The authors would like to acknowledge support from the CAMH Foundation, thanks to Michael and Sonja Koerner, the Kimel Family, and the Paul E. Garfinkel New Investigator Catalyst Award. This project was funded by the Fonds de Recherches Sant&#233; Qu&#233;bec, the Canadian Institutes of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada, the Weston Brain Institute, the Alzheimer&#39;s Society of Canada, and the Micheal J. Fox Foundation for Parkinson&#39;s Research (MMC), as well as CIHR, the Ontario Mental Health Foundation, NARSAD, and the National Institute of Mental Health (R01MH099167) (ANV). The authors would also like to thank Anusha Ravichandran for assistance acquiring the images.

Study Participants
The protocol in this manuscript was developed for five representative high-resolution images collected from healthy volunteers (3F, 2M; age 29-57, avg. 37) who were free of neurological and neuropsychiatric disorders and cases of severe head trauma. All subjects were recruited at the Centre for Addiction and Mental Health (CAMH). The study was approved by the CAMH Research Ethics Board and was conducted in keeping with the Declaration of Helsinki. All subjects provided written, informed consent for data acquisition and sharing. For details about the acquisition sequence used to collect these images, please refer to Winterburn et al., 2013 and Park et al., 2014.26,34 Images for all five subjects were checked for quality and retained. The hippocampus spanned an average of 118 coronal slices in these images.
1. Software Set-up
<ol>
	<li>Open Display: From the terminal using the following command: Display image_name.mnc -label label_name.mnc. The program will open 3 windows: 3D visualization window, 3-orientation image viewing window, and a navigation window. The terminal will also be used to run the program. Enlarge the coronal view, as the segmentations will be performed coronally. Zoom in on the hippocampus. Select F (Segmenting) in the navigation window. Select F (XY Radius:0.1). The terminal window will prompt for the user to &#8220;Enter xy brush size: &#8221;. Set to 0.1. This will set the size of your paintbrush. The user can now begin drawing the hippocampus onto the MR image.</li>
</ol>
2. Whole Hippocampus Manual Segmentation
<ol>
	<li>Set-up: Using a T1-weighted image, scroll to the anterior-most coronal slice of the hippocampus. To advance slices in the anterior direction, use the &#39;+&#39; key; use the &#39;-&#39; key to move in the posterior direction.</li>
	<li>Slice A: Anterior-Most Slice: Using the right-click on the mouse, draw the outer-most border of the hippocampal grey matter where it meets the surrounding temporal lobe white matter and use the high-intensity white matter of the alveus to assist with the superior border, where the hippocampus meets the amygdala12,22. Use the E (Label Fill) key in the segmentation menu of the navigation window to fill in the label inside the border. Continue to apply these borders throughout the anterior hippocampal head.</li>
	<li>Slice B: Hippocampal Head 1 (Figure 1B):
	<ol>
		<li>Superior, inferior, lateral, medial borders: Continue to draw the borders as described in step 2.2, using the white matter of the temporal lobe and alveus as a guide.</li>
		<li>Supero-medial border: For this, using the axial view, draw a horizontal line from the anterior edge of the lateral hippocampus29, and include anything below this line as hippocampus. NOTE: The supero-medial border becomes more ambiguous in these slices, where the grey matter of the hippocampus blends with the grey matter of the amygdala.</li>
	</ol>
	</li>
	<li>Slice C: Hippocampal Head 2 with Dentations: Depending on the subject, the dentations of the hippocampus may be visible for 3-4 slices (typically, they are more visible on T2-weighted versus T1-weighted images). In these slices, continue to use the white matter of the alveus and temporal lobe to guide border segmentation12,22. For further details, follow steps 2.5.1-2.5.2.</li>
	<li>Slice D: Hippocampal Head 3:
	<ol>
		<li>Superior, inferior, lateral, medial borders: Draw the inferior border of the hippocampus at the white matter of the temporal lobe, the lateral border at the inferior horn of the lateral ventricle, the superior border, following the curve of the dentations, at the white matter of the alveus/fimbria, and the medial border at the hypointense region of the ambient cistern12,22.</li>
		<li>Supero-medial and infero-medial borders: Continue to define the supero-medial border as described in step 2.3.2. Draw the inferior portion of the medial border where the hippocampus thins slightly and extends into the mildly hyperintense grey matter of the entorhinal cortex12,22.</li>
	</ol>
	</li>
	<li>Slice E: Hippocampal Head 4 with Uncus: Continue to draw the inferior, lateral, and superior borders described in steps 2.5.1-2.5.2. Include the uncus (which is located medal to the main body of the hippocampus and is surrounded by low-intensity CSF) in the hippocampal segmentation12,22.</li>
	<li>Slice F: Hippocampal Body: Continue to draw the inferior, lateral, medial, and superior borders described in steps 2.5.1-2.5.2. Draw the infero-medial border at the point where the hippocampus thins as it transitions to entorhinal cortex/para-hippocampal gyrus12,22. Do not include the low-intensity CSF of the vestigial hippocampal sulcus in the segmentation.</li>
	<li>Slice G: Hippocampal Tail 1: Begin segmenting hippocampal tail-type slices when the crus of the fornix is first visible. Exclude the fascicular gyrus (a grey matter structure which blends with the hippocampus in parts of the hippocampal tail) from the segmentation by extrapolating the shape of the fascicular gyrus into the hippocampal tail from more anterior slices12,22. This extrapolation is only possible for 2-3 slices, after which the two structures cannot be accurately distinguished; at this point, treat all visible grey matter in this area as hippocampus .</li>
