This work was supported by Consejo Nacional de Ciencia y Tecnolog&#237;a (CONACYT) through the following grants: infraestructura 226450 to VP-CIO, infraestructura 255277 to V.P., and FORDECYT-PRONACES/194171/2020 to V.H. We acknowledge the support of Juvenal Hern&#225;ndez Guevara at CIO in the video-making.

All procedures described were done in compliance with the laws and codes approved in the seventh title of the Regulation of the General Health Law Regarding Health Research of the Mexican Government (NOM-062-ZOO-1999) and in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Experimental Animals and were approved by the institutional committee of bioethics in research of the Universidad de Guanajuato and Benem&#233;rita Universidad Aut&#243;noma de Puebla.
1. Microscope settings
<ol>
	<li>Switch on the microscopy system.</li>
	<li>Turn on the pulsed laser to guarantee that it will be ready to lase at an optimal and steady power level after the sample extraction and preparation.</li>
	<li>Tune the laser to 810 nm. To study tissular microtubules, 10%-20% of the available laser power is used, which, in the described system, corresponds to 13-26 mW measured at the back focal plane of the objective.</li>
	<li>Make sure the microscope is K&#246;hler-aligned (Supplementary File 1), with the objective that will be used for the SHG imaging. 
	NOTE: This is important since, in the commercial setup used in this work, the detection is performed in transmission mode and through the condenser.</li>
	<li>Remove unwanted filters from the optical detection path (Figure 1A).</li>
	<li>Make sure the condenser diaphragm is fully opened to ensure that no light is unnecessarily stopped at this level.</li>
	<li>Prepare a 25x/0.8 numerical aperture (NA) oil-immersion objective with a small drop of immersion oil in the lens. 
	&#8203;NOTE: This objective was used to best match the condenser NA, which was 0.55 (ideally, NAcond&#8805; NAobj).</li>
</ol>
2. Microscope preliminary controls
NOTE: Perform the preliminary microscope controls once, unless the setup is modified.
<ol>
	<li>Image dry corn starch sandwiched between glass slides with SHG parameters to generate SHG images that reveal the laser polarization direction. Follow the steps reported in Supplementary File 2, except for the laser power, which is less than 5% for corn starch.</li>
	<li>Take one image of sparse corn starch grains, and mark the orientation of the SHG lobules in the x-y plane. This orientation corresponds to the laser orientation (Figure 2A).</li>
	<li>As a control, take another image of the same sample, inserting into the optical path a half-wave plate (position shown in Figure 1A) to alter the oscillation direction of the laser. The resulting image displays rotated SHG signal lobules (Figure 2B, C).</li>
	<li>If using a different type of bandpass filter for the SHG, make sure it has optimal transmission properties by comparing the images (Figure 2D, E) and the signal intensities pixel by pixel along a line-scan (Figure 2F).</li>
</ol>
3. Tissue extraction
NOTE: Always use clean tools to perform the surgical procedures.
<ol>
	<li>Use 6-12 month old taiep rats and age-matched Sprague-Dawley&#160;controls (WT).</li>
	<li>Anesthetize the animals with an i.p. injection of a mixture of ketamine-xylazine (0.125 mg/Kg and 5 mg/Kg) diluted in a 0.9% sterile NaCl solution.
	<ol>
		<li>Check the pain reflexes, and proceed only if they are absent.</li>
	</ol>
	</li>
	<li>Sacrifice the animal via decapitation.</li>
	<li>Once the head is isolated, open the bones of the cranial vault carefully along the midline, starting from the most caudal point at the top toward the nose, from the orbital cavities toward the midline, and then from the most caudal point to below the brain and cerebellum. 
	NOTE: Bone cutters and Rongeurs allow for proper removal of the head bones without damaging the brain structures.</li>
	<li>Release the brain and cerebellum from the bone. The two hemispheres could be separated. If the hemispheres are divided, use one half for SHG imaging, and fix the other half in 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min inside a chemical hood for subsequent sectioning and staining.</li>
</ol>
4. Vibratome sectioning
<ol>
