This work was supported by Florida Atlantic University Office of Undergraduate Research and Inquiry grant (M.J.S.), Florida Department of Health Ed and Ethel Moore Pilot Grant 20A17 (Q.Z.), Alzheimer&#39;s Association grant AARG-NTF-19-618710 (Q.Z.), and NIA R21 grant AG061656-01A1 (Q.Z.).

The following protocol includes (1) a simplified procedure for establishing mouse hippocampal and cortical cultures based on a well-established protocol22, (2) a brief introduction to an epifluorescence microscope setup for live neurons, (3) a detailed description of loading and imaging ND6 in mouse neurons, (4) a discussion about the quantification of membrane trafficking by ND6 signal. All procedures follow the biosafety and IACUC guidelines at the Florida Atlantic University. The synthesis of ND6 has been described previously20.
1. Preparation of mouse hippocampal and cortical cultures
NOTE: If not specified otherwise, all steps must be performed in a biosafety level 2 laminar flow hood. Sterilize all tools and materials.
<ol>
	<li>Use size 5 forceps to place glass coverslips in multiwell culture plates. 
	NOTE: For example, a 24-well plate is ideal for 12 mm <img alt="Equation 1" src="/files/ftp_upload/62717/62717eq01v2.jpg" /> coverslips.</li>
	<li>Add the appropriate volume of extracellular matrices to coat the cell culture surface (e.g., 75 &#956;L of basement membrane matrix solution per 12 mm <img alt="Equation 1" src="/files/ftp_upload/62717/62717eq01v2.jpg" /> coverslip; see the Table of Materials)</li>
	<li>Place the prepared plates in the tissue culture incubator (5% CO2, 100% humidity, and 37 &#176;C) for 1-4 h to allow basement membrane matrix crosslinking.</li>
	<li>Sacrifice the animals, open the skull, and transfer the whole brain to a 35 mm Petri dish with 3 mL of Hank&#39;s Balanced Salt Solution (HBSS) containing 20% fetal bovine serum (H+20). Cut the brain along the midline, and separate the cortices and hippocampi in a laminar flow dissection hood to reduce contamination.</li>
	<li>Use microscissors to cut the tissues into small pieces (~1 mm3). Transfer those tissues to a 15 mL conical tube containing 5 mL of H+20.</li>
	<li>Wash the tissues three times with 5 mL of H+20 and three times with 5 mL of HBSS.</li>
	<li>Add 1.5 mL of 1% trypsin with ethylenediamine tetraacetic acid and incubate at 37 &#176;C for 10 min for enzyme digestion.</li>
	<li>Repeat step 1.6 and use vacuum to aspirate HBSS in the end.</li>
	<li>Dissociate the tissues mechanically in 1 mL of HBSS containing 2.95 g/L MgSO4&#8226;7H2O using a fire-polished glass pipette.</li>
	<li>Centrifuge the cell suspension at 400 &#215; g, 4 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant and resuspend the cells in an appropriate volume of culture media&#160;to obtain a concentration of ~10,000,000 cells/ml.</li>
	<li>Plate the cells at ~1,000,000 cells/cm2 and place the culture in the incubator (5% CO2, 100% humidity, and 37 &#176;C) for 1-4 h to facilitate cell attachment to the glass coverslips.</li>
	<li>Add an appropriate volume of culture medium (e.g., 1 mL per well for a 24-well plate). Add the same volume of culture medium after 1 week. After 2 weeks, replace half of the existing culture medium with fresh media&#160;every week.</li>
</ol>
2. Microscope setup for live-cell fluorescence imaging
NOTE: An exemplary imaging setup (Figure 5) includes at least an inverted fluorescence microscope (see the Table of Materials), fluorescence light source with automatic shutter, fluorescence filter sets (e.g., for imaging ND6, use 405/10 BP for excitation, 495 LP for dichroic, and 510/20 BP for emission), and a high-sensitivity camera (Table of Materials), all of which are controlled by image acquisition software (Table of Materials).
<ol>
	<li>Prepare an imaging chamber that allows temperature control and solution input/output for live-cell fluorescence imaging. 
	NOTE: For example, a modified open-bath imaging chamber fixed on a heating platform was used in this protocol (Figure 6).</li>
	<li>Set up a programable device to switch the perfusion solutions and deliver the electric stimulus at defined time points during imaging. 
	NOTE: Hardware and software synchronization is necessary for quantitative analyses (Figure 7). For example, in this protocol, a trigger output from the imaging camera was used to start a computer program that controls an automatic switch device, which controls a multichannel perfusion system and a square-pulse stimulator.</li>
	<li>To co-image two or more fluorescent reporters, perform multichannel fluorescence imaging either by sequentially switching filter sets or by simultaneously splitting different fluorescence emissions and projecting to the same camera.</li>
</ol>
3. Loading and imaging ND6 in neuronal cultures
<ol>
	<li>Weigh out an appropriate amount of ND6, dissolve it in dimethylsulfoxide (DMSO), and allow it to solubilize at room temperature; sonicate briefly (e.g., 3 min). Filter the crude stock solution using a 0.22 &#956;m filter to remove large dye aggregates. After filtration, determine the dye concentration by absorption spectroscopy at 405 nm using a conventional or microvolume spectrophotometer using &#949; = 10,700 M-1 cm-1.</li>