	<li>Slice H: Hippocampal Tail 2: Segment the low-intensity grey matter of the posterior hippocampal tail from the surrounding high-intensity white matter.</li>
	<li>Slice I: Posterior-Most Slice: Segment the small remaining area of hippocampal grey matter from the surrounding white matter of the temporal lobe.</li>
</ol>
3. Hippocampal Subfield Manual Segmentation
<ol>
	<li>Set-up: Using a T2-weighted image, scroll to the anterior-most coronal slice of the hippocampus (as in step 2.1). To change the color of the paintbrush, select D (Set Paint Lbl:) on the segmenting menu in the navigation window. The command terminal will prompt: &#8220;Enter current paint label:&#8221;. Enter a number between 1 and 255. Each number corresponds to a different label color.</li>
	<li>Slice A: Anterior-Most Slice: Since subfield divisions are not yet visible in the anterior-most slice, draw a line dividing the visible hippocampal grey matter along its longest visible axis (which is not necessarily parallel to any of the cardinal axes) into two equal sections to approximate the true anatomy12,22. Label the superior of these two sections as CA1 and the inferior section as subiculum by choosing a different colored label for each subfield23,35.</li>
	<li>Slice B: Hippocampal Head 1: Label the low-intensity area in the middle of the hippocampal formation as SR/SL/SM13,37. When the bend along the inferior edge of the hippocampus becomes clear, use this landmark as the lateral border separating the subiculum from the CA112,22. Continue to follow the longest axis of the hippocampus to draw the CA1-subiculum border on the supero-medial tip37.</li>
	<li>Slice C: Hippocampal Head 2 with Dentations:
	<ol>
		<li>SR/SL/SM, CA4/DG, and subiculum: Label the SR/SL/SM, CA4/DG, and subiculum as described for slice D (step 3.5.1).</li>
		<li>CA2/CA3 and CA1: Define the border between CA1 and CA2/CA3 as a 45&#186; angle line extending in the supero-lateral direction from the most supero-lateral edge of the SR/SL/SM12,22. Extend the CA2/CA3 medially along the superior edge to the trough between the dentations12,22. Label the rest of the superior edge as CA112,22.</li>
	</ol>
	</li>
	<li>Slice D: Hippocampal Head 3
	<ol>
		<li>SR/SL/SM, CA4/DG, and subiculum: Label the dark SR/SL/SM band first, which will follow the curve of the CA137. Label any high-intensity grey matter inside of the SR/SL/SM as CA4/DG12,22,23,35,37. This may not be a continuous region, as in Figure 2C. Continue to define the subiculum-CA1 border using the bend in the inferior hippocampus12,22.</li>
		<li>CA2/CA3 and CA1: Continue to define the CA1 and CA2/CA3 border as in step 3.4.2. Extend the CA2/CA3 medially halfway along the superior edge of the hippocampus12,22 and label the other half of the superior edge as CA112,22.</li>
		<li>Supero-medial hippocampal head: In this slice, divide the supero-medial hippocampal head vertically in half. Label the medial half as SR/SL/SM12. Divide the lateral half in half again, this time horizontally. Label the superior portion as CA4/DG and the inferior portion as CA2/CA312.</li>
	</ol>
	</li>
	<li>Slice E: Hippocampal Head 4 with Uncus
	<ol>
		<li>Lateral hippocampal head (subiculum): In the lateral portion of these slices, define the subiculum-CA1 border as a vertical line extending in the inferior direction from the most medial edge of the CA4/DG12,22.</li>
		<li>Lateral hippocampal head (CA1, CA2/CA3, CA4/DG, SR/SL/SM.): Define the CA1-CA2/CA3 border in the same way as in step 3.4.2. Continue to label the SR/SL/SM as the low intensity region following the curve of the CA regions. Label the CA4/DG as the center cavity inside the SR/SL/SM, as in step 3.5.1.</li>
		<li>Uncal hippocampal head (SR/SL/SM): Label the uncus of the hippocampus for approximately 10 slices as the hippocampal head transitions into the hippocampal body. In the uncus, label the low intensity region in the center as SR/SL/SM (when this is difficult to see, approximate the anatomy by segmenting a line 2-3 voxels wide up the center of the uncus)12.</li>
		<li>Uncal hippocampal head (CA2/CA3, CA4/DG): Draw a line at the superior edge of the SR/SL/SM section along infero-lateral/supero-medial axis of the uncus. Label all grey matter above this line as CA2/CA312. Label any unlabeled grey matter below this line (on either side of the SR/SL/SM) as CA4/DG12.</li>
	</ol>
	</li>
	<li>Slice F: Hippocampal Body: Continue to apply the borders described in step 3.6.1-3.6.2.</li>
	<li>Slice G: Hippocampal Tail 1: Continue to apply the rules described in step 3.6.1-3.6.2. The subiculum-CA1 border becomes a 45&#186; angle line extending in the infero-medial direction from the medial edge of the CA4/DG12,22.</li>
	<li>Slice H: Hippocampal Tail 2: Once the fascicular gyrus can no longer be distinguished from the hippocampal formation, label the entire outer layer as CA1, the low-intensity area inside of this as SR/SL/SM (as in previous slices), and any remaining grey matter in the middle as CA4/DG12,22.</li>
	<li>Slice I: Posterior-Most Slice: Once the dark SR/SL/SM is no longer visible in the center of the hippocampal formation, label the entire structure as CA112,22.</li>
</ol>
4. Protocol Reliability
<ol>
	<li>Resegment either the right or left hippocampus of each subject after waiting approximately one month from performing the original segmentation. Segment all of the subfields along the entire anterior-posterior length of the hippocampus, trying to follow the protocol rules as consistently as is possible.</li>
	<li>Calculate the Dice&#8217;s kappa between the original and resegmented volumes: 
	<img alt="Equation 1" src="/files/ftp_upload/51861/51861eq1.jpg" /> 
	where k=Dice&#8217;s kappa and A and B are label volumes.</li>
</ol>

