This work was supported by Inserm, University of Lille 2, MEDIALZ, LabEx (excellence laboratory, program invest for the future) and DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease). F-J.F-G is currently a recipient fellowship of ANR (French National Research Agency/ NeuroSplice de Tau Projet ANR- 2010-BLAN-1114 01), but this work was also under the support of a grant from the JCCM (Spain).

1. Homogenization and Total Protein Extraction from Human and Mouse Brain Tissue
<ol>
	<li>Brain tissue homogenization. Homogenize the brain tissue 10% (w/v) in 8 M urea, 2 M thiourea and 1% w/v SDS buffer (UTS) using a potter glass (for human samples) or Teflon homogenizer (for mice samples). Sonicate at 60 Hz 30 pulses with a ultrasound generator applying 0.5 sec for total tissue disintegration13,14.
	<ol>
		<li>Determine protein concentration with Bradford assay, using BSA as standard. This UTS buffer extraction is totally compatible with one dimension SDS-PAGE as long as lysates are not over-heated to avoid carbamylation15.</li>
		<li>Add 1% (w/v) of Amberlite and incubate with gentle mixing during 10 min at room temperature. Thereafter filter onto 0.22 &#181;m syringe filters and finally add the SDS to the urea/thiourea solution. This step reduces the amount of uric acid and isocyanic acid, which are in equilibrium with urea in solution and facilitate its degradation.</li>
	</ol>
	</li>
	<li>Chloroform/methanol protein precipitation. Precipitate between 100-150 &#181;g of protein for 11 cm IPG Strips and 1-1.5 mg for 18 cm ones (independently on the pH range selected to perform the IEF).
	<ol>
		<li>Perform all the steps on ice or at 4 &#176;C. Cool chloroform and methanol at 4 &#176;C for 30 min before starting the precipitation procedure.</li>
		<li>Add three volumes of methanol and one volume of chloroform to one volume of UTS lysate. Shake manually the mixture and add three volumes of cold water. Vortex during 1 min and pellet the proteins by centrifugation at 12,000 x g for 30 min at 4 &#176;C.</li>
		<li>Discard the supernatant using a Pasteur pipette and add three volumes of cold MeOH. Vortex again and centrifuge at 12,000 x g during 30 min at 4 &#176;C. Finally discard supernatant and dry the pellet under N2.</li>
	</ol>
	</li>
	<li>Preparation of proteins for 2D analysis. Resuspend the protein-dried pellet in 2D-buffer (8 M urea, 2 M thiourea supplemented with 4% CHAPS). Wash the solution with Amberlite as indicated above and try to keep the proportion of 500 &#181;g of protein per 200 &#181;l of 2D buffer for 2D-DIGE. Note: 2D buffer can be stored at -80 &#176;C or kept at 4 &#176;C during one week (to prevent urea degradation).
	<ol>
		<li>Sonicate and store the samples at -80 &#176;C until carrying out the following step. Noteworthy, this protocol can be used after formic acid solubilization of protein aggregates10. Briefly, evaporate formic acid under N2 and resuspend the pellet as described.</li>
	</ol>
	</li>
</ol>
2. 2D-DIGE
<ol>
	<li>Samples test quality. Dose samples again using Bradford method. Dilute 15 &#181;g of proteins in LDS sample buffer, heat at 37 &#176;C15 and load onto 4-12% polyacrylamide gels.</li>
	<li>Coomassie staining. Stain polyacrylamide gel with 0.1% (w/v) Coomassie Blue G-250, 50% EtOH and 10% (v/v) acetic acid solution for at least 1 hr. Let background destain overnight in 7% acetic acid and 10% (v/v) ethanol solution.</li>
	<li>Pre-electrophoretic labeling of samples. Prior to performing cyanine dye labeling, verify the pH of the sample, which should be basic or even neutral since the N-hydroxysuccinimide substitution is more efficient at basic pH and given to be optimal at pH 8.6.
	<ol>
		<li>Label minimally the two samples of study with Cy3 or Cy5 fluorescent dyes with a ratio of 50 &#956;g protein/400 pmol dye and keep for 1 hr at 4 &#176;C in the darkness to facilitate the NHS ester reactive group of the cyanines with the unprotonated amine groups of lysines.</li>
		<li>Reduce sample variations making a cross-labeling with Cy3 and Cy5 dyes, avoiding a preferential coupling of one sample to a particular cyanine. Note that the cyanine-dye minimal labeling can be adapted for small amount of proteins with keeping the proportion of 1 &#181;g protein per 8 pmol cyanine-dye. If so, the mini-2DE system can be used instead of the large 2D gel system. This will enable the 2D-DIGE analysis for a small amount of brain tissue.</li>
		<li>Label a pool of both samples with the same amount of protein (50 &#181;g in total) with Cy2 fluorescent dye and used as internal standard. Use the same conditions as previously indicated.</li>
		<li>Complete to a total volume 350 &#956;l with 2D buffer containing 1.1% Destreak reagent, 1.2% of IPG buffer and traces of Bromophenol Blue the total 150 &#181;g of labeled proteins (50 &#181;g of Cy2, 50 &#181;g of Cy3 and 50 &#181;g of Cy2). Perform this step in quadruplicate using four independent strips. Moreover, prepare two additional non-labeled strips (one for each sample) with 350-450 &#181;g of protein for the preparative gels.</li>
		<li>Leave the six loaded strips covered with mineral oil overnight for passive rehydration.</li>
