Name: in vivo functional brain imaging approach based on bioluminescent calcium indicator gfp-aequorin
Uploaded: 2016-01-08T15:00:01
Description: Watch this Scientific Journal Video about In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin at JoVE.com

We are indebted to E. Karplus, from Sciences Wares, USA, for his precious and useful help and advices. We thank E. Carbognin, A. Avet-Rochex, M. Murmu, P. Pavot, D. Minocci, G. Vinatier, and J. Stinnakre for their contribution to the development and improvement of this technique. We also thank B. Pfeiffer and G. Rubin, Janelia Farm, H.H.M.I., Ashburn, USA, for the pJFRC65-13XLexAop2-IVS-G5A-BP line. This work was supported by the Chateaubriand Fellowship, French Embassy, Washington, USA to A. Lark, by the National Science Foundation (IOS 1352882) to T. Kitamoto, and by the French ANRs (ANR-05-NEUR-009 Drosaequorin, 2005, and FlyBrainImaging, 2011), the Conseil R&#233;gional Ile-De France (NeRF and DIM), the IFR-144, the Physique-Chimie-Biologie Interface Program of the CNRS (2009), and by the CNRS, France to JR Martin.

1. Preparation of Samples
<ol>
	<li>Preparation of solutions and set up
	<ol>
		<li>Maintain all Drosophila melanogaster lines at 24 &#176;C on standard food medium. Rear and keep them at low density in the vial to generate standardized size and weight of flies.
		<ol>
			<li>Add 10 virgin females with 10 males in a vial, let them mate and transfer them every 2 days to a fresh vial. Then 10 days later, when the flies start to eclose, harvest the flies every day. Keep a precise record of the age of the flies an...

