This work was supported in part by the Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control 26117004; to M.I. and T.M.). This research was partially supported by the Strategic Research Program for Brain Sciences from the Japan Agency for Medical Research and Development (AMED). All experiments were conducted in compliance with the ARRIVE guidelines.

Here, tissue samples were collected at the Tokyo Metropolitan Geriatric Hospital, which provides community-based medical services to the elderly population in Japan. The brain autopsy samples used in the study were registered to the Brain Bank for Aging Research (BBAR) with the informed consent of the deceased&#8217;s relatives. The BBAR is approved by the ethics committee of the Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology. For all brains registered at the brain bank, we obtained written informed consents for their use for medical research from the patients or their families. The patients were placed in a cold (4 &#176;C) room within 2 h after death to reduce postmortem changes in the brain. All methods described here have been approved by Doshisha University and the Brain Bank for Aging Research, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology.
1. Preparation of Tissue Sections for IMS
<ol>
	<li>Processing tissue specimen of a human autopsied brain 
	Note: Ensure that the sample preparation step for IMS preserves the original state of the tissue. Avoid contamination and post-mortem changes. The following step is crucial.
	<ol>
		<li>Obtain human cortical specimens for IMS from brains that were removed, processed, and stored at -80 &#176;C within 8 h postmortem. Take the brain specimen from the occipital cortex of AD patients and age-matched controls21.</li>
	</ol>
	</li>
	<li>Preparation of frozen tissue sections
	<ol>
		<li>Cut the tissue sections on a cryostat. Place conductive ITO-coated microscope glass slides inside the cryostat21.</li>
		<li>Warm the autopsy brain specimen from -80 &#176;C to -22 &#176;C inside the cryostat.</li>
		<li>Attach a new disposable blade to the cryostat for every experiment. Always try to use a clean part of the blade.</li>
		<li>Put the frozen autopsy brains on the stage along with a small amount of OCT compound (enough to cover the central area of the stage; see Table of Materials).</li>
		<li>Choose the conditions for thin sectioning. For IMS, use a thickness of 10&#8211;12 &#956;m for human brain sections. For IMS and IHC, cut five to six sections from each tissue sample.</li>
		<li>When the blade is just starting to cut the tissue, turn the wheel and &#8220;face&#8221; the block until all of the tissue is exposed. If there is a small streak or tear across the section, wait a little more in the cryostat until the temperature adjustment automatically fixes it. Count a few seconds before opening the anti-roll with a tissue underneath.</li>
		<li>Immediately place the tissue slice on the ITO-coated side of the glass slide. Thaw the tissue slice by putting a finger underneath the slide on the non-ITO-coated side. The tissue will stick to the slide; ensure that the tissue is as flat as possible with no wrinkles. Perform this step at room temperature. 
		Note: Wear gloves, masks, and a laboratory gown because the human samples may contain biological contaminants. When all sections have been made for the day, clean the cryostat, brushes, and chucks with laboratory wipes and 200 mL of 100% ethanol.</li>
	</ol>
	</li>
	<li>Rinsing the tissue sections
	<ol>
		<li>Immerse the samples in 40&#8211;100 mL of 70% ethanol for 30 s to remove endogenous lipids and inorganic salts. Use a glass staining jar.</li>
		<li>Wash the samples with 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of Carnoy&#8217;s solution for 3 min, 40&#8211;100 mL of 100% ethanol for 30 s, 40&#8211;100 mL of 0.1% trifluoroacetic acid (TFA) for 1 min, and 40&#8211;100 mL of 100% ethanol for 30 s. Use a glass staining jar. 
		Note: Carnoy&#39;s solution is a fixative composed of six parts ethanol, three parts acetic acid, and one part chloroform.</li>
		<li>Dry in a vacuum for 30 min.</li>
	</ol>
	</li>
	<li>Treatment of the tissue sections with a formic acid vapor for a better ionization of the A&#946; proteins from autopsy brain tissues
	<ol>
		<li>Prepare the oven and incubation glass slide to be used for the subsequent vaporization with 5 mL of 100% formic acid. To achieve a satisfactory acid treatment, keep the air humidity in the incubation glass dish at saturation level throughout this step and keep the temperature at 60 &#176;C. Place the tissue slides in the incubation glass dish while avoiding submersion in the formic acid and treat for 6 min.</li>
		<li>Take an optical image of the samples using a film scanner, gel scanner, or a digital microscope, etc. Perform this step at room temperature. The alignment of the optical image of the samples is necessary when the sample target is placed inside the instrument. Usually, it will not be possible to recognize the tissue section underneath the matrix layer. 
		NOTE: The most convenient way to take an optical image is to use an office scanner. It is a good idea to save the image in the imaging data storage folder.</li>
		<li>To correlate the optical images with the samples, make guide marks that are visible both in the optical image and underneath the matrix layer in the camera optic. The easiest way is to spot at least three correction fluid marks around the sample before taking the optical image.</li>
	</ol>
	</li>
	<li>Matrix application
