Name: measuring properties of the membrane periodic skeleton of the axon initial segment using 3d-structured illumination microscopy (3d-sim)
Uploaded: 2022-02-11T12:30:00
Description: Watch this Scientific Journal Video about Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM at JoVE.com

Dr. Pirta Hotulainen is acknowledged for her critical comments, invaluable for preparing this manuscript. Dr. Rimante Minkeviciene is acknowledged for her help in preparing the neuronal cultures used for the original experiments. All imaging was performed in the Biomedicum Imaging Unit. This work was supported by the Academy of Finland (D.M., SA 266351) and Doctoral Programme Brain &#38; Mind (A.A.)

Primary hippocampal neurons used in these experiments were harvested16 from embryonic day 17 Wistar rat embryos of either sex under the ethical guidelines of the University of Helsinki and Finnish law.
1. Sample preparation
<ol>
	<li>On high-fidelity glass coverslips, allow rat hippocampal neurons to grow in sparse culture conditions (~5,000-10,000 cells/cm2) for 14 days (14 days-in-vitro). 
	NOTE: Using sparse cultures reduces the cha...

<ol>
	<li>Leterrier, 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+Axon+initial+segment,+50+years+later:+A+nexus+for+neuronal+organization+and+function.">The Axon initial segment, 50 years later: A nexus for neuronal organization and function.</a> Current Topics in Membranes. 77, 185-233 (2016).</li><li>Leterrier, C., Dargent, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+pasaran!+Role+of+the+axon+initial+segment+in+the+regulation+of+protein+transport+and+the+maintenance+of+axonal+identity.">No pasaran! Role of the axon initial segment in the regulation of protein transport and the maintenance of axonal identity.</a> Seminars in Cell &amp; Developmental Biology. 27, 44-51 (2014).</li><li>Brachet, A., 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=Ankyrin+G+restricts+ion+channel+diffusion+at+the+axonal+initial+segment+before+the+establishment+of+the+diffusion+barrier.">Ankyrin G restricts ion channel diffusion at the axonal initial segment before the establishment of the diffusion barrier.</a> Journal of Cell Biology. 191 (2), 383-395 (2010).</li><li>Nakada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+anchored+proteins+forms+membrane+diffusion+barriers+during+neuronal+polarization.">Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.</a> Nature Cell Biology. 5 (7), 626-632 (2003).</li><li>Song, A. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selective+filter+for+cytoplasmic+transport+at+the+axon+initial+segment.">A selective filter for cytoplasmic transport at the axon initial segment.</a> Cell. 136 (6), 1148-1160 (2009).</li><li>Sun, X., 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=Selective+filtering+defect+at+the+axon+initial+segment+in+Alzheimer's+disease+mouse+models.">Selective filtering defect at the axon initial segment in Alzheimer's disease mouse models.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (39), 14271-14276 (2014).</li><li>Winckler, B., Forscher, P., Mellman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+diffusion+barrier+maintains+distribution+of+membrane+proteins+in+polarized+neurons.">A diffusion barrier maintains distribution of membrane proteins in polarized neurons.</a> Nature. 397 (6721), 698-701 (1999).</li><li>Kole, M. 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=Action+potential+generation+requires+a+high+sodium+channel+density+in+the+axon+initial+segment.">Action potential generation requires a high sodium channel density in the axon initial segment.</a> Nature Neuroscience. 11 (2), 178-186 (2008).</li><li>Xu, K., Zhong, G., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin,+spectrin,+and+associated+proteins+form+a+periodic+cytoskeletal+structure+in+axons.">Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons.</a> Science. 339 (6118), 452-456 (2013).</li><li>Leterrier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+architecture+of+the+axon+initial+segment+reveals+an+organized+and+robust+scaffold.">Nanoscale architecture of the axon initial segment reveals an organized and robust scaffold.</a> Cell Reports. 13 (12), 2781-2793 (2015).</li><li>Leterrier, 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+axon+initial+segment:+An+updated+viewpoint.">The axon initial segment: An updated viewpoint.