	</ol>
	</li>
	<li>Isoelectrofocusing. Carry out the IEF at 20 &#176;C. For a pH 3-11NL, 18 cm strip the protocol is: maximum current setting is 50 &#956;A/strip during 8 stages: 1) 150 V step for 1 hr, 2) 200 V step for 5 hr, 3) 500 V gradient for 2 hr, 4) 1,000 V gradient for 2 hr, 5) 2,000 V gradient for 2 hr, 6) 4,000 V gradient for 2 hr, 7) 8,000 V gradient for 2 hr and 8) for a total of 24,000 V&#183;hr step. After IEF, dispatch the excess of mineral oil using chromatography paper and store the strips at -20 &#176;C until the second dimension.</li>
	<li>IPG Strips equilibration. Equilibrate strips in 6 M urea, 2% SDS, 30% Glycerol, 50 mM Tris-HCl pH 8.6 buffer with 1% of DTT during 15 min and after with 4.7% of iodoacetamide in the same buffer but without DTT.</li>
	<li>Second dimension or SDS-PAGE electrophoresis. Make the six 12% SDS-PAGE gels (1.5 M Tris-HCl pH 8.6, 12% acrylamide/bisacrylamide, 0.1% (w/v) SDS, 0.072% (w/v) APS and 0.1% (v/v) TEMED) at the same time using a large 2D system, with four low-fluorescence glass plates for the strips with fluorescence samples and two other glass plates with 1.5 mm of thickness for preparative gels in order to excise the protein spots.
	<ol>
		<li>Load the six IPG Strips on the top of the gels and over layered with 0.5% (w/v) of agarose. Running buffer is composed of 25 mM Tris, 192 mM glycine and 0.1% (w/v) SDS. Carry out the migration at 2.5 W/gel overnight with a refrigeration system set to 4 &#176;C16,17.</li>
	</ol>
	</li>
	<li>Fluorescence images acquisition and analysis. Use a Typhoon 9400 scanner to obtain the fluorescence gels images (12 images for one experiment). Scan Cy2, Cy3 and Cy5 images for each gel at 488/520 nm, 532/580 nm and 630/670 nm excitation/emission wavelengths, respectively. Use informatic software to analyze the images. Data are representative of the changes of spot volume associated with the distribution of the signal across the four gels among the two samples studied.</li>
	<li>Coomassie staining for big gels. Fix the preparative gels in 50% (v/v) EtOH and 3% (v/v) orthophosphoric acid solution for at least 24 hr. Then after, wash twice with ultra pure water for 20 min. Perform coloration in 34% (v/v) MeOH, 17% (w/v) ammonium sulfate and 2% orthophosphoric acid for 1 hr before adding 0.1% (w/v) Coomassie Blue G-250. Keep the gels in coloration for at least 48 hr and decolorized in ultra pure water.
	<ol>
		<li>Spots for MS identification. Once fluorescence images are analyzed, the software provides a list of spots whose expression is statistically different. Manually excise these spots from the preparative gels. This procedure requires practice and some manual dexterity in order to avoid keratin contamination. Then samples can be kept in water at -20 &#176;C until sending to MS. Spot identification is perfectly compatible with Tandem mass spectrometry (MS/MS) and relevance of protein identities is judged according to the probability based Mowse score calculated with a p-value of 0.05 (p &#60; 0.05)18.</li>
	</ol>
	</li>
</ol>
3. Qualitative Mini-2DE Assay
<ol>
	<li>Resuspend maximum 100 &#181;g of protein sample in 2D buffer until a final volume of 200 &#181;l for a 11 cm IPG strip. In case of overexpression system or purified protein, the starting amount can be lowered to 20 &#181;g.</li>
	<li>Add 1.1% (v/v) Destreak Reagent, 0.55% (v/v) IPG buffer and traces of Bromophenol Blue to a final volume of 200 &#181;l. Then, vortex, make a fast spin and load into an IPG strip for passive rehydration (to bring the strip to its original thickness) overnight covered with mineral oil. Note: the proportion of IPG buffer is the same for all the interval of pH desired (pH 3-11, 4-7, 7-11, etc), but its composition varies in function of the pH range selected.</li>
	<li>Isoelectrofocalisation. Perform IEF at 20 &#176;C. The duration of the program depends on the pH interval selected. Noteworthy, parameters such as electroendosmosis may disturb the IEF profile and in these cases, optimization in electrofocalisation time is required19. Note: it is not necessarily a longer IEF that provides better results but shortening the time of IEF can also improve the resolution. After IEF, IPG strips can be stored at -20 &#176;C for months.</li>
	<li>IPG strips equilibration. Equilibrate the strips in a buffer containing 25 mM Tris-HCl pH 6.8, 20 mM DTT, 10% glycerol, 5% SDS, 0.05% Bromophenol Blue. Make three equilibration bathes for 15 min with changing the buffer solution between each bath.</li>
	<li>SDS-PAGE. Layer the IPG strip onto a precast gel (IPG+1 well, 11 cm IPG strip) with a percentage of polyacrylamide chosen depending on the MW of the protein of interest. Then, blot the gel onto a nitrocellulose/PVDF membrane at 100 mV during 33 min.</li>
	<li>Incubate overnight with the required antibody and visualize by peroxidase activity. Perform the alignments of immunoblots using unrelated polypeptides revealed by the secondary antibody. Reach a semi-quantitative analysis by densitometry of the volume and intensity for the traces/ spots with ImageJ software.</li>
</ol>