	<ol>
		<li>Preparation of the matrix solution
		<ol>
			<li>In an organic solvent-tolerant microtube, prepare a 10 mg/mL SA solution in 50% acetonitrile (ACN) and 0.1% TFA. Thoroughly dissolve the SA compound by vortexing or brief sonication for 10 min. Store the solution at room temperature until use. 
			NOTE: There are three different options for matrix spraying: using an airbrush, an ultrasonic sprayer, or an automatic sprayer.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an airbrush
		<ol>
			<li>Perform the operation at a constant room temperature (20&#8211;23 &#176;C) and humidity (40%&#8211;60%). The parameters to adjust for optimal spraying include the size of the droplet, the amount of mist, the angle and distance between the spray nozzle and the tissue section, and the laboratory temperature and humidity. Adjust these conditions by checking the microscopy results. 
			Note: As limiting factors include the crystal size and homogeneity of the matrix coverage and the undesirable migration/diffusion of analytes, smaller is better for the drop size. Homogeneity is also the point of inspection.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an ultrasonic sprayer
		<ol>
			<li>Remove the tissue to be sprayed from the desiccator and place it in the chamber. Make sure the tissue is not covering the sensor window.</li>
			<li>Start the preparation by pushing the Start button; usually, prep time is around 90 min. The preparation will be regulated automatically via the monitoring of the matrix layer thickness and wetness. After the preparation is complete, remove the slide and store it in the desiccator for 15 min before reading it in the MALDI instrument.</li>
			<li>Clean the sprayer with 2&#8211;3 mL of 100% MeOH until the spray head appears clean. 
			Note: A fine mist of matrix droplets is allowed to sink into the tissue by a gravitational sheet. An average droplet size of 20 &#956;m is generated; all droplet diameters are less than 50 &#956;m.</li>
		</ol>
		</li>
		<li>Spraying the matrix with an automatic sprayer
		<ol>
			<li>Spray the matrix solution on the tissue surface with an automatic sprayer. A constant &#64258;ow of heated sheath gas (N2, set at 10 psi and 75 &#176;C) will be delivered conjointly with the matrix solution spray. Use a solvent pump system (set at 10 psi and 0.15 mL/min) to deliver the matrix solution. 
			Note: Most importantly, work under a safety cabinet to avoid any inhalation of matrix aerosols. Control the room temperature and humidity to reproduce homogenous matrix crystallization.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. MALDI-IMS
<ol>
	<li>Perform ultra-high-speed mass spectrometry.
	<ol>
		<li>Perform high-throughput and high-spatial-resolution imaging experiments with MALDI-IMS equipped with a 10 kHz Nd:YAG (355 nm) laser.</li>
		<li>For mass spectrometry measurements, define the tissue areas using the MALDI control software and data analysis software.</li>
		<li>Acquire spectra in a positive linear mode with a mass range of m/z 2,000&#8211;20,000 and a spatial resolution of 20 and 100 &#181;m.</li>
		<li>To make the calibration standard, dissolve the peptide calibration standard and the protein calibration standard with a ratio of 1:4 with alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA) in TA30 solution (ACN:0.1% TFA = 30:70) and then dilute it 10x. Place 1 &#956;L of calibration standard on the slide at four different locations.</li>
	</ol>
	</li>
	<li>Using molecular histology software (see Table of Materials), overlay multiple single images to find the spatial correlation of various signals, such as different A&#946; peptides colocalizing in SPs and arterial walls.</li>
</ol>
3. Data Processing
<ol>
	<li>For spectral alignment, align all spectra to a selected m/z list from a previous publication21.</li>
	<li>Import the MALDI-IMS data set into statistical analysis software (see Table of Materials) with baseline subtraction.</li>
	<li>Trace regions of interest (ROIs) based on histological knowledge.
	<ol>
		<li>Perform a univariate analysis for the mean intensities, standard deviations, uncovering of discriminative m/z-markers (ROC analysis), hypothesis tests, and discovery of colocalized m/z values.</li>
	</ol>
	</li>
	<li>Create a segmentation map.
	<ol>
		<li>Perform an unsupervised multivariate analysis for the spatial segmentation of large datasets and a component analysis for the extraction of underlying trends.</li>
		<li>Select anatomical ROIs based on histological characteristics, such as parenchyma and the subarachnoid space.</li>
		<li>Assign structures as individual ROIs and correlate them with the processed MS data in order to identify the associated peptides that allowed the image segmentation of the region.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here we demonstrated a detailed protocol and results of the visualization of A&#946; and its isoforms from several autopsied brains with AD and CAA with MALDI-IMS. The deposition profile of A&#946;s was drastically changed from A&#946;1-42 to its N- and C-terminal variations. A&#946;1-41 was first identified and visualized in human brains with the current protocol and was further validated with IHC21. Considering that the morphology of deposited A&#946; by IMS with a high-resolution analysis (20 &#956;m) must be in good agreement with IHC, IMS and IHC equally contribute to distinguishing A&#946; deposits by their location and protein contents, as well as by their morphology. As the entire experiment described here was conducted in the occipital cortex, studying the localization of different beta-amyloid species across all brain regions will be generalized with future experiments using the current protocol.
