Received: 19 March 2020 | Accepted: 24 July 2020 DOI: 10.1111/ahe.12604 ORIGINAL ARTICLE Connections between the internal and the external capsules and the globus pallidus in the sheep: A dichromate stain X-ray microtomographic study Jorge Alfonso Murillo-González1 | Belen Notario2 | Estela Maldonado1 | Elena Martinez-Sanz1 | M. Carmen Barrio1 | Manuel Herrera1 1Department of Anatomy and Embryology, Faculty of Medicine, Complutense Abstract University of Madrid, Madrid, Spain Sheep are recognized as useful species for translational neurodegeneration research, 2 Microcomputed Tomography Lab, Centro in particular for the study of Huntington disease. There is a lack of information re- Nacional de Investigación sobre la Evolución Humana, CENIEH, Burgos, Spain garding the detailed anatomy and connections of the basal ganglia of sheep, in nor- mal myeloarchitectonics and in tract-tracing studies. In this work, the organization of Correspondence Belen Notario, Microcomputed Tomography the corticostriatal projections at the level of the putamen and globus pallidus (GP) are Laboratory, Centro Nacional de explored. For the first time, the myeloarchitectonic pattern of connections between Investigación sobre la Evolución Humana (CENIEH), Paseo Sierra de Atapuerca 3, the internal (IC) and the external (EC) capsules with the GP have been investigated Burgos 09002, Spain. in the sheep. Formaldehyde-fixed blocks of the striatum were treated with a metal- Email: [email protected] lic stain containing potassium dichromate and visualized using micro-CT (µ-CT). The trivalent chromium (Cr3+), attached to myelin phospholipids, imparts a differential contrast to the grey and white matter compartments, which allows the visualization of myelinated fascicles in µ-CT images. The fascicles were classified according to their topographical location in dorsal supreme fascicles (X, Y, apex) arising from the IC and EC; pre-commissurally, basal fascicles connecting the ventral part of the EC with the lateral zone of the ventral pallidum (VP) and, post-commissurally, superior (Z1), middle (Z2) and lower (Z3) fascicles, connecting at different levels the EC with the GP. The results suggest that the presumptive cortical efferent and afferent fibres to the pallidum could be organized according to a dorsal to ventrolateral topography in the sheep, similar to that seen in other mammals. The proposed methodology has the potential to delineate the myeloarchitectonic patterns of nervous systems and tracts. KEYWORDS basal ganglia, external capsule, internal capsule, putamen, sheep, X-ray microcomputed tomography 1 | INTRODUCTION extensive neuronal loss and astrogliosis (for a review, see Reiner, Dragatsis, & Dietrich, 2011). Sheep are recognized as useful species for translational neuro- The basal ganglia anatomy of sheep is similar to primates, but degeneration research, and in particular for Huntington disease there is a lack of studies describing their detailed anatomy and con- (Handley et al., 2016; Jacobsen et al., 2010; Pfister et al., 2018; nections. Studies that give anatomical information about the normal Reid et al., 2013; Van der Bom et al., 2013). In this neurological dis- appearance of basal ganglia subdivisions in sheep have been con- ease, the basal ganglia are dramatically affected by gross atrophy, ducted using MRI (Ella, Delgadillo, Chemineau, & Keller, 2017; Ella & 84 | © 2020 Wiley-VCH GmbH wileyonlinelibrary.com/journal/ahe Anat Histol Embryol. 2021;50:84–92. MURILLO-GONZÁLEZ ET AL. | 85 Keller, 2015; O´Conell et al., 2019); this is in addition to neurochemi- 2 | MATERIALS AND METHODS cal studies (Nelson, Marton, & Saper, 1993), including those focused on the substantia nigra (Murray et al., 2019). There are no sheep data The brains of five male lambs and five adult domestic sheep (Ovis relating to the corticostriatal projections and their termination in the orientalis aries) were used for this study. The samples belonged to striatum, using either tracer substances, MRI connectivity stud- the Department of Veterinary Sciences of our Institution and were ies or myelin staining techniques. It is widely known that the main obtained from the official slaughterhouses of the Autonomous input from the cortex to the striatum is the caudate-putamen and Community of Madrid following the required legal procedures. After the nucleus accumbens (Carman, Cowan, Powell, & Webster, 1965; skull excision in two halves, the samples were fixed in 4% formalde- Carpenter, 1976; Kemp & Powell, 1970; Nauta, 1979; Parent & hyde-1% calcium chloride (CaCl2) (pH = 5.5–6.0) for 24 hr. The brains Hazrati, 1995; Ranson, Ranson, & Mary Ranson, 1941). These fi- were then cut in 5 mm slabs and fixed in the same fixative for two bres arrive at the striatum via the