	<li>Prepare a vibratome buffer tray filled with warm (37 &#176;C) Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Fix the brain to the specimen plate using cyanoacrylate glue via a piece of masking tape. The most caudal portion/region contacts the glue so that the usable coronal sections from the opposite, rostral portion can be cut.
	<ol>
		<li>Allow ~10 s for the glue to polymerize to obtain effective attachment of the sample. 
		NOTE: The best results are obtained when the brain is oriented so that the blade cuts &#34;from top to bottom&#34; rather than &#34;from side to side&#34; (Figure 1B).</li>
	</ol>
	</li>
	<li>Transfer the specimen plate onto its magnetic support inside the buffer tray.</li>
	<li>Start cutting 300-500 &#181;m sections until the slices produced encompass the entire surface of the brain.</li>
	<li>At this point, reduce the thickness of the section to 160 &#181;m.</li>
	<li>Once a complete 160 &#181;m section is cut at the level of the corpus callosum, recover it with a modified glass Pasteur pipette with a large, flamed orifice (Figure 1C).</li>
	<li>Transfer it to a clean Petri dish with new warm HBSS or directly onto the glass support for microscopy (coverslip or glass-bottom dish). 
	&#8203;NOTE: For the described experimental conditions, adding a coverslip above the sample is detrimental to the imaging.</li>
</ol>
5. Transfer to the microscope
<ol>
	<li>If the microscope is in a different room, prepare an insulated box with warmed gel packs to transfer the tissue sections while maintaining the temperature. The box also serves to keep the sections warm until they are imaged.</li>
	<li>Put the sample under the microscope, and make sure it is positioned properly under the objective by direct observation through the oculars with transmitted light. 
	NOTE: The goal is to align the predicted emitting structures with the oscillation direction of the laser to maximize the emitted (and, therefore, detected) SHG signal.</li>
	<li>Remove the excess HBSS so that a thin liquid film covers the entire sample. Visually check the liquid film every few minutes to avoid excessive evaporation and drying of the sample. 
	NOTE: In the conditions described, a section is used for no more than 20 min. Drying of the sample causes drastic artifactual effects (Supplementary Figure 1A). Rather than adding more HBSS, periodic soaking of the section in warm, fresh medium is recommended if longer imaging times are needed. The use of perfusion chambers and glue or low-melting agarose to immobilize the tissue is also recommended when longer experiments are to be done.</li>
	<li>Prepare the microscope stage for non-descanned imaging, which might include closing all the doors of the dark incubation chamber or covering the incubation chamber with a black nylon polyurethane-coated fabric.</li>
</ol>
6. Imaging
<ol>
	<li>Select the &#34;non-descanned&#34; imaging mode along the transmission path. This way, the capture of the weak SH signal of tubulin will be optimized.</li>
	<li>Select the LCI Plan-Neofluar 25x/0.8 NA objective.</li>
	<li>Set a laser power between 13 mW and 26 mW, with a pixel dwell time of 12.6 &#181;s. Take images no bigger than 512 pixels x 512 pixels, with speed 5 and averaging 2, for an average acquisition time of about 15 s. 
	NOTE: Using higher laser powers and/or longer dwell times may damage the sample (Supplementary Figure 1B).</li>
	<li>Take images first using a 485 nm short-pass filter (SP485), and, in a second step, add a sharp 405 nm bandpass filter (BP405; Figure 3). Follow the steps reported in Supplementary File 2. 
	NOTE: Pseudo-brightfield images can be taken through the same detector using the residual laser light of a visible line (405 nm or 488 nm lasers work best).</li>
</ol>
7. Processing of the cerebellum
<ol>
	<li>First, cut the cerebellum with a scalpel into two hemispheres, and then glue them to the vibratome support by the middle portion (the flat part obtained after cutting).</li>
	<li>Section and image the cerebellum in the same way as was described for the brain (160 &#181;m sections, same vibratome settings, same blade, same microscope settings, etc.). 
	NOTE: Due to the anatomy of the cerebellum, its orientation at the moment of sectioning is not critical; in most of the slices generated away from the surface, there will be folia sectioned well into the white matter.</li>
</ol>