Critical steps within the protocol are tissue preparation steps to obtain an effective ionization of aggregated proteins in human brain tissues. A matrix layer is required to absorb the laser energy and induce desorption and ionization of analytes. In this process, an entire tissue section is homogeneously coated with small crystals. Homogenous cocrystallization of the analyte with the matrix is crucial for high-sensitivity and artifact-free imaging. Each of the three spray methods has its own advantages. Manual spray coating is one of the most frequently used methods. An airbrush is convenient; however, it requires skillful operation. As precise and reproducible experimental technique is essential, using an ultrasonic sprayer and/or an automatic sprayer as described in the current protocols is recommended. With respect to an ultrasonic sprayer, it will not be influenced by the humidity and temperature of the room because it is sprayed into a chamber. Meanwhile, with an automatic sprayer, relatively preserved spatial resolution with good reproducibility is obtained. Generally, the spatial resolution obtained with the three methods increase in the order of 1) airbrush, 2) automatic device, and 3) ultrasonic sprayer.
Most importantly, this protocol was originally generated to detect and visualize A&#946;s in human autopsied brain samples. The visualization of A&#946;s with MALDI-IMS for APP23 mice, which is generated as an animal model of AD based on the Swedish-type mutation of human APP, has been reported earlier by others with an existing method18,19. However, the former protocol applied to APP23 was not sufficient to visualize A&#946; in its lateral resolution and sensitivity. Earlier works discussed that high A&#946; concentrations outside the tissue boundary are clearly artifacts in APP23 imaging with their protocol18,19. That means the so-called &#39;blur&#39; between real SPs and IMS images due to the matrix extraction step was inevitable with MALDI-type imaging. However, in the current protocol, the blur disappeared and each spectrum represented every single SP in the brain parenchyma.
As shown here, we can trace A&#946;1-41 for the first time in AD and CAA brains with MALDI-IMS, as well as with IHC, with a specific antibody by our own generation21. According to the A&#946; processing model, A&#946;1-38 derives from A&#946;1-45 via A&#946;1-42, while A&#946;1-41 derives from A&#946;1-45 by &#947;-secretase stepwise cleavage27,28,29,30,31. This means that the current protocol supports this model. As for the limitations of this technique, we must consider the heterogeneity of the samples from human autopsy brain. The most critical step, in a sense, is to assess qualified autopsy brain tissue with ethical proof. With the current protocol on those qualified autopsy brain tissues, MALDI-IMS can individually track the whole distribution of the complex molecules having multiple modifications, as well as unknown factor(s) regulating the pathogenesis of AD, that are yet to be defined. Furthermore, in understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a standard approach, in combination with clinical, genetic, and pathological observations in neurological diseases.
Another critical step of MALDI-IMS is the data mining process from the obtained data set, which is always time-consuming. Manual data mining of each peak distribution requires the users to click through every image and look for distributions that may correlate to the morphology of the analyzed sample. Automatic spatial segmentation can be used as the first step of data mining, providing an overview of the data set and allowing the quick detection of prominent features. In this approach, similarities between spectra of a given region are statistically determined, and similar spectra are grouped into one cluster. All pixels are color-coded according to their cluster assignment (Figure 5). In the present AD/CAA study, the area of interest is the space of A&#946; depositions in parenchymal plaque and the subarachnoid and parenchymal vascular structures. The two distinctive peaks which were further validated with IHC were the m/z values from A&#946;1-40 and A&#946;1-4221. Thus, it is easy to find colocalized m/z with A&#946;1-40 and A&#946;1-42 which was already annotated to N- and C-terminal truncated A&#946;, as well as unknown peptides, for further analysis.
Weller and colleagues have reported that A&#946; accumulates in vessel walls, more around arteries than around veins21. Moreover, it has been proposed that the interstitial fluid (ISF) includes A&#946;s excreted from the cerebral parenchyma to the lymph node via a perivascular drainage pathway22,23,24,25,26. The current protocol to generate a segmentation map based on a MALDI-IMS data set at a 20 &#181;m resolution supports the possible existence of perivascular drainage pathways of the brain (Figure 5), which contribute significantly to CAA in AD21,32. Furthermore, we can discover marker proteins colocalized with plaque and subarachnoid vasculature by calculating the correlation of each m/z values. In understanding the overall pathogenesis of various neuropathology in aged human brains, it must be feasible to adopt MALDI-IMS as a powerful approach in combination with established IHC data of clinical, genetic, and pathological observations in neurological diseases.

