Brain Structure and Function (2020) 225:1055–1071 https://doi.org/10.1007/s00429-020-02058-x ORIGINAL ARTICLE Ex vivo difusion‑weighted MRI tractography of the Göttingen minipig limbic system Johannes Bech1 · Dariusz Orlowski1 · Andreas N. Glud1 · Tim B. Dyrby2,3 · Jens Christian H. Sørensen1 · Carsten R. Bjarkam4 Received: 25 September 2019 / Accepted: 18 March 2020 / Published online: 3 April 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020 Abstract The limbic system encompasses a collection of brain areas primarily involved in higher cognitive and emotional processing. Altered function in the limbic circuitry may play a major role in various psychiatric disorders. This study aims to provide a high-quality ex vivo difusion-weighted MRI (DWI) tractographic overview of the Göttingen minipig limbic system pathways, which are currently not well described. This may facilitate future translational large animal studies. The study used previously obtained post-mortem DWI scans in 3 female Göttingen minipigs aging 11–15 months. The tractography performed on the DWI data set was made using a probabilistic algorithm, and regions of interest (ROIs) were defned in accordance with a histological atlas. The investigated pathways included the fornix, mammillothalamic tract, stria terminalis, stria medullaris, habenulo-interpeduncular tract, and cingulum. All the investigated limbic connections could be visualized with a high detail yielding a comprehensive three-dimensional overview, which was emphasized by the inclusion of video material. The mini- pig limbic system pathways displayed using tractography closely resembled what was previously described in both human studies and neuronal tracing studies from other mammalian species. We encountered well-known inherent methodological challenges of tractography, e.g., partial volume efects and complex white matter regions, which may have contributed to derouted false-positive streamlines and the failure to visualize some of the minor limbic pathway ramifcations. This under- lines the importance of preexisting anatomical knowledge. Conclusively, we have, for the frst time, provided an overview and substantial insight of the Göttingen minipig limbic system. Keywords Cingulum · Fornix · Mammillothalamic tract · Stria medullaris · Stria terminalis · Sus scrofa Introduction Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0042 9-020-02058 -x) contains supplementary material, which is available to authorized users. The limbic system has been subjected to great interest as well as puzzlement, since Paul Broca described “le grand * Johannes Bech [email protected] lobe limbique” (Broca 1877). The strive to understand and identify the primary brain areas responsible for emotions 1 CENSE, Department of Neurosurgery, Department and higher cognition continued with Papez’ circuit of emo- of Clinical Medicine, Aarhus University Hospital, Aarhus tion (Papez 1937) and the later studies of Yakovlev (1948) University, Palle Juul-Jensens Boulevard 165, Entrance J, 8200 Aarhus N, Denmark and Maclean (1949, 1952). From an evolutionary perspec- tive at that time, however , a segregation of cognition and 2 Danish Research Centre for Magnetic Resonance, Center for Functional and Diagnostic Imaging and Research, emotion was present with the latter being deemed a more Copenhagen University Hospital Hvidovre, 2650 Hvidovre, primitive trait that could be ascribed to the limbic system, Denmark whereas more intellectual functions of mammalian spe- 3 Department of Applied Mathematics and Computer Science, cies were related to the neocortical areas (LeDoux 2000). Technical University of Denmark, 2800 Kongens Lyngby, Since then, the concept has evolved further; however, there Denmark is currently no broad scientifc consensus on which brain 4 Department of Neurosurgery, Department of Clinical structures constitute the limbic system, and some have even Medicine, Aalborg University Hospital, 9100 Aalborg, discussed if the term should be abandoned (Kotter and Denmark Vol.:(0123456789)1 3 1056 Brain Structure and Function (2020) 225:1055–1071 Stephan 1997). However, neuroscientifc research is increas- Tractography, derived from difusion-weighted mag- ingly continuing to both explore and understand the neuronal netic resonance imaging (DWI), is a non-invasive method circuitry and mechanisms underlying, e.g., memory, spatial used to investigate white matter structures and connectiv- orientation, emotion, fear, reward, and various psychiatric ity within the brain. It exploits the anisotropic difusion of conditions related to limbic brain areas (Catani et al. 2013; water molecules in biological tissues that provide insight Murray 2007; Mayberg 1997; LeDoux 2000; Aggleton and to the