<ol>
	<li>Palmiter, R. 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=Cell+lineage+ablation+in+transgenic+mice+by+cell-specific+expression+of+a+toxin+gene.">Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene.</a> Cell. 50 (3), 435-443 (1987).</li><li>Banerjee, 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=A+monoclonal+antibody+against+the+type+II+isotype+of+beta-tubulin.+Preparation+of+isotypically+altered+tubulin.">A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin.</a> The Journal of Biological Chemistry. 263 (6), 3029-3034 (1988).</li><li>Banerjee, A., Roach, M. C., Trcka, P., Luduena, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+a+monoclonal+antibody+specific+for+the+class+IV+isotype+of+beta-tubulin.+Purification+and+assembly+of+alpha+beta+II,+alpha+beta+III,+and+alpha+beta+IV+tubulin+dimers+from+bovine+brain.">Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.</a> The Journal of Biological Chemistry. 267 (8), 5625-5630 (1992).</li><li>Banerjee, 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=Localization+of+&#946;v+tubulin+in+the+cochlea+and+cultured+cells+with+a+novel+monoclonal+antibody.">Localization of &#946;v tubulin in the cochlea and cultured cells with a novel monoclonal antibody.</a> Cell Motility and the Cytoskeleton. 65 (6), 505-514 (2008).</li><li>Cross, A. R., Williams, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinky+microtubules:+Bending+and+breaking+induced+by+fixation+in+vitro+with+glutaraldehyde+and+formaldehyde.">Kinky microtubules: Bending and breaking induced by fixation in vitro with glutaraldehyde and formaldehyde.</a> Cell Motility and the Cytoskeleton. 20 (4), 272-278 (1991).</li><li>Van Steenbergen, V., 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=Molecular+understanding+of+label-free+second+harmonic+imaging+of+microtubules.">Molecular understanding of label-free second harmonic imaging of microtubules.</a> Nature Communications. 10 (1), 3530 (2019).</li><li>Campagnola, P. J., Clark, H. A., Mohler, W. A., Lewis, A., Loew, L. 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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=Measuring+microtubule+polarity+in+spindles+with+second-harmonic+generation.">Measuring microtubule polarity in spindles with second-harmonic generation.</a> Biophysical Journal. 106 (8), 1578-1587 (2014).</li><li>Bancelin, S., 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=Probing+microtubules+polarity+in+mitotic+spindles+in+situ+using+Interferometric+Second+Harmonic+Generation+Microscopy.">Probing microtubules polarity in mitotic spindles in situ using Interferometric Second Harmonic Generation Microscopy.</a> Scientific Reports. 7, 6758 (2017).</li><li>Dombeck, D. 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=Uniform+polarity+microtubule+assemblies+imaged+in+native+brain+tissue+by+second-harmonic+generation+microscopy.">Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (12), 7081-7086 (2003).</li><li>Psilodimitrakopoulos, S., 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=Estimation+of+the+effective+orientation+of+the+SHG+source+in+primary+cortical+neurons.">Estimation of the effective orientation of the SHG source in primary cortical neurons.</a> Optics Express. 17 (16), 14418 (2009).</li><li>Sharoukhov, D., Bucinca-Cupallari, F., Lim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+imaging+reveals+cytoskeletal+deficit+predisposing+the+retinal+ganglion+cell+axons+to+atrophy+in+DBA/2J.">Microtubule imaging reveals cytoskeletal deficit predisposing the retinal ganglion cell axons to atrophy in DBA/2J.</a> Investigative Opthalmology &amp; Visual Science. 59 (13), 5292 (2018).</li><li>Alata, M., Piazza, V., Eguibar, J. R., Cortes, C., Hernandez, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H-ABC+tubulinopathy+revealed+by+label-free+second+harmonic+generation+microscopy.">H-ABC tubulinopathy revealed by label-free second harmonic generation microscopy.</a> Scientific Reports. 12, 14417 (2022).</li><li>Duncan, I. D., Lunn, K. F., Holmgren, B., Urba-Holmgren, R., Brignolo-Holmes, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+taiep+rat:+A+myelin+mutant+with+an+associated+oligodendrocyte+microtubular+defect.">The taiep rat: A myelin mutant with an associated oligodendrocyte microtubular defect.</a> Journal of Neurocytology. 21 (12), 870-884 (1992).</li><li>Duncan, I. 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=A+mutation+in+the+Tubb4a+gene+leads+to+microtubule+accumulation+with+hypomyelination+and+demyelination:+Tubb4a+Mutation.">A mutation in the Tubb4a gene leads to microtubule accumulation with hypomyelination and demyelination: Tubb4a Mutation.</a> Annals of Neurology. 81 (5), 690-702 (2017).</li><li>Garduno-Robles, 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=MRI+features+in+a+rat+model+of+H-ABC+tubulinopathy.">MRI features in a rat model of H-ABC tubulinopathy.</a> Frontiers in Neuroscience. 14, 555 (2020).</li><li>Lopez-Juarez, 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=Auditory+impairment+in+H-ABC+tubulinopathy.">Auditory impairment in H-ABC tubulinopathy.</a> Journal of Comparative Neurology. 529 (5), 957-968 (2021).</li><li>Alata, M., 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=Longitudinal+evaluation+of+cerebellar+signs+of+H-ABC+tubulinopathy+in+a+patient+and+in+the+taiep+model.">Longitudinal evaluation of cerebellar signs of H-ABC tubulinopathy in a patient and in the taiep model.</a> Frontiers in Neurology. 12, 702039 (2021).</li><li>Parodi, V., 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=Nonlinear+optical+microscopy:+From+fundamentals+to+applications+in+live+bioimaging.">Nonlinear optical microscopy: From fundamentals to applications in live bioimaging.</a> Frontiers in Bioengineering and Biotechnology. 8, 585363 (2020).</li><li>Lefort, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+biomedical+multiphoton+microscopy+and+its+laser+sources.">A review of biomedical multiphoton microscopy and its laser sources.</a> Journal of Physics D: Applied Physics. 50 (42), 423001 (2017).</li><li>Campagnola, P. J., Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-harmonic+imaging+microscopy+for+visualizing+biomolecular+arrays+in+cells,+tissues+and+organisms.">Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms.</a> Nature Biotechnology. 21 (11), 1356-1360 (2003).</li><li>vander Knaap, M. S., 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=New+syndrome+characterized+by+hypomyelination+with+atrophy+of+the+basal+ganglia+and+cerebellum.">New syndrome characterized by hypomyelination with atrophy of the basal ganglia and cerebellum.</a> American Journal of Neuroradiology. 23 (9), 1466 (2002).</li><li>Stoller, P., Kim, B. -. M., Rubenchik, A. M., Reiser, K. M., Da Silva, L. 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B., 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=In+vivo+measurement+of+gene+expression,+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy.">In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nature Medicine. 7 (7), 864-868 (2001).</li><li>Chakraborti, S., Natarajan, K., Curiel, J., Janke, C., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+role+of+the+tubulin+code:+From+the+tubulin+molecule+to+neuronal+function+and+disease.">The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease.</a> Cytoskeleton. 73 (10), 521-550 (2016).</li></ol>

