## 2 Materials and Methods  
This review was designed in accordance with the PRISMA 2020 recommendations for systematic reviews, considering its adapted application to short review studies. The objective of this study was to provide a comprehensive overview of the current state-of-the-art and novel findings concerning the utilization of DTI and tractography for the investigation of SN connectivity in humans. A systematic search for relevant literature was performed in March 2025 in the PubMed, Web of Science, and ScienceDirect databases. Furthermore, the SciSpace tool was utilized, and the outputs were subjected to a rigorous process of validation to ensure their relevance and appropriateness for inclusion. The temporal scope of the search covered articles published up to March 2025, with the aim of including recent developments in the field.  
The following were included in the review: studies conducted on human subjects (including healthy volunteers and clinical populations), studies using DTI or tractography methods to investigate SN connectivity, and articles published in any language. Studies performed in animal models, post-mortem studies (ex vivo imaging), articles that did not make a relevant contribution to the topic of connectivity, and abstracts without available full text were excluded from the analysis.  
A single reviewer conducted the screening and selection process for the articles. The initial phase of this process involved searching for articles based on keywords (substantia nigra, tractography, connection) and evaluating the titles and abstracts. Following this preliminary assessment, a comprehensive full-text examination was performed.  
In the first step, a total of 150 records were identified, from the following databases: PubMed (\(n\) = 38), Web of Science (\(n\) = 28), ScienceDirect (\(n\) = 67) and SciSpace (\(n\) = 17).  
Figure 1: (**A**) Tractographic visualization of fiber pathways originating from the substantia nigra, derived from real diffusion MRI data. (**B**) Schematic representation of the nigrothalamic system. Reproduced from [PERSON] et al., 2025 [28]. Efferent connections of the SNc to the dorsal striatum (caudate and putamen) (**left**) and to thalamic nuclei (MD, IL, LP) (**right**), together with nigro-cortical fibers (dashed black line). Direct, indirect and hyperdirect pathways are represented on the left side. C, caudate; Pu, putamen; MD, medio dorsal nucleus; IL, intralaminar nuclei; LP, latero posterior nucleus; GPe, external globus pallidus; GPI, internal globus pallidus; STN, subthalamic nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata.  
Following the removal of duplicates (30 records), 120 articles were included in the preliminary screening.  
Following an evaluation of the titles and abstracts, 75 records were excluded based on the established criteria listed above. The primary reasons for exclusion included a lack of focus on SN, an absence of DTI methods, and overall irrelevance to the subject matter.  
A comprehensive full-text screening process was conducted on the remaining 45 articles, resulting in the exclusion of further 13 studies, primarily due to their emphasis on animal models, post-mortem data, or an indirect relevance to the subject matter.  
The remaining 32 studies were selected for the final review. In consideration of the nature of the review, it was deemed inappropriate to apply a formal tool for the purpose of assessing the quality of the included studies. The primary objective of this study was to produce a qualitative map of the predominant trends and novel methodological approaches in the domain of SN connectivity, utilizing DTI.