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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.
Even though detailed protocols for analysis of NM-MRI data have been discussed elsewhere, for completeness, we provide a brief summary of our previous work and recommendations for preprocessing of NM-MRI images and voxelwise analyses. This approach has been validated previously in conjunction with the acquisition protocol described in this paper. Previous studies discuss the advantages of this method in more detail and provide data supporting its reproducibility6,12,32. Note however that the standardized acquisition protocol described herein is applicable to any processing and analysis strategy (including ROI-based analysis in native or MNI space8,32) and not just the one described here.
For analysis of NM-MRI images, preprocessing can be performed to correct for motion and to spatially normalize individual subject data to a standard anatomical template. We recommend the following pipeline combining Statistical Parametric Mapping (SPM) and Advanced Normalization Tools (ANTs) to use the following tools in the following steps: (1) SPM-Realign to realign and correct separately acquired averages for motion, and SPM-ImCalc to average the realigned images; (2) antsBrainExtraction.sh for brain extraction of the T1w image; (3) antsRegistrationSyN.sh (rigid + affine + deformable syn) for spatial normalization of the brain-extracted T1w image to the MNI152NLin2009cAsym template space; (4) antsRegistrationSyN.sh (rigid) to coregister the NM-MRI image to the T1w (in native space) image; (5) antsApplyTransforms to combine the transformations estimated in steps 3 and 4 into a single-step transformation for spatial normalization of the NM-MRI images to MNI space; and (6) SPM-Smooth with a 1 mm full-width-at-half-maximum Gaussian kernel for spatial smoothing of the spatially normalized NM-MRI image. This processing pipeline was previously shown to achieve the highest test-retest reliability in the literature, with an average intra-class correlation coefficient (ICC) within the SN of ~0.9032. Furthermore, several previous studies have used similar preprocessing pipelines12,31,34,35,36,37.
After spatial normalization, the NM-MRI images should be analyzed by calculating the contrast-to-noise ratio at each voxel (CNRV). The CNR measures the percent signal difference between each voxel (IV) and a reference white matter region known to have little NM content12 (crus cerebri, ICC), given by the following formula: CNRV&#160;= {[IV&#160;- mode(ICC)] / mode(ICC)}*100. CNRV values can be averaged for each participant to determine the CNR of the entire SN or can be analyzed at the voxelwise level within the SN. Higher CNR values reflect increased NM content in that voxel or ROI. Unlike some other analysis methods that define the SN ROI as the hyperintense region in a NM-MRI image, this recommended method uses predefined template ROIs that can be obtained from the literature12 or drawn on the average of NM-MRI images in MNI space across all subjects in the study (using a study-specific template). Not only is this method fully automated, it also removes circularity in the analysis, accounts for heterogeneity within the SN-VTA complex, and does not limit analysis to the whole-ROI level.
When acquiring NM-MRI images, it is critical that the T1w images used to place the NM-MRI volume are aligned along the AC-PC line. Doing so will improve the reproducibility of the scans. It is also important to acquire the T1w images as close in time before acquiring the NM-MRI images as possible. Because the T1w image is used for NM-MRI volume placement, it is important that it accurately represents the location of the participant&#39;s head in the scanner. If the participant has moved between the T1w scan and the NM-MRI scan, then the NM-MRI volume will not be appropriately placed. Minimizing the amount of time between acquisition of the T1w images and the NM-MRI images will decrease the likelihood that the participant has moved between scans and therefore decrease the likelihood that part of the SN is not included in the NM-MRI volume.
Some modifications to the protocol may be required if issues with the NM-MRI acquisition arise. If the whole SN is not consistently covered, even after correcting the volume placement, then the number of slices in the NM-MRI protocol may need to be increased to capture the entire SN. Additionally, if the participant has difficulty staying still for the entirety of the NM-MRI scan, resulting in consistent motion artifacts, individual repetitions could be acquired and averaged offline. For example, instead of completing one 10-min scan that acquires five repetitions averaged online, five 2-min scans could be acquired and averaged offline. This would give the participant opportunities for breaks in between repetitions and may help them remain still for the duration of the individual scans.
One limitation of this protocol is that it does not provide full coverage of the LC with standard NM-MRI acquisition protocols, preventing the noradrenergic system from being thoroughly investigated using this method. While the LC is a structure that can be imaged using NM-MRI, including the LC in this protocol would increase the number of slices required to reliably capture both the SN and LC in their entirety. Increasing the number of slices would, in turn, increase the scan time for this protocol. Because these scans are sensitive to motion, an increase in scan time may produce lower-quality images as participants may find it more difficult to remain still for longer periods&#8212;particularly problematic in clinical populations. Thus, we chose not to include the LC in this protocol to minimize the potential for motion artifacts in the data. Future studies should investigate the reliability of NM-MRI protocols with a greater number of slices to simultaneously image the SN and LC.
A second limitation of this protocol is that the AC-PC alignment of the NM-MRI volume may not provide the optimal orientation for imaging the SN. While the AC-PC line is easy to identify, this orientation does not fully minimize partial volume effects as it is not perfectly perpendicular to the SN. Previous work has utilized an oblique axial section perpendicular to the floor of the fourth ventricle to image the SN38,39,40. While this volume placement, or one perpendicular to the cerebral aqueduct, may provide less partial volume effects than AC-PC alignment, we chose to use the AC-PC line given its clearly defined landmarks. The validity of this alignment was shown in previous work utilizing the protocol outlined above, in which excellent test-retest reliability was achieved32. AC-PC alignment has also been used in several other studies. Cassidy et al. found that patients with cocaine addiction had higher SN CNR values than controls35. In a study of patients with late-life depression, Wengler et al. found that psychomotor function was correlated with SN CNR values36. A third paper also found that Parkinson&#39;s patients had reduced CNR in the SN while patients with psychosis had increased CNR in the SN12.
However, no study has directly compared different volume placement methods, and this is an area that future research should explore to determine which method provides the best test-retest reliability across multiple acquisitions. 3D NM-MRI sequences could provide an alternative solution because they provide greater flexibility in reformatting after acquisition. Furthermore, 3D sequences achieve a higher signal-to-noise ratio than 2D sequences, potentially allowing for higher spatial resolution but come at the cost of increased sensitivity to motion. Currently, 2D-GRE MT is the only extensively validated NM-MRI sequence&#8212;the motivating factor for using it for this protocol. Future studies should compare NM-MRI signal from 3D sequences to NM concentration and striatal dopamine function, and reproducibility in comparison to 2D-GRE MT before widespread adoption.
This protocol has advantages over other NM-MRI protocols because it provides easily identifiable landmarks for NM-MRI volume placement, making it highly reproducible. It also provides online quality checks, which no other NM-MRI protocol has included. These quality checks allow the experimenter to reacquire images if they are of poor quality rather than simply excluding that subject from the analysis.
NM-MRI is a valuable tool that has been used to investigate several neuropsychiatric disorders. NM-MRI is a proxy measure of dopamine function in the nigrostriatal pathway12, thus offering a method of examining the in vivo dopaminergic system that does not require invasive procedures such as PET. Patients with schizophrenia have increased NM signal in the SN38,41, supporting previous studies that have revealed increased dopaminergic function in schizophrenia. NM-MRI signal in the SN also correlates with psychosis severity in patients with schizophrenia and those at high risk for schizophrenia12. Research has also shown that individuals with cocaine use disorder have increased NM-MRI signal in the ventrolateral regions of the SN35, and that in patients with late-life depression, lower NM-MRI signal in the SN is correlated with motor slowing36. Additionally, NM-MRI has been used to study dopaminergic cell loss in conditions such as Parkinson&#39;s disease.
Kitao and colleagues established that NM-MRI signal in the SN is correlated with the number of pigmented dopaminergic neurons in the SN11, and others have shown that NM-MRI signal in SN dopaminergic neurons is decreased in Parkinson&#39;s disease6,9,39,40. Further research in Parkinson&#39;s patients has used NM-MRI to map the topographical pattern of SN cell loss12 and the progression of SN cell loss over the course of the disease37. Altogether, this suggests that not only does NM-MRI provide insight into the underlying chemical components of neuropsychiatric disorders, but it may also be useful as a biomarker in predicting disease onset and severity. We hope that the standardized protocol presented here will facilitate future work to develop clinically useful biomarkers based on NM-MRI30.