	<li>Before application, dilute the stock solution to a concentration of 1 &#956;M&#160;using the bath solution. Keep the stock solution at room temperature in the dark.</li>
	<li>Labeling of endosomes and synaptic vesicles
	<ol>
		<li>To ubiquitously label endocytosed membrane compartments such as endosomes, add ND6 stock solution (e.g., 1 mM in DMSO) to the culture medium at the final concentration of 1 &#181;M, and incubate at 5% CO2, 100% humidity, and 37 &#176;C for 30 min. To reduce the extent of SV labeling, suppress the spontaneous neuronal activity pharmacologically. For example, use tetrodotoxin to block action potentials or 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) and D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) to inhibit excitatory transmission23.</li>
		<li>To selectively label SVs, use brief but strong stimulation to evoke presynaptic exo-/endocytosis. First, transfer the culture coverslip(s) to a 35 mm <img alt="Equation 1" src="/files/ftp_upload/62717/62717eq01v2.jpg" /> Petri dish containing 2 mL of normal Tyrode&#39;s solution at room temperature. Second, dilute the ND6 stock solution in high-K+ Tyrode&#39;s solution (90 mM KCl) at the final concentration of 1 &#956;M. Third, replace the normal Tyrode&#39;s solution in the Petri dish with ND6-containing high-K+ Tyrode&#39;s solution and incubate at room temperature for 2 min.</li>
	</ol>
	</li>
	<li>Transfer the ND6-labeled cell culture to the imaging chamber filled with prewarmed normal Tyrode&#39;s solution containing 10 &#956;M NBQX and 10 &#956;M D-AP5.</li>
	<li>Adjust the perfusion speed to ~0.2 mL/s (i.e., 1 drop per second) and start the perfusion of prewarmed normal Tyrode&#39;s solution containing NBQX and D-AP5 to remove excessive ND6 in the culture.</li>
	<li>Adjust the focus and locate the appropriate field of view containing healthy and well-spread neurons bearing connected neurites. Avoid areas containing unresolved dye colloids.</li>
	<li>Try imaging ND6-loaded cells with different exposure times to identify the best imaging settings. 
	NOTE: For the best exposure time, the highest pixel fluorescence intensity in the resulting image is about half of the bit range (e.g., for a 16-bit image, the bit range is from 0 to 65,535), which will allow a further increase in fluorescence when the acidic bath solution is applied. The selected exposure time should be used for all ND6 imaging.</li>
	<li>Set up the stimulation and perfusion protocol, frame interval, and total duration. For example, in the following protocol, use a 30 s baseline, a 10 s 30 Hz electric field stimulation, 50 s recovery, 120 s 90 mM K+, 60 s recovery, 60 s Tyrode&#39;s solution with 50-mM NH4Cl, 60 s Tyrode&#39;s solution at pH 5.5, and a frame interval of 3 s.</li>
	<li>Start the image acquisition accompanied by the synchronized stimulation and perfusion. Monitor the simulation and solution exchange during imaging.</li>
	<li>Stop the perfusion after the imaging ends, remove the coverslip, and clean the imaging chamber for the next trial.</li>
</ol>
4. Quantification of membrane trafficking by a change in ND6 fluorescence
<ol>
	<li>Back up and/or make an electronic copy of all image files.</li>
	<li>Choose an analysis program for data extraction, e.g., an open-source image analysis software such as ImageJ24 and/or FIJI25.</li>
	<li>Open or import an image stack to the analysis program.</li>
	<li>Set the first image as the reference and align the rest to it using functions/plugins such as Rigid Registration26, which will mitigate artifacts due to xy-drifting. Refer to Figure 8 for example images.</li>
	<li>Average all images acquired during the 30-s pre-baseline to produce a reference image. Save a copy of this image for future reference.</li>
	<li>Set the intensity threshold to generate a binary image from the baseline-averaged image.</li>
	<li>Use Watershed in ImageJ or similar functions in another program to separate connecting neurites or cells.</li>
	<li>Use the Analyze Particle function with appropriate area size and circularity to solicit regions of interest (ROIs) corresponding to cell membranes, endosomes, lysosomes, or synaptic boutons. Save all selected ROIs.</li>
	<li>Select four background ROIs in cell-free regions within the field of view.</li>
	<li>Measure the average pixel intensities for each ROI in every frame of the image stack, and export the results for statistical analysis.</li>
	<li>Calculate the mean intensity of the background ROIs as the baseline noise, which will be subtracted from the average pixel intensities for each ROI.</li>
	<li>Average the three highest average intensities for every selected ROI during the application of pH 5.5 Tyrode&#39;s solution to obtain the maximal fluorescence intensity, which is defined as 100% for normalization.</li>
	<li>Average the three lowest average intensities for every selected ROI during the application of 50 mM NH4Cl to set the minimal fluorescence intensity, which is defined as 0% for normalization.</li>
	<li>Calculate the relative fluorescence changes for every ROI based on its own 0% and 100% intensities. Derive mean fluorescence changes, change kinetics, and other values/plots from individual ROI data.</li>
</ol>

