The locus coeruleus (LC) is a small noradrenaline (NA)-producing structure located in the pons of the brainstem. Projections from the LC are widespread, with some LC neurons projecting down the spinal cord and some projecting into the forebrain. In the forebrain, the LC is the sole source of NA (Moore and Bloom, 1979) and acts as an important neuromodulator; it is believed to modulate the gain or responsivity of processing in cortical circuits critical for performance on decision making, sensorimotor processing, learning and memory (Aston-Jones and Cohen, 2005; Foote et al., 1991).

Despite its small size, the LC can be distinguished in the brainstem from its hyperintense contrast relative to the surrounding gray matter on a magnetization transfer (MT) prepared fast spin echo pulse sequence (Sasaki et al., 2006). This high contrast has historically been attributed to the neuromelanin that accumulates in LC cell bodies across the lifespan, as post-mortem histological analyses have confirmed that the high contrast seen in the LC in MRI corresponds to neuromelanin that has accumulated in tyrosine-hydroxylase (TH, a marker for catecholaminergic neurons) immunopositive LC neurons (Keren et al., 2015). However, more recent evidence suggests that the high MRI contrast in the LC is not solely from neuromelanin but also reflects the large size of LC neurons. The large cell size results in a large intra-cellular free water proton pool and lower macromolecular-to-free-water fraction within the LC that produces a brighter signal (Priovoulos et al., 2020; Trujillo et al., 2019; Watanabe, 2023; Watanabe et al., 2019).

While histologically-defined cell loss and MR-related structural integrity changes in the LC in diseases like Alzheimer’s and Parkinson’s diseases are well documented(Chu et al., 2022; Jacobs et al., 2021; Jacobs et al., 2023; Ohtsuka et al., 2014; Takahashi et al., 2015; Theofilas et al., 2017), how the LC is affected by normal aging remains somewhat controversial. A number of studies have utilized LC contrast or signal intensity as a measure of structural integrity, and these investigations of integrity of the LC in older adults have yielded mixed results. LC signal intensity is higher in older adults compared to young adults (Clewett et al., 2016, Keuken et al., 2017, Trujillo et al., 2019) and has been proposed to be due to increases in neuromelanin or changes in LC volume throughout the lifespan. Older adults generally show greater variance in LC signal intensity (Betts et al., 2017), and some studies report increases in signal intensity across the lifespan until around 60 years, and then significant decreases in older individuals (Liu et al., 2019; Shibata et al., 2006). Alternatively, some have posited that declines in LC contrast are observed only when tau or amyloid pathology is present (Jacobs et al., 2021). In older adults, reductions in LC MRI contrast are associated with lower cortical thickness in parietal, frontal, and occipital regions (Bachman et al., 2021) and lower arborization complexity in the LC, as indexed by lower orientation dispersion index (ODI) metrics, is associated with lower arborization complexity in frontotemporal regions (Beckers et al., 2024). Functional connectivity of the LC with these different cortical networks is integral for attentional and memory performance. For example, older adults show reduced functional connectivity between the LC and the salience network than do younger adults. This reduction in functional connectivity is associated with limited attentional and executive control responses (Lee et al., 2020; Neal et al., 2023; Pahl et al., 2024).

More recently, advances in neuroimaging methods have allowed for the analysis of LC projections to the forebrain through diffusion imaging and tractography (Solders et al., 2022; Sun et al., 2020). LC neurons are poorly myelinated, but analysis along the LC ascending projections to the forebrain that join the myelinated central tegmental tract (CTT) enables tractography analysis. One study revealed that older adults show a reduction in mean and radial diffusivity (MD, RD) in the CTT compared to younger adults (Langley et al., 2022). Another group also examined diffusivity not only within the LC-CTT projections but within the LC itself; they reported higher fractional anisotropy (FA) in the nucleus itself but lower FA in the ascending tract (Porat et al., 2022). Changes in diffusion MRI metrics are sensitive but not specific and may correspond to altered myelin, increased inflammation, axon structural changes, or any other cellular alteration that influences water diffusion within the voxels within the tract (Alexander et al., 2007; Jones et al., 2013; Stikov et al., 2011). In vivo MRI studies using LC tractography are informative, but are limited by spatial resolution and image quality challenges in the brainstem. Ex vivo MRI or MRI microscopy provides an important complementary approach as it allows full brain or more local coverage and has greater sensitivity due to longer scan times, smaller coils, and lack of movement artifacts (Mackenzie-Graham, 2012). The benefits of ex vivo MRI are especially apparent in deep brain regions such as the white matter projections of the brainstem, which are traditionally very difficult to resolve. Ex vivo MRI scans are also high quality and enable the development and application of quantitative mapping, including relaxometry based metrics such as T1, T2, and T2* values or related modeled metrics such as myelin water fraction (MWF) and bound pool fraction (BPF) (Does, 2018). Diffusion MRI for microstructural mapping can also be applied using ex vivo techniques, from conventional diffusion tensor imaging (DTI) to more advanced frameworks such as diffusion kurtosis imaging (DKI) and mean apparent propagator MRI (MAP-MRI) (Basser et al., 1994; Jensen et al., 2005; Özarslan et al., 2013).

A major caveat in many of the aforementioned MRI studies of human LC and LC projections during aging is the characterization of “normal agers”. In diseases such as Alzheimer’s and Parkinson’s diseases, pathological burden precedes cognitive and behavioral symptoms by many years, meaning that many older adults who appear to be “cognitively normal” may already have disruptions in brain networks such as the LC. To address the confounds of undiagnosed comorbidities on changes in LC microstructure over time, it is necessary to corroborate some of these findings in a true normative aging population. Nonhuman primates such as bonnet macaques are excellent models for this purpose, as they share many anatomical features with humans but do not spontaneously succumb to diseases like Alzheimer’s disease (e.g.,(Barnes et al., 2024, Walker and Jucker, 2017). In light of these advantages, we imaged fixed whole brain specimens from bonnet macaques with high quality MRI microstructural imaging and multivariate processing to generate comprehensive mappings of the LC and LC ascending projections using advanced imaging techniques. The goal of this paper is to determine both the microstructural environment and composition of the LC and its projections along the CTT in normative aging, and interpret these findings with respect to neurodegeneration, demyelination, and inflammation among other cellular changes.