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C., Walker, D. G., Lue, L., Sue, L. I., Beach, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+interval+effect+on+RNA+and+gene+expression+in+human+brain+tissue.">Postmortem interval effect on RNA and gene expression in human brain tissue.</a> Cell Tissue Bank. 12, 311-318 (2011).</li><li>Bell, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+a+twenty-first+century+brain+bank:+experience+in+the+BrainNet+Europe+consortium.">Management of a twenty-first century brain bank: experience in the BrainNet Europe consortium.</a> Acta Neuropathol. 115, 497-507 (2008).</li><li>Crecelius, 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=Assessing+quantitative+post-mortem+changes+in+the+gray+matter+of+the+human+frontal+cortex+proteome+by+2-D+DIGE.">Assessing quantitative post-mortem changes in the gray matter of the human frontal cortex proteome by 2-D DIGE.</a> Proteomics. 8, 1276-1291 (2008).</li><li>Swatton, J. E., Prabakaran, S., Karp, N. A., Lilley, K. S., Bahn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+profiling+of+human+postmortem+brain+using+2-dimensional+fluorescence+difference+gel+electrophoresis+(2-D+DIGE).">Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).</a> Mol Psychiatry. 9, 128-143 (2004).</li><li>Viswanathan, S., Unlu, M., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+difference+gel+electrophoresis.">Two-dimensional difference gel electrophoresis.</a> Nat Protoc. 1, 1351-1358 (2006).</li><li>Tannu, N. S., Hemby, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+fluorescence+difference+gel+electrophoresis+for+comparative+proteomics+profiling.">Two-dimensional fluorescence difference gel electrophoresis for comparative proteomics profiling.</a> Nat Protoc. 1, 1732-1742 (2006).</li></ol>

Authors have no conflict of interest to declare.