</a> The Journal of Neuroscience. 38 (9), 2135-2145 (2018).</li><li>Hamdan, 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=Mapping+axon+initial+segment+structure+and+function+by+multiplexed+proximity+biotinylation.">Mapping axon initial segment structure and function by multiplexed proximity biotinylation.</a> Nature Communications. 11 (1), 100 (2020).</li><li>Zhou, R., 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=Proteomic+and+functional+analyses+of+the+periodic+membrane+skeleton+in+neurons.">Proteomic and functional analyses of the periodic membrane skeleton in neurons.</a> bioRxiv. , (2020).</li><li>Valli, 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=Seeing+beyond+the+limit:+A+guide+to+choosing+the+right+super-resolution+microscopy+technique.">Seeing beyond the limit: A guide to choosing the right super-resolution microscopy technique.</a> Journal of Biological Chemistry. 297 (1), 100791 (2021).</li><li>Wang, 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=Radial+contractility+of+actomyosin+rings+facilitates+axonal+trafficking+and+structural+stability.">Radial contractility of actomyosin rings facilitates axonal trafficking and structural stability.</a> Journal of Cell Biology. 219 (5), 201902001 (2020).</li><li>Abouelezz, A., Stefen, H., Segerstr&#229;le, M., Micinski, D., Minkeviciene, R., Lahti, L., Hardeman, E., Gunning, P., Hoogenraad, C., Taira, T., Fath, T., Hotulainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tropomyosin+Tpm3.1+is+required+to+maintain+the+structure+and+function+of+the+axon+initial+segment.">Tropomyosin Tpm3.1 is required to maintain the structure and function of the axon initial segment.</a> iScience. 23 (5), 101053 (2020).</li><li>Muller, M., Monkemoller, V., Hennig, S., Hubner, W., Huser, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-source+image+reconstruction+of+super-resolution+structured+illumination+microscopy+data+in+ImageJ.">Open-source image reconstruction of super-resolution structured illumination microscopy data in ImageJ.</a> Nature Communications. 7, 10980 (2016).</li><li>Ball, 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=SIMcheck:+a+toolbox+for+successful+super-resolution+structured+illumination+microscopy.">SIMcheck: a toolbox for successful super-resolution structured illumination microscopy.</a> Scientific Reports. 5, 15915 (2015).</li><li>Stauffer, W., Sheng, H., Lim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EzColocalization:+An+ImageJ+plugin+for+visualizing+and+measuring+colocalization+in+cells+and+organisms.">EzColocalization: An ImageJ plugin for visualizing and measuring colocalization in cells and organisms.</a> Scientific Reports. 8 (1), 15764 (2018).</li><li>Dunn, K. W., Kamocka, M. M., McDonald, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+evaluating+colocalization+in+biological+microscopy.">A practical guide to evaluating colocalization in biological microscopy.</a> American Journal of Physiology-Cell Physiology. 300 (4), 723-742 (2011).</li><li>Lahti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+analysis+supplement+to+the+research+article+Abouelezz,+Stefen,+Segerstr&#229;le,+Micinski,+Minkeviciene,+Lahti,+Hardeman,+Gunning,+Hoogenraad,+Taira,+Fath,+&amp;+Hotulainen.">Data analysis supplement to the research article Abouelezz, Stefen, Segerstr&#229;le, Micinski, Minkeviciene, Lahti, Hardeman, Gunning, Hoogenraad, Taira, Fath, &amp; Hotulainen.</a> Tropomyosin Tpm3.1 Is Required to Maintain the Structure and Function of the Axon Initial Segment. , (2021).</li><li>Abouelezz, A., Micinski, D., Lipponen, A., Hotulainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sub-membranous+actin+rings+in+the+axon+initial+segment+are+resistant+to+the+action+of+latrunculin.">Sub-membranous actin rings in the axon initial segment are resistant to the action of latrunculin.</a> Journal of Biological Chemistry. 400 (9), 1141-1146 (2019).</li></ol>

The protocol described here provides a method for visualizing and measuring MPS proteins using the super-resolution technique. As actin rings and other MPS components display a periodicity of ~190 nm9,10, conventional diffraction-limited imaging approaches cannot reveal the details of the MPS. Several microscopy setups may resolve diffraction-limited structures in super-resolution, and SIM represents a robust and uncomplicated option. Importantly, SIM is compatib...