<ol>
	<li>Polo, S., Pece, S., Di Fiore, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+and+cancer.">Endocytosis and cancer.</a> Current Opinion in Cell Biology. 16 (2), 156-161 (2004).</li><li>Eckert, G. P., Wood, W. G., Muller, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+membranes+and+beta-amyloid:+a+harmful+connection.">Lipid membranes and beta-amyloid: a harmful connection.</a> Current Protein and Pept Science. 11 (5), 319-325 (2010).</li><li>Augustine, G. J., Burns, M. E., DeBello, W. M., Pettit, D. L., Schweizer, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytosis:+proteins+and+perturbations.">Exocytosis: proteins and perturbations.</a> Annual Review of Pharmacology and Toxicology. 36, 659-701 (1996).</li><li>Ammar, M. R., Kassas, N., Chasserot-Golaz, S., Bader, M. F., Vitale, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipids+in+regulated+exocytosis:+what+are+they+doing.">Lipids in regulated exocytosis: what are they doing.</a> Frontiers in Endocrinology. 4, 125 (2013).</li><li>Jahn, R., Lang, T., Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion.">Membrane fusion.</a> Cell. 112 (4), 519-533 (2003).</li><li>Chabanon, M., Stachowiak, J. C., Rangamani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems+biology+of+cellular+membranes:+a+convergence+with+biophysics.">Systems biology of cellular membranes: a convergence with biophysics.</a> Wiley Interdisciplinary Reviews. Systems Biology and Medicine. 9 (5), 1386 (2017).</li><li>Prosser, D. C., Wrasman, K., Woodard, T. K., O'Donnell, A. F., Wendland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+pHluorin+for+quantitative,+kinetic+and+high-throughput+analysis+of+endocytosis+in+budding+yeast.">Applications of pHluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast.</a> Journal of Visualized Experiments: JoVE. (116), e54587 (2016).</li><li>Burrone, J., Li, Z., Murthy, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+vesicle+cycling+in+presynaptic+terminals+using+the+genetically+encoded+probe+synaptopHluorin.">Studying vesicle cycling in presynaptic terminals using the genetically encoded probe synaptopHluorin.</a> Nature Protocols. 1 (6), 2970-2978 (2006).</li><li>Miesenb&#246;ck, 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 (6689), 192-195 (1998).</li><li>Chan, Y. -. H. M., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+membrane+systems+and+their+applications.">Model membrane systems and their applications.</a> Current Opinion in Chemical Biology. 11 (6), 581-587 (2007).</li><li>Demchenko, A. P., M&#233;ly, Y., Duportail, G., Klymchenko, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+biophysical+properties+of+lipid+membranes+by+environment-sensitive+fluorescent+probes.">Monitoring biophysical properties of lipid membranes by environment-sensitive fluorescent probes.</a> Biophysical Journal. 96 (9), 3461-3470 (2009).</li><li>Hoopmann, P., Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+synaptic+vesicle+recycling+by+staining+and+destaining+vesicles+with+FM+dyes.">Imaging synaptic vesicle recycling by staining and destaining vesicles with FM dyes.</a> Cold Spring Harbor Protocols. 2012 (1), 77-83 (2012).</li><li>Gaus, K., Zech, T., Harder, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+membrane+microdomains+by+Laurdan+2-photon+microscopy.">Visualizing membrane microdomains by Laurdan 2-photon microscopy.</a> Molecular Membrane Biology. 23 (1), 41-48 (2006).</li><li>Kahms, M., Klingauf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+pH-sensitive+lipid+based+exo-endocytosis+tracers+reveal+fast+intermixing+of+synaptic+vesicle+pools.">Novel pH-sensitive lipid based exo-endocytosis tracers reveal fast intermixing of synaptic vesicle pools.</a> Frontiers in Cellular Neuroscience. 12, 18 (2018).</li><li>Zhou, L., Xie, L., Liu, C., Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+trends+of+molecular+probes+based+on+the+fluorophore+4-amino-1,8-naphthalimide.">New trends of molecular probes based on the fluorophore 4-amino-1,8-naphthalimide.</a> Chinese Chemical Letters. 30 (10), 1799-1808 (2019).</li><li>Tomczyk, M. D., Walczak, K. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=l,8-Naphthalimide+based+DNA+intercalators+and+anticancer+agents.+A+systematic+review+from+2007+to+2017.">l,8-Naphthalimide based DNA intercalators and anticancer agents. A systematic review from 2007 to 2017.</a> European Journal of Medicinal Chemistry. 159, 393-422 (2018).</li><li>Ulla, 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=Blue+emitting+1,8-naphthalimides+with+electron+transport+properties+for+organic+light+emitting+diode+applications.">Blue emitting 1,8-naphthalimides with electron transport properties for organic light emitting diode applications.