Dr. Horga received support from the NIMH (R01-MH114965, R01-MH117323). Dr. Wengler received support from NIMH (F32-MH125540).

NOTE: The research conducted to develop this protocol was performed in compliance with New York State Psychiatric Institute Institutional Review Board guidelines (IRB #7655). One subject was scanned for recording the protocol video, and written informed consent was obtained. Refer to the Table of Materials for details about the MRI scanner used in this protocol.
1. MRI acquisition parameters
<ol>
	<li>Prepare to acquire high-resolution T1w images using a 3D magnetization prepared rapid acquisition gradient echo (MPRAGE) sequence with the following parameters: spatial resolution = 0.8 x 0.8 x 0.8 mm3; field-of-view (FOV) = 176 x 240 x 240 mm3; echo time (TE) = 3.43 ms; repetition time (TR) = 2462 ms; inversion time (TI) = 1060 ms; flip angle = 8&#176;; in-plane parallel imaging factor (ARC) = 2; through-plane parallel imaging factor (ARC) = 233; bandwidth = 208 Hz/pixel; total acquisition time = 6 min 39 s.</li>
	<li>Prepare to acquire NM-MRI images using a two-dimensional (2D) gradient recalled echo sequence with magnetization transfer contrast (2D GRE-MTC) with the following parameters: resolution = 0.43 x 0.43 mm2; FOV = 220 x 220 mm2; slice-thickness = 1.5 mm; 20 slices; slice gap = 0 mm; TE = 4.8 ms; TR = 500 ms; flip angle = 40&#176;; bandwidth = 122 Hz/pixel; MT frequency offset = 1.2 kHz; MT pulse duration = 8 ms; MT flip angle = 670&#176;; number of averages = 5; total acquisition time = 10 min 4 s. 
	NOTE: Although the displayed results used these MRI acquisition parameters, this protocol is valid for various T1w and NM-MRI imaging protocols. The NM-MRI protocol should cover ~25 mm in the inferior-superior direction to guarantee complete coverage of the SN.</li>
</ol>
2. Placement of NM-MRI volume
<ol>
	<li>Acquire a high-resolution T1w image (&#8804;1 mm isotropic voxel size). Use online reformatting directly after image acquisition to create high-resolution T1w images aligned to the anterior commissure-posterior commissure (AC-PC) line and the midline.
	<ol>
		<li>Carry out online reformatting using the vendor-provided software (e.g., if acquiring data on a GE scanner: MultiPlanar Reconstruction (MPR) in Planning; if acquiring data on a Siemens scanner: MPR in the 3D Task Card; if acquiring data on a Philips scanner: MPR in the Render Mode of the VolumeView Package).
		<ol>
			<li>Create multiplanar reconstructions of the 3D T1w image in the axial plane perpendicular to the AC-PC line to cover the whole brain with minimal slice-gap.</li>
			<li>Create multiplanar reconstructions of the 3D T1w image in the coronal plane perpendicular to the AC-PC line to cover the whole brain with minimal slice-gap.</li>
			<li>Create multiplanar reconstructions of the 3D T1w image in the sagittal plane parallel to the AC-PC line to cover the whole brain with minimal slice-gap.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Load the sagittal, coronal, and axial views of the reformatted high-resolution T1w image and ensure that reference lines depicting the location of each displayed slice are present.</li>
	<li>Identify the sagittal image that shows the largest separation between the midbrain and thalamus (Figure 1A). To do this, visually inspect the sagittal slices of the reformatted T1w image until the slice showing this greatest separation is identified.</li>
	<li>Using the sagittal image from the end of step 2.3, visually identify the coronal plane that delineates the most anterior aspect of the midbrain (Figure 1B).</li>
	<li>Using the coronal image from the end of step 2.4, visually identify the axial plane that delineates the inferior aspect of the third ventricle (Figure 1C).</li>
	<li>On the sagittal image from the end of step 2.3, align the superior boundary of the NM-MRI volume to the axial plane identified in step 2.5 (Figure 1D).</li>
	<li>Move the superior boundary of the NM-MRI volume 3 mm in the superior direction (Figure 1E).</li>
	<li>Align the NM-MRI volume to the midline in the axial and coronal images (Figure 1F).</li>
	<li>Acquire the NM-MRI images.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62493/62493fig01.jpg" /> 
