Connections Between the Internal and the External Capsules and the Globus Pallidus in the Sheep: a Dichromate Stain X-Ray Microtomographic Study

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

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 normal 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 &

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 special histological methods. vided into pre-commissural and post-commissural regions, according 86 | MURILLO-GONZÁLEZ et al.

(a) (b) (a) (b) (c)

(d) (e) (f)

FIGURE 1 Frontal two-dimensional images stained with the dichromate metallic stain and scanned under a laboratory-based µ-CT. (a) Overview of the striatum and adjacent regions at the level of the amygdaloid nuclei and the temporal cortex. Abbreviations: Am, amygdaloid region; AV, anteroventral thalamic nucleus; CN, caudate nucleus; CP, cerebral peduncle; CR, radiate crown; EC, external capsule; IC, internal capsule; EXT, extreme capsule; thalamus, LME, lamina medullaris externa; Pd, pallidal region; Put, FIGURE 2 Dichromate-stained CT frontal images from the putamen; Ret, reticular nucleus; TC, temporal cortex; VA, ventral µ middle to posterior parts of the putamen-pallidum complex. (a) anterior nucleus. (b) Detail of a dichromate-stained image at the Frontal view showing the medial and lateral sides of the external level of the lateral amygdaloid nucleus (Lat Amyg). The putamen (EC) and internal (IC) capsules and the IC-EC transitional zone (black nucleus (Put) is crossed by the myelinated fibres originating in the arrow). (b) Fascicles Y and X of the transitional zone on their way to external capsule (EC) or the pallidal region (Pd). The internal (IC) and the globus pallidum. (c) Black arrow points to the apex zone of the extreme (EXTR) capsules, as well as the foot of the corona radiata ogive. (d, e, f) fascicles Y (black arrows) cover the uppermost EC and (CRp) and the cerebral peduncle (CP), are well-identified. The grey end in the most posterior globus pallidus (GP) (e) and the internal zone lateral to the EC corresponds to the claustrum (Cl). Voxel size: 3 3 capsule (IC) (f). Voxel size: 10 µm (a to f bar: 1 mm) 10 µm (bar: 1 mm)

dorsal zone of the putamen and the GP (Figure 2a-c). The ogival to the location with respect to the anterior commissure. The pal- morphology is not always visible along the rostrocaudal sequence of lidum was divided into ventral pallidum (VP) and GP according to the putamen. At post-commissural rostral levels (optic chiasm, ros- the sheep Michigan State University atlas (Johnson, Sudheimer, tral part of the optic tract, fornix), the medial side of the ogive was Davis, Kerndt, & Winn, 1998). The fascicles were classified accord- less compact due to the exit of fibres (fascicles X, described below), ing to their topographical location with respect to their entry to the terminating in the GP (Figure 2a and b). The lateral side of the ogive putamen, from dorsal to ventral. A letter was assigned to each rec- is always identifiable. This pattern alternates with zones with clear ognized fascicle. The uppermost fascicles coming from the IC and EC ogive-like morphology. At the level of the amygdala and the uncus of were labelled the supreme fascicles X and Y, respectively. The fibres hippocampus, where the striatal nuclei are reduced in size, the ogival exiting between both capsules were labelled the apex fibres. Below morphology is no longer visible. The bundles that come from the the supreme fascicles, fascicles that connected the EC with the GP uppermost dorsal part of the EC thicken and cross the putamen to were named Z fascicles and, according to their position, classified in enter into the GP (Figure 2e and f). In the most posterior part of the superior (Z1), middle (Z2) and lower (Z3) fascicles. GP, with the ventral hippocampus visible, the thick bundles from the EC cross the dorsal putamen to enter the IC (Figure 2f). This fibrous pattern continues up to the end of the putamen. 3.1 | IC-EC transitional zone

Below the foot of the corona radiata, the separation of the IC, EC 3.2 | Systematization of the bundles and extreme capsule became visible. Between the IC and the EC, a fibrous zone was observed, whose medial and lateral sides bordered 3.2.1 | Supreme fascicles the uppermost dorsal area of the putamen (Figures 1b and 2a-f). This gives the zone an ogive-like morphology, where the IC fibres from its Supreme fascicles arise from the ogive, cross the neuropil of the medial curve, and the EC fibres the lateral one (Figures 1b and 2a-d). putamen and terminate in the GP (Figures 2a-c, 3a and b). At the From both sides of the ogive, as well as from their apex, myelinated level of the optic chiasm, fascicles X arising from the IC, fasci- fascicles (supreme fascicles, described below) traverse between the cles Y arising from the EC and the apex fibres are distinguishable. MURILLO-GONZÁLEZ et al. | 87