Discovering pathological expression changes of proteins, search for biomarkers and the modulation of potential pathways for pharmacological targets are among the aims of the neuroproteomics approaches30. Amid the emerging tools, the 2DE field adds a promising expectative. Nevertheless, a consensus should be reached in order to minimize variability and increase reproducibility in the experiments. In line of this idea a standardized protocol to perform 2D-DIGE (Figure 2) and the subsequent 2DE i...

Mental and neurological disorders represent 13% of the global burden of disease, new challenges like pathophysiological mechanisms, risk factors and prodromal biomarkers must be explored1. In line with this objective, proteomics studies of the human brain become indispensable to uncover molecular pathways involved in processes like memory, behavior, emotions and neuronal plasticity for instance, not only for physiological but also for pathological conditions. Therefore, the use of animal models and more specifically the transgenic mice, brings a wide range of possibilities to mimic the etiology of human neurodegenerative disorders2.
Proteomics approaches are nowadays available in order to accomplish these new perspectives in the neuroscience field. Two-dimensional gel electrophoresis (2DE) is a potent and likely simple method that enables to compare the proteome of a wide range of samples. Moreover, it is also a powerful method to isolate a protein from a complex mixture in order to identify and further analyze by mass spectrometry. This technique essentially consists in two successive steps: 1) protein separation according to their isoelectric point (pI) by isoelectrofocusing (IEF). More precisely, an electric potential is applied across the immobiline acrylamide strips among a pH gradient and then proteins will migrate and focus on a determined pI in function of their global net charge. 2) Isoelectrically focused proteins are denatured and negatively charged by the addition of sodium dodecyl sulfate (SDS), thereby proteins in their first structure are separated depending on their apparent molecular weight (MW) by SDS-PAGE3. These two distinct properties allow us to tackle a double value to go further in the study of the proteome. On one hand this approach offers the possibility to perform a quantitative analysis using minimal dyes 2D-DIGE method, and on the other hand a qualitative analysis by mini-2DE coupled to western blotting.
Quantitative analysis by 2D-DIGE bestows protein expression changes all over the sample proteome. Briefly, samples are labeled with three cyanines (Cy2, Cy3 and Cy5) emitting at three distinct wavelengths (blue, green and red). These fluor minimal dyes containing N-hydroxy succinimidyl ester group react with the &#949;-amino group of lysines residues of proteins resulting in covalent amide bonds4. Lysine residues are labeled only between 1-3% and thus preventing multiple labels addition per protein and major net charge modifications5,6. Cy3 and Cy5 are often used to label two independent samples while Cy2 tags a mix of equal proportion of the samples to compare. The two main advantages are that all labeled samples are mixed and IEF and SDS-PAGE are carried out in one gel at once for each step, avoiding the inter variability among experiments due to gels comparison. Moreover, it presents a high detection threshold of around 1 femtomole of protein7. Gels are scanned and 2D software compares the 2D gel fluorescence images, where Cy2 serves as an internal standard allowing for identification of statistical differences among the spots for their posterior identification by mass spectrometry. The 2D analysis software using the internal standard achieves a fast detection of less than 10% of differences between samples with more than 95% of statistical confidence8.
Qualitative analysis by mini-2DE is a crucial step for protein characterization. The principle is the same as previously described for pI and MW separation, but in this case proteins are transferred from a small polyacrylamide gel to a membrane and immunoblotting is performed afterwards. While one dimension gel electrophoresis provides changes in protein expression for one or several protein epitopes in function of the antibody, the information of mini-2DE endows with two additional parameters. Firstly, the protein isovariants change in function of the pI, indicating that post-translational modifications might take place. Secondly, mass spectrometry identification may be indicative of plausible zymogens and catabolic products of proteins. Therefore, modifications observed by 2D-DIGE are likely indicative of the mechanisms underlying changes in global proteome profile. Alignments of immunoblots among several samples for the same protein epitope/s by mini-2DE may reflect acidity changes shedding light into post-translational variations slightly observed or even not by monodimensional immunoblotting9,10. Moreover, this analysis informs about the potential cleavage sites due to the knowledge of the pI and MW of the metabolic residues11,12.
The combination of these two techniques provides a complementary proteomics analysis. On one hand 2D-DIGE affords a type of differences allowing a precise isolation of polypeptides whose expression is different. These differences consist essentially into the appearance or disappearance of a spot or increase/decrease of intensity of a given one by software analysis of the fluorescent gels. However, these observations by themselves are unlikely to explain the nature of the modification observed. For these reasons, once the polypeptide is isolated and identified by mass spectrometry, the use of mini-2DE enables to precisely confirm 1) the identity of the protein isolated and 2) the nature of the difference: change in the isovariant/isoform level of expression, post-translational modifications and cleavage processes for instance. However, it is necessary to develop a starting lysis buffer both compatible with 2D-DIGE and with mini-2DE in order to limit the potential dispersions resulting from the use of extraction protocols that are very dissimilar.
In the present article, we described an adapted protocol for the preparation, extraction and performance of 2D-DIGE and mini-2DE techniques for brain proteins coming from human and mouse tissue.