<ol>
	<li>Adler, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+the+human+hippocampus+in+3D+from+histology+and+high-resolution+ex-vivo+MRI.">Reconstruction of the human hippocampus in 3D from histology and high-resolution ex-vivo MRI.</a> IEEE Intl. Symp. on Biomed. Img. , 294-297 (2012).</li><li>Adler, D. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histology-derived+volumetric+annotation+of+the+human+hippocampal+subfields+in+postmortem+MRI.">Histology-derived volumetric annotation of the human hippocampal subfields in postmortem MRI.</a> NeuroImage. 84 (1), 505-523 (2014).</li><li>Amaral, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+golgi+study+of+cell+types+in+the+hilar+region+of+the+hippocampus+in+the+rat.">A golgi study of cell types in the hilar region of the hippocampus in the rat.</a> J. Comp. Neurol. 182 (4 Pt 2), 851-914 (1978).</li><li>Blumberg, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amygdala+and+Hippocampal+Volumes+in+Adolescents+and+Adults+With+Bipolar+Disorder.">Amygdala and Hippocampal Volumes in Adolescents and Adults With Bipolar Disorder.</a> Arch Gen Psychiatry. 60 (12), 1201-1208 (2003).</li><li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathol . 82 (4), 239-259 (1991).</li><li>Boccardi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survey+of+protocols+for+the+manual+segmentation+of+the+hippocampus:+preparatory+steps+towards+a+joint+EADC-ADNI+harmonized+protocol.">Survey of protocols for the manual segmentation of the hippocampus: preparatory steps towards a joint EADC-ADNI harmonized protocol.</a> J. Alzheimer's Dis. 26 (3), 61-75 (2011).</li><li>Chakravarty, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performing+label-fusion-based+segmentation+using+multiple+automatically+generated+templates.">Performing label-fusion-based segmentation using multiple automatically generated templates.</a> Hum. Brain Mapp. 34 (10), 2635-2654 (2013).</li><li>Chakravarty, M. M., Bertrand, G., Hodge, C. P., Sadikot, A. F., Collins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+creation+of+a+brain+atlas+for+image+guided+neurosurgery+using+serial+histological+data.">The creation of a brain atlas for image guided neurosurgery using serial histological data.</a> NeuroImage. 30 (2), 359-376 (2006).</li><li>Collins, D. L., Neelin, P., Peters, T. M., Evans, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+3D+intersubject+registration+of+MR+volumetric+data+in+standardized+Talairach+space.">Automatic 3D intersubject registration of MR volumetric data in standardized Talairach space.</a> J. Comput. Assist. Tomogr. 18 (2), 192-205 (1994).</li><li>Heijer, F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+diffusion+MRI+measures+of+the+hippocampus+and+memory+performance.">Structural and diffusion MRI measures of the hippocampus and memory performance.</a> NeuroImage. 63 (4), 1782-1789 (2012).</li><li>Duncan, K., Tompary, A., Davachi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associative+encoding+and+retrieval+are+predicted+by+functional+connectivity+in+distinct+hippocampal+area+ca1+pathways.">Associative encoding and retrieval are predicted by functional connectivity in distinct hippocampal area ca1 pathways.</a> The Journal of Neuroscience. 34 (34), 11188-11198 (2014).</li><li>Duvernoy, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Human Hippocampus: Functional Anatomy Vascularization, and Serial Sections with MRI. , (2005).</li><li>Fatterpekar, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoarchitecture+of+the+human+cerebral+cortex:+MR+microscopy+of+excised+specimens+at+9.4+Tesla.">Cytoarchitecture of the human cerebral cortex: MR microscopy of excised specimens at 9.4 Tesla.</a> Am. J. Neuroradiol. 23 (8), 1313-1321 (2002).</li><li>Frey, S., Pandya, D. N., Chakravarty, M. M., Bailey, L., Petrides, M., Collins, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+MRI+based+average+macaque+monkey+stereotaxic+atlas+and+space+(MNI+monkey+space).">An MRI based average macaque monkey stereotaxic atlas and space (MNI monkey space).</a> NeuroImage. 