In order to make the diagnosis and to understand the pathogenesis of neurodegenerative disorders, precise molecular identification of pathological deposits is essential1. In the course of AD, A&#946; is produced to make SPs in the cerebral parenchyma and deposited in vessels long before disease onset2,3,4,5,6. While A&#946;1-42 is the predominant peptide in the SP of AD brains, other A&#946; variants, such as N-terminal or C-terminal truncated or modified A&#946;s, are also identified in affected AD brains7,8,9,10. A full picture of the broad range of A&#946; species in human brains, especially with AD and cerebral amyloid angiopathy (CAA)11, will help scientists understand A&#946; production, metabolism, and deposition.
As the classical approach of neuropathology, immunohistochemistry (IHC) has been the most conclusive method to determine the location of A&#946;s in the brain tissues12,13,14,15. In general, IHC cannot distinguish molecules when several epitopes coexist simultaneously. By contrast, an emerging mass spectrometry-based proteomic analysis is a valuable approach especially for analyzing a variety of A&#946; species in brain tissues, which cannot be differentiated with antibodies16,17. The conventional mass spectrometry-based analysis of brain lysates and immuno-precipitated samples fails to detect minor A&#946; peptides and loses distribution information of A&#946; in the brain tissue.
In earlier works, visualizing A&#946; deposits in mouse brains has been successful using a transgenic animal model of AD, such as APP23. However, this process still needs technical advancements to compare IMS and IHC with respect to resolution and sensitivity18,19,20. AD neuropathology should be studied on human brains, and we used MALDI-IMS technology on human autopsy brain tissues to obtain comprehensive protein mapping21. For this purpose, we developed a protocol for an advanced type of mass spectrometry that has advantages in its rapidity, sensitivity, and reproducibility.

