In vivo quantification of amygdala subnuclei using 4.7 T fast spin echo imaging

Highlights

• The amygdala is an almond-shaped heterogeneous structure located in the medial temporal lobe.

• It is crucial to delineate and measure amygdala subnuclei using volumetric MRI.

• Ultra-high resolution MRI methods are needed to study amygdala subnuclei in-vivo

• Amygdala subnuclei can be measured reliably by a well-trained rater.

• Basolateral complex is the largest subnuclei group in the amygdala

Abstract

The amygdala (AG) is an almond-shaped heterogeneous structure located in the medial temporal lobe. The majority of previous structural Magnetic Resonance Imaging (MRI) volumetric methods for AG measurement have so far only been able to examine this region as a whole. In order to understand the role of the AG in different neuropsychiatric disorders, it is necessary to understand the functional role of its subnuclei. The main goal of the present study was to develop a reliable volumetric method to delineate major AG subnuclei groups using ultra-high resolution high field MRI.

38 healthy volunteers (15 males and 23 females, 21–60 years of age) without any history of medical or neuropsychiatric disorders were recruited for this study. Structural MRI datasets were acquired at 4.7 T Varian Inova MRI system using a fast spin echo (FSE) sequence.

The AG was manually segmented into its five major anatomical subdivisions: lateral (La), basal (B), accessory basal (AB) nuclei, and cortical (Co) and centromedial (CeM) groups. Inter-(intra-) rater reliability of our novel volumetric method was assessed using intra-class correlation coefficient (ICC) and Dice's Kappa.

Our results suggest that reliable measurements of the AG subnuclei can be obtained by image analysts with experience in AG anatomy. We provided a step-by-step segmentation protocol and reported absolute and relative volumes for the AG subnuclei. Our results showed that the basolateral (BLA) complex occupies seventy-eight percent of the total AG volume, while CeM and Co groups occupy twenty-two percent of the total AG volume. Finally, we observed no hemispheric effects and no gender differences in the total AG volume and the volumes of its subnuclei.

Future applications of this method will help to understand the selective vulnerability of the AG subnuclei in neurological and psychiatric disorders.

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.