55 (4), 1435-1442 (2011).</li><li>Goubran, M., Crukley, C., de Ribaupierre, S., Peters, T. M., Khan, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+registration+of+ex-vivo.+MRI+to+sparsely+sectioned+histology+of+hippocampal+and+neocortical+temporal+lobe+specimens.">Image registration of ex-vivo. MRI to sparsely sectioned histology of hippocampal and neocortical temporal lobe specimens.</a> NeuroImage. 83, 770-781 (2013).</li><li>Heckemann, R. A., Hajnal, J. V., Aljabar, P., Rueckert, D., Hammers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+anatomical+brain+MRI+segmentation+combining+label+propagation+and+decision+fusion.">Automatic anatomical brain MRI segmentation combining label propagation and decision fusion.</a> NeuroImage. 33 (1), 115-126 (2006).</li><li>Holmes, C. J., Hoge, R., Collins, L., Woods, R., Toga, A. W., Evans, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+MR+images+using+registration+for+signal+averaging.">Enhancement of MR images using registration for signal averaging.</a> J. Comput. Assist. Tomogr. 22 (2), 324-333 (1998).</li><li>Karnik-Henry, M. S., Wang, L., Barch, D. M., Harms, M. P., Campanella, C., Csernansky, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medial+temporal+lobe+structure+and+cognition+in+individuals+with+schizophrenia+and+in+their+non-psychotic+siblings.">Medial temporal lobe structure and cognition in individuals with schizophrenia and in their non-psychotic siblings.</a> Schizophrenia Research. 138 (2-3), 128-135 (2012).</li><li>Kim, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+3-D+extraction+and+evaluation+of+the+inner+and+outer+cortical+surfaces+using+a+Laplacian+map+and+partial+volume+effect+classification.">Automated 3-D extraction and evaluation of the inner and outer cortical surfaces using a Laplacian map and partial volume effect classification.</a> NeuroImage. 27 (1), 210-221 (2005).</li><li>La Joie, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effect+of+age+on+hippocampal+subfields+assessed+using+a+new+high-resolution+3T+MR+sequence.">Differential effect of age on hippocampal subfields assessed using a new high-resolution 3T MR sequence.</a> NeuroImage. 53 (2), 506-514 (2010).</li><li>Libby, L. A., Ekstrom, A. D., Ragland, J. D., Ranganath, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+connectivity+of+perirhinal+and+parahippocampal+cortices+within+human+hippocampal+subregions+revealed+by+high-resolution+functional+imaging.">Differential connectivity of perirhinal and parahippocampal cortices within human hippocampal subregions revealed by high-resolution functional imaging.</a> The Journal of Neuroscience. 32 (19), 6550-6560 (2012).</li><li>Mai, J. K., Paxinos, G., Voss, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of the Human Brain. , (2008).</li><li>Mueller, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hippocampal+subfields+and+age-related+changes+with+high+resolution+MRI+at+4T.">Measurement of hippocampal subfields and age-related changes with high resolution MRI at 4T.</a> Neurobiol Aging. 28 (5), 719-726 (2006).</li><li>Narr, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+specificity+of+hippocampal+volume+reductions+in+first-episode+schizophrenia.">Regional specificity of hippocampal volume reductions in first-episode schizophrenia.</a> NeuroImage. 21 (4), 1563-1575 (2004).</li><li>Olsen, R. K., Palombo, D. J., Rabin, J. S., Levine, B., Ryan, J. D., Rosenbaum, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetric+Analysis+of+Medial+Temporal+Lobe+Subregions+in+Development+Amnesia+using+High-Resolution+Magnetic+Resonance+Imaging.">Volumetric Analysis of Medial Temporal Lobe Subregions in Development Amnesia using High-Resolution Magnetic Resonance Imaging.</a> Hippocampus. 23 (10), 855-860 (2013).</li><li>Park, M. T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+high-resolution+MRI+atlases+of+the+human+cerebellum+at+3T+and+segmentation+using+multiple+automatically+generated+templates.">Derivation of high-resolution MRI atlases of the human cerebellum at 3T and segmentation using multiple automatically generated templates.