>
<li>Low melt agarose (Fisher, BP165-25)</li>
<li>Tricane methanesulfonate (Fisher, NC9435281)</li>
<li>N-phenylthiourea (Fisher, NC9968868)</li>
<li>Metronidazole (Fisher, AC21034-1000)</li>
</ul>
2. Supplies:
<ul>
<li>Deep petri-dishes (Fisher Scientific/Corning, 08-772-32)</li>
<li>Dumont #5 fine forceps (Fisher, NC9404145)</li>
</ul>
3. Equipment:
<ul>
<li>Zoom Stereoscope (Olympus, SZ51)</li>
<li>Fluorescence Zoom Stereoscope (Olympus, SZX16)</li>
<li>Confocal microscope (Olympus FV1000)</li>
<li>Digital block heater - Isotemp (Fisher, 11-715-125DQ)</li>
<li>Barnstead/Thermolyne Benchtop Incubator (Fisher, 11-702-4).</li>
</ul>

multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

Watch this Scientific Journal Video about Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation at JoVE.com

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

multicolor-time-lapse-imaging-transgenic-zebrafish-visualizing

Research

JoVE Journal

Biology

16.2K 次观看.  Medical College of Georgia. 在这个视频，多色共焦成像时间推移和有针对性的细胞消融技术。定时成像是用来监测多种细胞类型的利益的行为在体内。有针对性的细胞消融有利于研究神经回路的功能和细胞特异性神经再生范例。