Name: use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo
Uploaded: 2014-01-12T14:00:00
Description: Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

We thank Homaira Nawabi, Frederic Moret and Isabelle Sanyas for their help. This work is supported by CNRS, Association Francaise contre les Myopathies (AFM), ANR YADDLE, Labex DevWeCan, Labex Cortex, ERC YODA to V.C.; C.D-B and A.J are supported by a La Ligue contre le cancer and Labex DevWeCan fellowships, respectively.

1. Cloning Strategy to Tag PlexinA1 Receptor with pHluorin
<ol>
	<li>Choose an appropriate expression vector as a backbone (e.g.&#160;the mouse receptor plexinA1 expressing vector, a kind gift of Dr. Andreas Puschel11). 
	Note: This plexinA1 vector was engineered to achieve efficient HA- or VSV-tagged receptor insertion in the plasma membrane.</li>
	<li>Amplify by PCR the ecliptic pHluorin coding sequence using the adequate plasmid as a template (e.g.&#160;pHluorin-tagged GABA A recept...

<ol>
	<li>Winckler, B., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+guidance+receptors.">Trafficking guidance receptors.</a> Cold Spring Harb. Perspect. Biol. 2, (2010).</li><li>Jacob, T. 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=.">.</a> J. Neurosci. 25, 10469-10478 (2005).</li><li>Nawabi, H., Castellani, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+commissures+in+the+central+nervous+system:+how+to+cross+the+midline.">Axonal commissures in the central nervous system: how to cross the midline.</a> Cell Mol. Life Sci. 68, 2539-2553 (2011).</li><li>Keleman, K., Ribeiro, C., Dickson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comm+function+in+commissural+axon+guidance:+cell-autonomous+sorting+of+Robo+in+vivo.">Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo.</a> Nat. Neurosci. 8, 156-163 (2005).</li><li>Nawabi, H., 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=A+midline+switch+of+receptor+processing+regulates+commissural+axon+guidance+in+vertebrates.">A midline switch of receptor processing regulates commissural axon guidance in vertebrates.</a> Genes Dev. 24, 396-410 (2010).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapto-pHluorins:+genetically+encoded+reporters+of+synaptic+transmission.">Synapto-pHluorins: genetically encoded reporters of synaptic transmission.</a> Cold Spring Harb. Protoc.. 2012, 213-217 (2012).</li><li>Avraham, O., Zisman, S., Hadas, Y., Vald, L., Klar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+axonal+pathways+of+genetically+defined+groups+of+neurons+in+the+chick+neural+tube+utilizing+in+ovo+electroporation.">Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation.</a> J. Vis. Exp. (39), 1792-17 (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+ovo+electroporations+of+HH+stage+10+chicken+embryos.">In ovo electroporations of HH stage 10 chicken embryos.</a> J. Vis. Exp. (9), (2007).</li><li>Wilson, N. H., Stoeckli, E. 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+ovo+electroporation+of+miRNA-based+plasmids+in+the+developing+neural+tube+and+assessment+of+phenotypes+by+DiI+injection+in+open-book+preparations.">In ovo electroporation of miRNA-based plasmids in the developing neural tube and assessment of phenotypes by DiI injection in open-book preparations.</a> J. Vis. Exp. (68), (2012).</li><li>Rohm, B., Ottemeyer, A., Lohrum, M., Puschel, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plexin/neuropilin+complexes+mediate+repulsion+by+the+axonal+guidance+signal+semaphorin+3A.">Plexin/neuropilin complexes mediate repulsion by the axonal guidance signal semaphorin 3A.</a> Mech. Dev. 93, 95-104 (2000).</li><li>Hamburger, V., Hamilton, 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=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J. Vis. Exp. (8), (2007).</li><li>Alberts, 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=Cdc42+and+actin+control+polarized+expression+of+TI-VAMP+vesicles+to+neuronal+growth+cones+and+their+fusion+with+the+plasma+membrane.">Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane.</a> Mol. Biol. Cell. 17, 1194-1203 (2006).</li><li>Perret, E., Lakkaraju, A., Deborde, S., Schreiner, R., Rodriguez-Boulan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+endosomes:+how+many+varieties+and+why.">Evolving endosomes: how many varieties and why.</a> Curr. Opin. Cell Biol. 17, 423-434 (2005).</li><li>Li, Y., 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=Imaging+pHluorin-tagged+receptor+insertion+to+the+plasma+membrane+in+primary+cultured+mouse+neurons.">Imaging pHluorin-tagged receptor insertion to the plasma membrane in primary cultured mouse neurons.</a> J. Vis. Exp. (69), (2012).