</a> NeuroImage. 95, 217-231 (2014).</li><li>Pipitone, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-atlas+Segmentation+of+the+Whole+Hippocampus+and+Subfields+Using+Multiple+Automatically+Generated+Templates.">Multi-atlas Segmentation of the Whole Hippocampus and Subfields Using Multiple Automatically Generated Templates.</a> NeuroImage. 101, 494-512 (2014).</li><li>Pluta, J., Yushkevich, P., Das, S., Wolk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+hippocampal+subfield+atrophy+in+mild+cognitive+impairment+via+semi-automatic+segmentation+of+T2-weighted+MRI.Journal+of+Alzheimer's+Disease.">In vivo analysis of hippocampal subfield atrophy in mild cognitive impairment via semi-automatic segmentation of T2-weighted MRI.Journal of Alzheimer's Disease.</a> 31 (1), 85-99 (2012).</li><li>Pruessner, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volumetry+of+hippocampus+and+amygdala+with+high-resolution+MRI+and+three-+dimensional+analysis+software:+minimizing+the+discrepancies+between+laboratories.">Volumetry of hippocampus and amygdala with high-resolution MRI and three- dimensional analysis software: minimizing the discrepancies between laboratories.</a> Cereb Cortex. 10 (4), 433-442 (2000).</li><li>Sabuncu, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+cortical+and+hippocampal+atrophy+in+Alzheimer+disease.">The dynamics of cortical and hippocampal atrophy in Alzheimer disease.</a> Archives of Neurology. 68 (8), 1040-1048 (2011).</li><li>Scoville, W. B., Milner, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+recent+memory+after+bilateral+hippocampal+lesions.">Loss of recent memory after bilateral hippocampal lesions.</a> J. Neuropsych. and Clin. Neurosci. 12 (1), 103-113 (1957).</li><li>Toga, A. W., Thompson, P. M., Mori, S., Amunts, K., Zilles, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+multimodal+atlases+of+the+human+brain.">Towards multimodal atlases of the human brain.</a> Nat. Rev. Neurosci. 7 (12), 952-966 (2006).</li><li>van Leemput, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+segmentation+of+hippocampal+subfields+from+ultra-high+resolution+in+vivo.">Automated segmentation of hippocampal subfields from ultra-high resolution in vivo.</a> MRI. Hippocampus. 19 (6), 549-557 (2009).</li><li>Winterburn, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+in+vivo+atlas+of+human+hippocampal+subfields+using+high-resolution+3+T+magnetic+resonance+imaging.">A novel in vivo atlas of human hippocampal subfields using high-resolution 3 T magnetic resonance imaging.</a> NeuroImage. 74, 254-265 (2013).</li><li>Wisse, L. E. M., Gerritsen, L., Zwanenburg, J. J. M., Kuijf, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subfields+of+the+hippocampal+formation+at+7+T+MRI:+in+vivo.+volumetric+assessment.">Subfields of the hippocampal formation at 7 T MRI: in vivo. volumetric assessment.</a> NeuroImage. 61 (4), 1043-1049 (2012).</li><li>Yelnik, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional,+histological+and+deformable+atlas+of+the+human+basal+ganglia.+I.+Atlas+construction+based+on+immunohistochemical+and+MRI+data.">A three-dimensional, histological and deformable atlas of the human basal ganglia. I. Atlas construction based on immunohistochemical and MRI data.</a> NeuroImage. 34 (2), 618-638 (2007).</li><li>Yushkevich, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-resolution+computational+atlas+of+the+human+hippocampus+from+postmortem+magnetic+resonance+imaging+at+9.4+T.">A high-resolution computational atlas of the human hippocampus from postmortem magnetic resonance imaging at 9.4 T.</a> NeuroImage. 44 (2), 385-398 (2009).</li><li>Yushkevich, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Comparison+of+21+Protocols+for+Labeling+Hippocampal+Subfields+and+Parahippocampal+Subregions+in+In+Vivo+MRI:+Towards+a+Harmonized+Segmentation+Protocol.">Quantitative Comparison of 21 Protocols for Labeling Hippocampal Subfields and Parahippocampal Subregions in In Vivo MRI: Towards a Harmonized Segmentation Protocol.</a> NeuroImage. , (2015).</li></ol>

