The authors acknowledge the following funding sources, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Dorothy Pitts Research Fund (NG), and the Nicky and Thor Eaton Research Fund. The authors acknowledge Kevin DeSimone, and Aman Goyal and for their knowledge in MRI acquisition and analysis expertise.

1. Participant and Postmortem Brain Set-Up
NOTE: All images were acquired using a 3 T MRI scanner with a 32-channel head coil and all MRI scanning was performed at RT, approximately 20 &#176;C. All participants were right handed and gave written informed consent. Each participant was in good health with no history of neurological disorders. The experimental protocol was approved and follows the guidelines of York University Human Participants Review Committee.
<ol>
	<li>Ask each participant fill out and sign a patient consent form that details MRI safety guidelines and the neuro-imaging protocol.</li>
	<li>For each participant, place earplugs in each ear and secure their head with pads to minimize head motion.</li>
	<li>For post-mortem brain imaging, make sure the brain is fixed prior to neuroimaging and is contained within a bag or container that fits within the MRI head-coil. Place the postmortem brain in the head-coil with its z-axis (superior to inferior) aligned with the bore of the scanner. The brainstem (posterior) should face towards the foot of the scanner bed.</li>
	<li>Place vacuum cushion hands around the post-mortem brain for additional support.</li>
</ol>
2. Localizing and Prescribing the Subcortex
NOTE: The thalamus is a dual lobed structure located near the center of the brain situated between the midbrain and the cerebral cortex. Located within the dorsal thalamus, the human LGN is a small subcortical structure that extends a maximum of ~10 mm.
<ol>
	<li>To register a new participant, open the MRI imaging software and click on the Patient tab in the upper left hand corner. Then click on Register.</li>
	<li>Fill in the appropriate patient information, and then click on the Exam tab.</li>
	<li>To obtain a localizer scan, click on the Exam Explorer tab to create a new protocol. Observe the set-up window on the screen, click the Routine tab, and enter the following parameters: acquisition time 28 sec, acquisition matrix 160 &#215; 160, 1 slice, 1.6 mm thick isotropic voxel size, FOV = 260 mm, FoV phase = 100%, slice resolution = 69%, phase and slice partial phase Fourier = 6/8, TR = 3.15 ms, TE = 1.37 msec, Flip Angle = 8&#176;.</li>
	<li>Overlay the slice selection box used for acquiring the PD images over the localizer covering the subcortical nuclei within the thalamus as well as surrounding structures (Figure 4).</li>
</ol>
3. High-resolution Structural Parameters
<ol>
	<li>Create a new protocol for obtaining high-resolution PD-weighted scans. In the set-up window on the screen, click the Routine tab, and enter the following parameters in the coronal orientation: acquisition time 179 sec, acquisition matrix 512 &#215; 512, 0.3 &#215; 0.3 &#215; 1 mm3 voxel size, TR = 3.25 sec, TE = 32 msec, flip angle = 120&#176;, interleaved slice acquisition, FoV read = 160 mm, FoV phase = 100%, parallel imaging (GRAPPA) with an acceleration factor of 2.
	<ol>
		<li>Use a Turbo Spin Echo sequence, with an Echo Train Length of 5. The first echo at 32 msec is the effective echo for this sequence. Reduce the bandwidth (BW) to the minimum possible, 40 Hz/pixel, to maximize the SNR. To reduce scan duration, choose 18 slices, each 1 mm thick, with an FOV = 160 mm. This slab provides enough coverage of subcortical regions of interest. 
		NOTE: For reliable identification of subcortical structures, acquire 5 runs with the above parameters. The total scan duration is only ~15 min (Figure 5). Fat-saturation was not employed.</li>
	</ol>
	</li>
	<li>In post-mortem brain imaging, reliable identification of subcortical structures can be observed in just one scan with the total duration of only ~3 min following the same scanning protocol as in 3.1 (Figure 6).</li>
</ol>
4. Image Analysis
NOTE: To analyze the MRI data, use the freely available FMRIB&#39;s Software Library (FSL) package available for download at (<a href="https://www.fmrib.ox.ac.uk/fsl/">https://www.fmrib.ox.ac.uk/fsl/</a>).
<ol>