Figure 1: Images displaying the step-by-step NM-MRI volume placement procedure. Yellow lines indicate the location of the slices used for volume placement as described in the protocol. (A) First, the sagittal image with the greatest separation between the midbrain and thalamus is identified (step 2.3 of the protocol). (B) Second, using the image from A, the coronal plane delineating the most anterior aspect of the midbrain is identified (step 2.4). (C) Third, on the coronal image from the plane identified in B, the axial plane delineating the inferior aspect of the third ventricle is identified (step 2.5). (D) Fourth, the axial plane identified in C is displayed on the sagittal image from A (step 2.6). (E) Fifth, the axial plane from D is shifted 3 mm in the superior direction, and this plane indicates the superior boundary of the NM-MRI volume (step 2.7). (F) The final NM-MRI volume placement where the coronal image corresponds to C, the sagittal image corresponds to A, and the axial image corresponds to the axial plane in E. The NM-MRI volume is aligned to the brain midline in the coronal and axial images and the AC-PC line in the sagittal image (step 2.8). Part of this figure has been reprinted with permission from Elsevier from 30. Abbreviations: NM-MRI = neuromelanin-sensitive magnetic resonance imaging; AC-PC = anterior commissure-posterior commissure. <a href="https://www.jove.com/files/ftp_upload/62493/62493fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Quality control checks
<ol>
	<li>Ensure that the acquired NM-MRI images cover the entire SN and that the SN is visible in the central images but not in the most superior or most inferior images of the NM-MRI volume. Otherwise (Figure 2), repeat steps 2.3-2.9 to ensure correct NM-MRI volume placement. If the participant has moved significantly since the acquisition of the high-resolution T1w scan, repeat steps 2.1-2.9.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62493/62493fig02.jpg" /> 
Figure 2: Example of an NM-MRI acquisition that failed the first quality control check (step 3.1 of the protocol). Each of the 20 NM-MRI slices displayed from most inferior (top left image) to most superior (bottom right image); the image window/level was set to exaggerate the contrast between the substantia nigra and crus cerebri. The orange arrows in slices 15-19 show the location of the substantia nigra in those slices. The red arrow in the most superior slice (slice 20) shows that the substantia nigra is still visible in this slice, and thus, the acquisition fails the quality check. Abbreviation: NM-MRI = neuromelanin-sensitive magnetic resonance imaging. <a href="https://www.jove.com/files/ftp_upload/62493/62493fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Check for artifacts, particularly ones that go through the SN and the surrounding white matter, by visually inspecting each slice of the acquired NM-MRI scan.
	<ol>
		<li>Look for abrupt changes in signal intensity with a linear pattern that does not respect normal anatomical boundaries. For example, this may appear as a low-intensity region that is flanked by two high-intensity regions.</li>
		<li>If the artifact is the result of blood vessels (Figure 3A), retain the NM-MRI images because these artifacts will most likely always be present.</li>
		<li>If the artifacts are the result of participant head motion (Figure 3B), remind the participant to stay as still as possible and reacquire the NM-MRI images according to step 3.2.5.</li>
		<li>If the artifacts are ambiguous (Figure 3C), reacquire the NM-MRI images according to step 3.2.5. Upon reacquisition, if the artifacts remain present, proceed with these images as they are likely biological rather than a result of acquisition issues.</li>
		<li>If the NM-MRI images pass the quality control check in step 3.1, copy the previous NM-MRI volume placement. If the NM-MRI images fail the quality control check in step 3.1, repeat steps 2.3-2.9 to ensure correct NM-MRI volume placement (or steps 2.1-2.9 if the participant moved significantly).</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62493/62493fig03.jpg" /> 
Figure 3: Examples of NM-MRI acquisitions that failed the second quality control check (step 3.2 of the protocol). Only one representative slice is shown for each case. (A) An NM-MRI acquisition that fails the quality control check due to a blood vessel artifact (red arrows) that is the result of the blood vessel identified by the blue arrows. (B) An NM-MRI acquisition that fails the quality control check due to motion artifacts (red arrows). (C) An NM-MRI acquisition that fails the quality control check due to an ambiguous artifact (red arrows). Abbreviation: NM-MRI = neuromelanin-sensitive magnetic resonance imaging. <a href="https://www.jove.com/files/ftp_upload/62493/62493fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Drs Horga and Wengler each reported having patents for analysis and use of neuromelanin imaging in central nervous system disorders (WO2021034770A1, WO2020077098A1), licensed to Terran Biosciences, but have received no royalties.