<ol>
	<li>Martin, J. -. R. <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+brain+imaging:+fluorescence+or+bioluminescence,+which+to+choose.">In vivo brain imaging: fluorescence or bioluminescence, which to choose.</a> J Neurogenet. 22 (3), 285-307 (2008).</li><li>Shimomura, O., Johnson, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxidized+coelenterazine,+the+active+group+in+the+photoprotein+aequorin.">Peroxidized coelenterazine, the active group in the photoprotein aequorin.</a> Proc Natl Acad Sci USA. 75 (6), 2611-2615 (1978).</li><li>Shimomura, O., Johnson, F. H., Saiga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+data+on+the+bioluminescent+protein,+aequorin.">Further data on the bioluminescent protein, aequorin.</a> J. Cell. Comp. Physiol. 62 (1), 1-8 (1963).</li><li>Baubet, V., 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=Chimeric+green+fluorescent+protein-aequorin+as+bioluminescent+Ca2++reporters+at+the+single-cell+level.">Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.</a> Proc Natl Acad Sci USA. 97 (13), 7260-7265 (2000).</li><li>Cabrero, P., Richmond, L., Nitabach, M., Davies, S. A., Dow, J. A. 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+biogenic+amine+and+a+neuropeptide+act+identically:+tyramine+signals+through+calcium+in+Drosophila+tubule+stellate+cells.">A biogenic amine and a neuropeptide act identically: tyramine signals through calcium in Drosophila tubule stellate cells.</a> Proc. Biol. Sci. 280 (1757), 20122943 (2013).</li><li>Leclerc, C., Webb, S. E., Daguzan, C., Moreau, M., Miller, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+patterns+of+calcium+transients+during+neural+induction+in+Xenopus+laevis+embryos.">Imaging patterns of calcium transients during neural induction in Xenopus laevis embryos.</a> J. Cell Sci. 113 (19), 3519-3529 (2000).</li><li>Martin, J. -. R., Rogers, K. L., Chagneau, C., Br&#251;let, P. <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+bioluminescence+imaging+of+Ca2++signalling+in+the+brain+of+Drosophila.">In vivo bioluminescence imaging of Ca2+ signalling in the brain of Drosophila.</a> PLoS One. 2 (3), e275 (2007).</li><li>Murmu, M. S., Stinnakre, J., Martin, J. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+Ca2++stores+contribute+to+odor-induced+responses+in+Drosophila+olfactory+receptor+neurons.">Presynaptic Ca2+ stores contribute to odor-induced responses in Drosophila olfactory receptor neurons.</a> J. Exp. Biol. 213 (24), 4163-4173 (2010).</li><li>Murmu, M. S., Stinnakre, J., R&#233;al, E., Martin, J. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium-stores+mediate+adaptation+in+axon+terminals+of+Olfactory+Receptor+Neurons+in+Drosophila.">Calcium-stores mediate adaptation in axon terminals of Olfactory Receptor Neurons in Drosophila.</a> BMC Neurosci. 12, 105 (2011).</li><li>Pavot, P., Carbognin, E., Martin, J. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKA+and+cAMP/CNG+channels+independently+regulate+the+cholinergic+Ca2+-response+of+Drosophila+mushroom+body+neurons.">PKA and cAMP/CNG channels independently regulate the cholinergic Ca2+-response of Drosophila mushroom body neurons.</a> eNeuro. 2 (2), 0054-0068 (2015).</li><li>Minocci, D., Carbognin, E., Murmu, M. S., Martin, J. -. R. <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+functional+calcium+imaging+of+induced+or+spontaneous+activity+in+the+fly+brain+using+a+GFP-apoaequorin-based+bioluminescent+approach.">In vivo functional calcium imaging of induced or spontaneous activity in the fly brain using a GFP-apoaequorin-based bioluminescent approach.</a> BBA-Mol Cell Res. 1833 (7), 1632-1640 (2013).</li><li>Lima, S. Q., Miesenb&#246;ck, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+behavior+through+genetically+targeted+photostimulation+of+neurons.">Remote control of behavior through genetically targeted photostimulation of neurons.</a> Cell. 121 (1), 141-152 (2005).</li><li>Shimomura, O., Musicki, B., Kishi, Y., Inouye, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+properties+of+recombinant+semisynthetic+aequorins+and+recombinant+fluorescein-conjugated+aequorin+for+measuring+cellular+calcium.">Light-emitting properties of recombinant semisynthetic aequorins and recombinant fluorescein-conjugated aequorin for measuring cellular calcium.</a> Cell Calcium. 14 (5), 373-378 (1993).</li><li>Berridge, M. J., Bootman, M. D., Roderick, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signalling:+dynamics,+homeostasis+and+remodelling.">Calcium signalling: dynamics, homeostasis and remodelling.</a> Nat Rev Mol Cell Bio. 4 (7), 517-529 (2003).</li><li>Fiala, A., Spall, T. <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+calcium+imaging+of+brain+activity+in+Drosophila+by+transgenic+cameleon+expression.">In vivo calcium imaging of brain activity in Drosophila by transgenic cameleon expression.</a> Sci STKE. 2003 (174), PL6 (2003).</li><li>Wang, J. W., Wong, A. M., Flores, J., Vosshall, L. B., Axel, R. <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+calcium+imaging+reveals+an+odor-evoked+map+of+activity+in+the+fly+brain.">Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain.</a> Cell. 112 (2), 271-282 (2003).</li><li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+Olfactory+Representations+in+the+Drosophila+Antennal+Lobe.">Transformation of Olfactory Representations in the Drosophila Antennal Lobe.</a> Science. 303 (5656), 366-370 (2004).</li></ol>

The recently developed bioluminescent-based GFP-aequorin approach presented here allows in vivo functional recording of Ca2+-activity in different neurons, as well as if desired, in other kind of cells such as kidney stellate cells as reported in Cabrero et al., 20135.
Modifications, trouble shooting, additional components, and critical steps
Drosophila Husbandry
<p class...