	<li>Open a terminal window, and convert the raw DICOM files from the scanner for each PD volume to a NIfTI format with a DICOM to NIfTI converter. A number of which are freely available for download (e.g.,&#160;<a href="https://www.nitrc.org/projects/mricron">https://www.nitrc.org/projects/mricron</a>). In the command line, type dcm2nii followed by the directory of each PD weighted image run.</li>
	<li>In a terminal window obtain the parameters of the original PD scan. Type fslinfo in the command line followed by the PD scan in NIfTI format.</li>
	<li>Create a high-resolution blank image target volume that has twice the resolution and half the voxel size given by the parameters from fslinfo from the original PD scan. The order of data inputs for this command are as follows: 
	fslcreatehd &#60;xsize&#62; &#60;ysize&#62; &#60;zsize&#62; &#60;tsize&#62; &#60;xvoxsize&#62; &#60;yvoxsize&#62; &#60;zvoxsize&#62; &#60;tr&#62; &#60;xorigin&#62; &#60;yorigin&#62; &#60;zorigin&#62; &#60;datatype&#62; &#60;headername&#62; 
	NOTE: For example, if the original PD scan with the following parameters as described in 3.1 are collected (i.e., 512 &#215; 512 matrix, 18 slice, 0.3 &#215; 0.3 &#215; 1 mm3 voxel size, TR = 3.25 s), type the following into the command window: 
	fslcreatehd 1024 1024 36 1 0.15 0.15 0.5 3.25 0 0 0 4 blankhr.nii.gz</li>
	<li>Define the transformation using an identity matrix. Type in any text editor program a text file saved as &#39;identity.mat&#39; that looks like this: 
	0 0 0 
	1 0 0 
	0 1 0 
	0 0 1</li>
	<li>Use the flirt command to apply the transformation, upsampling each original PD weighted run to double the total resolution from a 512 to a 1024 matrix, and halve the voxel size in each dimension resulting in a resolution of 0.15 &#215; 0.15 &#215; 0.5 mm3. In a terminal window for each PD volume, type the following flirt command changing the original and output names per run: 
	flirt -interp sinc -in originalPD.nii.gz -ref blankhr.nii.gz -applyxfm -init identity.mat -out highresPD.nii.gz 
	NOTE: Where originalPD.nii.gz is the source volume, blankhr.nii.gz is the desired output resolution, and highresPD.nii.gz is the name of the output volume.</li>
	<li>Move all the high-resolution images to a new folder, and navigate to it in a terminal window.</li>
	<li>For each participant, concatenate all the upsampled PD images into a single 4D file using fslmerge. In a terminal window type: 
	fslmerge -t concat_highresPD *.nii.gz 
	NOTE: This creates a 4D file called concat_highresPD.nii.gz.</li>
	<li>Motion correct the concatenated file using mcflirt 10 . This tool allows for an automated robust registration for linear (affine) inter and inter-modal brain images. Select a 4-stage correction, which utilizes sinc interpolation (internally) as a further optimization pass for greater accuracy. In a terminal window type: 
	mcflirt -in concat_highresPD -out mcf_concat_highresPD.nii.gz -stages 4 -plots 
	NOTE: This creates a 4D file called mcf_concat_highresPD.nii.gz.</li>
	<li>Finally, create the 3D mean image using fslmaths. In a terminal window type: 
	fslmaths mcf_concat_highresPD.nii.gz -Tmean mean_highresPD.nii.gz 
	NOTE: This creates a 3D file called mean_highresPD.nii.gz that is of high quality</li>
	<li>Visualize the final outcome 3D high-resolution image using the fslview command. In the directory of where your image is, type the following in a terminal window: 
	fslview mean_highresPD.nii.gz.&#34;</li>
	<li>Inspect intensity profiles of ROIs in question. Create an ROI using fslview (this can be a vertical line drawn across a region of the LGN for example). In fslview load the high-resolution PD image. Click on the tools tab, then click on the single image tab to enlarge the image for drawing ROIs. Then, click the File tab followed by the Create Mask tab. Draw a line in the ROI of interest. Save the ROI by clicking File, then the Save As. Repeat the line masks for multiple areas within the ROI for intensity comparisons and other ROIs in question.</li>
	<li>Use AFNI&#39;s 3dmaskdump command to analyze the resulting intensity of the image. In the directory of where the images are, use the following command in a terminal window to extract the image intensities and location (given as result_mask.txt) of your ROI mask: 
	3dmaskdump -o result_mask.txt -noijk -xyz -mask ROI_linemask.nii.gz PDaverage_image.nii.gz</li>
</ol>

