This material is based upon work supported by the National Science Foundation under Grant BCS 1063774.

1. Polar-angle Topography Stimulus and Psychophysics
To obtain a polar-angle retinotopic map in the SC, we use a 90&deg; wedge of moving dots as the stimulus (eccentricity 2-9&deg; of visual angle, mean dot-speed 4&deg; / sec) (Fig. 1). It is known that activity in the SC is enhanced by applying covert attention5, hence we use an attention task in our paradigm to increase available signal. On each 2 s trial, subjects are instructed to covertly attend to the entire wedge and perform a speed discrimination task while maintaining fixation. The wedge is divided into 2 &times; 3 virtual sectors with dots in one of the sectors, randomly chosen on each trial, moving slower or faster than all other dots. After each trial, the wedge is rotated 30&deg; around fixation so that the stimulus rotates fully with a 24 sec period. Each run consists of 9.5 rotations of the stimulus (228 sec), and experimental sessions include 16-18 runs.
To maintain subject's performance on this task throughout the duration of each run, the difficulty of the task is adjusted using two randomly interleaved two-up-one-down staircases. After every two consecutive correct trials the speed difference is reduced by 8%, and for every incorrect trial, the difference is increased by 8%.
Prior to scanning, all subjects practice the visual task outside the scanner until they achieve a stable level of performance; this requires 3-4 20 minute duration practice sessions. Typical discrimination thresholds are in the range of 1&deg;-1.5&deg; / sec.
2. Subject Preparation
<ol>
 <li>Subjects heads are secured with pads to minimize head motion before they are positioned inside the scanner bore. At these high spatial resolutions, fMRI is particularly sensitive to motion artifacts, so head stabilization is critical.</li>
 <li>Subjects are given an MRI-compatible button pad in one hand and instructed about which button to press to indicate their judgments about dot speed.</li>
</ol>
3. Localizing and Prescribing the SC
<ol>
 <li>Human SC is a small but distinct structure, ~9 mm in diameter, located on the roof of the midbrain. When using a small number of fMRI slices, multiple localizer imaging series are required for its precise localization. We run these series along sagittal, axial, and coronal planes.</li>
 <li>We then use these localizer images to precisely prescribe the SC with 8-10 contiguous slices, 1.2 mm thick, field-of-view (FOV), 170 mm in an oblique, quasi-axial plane.</li>
 <li>Next, high-resolution T1-weighted structural images are obtained using a three-dimensional (3D) RF-spoiled GRASS (SPGR) sequence (15&deg; flip angle, 1.2 mm voxels) once prior to collecting the functional data and once after. These images are used as a reference to align the fMRI data to a high-resolution structural reference volume obtained in a separate session that we describe later.</li>
</ol>
4. Functional MRI Parameters
All imaging was performed on a GE Signa HD12 3T MRI scanner using the GE-supplied 8-channel, head coil. Excitation was a 6.4 ms windowed-sinc pulse applied using the scanner body coil.
To obtain 1.2 mm sampling in human SC, we use a three-shot spiral-trajectory acquisition6,7. Three shots are needed for several reasons. In particular, single-shot acquisition for our scanner and FOV requires &gt; 77 msec, too long to be practical. The multiple shots are combined together after correction by subtracting the initial value and linear trend of the phase. TE is incremented by 2 ms on the first frame to estimate a field map from the first two volumes acquired, and this map is used for linear correction. Reconstructed images had an SNR of ~20. Temporal power spectra in SC voxels typically showed little of the structure associated with physiological noise; the use of a 3-shot acquisition had a strong filtering effect on the comparatively high-frequency effects of cardiac pulse and respiration. Other noise-reduction techniques are problematic in this high-resolution context. For example, retrospective correction methods such as RETROICOR6 are not applicable to multi-shot data, and cardiac gating introduces noise and artifact associated with disturbance of T1 equilibrium.
Echo time, TE = 40 msec, is longer than typically used in cortex (30 msec) because we measured a correspondingly longer T2* in SC tissue (~60 msec) than observed in cortical gray matter (~45 msec).
Acquisition bandwidth is limited to 62.5 kHz to reduce peak gradient current that causes unwanted heating on our scanner. We choose TR = 1 sec, so with three shots a volume is acquired every 3 sec.
5. Structural MRI and 3D Modeling
In a single separate session for each subject, we obtain a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast (3D SPGR, 15&deg; flip angle, inversion prepared with TI = 450 msec, 2 excitations, ~28 minute duration, 0.7 mm voxels).
In this reference volume, we segment the tissue of the midbrain, brainstem, and portions of the thalamus (Fig. 2A) using a combination of automatic and manual techniques provided by the ITK-SNAP application2. In particular, we use an automatic segmentation tool in which the user seeds multiple points within each brainstem; the software then automatically expands the segmentation around the seed points constrained within a region defined by contrast and intensity criteria. This automatic segmentation is then adjusted, if necessary, using manual, "paint-like" voxel tools.
The cerebrospinal fluid-tissue interface of the SC is interpolated from the segmentation using isodensity surface tessellation, and this initial surface is refined to reduce aliasing artifacts (Fig. 2, B and D) using a variational deformable-surface algorithm3. This surface provides vertices and outward normal vectors to be used as a reference for the laminar calculations (described below) as well as a means to visualize functional data.
6. Image Analysis
To analyze the fMRI data, we use the mrVista software package (available for download at <a href="http://white.stanford.edu/mrvista.php" target="_blank">http://white.stanford.edu/mrvista.php)</a> as well as tools developed on the mrVista framework in our lab. In the next few steps we use standard mrVista package tools:
<ol>
