In vivo quantification of amygdala subnuclei using 4.7 T fast spin echo imaging
Introduction
The amygdala (AG) is an almond-shaped heterogeneous structure located in the medial temporal lobe (MTL). The AG is involved in neuronal circuits of fear and reward learning, as well as aggressive, maternal, sexual, and feeding behaviors (LeDoux, 2007). Moreover, through its extensive connections with cortical and subcortical areas, the AG plays an important role in stress response (Davis and Whalen, 2001), goal-directed behavior (Hampton et al., 2007), social behavior (Adolphs, 2009), attention and perception (Vuilleumier, 2009), processing of facial expression (Tottenham et al., 2009), motivation and explicit memory (LeDoux, 2007).
Alterations in the AG structure and function have been reported in different psychiatric and neurological disorders including affective disorders (Hajek et al., 2009), anxiety disorders (LeDoux, 2007), Alzheimer's disease (Kromer et al., 1990, Scott et al., 1991), and Parkinson's disease (Braak et al., 1994).
In human and animal studies the AG is subdivided into at least thirteen different subnuclei and cortical areas (Amaral et al., 1992, Pitkänen et al., 1997, Sah et al., 2003). These subnuclei are further grouped into two major divisions: (1) the cortico-medial region consisting of the cortical (Co), medial (Me), and central (Ce) nuclei; and (2) the basolateral complex (BLA) consisting of the lateral (La), basal (B), and accessory basal (AB) nuclei (LeDoux, 2007). Animal studies have demonstrated that these subnuclei – via their unique connectivity patterns – appear to have specialized roles in the expression of fear responses as well as the acquisition and storage of a memory for the conditioning experience (Phelps and LeDoux, 2005). In addition, patients with focal lesions to the BLA produce hypervigilance to subliminal fearful facial expressions (Terburg et al., 2012), while bilateral destruction of the entire AG impairs the processing of the fearful facial expression as demonstrated by an insensitivity to the intensity of fear expressed by faces (Adolphs et al., 1995). Recent high-resolution functional Magnetic Resonance Imaging (fMRI) studies of the AG provide further support for functional specialization of AG subnuclei (Bach et al., 2011, Boll et al., 2013, Hrybouski et al., 2016, Prévost et al., 2013).
The majority of previous structural MRI volumetric methods for AG measurement have so far only been able to examine this region as a whole (Achten et al., 1998, Bonilha et al., 2004, Convit et al., 1999, Makris et al., 1999, Malykhin et al., 2007, Matsuoka et al., 2003, Pruessner et al., 2000, Watson et al., 1992). For a better understanding of AG subnuclei function, it is crucial to delineate and measure these structures using volumetric MRI. This could only be achieved by developing new methods for measuring the AG subnuclei in-vivo.
Improved spatial resolution and contrast of MRI images due to the continued increase in magnetic field strength have allowed researchers to study very small details of brain anatomy that previously were not visible on conventional MR images (Duyn et al., 2012). Different MRI techniques with high spatial resolution have been developed for high-field MRI magnets to visualize different MTL structures, particularly hippocampal subfields: 3 T (Bonnici et al., 2012, La Joie et al., 2010, Raz et al., 2014, Winterburn et al., 2013), 4 T (Mueller et al., 2007), 4.7 T (Malykhin et al., 2010), 7 T (Boutet et al., 2014, Henry et al., 2011, Goubran et al., 2014, Kerchner et al., 2012, Parekh et al., 2015, Wisse et al., 2014) and 9.4 T (Fatterpekar et al., 2002, Yushkevich et al., 2009). Since visualization of different cell types in-vivo at this resolution is impossible, all methods for segmentation of the hippocampal subfields are based on visibility of the white matter band called the stratum lacunosum-moleculare (SLM) (Duvernoy et al., 2005) and combination of known anatomical landmarks with different geometrical rules to define the boundaries between neighboring substructures (Yushkevich et al., 2015).
Unfortunately, since the AG has low myelin sheath content (Solano-Castiella et al., 2011), visualization of the intra-white matter borders between AG subnuclei using in-vivo MRI is problematic. As a result, various strategies and methods have been utilized to segment the AG as a whole structure or into different subnuclei: manual segmentation (Entis et al., 2012, Prévost et al., 2011), automated segmentation (Morey et al., 2009, Schoemaker et al., 2016), voxel intensity based segmentation (Solano-Castiella et al., 2011), spectral clustering algorithm of diffusion tensor based segmentation (Solano-Castiella et al., 2010), atlas based segmentation (Amunts et al., 2005), diffusion probabilistic tractography based segmentation (Bach et al., 2011, Saygin et al., 2011), and resting-state functional connectivity with cortical regions based segmentation (Bickart et al., 2012).
