NeuroImage 60 (2012) 1226–1235 Contents lists available at SciVerse ScienceDirect NeuroImage journal homepage: www.elsevier.com/locate/ynimg A reliable protocol for the manual segmentation of the human amygdala and its subregions using ultra-high resolution MRI Jonathan J. Entis a, Priya Doerga f, Lisa Feldman Barrett d,e,g,1, Bradford C. Dickerson b,c,d,g,⁎,1 a Department of Psychology, Boston College, USA b Frontotemporal Disorders Unit, Massachusetts Alzheimer's Disease Research Center, USA c Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA d Department of Psychiatry, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA e Department of Psychology, Northeastern University, Boston, MA, USA f Department of Anatomy and Neuroscience, VU University Amsterdam, The Netherlands g Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA article info abstract Article history: The measurement of the volume of the human amygdala in vivo has received increasing attention over the Received 6 May 2011 past decade, but existing methods face several challenges. First, due to the amorphous appearance of the Revised 9 December 2011 amygdala and the difficulties in interpreting its boundaries, it is common for protocols to omit sizable sec- Accepted 29 December 2011 tions of the rostral and dorsal regions of the amygdala comprising parts of the basolateral complex (BL) Available online 5 January 2012 and central nucleus (Ce), respectively. Second, segmentation of the amgydaloid complex into separate sub- Keywords: divisions is challenging due to the resolution of routinely acquired images and the lack of standard protocols. Amygdala Recent advances in technology have made ultra-high resolution MR images available, and in this study we Segmentation provide a detailed segmentation protocol for manually tracing the whole amygdala that incorporates a great- MRI er portion of the rostral and dorsal sections with techniques illustrated in detail to maximize reproducibility. Subnuclei In addition, we propose a geometrically-based protocol for segmenting the amygdala into four component Parcellation subregions of interest (sROI), which correspond largely to amygdala subnuclear divisions: the BL sROI, cen- tromedial (CM) sROI, basomedial (BM) sROI, and the amygdaloid cortical (ACo) sROI. We performed an intra- and inter-rater reliability study of our methods in 10 adults (5 young adults and 5 older adults). The results indicate that both protocols can be implemented with a high degree of reliability (the majority of intra-rater and inter-rater correlations were >0.81). This protocol should aid further research into the alter- ations in amygdala anatomy, connectivity, and function that accompany normal aging and pathology associ- ated with neuropsychiatric disorders. © 2012 Elsevier Inc. All rights reserved. Introduction its dense connections with many regions of the brain (for a review, see Swanson and Petrovich, 1998). The amygdala is an amorphous gray matter structure within the In living humans, investigations of amygdala function and dys- rostral medial temporal lobe. It has been implicated in many psycho- function have primarily employed structural and functional neuroim- logical phenomena, including emotion and affect (e.g. Adolphs et al., aging tools (in addition to studies of patients with lesions). Although 1994; Barrett et al., 2007; Lanteaume et al., 2007; LeDoux et al., a number of studies have been performed on the relationship of 1988; Sharot et al., 2007), social behavior (e.g. Amaral, 2003; amygdala volume to specific behaviors and in aging and neuropsychi- Machado et al., 2008; Rosvold et al., 1954), attention (e.g. Pessoa et atric disorders (for reviews, see Anand and Shekhar, 2003; Phelps and al., 2002a, 2002b; Ursin and Kaada, 1960), perception (e.g. Sander LeDoux, 2005; Wright, 2009), heterogeneous results are present like- and Scheich, 2001; Whalen et al., 1998), learning (e.g. Gaffan et al., ly in part because of the variety of approaches to volumetric measure- 1989; Hooker et al., 2006; Morris et al., 1998), and memory (e.g. ment of the amygdala. Fadok et al., 2010; Packard et al., 1994). This is not surprising given Over the past 20 years, a number of manual tracing protocols have been published that provide methods for measuring human amygda- lar volume (Achten et al., 1998; Bonilha et al., 2004; Convit et al., ⁎ Corresponding author at: 149 13th St., Suite 2691, Charlestown, MA 02129, USA. 1999; Makris et al., 1999; Matsuoka et al., 2003; Pruessner et al., E-mail addresses: [email protected] (J.J. Entis), [email protected] 2000; Watson et al., 1992). Although these approaches have been (P. Doerga), [email