</a> Journal of Molecular Structure. 1143 (5), 344-354 (2017).</li><li>Duke, R. M., Veale, E. B., Pfeffer, F. M., Kruger, P. E., Gunnlaugsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+and+fluorescent+anion+sensors:+An+overview+of+recent+developments+in+the+use+of+1,8-naphthalimide-based+chemosensors.">Colorimetric and fluorescent anion sensors: An overview of recent developments in the use of 1,8-naphthalimide-based chemosensors.</a> Chemical Society Reviews. 39 (10), 3936-3953 (2010).</li><li>Panja, S. K., Dwivedi, N., Saha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+the+intramolecular+charge+transfer+(ICT)+process+in+push-pull+systems:+Effect+of+nitro+groups.">Tuning the intramolecular charge transfer (ICT) process in push-pull systems: Effect of nitro groups.</a> RSC Advances. 6 (107), 105786-105794 (2016).</li><li>Thomas, D., 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=Solvatochromic+and+pH-sensitive+fluorescent+membrane+probes+for+imaging+of+live+cells.">Solvatochromic and pH-sensitive fluorescent membrane probes for imaging of live cells.</a> ACS Chemical Neuroscience. 12 (4), 719-734 (2021).</li><li>Leslie, K. G., Jacquemin, D., New, E. J., Jolliffe, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+breadth+of+4-amino-1,8-naphthalimide+photophysical+properties+through+substitution+of+the+naphthalimide+core.">Expanding the breadth of 4-amino-1,8-naphthalimide photophysical properties through substitution of the naphthalimide core.</a> Chemistry - A European Journal. 24 (21), 5569-5573 (2018).</li><li>Liu, G. S., 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=Properties+of+synaptic+transmission+at+single+hippocampal+synaptic+boutons.">Properties of synaptic transmission at single hippocampal synaptic boutons.</a> Nature. 375 (6530), 404-408 (1995).</li><li>Zhang, Q., Cao, Y. -. Q., 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=Quantum+dots+provide+an+optical+signal+specific+to+full+collapse+fusion+of+synaptic+vesicles.">Quantum dots provide an optical signal specific to full collapse fusion of synaptic vesicles.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 104 (45), 17843-17848 (2007).</li><li>Rueden, C. 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=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18 (1), 529 (2017).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Transactions on Image Processing. 7 (1), 27-41 (1998).</li><li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synaptic+vesicle+cycle.">The synaptic vesicle cycle.</a> Annual Review of Neuroscience. 27, 509-547 (2004).</li><li>Lazarenko, R. M., DelBove, C. E., Strothman, C. E., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium+chloride+alters+neuronal+excitability+and+synaptic+vesicle+release.">Ammonium chloride alters neuronal excitability and synaptic vesicle release.</a> Scientific Reports. 7 (1), 5061 (2017).</li><li>Wilhelm, B. G., 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=Composition+of+isolated+synaptic+boutons+reveals+the+amounts+of+vesicle+trafficking+proteins.">Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins.</a> Science. 344 (6187), 1023-1028 (2014).</li><li>Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools.">Synaptic vesicle pools.</a> Nature Reviews. Neuroscience. 6 (1), 57-69 (2005).</li><li>DelBove, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+modulation+between+amyloid+precursor+protein+and+synaptic+membrane+cholesterol+revealed+by+live+cell+imaging.">Reciprocal modulation between amyloid precursor protein and synaptic membrane cholesterol revealed by live cell imaging.</a> Neurobiology of Disease. 127, 449-461 (2019).</li><li>Dason, J. S., Smith, A. J., Marin, L., Charlton, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicular+sterols+are+essential+for+synaptic+vesicle+cycling.">Vesicular sterols are essential for synaptic vesicle cycling.</a> Journal of Neuroscience. 30 (47), 15856-15865 (2010).</li><li>Chanaday, N. L., Cousin, M. A., Milosevic, I., Watanabe, S., Morgan, 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=The+synaptic+vesicle+cycle+revisited:+New+insights+into+the+modes+and+mechanisms.">The synaptic vesicle cycle revisited: New insights into the modes and mechanisms.</a> Journal of Neuroscience. 39 (42), 8209-8216 (2019).</li><li>Betz, W. J., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255 (5041), 200 (1992).</li><li>Afuwape, O. A., Kavalali, 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=Imaging+synaptic+vesicle+exocytosis-endocytosis+with+pH-sensitive+fluorescent+proteins.">Imaging synaptic vesicle exocytosis-endocytosis with pH-sensitive fluorescent proteins.</a> Methods in Molecular Biology. 1474, 187-200 (2016).</li></ol>