</li><li>Tojima, T., Itofusa, R., Kamiguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+clathrin-mediated+endocytosis+drives+repulsive+growth+cone+guidance.">Asymmetric clathrin-mediated endocytosis drives repulsive growth cone guidance.</a> Neuron. 66, 370-377 (2010).</li><li>Matsui, A., Yoshida, A. C., Kubota, M., Ogawa, M., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+in+utero+electroporation:+controlled+spatiotemporal+gene+transfection.">Mouse in utero electroporation: controlled spatiotemporal gene transfection.</a> J. Vis. Exp. (54), 3024-30 (2011).</li><li>Falk, J., 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=Electroporation+of+cDNA/Morpholinos+to+targeted+areas+of+embryonic+CNS+in+Xenopus.">Electroporation of cDNA/Morpholinos to targeted areas of embryonic CNS in Xenopus.</a> BMC Dev. Biol. 7 (107), (2007).</li><li>Holzhausen, L. C., Lewis, A. A., Cheong, K. K., Brockerhoff, 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=Differential+role+for+synaptojanin+1+in+rod+and+cone+photoreceptors.">Differential role for synaptojanin 1 in rod and cone photoreceptors.</a> J. Comp. Neurol. 517, 633-644 (2009).</li><li>Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M., Miesenbock, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+local+circuits+and+their+implications+for+olfactory+processing+in+the+fly+antennal+lobe.">Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe.</a> Cell. 128, 601-612 (2007).</li><li>Dittman, J. S., Kaplan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+regulating+the+abundance+and+localization+of+synaptobrevin+in+the+plasma+membrane.">Factors regulating the abundance and localization of synaptobrevin in the plasma membrane.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11399-11404 (2006).</li><li>Bozza, T., McGann, J. P., Mombaerts, P., Wachowiak, M. <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+of+neuronal+activity+by+targeted+expression+of+a+genetically+encoded+probe+in+the+mouse.">In vivo imaging of neuronal activity by targeted expression of a genetically encoded probe in the mouse.</a> Neuron. 42, 9-21 (2004).</li><li>Sankaranarayanan, S., Ryan, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurements+of+vesicle-SNARE+recycling+in+synapses+of+the+central+nervous+system.">Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system.</a> Nat. Cell. Biol. 2, 197-204 (2000).</li><li>Stark, D. A., Kasemeier-Kulesa, J. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivation+cell+labeling+for+cell+tracing+in+avian+development.">Photoactivation cell labeling for cell tracing in avian development.</a> CSH Protoc.. 2008, (2008).</li><li>Hildick, K. L., Gonzalez-Gonzalez, I. M., Jaskolski, F., Henley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+and+exocytosis+of+membrane+proteins+in+cultured+neurons+assessed+using+fluorescence+recovery+and+fluorescence-loss+photobleaching.">Lateral diffusion and exocytosis of membrane proteins in cultured neurons assessed using fluorescence recovery and fluorescence-loss photobleaching.</a> J. Vis. Exp. (60), (2012).</li><li>Hanson, G. T., 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=Green+fluorescent+protein+variants+as+ratiometric+dual+emission+pH+sensors.+1.+Structural+characterization+and+preliminary+application.">Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application.</a> Biochemistry. 41, 15477-15488 (2002).</li><li>Rose, T., Schoenenberger, P., Jezek, K., Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+refinement+of+vesicle+cycling+at+schaffer+collateral+synapses.">Developmental refinement of vesicle cycling at schaffer collateral synapses.</a> Neuron. 77, 1109-1121 (2013).</li><li>Li, Y., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pHTomato,+a+red,+genetically+encoded+indicator+that+enables+multiplex+interrogation+of+synaptic+activity.">pHTomato, a red, genetically encoded indicator that enables multiplex interrogation of synaptic activity.</a> Nat. Neurosci. 15, 1047-1053 (2012).</li><li>de Wit, J., Toonen, R. F., Verhage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-dependent+local+retention+of+secretory+vesicle+cargo+in+cortical+neurons.">Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.</a> J. Neurosci. 29, 23-37 (2009).</li></ol>

This protocol provides a step-by-step procedure to follow the dynamics of an axon guidance receptor both in cell culture and in the developmental context of the chick embryo spinal cord.
To design a de novo pHluorin tagged protein, two points need to be considered regarding the cloning strategy. First, the pHluorin tag should be exposed to the lumen of the acidic endosomes, and consequently, to the extracellular compartment in order to visualize the plasma membrane receptor pool. Thus...