 <li>Initialize the session in mrVista, choosing the option to spatially normalize the intensity of the averaged data to reduce the effects of coil inhomogeneity. The normalization uses a homomorphic method, that is, dividing by a low-pass filtered version of the temporally averaged volume image intensities with an additive robust correction for estimated noise. The first half cycle of images (12 sec) is discarded to avoid transient MR equilibrium and hemodynamic effects.</li>
 <li>Correct within-run motion. Motion is estimated from the time series of volumes. However, because of the relatively low signal-to-noise ratio (SNR) of the images, we first perform a 5-sample boxcar smoothing upon the time series. Each volume is then registered to the average of the last 5 samples. Note that smoothing is only used to estimate the motion, and the actual data is not smoothed.</li>
 <li>Form temporal averages of each run's motion-corrected image data, and use these averages to correct the between-run motion using the last run as reference.</li>
 <li>Perform a slice-timing correction. We use sequential slice acquisition, so errors due to performing timing correction after the motion correction will be small (~125 msec).</li>
 <li>Average the multiple runs recorded within each session to improve SNR.</li>
 <li>Align the structural data from the fMRI session to the reference structural volume using a robust intensity-based registration algorithm8. Load the alignment and segmentation into mrVista.</li>
 <li>Transform the functional time series data to the segmented reference volume. In the following steps we use tools developed in our lab to perform further analysis.</li>
 <li>Compute a distance map by calculating the distance between each SC tissue voxels and its nearest vertex on the SC surface. These distances are used to measure laminar position within the reference volume.</li>
 <li>Perform a laminar segmentation process to enable depth averaging of time-series data to improve the SNR. Small (1.6 mm diameter) disks of tissue are associated with each vertex of the surface model along the entire superficial SC surface, and each disk is then extended both inward and outward from the SC tissue using the local surface normals to form an individual laminar neighborhood4.</li>
 <li>For each point on the SC surface, we use these laminar associations to average the time series over a specified depth range. Since, visually responsive neurons are primarily present in the superficial layers of the SC, for the visual stimulation experiment we used a depth range of 0-1.8 mm.</li>
</ol>
7. Topographic Mapping Analysis
For analyzing the topographic representation of the data, Coherence analysis is performed on the depth-averaged time series by fitting a sinusoid at the stimulus repetition frequency (i.e., 1/24 Hz for the 24 sec stimulus described above) for each voxel. From this fit, we derive surface maps of response amplitude, coherence, and phase. This analysis is performed in the frequency domain, and is a common technique to quantify retinotopic maps in visual cortex9,10.
The phase of the sinusoidal fit measures the position of the stimulus. Zero-phase corresponds to the upper vertical meridian (Fig. 3). The stimulus then rotates clockwise, so a &pi;/2 phase corresponds to when the stimulus has rotated to the horizontal meridian in the right visual field. After &pi; radians of phase, the stimulus crosses into the left visual field, and so forth.
We also obtained boundaries of the entire superficial extent of the SC using manual inspection of the high-resolution T1-weighted volume anatomy. These boundaries are marked in Fig. 3 by red dashed lines.
fMRI does not directly measure neural activity, but rather a blood-flow response that is tightly but sluggishly coupled to neural activity. This adds a hemodynamic delay to the phase response. The delay is estimated by taking all voxels above our coherence threshold from each SC region-of-interest, and centering their means around &pi; in the complex plane. In our SC data, these delays are quite small, of the order of 2-4 sec. We remove this delay by rotating the colormap counter-clockwise by 45 degrees (corresponding to 3 sec) (Fig. 3).
8. Representative Results
Phase data overlaid on a 3D surface of the SC (Fig. 3) shows that the response to visual stimulation is represented contralaterally in the SC, i.e., the left visual field is represented on the right SC and vice-a-versa.
There is also a retinotopic organization of the activity. The right upper visual field is represented medially on the left colliculus (blue-magenta), and the lower field is represented laterally (red-yellow). Similarly, the left upper visual field is represented medially on the right colliculus (blue-cyan) and lower is represented laterally (green-yellow).
This topography is consistent with the results of non-human primate studies of SC responses: 1) to visual stimulation using single-unit electrophysiology, and 2) the mapping of microstimulation induced saccadic eye movements11,12.
<img alt="Figure 1" src="/files/ftp_upload/3746/3746fig1.jpg" /> 
Figure 1. A) Slice prescription viewed on mid-sagittal image. B) Temporal mean (from 1-run) of functional images obtained from central slice marked in red in A).
<img alt="Figure 2" src="/files/ftp_upload/3746/3746fig2.jpg" /> 
Figure 2. Polar-angle topography stimulus. A 90&deg; wedge of moving black-and-white dots on a gray background rotated slowly around fixation. The wedge was divided into an array of 6 virtual sectors (gray lines added to emphasize sectors) to allow the subject to perform a speed discrimination task in a randomly chosen sector.
<img alt="Figure 3" src="/files/ftp_upload/3746/3746fig3.jpg" /> 
Figure 3. Segmentation and surface modeling. A) The midbrain, brainstem, and portions of the thalamus were segmented from high-resolution MRI anatomy volumes. B) A surface was created at the edge of segmented region. C) Sinusoidal-fit phase data viewed on an inplane slice (coherence &gt; 0.25). D) A rotated and enlarged view of the brainstem surface model was used to visualize phase data on the SC.
<img alt="Figure 4" src="/files/ftp_upload/3746/3746fig4.jpg" /> 
Figure 4. Polar-angle maps. fMRI phase maps in two subjects that encode visual polar angle. Coherence thresholds for each map are provided on the bottom right. The color wheel relates the overlaid phases of the stimulus in polar angle to their visual field positions.