<table>
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>Indium tin oxide (ITO)-coated microscope glass slide</td>
			<td>Bruker Daltonics</td>
			<td>#237001</td>
			<td></td>
		</tr>
		<tr>
			<td>Blade (disposable)</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>#117394</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>FSC 22 Blue</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanner</td>
			<td>EPSON</td>
			<td>GT-980</td>
			<td></td>
		</tr>
		<tr>
			<td>Air-Brush</td>
			<td>GSI Creos</td>
			<td>PS274</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressor</td>
			<td>MRHOBBY</td>
			<td>Mr.Linear compressor L5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic sprayer</td>
			<td>Bruker Daltonics</td>
			<td>ImagePrep</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic sprayer</td>
			<td>HTX Technologies</td>
			<td>TM Sprayer</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal-laser-scanning-microscope</td>
			<td>Carl Zeiss Inc.</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra high speed MALDI instrument</td>
			<td>Bruker Daltonics</td>
			<td>rapifleX MALDI Tissuetyper</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI control software</td>
			<td>Bruker Daltonics</td>
			<td>FlexControl 3.8</td>
			<td></td>
		</tr>
		<tr>
			<td>Data analysis software</td>
			<td>Bruker Daltonics</td>
			<td>FlexImaging 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular histology software</td>
			<td>SCiLS, Bremen, Germany</td>
			<td>SciLS Lab 2016a</td>
			<td></td>
		</tr>
		<tr>
			<td>Statistical software</td>
			<td>SCiLS, Bremen, Germany</td>
			<td>SciLS Lab 2016a</td>
			<td></td>
		</tr>
		<tr>
			<td>Sinapinic acid (SA)</td>
			<td>Nacalai tesque</td>
			<td>30494-91</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha-Cyano-4-hydroxyl-cinnamic acid (CHCA)</td>
			<td>Wako</td>
			<td>037-19261</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibration standard</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse IgG antibodies</td>
			<td>Vector Laboratories, Inc., Burlingame, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-rabbit IgG antibodies</td>
			<td>Vector Laboratories, Inc., Burlingame, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin and biotinylated HRP complex.</td>
			<td>Vector Laboratories, Inc., Burlingame, CA</td>
			<td>Vectastain Elite ABC kit</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3-diaminobenzidine (DAB)</td>
			<td>Vector Laboratories, Inc., Burlingame, CA</td>
			<td>Vectastain Elite ABC kit</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of amyloid &#946; deposits in the human brain with matrix-assisted laser desorption/ionization imaging mass spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Sporadic AD patients with CAA (n = 5; mean age = 83.2 y) and aged subjects with senile plaque free (SP O) and CAA (n = 5; mean age = 77.2 y) were analyzed (Table 1). The CAA phenotypes of patient #3 were most prominent in this study. Distributions of A&#946;1-40 and A&#946;1-42 deposits in the brain tissue from patient #3 were visualized with MALDI-IMS (Figure 1). There were no significant signals in nonpathological control brains (patients #9 and #10), as shown in Figure 1. Here, MALDI-IMS clearly visualized that A&#946;1-42 was preferentially deposited as SPs in the cerebral parenchyma. By contrast, shorter A&#946;s, such as A&#946;1-36 to 1-41, were preferentially deposited on the leptomeningeal vascular areas (Figure 1).
Distributions of A&#946;40 and A&#946;42 were further validated with IHC using adjacent frozen sections of the tissues. The anti-A&#946;40 antibody labeled CAA (arrows in Figure 2B), which is in clear contrast to the distribution of A&#946;42 in the cerebral parenchyma as SPs. For A&#946;1-41, we are the first to detect this fragment in human brains, and we have generated specific antibodies that can differentiate A&#946;41 from A&#946;40 andA&#946;4221.
The spatial resolution of the MALDI-IMS is generally the most important factor to be improved upon. In Figure 1 and Figure 2 show MALDI-IMS with a 100 &#181;m pitch resolution and obtain an overall distribution profile for a relatively wide area21. It is difficult to define amyloid deposition exactly at the subarachnoid space, including vascular structure. To portray fine tissue structures of subarachnoid vessels and the surface of the cortex, high-resolution MALDI imaging (40 &#181;m: Figure 3; 20 &#181;m: Figure 4 and Figure 5) was performed. As a result, MALDI-IMS clearly demonstrated that shorter A&#946;s, such as A&#946;1-36 to 1-41, are distributed in the walls of arteries, which is comparable with IHC.
MALDI-IMS demonstrated the detailed distributions of both A&#946;x-40 and A&#946;x-42 (x = 2, 4, 5, 6, 7, 8, 9, and 11pE) in AD accompanied with moderate CAA brain. For example, while A&#946;x-40 species showed a similar distribution profile to A&#946;1-40, A&#946;x-42 species showed a different distribution pattern with A&#946;1-4221. By the current protocol, single ion images of the individual A&#946; peptides observed with MALDI-IMS are assigned to A&#946; species. By contrast, acquired image data can be investigated using unsupervised multivariate statistics in order to obtain image segmentation of anatomical regions of interest for the further analysis of undefined proteins. Figure 5 shows a segmentation map obtained with a bisecting k-means analysis applied to the same section from patient #321. This clustering method successfully identified plaque-like structures in the parenchyma (blue in Figure 5C) and vascular structures in the subarachnoid space (green in Figure 5C). It is interesting to find a small circular area in the parenchyma, which is detected just around a small arteriole in the parenchyma, as well as in the subarachnoid space (purple in Figure 5C). This is verified by single ion images of these individual A&#946; peptides in Figure 5D-5F.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57645/57645fig1.jpg" /> 