The authors declare no competing financial interests.

Lipid-based dyes, such as 1,1&#8242;-dioctadecyl-3,3,3&#8242;,3&#8242;-tetramethylindocarbocyanine (DiI) and 3,3&#8242;-Dioctadecyloxacarbocyanine perchlorate (DiO), have long been used to illustrate cell morphology and track cellular processes such as the axon projections of neurons. Styryl dyes, such as FM1-43, have been invented and used successfully for the study of exocytosis34. Due to their low membrane affinity, they selectively label endocytosed vesicles where they are trapped while dyes r...

The lipid bilayer is one of the most common biomolecule assemblies and is indispensable for all cells. Outside cells, it forms the plasma membrane interfacing cells and their environment; inside cells, it compartmentalizes various organelles specialized for designated functionalities. Rather dynamic than still, lipid membranes constantly experience fusion and fission, which mediates biomaterial transport, organelle reform, morphology change, and cellular communication. Undoubtedly, the lipid membrane is the physical foundation for almost all cellular processes, and its dysfunction plays a crucial role in various disorders ranging from cancer1 to Alzheimer&#39;s disease2. Although lipid molecules are far less diverse than proteins, membrane research so far has mainly been protein-centric. For example, a lot more is known about protein machinery than about lipids in exocytosis3,4,5. Moreover, the organization, distribution, dynamics, and homeostasis of lipids across surface and intracellular membranes largely remain unexplored in comparison to membrane proteins6.
This is not surprising as modern molecular biology techniques, such as mutagenesis, provide a methodological advantage for studying proteins rather than lipids. For example, transgenic tagging of pH-sensitive green fluorescent protein (a.k.a., pHluorin) to vesicular proteins facilitates the quantitative measurement of the amount and rate of vesicular protein turnover during exo-/endocytosis7,8,9. However, it is almost impossible to genetically modify membrane lipids in vivo. Moreover, qualitative and even quantitative manipulations of protein amounts and distributions are much more feasible than those of lipids10. Nevertheless, native and synthetic fluorescent lipids have been isolated and developed to simulate endogenous membrane lipids in vitro and in vivo11. One group of widely used fluorescent lipids are styryl dyes, e.g., FM1-43, which exhibit membrane-enhanced fluorescence and are a powerful tool in studying synaptic vesicle (SV) release in neurons12. Lately, environment-sensitive lipid dyes have been invented and widely used as a new class of reporters to study various cell membrane properties, including membrane potential11, phase order13, and secretion14.
A new class of lipid mimetics whose fluorescence is both pH-sensitive (e.g., pHluorin) and membrane-sensitive (e.g., FM1-43) was developed to directly measure the lipid distribution in the plasma membrane and endosomes/lysosomes and the lipid traffic during exo-/endocytosis. The well-known solvatochromic fluorophores exhibiting push-pull characteristics due to intramolecular charge transfer were selected for this purpose. Among existing solvatochromic fluorophores, the 1,8-naphthalimide (ND) scaffold is relatively easy to modify, versatile for tagging, and is unique in photo-physics15 and has therefore been used in DNA intercalators, organic light-emitting diodes, and biomolecule sensors16,17,18.
Attaching an electron-donating group to the C4 position of the ND scaffold generates a push-pull structure, which leads to an increased dipole moment by redistributing the electron density in the excited state19,20. Such an intramolecular charge transfer produces large quantum yields and Stokes shifts, resulting in bright and stable fluorescence21. This group has recently developed a series of solvatochromic lipid analogs based on the ND scaffold and obtained them with good synthetic yields20.