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			<td>Reference in function of the pH interval/size</td>
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			<td height="21" style="height:21px;width:311px;">MILLEX GS Filter</td>
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			<td height="22" style="height:22px;width:311px;">Criterion XT Precast Gels 13.3 x 8.7 cm (W x L)</td>
			<td style="width:113px;">Bio-Rad</td>
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			<td>Reference in function of the MW to separate</td>
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			<td height="21" style="height:21px;width:311px;">IPGphor III Isoelectric Focusing Unit</td>
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</table>

consensus brain-derived protein, extraction protocol for the study of human and murine brain proteome using both 2d-dige and mini 2de immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

Proteomics on brain tissue remains challenging since no ideal buffer exists to recover 100% of proteins, especially membrane-associated or cytoskeleton proteins. The first set of experiments were focused on the search for a suitable lysis buffer compatible with the two approaches and which enables to recover a large panel of protein. Thus, three lysis buffers were assayed to determine the most appropriate one. Firstly, it was used the common biochemical and molecular biology buffer Tris-HCl20 at 10 mM with 1% ...

Watch this Scientific Journal Video about Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting at JoVE.c

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

consensus-brain-derived-protein-extraction-protocol-for-study-human

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Neuroscience

16.0K Aufrufe.  Inserm UMR 837.  Eine gemeinsame Proteinextraktionsprotokoll unter Verwendung von Harnstoff / Thioharnstoff / SDS Puffer für Mensch und Maus Hirngewebe ermöglicht indentification von Proteinen mittels 2D-DIGE und ihre anschließende Charakterisierung von Mini 2DE Immunoblotting. Diese Methode ermöglicht es, mehr reproduzierbare und zuverlässige Ergebnisse aus menschlichen Biopsien und experimentellen Modellen zu erhalten. 

Serie: Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

New Methods to Study Gustatory Coding

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons (GRNs) detect tastant molecules, they encode information about the identity and concentration of the tastant as patterns of electrical activity that then propagate to follower neurons in the brain. These patterns constitute internal representations of the tastant, which then allow the animal to select actions and form memories. The use of relatively simple animal models has been a powerful tool to study basic principles in sensory coding. Here, we propose three new methods to study gustatory coding using the moth Manduca sexta. First, we present a dissection procedure for exposing the maxillary nerves and the subesophageal zone (SEZ), allowing recording of the activity of GRNs from their axons. Second, we describe the use of extracellular electrodes to record the activity of multiple GRNs by placing tetrode wires directly into the maxillary nerve. Third, we present a new system for delivering and monitoring, with high temporal precision, pulses of different tastants. These methods allow the characterization of neuronal responses in vivo directly from GRNs before, during and after tastants are delivered. We provide examples of voltage traces recorded from multiple GRNs, and present an example of how a spike sorting technique can be applied to the data to identify the responses of individual neurons. Finally, to validate our recording approach, we compare extracellular recordings obtained from GRNs with tetrodes to intracellular recordings obtained with sharp glass electrodes.