The axon initial segment (AIS) is a short, uniquely specialized region of the proximal axon of vertebrate neurons1. The AIS comprises a transport filter and diffusion barrier essential in maintaining neuronal polarity by sorting somato-dendritic cargo2,3,4,5,6,7. In addition, the unique structure of the AIS allows it to accommodate clusters of voltage-gated ion channels that facilitate its function as the site of action potential initiation8. A highly stable structural complex underlies the unique functions of the AIS. Research within the last decade has revealed the presence of a membrane periodic skeleton (MPS) containing actin rings connected by spectrin and providing a scaffold for anchoring various AIS proteins9,10.
The distance between actin rings in the MPS (~190 nm)9,10 is under the resolution limit of conventional light microscopy. Early attempts to use electron microscopy to visualize the MPS were not successful, as the harsh preparation procedures involved failed to preserve the structure of the MPS. Thus, super-resolution microscopy techniques have proven invaluable in elucidating some of the structural details of the MPS11. However, the understanding of the AIS structural complex, the identity and functions of its components, and its spatiotemporal regulation are still incomplete. Recent proteomic studies succeeded in creating a sizeable list of proteins that localize to the AIS close to structural components of the AIS12,13. Still, details of their function and precise place in the AIS complex are lacking. Thus, super-resolution microscopy techniques serve as an essential tool to examine the accurate positions of these proteins relative to other MPS components and investigate their functions. Several light microscopy techniques can achieve resolutions higher than the diffraction limit of light, some even capable of localizing single molecules. However, many of these techniques typically require specialized fluorophores or imaging buffers, and image acquisition is often time-intensive14.
3D structured illumination microscopy (3D-SIM), owing to its ease of use and simple sample preparation requirements, requires no special reagents for imaging or sample preparation, works well with a wide array of fluorophores and samples, can be readily implemented in multiple colors, and is capable of live-cell imaging15. While the best possible resolution SIM offers (~120 nm) is low compared to many other super-resolution techniques, it is sufficient for many applications (for example, for resolving the components of the MPS in neurons). Thus, it is crucial to consider the requirement for specific applications to determine if SIM is a suitable choice or if an even higher resolution is necessary. Here, a protocol is described for using cultured hippocampal neurons and 3D-structured illumination microscopy (3D-SIM) to examine the position and organization of putative AIS proteins relative to actin rings in the MPS, as implemented in Abouelezz&#160;et al.16

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plates</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-488 Phalloidin</td>
			<td>ThermoFisher</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-647 anti-mouse</td>
			<td>ThermoFisher</td>
			<td>A31571</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ankyrin G antibody</td>
			<td>UC Davis/NIH NeuroMab Facility, Clone 106/36</td>
			<td>75-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-MAP2 antibody</td>
			<td>Merck Millipore</td>
			<td>AB5543</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>BioWest</td>
			<td>P6154</td>
			<td></td>
		</tr>
		<tr>
			<td>Deltavision OMX SR</td>
			<td>GE Healthcare Life Sciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software package</td>
			<td>ImageJ</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GNU Octave</td>
			<td>GNU</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High performance coverslips</td>
			<td>Marienfeld</td>
			<td>117530</td>
			<td></td>
		</tr>
		<tr>
			<td>Immersion Oil Calculator</td>
			<td>Cytiva Life Sciences</td>
			<td></td>
			<td>https://tinyurl.com/ImmersionOilCalculator</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>VWR</td>
			<td>ICNA1680149</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB R2020a</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OMX SR</td>
			<td>Delta Vision OMX</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>InvivoGen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold mounting media</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>softWoRx Deconvolution</td>
			<td>Cytiva Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Slides</td>
			<td>Epredia</td>
			<td>ISO 8037/1</td>
			<td></td>
		</tr>
		<tr>
			<td>TetraSpeck microspheres 0.1 &#181;m</td>
			<td>ThermoFisher</td>
			<td>T7279</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring properties of the membrane periodic skeleton of the axon initial segment using 3d-structured illumination microscopy (3d-sim)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Using cultured rat hippocampal neurons and 3D-SIM, a protocol is described to visualize and measure actin rings and other components of the MPS in the AIS. Reconstructions of image stacks showed clear periodicity of actin rings (Figure 2A). In our hands, the mean inter-peak distance of actin rings in the MPS, visualized using Alexa 488-tagged phalloidin, was 190.36 &#177; 1.7 nm (mean &#177; SEM). This is in line with the previously reported average distance of ~190 nm between actin rings in...