Spectroscopic characterization shows that among those products, ND6 possesses the best fluorescence properties (Figure 1)20. First, it has well-separated excitation and emission peaks (i.e., ~400 nm and ~520 nm, respectively, in Figure 2A,B) compared to popular fluorophores such as fluorescein isothiocyanate, rhodamine, or GFP, making it spectrally separatable from them and thus useful for multicolor imaging. Second, ND6 fluorescence exhibits a more than eight-fold increase in its fluorescence in the presence of micelles (Figure 2C), suggesting a strong membrane-dependency. Prior live-cell fluorescence imaging studies with different types of cells showed excellent membrane staining by ND620. Third, when the solution&#39;s pH is decreased from 7.5 (commonly found in extracellular or cytosolic environments) to 5.5 (commonly found in endosomes and lysosomes), ND6 shows an approximately two-fold increase in fluorescence (Figure 2D), showing its pH-sensitivity. Moreover, molecular dynamics simulation indicates that ND6 readily integrates into the lipid bilayer with its ND scaffold facing out of the membrane and piperazine residue showing strong interactions with phospholipid head groups (Figure 3). Altogether, these features make ND6 an ideal fluorescent lipid analog to visualize and measure membrane lipid turnover during exo-/endocytosis.
This paper presents a method to study the turnover rate and dynamics of SV lipids using cultured mouse hippocampal neurons. By stimulating neurons with high K+ Tyrode&#39;s solutions, SVs and the plasma membrane were loaded with ND6 (Figure 4A,B). Subsequently, neurons were re-stimulated with different stimuli followed by NH4Cl-containing and pH 5.5 Tyrode&#39;s solutions (Figure 4D). This protocol facilitates the quantitative measurement of the assembled exocytosis and endocytosis rates under different circumstances (Figure 4C).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digidata 1440A Data Acquistion System</td>
			<td>Molecular Devices</td>
			<td>Digidata 1440A</td>
			<td>For synchronized stimulation and solution exchange</td>
		</tr>
		<tr>
			<td>Dual Channel Temperature Controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>OMEGA Scientific</td>
			<td>FB-01</td>
			<td>For making H+20 solution used in dissection and tissue culture</td>
		</tr>
		<tr>
			<td>Hamamatsu Flash4.0 sCOMS camera</td>
			<td>Hamamatsu Inc.</td>
			<td>C13440-20CU</td>
			<td>high-sensitivity camera</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution</td>
			<td>Sigma</td>
			<td>H6648</td>
			<td>For making H+20 solution used in dissection and tissue culture</td>
		</tr>
		<tr>
			<td>Heated Platform</td>
			<td>Warner Instruments</td>
			<td>PH-1</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Micro-G Vibration Isolation Table</td>
			<td>TMC</td>
			<td>63-564</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Micro-manager</td>
			<td>https://micro-manager.org/</td>
			<td>NA</td>
			<td>For image acquisition control</td>
		</tr>
		<tr>
			<td>Multi-Line In-Line Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SHM-6</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Neurobasal Plus Medium</td>
			<td>THermoFisher Scientific</td>
			<td>A3582901</td>
			<td>For tissue culture</td>
		</tr>
		<tr>
			<td>Nikon Ti-E Inverted Microscope</td>
			<td>Nikon</td>
			<td>Ti-E/B</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>ORCA-Flash4.0 Digital CMOS camera</td>
			<td>Hamamatsu</td>
			<td>C1340-20CU</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Perfusion Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26G</td>
			<td>For live-cell imaging</td>
		</tr>
		<tr>
			<td>Six-Channel Valve Control Perfusion System</td>
			<td>Warner Instruments</td>
			<td>VC-6</td>
			<td>For solution exchange</td>
		</tr>
		<tr>
			<td>Square Pulse Stimulator</td>
			<td>Grass Instrument</td>
			<td>SD9</td>
			<td>For electric field stimulation</td>
		</tr>
	</tbody>
</table>