The authors have no conflicts of interest to declare.

Hippocampal subfield segmentation in MR images is well-represented in the literature. However, existing protocols exclude portions of the hippocampus20,23,33,35, apply only to fixed images37, or require ultra-high field scanners for image acquisition35,37. This manuscript offers a segmentation protocol that includes five major subdivisions (CA1, CA2/CA3, CA4/dentate gyrus, SR/SL/SM, and subiculum) of the hippocampus and spans the entire anterior-posterior length of the structure. The comp...

The hippocampus is a widely studied medial temporal lobe structure that is associated with episodic memory, spatial navigation, and other cognitive functions10,31. Its role in neurodegenerative and neuropsychiatric disorders such as Alzheimer&#8217;s disease, schizophrenia, and bipolar disorder is well-documented4,5,18,24,30. The goal of this manuscript is to provide additional detail to the manual segmentation protocol published previously34 for human hippocampal subfields on high-resolution magnetic resonance (MR) images acquired at 3T. Additionally, the video component accompanying this manuscript will provide further assistance to researchers who wish to implement the protocol on their own datasets.
The hippocampus can be divided into subfields based on cytoarchitectonic differences observed in histologically-prepared post-mortem specimens12,22. Such post-mortem specimens define the ground truth for the identification and study of hippocampal subfields; however preparations of this nature require specialized skills and equipment for staining, and are limited by the availability of fixed tissue, especially in diseased populations. In vivo imaging has the advantage of a much larger pool of subjects, and also presents the opportunity for follow-up studies and observing changes in populations. Although it has been shown that signal intensities in T2-weighted ex vivo MR images reflect cellular density13, it is still difficult to identify undisputed borders between subfields using solely MR signal intensities. As such, a number of different approaches for identifying histology-level detail on MR images have been developed.
Some groups have made efforts to reconstruct and digitize histological datasets and then use these reconstructions along with image registration techniques to localize hippocampal subfield neuroanatomy on in vivo MR1,2,8,9,14,15,17,32. Although this is an effective technique for mapping a version of the histological ground truth directly onto MR images, reconstructions of this nature are difficult to complete. Projects such as these are limited by the availability of intact medial temporal lobe specimens, histological techniques, data loss during histological processing, and the fundamental morphological inconsistencies between fixed and in vivo brains. Other groups have used high-field scanners (7T or 9.4T) in an effort to acquire in vivo or ex vivo images with a small enough (0.20-0.35 mm isotropic) voxel size to visualize spatially localized differences in image contrast that are used to infer boundaries between subfields35,37. Even at 7T-9.4T and with such a small voxel size, the cytoarchitectonic characteristics of hippocampal subfields are not visible. As such, manual segmentation protocols have been developed that approximate the known histological boundaries on MR images. These protocols determine subfield boundaries by interpreting local image contrast differences and defining geometric rules (such as straight lines and angles) relative to visible structures. Although images taken at a high field strength are able to offer detailed insight into hippocampal subfields, high-field scanners are not yet common in clinical or research settings, so 7T and 9.4T protocols currently have limited applicability. Similar protocols have been developed for images collected on 3T and 4T scanners11,20,21,23,24,25,28,33. Many of these protocols are based on images with sub-1mm voxels voxel dimensions in the coronal plane, but have large slice thicknesses (0.8-3 mm)11,20,21,23,25,28,33 or large inter-slice distances20,28, both of which result in a significant measurement bias in the estimation of volumes of the individual subfields. Additionally, many of the existing 3T protocols exclude subfields in all or part of the hippocampal head or tail20,23,25,33 or do not provide detailed segmentations of important substructures (i.e., combine the DG with CA2/CA3 or do not include the strata radiatum/lacunosum/moleculare of the CA)11,20,21,23,24,25,28,33. There is therefore a need in the field for a detailed description of a protocol that can reliably identify relevant subfields throughout the head, body, and tail of the hippocampus that is based on a scanner commonly available in clinical and research settings. Efforts are currently underway by the Hippocampal Subfields Group (www.hippocampalsubfields.com) to harmonize the hippocampal subfield segmentation process between laboratories, similar to an existing harmonization effort for whole hippocampal segmentation6, and an initial paper comparing 21 existing protocols was recently published38. The work from this group will further elucidate optimal segmentation procedures.
This manuscript provides detailed written and video instructions for reliably implementing the hippocampal subfield segmentation protocol described previously by Winterburn and colleagues34 on high-resolution 3T MR images. The protocol has been implemented on five images of healthy controls for the whole hippocampus and five hippocampal subfields (CA1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum). These segmented images are available to the public online (cobralab.ca/atlases/Hippocampus). The protocol and the segmented images will be useful for groups who wish to study detailed hippocampal neuroanatomy in MR images.