The fundamental patterning and function of activity within the brain and its discrete structures have long been an area of intense study within the field of neuroscience. Perhaps some of the earliest successful approaches used to address this issue were direct physiological measurement of the change in electrical activity &#8212; however alternate methods which allow live imaging of changes in voltage, pH or calcium concentrations (as proxy for activity) have brought additional capabilities to the study of neuronal activity 1. Imaging techniques carry a wide array of advantages such as reduced invasiveness and the ability to monitor activity within whole brain structures. Furthermore in animals with tractable genetics including the fruit fly Drosophila, development of genetically encoded calcium indicators, such as cameleon, GCaMPs and others1 have allowed researchers to monitor single neurons, neuronal populations and whole brain structures. While more traditional fluorescent calcium imaging methods using GCaMPs (GFP fused to calmodulin) or FRET (CFP and YFP fused to calmodulin) have proved to be useful techniques providing good spatial and temporal resolution, the underlying process of light excitation engenders some limitations on its experimental applications. Due to the nature of light excitation, autofluorescence, photo-bleaching, and phototoxicity are unavoidably produced, which consequently limits the depth of the structures that can be imaged, the duration of recordings as well as the real time continuity of recording. In other words, recordings must often be performed intermittently to avoid these undesired side effects.
Because of these challenges, additional alternative methods of calcium imaging have been developed. One of the most promising methods is bioluminescent imaging, which relies on light produced through an enzymatic reaction after calcium binding. Bioluminescent imaging does not require light excitation and consequently does not experience the same difficulties as conventional calcium imaging. The bioluminescent reporter Aequorin &#8212; isolated from Aequorea victoria, the same Jellyfish from which GFP was also isolated- binds calcium via its three EF hand structures and undergoes a conformational change, leading to the oxidation of its cofactor coelenterazine and the release of blue light (&#955;=469 nm)2,3. Aequorin has a very low affinity for calcium (Kd = 10 &#181;M) which makes it ideal for monitoring calcium transients because it produces very little background noise and does not interfere with the intracellular calcium buffering system4. Although aequorin has been previously used to monitor the process of neural induction in the Xenopus egg5 the light produced is too dim to use for real time imaging of neuronal activity. In an effort to address this challenge, Philippe Br&#251;let and colleagues at the Pasteur Institute created a genetic construct fusing GFP and aequorin genes, mimicking the native state within the jellyfish4. This fusion gene product binds calcium again via the three EF hand structures of aequorin, which undergoes a conformational change leading to oxidation of the coelenterazine, which transfers energy non-radiatively to the GFP. This energy transfer results in the release of green light (&#955;=509 nm) from GFP, instead of blue light from aequorin. The fusion of GFP and aequorin also confers greater protein stability helping the fusion protein produce light 19 to 65 times brighter than aequorin alone4. Using a bioluminescent approach within the brain expands the current experimental application of calcium imaging both in terms of the duration of continuous recording and the number of structures within the brain that can be recorded. Broadly in the context of previous work it means that both global and a greater variety of regionalized &#8220;activity&#8221; of the brain can be monitored (through the proxy of calcium transients). More importantly activity can be monitored for longer, in a real time and continuous basis, which readily lends itself to the pursuit of a complete understanding of basal function in the brain.
In the last few years, our lab and others have developed transgenic flies expressing the GFP-aequorin under the control of the binary expression system (GAL4/UAS-GFP-aequorin)5,6. We have used GFP-aequorin bioluminescence to record activity produced by natural stimuli such as scent7,8, as well as studied the cellular components modulating Ca2+-activity in the mushroom bodies following acetylcholine receptor stimulation by direct nicotinic application9. In addition, we have also used GFP-aequorin to address a number of experimental questions that have not been able to be addressed previously, like long-term imaging of spontaneous calcium transients and visualization of deeper brain structures10. More recently, we have used the GAL4/UAS system to express the mammalian ATP receptor P2X211 a cation channel, in the projection neurons (PNs) and used the LexA system to express GFP-aequorin in the mushroom bodies (MB247-LexA,13XLexAop2-IVS-G5A-BP) (a courtesy of B. Pfeiffer, Janelia Farm, USA). This has allowed us to activate the PNs by application of ATP, which in turn activates the mushroom bodies (a downstream target of the PNs), mimicking more natural conditions, such as the response to an odor stimulus. Overall this technique combining P2X2 and GFP-aequorin expands the experimental possibilities. Broadly it offers the opportunity to better understand the patterns of activity in the brain, initiating new studies about how different stimuli and perturbations can alter the basal patterning of activity.