measuring membrane lipid turnover with the ph-sensitive fluorescent lipid analog nd6

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized cells use this process to execute vital body functions such as insulin secretion from &#946; cells and neurotransmitter release from chemical synapses. Owing to its physiological significance, exo-/endocytosis has been one of the most studied topics in cell biology. Many tools have been developed to study this process at the gene and protein level, because of which much is known about the protein machinery participating in this process. However, very few methods have been developed to measure membrane lipid turnover, which is the physical basis of exo-/endocytosis.
This paper introduces a class of new fluorescent lipid analogs exhibiting pH-dependent fluorescence and demonstrates their use to trace lipid recycling between the plasma membrane and the secretory vesicles. Aided by simple pH manipulations, those analogs also allow the quantification of lipid distribution across the surface and the intracellular membrane compartments, as well as the measurement of lipid turnover rate during exo-/endocytosis. These novel lipid reporters will be of great interest to various biological research fields such as cell biology and neuroscience.

SVs are specialized for neurotransmitter release via evoked exo-/endocytosis27. SVs have highly acidic lumen (i.e., pH 5.5), which is ideal for ND6. We used high K+ stimulation to evoke SV exo-/endocytosis in order to allow ND6 to access SV. Expectedly, bright green fluorescent puncta along neuronal processes showed up after loading (Figure 9A). The line profile shown in Figure 4B demonstrated a strong overlap between ND6 (green curve) and ...