During their navigation, axons integrate multiple environmental cues that guide them towards their target. These cues activate guidance receptors at the surface of axon terminals, the growth cones, which in turn initiate an appropriate signaling pathway. Thus, the temporal and spatial regulation of the cell surface distribution of the receptors is critical to set the sensitivity of the growth cone1. In this context, midline crossing by commissural axons is an excellent model to investigate the regulation of receptor cell surface levels. In the developing spinal cord, commissural axons are initially attracted towards the ventral floor plate where they cross the midline. After crossing, they lose their responsiveness to the floor plate attractants and gain response to floor plate repellents so that they can exit the floor plate and navigate towards their final destination in the contralateral side of the nervous system2,3. Regulation of receptor availability at the growth cone surface is one of the mechanisms underlying the switch of responsiveness to midline cues4,5. Thus, selective monitoring of the receptors present at the plasma membrane of growth cones is of prime importance. We describe here a method based on the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically visualize the axon guidance receptors that are addressed to the plasma membrane in vitro and in vivo, in the developing chick spinal cord.
Rothman and colleagues engineered by point mutations pH-sensitive variants of GFP including the ecliptic pHluorin6. Ecliptic pHluorin has the property of being nonfluorescent when exposed to acidic pH (&#60;6), while being fluorescent at neutral pH. This allows distinguishing nonfluorescent receptors localized in intracellular acidic compartments (i.e.&#160;endosomes, trafficking vesicles)&#160;from fluorescent receptors incorporated to the plasma membrane and thus exposed to the extracellular neutral pH7. We took advantage of this to monitor the plasma membrane localization of plexinA1, an axon guidance receptor mediating the growth cone response to the midline repellent semaphorin 3B5 (Figure 1A). We describe here the in vitro characterization of a pHluorin-plexinA1 construct, along with in ovo electroporation8-10 of this construct in the developing chick spinal cord followed by the microscopic analysis of cryosections which enable to follow the axon guidance receptor dynamics in vivo with both spatial and temporal resolutions.

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			<td height="21" style="height:21px;width:264px;">COS7 cells</td>
			<td style="width:147px;">ATCC</td>
			<td style="width:219px;">CRL-1651</td>
			<td style="width:379px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">DMEM GlutaMAX</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">61965-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sodium pyruvate</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">11360-039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fetal bovine serum</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">10270-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:264px;">Exgen500 reagent</td>
			<td style="width:147px;">Euromedex Fermentas</td>
			<td style="width:219px;">ET0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">PBS -Ca2+ -Mg2+</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">14190-094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">F7252</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">32% Paraformaldehyde aqueous solution</td>
			<td style="width:147px;">Electron Microscopy</td>
			<td style="width:219px;">15714-S</td>
			<td>Dilute extemporaneously in PBS to achieve a 4% solution</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Gelatin from cold water fish skin</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">G7041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Sucrose</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">S0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryomount</td>
			<td style="width:147px;">Histolab</td>
			<td style="width:219px;">00890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Hoechst 34580</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:219px;">H21486</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Mowiol 4-88</td>
			<td style="width:147px;">Fluka</td>
			<td style="width:219px;">81381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Bottom-glass 35 mm dish</td>
			<td style="width:147px;">MatTek</td>
			<td style="width:219px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">SS-05S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
			<td style="width:147px;">Terumo</td>
			<td style="width:219px;">NN-2025R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Capillaries</td>
			<td style="width:147px;">CML</td>
			<td style="width:219px;">PP230PO</td>
			<td>capillaries are stretched manually in the flame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td>4951PLUS</td>
			<td></td>
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			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Curved scissors</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">129-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Microscalpel</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">10316-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Forceps</td>
			<td style="width:147px;">FST</td>
			<td style="width:219px;">Dumont #5 REF#11254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:1008px;">Equipment/software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:219px;">Observer 1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">CO2 module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Metamorph software</td>
			<td style="width:147px;">Metamorph</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:264px;">Eggs incubator</td>
			<td style="width:147px;">Sanyo</td>
			<td style="width:219px;">MIR154</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electroporator apparatus</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Electrodes</td>
			<td style="width:147px;">Nepa Gene CO., LTD</td>
			<td style="width:219px;">CUY611P7-4</td>
			<td>4 mm platinum electrodes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluorescence stereomicroscope</td>
			<td style="width:147px;">LEICA</td>
			<td style="width:219px;">MZ10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Cryostat</td>
			<td style="width:147px;">MICROM</td>
			<td style="width:219px;">HM550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Confocal microscope</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;">FV1000, X81</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Fluoview software</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CLC Main Workbench software</td>
			<td style="width:147px;">CLC Bio</td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

use of phluorin to assess the dynamics of axon guidance receptors in cell culture and in the chick embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50883/50883fig1highres.jpg" src="/files/ftp_upload/50883/50883fig1.jpg" /> 
Figure 1.&#160; A. Scheme of the pHluorin-plexinA1 fluorescence properties in a cellular context. PHluorin is nonfluorescent in intracellular compartments where the pH is acidic (&#60;6) such as in trafficking vesicules or in endosomes and is fluorescent when exposed to the extracellular medium where th...

Watch this Scientific Journal Video about Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo at JoVE.com

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Research

JoVE Journal

Neuroscience

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

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Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

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The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...