<table border="0">
	<tbody>
		<tr>
			<td>Discovery MR750 3T</td>
			<td>GE</td>
			<td>NA</td>
			<td>Or equivalent 3T scanner</td>
		</tr>
		<tr>
			<td>Minc Tool Kit</td>
			<td>McConnell Brain Imaging Center, Montreal Neurological Institute</td>
			<td>NA</td>
			<td>Open source: http://www.bic.mni.mcgill.ca/ServicesSoftware/ServicesSoftwareMincToolKit</td>
		</tr>
	</tbody>
</table>

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.

Results from the protocol reliability test are summarized in Table 2. For the whole bilateral hippocampus, mean spatial overlap as measured by Dice&#8217;s kappa is 0.91 and ranges from 0.90 - 0.92. Subfield kappa values range from 0.64 (CA2/CA3) to 0.83 (CA4/dentate gyrus). Mean volumes for all subfields and the whole hippocampus are reported in Table 3. Volumes for the whole hippocampus range from 2456.72-3325.02 mm3. The CA2/CA3 is the smallest subfield at 208.33 mm3</...

Watch this Scientific Journal Video about High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging at JoVE.com

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.

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 ...

high-resolution-vivo-manual-segmentation-protocol-for-human

Research

JoVE Journal

Neuroscience

9.2K Views.  University of Toronto. 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. 