Despite some limitations of manual segmentation methods, it is still commonly considered the “gold standard” in neuroimaging research (Despotović et al., 2015). In contrast to a large number of protocols for segmentation of hippocampal subfields in-vivo (Yushkevich et al., 2015), there are only a few studies that aimed to delineate the AG subnuclei in humans and report their volumetric measurements. In the first study (Amunts et al., 2005) architectonic probabilistic maps for the BLA, centromedial (CeM) and superficial AG parcellation were developed using ex-vivo histological data from ten individuals. In a second study (Bach et al., 2011), AG was segmented into anterior/inferior/lateral and posterior/superior/medial clusters corresponding to the BLA and the cortico-medial regions respectively. AG subregions were outlined using clustering methods based on differences in the connectivity profile of the clusters. In a third study (Saygin et al., 2011) AG was segmented into La, BA (B and AB), superficial (Co and Me) and Ce nuclei using the differential connectivity patterns of the AG subregions. The last study used high-resolution T1-weighted Magnetization Prepared Rapid Gradient-Echo (MPRAGE) MRI scans to manually segment the BL group (consisting of the La and B nuclei), basomedial, CeM and Co AG using external landmarks and geometrical rules (Entis et al., 2012).
Ultra–high resolution T2-weighted images have not been used so far for manual segmentation of the AG and its subnuclei despite the fact that they provide improved resolution and contrast to delineate MTL structures compared to T1-weighted images (Malykhin et al., 2010, Wisse et al., 2014). Moreover, the accuracy of atlas-based methods crucially depends on the registration methods (Despotović et al., 2015) and atlases registration to the MNI reference space omits individual neuroanatomy differences (Saygin et al., 2011).
Therefore, the main goal of the present study was to develop a reliable volumetric method to delineate five major AG subnuclei groups using ultra-high resolution T2-weighted images acquired with a high-field MRI magnet. The second goal was to provide in-vivo volumetric measurements from the larger cohort of healthy individuals in order to ensure consistency of our AG subnuclei methodology with histological measurements from post-mortem literature.
Section snippets
Study participants
Thirty-eight healthy volunteers without any history of medical or psychiatric disorders were recruited for this study. The sample consisted of 15 males and 23 females, 21–60 years of age, with a mean age of 34.7 years (SD: 12.4). Our participants were all right-handed. Handedness was assessed using a 20-item Edinburgh Handedness Inventory and individuals with laterality quotient ≥ +80 were determined as right-handed (Oldfield, 1971). Participants were excluded if they had any history of
Reliability tests
Both inter/intra-rater ICCs were high (> 0.84) (Table 2). The inter (intra)-rater Dice's Kappa values were above 0.81 for the total AG and the La nucleus which is the largest nucleus of the AG (Table 2). However, for the smaller subnuclei it was somewhat lower (0.71–0.81). The significance level for all ICCs was less than 0.0005.
AG subnuclei volumes
Raw (before ICV correction) and ICV adjusted volumes of the AG and its subnuclei are shown in Table 3, including volumes for the left and the right AG, together with
Discussion
Using ultra-high resolution structural T2-weighted MR images, we developed a reliable segmentation method of the human AG into its five major subnuclei in-vivo for the first time. Our results suggest that consistent measurements of the AG subnuclei can be obtained by experienced MTL image analysts in 2 h. The strength of our method is that it relies on the internal AG landmarks, not arbitrary boundaries, which are often insensitive to individual brain variations. Finally, we provided a
Limitations
4.7 T FSE protocol can provide very high resolution images in relatively short time. However, it is very sensitive to head motion which causes imaging artefacts and therefore has very limited application in individuals with reduced cooperation. Current acquisition protocol was designed with the main goal of the visualization of hippocampal subfields in-vivo across the entire hippocampal formation regardless of the brain size (9 cm coverage) in a reasonable scan time (13.5 min). Achieving higher
Acknowledgments
This work was supported by the Canadian Institutes of Health Research (CIHR MOP 115011 and MOP 111049 to NM). We thank Stanislau Hrybouski for assisting with Dice's Kappa calculations and Melanie MacGillivray and Scott G. Travis for editing the manuscript.
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