<ol>
	<li>Devlin, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliable+identification+of+the+auditory+thalamus+using+multi-modal+structural+analyses.">Reliable identification of the auditory thalamus using multi-modal structural analyses.</a> NeuroImage. 30 (4), 1112-1120 (2006).</li><li>Fellner, F., 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=True+proton+density+and+T2-weighted+turbo+spin-echo+sequences+for+routine+MRI+of+the+brain.">True proton density and T2-weighted turbo spin-echo sequences for routine MRI of the brain.</a> Neuroradiology. 36 (8), 591-597 (1994).</li><li>Schumann, C. M., Buonocore, M. H., Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+of+the+post-mortem+autistic+brain.">Magnetic resonance imaging of the post-mortem autistic brain.</a> J Autism Dev Disord. 31 (6), 561-568 (2001).</li><li>Tovi, M., Ericsson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+T1+and+T2+over+time+in+formalin-fixed+human+whole-brain+specimens.">Measurements of T1 and T2 over time in formalin-fixed human whole-brain specimens.</a> Acta Radiol. 33 (5), 400-404 (1992).</li><li>Fujita, N., 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=Lateral+geniculate+nucleus:+anatomic+and+functional+identification+by+use+of+MR+imaging.">Lateral geniculate nucleus: anatomic and functional identification by use of MR imaging.</a> Am J Neuroradiol. 22 (9), 1719-1726 (2001).</li><li>Bridge, H., Thomas, O., Jbabdi, S., Cowey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+connectivity+after+visual+cortical+brain+damage+underlie+altered+visual+function.">Changes in connectivity after visual cortical brain damage underlie altered visual function.</a> Brain. 131 (6), 1433-1444 (2008).</li><li>Gupta, N., 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=Atrophy+of+the+lateral+geniculate+nucleus+in+human+glaucoma+detected+by+magnetic+resonance+imaging.">Atrophy of the lateral geniculate nucleus in human glaucoma detected by magnetic resonance imaging.</a> Br J Opthalmol. 93 (1), 56-60 (2009).</li><li>Dai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lateral+geniculate+nucleus+atrophy+with+3T+MR+imaging+and+correlation+with+clinical+stage+of+glaucoma.">Assessment of lateral geniculate nucleus atrophy with 3T MR imaging and correlation with clinical stage of glaucoma.</a> Am J Neuroradiol. 32 (7), 1347-1353 (2011).</li><li>McKetton, L., Kelly, K. R., Schneider, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+lateral+geniculate+nucleus+and+optic+chiasm+in+human+albinism.">Abnormal lateral geniculate nucleus and optic chiasm in human albinism.</a> J Comp Neurol. 522 (11), 2680-2687 (2014).</li><li>Jenkinson, M., Bannister, P., Brady, M., Smith, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+optimization+for+the+robust+and+accurate+linear+registration+and+motion+correction+of+brain+images.">Improved optimization for the robust and accurate linear registration and motion correction of brain images.</a> NeuroImage. 17 (2), 825-841 (2002).</li><li>Dietrich, O., Raya, J. G., Reeder, S. B., Reiser, M. F., Schoenberg, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+signal-to-noise+ratios+in+MR+images:+influence+of+multichannel+coils,+parallel+imaging,+and+reconstruction+filters.">Measurement of signal-to-noise ratios in MR images: influence of multichannel coils, parallel imaging, and reconstruction filters.</a> J Magn Reson Imaging. 26 (2), 375-385 (2007).</li><li>Andrews, T. J., Halpern, S. D., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+size+variations+in+human+visual+cortex,+lateral+geniculate+nucleus,+and+optic+tract.">Correlated size variations in human visual cortex, lateral geniculate nucleus, and optic tract.</a> J Neurosci. 17 (8), 2859-2868 (1997).</li><li>Pfefferbaum, A., Sullivan, E. V., Adalsteinsson, E., Garrick, T., Harper, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+MR+imaging+of+formalin-fixed+human+brain.">Postmortem MR imaging of formalin-fixed human brain.</a> NeuroImage. 21 (4), 1585-1595 (2004).</li></ol>