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			<td>HEPES</td>
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			<td>KCL&#160;</td>
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			<td>Corning Incorporated</td>
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			<td>100-1000&#181;L Univesal Pipette Tip</td>
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			<td>N/A</td>
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			<td>immersion objective lens</td>
			<td>Nikon</td>
			<td>N/A</td>
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			<td>dissection microscope with fluorescence</td>
			<td>Leica MZ Fl III</td>
			<td>N/A</td>
			<td>leica dissection scope and florescent lamp</td>
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			<td>tight dark box</td>
			<td>Science Wares</td>
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			<td>Custom built by Science Wares</td>
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			<td>peristaltic pump&#160;</td>
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			<td>N/A</td>
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			<td>Warner</td>
			<td>64-0135 &#160;(VC-66CS)</td>
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			<td>perfusion regulators</td>
			<td>Leventon</td>
			<td>201108</td>
			<td>Dosi-flow 3</td>
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		<tr>
			<td>measurement and automation explorer</td>
			<td>Sciences Wares &#38; National Instruments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
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		<tr>
			<td>photon imager&#160;</td>
			<td>Science Wares &#38; National Instruments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
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		<tr>
			<td>photon viewer</td>
			<td>Science Wares &#38; National Instuments</td>
			<td>N/A</td>
			<td>software http://sine.ni.com/nips/cds/view/p/lang/en/nid/1380</td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>software https://www.microsoft.com</td>
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		<tr>
			<td>virtual dub</td>
			<td>open access&#160;</td>
			<td>N/A</td>
			<td>software http://virtualdub.sourceforge.net/</td>
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		<tr>
			<td>UAS-GA2</td>
			<td>Martin et al., 2007, Ref. 1&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</td>
		</tr>
		<tr>
			<td>pJFRC65-13XLexAop2-IVS-G5A-BP&#160;&#160;</td>
			<td>B. Pfeiffer, Janelia Farms, Ashburn USA.&#160;</td>
			<td>(STOCK #1117340)</td>
			<td>pfeifferb@janelia.hhmi.org</td>
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		<tr>
			<td>P2X2</td>
			<td>Lima and Misenboeck, Ref. 11&#160;</td>
			<td>N/A</td>
			<td>&#160;No stocks {currently} publicly available&#160;</td>
		</tr>
		<tr>
			<td>OK107</td>
			<td>Bloomington stock center</td>
			<td>854</td>
			<td>http://flystocks.bio.indiana.edu/</td>
		</tr>
	</tbody>
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in vivo functional brain imaging approach based on bioluminescent calcium indicator gfp-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

The fusion of the GFP to aequorin allows us to visualize our region of interest prior to bioluminescent imaging through excitation of GFP in fluorescent mode on the microscope (Figure 3A, 4A, 6A, 6E). One of the simplest ways to stimulate the mushroom bodies is through activation of the ionotropic nicotinic acetylcholine receptors. Although acetylcholine is the endogenous ligand of this receptor we have found that nicotine produces more reproducible responses in the mushroom bodies. This is in part becau...

Watch this Scientific Journal Video about In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin at JoVE.com

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

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Ãƒâ€šÃ‚ÂµTongue: A Microfluidics-Based Functional Imaging Platform for the Tongue In Vivo

Intravital fluorescence microscopy is a tool used widely to study multicellular dynamics in a live animal. However, it has not been successfully used in ...

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

Stereotaxic Viral Injection and Gradient-Index Lens Implantation for Deep Brain In Vivo Calcium Imaging

A miniature fluorescence microscope (miniscope) is a potent tool for in vivo calcium imaging from freely behaving animals. It offers several advantages ...