Figure 1: MALDI-IMS of a frozen AD/CAA brain section. Various C-terminal truncated A&#946; peptides in AD accompanying severe CAA (patient #3) are visualized in the left panel and controls (patient #9 on the right and patient #10 on the left in all images) in the right panel. A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques in case #3, while there was no signal in the control patients&#39; brains (cases #9 and #10). Resolution = 100 &#956;m. Scale bar = 5 mm. This figure has been modified with permission from Kakuda et al.21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57645/57645fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57645/57645fig2.jpg" /> 
Figure 2: MALDI-IMS of frozen AD/CAA brain sections and adjacent sections of the occipital cortex from AD brains. (A - C) The frozen AD/CAA brain sections and (D and E) adjacent sections of the occipital cortex from AD brains were immune-stained and focused on arteriole and cerebral parenchyma,&#160;using antibodies against A&#946;40 (D: BA27) or A&#946;42 (E: anti-A&#946;42 polyclonal). Both analyses demonstrated that A&#946;40 is preferentially deposited in leptomeningeal blood vessels (arrows in panel D) and arterioles in the subarachnoid space and the cerebral parenchyma forming CAA. In contrast, A&#946;42 is mainly deposited in SPs. For IMS, Resolution = 100 &#956;m. Scale bar = 5 mm (A, B, and C) = 5 mm, 500 (D and E). This figure has been modified with permission from Kakuda et al.21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57645/57645fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57645/57645fig3.jpg" /> 
Figure 3: MALD-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 40 &#181;m. Various C-terminal and N-terminal truncated and modified A&#946; peptides in AD accompanying severe CAA (patient #3). A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 1 mm (A-B),&#160;2 mm (upper left). Resolution = 40 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57645/57645fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57645/57645fig4.jpg" /> 
Figure 4: MALDI-IMS of frozen AD/CAA brain sections (patient #3) at a resolution of 20 &#181;m. This panel shows various C-terminal and N-terminal truncated and modified A&#946; peptides in AD accompanying severe CAA (patient #3). A&#946;1-36 to A&#946;1-41 are preferentially deposited in leptomeningeal blood vessels, while A&#946;1-42 and A&#946;1-43 are deposited in the cerebral parenchyma as senile plaques. Scale bars = 200 &#956;m (a, b, c), 1 mm (upper left). Resolution = 20 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57645/57645fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57645/57645fig5.jpg" /> 
Figure 5: Segmentation map, obtained by MALDI-IMS of a frozen AD brain section, reveals putative senile plaque, large subarachnoid vessel structures, and small parenchymal arterioles. (A) Segmentation map obtained from a multivariate image analysis of MALDI-IMS data. (B) Bisecting k-means based clustering analysis identified plaque-like and vessel-like structures in the occipital cortex. The clusters and substructures and their relations are shown as nodes (e.g.,1-0-0). (C) Distinct A&#946; peptide localization patterns resembling plaques (blue), subarachnoid vessels (green), and arteriole (red) structures. Note that a few red clusters are also distributed in the subarachnoid space. This is verified by single ion images of these individual A&#946; peptides at a 20 &#181;m resolution: (D) A&#946; 1-40, (E) A&#946; 1-41, and (F) A&#946; 1-42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57645/57645fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Case</td>
			<td>Gender</td>
			<td>Age at death</td>
			<td>Braak SP</td>
			<td>CAA</td>
		</tr>
		<tr>
			<td>1</td>
			<td>M</td>
			<td>83</td>
			<td>C</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>2</td>
			<td>M</td>
			<td>88</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>3</td>
			<td>M</td>
			<td>84</td>
			<td>C</td>
			<td>2</td>
		</tr>
		<tr>
			<td>4</td>
			<td>M</td>
			<td>78</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>5</td>
			<td>M</td>
			<td>83</td>
			<td>C</td>
			<td>1</td>
		</tr>
		<tr>
			<td>6</td>
			<td>M</td>
			<td>84</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>7</td>
			<td>M</td>
			<td>78</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>8</td>
			<td>M</td>
			<td>70</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>9</td>
			<td>M</td>
			<td>73</td>
			<td>O</td>
			<td>0</td>
		</tr>
		<tr>
			<td>10</td>
			<td>M</td>
			<td>81</td>
			<td>O</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Clinical and pathological data of AD with CAA cases and aged SP O subjects. Human cortical specimens for IMS and IHC were obtained from the Brain Bank at Tokyo Metropolitan Institute of Gerontology. Each brain specimen was taken from the occipital cortex of five AD patients and five age-matched controls. The extent of amyloid deposition as shown by an A&#946; monoclonal antibody was defined by Braak SP (amyloid) stages. At stage O, there are almost no senile plaques throughout the isocortex. At stage C, virtually all the isocortical areas are affected. AD brains are invariable at stage C. This table has been modified with permission from Kakuda et al.21.