The authors have nothing to disclose.

This study describes an optimized protocol in acquisition and analysis technique in order to obtain high-resolution PD weighted images of subcortical regions. A number of scanning parameters were tested and modified with the most significant ones pertaining to matrix size, voxel size, and bandwidth to increase the SNR and decrease the number of acquisitions, a critical step in being able to determine high-resolution subcortical structures. In conjunction with finding the optimal parameters within living humans, this rese...

The purpose of this research was to test the resolution limits of structural MRI in the absence of physiological noise. Proton density (PD) weighted images were acquired in a postmortem brain over a long duration (two ~24 hr sessions) to determine the minimum number of images that needed to be registered and averaged to resolve the subcortical structures. For comparison, PD weighted images were also acquired in living humans over a number of sessions. In particular, the objective was to ascertain whether it would be possible in a best-case scenario to resolve all six individual layers of the human LGN, which are approximately 1 mm thick (Figure 1).



<img alt="Figure 1" src="/files/ftp_upload/53309/53309fig1.jpg" /> 
Figure 1.&#160;Human Lateral Geniculate Nucleus layers. Schematic of the laminar structure of the LGN. Magnocellular (M) layers are comprised of larger neuronal cell size and smaller cell density that are responsible for resolving motion and course outlines (layers 1-2, depicted as dark grey). Parvocellular layers (P) are comprised of smaller neuronal cell size and larger cell density that are responsible for resolving fine-form and colour (layers 4-6, depicted as light grey). Scale bar 1 mm. Figure based on stained human LGN 12.
Spatial resolution in MRI is improved when the matrix size is increased, and when field-of view (FOV) and slice thickness are decreased. However, increased resolution decreases the signal to noise ratio (SNR), which is proportional to the voxel volume. SNR is also proportional to the square root of the number of measurements. In living humans, although multiple images can be acquired over a number of separate imaging sessions, the ultimate resolution is limited by physiological noise, such as respiration, circulatory pulsations and head movement.
High-resolution (0.35 mm in-plane voxels) PD weighted scans were acquired. PD scans enhance grey and white contrast in the thalamus1, and result in images that minimize T1 and T2 effects. Its image is dependent on the density of protons in the form of water and macromolecules such as proteins and fat in the imaging volume. The increased numbers of protons in a tissue results in a brighter signal on the image due to the higher longitudinal component of magnetization2.
PD-weighted scans were collected since they provide a higher contrast of subcortical structures with surrounding tissue. Other contrasts, such as T1- and T2-weighted images result in difficulty in delineating subcortical structures like the LGN due to smaller contrast-to-noise ratios, as determined &#402; 1,3.
Likewise, earlier studies found that PD-weighted images of formalin fixed post-mortem brains resulted in higher contrast differences between gray and white matter as compared to T1- and T2-weighted images that had similar grey and white matter image intensities 3,4. The underlying biophysical determinants can explain these differences. T1 (longitudinal) and T2 (transverse) relaxation times of hydrogen protons depend on how water moves within the tissue. Fixatives such as formalin work by cross-linking proteins. The differences between water mobility are reduced between different tissue types when fixatives are used. Reduced T1 tissue contrast has been observed after fixation, whereas the differences in the relative density of protons within brain tissues increased with fixation, providing better contrast differentiation 3, 4.
Previous studies have identified the LGN in PD-weighted scans using a 1.5 T 5,6,7, and at 3 T scanner 8,9. It is critical to obtain these scans to be able to precisely outline the extent of the LGN. To maintain full coverage of the subcortical nuclei, 18 PD-weighted slices were obtained within the thalamus. Each volume was re-sampled to twice the resolution 1024 matrix, (0.15 mm in-plane voxel size), concatenated, motion corrected and averaged to produce a high-resolution 3D image of subcortical structures. The optimum number of PD images required for the following slice prescription was 5, reducing scan time to less than 15 min&#160;in living humans. Only 1 PD image was required to clearly demarcate subcortical regions in the postmortem brain, reducing scan time to less than 3 min&#160;(Figure 2 and 3).
A whole formalin-fixed postmortem brain specimen was scanned from a woman who had died of cardiopulmonary arrest at age 82 years. Review of medical records revealed that she had: chronic obstructive pulmonary disease, angina, triple bypass surgery 8 years prior to death, uterine cancer treated with hysterectomy 7 years prior to death, hyperlipidemia, glaucoma, and cataract surgery. The postmortem brain specimen was immersion-fixed in 10% neutral buffered formalin for at least 3 weeks at 4 &#176;C.The postmortem brain was scanned with the same imaging protocol as well as with other parameters over the course of many hours for image quality comparisons. Only the optimized parameters will be described for the protocol.

<table>
	<tbody>
		<tr>
			<td>Magnetom Trio 3T&#160; MRI</td>
			<td>Siemens (Erlangen, Germany).</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum cushion hand</td>
			<td>Siemens</td>
			<td>Mat No: 4765454</td>
			<td>Manufactured by: Johannes-Stark-Stk. 8 D-92224 Amberg</td>
		</tr>
	</tbody>
</table>

high-resolution structural magnetic resonance imaging of the human subcortex in vivo and postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

Once the subcortex is prescribed within the thalamus, PD weighted images are collected within the slice selection box (Figure 4). The SNR improved by increasing the number of averages in both postmortem and in vivo scans. To determine image quality, the SNR from different scan averages was compared by dividing the signal of the mean brain region by the standard deviation in some area outside the brain. The SNR was calculated as SNR = 0.655 * &#181;tissue/&#963;a...