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No conflicts of interest declared.

Our acquisition and data-analysis techniques enable the measurement of neural activity in subcortical human brain structures at high resolution (1.2 mm voxels). The 3-shot spiral acquisition reduces the physiological noise that is particularly detrimental to fMRI measurements around the midbrain. In addition, our laminar segmentation of the tissue allows us to perform depth averaging of the data that helps improve the SNR. We have used these methods to show precise polar-angle topographic maps of visual stimulation and covert visual attention in the human SC1. The laminar segmentation also enables the analysis of depth profiles of functional activity that vary under experimental control1.
Our imaging methods open up new avenues for neuroscience experimentation in human subcortical structures. These methods can enable translation of fine-scale research done on animals in subcortical areas to humans, e.g., investigating the organization of auditory responses in structures such as the inferior colliculus and the cochlear nucleus13-15, or visual and multisensory responses in thalamic nuclei such as pulvinar16,17. Finally, these techniques could provide functional localization for small structures such as the subthalamic nucleus and globus pallidus, which are often targets for deep brain stimulation in patients with Parkinson's disease, dystonia or chronic pain18-21.

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.

Watch this Scientific Journal Video about High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain at JoVE.com

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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 ...

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12.8K Views.  The University of Texas at Austin. 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.

Video: High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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 ...

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 ...

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.

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.

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 ...

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 ...

Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the Substantia Nigra

The dopaminergic system plays a crucial role in healthy cognition (e.g., reward learning and uncertainty) and neuropsychiatric disorders (e.g., Parkinson's disease and schizophrenia). Neuromelanin is a byproduct of dopamine synthesis that accumulates in dopaminergic neurons of the substantia nigra. Neuromelanin-sensitive magnetic resonance imaging (NM-MRI) is a noninvasive method for measuring neuromelanin in those dopaminergic neurons, providing a direct measure of dopaminergic cell loss in the substantia nigra and a proxy measure of dopamine function. Although NM-MRI has been shown to be useful for studying various neuropsychiatric disorders, it is challenged by a limited field-of-view in the inferior-superior direction resulting in the potential loss of data from the accidental exclusion of part of the substantia nigra. In addition, the field is lacking a standardized protocol for the acquisition of NM-MRI data, a critical step in facilitating large-scale multisite studies and translation into the clinic. This protocol describes a step-by-step NM-MRI volume placement procedure and online quality control checks to ensure the acquisition of good-quality data covering the entire substantia nigra.

This protocol shows how to acquire neuromelanin-sensitive magnetic resonance imaging data of the substantia nigra.

The dopaminergic system plays a crucial role in healthy cognition (e.g., reward learning and uncertainty) and neuropsychiatric disorders (e.g., ...

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 ...