The authors have no competing financial interests.

SHG microscopy is part of a group of non-linear optics techniques, which include two-photon excitation microscopy, third harmonic generation microscopy, and coherent anti-Stokes Raman scattering microscopy, that have contributed to expanding the range of applications of conventional optical microscopy to the life sciences20.
Specifically, the major strength and weakness of SHG microscopy relate to the same condition: the signal generator is non-centrosymmetric<sup class...

The optical imaging of cytoskeletal structures in tissues and organ preparations is not an easy task. Cytoskeletal filaments are ubiquitous, so if generic staining is performed, for example, against alpha-tubulin or beta-actin or potentially keratin in an epithelial sample, the signal will likely be distributed rather homogeneously all over the sample. To restrict the staining to a more meaningful subset of cellular components, one can either use transgenic mice with targeted expression1 or plan to use isoform-specific antibodies. While very few of the latter are on the market (and very few exist at all2,3,4), a transgenic animal model might be available. However, it needs to be acquired by the lab and properly housed, with all the expenses involved in the process. Certain antibodies or chemicals, for example, fluorophore-conjugated drugs like phalloidin or paclitaxel, may be partially or fully incompatible with use in living cells or tissues, thus limiting their applicability to only studies of fixed samples.
In the case of tubulin, an additional aspect has to be taken into consideration, which is the sensitivity of the polymer to fixation. Conventional chemical fixation with formaldehyde is known for not being adequate for optimally preserving the integrity of microtubules5. Additionally, a recent report confirms that formaldehyde crosslinking induces subtle changes in the ultrastructure of the microtubule, similar to what happens with the binding of some drugs or physiological molecules such as GTP6.
The direct visualization of microtubules in unstained, unfixed samples is, therefore, often desirable. To achieve this, one technical solution is second harmonic generation (SHG) microscopy7, which is based on the ability of bundles of parallel microtubules to act as harmonophores and to emit frequency-doubled light when properly illuminated with an intense, pulsed infrared laser. Although a stronger and more stable second harmonic signal can be generated from collagen and myosin, which are the only other two biological materials known to be capable of frequency-doubling, the signal from tubulin has been used so far mostly to study mitotic spindle rearrangements8,9,10 and axonal microtubule morphology11,12,13.
In this work, we introduce a novel use of SHG microscopy as a diagnostic tool to distinguish central nervous system (CNS) tissues affected by tubulin beta 4 A (TUBB4A) tubulinopathy from their healthy counterparts14. Some of the mutations occurring in this predominantly neural isoform of tubulin, like those causing hypomyelination and atrophy of the basal ganglia and cerebellum (H-ABC), induce microtubule overfilling in the oligodendrocytes15,16; the cytoskeletal alterations, in turn, are associated with downstream effects like dysmyelination, with profound impairment of the motor and sensory pathways16,17,18,19. The taiep murine model used in this work displays abnormal microtubule content in the oligodendrocytes and recapitulates most of the sensory-motor symptoms of H-ABC patients17. The protocol explains how to image structures as the corpus callosum and the cerebellum, which are usually highly myelinated and which are severely affected in human patients as well as in the taiep rat19, to highlight the differences in SH signals between healthy and mutant tissues.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>405/10 nm BrightLine(R) single-band bandpass filter&#160;</td>