FIGURE 3 (a, b, c) Supreme fascicles (a) (b) (c) (d) (X, Y) arising from the ogive zone and ending in the globus pallidus (GP). (d) Fascicles Y (black arrow) become thicker, hiding the upper part of the external capsule and cross the putamen in the direction of the most posterior part of the globus pallidus (GP). (e-h) Non- consecutive two-dimensional µCT images of a pre-commissural basal fascicle (white arrows) extending between the ventral part of the external capsule (EC) and the lateral zone of the ventral pallidum (VP)

(e) (f)

(g) (h)

Fascicles Y are seen in the anteroposterior direction of the puta- 3.2.2 | Pre-commissural fascicles men, connecting the EC with the GP (Figure 2b-d). Fascicles X enter the dorsal zone of GP and are recognized up to the posterior levels Ventral fascicles, approximately four to five per sample, and situated of the fornix and mammillothalamic tract (Figures 2b, c and 3a-c). in a similar position in the anteroposterior direction, extend between Some fascicles X and apex fascicles traverse the medial part of the ventral part of the EC and the lateral zone of the VP (Figures 3e- the GP and join the ventral part of the IC (Figure 2f). At posterior h, 4a and b). levels, where the hippocampal uncus, the fornix and the amygdala are visible, only apex and fascicles Y are observed. Fascicles X are not differentiated from the rest of the IC, and they are compact 3.2.3 | Post-commissural fascicles (Z) here (Figure 2d-f). As described above, the thickened fascicles Y hide the upper part of the EC, but cross the putamen and arrive at These fascicles are recognized as aggrupations of myelinated fibres the GP (Figures 2d and 3d). with a radiate appearance, separated by zones of putaminal neu- At the most posterior level of the putamen, fascicles Y cross ropil apparently devoid of, or sparse in, fibres. They connect the its dorsal zone and enter the IC (Figure 2e, f). upper, middle and lower parts of the EC with the GP. The fascicles 88 | MURILLO-GONZÁLEZ et al.

(a) (b) (c)

(d) (e) (f) (g)

FIGURE 4 (a-c) General overview of the sheep basal ganglia, at the (a) caudal pre-commissural, (b) commissural and (c) post-commissural levels. Z fascicles arising from the external capsule (EC) are seen at the post-commissural level (c, white circles). (d-g) Post-commissural fascicles (Z). (d-g) superior fascicles (Z1, black arrows) (e-g) middle fascicles (Z2) (white arrow). (g) Lower fascicle (Z3) (white arrowhead). Abbreviations: AC, anterior commissure; AD, anterodorsal thalamic nucleus; AV, anteroventral thalamic nucleus; Bv, blood vessel; Clx, claustrocortex; CN, caudate nucleus; CR, corona radiata; EC, external capsule; Fo, fornix; GP, globus pallidus; IC, internal capsule; LV, lateral ventricle; Mt, mammillothalamic tract; OT, optic tract; PCx, pyriform cortex; Put, Putamen; Pyr, pyriform lobe; Sep, lateral septal nucleus; VP, 3 ventral pallidum. Dichromate-µCT method. Voxel size: 10 µm . Bars: a-c, 3 mm; d-g, 1 mm are more evident and robust from the middle to posterior parts of imparted to the grey and white matter compartments allows the the GP (Figure 4c-g). The following fascicles are distinguishable: (a) identification of the putamen and the GP in the images, and their superior fascicles (Z1), connecting the EC with the middle zone of relationships with the IC and EC; it also and facilitates the observa- the GP (Figure 4d-g); (b) middle fascicles (Z2), connecting the EC tion of bundle trajectories. with the middle zone of the GP (Figure 4e-g); and (c) lower fascicles Studies regarding the myeloarchitecture of striatum in the sheep

