23Evaluation of brainstem pathways with diffusion tensor imaging (DTI) and tractography 24 may provide insights into pathophysiologies associated with dysfunction of key brainstem 25 circuits. However, identification of these tracts has been elusive, with relatively few in vivo 26 human studies to date. In this paper we proposed an automated approach for reconstructing nine 27 brainstem fiber trajectories of pathways that might be involved in pain modulation. We first 28 performed native-space manual tractography of these fibers in a small normative cohort of 29 participants and confirmed the anatomical precision of the results using existing anatomical 30 literature. Second, region-of-interest pairs were manually defined at each extracted fiber's 31 termini and nonlinearly warped to a standard anatomical brain template to create an atlas of the 32 region-of-interest pairs. The resulting atlas was then transformed non-linearly into the native 33 space of 17 veteran patients' brains for automated brainstem tractography. Lastly, we assessed 34 the relationships between the integrity levels of the obtained fiber bundles and pain severity 35 levels. Fractional anisotropy (FA) measures derived using automated tractography reflected the 36 respective tracts' FA levels obtained via manual tractography. A significant inverse relationship 37 between FA and pain levels was detected within the automatically derived dorsal and medial 38 longitudinal fasciculi of the brainstem. This study demonstrates the utility of DTI in exploring 39 brainstem circuitries involved in pain processing. In this context, the described automated 40 approach is a viable alternative to the time-consuming manual tractography. The physiological 41 and functional relevance of the measures derived from automated tractography is evidenced by 42 their relationships with individual pain severities. 3 43 44 48 regulation, sleep and alertness, pain, posture, mood, and mnemonic functions. It is crucial to 49 understand how structural changes in small brainstem regions and circuitries may cause/alter 50 pathologies. 51 Studies of brainstem substructures using anatomical brain MRI have been complicated by 52 difficulties in detecting neuronal loss due to lack of sufficient contrast to delineate small internal 53 substructures in intensity-based images. Diffusion tensor imaging (DTI) is a noninvasive MRI 54 imaging technique that measures changes of water diffusion in white matter microstructures. 55 Fractional anisotropy (FA), one of the standard DTI indices, is known to be sensitive to detect 56 damages in orientationally organized structures (e.g. white matter fibers). Furthermore, based on 57 computing the directional information in each voxel of DTI, tractography is used to reconstruct 58 trajectories of white matter tracts that correspond to known neuroanatomy in 3-dimensional 59 space [1,2]. Diffusion tensor tractography has orientation-based contrasts and thus permits not 60 only the anatomical illustration of neural pathways, but also examines the ...