We present three new methods to study gustatory coding. Using a simple animal, the moth Manduca sexta (Manduca), we describe a dissection protocol, the use of extracellular tetrodes to record the activity of multiple gustatory receptor neurons, and a system for delivering and monitoring precisely timed pulses of tastants.

The sense of taste allows animals to detect chemicals in the environment, giving rise to behaviors critical for survival. When Gustatory Receptor Neurons ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

Laser Microdissection-Based Protocol for the LC-MS/MS Analysis of the Proteomic Profile of Neuromelanin Granules

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. Besides neuromelanin, NMGs contain a variety of proteins, lipids, and metals. Although NMGs-containing dopaminergic neurons are preferentially lost in neurodegenerative diseases like Parkinson&#39;s disease and dementia with Lewy bodies, only little is known about the mechanism of NMG formation and the role of NMGs in health and disease. Thus, further research on the molecular characterization of NMGs is essential. Unfortunately, standard protocols for the isolation of proteins are based on density gradient ultracentrifugation and therefore require high amounts of human tissue. Thus, an automated laser microdissection (LMD)-based protocol is established here which allows the collection of NMGs and surrounding substantia nigra (SN) tissue using minimal amounts of tissue in an unbiased, automatized way. Excised samples are subsequently analyzed by mass spectrometry to decipher their proteomic composition. With this workflow, 2,079 proteins were identified of which 514 proteins were exclusively identified in NMGs and 181 in SN. The present results have been compared with a previous study using a similar LMD-based approach reaching an overlap of 87.6% for both proteomes, verifying the applicability of the revised and optimized protocol presented here. To validate current findings, proteins of interest were analyzed by targeted mass spectrometry, e.g., parallel reaction monitoring (PRM)-experiments.

A robust protocol is presented here for isolating neuromelanin granules from human post-mortem substantia nigra pars compacta tissue via laser microdissection. This revised and optimized protocol massively minimizes the required time for sample collection, reduces the required sample amount, and enhances the identification and quantification of proteins by LC-MS/MS analysis.

Neuromelanin is a black-brownish pigment, present in so-called neuromelanin granules (NMGs) in dopaminergic neurons of the substantia nigra pars compacta. ...

Revolutionizing fNIRS Studies&amp;#58; A Novel Approach to Neural Coupling

New Framework for Understanding Cross-Brain Coherence in Functional Near-Infrared Spectroscopy (fNIRS) Hyperscanning Studies

Despite the growing body of functional near-infrared spectroscopy (fNIRS) hyperscanning studies, the assessment of coupling between two neural signals using wavelet transform coherence (WTC) seems to ignore the directionality of the interaction. The field is currently lacking a framework that allows researchers to determine whether a high coherence value obtained using a WTC function reflects in-phase synchronization (i.e., neural activation is seen in both members of the dyad at the same time), lagged synchronization (i.e., neural activation is seen in one member of the dyad prior to the other member), or anti-phase synchronization (i.e., neural activation is increased in one member of the dyad and decreased in the other). To address this need, a complementary and more sensitive approach for analyzing the phase coherence of two neural signals is proposed in this work. The toolbox allows investigators to estimate the coupling directionality by classifying the phase angle values obtained using traditional WTC into in-phase synchronization, lagged synchronization, and anti-phase synchronization. The toolbox also allows researchers to assess how the dynamics of interactions develop and change throughout the task. Using this novel WTC approach and the toolbox will advance our understanding of complex social interactions through their uses in fNIRS hyperscanning studies.

Author Spotlight: Unlocking New Insights in fNIRS Studies -  A Novel Framework for Inter-Brain Synchrony Analysis 

Wavelet transform coherence (WTC) is a common methodology for assessing the coupling between signals that is used in functional near-infrared spectroscopy (fNIRS) hyperscanning studies. A toolbox for assessing the directionality of the signal interaction is presented in this work.

Despite the growing body of functional near-infrared spectroscopy (fNIRS) hyperscanning studies, the assessment of coupling between two neural signals ...