Watch this Scientific Journal Video about Visualization of Amyloid ÃŽÂ² Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry at JoVE.com

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

visualization-amyloid-deposits-human-brain-with-matrix-assisted-laser

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.5K Views.  Doshisha University. Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid β protein on brain tissues of Alzheimer's disease and cerebral amyloid angiopathy samples using MALDI-IMS.

Video: Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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

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

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

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

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

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

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). ...

Measuring and Manipulating Functionally Specific Neural Pathways in the Human Motor System with Transcranial Magnetic Stimulation

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has provided important insights into fundamental processes of the brain like cognition, learning, and motor control. However, this approach cannot provide causal evidence for the involvement of brain areas of interest. Transcranial magnetic stimulation (TMS) is a powerful, noninvasive tool for studying the human brain that can overcome this limitation by transiently modifying brain activity. Here, we highlight recent advances using a paired-pulse, dual-site TMS method with two coils that causally probes cortico-cortical interactions in the human motor system during different task contexts. Additionally, we describe a dual-site TMS protocol based on cortical paired associative stimulation (cPAS) that transiently enhances synaptic efficiency in two interconnected brain areas by applying repeated pairs of cortical stimuli with two coils. These methods can provide a better understanding of the mechanisms underlying cognitive-motor function as well as a new perspective on manipulating specific neural pathways in a targeted fashion to modulate brain circuits and improve behavior. This approach may prove to be an effective tool to develop more sophisticated models of brain-behavior relations and improve diagnosis and treatment of many neurological and psychiatric disorders.

This article describes new approaches to measure and strengthen functionally specific neural pathways with transcranial magnetic stimulation. These advanced noninvasive brain stimulation methodologies can provide new opportunities for the understanding of brain-behavior relations and development of new therapies to treat brain disorders.

Understanding interactions between brain areas is important for the study of goal-directed behavior. Functional neuroimaging of brain connectivity has ...