2.1.1. Region of Interest (ROI) Masks

All binary ROIs had a 1 mm³ isotropic voxel size and were oriented in the Montreal Neurological Institute (MNI) space as the processed T1 (GM + WM) images. The six ROI masks were obtained by previously published atlases. The technical details and theoretical justifications for the specific ROI definitions are described in the following section for each neuromodulator seed. Further details are provided in the Supplementary Materials.

Accurate MRI localization of the LC in the human brain is still lacking wide-spread agreement [34]. In the last few years, several probabilistic maps of the LC have been released; however, these probabilistic maps are inconsistent in both localization and volume extent within the MNI space. Indeed, different sample sizes have been recruited and this exacerbates the limitations due to different methodologies involved. These differences reflect a large anatomical variability of the samples scanned, suggesting that the LC varies across the general population.

In order to perform volumetric analyses appropriate to the present research and to attempt to resolve these differences, it was necessary to define a common space that included all the previous maps as to increase the likelihood of inclusion of the entirety of the LC, given the probable increase in between-subject anatomical variability in the present ADNI populations (n = 395 HC, n = 156 MCI, n = 135 AD).

Therefore, a new symmetrical “omni-comprehensive” LC mask in the MNI space was created in order to include the whole LC rostro-caudal extent (see Figure 1). Indeed, it was observed that with increasing age, the LC signal intensity tends to shift from the rostral to the caudal portion [34,102]. This process might be influenced by manifold variables, such as ageing, the degree of biological brain maintenance and even dementia progression, which is likely to exacerbate this “caudal-shifting” process. Moreover, it is acknowledged how the noradrenergic system is susceptible to compensatory changes across the brain involving the caudal portion of the LC and peri-coeruleus/LC-peri-dendritic regions (Epi-coeruleus and Sub-coeruleus) [14,54,55,61,103,104,105,106]. Therefore, a larger area rather than a very specific and concise region would be more informative and appropriate while investigating the LC–NA system on different groups, particularly known the heterogeneity of Alzheimer’s disease. The new “omni-comprehensive” LC mask included the six LC MNI atlases previously published: (1) Keren et al. (2009) [102,107], (2) Tona et al. (2017) [108], (3) Betts et al. (2017) [109], (4) Dahl et al. (2019) [33], (5) Liu et al. (2019) [110] and (6) Rong Ye et al. (2020) [111] without encroaching the median raphe (MR) and the dorsal raphe (DR) defined by Beliveau et al. (2015) [112] and the cerebellar white matter. Additionally, the new created LC “omni-comprehensive” mask included the LC meta-mask developed by Dahl et al. (2021) [70], but with a larger and symmetrical rostro-caudal extent to avoid induced lateralization biases in the analyses. Indeed, as pointed out by Betts et al. [109], the LC asymmetries reported in certain MRI studies could be caused by MRI biases of how radiofrequencies are transmitted and received in the scanner. In fact, post-mortem histological studies consistently revealed symmetrical distribution of LC cells [14,113,114,115,116,117].

The spatial resolution of the new created symmetrical “omni-comprehensive” LC mask in comparison with the previously published LC MRI atlases and masks.

MR and DR 1 mm³ MNI masks were generously provided by Beliveau et al. (2015) [112]. The probabilistic maps and masks were obtained by analysing 232 PET scans matched with high-res 3T structural MRI of healthy subjects between 18 and 45 years old.

The VTA mask was obtained by downloading the VTA MNI probabilistic map from the atlas made by Pauli et al. 2018 [118] from the NeuroVault website (https://neurovault.org/ accessed on 15 December 2018). The atlas was made using the MRI data from the Human Connectome Project (HCP) and was derived from a selected sample size of 168 healthy subjects between 22 and 35 years old. The localisation of the substantia innominata (SI)/NBM was more controversial than the previous nuclei, as there are no specific maps available in MNI space. Albeit, probabilistic MNI maps of the acetylcholine cells of the Forebrain are provided by SPM Anatomy Toolbox 2.2c (https://www.fzjuelich.de/inm/inm1/EN/Forschung/_docs/SPMAnatomyToolbox/SPMAnatomyToolbox_node.html accessed on 15 December 2018) [119]. However, the probabilistic map referring to the SI/NBM (4ch.nii) defined by Zaborszky et al. (2008) [119] overlaps several subcortical nuclei delineated in other atlases [118,120,121,122,123,124,125,126]. Therefore, the “4ch.nii” was used as main reference to delineate the SI/NBM, but was adjusted by excluding the subcortical nuclei identified by other atlases while accounting for the probabilistic localisation of the SI/NBM delineated in previous works [81,82,127,128,129]. In order to control for a “neuromodulator-free” brainstem’s region, a squared binary ROI not referring to any anatomical nuclei was drawn in the ventro-rostral portion of the pons. A greater number of voxels were used in order to obtain a control region similar in voxel-size well-suited for the detection of false positives. The six ROIs are displayed in Figure 2 below.

The neuromodulators’ seeds and the neuromodulator-free control region investigated in the VBM analyses. Blue: Locus Coeruleus (LC—“omni-comprehensive”)—Noradrenaline (NA)—(714 voxels). Red: Dorsal Raphe Nuclei (DR)—Serotonin (5HT)—(174 voxels). Orange: Median Raphe Nuclei (MR)—Serotonin (5HT)—(108 voxels). Green: Ventral Tegmental Area (VTA)—Dopamine (DA)—(252 voxels). Purple: Nucleus Basalis of Meynert (NBM)—Acethylcholine (Ach)—(492 voxels). White: Control Pontine ROI (“neuromodulator-free” region)—(906 voxels). All the ROI binary masks were symmetrical with a 1 mm3 isotropic voxel size, and there were no overlapping boundaries between the masks. Image (a) displays the anatomical localization of the six ROIs in sagittal view of the Cerebellum, Brainstem and Diencephalon. Image (b) displays the coronal view. Image (c) shows a fronto-lateral view of the whole brain and the anatomical localization of the six ROIs.