Watch this Scientific Journal Video about High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem at JoVE.com

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

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15.3K Views.  York University. Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise. 

Video: High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

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 ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

A Magnetic Resonance Imaging Protocol for Stroke Onset Time Estimation in Permanent Cerebral Ischemia

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat model of ischemic stroke, differences in quantitative T1 and T2 MRI relaxation times (qT1 and qT2) between the ischemic lesion (delineated by low diffusion) and the contralateral non-ischemic hemisphere increase with time from stroke onset. The time dependency of MRI relaxation time differences is heuristically described by a linear function and thus provides a simple estimate of stroke onset time. Additionally, the volumes of abnormal qT1 and qT2 within the ischemic lesion increase linearly with time providing a complementary method for stroke timing. A (semi)automated computer routine based on the quantified diffusion coefficient is presented to delineate acute ischemic stroke tissue in rat ischemia. This routine also determines hemispheric differences in qT1 and qT2 relaxation times and the location and volume of abnormal qT1 and qT2 voxels within the lesion. Uncertainties associated with onset time estimates of qT1 and qT2 MRI data vary from &plusmn; 25 min to &plusmn; 47 min for the first 5 hours of stroke. The most accurate onset time estimates can be obtained by quantifying the volume of overlapping abnormal qT1 and qT2 lesion volumes, termed 'Voverlap' (&plusmn; 25 min) or by quantifying hemispheric differences in qT2 relaxation times only (&plusmn; 28 min). Overall, qT2 derived parameters outperform those from qT1. The current MRI protocol is tested in the hyperacute phase of a permanent focal ischemia model, which may not be applicable to transient focal brain ischemia.

A protocol for stroke onset time estimation in a rat model of stroke exploiting quantitative magnetic resonance imaging (qMRI) parameters is described. The procedure exploits diffusion MRI for delineation of the acute stroke lesion and quantitative T1 and T2 (qT1 and qT2) relaxation times for timing of stroke.

MRI provides a sensitive and specific imaging tool to detect acute ischemic stroke by means of a reduced diffusion coefficient of brain water. In a rat ...

Diffusion Tensor Magnetic Resonance Imaging in Chronic Spinal Cord Compression

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic resonance imaging (MRI) plays an important role in both confirming the diagnosis and evaluating the degree of compression. However, the anatomical detail provided by conventional MRI is not sufficient to accurately estimate neuronal damage and/or assess the possibility of neuronal recovery in chronic spinal cord compression patients. In contrast, diffusion tensor imaging (DTI) can provide quantitative results according to the detection of water molecule diffusion in tissues. In the present study, we develop a methodological framework to illustrate the application of DTI in chronic spinal cord compression disease. DTI fractional anisotropy (FA), apparent diffusion coefficients (ADCs), and eigenvector values are useful for visualizing microstructural pathological changes in the spinal cord. Decreased FA and increases in ADCs and eigenvector values were observed in chronic spinal cord compression patients compared to healthy controls. DTI could help surgeons understand spinal cord injury severity and provide important information regarding prognosis and neural functional recovery. In conclusion, this protocol provides a sensitive, detailed, and noninvasive tool to evaluate spinal cord compression.

Here, we present a protocol for the application of diffusion tensor imaging parameters to evaluate spinal cord compression.

Chronic spinal cord compression is the most common cause of spinal cord impairment in patients with nontraumatic spinal cord damage. Conventional magnetic ...

Acquisition of Resting-State Functional Magnetic Resonance Imaging Data in the Rat

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task state. This protocol describes a preclinical survival method for obtaining rs-fMRI data. Combining low dose isoflurane with continuous infusion of the &#945;2 adrenergic receptor agonist dexmedetomidine provides a robust option for stable, high-quality data acquisition while preserving brain network function. Furthermore, this procedure allows for spontaneous breathing and near-normal physiology in the rat. Additional imaging sequences can be combined with resting-state acquisition creating experimental protocols with anesthetic stability of up to 5 h using this method. This protocol describes the setup of equipment, monitoring of rat physiology during four distinct phases of anesthesia, acquisition of resting-state scans, quality assessment of data, recovery of the animal, and a brief discussion of post-processing data analysis. This protocol can be used across a wide variety of preclinical rodent models to help reveal the resulting brain network changes that occur at rest.

This protocol describes a method for obtaining stable resting-state functional magnetic resonance imaging (rs-fMRI) data from a rat using low dose isoflurane in combination with low dose dexmedetomidine.

Resting-state functional magnetic resonance imaging (rs-fMRI) has become an increasingly popular method to study brain function in a resting, non-task ...