Watch this Scientific Journal Video about Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6 at JoVE.com

Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

Exo-/endocytosis is a common process mediating the exchange of biomolecules between cells and their environment and among different cells. Specialized ...

measuring-membrane-lipid-turnover-with-ph-sensitive-fluorescent-lipid

Research

JoVE Journal

Neuroscience

1.5K Views.  The Brain Institute at Florida Atlantic University. This protocol presents a fluorescence imaging method that uses a class of pH-sensitive lipid fluorophores to monitor lipid membrane trafficking during cell exocytosis and the endocytosis cycle.

Video: Measuring Membrane Lipid Turnover with the pH-sensitive Fluorescent Lipid Analog ND6

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of F&#246;rster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.

A method for imaging changes in membrane potential using genetically encoded voltage indicators is described.

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

A Micro-agar Salt Bridge Electrode for Analyzing the Proton Turnover Rate of Recombinant Membrane Proteins

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of membrane carrier proteins reconstituted in lipid bilayer membranes is a powerful but delicate method for the assessment of their physicochemical and pharmacological properties. The substrate turnover number is a unique parameter that allows the comparison of the activity of different membrane proteins. In an electrogenic transport, the gradient of the translocated substrate creates a membrane potential that directly correlates to the substrate turnover rate of the protein. By using silver chloride electrodes, a diffusion potential, also called liquid junction potential, is induced, which alters electrode potential and significantly disturbs precise membrane potential measurements. Diffusion potential can be minimized by a salt bridge, which balances electrode potential. In this article, a micro-agar salt bridge is designed to improve the electrophysiological set-up, which uses micropipettes for the membrane formation. The salt solution is filled into a microcapillary pipette tip, stabilized by the addition of agarose, and can be easily mounted to a standard electrode. The electrode potential of a micro-salt bridge electrode is more stable compared to a standard electrode. The implementation of this system stabilizes electrode potential and allows more precise measurements of membrane potential generated by a pH gradient. Using this system, the proton turnover rates of the mitochondrial carriers UCP1 and UCP3 are reinvestigated and compared to earlier measurements.

In electrophysiological measurements, the presence of a diffusion potential disturbs the precise measurement of the reverse potential by altering the electrode potential. Using a micro-agar salt bridge, the impact of the diffusion potential is minimized, which allows a more precise measurement of substrate turnover numbers of reconstituted recombinant membrane proteins.

To date, more than 50% of all pharmacological drugs target the transport kinetics of membrane proteins. The electrophysiological characterization of ...

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions under various membrane lipid compositions. Among them, the droplet interface bilayer has gained popularity; however, the large membrane size hinders the recording of low electrical background noise. We have established a contact bubble bilayer (CBB) method that combines the benefits of planar lipid bilayer and patch-clamp methods, such as the ability to vary the lipid composition and to manipulate the bilayer mechanics, respectively. Using the setup for conventional patch-clamp experiments, CBB-based experiments can be readily performed. In brief, an electrolyte solution in a glass pipette is blown into an organic solvent phase (hexadecane), and the pipette pressure is maintained to obtain a stable bubble size. The bubble is spontaneously lined with a lipid monolayer (pure lipids or mixed lipids), which is provided from liposomes in the bubbles. Next, the two monolayer-lined bubbles (~50 &#181;m in diameter) at the tip of the glass pipettes are docked for bilayer formation. Introduction of channel-reconstituted liposomes into the bubble leads to the incorporation of channels in the bilayer, allowing for single-channel current recording with a signal-to-noise ratio comparable to that of patch-clamp recordings. CBBs with an asymmetric lipid composition are readily formed. The CBB is renewed repeatedly by blowing out the previous bubbles and forming new ones. Various chemical and physical perturbations (e.g., membrane perfusion and bilayer tension) can be imposed on the CBBs. Herein, we present the basic procedure for CBB formation.

Here, we present a protocol for the formation of lipid bilayers using a contact bubble bilayer method. A water bubble is blown into an organic solvent, whereby a monolayer is formed at the water-oil interface. Two pipettes are manipulated to dock the bubbles to form a bilayer.

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...