The dopaminergic system plays a crucial role in healthy cognition and neuropsychiatric disorders. The development of noninvasive methods that can be used to repeatedly investigate the dopaminergic system in vivo is critical for the development of clinically meaningful biomarkers. The protocol described here supplies step-by-step instructions for acquiring good-quality NM-MRI images of the SN, including placement of the NM-MRI volume and quality control checks to ensure usable data.
Ev...

Neuromelanin (NM) is a dark pigment found in dopaminergic neurons of the substantia nigra (SN) and noradrenergic neurons of the locus coeruleus (LC)1,2. NM is synthesized by the iron-dependent oxidation of cytosolic dopamine and norepinephrine and is stored in autophagic vacuoles in the soma3. It first appears in humans around 2-3 years of age and accumulates with age1,4,5.
Within the NM-containing vacuoles of SN and LC neurons, NM forms complexes with iron. These NM-iron complexes are paramagnetic, allowing for noninvasive visualization of NM using magnetic resonance imaging (MRI)6,7. MRI scans that can visualize NM are known as NM-sensitive MRI (NM-MRI) and use either direct or indirect magnetization transfer effects to provide contrast between regions with high NM concentration (e.g., the SN) and the surrounding white matter8,9.
Magnetization transfer contrast is the result of the interaction between macromolecular-bound water protons (which are saturated by the magnetization transfer pulses) and the surrounding free water protons. In NM-MRI, it is believed that the paramagnetic nature of NM-iron complexes shortens the T1 of the surrounding free water protons, resulting in reduced magnetization-transfer effects so that regions with higher NM concentration appear hyperintense on NM-MRI scans10. Conversely, the white matter surrounding the SN has a high macromolecular content, resulting in large magnetization-transfer effects so that these regions appear hypointense on NM-MRI scans, thus providing high contrast between the SN and surrounding white matter.
In the SN, NM-MRI can provide a marker of dopaminergic cell loss11 and dopamine system function12. These two processes are relevant for several neuropsychiatric disorders and are supported by a vast body of clinical and preclinical work. For example, abnormalities in dopamine function have been widely observed in schizophrenia; in vivo studies using positron emission tomography (PET) have shown increased striatal dopamine release13,14,15,16 and increased dopamine synthesis capacity17,18,19,20,21,22. Furthermore, post-mortem studies have shown that patients with schizophrenia have increased levels of tyrosine hydroxylase&#8212;the rate-limiting enzyme involved in dopamine synthesis&#8212;in the basal ganglia23 and SN24,25.
Several studies have investigated patterns of dopaminergic cell loss, particularly in Parkinson&#39;s disease. Post-mortem studies have revealed that the pigmented dopaminergic neurons of the SN are the primary site of neurodegeneration in Parkinson&#39;s disease26,27, and that, while SN cell loss in Parkinson&#39;s disease is not correlated with cell loss in normal aging28, it is correlated with the duration of the disease29. Unlike most methods for investigating the dopaminergic system, the non-invasiveness, cost-effectiveness, and lack of ionizing radiation make NM-MRI a versatile biomarker30.
The NM-MRI protocol described in this paper was developed to increase both within-subject and across-subject reproducibility of NM-MRI. This protocol ensures full coverage of the SN despite the limited coverage of NM-MRI scans in the inferior-superior direction. The protocol makes use of sagittal, coronal, and axial three-dimensional (3D) T1-weighted (T1w) images, and the steps should be followed to achieve proper slice stack placement. The protocol outlined in this paper has been utilized in multiple studies31,32 and was extensively tested. Wengler et al. completed a study of the reliability of this protocol in which NM-MRI images were acquired twice in each participant across multiple days32. Intra-class correlation coefficients demonstrated excellent test-retest reliability of this method for region of interest (ROI)-based and voxelwise analyses, as well as high contrast in the images.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3T Magnetic Resonance Imaging</td>
			<td>General Electric</td>
			<td></td>
			<td>GE SIGNA Premier with 48-channel head coil</td>
		</tr>
	</tbody>
</table>

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.

Figure 4 shows the representative results from a 28-year-old female participant with no psychiatric or neurological disorders. The NM-MRI protocol ensures complete coverage of the SN, achieved by following step 2 of the protocol&#160;outlined in Figure 1, and satisfactory NM-MRI images by following step 3 of the protocol. Excellent contrast between the SN and neighboring white matter regions with negligible NM concentration (i.e., crus cerebri) can be seen. Thes...

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Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the 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., ...

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3.1K Views.  New York State Psychiatric Institute. This protocol shows how to acquire neuromelanin-sensitive magnetic resonance imaging data of the substantia nigra.

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

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 ...

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 ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

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 ...

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 ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

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 ...