			<td>Semrock</td>
			<td>FF01-405/10-25</td>
			<td>32 mm diameter, with housing ring</td>
		</tr>
		<tr>
			<td>Black Nylon, Polyurethane-Coated Fabric</td>
			<td>Thorlabs</td>
			<td>BK5</td>
			<td>5&#39; x 9&#39; (1.5 m x 2.7 m) x 0.005&#34; (0.12 mm) Thick&#160;</td>
		</tr>
		<tr>
			<td>Blades for vibratome</td>
			<td>any commercial; e.g. Wilkinson Sword</td>
			<td></td>
			<td>&#160;Classic stainless steel double edge razor blades</td>
		</tr>
		<tr>
			<td>Cell culture dishes, 35 mm</td>
			<td>any commercial; e.g. Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM710NLO AxioObserver Z1</td>
			<td>Inverted microscope, objective used is LCI Plan-Neofluar 25x/0.8 NA&#160;</td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>any commercial</td>
			<td></td>
			<td>Any insulated, polystyrene box could work, to mantain the sample at about 37 &#176;C</td>
		</tr>
		<tr>
			<td>Corn stach</td>
			<td>e.g. Maizena</td>
			<td></td>
			<td>From the supermarket</td>
		</tr>
		<tr>
			<td>Coverslips #1.5</td>
			<td>any commercial</td>
			<td></td>
			<td>Rectangular</td>
		</tr>
		<tr>
			<td>Cyanoacrylate glue</td>
			<td>e.g. Loctite</td>
			<td></td>
			<td>To glue the brain to the masking tape</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>fine science tools</td>
			<td>11412-11</td>
			<td>To manipulate tissue sections by handling from the meninges</td>
		</tr>
		<tr>
			<td>Fine scissors</td>
			<td>fine science tools</td>
			<td>14370-22</td>
			<td>To cut the skin&#160;</td>
		</tr>
		<tr>
			<td>Fine scissors curved tip</td>
			<td>fine science tools</td>
			<td>14061-09</td>
			<td>To cut along the midline</td>
		</tr>
		<tr>
			<td>Formaldehyde 37%</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>To dilute 1:10 in PBS</td>
		</tr>
		<tr>
			<td>Friedman Rongeur</td>
			<td>fine science tools</td>
			<td>16000-14</td>
			<td>To cut the bone</td>
		</tr>
		<tr>
			<td>Gel packs</td>
			<td>any commercial</td>
			<td></td>
			<td>Prewarmed to 37 &#176;C, to help mantaining the temperature inside the cooler</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette, modified</td>
			<td>any commercial</td>
			<td></td>
			<td>To transfer the tissue section</td>
		</tr>
		<tr>
			<td>Hanks&#8242; Balanced Salt solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-076</td>
			<td>Could be prepared from powders</td>
		</tr>
		<tr>
			<td>Kelly hemostats</td>
			<td>fine science tools</td>
			<td>13018-14</td>
			<td>To separate the bone&#160;</td>
		</tr>
		<tr>
			<td>Masking tape</td>
			<td>any commercial</td>
			<td></td>
			<td>To protect th surface of the specimen plate</td>
		</tr>
		<tr>
			<td>NDD module, type C</td>
			<td>Zeiss</td>
			<td>000000-1410-101</td>
			<td>To detect the signal, reducing light loss. Housing the 000000-1935-163 filter set with the SP485</td>
		</tr>
		<tr>
			<td>Offset bone nippers</td>
			<td>fine science tools</td>
			<td>16101-10</td>
			<td>To cut the bone</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td>Could be prepared from powders or tabs</td>
		</tr>
		<tr>
			<td>Pulsed laser</td>
			<td>Coherent</td>
			<td>Chameleon Vision II</td>
			<td>680&#8211;1080 nm tunable laser</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>any commercial</td>
			<td></td>
			<td>Straight blade with sharp point</td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>fine science tools</td>
			<td>11000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>fine science tools</td>
			<td>15018-10</td>
			<td>To cut meninges that remain joined to both the slice obtained from vibratome cutting and the section glued to the specimen plate.</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>any commercial; e.g. Leica</td>
			<td>VT1200</td>
			<td></td>
		</tr>
	</tbody>
</table>