underlying white matter anatomy (Basser et al. Brown 1999; Seo et al. 2018; Gomes et al. 2018). Thus, 1994). Previously, tractography has been used in human treatment-resistant depression has been linked to a patholog- studies of limbic white matter tracts, but has been reported ical activity within limbic–cortical circuits, previously mod- to sufer from low resolution and partial volume efects ulated using deep-brain stimulation (DBS) of the subgenual (Concha et al. 2005; Kamali et al. 2016), where the lat- cingulate white matter (Mayberg et al. 2005). Likewise, a ter results from diferent tissue properties averaged within pathological information fow between the amygdala and one voxel. As this can reduce the anisotropy within, for the accumbens nucleus has been suggested to be involved instance, the juxtaventricular voxels, streamlines may ter- in anxiety disorders and obsessive–compulsive disorder minate prematurely. The use of ex vivo DWI has, however, (OCD), also potentially treatable with DBS (Sturm et al. yielded high-quality data (Dyrby et al. 2011) with a higher 2003; Huf et al. 2010). Other white matter abnormalities resolution than can normally be obtained in human clinical have been observed in patients sufering from bipolar disor- settings and an advantageous signal-to-noise ratio. Moreo- der or schizophrenia, where tractography disclosed altered ver, current advanced models have addressed the previous connectivity in the anterior thalamic projections and the issues with resolving fbers that traverse complex white uncinate fasciculus (McIntosh et al. 2008). Another white matter regions using multi-fber reconstruction methods matter structure, the habenulo-interpeduncular pathway (fas- and probabilistic algorithms (Tournier et al. 2010, 2012; ciculus retrofexus), has been linked to nicotine addiction Behrens et al. 2007), and tractography has achieved great (Antolin-Fontes et al. 2015). These examples indicate the accordance with the “gold standard” method of display- importance and relevance of understanding the pathological ing neuronal connectivity, i.e., invasive neuronal tracing mechanisms involving limbic structures. To this end, trans- (Bech et al. 2018), although recently it has been discussed lational studies are generally valuable, since they permit the if invasive neuronal tracing is becoming more of a “sil- use of invasive methods, various research setups, and post- ver standard” (Dyrby et al. 2018) with new emerging mortem analyses (Glud et al. 2011; Sorensen et al. 2011). To techniques. perform such studies, it is, however, a prerequisite to have This study aims to investigate and describe the neuro- the sufcient prior anatomical understanding. anatomy of the major pathways within the Göttingen minipig The Göttingen minipig is an excellent candidate for trans- limbic system by the use of high-quality ex vivo tractogra- lational large animal models due to several factors. It has a phy. These limbic system pathways have not yet been well human-resembling physiology, and a relatively large gyren- described in the minipig to the authors’ best knowledge. We cephalic brain relative to its body size, which still permits hope to provide a new anatomical insight in this animal. the use of clinical surgical equipment as well as clinical Our data will be compared with previous neuronal tracing scanners (Bjarkam et al. 2008; Lind et al. 2007; Fang et al. data from other species for evaluation. Moreover, we wish 2012; Dolezalova et al. 2014; Orlowski et al. 2017). Moreo- to provide a spatially comprehendable three-dimensional ver, the minipig is easily handled and can be trained for both overview of this system’s anatomy through the inclusion of neurologic testing and behavioral analyses (Glud et al. 2010; video material. Such spatial understanding may otherwise be Christensen et al. 2018; Lillethorup et al. 2018), and it is a difcult to obtain merely through viewing two-dimensional more economical and ethically feasible animal model than histological sections. In doing this, we wish to make the non-human primates (Bjarkam et al. 2009; Sorensen et al. connectivity within this complex system easier to recognize. 2011). The neuroanatomical insight and histological knowl- By increasing the neuroanatomical knowledge of the minipig edge of the minipig have increased over the recent years, and limbic system, we hope to facilitate future translational stud- several structures and features are already well described ies on the limbic circuitry in health and disease in porcine including the telencephalon (Bjarkam et al. 2017a), the large animal models. accumbens nucleus (Meidahl et al. 2016), the corticospinal tract (Bech et al. 2018), and the hypothalamus (Ettrup et al. 2010), as well as