(Z3) (Figure 4g), seldom observed, connecting the EC to the lower were not found in the literature, in comparison with studies found part of the GP. on other mammals including humans (Dejerine, 1980; Papez, 1941; Quinn & Graybiel, 1994; Schaltenbrand & Wahren, 1977; Sych, 1960; Wilson, 1914). The myeloarchitecture of the putamen has been mainly 4 | DISCUSSION concerned with the Wilson's bundles (Papez, 1938; Wilson, 1914). However, a topographical systematization of these bundles has not The myeloarchitecture of the putamen in the sheep has been studied been found in previous works. In the study described here, a topo- in relationship to the fibre pathways that come from cerebral cortex graphical systematization in bundles of the incoming fibres from the through the IC and EC to this nucleus. Also, GP-EC fibre connections IC and the EC, as well as the GP-EC connections, has been made. have been identified. For this purpose, the approach using the di- Since no previous studies were found in the sheep, the comparison of chromate-staining protocol under a laboratory-based µ-CT (Herrera tracts and fascicles identified here is based on normal myelin-stained et al., 2018) allows the three-dimensional visualization of myelinated material as well as experimental studies of the corticostriatal, cort- bundles in a non-destructive manner. Also, the differential contrast icopallidal and pallidocortical pathways in gyrencephalic mammals. MURILLO-GONZÁLEZ et al. | 89