label-free non-linear optics for the study of tubulin-dependent defects in central myelin

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, microfilaments, and intermediate filaments in all the neural tissues, together with the variability in the outcomes of fluorescent protein fusion strategies and their limited applicability to dynamic studies of antibodies and drugs as chromophore vehicles, make classical optical approaches not as effective as for other proteins. When tubulin needs to be studied, the label-free generation of second harmonics is a very suitable option due to the non-centrosymmetric organization of the molecule. This technique, when conjugated to microscopy, can qualitatively describe the volumetric distribution of parallel bundles of microtubules in biological samples, with the additional advantage of working with fresh tissues that are unfixed and unpermeabilized. This work describes how to image tubulin with a commercial second harmonic generation microscopy setup to highlight microtubules in the tubulin-enriched structures of the oligodendrocytes, as&#160;in hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) tubulinopathy, a recently described myelin disorder.

The images obtained with this methodology have an intrinsic low background level due to the very limited number of harmonophores present in biological tissues, which is one of the significant advantages of the method.
When the fibers of the corpus callosum are imaged, fiber-like short structures and rounded elements can be consistently found in the taiep brain (Figure 3B), while the corpus callosum of the control brain shows a much more heterogeneous and ...

Watch this Scientific Journal Video about Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin at JoVE.com

Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

In this article, we present a protocol to detect microtubule-loaded oligodendrocytes in a model of tubulinopathy through a simple, innovative second harmonic generation microscopy approach.

The satisfactory visualization of cytoskeletal components in the brain is challenging. The ubiquitous distribution of the networks of microtubules, ...

label-free-non-linear-optics-for-study-tubulin-dependent-defects

Research

JoVE Journal

Neuroscience

1.8K Views.  Centro de Investigaciones en Óptica A.C. (CIO). In this article, we present a protocol to detect microtubule-loaded oligodendrocytes in a model of tubulinopathy through a simple, innovative second harmonic generation microscopy approach.