IC and EC (Carman et al., 1965; weeks at 18–22ºC. Then, the slabs were washed for 24 hr in bidis- Cowan & Powell, 1966; Sych, 1960), which are in close relationship tilled water with 2% CaCl2 (pH = 6.8) and immersed progressively to the putamen. Connections between the internal capsule (IC), ex- in 1% to 4% potassium dichromate (K2Cr2O7, DK)-1% CaCl2 aque- ternal capsule (EC) and the pallidum (globus pallidus, GP) have been ous solutions in intervals of 96 hr. Finally, the slabs were immersed described by several authors (Berke, 1960; Carman et al., 1965; in a 1% CaCl2-5% DK solution (pH = 3.9) for five weeks at labora- Carpenter, 1976; Déjerine & Déjerine-Klumpke, 1895; Nauta, 1979; tory temperature (18–23°C). After the staining period, the samples Sych, 1960). were washed in bidistilled water until they did not release any more The main output source from the putamen is the GP (Cowan & DK, transferred to a cylindrical µ-CT sample holder and scanned in Powell, 1966; Graybiel & Ragsdale, 1975; Hazrati & Parent, 1992; wet cotton. For a more detail description of the methods used, see Nauta, 1979). These fibres cross the putamen through the pal- Herrera et al., (2018). lidal segments, forming “Wilson´s pencils” (Horn et al., 2019; Wilson, 1914). Projections from the GP to the striatum have been described (Voorn, 2010). 2.1 | CT scanning and analysis The use of micro-CT (µ-CT) has been recommended to charac- terize microstructural features of bone and soft tissues to obtain, The analyses were performed using a V|Tome|X s 240 µ-CT from GE in a non-destructive manner, accurate 2D images in invertebrate Sensing & Inspections Technologies (GE, Hürth, Germany). A final and vertebrate samples (de Bournonville, Vangrunderbeeck, & filament voltage of 50 KV, a current of 500 µA, and an acquisition Kerckhofs, 2019; de Crespigny et al., 2008; Descamps et al., 2014; time of 500 ms were used in all cases. Typically, 700 images covering 3 Gignac & Kley, 2014; Heimel et al., 2019; Koç, Aslan, Kao, & 360º, with a voxel size of 10 × 10 × 10 µm , were scanned. Output Barber, 2019; Metscher, 2009; Pauwels, Van Loo, Cornillie, Brabant, images were then processed with a median and a ring artefact filter & Van Hoorebeke, 2013; Rüegsegger, Koller, & Müller, 1996; Shearer, to improve image contrast and to reduce noise. Bradley, Hidalgo, Sherratt, & Cartmell, 2016; Swain & Xue, 2009). In all the samples stained with DK, the white matter generally In veterinary research, µ-CT studies have mainly focused on the showed high X-ray absorption, while grey matter showed moderate locomotor apparatus and associated diseases (Dedrick et al., 1993; absorption (Figure 1). The identification of myelinated bundles in Horbal, Smith, & Dixon, 2019; Lill, Gerlach, Eckhardt, Goldhahn, & the images is the result of the reduced trivalent chromium (Cr3+) Schneider, 2002; Salmon, 2020). bounded to myelin phospholipids. Three-dimensional reconstruc- In the last decade, important advances have been made visual- tions of the bundles were not made due to the intrinsic inhomoge- ization of the nervous system using contrast-enhanced techniques neities given by its grey values, as the consequence of the spatial with an X-ray source (deCrespigny et al., 2008; Gignac & Kley, 2018; resolution obtained. Therefore, the observations are based on com- Metscher, 2009), permitting visualization of the grey and white mat- plete sequences of 2D-images. ter compartments of the brain. However, the majority of the media used are non-specific for the white matter compartment, with the exception of osmium tetroxyde (Metscher, 2009) and potassium di- 3 | RESULTS chromate (Herrera, Notario, Barrio, Metscher, & Murillo, 2018). The use of µ-CT methods, using contrast-enhanced media, to facilitate Dichromate-stained samples showed myelin-rich structures with a study of the normal myeloarchitecture in animals can facilitate the high-to-medium white signal, whereas the cortical and nuclear areas extrapolation of experimental results to human studies. In the pres- were identified with a medium-to-low grey signal (Figure 1a and b). ent study, the myeloarchitectonic pattern of connections between At the level of the striatum, all the relevant structures studied were the IC, EC and the GP are shown using the combination of a my- recognized, as well as the myelinated tracts and fascicles that they elin-specific stain and an X-ray tomographic system. The arrange- surround or traverse (Figure 1b). ment of myelin fascicles that connects the EC and the IC with the GP The zone studied was from below the foot of the corona radiata is demonstrated, and the results obtained are compared with those to the basal part of the putamen (Figure 1a). The striatum was di- previously described in mammals using