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 receptor, a kind gift of Dr. Jacob2). If necessary, add a restriction site to the 5&#8217; end of the primer in order to facilitate the cloning step in the backbone.</li>
	<li>Insert the pHluorin sequence in frame between the signal peptide and the receptor coding sequence using restriction sites (e.g.&#160;NruI/Kpn2I restriction sites as described in Figure 1B). 
	Note: Because the signal peptide that ensures the correct targeting of the receptors is cleaved, the pHluorin should be placed after it. This warrants the recognition of the signal peptide and prevents cleavage of the pHluorin from the receptor of interest.</li>
	<li>Sequence the constructs obtained to ensure that no mutation was introduced by PCR.</li>
</ol>
2. Characterization of pHluorin-tagged Receptor In vitro in COS7 Cells
The ability of the fusion protein to reach the plasma membrane and its reversible loss of fluorescence as pH is lowered can be confirmed using the following procedure.
<ol>
	<li>Day 1. Plate 1.5 x 105 COS7 cells in a glass-bottom 35 mm dish in 2 ml&#160;of complete Dulbecco&#8217;s Modified Eagle Medium (DMEM - 10% Fetal Bovine Serum - 1 mM sodium pyruvate - 25 U/ml penicillin/stretomycin - 2.5 &#181;g/ml&#160;Amphotericin B - pH 7.4).</li>
	<li>Day 2. Transfect cells: 
	Note: Cells should be 70-80% confluent.
	<ol>
		<li>Prepare 200 &#181;l&#160;NaCl 150 mM and add 3 &#181;g DNA i.e.&#160;the vector encoding pHluorin-tagged receptor. Gently vortex and spin down briefly. 
		Note: A map of the pHluorin-plexinA1 vector used in these experiments is shown in Figure 1B.</li>
		<li>Add 10 &#181;l&#160;of transfection reagent (or the appropriate amount of the reagent used). Vortex immediately.</li>
		<li>Incubate 10 min&#160;at RT.</li>
		<li>Add 200 &#181;l&#160;of the transfection reagent/DNA mix to the cells.</li>
		<li>Move the plate gently to achieve repartition of the mix and place the cells back to the 37 &#176;C incubator.</li>
	</ol>
	</li>
	<li>Day 3. Remove transfection medium and replace it by 2 ml&#160;of fresh complete DMEM.</li>
	<li>Day 4. Perform live cell imaging of COS7 pHluorin-plexinA1 transfected cells:
	<ol>
		<li>Prepare two aliquots of DMEM complete medium and adjust pH to 3.5 and to 9.5, respectively. 
		Note: For one 35 mm plate, 1.5 ml&#160;of each solution is needed to perform the experiment.</li>
		<li>Remove cell medium and replace it by 1 ml&#160;of DMEM complete medium pH 7.4.</li>
		<li>Prepare a 5 ml&#160;syringe with an appropriate type of tubing in order to inject various components directly into the cell culture medium without opening the microscope chamber.</li>
		<li>Use a module that allows maintenance of a 37 &#176;C,&#160;5% CO2 humid working atmosphere. 
		Note: An alternate approach to the use of a CO2 chamber is to use HEPES-buffered medium (usually in the range of 10-25 mM according to the cell type).</li>
		<li>Place cells in the chamber and adjust the tubing and the syringe. 
		Note: The microscope should be equilibrated before starting to avoid mechanical drift during the recording.</li>
		<li>Open the imaging software and select the multidimensional acquisition program.</li>
		<li>Find transfected COS7 cells with the 40X&#160;objective and mark position in the software for each of them.</li>
		<li>Configure Z stack in order to have a 15 &#181;m depth acquisition (the focus can change when adding media in the plate).</li>
		<li>Set up exposure for the GFP filter and the Phase.</li>
		<li>Configure acquisition timing. 
		Note: For the whole experiment with 5 fields of interest, acquisition every 20 sec&#160;for 10 min&#160;should be sufficient.</li>
		<li>Start acquisition and take 5 control images in DMEM pH 7.4 medium.</li>
		<li>Pause acquisition, inject 1.25 ml&#160;of pH 3.5 complete DMEM to achieve a pH of 5.5 in the culture medium, mark event in the software and resume acquisition for 5 more time points. 
		Note: Green fluorescence should progressively disappear.</li>
		<li>Pause acquisition, inject 1.2 ml&#160;of pH 9.5 complete DMEM to achieve a pH 7.4 in the culture medium, mark event in the software and resume acquisition for 5 more time points. 
		Note: Green fluorescence should reappear at the plasma membrane.</li>
		<li>Analyze images. 
		Figure 2 shows representative images obtained with such a protocol with the pHluorin-plexinA1 construct.</li>
	</ol>
	</li>
</ol>
3. In ovo&#160;Electroporation of pHluorin-plexinA1 Construct
<ol>
	<li>Handling of eggs before electroporation:
	<ol>
		<li>Store fertilized eggs in a fridge at 14 &#176;C up to one week prior to incubation.</li>
		<li>Incubate eggs at 38.5 &#176;C (101.3 &#176;F) in an incubator with saturated humidity for 50-52 hr&#160;until the embryos reach stage HH1412. 
		Note: The eggs must be placed horizontally during incubation so that the embryo is properly positioned for electroporation, floating on the top of the yolk. HH14 stage is suitable to obtain expression of the plasmids in differentiated neurons in the spinal cord and in the dorsal root ganglion with an appropriate survival rate.</li>
	</ol>
	</li>
	<li>Electroporate embryos8-10:
	<ol>
		<li>Prepare electroporation:
		<ol>
			<li>Prepare endotoxin-free DNA plasmids with a concentration superior to 2 &#181;g/&#181;l, to be able to dilute it as the desired concentration.</li>
			<li>Pull enough glass capillaries to inject the different DNA solutions.</li>
			<li>Prepare sterile PBS (-Ca2+; -Mg2+) - 100 U/ml penicillin/streptomycin and equilibrate at 38.5 &#176;C.</li>
			<li>Sterilize the hood, curved scissors and fine forceps.</li>
			<li>Control the electrode spacing. 
			Note: A 4 mm space between the electrodes is generally used.</li>
		</ol>
		</li>
		<li>Window the egg13 (Figure 3A):
		<ol>
			<li>Use curved scissors to pierce the shell on the blunt side of the egg.</li>
			<li>Remove 2 ml of albumen using a 0.9 mm x 25 mm needle and a 5 ml&#160;syringe. Orient the needle vertically in order to avoid damaging the yolk sac.</li>
			<li>Cover the top of the egg with tape to maintain the integrity of the shell.</li>
