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 chances of overlapping neurites, which helps quantify the MPS.</li>
	<li>Fix neurons using 4% paraformaldehyde for 12 min at room temperature. Wash the coverslip once in 0.2% BSA in PBS (BSA-PBS), then incubate for 10 min in a 1% solution of Triton-X in PBS at room temperature. Wash once in BSA-PBS.</li>
	<li>Dilute anti-ankyrin G antibodies (1:200) in BSA-PBS. Add antibody solution to the coverslip and incubate overnight at 4 &#176;C. Optionally, add chicken anti-MAP2 antibodies (see Table of Materials) to the antibody solution to demarcate the somato-dendritic domain.</li>
	<li>Wash cells once in BSA-PBS, followed by 0.1% Triton-X in PBS, then do a final wash in BSA-PBS.</li>
	<li>Dilute fluorophore-tagged anti-mouse secondary antibodies (1:200) (see Table of Materials) in BSA-PBS, add to the coverslip, and incubate for 1 h at room temperature. Wash the cells once in BSA-PBS, once in 0.1% Triton-X in PBS, then once in PBS.</li>
	<li>Prepare a 1 &#181;M solution of tagged phalloidin in PBS (see Table of Materials) and add it to the cells. Incubate for 2 h at room temperature. Wash cells once in PBS, once in 0.1% Triton-X in PBS, then once in PBS. 
	&#8203;NOTE: AlexaFluor488-tagged phalloidin was used here, but other tags will also work.</li>
	<li>For mounting the coverslip on a glass slide, apply a drop of mounting media on a glass slide, dip the coverslip in deionized water, and dab using a soft paper towel (to remove excess water).
	<ol>
		<li>Place the coverslip on the glass slide. Incubate at room temperature for 24 h. 
		&#8203;NOTE: No adverse effects on the sample were observed using hard-setting mounting media (refractive index 1.47 after curing).</li>
	</ol>
	</li>
</ol>
2. Imaging
<ol>
	<li>If possible, consult with the microscopy facility&#39;s personnel before imaging. Use an immersion oil calculator (see Table of Materials) to select the immersion oil suitable for the sample.</li>
	<li>Once the samples are ready, ensure the coverslips are clean and clear of any residue or excess mounting media. If necessary, use a cotton tip dipped in water or ethanol to clean. Place the sample in a 3D SIM-capable microscope (see Table of Materials) and find a cell to image.</li>
	<li>Adjust the power of the relevant laser lines and the exposure times to maximize the signal-to-noise ratio without significantly bleaching the sample. 
	NOTE: A good signal-to-noise ratio will show a clear grid-like appearance focused on the sample and lead to accurate high-resolution reconstructions. 488 nm and 640 nm laser lines were used to visualize F-actin and ankyrin G, respectively.</li>
	<li>Set the upper and lower limits of the sample in the z-dimension and proceed to acquire a stack. 
	NOTE: z-step size of 125 nm was used here.</li>
	<li>For successful SIM reconstruction, ensure that the microscopy software (see Table of Materials) has an optical transfer file (OTF) suitable for the used dyes. 
	NOTE: These are typically created and maintained by specialized personnel. In the absence of a functional OTF, open-source tools are also available to perform reconstructions based on estimates17. In this work, up-to-date OTF files were used controlled by specialized personnel.</li>
	<li>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. 
	NOTE: The default algorithm parameters typically give accurate results. Many of these artifacts involve some repeating patterns: striped lines along with multiple directions on one z-plane, &#39;ghosting&#39; (repeated features appearing in various z-planes), or a hexagonal &#39;honeycomb&#39; pattern emerging in some areas. These artifacts can often be fixed with better sample preparation, a better signal-to-noise ratio, adjusting the parameters of the reconstruction algorithm, or ensuring the refractive index of the chosen immersion oil is suitable for the mounting medium and sample. For a more detailed discussion of SIM artifacts and a freely-available tool to check for the presence of artifacts, see Ball et al.18</li>
	<li>Compare the SIM reconstruction to a wide-field image to observe improvements in resolution.</li>
	<li>When performing multi-color SIM, use the alignment algorithm to align the different channels correctly once a satisfactory reconstruction is ready. 
	&#8203;NOTE: The alignment algorithm uses an alignment reference file generated using a microsphere bead preparation as per the instruction of the microscope manufacturer.</li>
</ol>
3. Image analysis
<ol>
	<li>Actin rings in the MPS have a distinctive periodic appearance. Using image analysis software (see Table of Materials), create a maximum intensity projection image using all the focal planes where actin rings are visible.</li>
	<li>On the maximum intensity projection image, draw a perpendicular line across visible adjacent rings and record the fluorescence intensity along with the software&#39;s line &#39;Plot Profile&#39; function.</li>
	<li>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. 
	NOTE: This can be easily calculated, for example, using the &#39;findpeaks&#39; function in MATLAB or the (open-source) GNU Octave platform.</li>
	<li>To evaluate the colocalization of different proteins with actin rings in the MPS, run a colocalization analysis procedure19,20,21 on SIM reconstructions of actin and the candidate protein. Manually draw a selection to define the AIS as a region of interest for the EzColocalization plugin in the software platform19 and run the analysis to calculate the Pearson&#39;s coefficient of correlation (PCC) of co-localization19,20,21. A PCC value close to 1 indicates strong colocalization. 
	NOTE: This protocol is summarized in Figure 1.</li>
</ol>