Watch this Scientific Journal Video about Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM at JoVE.com

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

This protocol allows resolving the membrane periodic skeleton in the axon initial segment of cultured neurons. By examining the localization of proteins insight can be gained into the function in the membrane periodic skeleton. The main advantage of this technique is its robustness and ease of use.Structured illumination microscopy is capable of resolving the membrane periodic skeleton and sample preparation is easy and straightforward. Anyone with previous experience in fluorescence microscopy can readily implement this technique. This protocol is designed to maximize signal to noise ratio during sample preparation and imaging.To begin, fix the neurons using 4%Paraformaldehyde for 12 minutes at room temperature. Wash the cover slip once in 0.2%BSA in phosphate buffer solution and incubate for 10 minutes in a 1%solution of Triton X in PBS at room temperature. Wash the cover slip with BSA in phosphate buffer solution, dilute the anti ankyrin G antibodies using BSA in a phosphate buffer solution at one to 200 ratio.Add antibody solution to the cover slip and incubate overnight at four degrees Celsius. Wash the cells once in BSA phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution and once in phosphate buffer solution. Dilute Fluoro 4 tagged, add more secondary antibodies in BSA phosphate buffer solution and add to the cover slip.Wash the cells once in BSA phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution, then once in phosphate buffer solution. Prepare a one micromolar solution of tagged Ferodin in phosphate buffer solution and add it to the cells. Wash the cells once in Bovine Serum Albumin phosphate buffer solution once in 0.1%Triton X in phosphate buffer solution, then once in phosphate buffer solution.For mounting the cover slip on a glass slide, apply a drop of mounting media on a slide, dip the cover slip in deionized water, and tap it with a soft paper towel to remove excess water. Place the cover slip on the glass slide and incubate at room temperature for 24 hours. If possible, consult with the personnel from the microscopy facility before imaging and use an immersion oil calculator to select the immersion oil suitable for the sample.Once the samples are ready, ensure the cover slips are clean and clear of any residue by cleaning them with a cotton tip dipped in water or ethanol. Place the sample in a 3D-SIM microscope and find the cell to capture the image. Adjust the power of the relevant laser lines.And the exposure times to maximize the signal to noise ratio without significantly bleaching the sample. Set the upper and lower limits of the sample in the Z dimension and proceed to acquire a stack. Run the reconstruction algorithm on the stack to obtain a super resolved reconstruction.Check the reconstructions for known artifacts and if necessary, adjust the parameters to correct them. Compare the SIM reconstruction to a wide field image to observe improvements in resolution. When performing multicolor structured illumination microscopy, use the alignment algorithm to align the different channels correctly, once a satisfactory reconstruction is ready.Actin rings in the membrane periodic skeleton have a distinctive periodic appearance. Create a maximum intensity projection image in image analysis software using all the focal planes where the actin rings are visible. On the maximum intensity projection image, draw a perpendicular line across visible adjacent rings and record the fluorescence intensity along with the software's line plot profile function.To calculate the mean inter peak distance, note the local maxima in the line profile and measure the distance between individual adjacent fluorescent intensity peaks. To evaluate the colocalization of different proteins with actin rings in the membrane periodic skeleton, run a colocalization analysis procedure on SIM reconstructions of actin and the candidate protein. Manually draw a selection to define the axon initial segment as a region of interest, for the easy colocalization plugin in the software platform, and run the analysis to calculate the Pearson's coefficient of correlation for colocalization.During SIM image analysis, the reconstructions of image stacks, showed clear periodicity of actin rings. An anti ankyrin G antibody was used to visualize ankyrin G.The colocalization of ankyrin G and actin rings was tested in the axon initial segment using a colocalization analysis procedure to calculate Pearson's coefficient of correlation. The Pearson's coefficient of correlation for colocalization of ankyrin G and actin rings fluorescence was 0.36 plus or minus 0.03.It is essential to use the right immersion oil and adjust the laser power and exposure time to make the grid lines visible. Once it is determined that protein is part of the membrane periodic skeleton, it's function can be investigated and established in maintaining the membrane periodic skeleton. For example, the effects of knocking down the protein on actin rings can be analyzed.The development of super resolution microscopy techniques is the reason we know the axon initial segment contains membrane periodic skeleton comprising of actin rings, spectrum and ankyrin. SIM has enabled researchers to easily visualize membrane periodic skeleton components and look at actin rings in live cells.

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