Video: Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Preparation of Drosophila Central Neurons for in situ Patch Clamping

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience research. Ion channels are the basis of all behavior since they mediate neuronal excitability. The first voltage gated ion channel cloned was the Drosophila voltage gated potassium channel Shaker1,2. Toward understanding the role of ion channels and membrane excitability for nervous system function it is useful to combine powerful genetic tools available in Drosophila with in situ patch clamp recordings. For many years such recordings have been hampered by the small size of the Drosophila CNS. Furthermore, a robust sheath made of glia and collagen constituted obstacles for patch pipette access to central neurons. Removal of this sheath is a necessary precondition for patch clamp recordings from any neuron in the adult Drosophila CNS. In recent years scientists have been able to conduct in situ patch clamp recordings from neurons in the adult brain3,4 and ventral nerve cord of embryonic5,6, larval7,8,9,10, and adult Drosophila11,12,13,14. A stable giga-seal is the main precondition for a good patch and depends on clean contact of the patch pipette with the cell membrane to avoid leak currents. Therefore, for whole cell in situ patch clamp recordings from adult Drosophila neurons must be cleaned thoroughly. In the first step, the ganglionic sheath has to be treated enzymatically and mechanically removed to make the target cells accessible. In the second step, the cell membrane has to be polished so that no layer of glia, collagen or other material may disturb giga-seal formation. This article describes how to prepare an identified central neuron in the Drosophila ventral nerve cord, the flight motoneuron 5 (MN515), for somatic whole cell patch clamp recordings. Identification and visibility of the neuron is achieved by targeted expression of GFP in MN5. We do not aim to explain the patch clamp technique itself.

Preparation of Drosophila Central Neurons for in situ Patch Clamping

In situ patch clamp recordings are used for electrophysiological characterization of neurons in intact circuitry. In the Drosophila genetic model patch clamping is difficult because the CNS is small and surrounded by a robust sheath. This article describes the procedure to remove the sheath and clean neurons for subsequent patch clamp recordings.

Short generation times and facile genetic techniques make the fruit fly Drosophila melanogaster an excellent genetic model in fundamental neuroscience ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

A Computerized Test Battery to Study Pharmacodynamic Effects on the Central Nervous System of Cholinergic Drugs in Early Phase Drug Development

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for further development of new compounds. A computerized and thoroughly validated battery of neuropsychological and neurophysiological tests has been shown to be sensitive to detect drug-induced effects of multiple new and existing compounds. The test battery covers the main CNS domains, which have been shown to respond to drug effects and can be repeatedly administered following drug administration to characterize the concentration-effect profile of a drug.
The standard tests in the battery are saccadic eye movement, smooth pursuit eye movement, the Bowdle visual analog scale (VAS), the Bond and Lader VAS, body sway, adaptive tracking, visual verbal learning, and quantitative electroencephalography (qEEG). However, the test battery is adaptive in nature, meaning that it can be composed and adjusted with tests fit to investigate specific drug classes, or even specific receptors.
Showing effects of new cholinergic drugs designed to have a pro-cognitive outcome has been difficult. The pharmacological challenge model is a tool for early proof-of-pharmacology. Here, a marketed drug is used to induce temporary and reversible disease-like symptoms in healthy subjects, via a pharmacological mechanism related to the disease that is targeted as indication for the new compound. The test battery was implemented to investigate the potential of the nicotinic receptor antagonist mecamylamine to be used as a challenge model for cholinergic dysfunction, as seen in neurodegenerative disorders.
A worsening of scores in a dose dependent manner on the visual verbal learning test (VVLT; a test for learning and memory abilities) and the adaptive tracking test (a measure of visuomotor control and arousal), in particular, showed that the test battery is sensitive to showing acute pharmacodynamic effect after administration of anti-cholinergic drugs.

A validated computerized battery of neuropsychological and neurophysiological tests is used to study pharmacodynamic effects on the central nervous system of newly developed drugs in early phase development. To demonstrate the test battery, the acute effects of mecamylamine and the reversal of these effects by two agonist drugs are described.

Investigating potential pharmacodynamic effects in an early phase of central nervous system (CNS) drug research can provide valuable information for ...

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...