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><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. These images were checked immediately after acquisition to ensure the proper coverage of the SN and to check for artifacts. Because full coverage of the SN was achieved without any artifacts, the scan passed the quality checks and did not need to be repeated.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62493/62493fig04.jpg" /> 
Figure 4: Example of a representative NM-MRI acquisition. 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 from a 28-year-old female participant with no psychiatric or neurological disorders. The NM-MRI protocol ensures complete coverage of the substantia nigra, partial coverage of the locus coeruleus, and satisfactory NM-MRI images. Excellent contrast between the substantia nigra and neighboring white matter regions with no neuromelanin concentration (i.e., crus cerebrus) can be seen on slices 9-16. The image at the bottom shows a zoomed-in view of the midbrain from slice 13. Abbreviation: NM-MRI = neuromelanin-sensitive magnetic resonance imaging. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62493/62493fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2 shows the representative results from a 28-year-old female participant with no psychiatric or neurological disorders whose images failed the first quality control check (step 3.1). The SN is visible in the most superior slice (slice 20), indicating that full coverage of the SN was not achieved. In this instance, the data must be reacquired by repeating steps 2.3-2.9 of the protocol, as shown in Figure 1. If the participant has moved significantly since the acquisition of the initial T1w image, then the researcher should return to step 2.1 to reacquire the T1w image.
Figure 3 shows example images that failed the second quality control check (step 3.2). As outlined in step 3.2, scans containing artifacts due to blood vessels (Figure 3A) do not need to be repeated, as those artifacts will likely be present in every acquisition. Scans that contain artifacts resulting from motion (Figure 3B) or ambiguous artifacts (Figure 3C) should be repeated. In the case of ambiguous artifacts, if&#160;the artifacts remain present after reacquisition, then the scan does not need to be reacquired further as the artifacts are likely biological and therefore, will be present in every acquisition.

Watch this Scientific Journal Video about Standardized Data Acquisition for Neuromelanin-Sensitive Magnetic Resonance Imaging of the Substantia Nigra at JoVE.com

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., ...

standardized-data-acquisition-for-neuromelanin-sensitive-magnetic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring 

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

Bioengineering

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Medicine

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

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 ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

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 ...

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

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

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

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

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

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Chapters in this video

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

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

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

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

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

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

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