			<li>Using curved scissors, pierce the shell in the middle of the tape to equalize the pressure when removing 2 ml of albumen from the egg. Then, cut a window large enough to visualize the embryo and be able to work on it.</li>
			<li>Add ~2 ml&#160;sterile warm PBS (-Ca2+; -Mg2+) - 100 U/ml penicillin/streptomycin to avoid dehydration of the embryo and make it more accessible to the manipulator.</li>
		</ol>
		</li>
		<li>Inject DNA and electroporate the embryo
		<ol>
			<li>Dilute plasmid in PBS (-Ca2+; -Mg2+) to a concentration between 0.5-2 &#181;g/&#181;l and add Fast green dye to reach a final 0.025% concentration. Load the DNA mix in a capillary. The use of an injector is recommended. 
			Note: Check that the capillary resistance is neither too large (meaning there could be difficulties when injecting embryos) nor too small (meaning that the capillary could be too big and could damage the embryo). Also, concentration of nucleic acids higher than 2 &#181;g/&#181;l may cause unspecific effects and needs to be controlled.</li>
			<li>Puncture the yolk-sac and the neural tube at the caudal side with the loaded capillary. Enter the neural tube with a shallow angle and fill the lumen from the tail to the head with the DNA mix (Figure 3B). 
			Note: Fast green allows controlling the accuracy of the injection.</li>
			<li>Quickly place the 4 mm platinum electrodes on either side of the neural tube at the level you wish to electroporate and apply 3 pulses at 31 V for 50 msec with 500 msec intervals (Figure 3C). 
			Note: Avoid placing the electrodes on the heart or on big extra embryonic vessels to avoid damaging the developing embryo. Bubbles should form on the electrodes.</li>
			<li>With the needle, remove 2 ml of albumen to decrease the level in the shell.</li>
			<li>Seal the window and the blunt side hermetically with tape.</li>
			<li>Put the eggs back at 38.5 &#176;C in the incubator until they reach the desired stage.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. Embryos Embedding and Cryosectioning
<ol>
	<li>48 hr&#160;after electroporation, carefully harvest electroporated embryos (HH24 stage). Cut the tape and half of the chorioallantoid membrane. To prevent embryos from sinking into the yolk, position a colander under the embryo and cut the second half of the chorioallantoid membrane.</li>
	<li>Transfer the embryo to a dissection dish filled with ice cold PBS.</li>
	<li>Check electroporation efficiency by looking for fluorescence in the neural tube with a fluorescence stereo microscope. 
	Note : Coelectroporation of a control plasmid encoding the RFP may help to visualize the electroporated area.</li>
	<li>Dissect embryos using a microscalpel in order to select the electroporated area of the spinal cord.</li>
	<li>Transfer dissected embryos to a 24-well plate and fix in pH 7.4 4% Paraformaldehyde (PFA) - Phosphate Buffer Saline (PBS), O/N&#160;at 4 &#176;C. 
	Note: Fixation step is crucial to allow the stabilization of pHluorin in its &#8220;live&#8221; conformation and thus to be able to use pHluorin in fixed/permeabilized tissue. Although fixation slows drastically pH-dependent change of fluorescence, one must take into account that conformational/protonation changes can still occur after fixation. Thus, the following protocol (embedding, cryosections and observation) has to be performed within 3 days after the fixation step, with all buffers at pH 7. If fixation is not required, performing observation on live tissue sections is recommended.</li>
	<li>Remove 4% PFA and wash embryos in pH 7.4 PBS.</li>
	<li>Incubate embryos in PBS-15% sucrose and keep at 4 &#176;C until the embryos sink.</li>
	<li>Incubate fixed embryos in pH 7.4 7.5% gelatin- 15% sucrose for 45 min&#160;at 37 &#176;C so&#160;embryos are completely embedded.</li>
	<li>Place embedding molds on ice and add 400 &#181;l&#160;of pH 7.4 7.5% gelatin- 15% sucrose to achieve a solid 2 mm base.</li>
	<li>Aspirate the embedded embryo with a cut tip and place the embryo on the solid gelatin base.</li>
	<li>Cover with pH 7.4 7.5% gelatin- 15% sucrose and position the embryo with forceps before the gelatin solidifies.</li>
	<li>Once the gelatin is solid, prepare a -40 &#176;C isopropanol bath (use dry ice or liquid nitrogen) and freeze the gelatin block for 5 min.</li>
	<li>Keep the frozen blocks at -80 &#176;C.</li>
	<li>Place the frozen block at -20 &#176;C for 1 hr.</li>
	<li>Remove the mold and fix the block on a chuck with a polyethylene glycol medium.</li>
	<li>Once the block is tightly fixed, place the chuck in the cryostat system. 
	Note: Use coated slides to avoid tissue loss during staining.</li>
	<li>Perform serial cryosections (20 &#181;m cryosections are usually performed).</li>
	<li>Let the cryosections dry for 15 min&#160;at RT. 
	Note: cryosections should be protected from unnecessary light exposure in order to avoid bleaching of the GFP fluorescence.</li>
</ol>
5. Microscopic Analysis of Cryosections
<ol>
	<li>Rehydrate cryosections in pH 7.4 PBS at RT for 10 min. 
	Note: If needed, nuclei can be stained with Hoechst.</li>
	<li>Use a 0.5 &#181;g/ml&#160;Hoechst solution in PBS and incubate cryosections for 15 min.</li>
	<li>Rinse the slides 3x with pH 7.4 PBS for 5 min.</li>
	<li>Proceed to slide mounting. A pH 7.4 (or more basic) polyvinyl alcohol mounting solution that hardens O/N&#160;can be used: carefully position the coverslip to avoid the formation of air bubbles between the slide and the coverslip.</li>
	<li>Let the mounting medium harden O/N at 4 &#176;C in the dark.</li>
	<li>Use an inverted confocal microscope to precisely visualize the pHluorin-plexinA1 fusion protein: perform z-stack at the optimal pinhole and optical resolution and use a 20X (NA 0.75) or 40X (NA 1.3) lenses. 
	Note: pHluorin is detectable with the same parameters used to detect GFP (i.e. emission peak at 509 nm). Wavelength excitation and detection filter settings are optimally defined by the imaging software. Hoechst is detected between 425-460 nm (excitation is at 405 nm), GFP or pHluorin is detected between 485-545 nm (excitation is at 473 nm) and RFP is detected between 575-675 nm (excitation is at 559 nm).</li>
</ol>
Representative images of pHluorin-plexinA1 and eGFP expression in the chick embryo spinal cord are shown in Figure 4.