<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 authors have nothing to disclose.

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 ...

measuring-properties-membrane-periodic-skeleton-axon-initial-segment

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2.0K Views.  Minerva Foundation Institute for Medical Research. 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).

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

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 ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

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.

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 ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

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 ...

Characterizing Multiscale Mechanical Properties of Brain Tissue Using Atomic Force Microscopy, Impact Indentation, and Rheometry

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain tissue itself must be well characterized at various length and time scales. Like many biological tissues, brain tissue exhibits a complex, hierarchical structure. However, in contrast to most other tissues, brain is of very low mechanical stiffness, with Young&#39;s elastic moduli E on the order of 100s of Pa. This low stiffness can present challenges to experimental characterization of key mechanical properties. Here, we demonstrate several mechanical characterization techniques that have been adapted to measure the elastic and viscoelastic properties of hydrated, compliant biological materials such as brain tissue, at different length scales and loading rates. At the microscale, we conduct creep-compliance and force relaxation experiments using atomic force microscope-enabled indentation. At the mesoscale, we perform impact indentation experiments using a pendulum-based instrumented indenter. At the macroscale, we conduct parallel plate rheometry to quantify the frequency dependent shear elastic moduli. We also discuss the challenges and limitations associated with each method. Together these techniques enable an in-depth mechanical characterization of brain tissue that can be used to better understand the structure of brain and to engineer bio-inspired materials.

We present a set of techniques to characterize the viscoelastic mechanical properties of brain at the micro-, meso-, and macro-scales.

To design and engineer materials inspired by the properties of the brain, whether for mechanical simulants or for tissue regeneration studies, the brain ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Visualization of Motor Axon Navigation and Quantification of Axon Arborization In Mouse Embryos Using Light Sheet Fluorescence Microscopy

Spinal motor neurons (MNs) extend their axons to communicate with their innervating targets, thereby controlling movement and complex tasks in vertebrates. Thus, it is critical to uncover the molecular mechanisms of how motor axons navigate to, arborize, and innervate their peripheral muscle targets during development and degeneration. Although transgenic Hb9::GFP mouse lines have long served to visualize motor axon trajectories during embryonic development, detailed descriptions of the full spectrum of axon terminal arborization remain incomplete due to the pattern complexity and limitations of current optical microscopy. Here, we describe an improved protocol that combines light sheet fluorescence microscopy (LSFM) and robust image analysis to qualitatively and quantitatively visualize developing motor axons. This system can be easily adopted to cross genetic mutants or MN disease models with Hb9::GFP lines, revealing novel molecular mechanisms that lead to defects in motor axon navigation and arborization.

Here, we describe a protocol for visualizing motor neuron projection and axon arborization in transgenic Hb9::GFP mouse embryos. After immunostaining for motor neurons, we used light sheet fluorescence microscopy to image embryos for subsequent quantitative analysis. This protocol is applicable to other neuron navigation processes in the central nervous system.