protected] (L.F. Barrett), [email protected] (B.C. Dickerson). very useful for identifying a variety of interesting effects, we believe 1 These authors contributed equally to the research. that they and automated segmentation protocols based on similar 1053-8119/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2011.12.073 J.J. Entis et al. / NeuroImage 60 (2012) 1226–1235 1227 Box 1 basomedial complex (BM), and the amygdaloid cortical complex (ACo) (Paxinos and Mai, 2004). ac, anterior commissure; ACo, amygdaloid cortical nucleus; Amunts and colleagues published the first study to match histolog- ACoD, anterior cortical amygdaloid nucleus, dorsal part; ACoV, ical and MRI data in an attempt to develop a histologically-based prob- anterior cortical amygdaloid nucleus, ventral part; AG, ambiens abilistic atlas of subdivisions of the amygdala (Amunts et al., 2005). gyrus; Ai, amygdaloid island; alv, alveus; AStr, amygdalostria- Their protocol involved generating 3D reconstructions of digital pho- tal transition area; BC, basal nucleus, compact part; BLD, baso- tomicrographs of the histological samples from 10 post-mortem lateral amygdaloid nucleus, dorsal (magnocellular) part; BLI, brains and manually tracing three subdivisions (the CM group, the basolateral amygdaloid nucleus, intermediate part; BLPL, baso- BL group, and the superficial group) directly onto the photomicro- lateral amygdaloid nucleus, paralaminar part; BLVL, basolateral graph images. The ten 3D-reconstructed images were registered to amygdaloid nucleus, ventrolateral part; BLVM, basolateral MNI template space to produce probabilistic maps of the amygdala amygdaloid nucleus, ventromedial part; BM, basomedial amyg- subregions. The application of this protocol necessitates manipulation daloid nucleus; CA1, CA1 field of hippocampus; Ce, central of individual subjects' MRIs into standard template (MNI) space, a amygdaloid nucleus; CeL, central amygdaloid nucleus, lateral process that can produce inaccuracies due to the deformations re- part; CeM, central amygdaloid nucleus, medial part; CM, cen- quired (Yassa and Stark, 2009). Moreover, the probabilistic nature of tromedial complex; DiCl, diffuse insular claustrum; Ent, entorhi- the method requires that the user measures subnuclear volume in nal cortex; HiH, hippocampal head; La, lateral amygdaloid areas in which voxels of different subnuclei do not overlap (e.g., > nucleus; LaDA, lateral amygdaloid nucleus, dorsal anterior part; 50–75% probability), which underestimates the full extent of the vol- LaDL, lateral amygdaloid nucleus, dorsolateral part; LaDM, lat- ume of each subnuclear region in any individual subject. eral amygdaloid nucleus, dorsomedial part; Lal, lateral amygda- Newer methods using diffusion tensor imaging (DTI) to segment loid nucleus, intermediate part; LaV, lateral amygdaloid the amygdala have added a perspective from connectional anatomy nucleus, anterior part; LiCl, limitans claustrum; Me, medial to parcellation endeavors (Bach et al., 2011; Saygin et al., 2011; amygdaloid nucleus; MeA, medial amygdaloid nucleus, ant. Solano-Castiella et al., 2010). Solano-Castiella et al. (2010) identified part; opt, optic tract; PaCl, preamygdalar claustrum; PAM, peri- two primary diffusion directions of amygdala voxels and used this in- amygdaloid cortex; PCo, post. cortical amygdaloid nucleus; formation to separate the amygdala into medial and lateral subdivi- PHA, parahippocampal–amygdaloid transition area; PHG, para- sions. Further work has shown that diffusion-based segmentations hippocampal gyrus; PirT, piriform cortex, temporal area; Pu, pu- correspond to anatomical connectivity (Bach et al., 2011). One chal- tamen; PuV, ventral putamen; S, subiculum; sas, semiannular lenge in using DTI for amygdala subnuclear segmentation is its low sulcus; SEpS, subependymal stratum; SLG, semilunar gyrus; spatial resolution (although the data in Solano-Castiella et al., 2010 st, stria terminalis; TCl, temporal claustrum; TLV, temporal is relatively high resolution for DTI, at 1.7 mm in-plane and 1.7 mm horn of lateral ventricle; Un, uncus; unc, uncinate fasciculus; thickness). Similar resolution issues exist for resting-state fMRI- us, uncal sulcus; VCl, ventral claustrum. based connectivity analysis (3 mm slice thickness in Roy et al., 2009). Here we used the clarity of ultra high-resolution scans to create a de- tailed approach to the segmentation of the amygdala using a widely available histological atlas as our guide. Such high-resolution MR im- ages have not been used before for amygdala segmentation, although they have been used previously in a study of hippocampal subregions manual tracings (e.g., Freesurfer's