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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>

The authors have nothing to disclose.

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;">DMEM GlutaMAX</td>
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			<td style="width:219px;">11360-039</td>
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			<td height="21" style="height:21px;width:264px;">Amphotericin B</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:219px;">A2942</td>
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		<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>
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			<td height="21" style="height:21px;width:264px;">Penicillin/Streptomycin</td>
			<td style="width:147px;">GIBCO</td>
			<td style="width:219px;">15140-122</td>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:264px;">Fast green dye</td>
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			<td style="width:219px;">F7252</td>
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		<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>
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			<td height="21" style="height:21px;width:264px;">Cryomount</td>
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			<td style="width:219px;">81381</td>
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			<td colspan="4" height="21" style="height:21px;width:1008px;">Consumables</td>
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			<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>
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			<td height="21" style="height:21px;width:264px;">5 ml Syringe</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:264px;">Needles 0.9 mm x 25 mm</td>
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			<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>
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			<td height="21" style="height:21px;width:264px;">Superfrost Plus Slides</td>
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			<td>4951PLUS</td>
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			<td colspan="4" height="21" style="height:21px;width:1008px;">Material</td>
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		<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>
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		<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>
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			<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>
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			<td height="21" style="height:21px;width:264px;">Time lapse microscope</td>
			<td style="width:147px;">Zeiss</td>
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			<td height="20" style="height:20px;width:264px;">Temp module S</td>
			<td style="width:147px;">PECON for Zeiss</td>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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 ...

use-phluorin-to-assess-dynamics-axon-guidance-receptors-cell-culture

Research

JoVE Journal

Neuroscience

8.2K vistas.  University of Lyon. Se describe aquí el uso de una variante de proteína fluorescente verde sensible al pH, pHluorin, para estudiar la dinámica espacio-temporal de los receptores de guía de axones que se trafficking en la superficie celular. El receptor marcado con pHluorin se expresa tanto en cultivo celular como in vivo,utilizando la electroporación del embrión de pollito.

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

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 insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

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

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

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 undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

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, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

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 eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

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