The supreme fascicles leave the lateral and medial sides of the originating in the MI, the SMA, and a part of the SI is described in the ogive and appear to terminate predominantly in the GP. Although rat (Naito & Kita, 1994) as well as primates (Leichnetz & Astruc, 1977). not described as a specific anatomical zone, the ogive is repre- Also, Akkal, Dum, and Strick (2007) and Zhang, Ide, and Li (2012) re- sented in the drawings of studies in normal and experimental mate- ported connections between the pre-SMA and GP, the zone of entry rials (Dejerine, 1980; Leichnetz & Astruc, 1977; Papez, 1938, 1941; corresponding the dorsolateral zone of the IC. Milardi et al., (2015), Schaltenbrand & Wahren, 1977; Schmahmann & Pandya, 2006; using MRI-advanced techniques in humans, traced a direct pathway Sych, 1960; Webster, 1961) which studied the corticostriatal and connecting the cortex to the GP, but the zone of entry to the GP was corticopallidal projections. This study reveals that the ogive region not specified. is a topographical zone where subsets of the corticostriatal projec- It has been demonstrated in this work that fascicles arising from tions pass through to their target areas. It is well-established that the EC connect with the GP, which suggests there are Wilson's bun- all cerebral lobes contribute, together with striatal bundles, to the dles. However, in the sheep, it seems these are restricted to the cau- putamen (Heilbronner, Rodriguez-Romaguera, Quirk, Groenewegen, dal third of the GP, and they appear more compact than in humans. & Haber, 2016; Schmahmann & Pandya, 2006), but the contribu- The pallidocortical projection was first described by Nauta (1979) tions of the motor and parietal cortices exceeds the other lobes. in cats, where the fibres, on their way to the cortex, traverse the Corticostriatal fibres have two major components: the fasciculus dorsal putamen and enter the EC; according to his drawings, the subcallosus (Muratoff, 1893; Schmahmann & Pandya, 2006), which dorsal putaminal route in cats is located in a similar ogive-like re- mainly target the caudate nucleus and the putamen, and the EC, gion as that seen in sheep. This projection was confirmed in cats targeting the ventral part of the caudate nucleus, the putamen and (Jayaraman, 1980; Reinoso-Suarez, Llamas, & Avendaño, 1982; claustrum. (Kemp & Powell, 1971; Schmahmann & Pandya, 2006). Shinonaga et al., 1991) and their origin was identified from prefron- Schmahmann and Pandya (2006) described bundles that from the tal, motor and auditory areas, including the temporal polar gyrus. superior and inferior parietal lobes and enter through the ogive re- Fibres that leave the EC and end in the GP were not previously gion in their drawings, but not from the other parietal regions. This reported in the literature. However, the EC is one of the preferen- is supported by the descending projection and trajectory of degen- tial routes to the putamen for fibres coming from all cerebral lobes erating fibres from the primary somatosensory (SI) area (Carman (Schmahmann & Pandya, 2006; Tanaka, Gorska, & Dutkiewicz, 1981; et al., 1965; Kemp & Powell, 1970). These results suggest that a part Van Hoesen et al., 1981; Webster, 1961), including neurons situated of the Y fascicles identified here are the candidates to be SI fibres in the EC proper (Borra, Luppino, Gerbella, Rozzi, & Rockland, 2019). in the sheep, carrying somatosensory information to the putamen In primates, fibres from the parietal association cortices course and the GP. The same is true in monkeys for area 4 (MI), where pro- through the EC and terminate exclusively in the putamen. This fact jections from the trunk, hand and foot regions and the SMA (MII) contrasts with the zones of termination of the other parietal areas region, course through the ogive region and upper dorsolateral entering through the ogive region (Schmahmann & Pandya, 2006; EC (Jones & Powell, 1969; Kemp & Powell, 1970; Schmahmann & Webster, 1961). The same occurs for temporal areas, whose routes Pandya, 2006). Prefrontal projections are segregated into dorsal are situated ventrally in the EC, where they enter and end in the ven- and ventral trajectories. Through the ventral EC, fibres enter and tral putamen (Van Hoesen et al., 1981). Taken together, the evidence innervate heavily the rostral putamen and sparsely the caudal from previous sections suggests that the superior and middle fasci- putamen, with the incoming fibres through the medial side of the cles, and the inferior fibres described in the sheep could represent ogive being very sparse (Leichnetz & Astruc, 1977; Schmahmann & the sum of pallidocortical and corticoputaminal (via the EC route) Pandya, 2006; Webster, 1961). Van Hoesen, Yeterian, and Lavizzo- fibres. This topic needs further study, including an experimental con- Mourey (1981) described fibres from the temporal cortex entering firmation in sheep. the EC and ending in the caudate and putamen. In the putamen, the Therefore, in this study myelinated fascicles have been iden- fibres terminate mainly in the rostral and ventrocaudal putamen, as tified and the µ-CT images have showed a myelin-like picture confirmed by Schmahmann and Pandya (2006). Occipital fibres from similar to those obtained by using Weigert-type myelin stains the preoccipital gyrus enter through the apex and medial side of the (Déjerine & Déjerine-Klumpke, 1895; Papez, 1941; Schaltenbrand & ogive, while those from area V4 enter through the ventrorostral part Wahren, 1977; Sych, 1960). However, the fine striatopallidal fibres of the EC. Taking together the results obtained experimentally in between the putamen and the GP (Cowan & Powell, 1966) have not other mammals, corticostriatal fibres from the parietal and frontal been clearly identified. regions enter mainly through the ogive region, while those of the temporal, occipital and prefrontal lobes enter the putamen through the middle and ventral zones of the EC. 5 | CONCLUSION In this study, X fascicles from the medial part of the ogive leave from this zone and enter the GP, giving the zone an inhomogeneous The general myeloarchitectonic pattern of connections between appearance. This corroborates the observations of Webster (1961) the IC and EC and the GP has been demonstrated utilizing a novel in the rat. In the sheep, the IC-pallidal fibres appear visible only in laboratory-based µ-CT methodology. The present findings suggest the rostral half of the GP. A consistent corticopallidal projection that in the sheep presumptive corticostriatal, corticopallidal and 90 | MURILLO-GONZÁLEZ et al. pallidocortical fibres arrive at the pallidum, crossing the putamen ac- Dejerine, J. (1980). Anatomie des Centres Nerveux. Paris, France: Masson, SA. cording to dorsal, dorsolateral and ventrolateral topographies. The Descamps, E., Sochacka, A., de Kegel, B., Van Loo, D., Van Hoorebeke, L., topography and trajectories of fascicles in sheep are similar to other & Adriaens, D. (2014). Soft tissue discrimination with contrast agents investigated mammals. The non-invasive methodology used here is a using micro-CT scanning. Belgian Journal of Zoology, 144, 20–40. promising way to delineate the myeloarchitectonic patterns of nerv- https://doi.org/10.26496/bjz.2014.63 ous systems and tracts. Ella, A., Delgadillo, J. A., Chemineau, P., & Keller, M. (2017). Computation of a high-resolution MRI 3D stereotaxic atlas of the sheep brain. The Journal of Comparative Neurology, 525, 676–692. https://doi. ACKNOWLEDGEMENTS org/10.1002/cne.24079 We thank Dr. Isabel Sarró (Centro Nacional de Investigación sobre la Ella, A., & Keller, M. (2015). Construction of a MRI 3D high resolution Evolución Humana [CENIEH], Burgos, Spain) for the assistance given sheep brain template. Magnetic Resonance Imaging, 33, 1329–1337. https://doi.org/10.1016/j.mri.2015.09.001 to this work. Gignac, P. M., & Kley, N. J. (2014). Iodine-enhanced micro-CT imaging: Methodological refinements for the study of the soft-tissue anatomy CONFLICT OF INTEREST of post-embryonic vertebrates. Journal of Experimental Zoology Part This research did not receive any specific grant from funding agen- B, Molecular and Developmental Evolution, 322, 166–176. https://doi. org/10.1002/jez.b.22561 cies. The authors declare that there is no conflict of interest regard- Gignac, P. M., & Kley, N. J. (2018). 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