Name: label-free non-linear optics for the study of tubulin-dependent defects in central myelin
Uploaded: 2023-03-24T12:20:00
Description: Watch this Scientific Journal Video about Label-Free Non-Linear Optics for the Study of Tubulin-Dependent Defects in Central Myelin at JoVE.com

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.
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<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. 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+of+living+cells.">Second-harmonic imaging microscopy of living cells.</a> Journal of Biomedical Optics. 6 (3), 277 (2001).</li><li>Campagnola, P. 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=Three-dimensional+high-resolution+second-harmonic+generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.</a> Biophysical Journal. 82 (1), 493-508 (2002).</li><li>Yu, C. -. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization-dependent+optical+second-harmonic+imaging+of+a+rat-tail+tendon.">Polarization-dependent optical second-harmonic imaging of a rat-tail tendon.</a> Journal of Biomedical Optics. 7 (2), 205 (2002).</li><li>Brown, E. 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>

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, ...

Second-Harmonic Generation imaging of biological samples is an optical technique that allows the visualization of non-centrosymmetric molecules and their assemblies without the need of a tag or a dye. The images generated with this method have low background since very few biological molecules could act as a harmonal force. This protocol describes the application of the technique for diagnostic imaging of Tubulin beta-4A in the taiep animal model.Tubulinopathies are recently described diseases. For this reason, a limited amount of information is available about the basic mechanisms underlying the pathophysiology at the cellular level. To begin, turn on the pulse laser to guarantee it will be ready to lase at an optimal and steady power level.To study tissular microtubules, 10 to 20%of the available laser power is used, which in the described system corresponds to 13 to 26 milliwatts measured at the back focal plane of the objective. Prior to imaging, turn the laser to 810 nanometers. Then ensure that the microscope is Koehler-aligned with the objective used for the Second-Harmonic Generation, or SHG imaging.Remove unwanted filters from the optical detection path and ensure that the condenser diaphragm is fully opened and no light is unnecessarily stopped. Prepare a 25x by 0.8 numerical aperture oil immersion objective with a small drop of immersion oil in the lens. Following the steps reported in the table here, except for the laser power which is less than 5%for cornstarch.Image dry cornstarch sandwiched between glass slides. with SHG parameters to generate SHG images, they reveal the laser polarization direction. Capture one image of sparse cornstarch grains and mark the orientation of the SHG lobules in the XY plane corresponding to the laser polarization orientation.For control, take another image of the same sample inserting a half-wave plate into the optical path to alter the laser's oscillation direction. The resulting image displays rotated SHG signal lobules. If using a different bandpass filter for the SHG, ensure that it has optimal transmission properties by comparing the images and the signal intensities pixel-by-pixel along the scan line.Prepare a vibratome buffer tray filled with a warm Hanks'Balanced Salt Solution or HBSS. Fix the brain to the specimen plate using cyanoacrylate glue via a piece of masking tape. The most caudal portion contacts the glue so that the usable coronal sections from the opposite rostral portion can be cut.Transfer the specimen plate onto its magnetic support inside the buffer tray. Start cutting 300 to 500 micron sections until the slices encompass the entire brain surface. Then reduce the thickness of the section to 160 microns.Once a 160 micron section is cut at the level of the corpus callosum, recover it with a modified glass Pasteur pipette with a large flanged orifice. Transfer the section to a clean Petri dish with new warm HBSS or directly onto the cover slip or glass bottom dish for microscopy. Place the sample under the microscope and position it appropriately under the objective by direct observation through the oculars with transmitted light.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. Prepare the microscope stage for non-descanned imaging, which includes closing all the doors of the dark incubation chamber or covering the incubation chamber with a black nylon polyurethane coated fabric.Select the non-descanned imaging mode along the transmission path. This way, the capture of the weak SH signal of tubulin will be optimized. Then select the objective.Next, set a laser power with a pixel-dwell time of 12.6 microseconds. Take images no bigger than 512 by 512 pixels with speed five and averaging two for an average acquisition time of 15 seconds. Capture images first using a 485 nanometer shortpass filter, and in a second step, add a sharp 405 nanometer bandpass filter.Cut the cerebellum with a scalpel into two hemispheres and fix them to the vibratome support by the middle portion. Section and image the cerebellum using the same vibratome, blade, and microscope settings as described for the brain. When the fibers of the corpus callosum are imaged, fiber-like short structures and rounded elements are observed in the taiep brain.In contrast, the corpus callosum of the control brain shows a much more heterogeneous and isotropic signal throughout the brain region. The origin of the differential signal lies specifically in the Second-Harmonic Generation phenomenon since adding the narrow bandpass filter only decreases the non-specific signal intensity from the control images, while selectively removing this low diffuse signal from around the soma-like and the short elongated structures in the taiep images which always generate intense SH light. In cerebellar white matter control tissue, the complete absence of SH signal was observed.The Purkinje cells are barely visible when using the short pass filter, and the elongated and rounded structures persist in the SH image from the taiep tissue. A critical step to obtain the best image is to cut tissue sections as thin as possible. Moreover, working with acute tissue sections requires fast protocols to prevent tissue deterioration.Therefore, it is crucial to set up and check the optical system beforehand. Second-harmonic signal from microtubules is weaker than second-harmonic signal from starch. Therefore, more laser power has to be used for imaging microtubules.The laser power should be increased carefully because, as a high numerical aperture objective is used for imaging, the intensity at the sample would rapidly increase With Second-Harmonic Generation imaging, the pathological changes in the central myelinomatic of the tubulinopathic model are quickly and easily identified. Potential applications of this technique include its use for the study of the basic mechanisms of H-ABC tubulinopathy and for the assessment of pharmacological therapies to treat patients. On the long term, the technique has the potential to become a complimentary diagnostic tool to be used intracranially or on fresh biopsies.

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

Research

JoVE Journal

Neuroscience

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 ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...